Bio

Academic Appointments


Research & Scholarship

Current Research and Scholarly Interests


Dr. Helms is a Professor in the Department of Surgery at Stanford University.

Her research interests center around regenerative medicine and craniofacial development.


Regenerative medicine

Every adult tissue harbors stem cells, which potentially could be used to regenerate damaged or diseased tissues. In my laboratory, one of our goals is to understand the regulatory pathways that control stem cell self-renewal, proliferation, and differentiation. We have focused on two signaling pathways whose activities seem to be an essential feature of tissue healing. Wnts and Hedgehog proteins are both lipid-modified growth factors that have well documented- and essential- roles in embryonic development. We have found that both pathways are active during the repair of bones, muscle, skin, heart, brain, and retina, and that repair of most or all of these tissues is impeded when these two pathways are blocked.
We have developed a novel packaging method whereby the biological activity of lipidated Wnt and Hedgehog proteins can be preserved in the in vivo wound environment. Using these and other tools developed by our collaborator Roel Nusse, we have embarked on experiments to first understand the mechanisms of action of these growth factors in the healing wound, and second, to use this information in biomimetic strategies to accelerate tissue repair.

Craniofacial morphogenesis

The long-term goal of this component of my research program is to elucidate the molecular and cellular mechanisms regulating normal and abnormal craniofacial development. We use a variety of animal models (mice, chicks, quails, ducks, pigeons) and approaches (genetic approaches, experimental manipulation of embryos) to understand how the face is patterned.


Prior to Stanford, Dr. Helms spent 8 years at the University of California at San Francisco, where she was the Director of the Molecular and Cellular Biology Laboratory in the Department of Orthopaedic Surgery. Dr. Helms received her dental degree from the University of Minnesota, and her residency certificate and Ph.D from the University of Connecticut Health Sciences Center.

Dr. Helms reviews manuscripts for leading journals such as Nature, Science, and Development and reviews grants for the NIH, NASA, March of Dimes, and a number of other non-profit organizations. She is President of the American Society of Craniofacial Genetics.

Dr. Helms is also an active teacher in both craniofacial and stem cell biology. She teaches undergraduate, graduate, and continuing education classes at Stanford. She mentors undergraduate and graduate students, dental and medical students, residents, and fellows and has been an advisor for Master’s and Ph.D candidates.

Teaching

2013-14 Courses


Postdoctoral Advisees


Publications

Journal Articles


  • Improving oral implant osseointegration in a murine model via Wnt signal amplification. Journal of clinical periodontology Mouraret, S., Hunter, D. J., Bardet, C., Popelut, A., Brunski, J. B., Chaussain, C., Bouchard, P., Helms, J. A. 2014; 41 (2): 172-180

    Abstract

    To determine the key biological events occurring during implant failure and then we use this knowledge to develop new biology-based strategies that improve osseointegration.Wild-type and Axin2(LacZ/LacZ) adult male mice underwent oral implant placement, with and without primary stability. Peri-implant tissues were evaluated using histology, alkaline phosphatase (ALP) activity, tartrate resistant acid phosphatase (TRAP) activity and TUNEL staining. In addition, mineralization sites, collagenous matrix organization and the expression of bone markers in the peri-implant tissues were assessed.Maxillary implants lacking primary stability show histological evidence of persistent fibrous encapsulation and mobility, which recapitulates the clinical problems of implant failure. Despite histological and molecular evidence of fibrous encapsulation, osteoblasts in the gap interface exhibit robust ALP activity. This mineralization activity is counteracted by osteoclast activity that resorbs any new bony matrix and consequently, the fibrous encapsulation remains. Using a genetic mouse model, we show that implants lacking primary stability undergo osseointegration, provided that Wnt signalling is amplified.In a mouse model of oral implant failure caused by a lack of primary stability, we find evidence of active mineralization. This mineralization, however, is outpaced by robust bone resorption, which culminates in persistent fibrous encapsulation of the implant. Fibrous encapsulation can be prevented and osseointegration assured if Wnt signalling is elevated at the time of implant placement.

    View details for DOI 10.1111/jcpe.12187

    View details for PubMedID 24164629

  • A pre-clinical murine model of oral implant osseointegration BONE Mouraret, S., Hunter, D. J., Bardet, C., Brunski, J. B., Bouchard, P., Helms, J. A. 2014; 58: 177-184

    Abstract

    Many of our assumptions concerning oral implant osseointegration are extrapolated from experimental models studying skeletal tissue repair in long bones. This disconnect between clinical practice and experimental research hampers our understanding of bone formation around oral implants and how this process can be improved. We postulated that oral implant osseointegration would be fundamentally equivalent to implant osseointegration elsewhere in the body. Mice underwent implant placement in the edentulous ridge anterior to the first molar and peri-implant tissues were evaluated at various timepoints after surgery. Our hypothesis was disproven; oral implant osseointegration is substantially different from osseointegration in long bones. For example, in the maxilla peri-implant pre-osteoblasts are derived from cranial neural crest whereas in the tibia peri-implant osteoblasts are derived from mesoderm. In the maxilla, new osteoid arises from periostea of the maxillary bone but in the tibia the new osteoid arises from the marrow space. Cellular and molecular analyses indicate that osteoblast activity and mineralization proceeds from the surfaces of the native bone and osteoclastic activity is responsible for extensive remodeling of the new peri-implant bone. In addition to histologic features of implant osseointegration, molecular and cellular assays conducted in a murine model provide new insights into the sequelae of implant placement and the process by which bone is generated around implants.

    View details for DOI 10.1016/j.bone.2013.07.021

    View details for Web of Science ID 000328304000023

    View details for PubMedID 23886841

  • CXCR4 Antagonism Attenuates Load-Induced Periosteal Bone Formation in Mice JOURNAL OF ORTHOPAEDIC RESEARCH Leucht, P., Temiyasathit, S., Russell, A., Arguello, J. F., Jacobs, C. R., Helms, J. A., Castillo, A. B. 2013; 31 (11): 1828-1838

    Abstract

    Mechanical loading is a key anabolic regulator of bone mass. Stromal cell-derived factor-1 (SDF-1) is a stem cell homing factor that is important in hematopoiesis, angiogenesis, and fracture healing, though its involvement in skeletal mechanoadaptation is virtually unknown. The objective of this study was to characterize skeletal expression patterns of SDF-1 and CXCR4, the receptor for SDF-1, and to determine the role of SDF-1 signaling in load-induced periosteal bone formation. Sixteen-week-old C57BL/6 mice were treated with PBS or AMD3100, an antagonist against CXCR4, and exposed to in vivo ulnar loading (2.8 N peak-to-peak, 2 Hz, 120 cycles). SDF-1 was expressed in cortical and trabecular osteocytes and marrow cells, and CXCR4 was primarily expressed in marrow cells. SDF-1 and CXCR4 expression was enhanced in response to mechanical stimulation. The CXCR4 receptor antagonist AMD3100 significantly attenuated load-induced bone formation and led to smaller adaptive changes in cortical geometric properties as determined by histomorphometric analysis. Our data suggest that SDF-1/CXCR4 signaling plays a critical role in skeletal mechanoadaptation, and may represent a unique therapeutic target for prevention and treatment of age-related and disuse bone loss. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res XX:XXX-XXX, 2013.

    View details for DOI 10.1002/jor.22440

    View details for Web of Science ID 000324930500022

    View details for PubMedID 23881789

  • Wnt3a Reestablishes Osteogenic Capacity to Bone Grafts from Aged Animals JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Leucht, P., Jiang, J., Cheng, D., Liu, B., Dhamdhere, G., Fang, M. Y., Monica, S. D., Urena, J. J., Cole, W., Smith, L. R., Castillo, A. B., Longaker, M. T., Helms, J. A. 2013; 95A (14): 1278-1288
  • Wnt3a reestablishes osteogenic capacity to bone grafts from aged animals. journal of bone and joint surgery. American volume Leucht, P., Jiang, J., Cheng, D., Liu, B., Dhamdhere, G., Fang, M. Y., Monica, S. D., Urena, J. J., Cole, W., Smith, L. R., Castillo, A. B., Longaker, M. T., Helms, J. A. 2013; 95 (14): 1278-1288

    Abstract

    Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity.Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]).Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls.Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals.We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

    View details for DOI 10.2106/JBJS.L.01502

    View details for PubMedID 23864176

  • Micromotion-induced strain fields influence early stages of repair at bone-implant interfaces. Acta biomaterialia Wazen, R. M., Currey, J. A., Guo, H., Brunski, J. B., Helms, J. A., Nanci, A. 2013; 9 (5): 6663-6674

    Abstract

    Implant loading can create micromotion at the bone-implant interface. The interfacial strain associated with implant micromotion could contribute to regulating the tissue healing response. Excessive micromotion can lead to fibrous encapsulation and implant loosening. Our objective was to characterize the influence of interfacial strain on bone regeneration around implants in mouse tibiae. A micromotion system was used to create strain under conditions of (1) no initial contact between implant and bone and (2) direct bone-implant contact. Pin- and screw-shaped implants were subjected to displacements of 150 or 300 ?m for 60 cycles per day for 7 days. Pin-shaped implants placed in five animals were subjected to three sessions of 150 ?m displacement per day, with 60 cycles per session. Control implants in both types of interfaces were stabilized throughout the healing period. Experimental strain analyses, microtomography, image-based displacement mapping, and finite element simulations were used to characterize interfacial strain fields. Calcified tissue sections were prepared and Goldner trichrome stained to evaluate the tissue reactions in higher and lower strain regions. In stable implants bone formation occurred consistently around the implants. In implants subjected to micromotion bone regeneration was disrupted in areas of high strain concentrations (e.g. >30%), whereas lower strain values were permissive of bone formation. Increasing implant displacement or number of cycles per day also changed the strain distribution and disturbed bone healing. These results indicate that not only implant micromotion but also the associated interfacial strain field contributes to regulating the interfacial mechanobiology at healing bone-implant interfaces.

    View details for DOI 10.1016/j.actbio.2013.01.014

    View details for PubMedID 23337705

  • Primary cilia act as mechanosensors during bone healing around an implant MEDICAL ENGINEERING & PHYSICS Leucht, P., Monica, S. D., Temiyasathit, S., Lenton, K., Manu, A., Longaker, M. T., Jacobs, C. R., Spilkere, R. L., Guo, H., Brunski, J. B., Helms, J. A. 2013; 35 (3): 392-402

    Abstract

    The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

    View details for DOI 10.1016/j.medengphy.2012.06.005

    View details for Web of Science ID 000315931400013

    View details for PubMedID 22784673

  • Wnt Signaling Promotes Muller Cell Proliferation and Survival after Injury INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Liu, B., Hunter, D. J., Rooker, S., Chan, A., Paulus, Y. M., Leucht, P., Nusse, Y., Nomoto, H., Helms, J. A. 2013; 54 (1): 444-453

    Abstract

    Müller glia respond to retinal injury by a reactive gliosis, but only rarely do mammalian glial cells re-enter the cell cycle and generate new neurons. In the nonmammalian retina, however, Müller glia act as stem/progenitor cells. Here, we tested the function of Wnt signaling in the postinjury retina, focusing on its ability to influence mammalian Müller cell dedifferentiation, proliferation, and neurogenesis.A 532 nm frequency doubled neodymium-doped yttrium aluminum garnet (ND:YAG) laser was used to create light burns on the retina of Axin2(LacZ/+) Wnt reporter mice. At various time points after injury, retinas were analyzed for evidence of Wnt signaling as well as glial cell response, proliferation, and apoptosis. Laser injuries also were created in Axin2(LacZ/LacZ) mice, and the effect of potentiated Wnt signaling on retinal repair was assessed.A subpopulation of mammalian Müller cells are Wnt responsive and, when Wnt signaling is increased, these cells showed enhanced proliferation in response to injury. In an environment of heightened Wnt signaling, caused by the loss of the Wnt negative regulator Axin2, Müller cells proliferated after injury and adopted the expression patterns of retinal progenitor cells (RPCs). The Wnt-responsive Müller cells also exhibited long-term survival and, in some cases, expressed the rod photoreceptor marker, rhodopsin.The Wnt pathway is activated by retinal injury, and prolonging the endogenous Wnt signal causes a subset of Müller cells to proliferate and dedifferentiate into RPCs. These data raised the possibility that transient amplification of Wnt signaling after retinal damage may unlock the latent regenerative capacity long speculated to reside in mammalian neural tissues.

    View details for DOI 10.1167/iovs.12-10774

    View details for Web of Science ID 000314338400055

    View details for PubMedID 23154457

  • Wntless functions in mature osteoblasts to regulate bone mass PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhong, Z., Zylstra-Diegel, C. R., Schumacher, C. A., Baker, J. J., Carpenter, A. C., Rao, S., Yao, W., Guan, M., Helms, J. A., Lane, N. E., Lang, R. A., Williams, B. O. 2012; 109 (33): E2197-E2204

    Abstract

    Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

    View details for DOI 10.1073/pnas.1120407109

    View details for Web of Science ID 000307807000005

    View details for PubMedID 22745162

  • Wnt Signaling and Injury Repair COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Whyte, J. L., Smith, A. A., Helms, J. A. 2012; 4 (8)

    Abstract

    Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. In this review we summarize recent data elucidating the roles that the Wnt pathway plays in the injury repair process. These data provide a foundation for potential Wnt-based therapeutic strategies aimed at stimulating tissue regeneration.

    View details for DOI 10.1101/cshperspect.a008078

    View details for Web of Science ID 000308030500007

    View details for PubMedID 22723493

  • Differentiation of multipotent vascular stem cells contributes to vascular diseases NATURE COMMUNICATIONS Tang, Z., Wang, A., Yuan, F., Yan, Z., Liu, B., Chu, J. S., Helms, J. A., Li, S. 2012; 3

    Abstract

    It is generally accepted that the de-differentiation of smooth muscle cells, from the contractile to the proliferative/synthetic phenotype, has an important role during vascular remodelling and diseases. Here we provide evidence that challenges this theory. We identify a new type of stem cell in the blood vessel wall, named multipotent vascular stem cells. Multipotent vascular stem cells express markers, including Sox17, Sox10 and S100?, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell-like cells that subsequently differentiate into smooth muscle cells. On the other hand, we perform lineage tracing with smooth muscle myosin heavy chain as a marker and find that multipotent vascular stem cells and proliferative or synthetic smooth muscle cells do not arise from the de-differentiation of mature smooth muscle cells. In response to vascular injuries, multipotent vascular stem cells, instead of smooth muscle cells, become proliferative, and differentiate into smooth muscle cells and chondrogenic cells, thus contributing to vascular remodelling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of multipotent vascular stem cells, rather than the de-differentiation of smooth muscle cells, contributes to vascular remodelling and diseases.

    View details for DOI 10.1038/ncomms1867

    View details for Web of Science ID 000306099900002

    View details for PubMedID 22673902

  • A Cross-Species Analysis of MicroRNAs in the Developing Avian Face PLOS ONE Powder, K. E., Ku, Y., Brugmann, S. A., Veile, R. A., Renaud, N. A., Helms, J. A., Lovett, M. 2012; 7 (4)

    Abstract

    Higher vertebrates use similar genetic tools to derive very different facial features. This diversity is believed to occur through temporal, spatial and species-specific changes in gene expression within cranial neural crest (NC) cells. These contribute to the facial skeleton and contain species-specific information that drives morphological variation. A few signaling molecules and transcription factors are known to play important roles in these processes, but little is known regarding the role of micro-RNAs (miRNAs). We have identified and compared all miRNAs expressed in cranial NC cells from three avian species (chicken, duck, and quail) before and after species-specific facial distinctions occur. We identified 170 differentially expressed miRNAs. These include thirty-five novel chicken orthologs of previously described miRNAs, and six avian-specific miRNAs. Five of these avian-specific miRNAs are conserved over 120 million years of avian evolution, from ratites to galliforms, and their predicted target mRNAs include many components of Wnt signaling. Previous work indicates that mRNA gene expression in NC cells is relatively static during stages when the beak acquires species-specific morphologies. However, miRNA expression is remarkably dynamic within this timeframe, suggesting that the timing of specific developmental transitions is altered in birds with different beak shapes. We evaluated one miRNA:mRNA target pair and found that the cell cycle regulator p27(KIP1) is a likely target of miR-222 in frontonasal NC cells, and that the timing of this interaction correlates with the onset of phenotypic variation. Our comparative genomic approach is the first comprehensive analysis of miRNAs in the developing facial primordial, and in species-specific facial development.

    View details for DOI 10.1371/journal.pone.0035111

    View details for Web of Science ID 000305345000043

    View details for PubMedID 22523571

  • Mechanosensing by the Primary Cilium: Deletion of Kif3A Reduces Bone Formation Due to Loading PLOS ONE Temiyasathit, S., Tang, W. J., Leucht, P., Anderson, C. T., Monica, S. D., Castillo, A. B., Helms, J. A., Stearns, T., Jacobs, C. R. 2012; 7 (3)

    Abstract

    Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Col?1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Col?1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Col?1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.

    View details for DOI 10.1371/journal.pone.0033368

    View details for Web of Science ID 000302381500135

    View details for PubMedID 22428034

  • Vascular endothelial growth factor improves bone repair in a murine nonunion model. The Iowa orthopaedic journal Ogilvie, C. M., Lu, C., Marcucio, R., Lee, M., Thompson, Z., Hu, D., Helms, J. A., Miclau, T. 2012; 32: 90-94

    Abstract

    Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays an important role during skeletal development and fracture healing. Previous experimental studies have shown that VEGF applied immediately after injury can stimulate bone repair in animal fracture nonunion models. However, the effectiveness of VEGF on an established fracture non-union has not been determined. the goal of this work was to test the ability of VEGF applied at a later stage on the healing of fracture nonunions.In this study, a murine non-union model was induced by rapid distraction of a tibia osteotomy. this model exhibits radiological and histological evidence of impaired fracture healing at 7 days after the completion of distraction. VEGF (10 µg in 20 µl Pbs/day, n=10) or control (20 µl Pbs/day, n=10) was injected directly into the distraction gap through the posterior musculature on three consecutive days (7, 8, and 9 days after completing distraction). A third group of animals (n=10) with rapid distraction, but no injections, served as non-treated controls. Fracture healing was analyzed by x-ray, histology, and histomorphometry at 27 days after the last round of distraction.radiographs showed that half of the VEGF treated animals (5/10) achieved bony healing whereas the majority of Pbs treated (7/10) and non-treated controls (8/10) did not exhibit bone bridging. Histological and histomorphometric analyses demonstrated that VEGF increased, but not significantly, the amount of bone formed in the distraction gap (1.35 ± 0.35 mm(3)), compared to the saline treated (0.77 ± 0.25 mm(3), p=0.19) and non-treated animals (0.79 ± 0.23mm(3), p=0.12).Results from this study demonstrate that VEGF potentially promotes bone repair, warranting further research in this direction.

    View details for PubMedID 23576927

  • Wnt/ß-catenin signaling and Msx1 promote outgrowth of the maxillary prominences. Frontiers in physiology Medio, M., Yeh, E., Popelut, A., Babajko, S., Berdal, A., Helms, J. A. 2012; 3: 375-?

    Abstract

    Facial morphogenesis requires a series of precisely orchestrated molecular events to promote the growth and fusion of the facial prominences. Cleft palate (CP) results from perturbations in this process. The transcriptional repressor Msx1 is a key participant in these molecular events, as demonstrated by the palatal clefting phenotype observed in Msx1(-/-) embryos. Here, we exploited the high degree of conservation that exists in the gene regulatory networks that shape the faces of birds and mice, to gain a deeper understanding of Msx1 function in CP. Histomorphometric analyses indicated that facial development was disrupted as early as E12.5 in Msx1(-/-) embryos, long before the palatal shelves have formed. By mapping the expression domain of Msx1 in E11.5 and E12.5 embryos, we found the structures most affected by loss of Msx1 function were the maxillary prominences. Maxillary growth retardation was accompanied by perturbations in angiogenesis that preceded the CP phenotype. Experimental chick manipulations and in vitro assays showed that the regulation of Msx1 expression by the Wnt/?-catenin pathway is highly specific. Our data in mice and chicks indicate a conserved role for Msx1 in regulating the outgrowth of the maxillary prominences, and underscore how imbalances in Msx1 function can lead of growth disruptions that manifest as CP.

    View details for DOI 10.3389/fphys.2012.00375

    View details for PubMedID 23055979

  • Indian Hedgehog Positively Regulates Calvarial Ossification and Modulates Bone Morphogenetic Protein Signaling GENESIS Lenton, K., James, A. W., Manu, A., Brugmann, S. A., Birker, D., Nelson, E. R., Leucht, P., Helms, J. A., Longaker, M. T. 2011; 49 (10): 784-796

    Abstract

    Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh-/- phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh-/- mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro-osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh-/- calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification.

    View details for DOI 10.1002/dvg.20768

    View details for Web of Science ID 000296420300003

    View details for PubMedID 21557453

  • A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression NATURE Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. 2011; 472 (7341): 120-U158

    Abstract

    The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.

    View details for DOI 10.1038/nature09819

    View details for Web of Science ID 000289199400049

    View details for PubMedID 21423168

  • The acceleration of implant osseointegration by liposomal Wnt3a BIOMATERIALS Popelut, A., Rooker, S. M., Leucht, P., Medio, M., Brunski, J. B., Helms, J. A. 2010; 31 (35): 9173-9181

    Abstract

    The strength of a Wnt-based strategy for tissue regeneration lies in the central role that Wnts play in healing. Tissue injury triggers local Wnt activation at the site of damage, and this Wnt signal is required for the repair and/or regeneration of almost all tissues including bone, neural tissues, myocardium, and epidermis. We developed a biologically based approach to create a transient elevation in Wnt signaling in peri-implant tissues, and in doing so, accelerated bone formation around the implant. Our subsequent molecular and cellular analyses provide mechanistic insights into the basis for this pro-osteogenic effect. Given the essential role of Wnt signaling in bone formation, this protein-based approach may have widespread application in implant osseointegration.

    View details for DOI 10.1016/j.biomaterials.2010.08.045

    View details for Web of Science ID 000284393300004

    View details for PubMedID 20864159

  • Craniofacial Ciliopathies: A New Classification for Craniofacial Disorders AMERICAN JOURNAL OF MEDICAL GENETICS PART A Brugmann, S. A., Cordero, D. R., Helms, J. A. 2010; 152A (12): 2995-3006

    Abstract

    Craniofacial anomalies are some of the most variable and common defects affecting the population. Herein, we examine a group of craniofacial disorders that are the result of defects in primary cilia; ubiquitous, microtubule-based organelles that transduce molecular signals and facilitate the interactions between the cell and its environment. Based on the frequent appearance of craniofacial phenotypes in diseases born from defective primary cilia (ciliopathies) we propose a new class of craniofacial disorders referred to as craniofacial ciliopathies. We explore the most frequent phenotypes associated with ciliopathic conditions and the ciliary gene mutations responsible for craniofacial defects. Finally, we propose that some non-classified disorders may now be classified as craniofacial ciliopathies.

    View details for DOI 10.1002/ajmg.a.33727

    View details for Web of Science ID 000285251800008

    View details for PubMedID 21108387

  • Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A James, A. W., Leucht, P., Levi, B., Carre, A. L., Xu, Y., Helms, J. A., Longaker, M. T. 2010; 16 (8): 2605-2616

    Abstract

    Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mouse ASCs, by all markers examined (*p < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*p < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using quantitative real-time polymerase chain reaction and in situ hybridization, we found that skeletal injury (a monocortical 1 mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mouse ASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration.

    View details for DOI 10.1089/ten.tea.2010.0048

    View details for Web of Science ID 000280648700018

    View details for PubMedID 20367246

  • rBMP Represses Wnt Signaling and Influences Skeletal Progenitor Cell Fate Specification During Bone Repair JOURNAL OF BONE AND MINERAL RESEARCH Minear, S., Leucht, P., Miller, S., Helms, J. A. 2010; 25 (6): 1196-1207

    Abstract

    Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well-documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously suppress the differentiation of skeletal progenitor cells in the endosteum and bone marrow cavity to an osteoblast lineage. Both the prochondrogenic and antiosteogenic effects are achieved because rBMP inhibits endogenous beta-catenin-dependent Wnt signaling. In the injured periosteum, this repression of Wnt activity results in sox9 upregulation; consequently, cells in the injured periosteum adopt a chondrogenic fate. In the injured endosteum, rBMP also inhibits Wnt signaling, which results in the runx2 and collagen type I downregulation; consequently, cells in this region fail to differentiate into osteoblasts. In muscle surrounding the skeletal injury site, rBMP treatment induces Smad phosphorylation followed by exuberant cell proliferation, an increase in alkaline phosphatase activity, and chondrogenic differentiation. Thus different populations of adult skeletal progenitor cells interpret the same rBMP stimulus in unique ways, and these responses mirror the pleiotropic effects of BMPs during fetal skeletogenesis. These mechanistic insights may be particularly useful for optimizing the reparative potential of rBMPs while simultaneously minimizing their adverse outcomes.

    View details for DOI 10.1002/jbmr.29

    View details for Web of Science ID 000279441300002

    View details for PubMedID 20200943

  • Molecular control of facial morphology SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Liu, B., Rooker, S. M., Helms, J. A. 2010; 21 (3): 309-313

    Abstract

    We present a developmental perspective on the concept of phylotypic and phenotypic stages of craniofacial development. Within orders of avians and mammals, a phylotypic period exists when the morphology of the facial prominences is minimally divergent. We postulate that species-specific facial variations arise as a result of subtle shifts in the timing and the duration of molecular pathway activity (e.g., heterochrony), and present evidence demonstrating a critical role for Wnt and FGF signaling in this process. The same molecular pathways that shape the vertebrate face are also implicated in craniofacial deformities, indicating that comparisons between and among animal species may represent a novel method for the identification of human craniofacial disease genes.

    View details for DOI 10.1016/j.semcdb.2009.09.002

    View details for Web of Science ID 000275805500011

    View details for PubMedID 19747977

  • Wnt Proteins Promote Bone Regeneration SCIENCE TRANSLATIONAL MEDICINE Minear, S., Leucht, P., Jiang, J., Liu, B., Zeng, A., Fuerer, C., Nusse, R., Helms, J. A. 2010; 2 (29)

    Abstract

    The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wnt ligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bone healing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair through the use of the mouse strain Axin2(LacZ/LacZ) in which the cellular response to Wnt is increased. We found that bone healing after injury is accelerated in Axin2(LacZ/LacZ) mice, a consequence of more robust proliferation and earlier differentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increase the duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in liposomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitor cells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end result was faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

    View details for DOI 10.1126/scitranslmed.3000231

    View details for Web of Science ID 000277304700001

    View details for PubMedID 20427820

  • A primary cilia-dependent etiology for midline facial disorders HUMAN MOLECULAR GENETICS Brugmann, S. A., Allen, N. C., James, A. W., Mekonnen, Z., Madan, E., Helms, J. A. 2010; 19 (8): 1577-1592

    Abstract

    Human faces exhibit enormous variation. When pathological conditions are superimposed on normal variation, a nearly unbroken series of facial morphologies is produced. When viewed in full, this spectrum ranges from cyclopia and hypotelorism to hypertelorism and facial duplications. Decreased Hedgehog pathway activity causes holoprosencephaly and hypotelorism. Here, we show that excessive Hedgehog activity, caused by truncating the primary cilia on cranial neural crest cells, causes hypertelorism and frontonasal dysplasia (FND). Elimination of the intraflagellar transport protein Kif3a leads to excessive Hedgehog responsiveness in facial mesenchyme, which is accompanied by broader expression domains of Gli1, Ptc and Shh, and reduced expression domains of Gli3. Furthermore, broader domains of Gli1 expression correspond to areas of enhanced neural crest cell proliferation in the facial prominences of Kif3a conditional knockouts. Avian Talpid embryos that lack primary cilia exhibit similar molecular changes and similar facial phenotypes. Collectively, these data support our hypothesis that a severe narrowing of the facial midline and excessive expansion of the facial midline are both attributable to disruptions in Hedgehog pathway activity. These data also raise the possibility that genes encoding ciliary proteins are candidates for human conditions of hypertelorism and FNDs.

    View details for DOI 10.1093/hmg/ddq030

    View details for Web of Science ID 000276544000017

    View details for PubMedID 20106874

  • Comparative gene expression analysis of avian embryonic facial structures reveals new candidates for human craniofacial disorders HUMAN MOLECULAR GENETICS Brugmann, S. A., Powder, K. E., Young, N. M., Goodnough, L. H., Hahn, S. M., James, A. W., Helms, J. A., Lovett, M. 2010; 19 (5): 920-930

    Abstract

    Mammals and birds have common embryological facial structures, and appear to employ the same molecular genetic developmental toolkit. We utilized natural variation found in bird beaks to investigate what genes drive vertebrate facial morphogenesis. We employed cross-species microarrays to describe the molecular genetic signatures, developmental signaling pathways and the spectrum of transcription factor (TF) gene expression changes that differ between cranial neural crest cells in the developing beaks of ducks, quails and chickens. Surprisingly, we observed that the neural crest cells established a species-specific TF gene expression profile that predates morphological differences between the species. A total of 232 genes were differentially expressed between the three species. Twenty-two of these genes, including Fgfr2, Jagged2, Msx2, Satb2 and Tgfb3, have been previously implicated in a variety of mammalian craniofacial defects. Seventy-two of the differentially expressed genes overlap with un-cloned loci for human craniofacial disorders, suggesting that our data will provide a valuable candidate gene resource for human craniofacial genetics. The most dramatic changes between species were in the Wnt signaling pathway, including a 20-fold up-regulation of Dkk2, Fzd1 and Wnt1 in the duck compared with the other two species. We functionally validated these changes by demonstrating that spatial domains of Wnt activity differ in avian beaks, and that Wnt signals regulate Bmp pathway activity and promote regional growth in facial prominences. This study is the first of its kind, extending on previous work in Darwin's finches and provides the first large-scale insights into cross-species facial morphogenesis.

    View details for DOI 10.1093/hmg/ddp559

    View details for Web of Science ID 000274341400015

    View details for PubMedID 20015954

  • Role of Wnt Signaling in the Biology of the Periodontium DEVELOPMENTAL DYNAMICS Rooker, S. M., Liu, B., Helms, J. A. 2010; 239 (1): 140-147

    Abstract

    Continuously erupting teeth have associated with them a continuously regenerating periodontal ligament, but the factors that control this amazing regenerative potential are unknown. We used genetic strategies to show that the periodontal ligament arises from the cranial neural crest. Despite their histological similarity, the periodontal ligament of continuously erupting incisor teeth differs dramatically from the periodontal ligament of molar teeth. The most notable difference was in the distribution of Wnt responsive cells in the incisor periodontal ligament, which coincided with regions of periodontal ligament cell proliferation. We discuss these findings in the context of dental tissue regeneration.

    View details for DOI 10.1002/dvdy.22003

    View details for Web of Science ID 000273703900012

    View details for PubMedID 19530172

  • Regeneration, repair and remembering identity: the three Rs of Hox gene expression TRENDS IN CELL BIOLOGY Wang, K. C., Helms, J. A., Chang, H. Y. 2009; 19 (6): 268-275

    Abstract

    Hox genes encode transcription factors that specify embryonic positional identity in cells and guide tissue differentiation. Recent advances have greatly increased our understanding of the epigenetic mechanisms that ensure the faithful expression of Hox genes in adult cells and which involve the interplay of histone methylation, demethylation and intergenic transcription of long non-coding RNAs. The transcriptional memory of Hox genes poses both an opportunity and a challenge for regenerative medicine. Matching the positional identity of transplanted stem cells with that of the host environment, as reflected by their respective Hox profiles, is likely to be required to achieve regenerative healing. Strategies to manipulate the plasticity of Hox gene expression will probably become a major focus in regenerative medicine.

    View details for DOI 10.1016/j.tcb.2009.03.007

    View details for Web of Science ID 000267449500004

    View details for PubMedID 19428253

  • Endochondral ossification is required for haematopoietic stem-cell niche formation NATURE Chan, C. K., Chen, C., Luppen, C. A., Kim, J., DeBoer, A. T., Wei, K., Helms, J. A., Kuo, C. J., Kraft, D. L., Weissman, I. L. 2009; 457 (7228): 490-U9

    Abstract

    Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

    View details for DOI 10.1038/nature07547

    View details for Web of Science ID 000262519200049

    View details for PubMedID 19078959

  • CONTROLLING THE IN VIVO ACTIVITY OF WNT LIPOSOMES METHODS IN ENZYMOLOGY LIPOSOMES, PT G Zhao, L., Rooker, S. M., Morrell, N., Leucht, P., Simanovskii, D., Helms, J. A. 2009; 465: 331-347

    Abstract

    Liposomes offer a method of delivering small molecules, nucleic acids, and proteins to sites within the body. Typically, bioactive materials are encapsulated within the liposomal aqueous core and liposomal phase transition is elicited by pH or temperature changes. We developed a new class of liposomes for the in vivo delivery of lipid-modified proteins. First, we show that the inclusion of a chromophore into the liposomal or vesosomal membrane renders these lipid vesicles extremely sensitive to very small (muJ) changes in energy. Next, we demonstrate that the lipid-modified Wnt protein is not encapsulated within a liposome but rather is tethered to the exoliposomal surface in an active configuration. When applied to intact skin, chromophore-modified liposomes do not penetrate past the corneal layer of the epidermis, but remain localized to the site of application. Injury to the epidermis allows rapid penetration of liposomes into the dermis, which suggests that mild forms of dermabrasion will greatly enhance transdermal delivery of liposome-packaged molecules. Finally, we demonstrate that topical application of Wnt3a liposomes rapidly stimulates proliferation of cells in the corneal layer, resulting in a thicker, more fibrillous epidermis.

    View details for DOI 10.1016/S0076-6879(09)65017-5

    View details for Web of Science ID 000272400200018

    View details for PubMedID 19913175

  • Translating insights from development into regenerative medicine: The function of Wnts in bone biology SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Leucht, P., Minear, S., Ten Berge, D., Nusse, R., Helms, J. A. 2008; 19 (5): 434-443

    Abstract

    The Wnt pathway constitutes one of the most attractive candidates for modulating skeletal tissue regeneration based on its functions during skeletal development and homeostasis. Wnts participate in every stage of skeletogenesis, from the self-renewal and proliferation of skeletal stem cells to the specification of osteochondroprogenitor cells and the maturation of chondrocytes and osteoblasts. We propose that the function of Wnts depend upon a skeletogenic cell's state of differentiation. In this review we summarize recent data with a focus on the roles of Wnt signaling in mesenchymal stem cell fate, osteoprogenitor cell differentiation, chondrocyte maturation, bone remodeling, and bone regeneration.

    View details for DOI 10.1016/j.semcdb.2008.09.002

    View details for Web of Science ID 000262670500003

    View details for PubMedID 18824114

  • Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development DEVELOPMENT ten Berge, D., Brugmann, S. A., Helms, J. A., Nusse, R. 2008; 135 (19): 3247-3257

    Abstract

    A fundamental question in developmental biology is how does an undifferentiated field of cells acquire spatial pattern and undergo coordinated differentiation? The development of the vertebrate limb is an important paradigm for understanding these processes. The skeletal and connective tissues of the developing limb all derive from a population of multipotent progenitor cells located in its distal tip. During limb outgrowth, these progenitors segregate into a chondrogenic lineage, located in the center of the limb bud, and soft connective tissue lineages located in its periphery. We report that the interplay of two families of signaling proteins, fibroblast growth factors (FGFs) and Wnts, coordinate the growth of the multipotent progenitor cells with their simultaneous segregation into these lineages. FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification. Withdrawal of both signals results in cell cycle withdrawal and chondrogenic differentiation. Continued exposure to Wnt, however, maintains proliferation and re-specifies the cells towards the soft connective tissue lineages. We have identified target genes that are synergistically regulated by Wnts and FGFs, and show how these factors actively suppress differentiation and promote growth. Finally, we show how the spatial restriction of Wnt and FGF signals to the limb ectoderm, and to a specialized region of it, the apical ectodermal ridge, controls the distribution of cell behaviors within the growing limb, and guides the proper spatial organization of the differentiating tissues.

    View details for DOI 10.1242/dev.023176

    View details for Web of Science ID 000258989400009

    View details for PubMedID 18776145

  • Primary cilia: Cellular sensors for the skeleton ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY Anderson, C. T., Castillo, A. B., Brugmann, S. A., Helms, J. A., Jacobs, C. R., Stearns, T. 2008; 291 (9): 1074-1078

    Abstract

    The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions.

    View details for DOI 10.1002/ar.20754

    View details for Web of Science ID 000259324900004

    View details for PubMedID 18727074

  • Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration DEVELOPMENT Leucht, P., Kim, J., Amasha, R., James, A. W., Girod, S., Helms, J. A. 2008; 135 (17): 2845-2854

    Abstract

    The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

    View details for DOI 10.1242/dev.023788

    View details for Web of Science ID 000258395500003

    View details for PubMedID 18653558

  • Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity PLOS ONE Morrell, N. T., Leucht, P., Zhao, L., Kim, J., Ten Berge, D., Ponnusamy, K., Carre, A. L., Dudek, H., Zachlederova, M., McElhaney, M., Brunton, S., Gunzner, J., Callow, M., Polakis, P., Costa, M., Zhang, X. M., Helms, J. A., Nusse, R. 2008; 3 (8)

    Abstract

    Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

    View details for DOI 10.1371/journal.pone.0002930

    View details for Web of Science ID 000264412600016

    View details for PubMedID 18698373

  • Beta-catenin-dependent Wnt signaling in mandibular bone regeneration. journal of bone and joint surgery. American volume Leucht, P., Kim, J., Helms, J. A. 2008; 90: 3-8

    Abstract

    Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.

    View details for DOI 10.2106/JBJS.G.01136

    View details for PubMedID 18292349

  • A dermal HOX transcriptional program regulates site-specific epidermal fate GENES & DEVELOPMENT Rinn, J. L., Wang, J. K., Allen, N., Brugmann, S. A., Mikels, A. J., Liu, H., Ridky, T. W., Stadler, H. S., Nusse, R., Helms, J. A., Chang, H. Y. 2008; 22 (3): 303-307

    Abstract

    Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

    View details for DOI 10.1101/gad.1610508

    View details for Web of Science ID 000253170400005

    View details for PubMedID 18245445

  • Periosteal biaxial residual strains correlate with bone specific growth rates in chick embryos COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING Chen, J. C., Zhao, B., Longaker, M. T., Helms, J. A., Carter, D. R. 2008; 11 (5): 453-461

    Abstract

    It has been proposed that periosteal residual tensile strains influence periosteal bone apposition and endochondral ossification. The role of bone growth rates on the development of residual strains is not well known. This study examined the relationships between specific growth rate and residual strains in chick tibiotarsi. We measured length and circumference during embryonic days 11-20 using microCT. Bones grew faster in length, with longitudinal and circumferential specific growth rates decreasing from 17 to 9% and 14 to 8% per day, respectively. To calculate residual strains, opening dimensions of incisions through the periosteum were analysed using finite element techniques. Results indicate that Poisson's ratio for an isotropic material model is between 0 and 0.04. For the model with Poisson's ratio 0.03, longitudinal and circumferential residual strains decreased from 46.2 to 29.3% and 10.6 to 3.9%, respectively, during embryonic days 14-20. Specific growth rates and residual strains were positively correlated (p<0.05).

    View details for DOI 10.1080/10255840802129817

    View details for Web of Science ID 000260457900004

    View details for PubMedID 18608339

  • Bone regeneration is regulated by Wnt signaling JOURNAL OF BONE AND MINERAL RESEARCH Kim, J., Leucht, P., Lam, K., Luppen, C., Ten Berge, D., Nusse, R., Helms, J. A. 2007; 22 (12): 1913-1923

    Abstract

    Tissue regeneration is increasingly viewed as reactivation of a developmental process that, when misappropriated, can lead to malignant growth. Therefore, understanding the molecular and cellular pathways that govern tissue regeneration provides a glimpse into normal development as well as insights into pathological conditions such as cancer. Herein, we studied the role of Wnt signaling in skeletal tissue regeneration.Some adult tissues have the ability to regenerate, and among these, bone is one of the most remarkable. Bone exhibits a persistent, lifelong capacity to reform after injury, and continual bone regeneration is a prerequisite to maintaining bone mass and density. Even slight perturbations in bone regeneration can have profound consequences, as exemplified by conditions such as osteoporosis and delayed skeletal repair. Here, our goal was to determine the role of Wnts in adult bone regeneration.Using TOPgal reporter mice, we found that damage to the skeleton instigated Wnt reporter activity, specifically at the site of injury. We used a skeletal injury model to show that Wnt inhibition, achieved through adenoviral expression of Dkk1 in the adult skeleton, prevented the differentiation of osteoprogenitor cells.As a result, injury-induced bone regeneration was reduced by 84% compared with controls. Constitutive activation of the Wnt pathway resulting from a mutation in the Lrp5 Wnt co-receptor results in high bone mass, but our experiments showed that this same point mutation caused a delay in bone regeneration. In these transgenic mice, osteoprogenitor cells in the injury site were maintained in a proliferative state and differentiation into osteoblasts was delayed.When considered together, these data provide a framework for understanding the roles of Wnt signaling in adult bone regeneration and suggest a feasible approach to treating clinical conditions where enhanced bone formation is desired.

    View details for DOI 10.1359/JBMR.070802

    View details for Web of Science ID 000251292400012

    View details for PubMedID 17696762

  • Cross-regulatory interactions between Fgf8 and Shh in the avian frontonasal prominence. Congenital anomalies Abzhanov, A., Cordero, D. R., Sen, J., Tabin, C. J., Helms, J. A. 2007; 47 (4): 136-148

    Abstract

    The frontonasal prominence of the developing avian embryo contains an organizing center, defined by juxtaposition of the Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. This molecular interface presages any detectable growth of the frontonasal prominence, and experiments involving transplantation of this boundary epithelium have demonstrated it is a source of dorsal-ventral and rostral-caudal patterning information for the neural crest-derived mesenchyme of the upper beak. We explored the ontogeny of this organizing center by mapping the expression domains of both genes and their receptors and downstream targets. We tested the extent to which Shh and Fgf8 regulate each other's expression in this frontonasal organizer by either blocking or ectopically activating these pathways. Our experiments revealed mutual antagonism between the two molecules, which aids in establishing and maintaining a molecular boundary that subsequently influences patterning and growth of the middle and upper face.

    View details for PubMedID 17988255

  • Bone voyage: An expedition into the molecular and cellular parameters affecting bone graft fate BONE Helms, J. A., Amasha, R. R., Leucht, P. 2007; 41 (4): 479-485

    Abstract

    The demand for bone grafts in orthopaedic and craniofacial surgery is steadily increasing. Estimations suggest that about 500,000 are performed annually in the United States that include bone grafting as a component of the surgery, and the majority of these surgeries employ autografts. This perspective focuses on the biological events that occur during osseointegration of such bone grafts. Here, three key factors of graft osseointegration--the embryonic origin, the inclusion of skeletal progenitor cells, and the integrity of the recipient site--are discussed. Altogether, they form the foundation for survival of the bone graft and eventually for a positive clinical outcome of the procedure.

    View details for DOI 10.1016/j.bone.2007.06.023

    View details for Web of Science ID 000249800800001

    View details for PubMedID 17692586

  • Shaping up and shipping out: the role of cilia in growth and patterning. Journal of musculoskeletal & neuronal interactions Brugmann, S., Helms, J. 2007; 7 (4): 300-?

    View details for PubMedID 18094481

  • Wnt signaling mediates regional specification in the vertebrate face DEVELOPMENT Brugmann, S. A., Goodnough, L. H., Gregorieff, A., Leucht, P., Ten Berge, D., Fuerer, C., Clevers, H., Nusse, R., Helms, J. A. 2007; 134 (18): 3283-3295

    Abstract

    At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

    View details for DOI 10.1242/dev.005132

    View details for Web of Science ID 000249013700006

    View details for PubMedID 17699607

  • Molecular analysis of healing at a bone-implant interface JOURNAL OF DENTAL RESEARCH Colnot, C., Romero, D. M., Huang, S., Rahman, J., Currey, J. A., Nanci, A., Brunski, J. B., Helms, J. A. 2007; 86 (9): 862-867

    Abstract

    While bone healing occurs around implants, the extent to which this differs from healing at sites without implants remains unknown. We tested the hypothesis that an implant surface may affect the early stages of healing. In a new mouse model, we made cellular and molecular evaluations of healing at bone-implant interfaces vs. empty cortical defects. We assessed healing around Ti-6Al-4V, poly(L-lactide-co-D,L,-lactide), and 303 stainless steel implants with surface characteristics comparable with those of commercial implants. Our qualitative cellular and molecular evaluations showed that osteoblast differentiation and new bone deposition began sooner around the implants, suggesting that the implant surface and microenvironment around implants favored osteogenesis. The general stages of healing in this mouse model resembled those in larger animal models, and supported the use of this new model as a test bed for studying cellular and molecular responses to biomaterial and biomechanical conditions.

    View details for Web of Science ID 000249013200011

    View details for PubMedID 17720856

  • Stage-dependent craniofacial defects resulting from Sprouty2 overexpression DEVELOPMENTAL DYNAMICS Goodnough, L. H., Brugmann, S. A., Hu, D., Helms, J. A. 2007; 236 (7): 1918-1928

    Abstract

    Sprouty genes encode intracellular regulators of receptor tyrosine kinases that function in a variety of developmental events. Although mice carrying null mutations in Sprouty genes exhibit craniofacial anomalies, the precise role of these regulatory proteins in facial development remains unclear. Here, we show that overexpression of spry2 at the initiation of craniofacial development results in a dramatic arrest in outgrowth of the facial prominences. Although endogenous spry2 and fibroblast growth factor 8 (fgf8) are coexpressed throughout much of craniofacial development, overexpression of spry2 did not alter the spatiotemporal patterns of fgf target gene expression. The morphological consequences of spry2 overexpression were specific: all of the facial prominences were truncated, but despite this gross malformation, the programs of osteogenesis and chondrogenesis were not impaired. Collectively, these data suggest that Sprouty2 plays a role in the outgrowth of facial prominences independent of canonical Fgf signaling.

    View details for DOI 10.1002/dvdy.21195

    View details for Web of Science ID 000248041000018

    View details for PubMedID 17576140

  • Accelerated bone repair after plasma laser corticotomies ANNALS OF SURGERY Leucht, P., Lam, K., Kim, J., Mackanos, M. A., Simanovskii, D. M., Longaker, M. T., Contag, C. H., Schwettman, H. A., Helms, J. A. 2007; 246 (1): 140-150

    Abstract

    To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal.Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing.Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling.Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery.In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

    View details for DOI 10.1097/01.sla.0000258559.07435.b3

    View details for Web of Science ID 000247672300022

    View details for PubMedID 17592303

  • Reconciling the roles of FAK in osteoblast differentiation, osteoclast remodeling, and bone regeneration BONE Kim, J., Leuch, P., Luppen, C. A., Park, Y. J., Beggs, H. E., Damsky, C. H., Helms, J. A. 2007; 41 (1): 39-51

    Abstract

    Integrins link the inside of a cell with its outside environment and in doing so regulate a wide variety of cell behaviors. Integrins are well known for their roles in angiogenesis and cell migration but their functions in bone formation are less clear. The majority of integrin signaling proceeds through focal adhesion kinase (FAK), an essential component of the focal adhesion complex. We generated transgenic mice in which FAK was deleted in osteoblasts and uncovered a previously unknown role in osteoblast differentiation associated with bone healing. FAK mutant cells migrated to the site of skeletal injury and angiogenesis was unaffected yet the transgenic mice still exhibited numerous defects in reparative bone formation. Osteoblast differentiation itself was unperturbed by the loss of FAK, whereas the attachment of osteoclasts to the bone matrix was disrupted in vivo. We postulate that defective bi-directional integrin signaling affects the organization of the collagen matrix. Finally, we present a compensatory candidate molecule, Pyk2, which localized to the focal adhesions in osteoblasts that were lacking FAK.

    View details for DOI 10.1016/j.bone.2007.01.024

    View details for Web of Science ID 000247854900006

    View details for PubMedID 17459803

  • Functional demarcation of active and silent chromatin domains in human HOX loci by Noncoding RNAs CELL Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., Chang, H. Y. 2007; 129 (7): 1311-1323

    Abstract

    Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

    View details for DOI 10.1016/j.cell.2007.05.022

    View details for Web of Science ID 000247911400017

    View details for PubMedID 17604720

  • FAK-Mediated Mechanotransduction in Skeletal Regeneration PLOS ONE Leucht, P., Kim, J., Currey, J. A., Brunski, J., Helms, J. A. 2007; 2 (4)

    Abstract

    The majority of cells are equipped to detect and decipher physical stimuli, and then react to these stimuli in a cell type-specific manner. Ultimately, these cellular behaviors are synchronized to produce a tissue response, but how this is achieved remains enigmatic. Here, we investigated the genetic basis for mechanotransduction using the bone marrow as a model system. We found that physical stimuli produced a pattern of principal strain that precisely corresponded to the site-specific expression of sox9 and runx2, two transcription factors required for the commitment of stem cells to a skeletogenic lineage, and the arrangement and orientation of newly deposited type I collagen fibrils. To gain insights into the genetic basis for skeletal mechanotransduction we conditionally inactivated focal adhesion kinase (FAK), an intracellular component of the integrin signaling pathway. By doing so we abolished the mechanically induced osteogenic response and thus identified a critical genetic component of the molecular machinery required for mechanotransduction. Our data provide a new framework in which to consider how physical forces and molecular signals are synchronized during the program of skeletal regeneration.

    View details for DOI 10.1371/journal.pone.0000390

    View details for Web of Science ID 000207445600004

    View details for PubMedID 17460757

  • Effect of mechanical stimuli on skeletal regeneration around implants BONE Leucht, P., Kim, J., Wazen, R., Currey, J. A., Nanci, A., Brunski, J. B., Heims, J. A. 2007; 40 (4): 919-930

    Abstract

    Due to the aging population and the increasing need for total joint replacements, osseointegration is of a great interest for various clinical disciplines. Our objective was to investigate the molecular and cellular foundation that underlies this process. Here, we used an in vivo mouse model to study the cellular and molecular response in three distinct areas of unloaded implants: the periosteum, the gap between implant and cortical bone, and the marrow space. Our analyses began with the early phases of healing, and continued until the implants were completely osseointegrated. We investigated aspects of osseointegration ranging from vascularization, cell proliferation, differentiation, and bone remodeling. In doing so, we gained an understanding of the healing mechanisms of different skeletal tissues during unloaded implant osseointegration. To continue our analysis, we used a micromotion device to apply a defined physical stimulus to the implants, and in doing so, we dramatically enhanced bone formation in the peri-implant tissue. By comparing strain measurements with cellular and molecular analyses, we developed an understanding of the correlation between strain magnitudes and fate decisions of cells shaping the skeletal regenerate.

    View details for DOI 10.1016/j.bone.2006.10.027

    View details for Web of Science ID 000245419800015

    View details for PubMedID 17175211

  • Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm DEVELOPMENTAL BIOLOGY Haworth, K. E., Wilson, J. M., Grevellee, A., Cobourne, M. T., Healy, C., Helms, J. A., Sharpe, P. T., Tucker, A. S. 2007; 303 (1): 244-258

    Abstract

    Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.

    View details for DOI 10.1016/j.ydbio.2006.11.009

    View details for Web of Science ID 000244542800021

    View details for PubMedID 17187772

  • Visualizing in vivo liposomal drug delivery in real-time JOURNAL OF DRUG TARGETING Kim, J., Leucht, P., Morrell, N. T., Schwettman, H. A., Helms, J. A. 2007; 15 (9): 632-639

    Abstract

    Liposomes have tremendous potential for efficient small molecule delivery. Previous studies, however, have been hampered by an inability to monitor their distribution and release of contents. Here, the authors demonstrate the real time monitoring of small molecule delivery using luciferin as a model. To monitor the release of luciferin in vivo, luciferin was packaged in thermosensitive liposomes and delivered into transgenic mice that constitutively express luciferase. Their experiments show the thermally induced release of the liposomal content in real time. In addition, the model provides evidence that the thermosensitive liposomes are stable over a long period of time ( approximately 3 weeks), and still release their content upon heating. These data present a strategy to monitor liposomal drug delivery in vivo with luciferin.

    View details for DOI 10.1080/10611860701538651

    View details for Web of Science ID 000251334600007

    View details for PubMedID 17968717

  • Rapid growth of cartilage rudiments may generate perichondrial structures by mechanical induction BIOMECHANICS AND MODELING IN MECHANOBIOLOGY Henderson, J. H., De la Fuente, L., Romero, D., Colnot, C. I., Huang, S., Carter, D. R., Helms, J. A. 2007; 6 (1-2): 127-137

    Abstract

    Experimental and theoretical research suggest that mechanical stimuli may play a role in morphogenesis. We investigated whether theoretically predicted patterns of stress and strain generated during the growth of a skeletal condensation are similar to in vivo expression patterns of chondrogenic and osteogenic genes. The analysis showed that predicted patterns of compressive hydrostatic stress (pressure) correspond to the expression patterns of chondrogenic genes, and predicted patterns of tensile strain correspond to the expression patterns of osteogenic genes. Furthermore, the results of iterative application of the analysis suggest that stresses and strains generated by the growing condensation could promote the formation and refinement of stiff tissue surrounding the condensation, a prediction that is in agreement with an observed increase in collagen bundling surrounding the cartilage condensation, as indicated by picro-sirius red staining. These results are consistent with mechanical stimuli playing an inductive or maintenance role in the developing cartilage and associated perichondrium and bone collar. This theoretical analysis provides insight into the potential importance of mechanical stimuli during the growth of skeletogenic condensations.

    View details for DOI 10.1007/s10237-006-0038-x

    View details for Web of Science ID 000243873500014

    View details for PubMedID 16691413

  • Looking different: Understanding diversity in facial form AMERICAN JOURNAL OF MEDICAL GENETICS PART A Brugmann, S. A., Kim, J., Helms, J. A. 2006; 140A (23): 2521-2529

    Abstract

    The face is perhaps the most distinguishing feature of the vertebrate body. Six billion human faces decorate the earth, each of them unique and exceptional in their own way. Likewise, facial variation is the cornerstone of species-specific diversity within the animal kingdom. Yet despite this multiplicity in form, the underlying architecture of the vertebrate face is remarkably conserved. If early embryos of different species first resemble one another, then how is this facial diversity generated? Our primary goal is to elucidate the molecular origins of species-specific craniofacial morphogenesis. We examined one facial primordia, the frontonasal prominence, of phylogenetically related (chick vs. quail vs. duck) and distant (mouse vs. chick) embryos and asked how such drastically different forms (e.g., beak, bill, or muzzle) could be generated from a once-similar entity. We examined the morphological ontogeny and a number of molecular expression patterns in an attempt to shed light on when species-specific variations occur and what molecules (BMPs, FGFs, etc.) are implicated in its differential growth. We hypothesize that subtle changes in the signaling of these morphogens can reproducibly alter the morphology of the frontonasal prominence. Taken together, these data facilitate our fledgling understanding of the process by which facial morphogenesis is regulated.

    View details for DOI 10.1002/ajmg.a.31361

    View details for Web of Science ID 000242466100005

  • Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Colnot, C., Huang, S., Helms, J. 2006; 350 (3): 557-561

    Abstract

    The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.

    View details for DOI 10.1016/j.bbrc.2006.09.079

    View details for Web of Science ID 000241584300009

    View details for PubMedID 17022937

  • Craniofacial tissue engineering by stem cells JOURNAL OF DENTAL RESEARCH Mao, J. J., Giannobile, W. V., Helms, J. A., Hollister, S. J., Krebsbach, P. H., Longaker, M. T., Shi, S. 2006; 85 (11): 966-979

    Abstract

    Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

    View details for Web of Science ID 000241520600001

    View details for PubMedID 17062735

  • The molecular origins of species-specific facial pattern CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 73 Brugmann, S. A., Tapadia, M. D., Helms, J. A. 2006; 73: 1-?

    Abstract

    The prevailing approach within the field of craniofacial development is focused on finding a balance between tissues (e.g., facial epithelia, neuroectoderm, and neural crest) and molecules (e.g., bone morphogenetic proteins, fibroblast growth factors, Wnts) that play a role in sculpting the face. We are rapidly learning that neither these tissues nor molecular signals are able to act in isolation; in fact, molecular cues are constantly reciprocating signals between the epithelia and the neural crest in order to pattern and mold facial structures. More recently, it has been proposed that this crosstalk is often mediated and organized by discrete organizing centers within the tissues that are able to act as a self-contained unit of developmental potential (e.g., the rhombomere and perhaps the ectomere). Whatever the molecules are and however they are interpreted by these tissues, it appears that there is a remarkably conserved mechanism for setting up the initial organization of the facial prominences between species. Regardless of species, all vertebrates appear to have the same basic bauplan. However, sometime during mid-gestation, the vertebrate face begins to exhibit species-specific variations, in large part due to differences in the rates of growth and differentiation of cells comprising the facial prominences. How do these differences arise? Are they due to late changes in molecular signaling within the facial prominences themselves? Or are these late changes a reflection of earlier, more subtle alterations in boundaries and fields that are established at the earliest stages of head formation? We do not have clear answers to these questions yet, but in this chapter we present new studies that shed light on this age-old question. This chapter aims to present the known signals, both on a molecular and cellular level, responsible for craniofacial development while bringing to light the events that may serve to create difference in facial morphology seen from species to species.

    View details for DOI 10.1016/S0070-2153(05)73001-5

    View details for Web of Science ID 000238776000001

    View details for PubMedID 16782454

  • It's all in your head: new insights into craniofacial development and deformation JOURNAL OF ANATOMY Tapadia, M. D., Cordero, D. R., Helms, J. A. 2005; 207 (5): 461-477

    View details for Web of Science ID 000232997900004

    View details for PubMedID 16313388

  • Molecular interactions coordinating the development of the forebrain and face DEVELOPMENTAL BIOLOGY Marcucio, R. S., Cordero, D. R., Hu, D., Helms, J. A. 2005; 284 (1): 48-61

    Abstract

    From an architectural point of view, the forebrain acts as a framework upon which the middle and upper face develops and grows. In addition to serving a structural role, we present evidence that the forebrain is a source of signals that shape the facial skeleton. In this study, we inhibited Sonic hedgehog (Shh) signaling from the neuroectoderm then examined the molecular changes and the skeletal alterations resulting from the treatment. One of the first changes we noted was that the dorsoventral polarity of the forebrain was disturbed, which manifested as a loss of Shh in the ventral telencephalon, a reduction in expression of the ventral markers Nkx2.1 and Dlx2, and a concomitant expansion of the dorsal marker Pax6. In addition to changes in the forebrain neuroectoderm, we observed altered gene expression patterns in the facial ectoderm. For example, Shh was not induced in the frontonasal ectoderm, and Ptc and Gli1 were reduced in both the ectoderm and adjacent mesenchyme. As a consequence, a signaling center in the frontonasal prominence was disrupted and the prominence failed to undergo proximodistal and mediolateral expansion. After 15 days of development, the upper beaks of the treated embryos were truncated, and the skeletal elements were located in more medial and proximal locations in relation to the skeletal elements of the lower jaw elements. These data indicate that a role of Shh in the forebrain is to regulate Shh expression in the face, and that together, these Shh domains mediate patterning within the frontonasal prominence and proximodistal outgrowth of the middle and upper face.

    View details for DOI 10.1016/j.ydbio.2005.04.030

    View details for Web of Science ID 000231352800005

    View details for PubMedID 15979605

  • Head, shoulders, knees, and toes DEVELOPMENTAL BIOLOGY De la Fuente, L., Helms, J. A. 2005; 282 (2): 294-306

    View details for DOI 10.1016/j.ydbio.2005.03.036

    View details for Web of Science ID 000230005900002

    View details for PubMedID 15950599

  • The fickle finger of fate JOURNAL OF CLINICAL INVESTIGATION De la Fuente, L., Helms, J. A. 2005; 115 (4): 833-836

    Abstract

    In this issue of the JCI, Niedermaier and colleagues demonstrate that a chromosomal inversion in mice results in dysregulation of Sonic hedgehog (Shh), such that Shh is ectopically expressed in a skeletogenic domain typically occupied by Indian hedgehog (Ihh). This molecular reversal eliminates phalangeal joint spaces, and consequently, Short digits (Dsh) heterozygotes (Dsh/+) have brachydactyly (shortened digits). Ihh is normally downregulated in regions that will become the joint space, but in Dsh/+ mice, Shh bypasses this regulatory control and persists; accordingly, cells maintain their chondrogenic fate and the developed digits are shorter than normal. The significance of these data extends far beyond the field of skeletal biology: they hint at the very real possibility that the endogenous Shh regulatory region contains a repressor designed to segregate the activity of Shh from Ihh. The existence of such a repressor provides a window into the distant past, revealing that Shh and Ihh must once have shared responsibilities in establishing tissue boundaries and orchestrating vertebrate tissue morphogenesis.

    View details for DOI 10.1172/JCI200524840

    View details for Web of Science ID 000228145700012

    View details for PubMedID 15841172

  • New insights into craniofacial morphogenesis DEVELOPMENT Helms, J. A., Cordero, D., Tapadia, M. D. 2005; 132 (5): 851-861

    Abstract

    No region of our anatomy more powerfully conveys our emotions nor elicits more profound reactions when disease or genetic disorders disfigure it than the face. Recent progress has been made towards defining the tissue interactions and molecular mechanisms that control craniofacial morphogenesis. Some insights have come from genetic manipulations and others from tissue recombinations and biochemical approaches, which have revealed the molecular underpinnings of facial morphogenesis. Changes in craniofacial architecture also lie at the heart of evolutionary adaptation, as new studies in fish and fowl attest. Together, these findings reveal much about molecular and tissue interactions behind craniofacial development.

    View details for DOI 10.1242/dev.01705

    View details for Web of Science ID 000228093300001

    View details for PubMedID 15705856

  • Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development DEVELOPMENT Colnot, C., De la Fuente, L., Huang, S., Hu, D., Lu, C. Y., St-Jacques, B., Helms, J. A. 2005; 132 (5): 1057-1067

    Abstract

    A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh(-/-) chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh(-/-) skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh(-/-) cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh(-/-) cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh(-/-) cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage -- comprising osteoblast precursors and endothelial cells -- as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh(-/-) embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

    View details for DOI 10.1242/dev.01649

    View details for Web of Science ID 000228093300019

    View details for PubMedID 15689378

  • Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes JOURNAL OF CLINICAL INVESTIGATION Cordero, D., Marcucio, R., Hu, D., Gaffield, W., Tapadia, M., Helms, J. A. 2004; 114 (4): 485-494

    Abstract

    One of the most perplexing questions in clinical genetics is why patients with identical gene mutations oftentimes exhibit radically different clinical features. This inconsistency between genotype and phenotype is illustrated in the malformation spectrum of holoprosencephaly (HPE). Family members carrying identical mutations in sonic hedgehog (SHH) can exhibit a variety of facial features ranging from cyclopia to subtle midline asymmetries. Such intrafamilial variability may arise from environmental factors acting in conjunction with gene mutations that collectively reduce SHH activity below a critical threshold. We undertook a series of experiments to test the hypothesis that modifying the activity of the SHH signaling pathway at discrete periods of embryonic development could account for the phenotypic spectrum of HPE. Exposing avian embryos to cyclopamine during critical periods of craniofacial development recreated a continuum of HPE-related defects. The craniofacial malformations included hypotelorism, midfacial hypoplasia, and facial clefting and were not the result of excessive crest cell apoptosis. Rather, they resulted from molecular reprogramming of an organizing center whose activity controls outgrowth and patterning of the mid and upper face. Collectively, these data reveal one mechanism by which the variable expressivity of a disorder such as HPE can be produced through temporal disruption of a single molecular pathway.

    View details for DOI 10.1172/JCI200419596

    View details for Web of Science ID 000223287500008

    View details for PubMedID 15314685

  • Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development DEVELOPMENTAL BIOLOGY Colnot, C., Lu, C. Y., Hu, D., Helms, J. A. 2004; 269 (1): 55-69

    Abstract

    During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.

    View details for DOI 10.1016/j.ydbio.2004.01.011

    View details for Web of Science ID 000221034400005

    View details for PubMedID 15081357

  • Altered fracture repair in the absence of MMP9 DEVELOPMENT Colnot, C., Thompson, Z., Miclau, T., WERB, Z., Helms, J. A. 2003; 130 (17): 4123-4133

    Abstract

    The regeneration of adult skeletal tissues requires the timely recruitment of skeletal progenitor cells to an injury site, the differentiation of these cells into bone or cartilage, and the re-establishment of a vascular network to maintain cell viability. Disturbances in any of these cellular events can have a detrimental effect on the process of skeletal repair. Although fracture repair has been compared with fetal skeletal development, the extent to which the reparative process actually recapitulates the fetal program remains uncertain. Here, we provide the first genetic evidence that matrix metalloproteinase 9 (MMP9) regulates crucial events during adult fracture repair. We demonstrate that MMP9 mediates vascular invasion of the hypertrophic cartilage callus, and that Mmp9(-/-) mice have non-unions and delayed unions of their fractures caused by persistent cartilage at the injury site. This MMP9- dependent delay in skeletal healing is not due to a lack of vascular endothelial growth factor (VEGF) or VEGF receptor expression, but may instead be due to the lack of VEGF bioavailability in the mutant because recombinant VEGF can rescue Mmp9(-/-) non-unions. We also found that Mmp9(-/-) mice generate a large cartilage callus even when fractured bones are stabilized, which implicates MMP9 in the regulation of chondrogenic and osteogenic cell differentiation during early stages of repair. In conclusion, the resemblance between Mmp9(-/-) fetal skeletal defects and those that emerge during Mmp9(-/-) adult repair offer the strongest evidence to date that similar mechanisms are employed to achieve bone formation, regardless of age.

    View details for DOI 10.1242/dev.00559

    View details for Web of Science ID 000187398500019

    View details for PubMedID 12874132

  • Cranial skeletal biology NATURE Helms, J. A., SCHNEIDER, R. A. 2003; 423 (6937): 326-331

    Abstract

    To artists, the face is a mirror of the soul. To biologists, the face reflects remarkable structural diversity--think of bulldogs and wolfhounds or galapagos finches. How do such variations in skeletal form arise? Do the same mechanisms control skeletogenesis elsewhere in the body? The answers lie in the molecular machinery that generates neural crest cells, controls their migration, and guides their differentiation to cartilage and bone.

    View details for DOI 10.1038/nature01656

    View details for Web of Science ID 000182853100052

    View details for PubMedID 12748650

  • A zone of frontonasal ectoderm regulates patterning and growth in the face DEVELOPMENT Hu, D., Marcucio, R. S., Helms, J. A. 2003; 130 (9): 1749-1758

    Abstract

    A fundamental set of patterning genes may define the global organization of the craniofacial region. One of our goals has been to identify these basic patterning genes and understand how they regulate outgrowth of the frontonasal process, which gives rise to the mid and upper face. We identified a molecular boundary in the frontonasal process ectoderm, defined by the juxtaposed domains of Fibroblast growth factor 8 and Sonic hedgehog, which presaged the initial site of frontonasal process outgrowth. Fate maps confirmed that this boundary region later demarcated the dorsoventral axis of the upper beak. Ectopic transplantation of the ectodermal boundary region activated a cascade of molecular events that reprogrammed the developmental fate of neural crest-derived mesenchyme, which resulted in duplications of upper and lower beak structures. We discuss these data in the context of boundary/morphogen models of patterning, and in view of the recent controversy regarding neural crest pre-patterning versus neural crest plasticity.

    View details for DOI 10.1242/dev.00397

    View details for Web of Science ID 000182811600003

    View details for PubMedID 12642481

  • Molecular ontogeny of the skeleton. Birth defects research. Part C, Embryo today : reviews Eames, B. F., de la Fuente, L., Helms, J. A. 2003; 69 (2): 93-101

    Abstract

    From a traditional viewpoint, skeletal elements form by two distinct processes: endochondral ossification, during which a cartilage template is replaced by bone, and intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts. There are inherent difficulties with this historical classification scheme, not the least of which is that bones typically described as endochondral actually form bone through an intramembranous process, and that some membranous bones may have a transient chondrogenic phase. These innate contradictions can be circumvented if molecular and cellular, rather than histogenic, criteria are used to describe the process of skeletal tissue formation. Within the past decade, clinical examinations of human skeletal syndromes have led to the identification and subsequent characterization of regulatory molecules that direct chondrogenesis and osteogenesis in every skeletal element of the body. In this review, we survey these molecules and the tissue interactions that may regulate their expression. What emerges is a new paradigm, by which we can explain and understand the process of normal- and abnormal-skeletal development.

    View details for PubMedID 12955855

  • The cellular and molecular origins of beak morphology SCIENCE SCHNEIDER, R. A., Helms, J. A. 2003; 299 (5606): 565-568

    Abstract

    Cellular and molecular mechanisms underlying differences in beak morphology likely involve interactions among multiple embryonic populations. We exchanged neural crest cells destined to participate in beak morphogenesis between two anatomically distinct species. Quail neural crest cells produced quail beaks in duck hosts and duck neural crest produced duck bills in quail hosts. These transformations involved morphological changes to non-neural crest host beak tissues. To achieve these changes, donor neural crest cells executed autonomous molecular programs and regulated gene expression in adjacent host tissues. Thus, neural crest cells are a source of molecular information that generates interspecific variation in beak morphology.

    View details for Web of Science ID 000180559800050

    View details for PubMedID 12543976

  • Prenatal morphogenesis of the human mental foramen EUROPEAN JOURNAL OF ORAL SCIENCES Radlanski, R. J., Renz, H., Muller, U., Schneider, R. S., Marcucio, R. S., Helms, J. A. 2002; 110 (6): 452-459

    Abstract

    The mental foramen, at first glance, merely looks like a hole where the mental nerve and the vascular bundle runs through. From a morphogenetic point of view, however, the mental foramen is a suitable model to study the development of a structure where different components are involved. To understand this developmental process, a three-dimensional description at different developmental stages first has to be given. From histological serial sections of human embryos and fetuses, ranging in size from 19 to 117 mm crown rump length (CRL), three-dimensional reconstructions of the foraminar regions were made. Outline and form of the developing foramen, size, course of the mental nerve and the adjacent blood vessels could be shown in detail. In this way, the formation of these structures became concrete in three dimensions. In the future, to understand the mechanisms regulating this complex system, where a nerve and blood vessels became successively surrounded by bone, molecular biological data have to be correlated with morphological findings.

    View details for Web of Science ID 000179381400008

    View details for PubMedID 12507219

  • A model for intramembranous ossification during fracture healing JOURNAL OF ORTHOPAEDIC RESEARCH Thompson, Z., Miclau, T., Hu, D., Helms, J. A. 2002; 20 (5): 1091-1098

    Abstract

    We have developed a method to study the molecular basis of intramembranous fracture healing. Unlike intramedullary rods that permit rotation of the fractured bone segments, our murine model relies on an external fixation device to provide stabilization. In this study we compare stabilized fracture callus tissues with callus tissues from non-stabilized fractures during the inflammatory, soft callus, hard callus, and remodeling stages of healing. Histological analyses indicate that stabilized fractures heal with virtually no evidence of cartilage whereas non-stabilized fractures produce abundant cartilage at the fracture site. Expression patterns of collagen type IIa (colIIa) and osteocalcin (oc) reveal that mesenchymal cells at the fracture site commit to either a chondrogenic or an osteogenic lineage during the earliest stages of healing. The mechanical environment influences this cell fate decision, since mesenchymal cells in a stabilized fracture express oc and fail to express colIIa. Future studies will use this murine model of intramembranous fracture healing to explore, at a molecular level, how the mechanical environment exerts its influence on healing of a fracture.

    View details for Web of Science ID 000178383400028

    View details for PubMedID 12382977

  • Local retinoid signaling coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH DEVELOPMENT SCHNEIDER, R. A., Hu, D., Rubenstein, J. L., Maden, M., Helms, J. A. 2001; 128 (14): 2755-2767

    Abstract

    Correlations between facial anomalies and brain defects are well characterized throughout the clinical literature, yet a developmental basis for this association has not been identified. We demonstrate that the frontonasal process, which gives rise to the mid- and upper face, and the forebrain are linked early in their morphogenesis by a local retinoid signaling event that maintains the expression of key regulatory molecules. First, we show that aldehyde dehydrogenase 6, which synthesizes the ligand, retinoic acid, is localized to the ventral epithelium of the presumptive frontonasal process of chick embryos. At least two retinoid receptors are expressed in adjacent populations of mesenchyme. Second, using synthetic pan-specific retinoid antagonists, we transiently inhibit the ability of retinoid receptors to bind retinoic acid in the rostral head and we generate embryos with a hypoplastic forebrain, fused eyes, and no frontonasal process-derived structures such as the upper beak. These defects are not due to eliminating mesenchymal progenitors, as neural crest cells still migrate into the frontonasal process, despite disruptions to retinoid signaling. Rather, these malformations result from loss of fibroblast growth factor 8 and sonic hedgehog expression, which leads to increased programmed cell death and decreased proliferation in the forebrain and frontonasal process. Most significantly, we can rescue the morphological defects by re-introducing retinoic acid, or fibroblast growth factor and sonic hedgehog proteins into antagonist-treated embryos. We propose that the local source of retinoic acid in the rostral head initiates a regulatory cascade that coordinates forebrain and frontonasal process morphogenesis.

    View details for Web of Science ID 000170209900012

    View details for PubMedID 11526081

  • A molecular analysis of matrix remodeling and angiogenesis during long bone development MECHANISMS OF DEVELOPMENT Colnot, C. I., Helms, J. A. 2001; 100 (2): 245-250

    Abstract

    The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.

    View details for Web of Science ID 000166837600008

    View details for PubMedID 11165481

  • Genetic and teratogenic approaches to craniofacial development CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE Young, D. L., SCHNEIDER, R. A., Hu, D., Helms, J. A. 2000; 11 (3): 304-317

    Abstract

    Craniofacial malformations are the most common birth defects that occur in humans, with facial clefting representing the majority of these defects. Facial clefts can arise at any stage of development due to perturbations that alter the extracellular matrix as well as affect the patterning, migration, proliferation, and differentiation of cells. In this review, we focus on recent advances in the understanding of the developmental basis for facial clefting through the analysis of the effects of gene disruption experiments and treatments with teratogens in both chickens and mice. Specifically, we analyze the results of disruptions to genes such as Sonic hedgehog (Shh), epidermal growth factor receptor (EGFR), Distal-less (Dlx), and transforming growth factor beta 3 (TGFbeta3). We also describe the effects that teratogens such as retinoic acid, jervine, and cyclopamine have on facial clefting and discuss mechanisms for their action. In addition to providing insight into the bases for abnormal craniofacial growth, genetic and teratogenic techniques are powerful tools for understanding the normal developmental processes that generate and pattern the face.

    View details for Web of Science ID 000175180100002

    View details for PubMedID 11021632

  • The role of Sonic hedgehog in normal and abnormal craniofacial morphogenesis DEVELOPMENT Hu, D., Helms, J. A. 1999; 126 (21): 4873-4884

    Abstract

    There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.

    View details for Web of Science ID 000083881600018

    View details for PubMedID 10518503

  • Does adult fracture repair recapitulate embryonic skeletal formation? MECHANISMS OF DEVELOPMENT Ferguson, C., Alpern, E., Miclau, T., Helms, J. A. 1999; 87 (1-2): 57-66

    Abstract

    Bone formation is a continuous process that begins during fetal development and persists throughout life as a remodeling process. In the event of injury, bones heal by generating new bone rather than scar tissue; thus, it can accurately be described as a regenerative process. To elucidate the extent to which fetal skeletal development and skeletal regeneration are similar, we performed a series of detailed expression analyses using a number of genes that regulate key stages of endochondral ossification. They included genes in the indian hedgehog (ihh) and core binding factor 1 (cbfa1) pathways, and genes associated with extracellular matrix remodeling and vascular invasion including vascular endothelial growth factor (VEGF) and matrix metalloproteinase 13 (mmp13). Our analyses suggested that even at the earliest stages of mesenchymal cell condensation, chondrocyte (ihh, cbfa1 and collagen type II-positive) and perichondrial (gli1 and osteocalcin-positive) cell populations were already specified. As chondrocytes matured, they continued to express cbfa1 and ihh whereas cbfa1, osteocalcin and gli1 persisted in presumptive periosteal cells. Later, VEGF and mmp13 transcripts were abundant in chondrocytes as they underwent hypertrophy and terminal differentiation. Based on these expression patterns and available genetic data, we propose a model where Ihh and Cbfa1, together with Gli1 and Osteocalcin participate in establishing reciprocal signal site of injury. The persistence of cbfa1 and ihh, and their targets osteocalcin and gli1, in the callus suggests comparable processes of chondrocyte maturation and specification of a neo-perichondrium occur following injury. VEGF and mmp13 are expressed during the later stages of healing, coincident with the onset of vascularization of the callus and subsequent ossification. Taken together, these data suggest the genetic mechanisms regulating fetal skeletogenesis also regulate adult skeletal regeneration, and point to important regulators of angiogenesis and ossification in bone regeneration.

    View details for Web of Science ID 000082828000006

    View details for PubMedID 10495271

  • From head to toe: conservation of molecular signals regulating limb and craniofacial morphogenesis CELL AND TISSUE RESEARCH SCHNEIDER, R. A., Hu, D., Helms, J. A. 1999; 296 (1): 103-109

    Abstract

    Recent evidence indicates that many molecules involved in generating and patterning the limbs also play a role during craniofacial morphogenesis. On the surface, this is an unexpected finding given that these regions of the body have separate evolutionary origins, are composed of different embryonic tissues, and are quite dissimilar in their anatomy. Results from several experiments involving Sonic hedgehog and retinoic acid point to a remarkable conservation of the signaling pathways mediated by these morphogens across multiple organ systems. Moreover, mutants such as the extra-toes and doublefoot mouse, and the talpid chicken also provide insights on common developmental processes that underlie the formation of the limbs and face. The identification of highly conserved aspects of morphogenesis is important for understanding fundamental mechanisms of development, as well as for revealing the common denominator of countless birth defects and providing new strategies for their prevention and cure.

    View details for Web of Science ID 000079824500012

    View details for PubMedID 10199970

  • LMP-1, a LIM-domain protein, mediates BMP-6 effects on bone formation ENDOCRINOLOGY Boden, S. D., Liu, Y. S., Hair, G. A., Helms, J. A., Hu, D., Racine, M., Nanes, M. S., Titus, L. 1998; 139 (12): 5125-5134

    Abstract

    Glucocorticoids can promote osteoblast differentiation from fetal calvarial cells and bone marrow stromal cells. We recently reported that glucocorticoid specifically induced bone morphogenetic protein-6 (BMP-6), a glycoprotein signaling molecule that is a multifunctional regulator of vertebrate development. In the present study, we used fetal rat secondary calvarial cultures to determine genes induced during early osteoblast differentiation as initiated by glucocorticoid treatment. Glucocorticoid, and subsequently BMP-6, was found to induce a novel rat intracellular protein, LIM mineralization protein-1 (LMP-1), that in turn resulted in synthesis of one or more soluble factors that could induce de novo bone formation. Blocking expression of LMP-1 using antisense oligonucleotide prevented osteoblast differentiation in vitro. Overexpression of LMP-1 using a mammalian expression vector was sufficient to initiate de novo bone nodule formation in vitro and in sc implants in vivo. These data demonstrate that LMP-1 is an essential positive regulator of the osteoblast differentiation program as well as an important intermediate step in the BMP-6 signaling pathway.

    View details for Web of Science ID 000077104000043

    View details for PubMedID 9832452

  • Mechanobiology of skeletal regeneration CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Carter, D. R., Beaupre, G. S., Giori, N. J., Helms, J. A. 1998: S41-S55

    Abstract

    Skeletal regeneration is accomplished by a cascade of biologic processes that may include differentiation of pluripotential tissue, endochondral ossification, and bone remodeling. It has been shown that all these processes are influenced strongly by the local tissue mechanical loading history. This article reviews some of the mechanobiologic principles that are thought to guide the differentiation of mesenchymal tissue into bone, cartilage, or fibrous tissue during the initial phase of regeneration. Cyclic motion and the associated shear stresses cause cell proliferation and the production of a large callus in the early phases of fracture healing. For intermittently imposed loading in the regenerating tissue: (1) direct intramembranous bone formation is permitted in areas of low stress and strain; (2) low to moderate magnitudes of tensile strain and hydrostatic tensile stress may stimulate intramembranous ossification; (3) poor vascularity can promote chondrogenesis in an otherwise osteogenic environment; (4) hydrostatic compressive stress is a stimulus for chondrogenesis; (5) high tensile strain is a stimulus for the net production of fibrous tissue; and (6) tensile strain with a superimposed hydrostatic compressive stress will stimulate the development of fibrocartilage. Finite element models are used to show that the patterns of tissue differentiation observed in fracture healing and distraction osteogenesis can be predicted from these fundamental mechanobiologic concepts. In areas of cartilage formation, subsequent endochondral ossification normally will proceed, but it can be inhibited by intermittent hydrostatic compressive stress and accelerated by octahedral shear stress (or strain). Later, bone remodeling at these sites can be expected to follow the same mechanobiologic adaptation rules as normal bone.

    View details for Web of Science ID 000077173200007

    View details for PubMedID 9917625

  • Histochemical and molecular analyses of distraction osteogenesis in a mouse model JOURNAL OF ORTHOPAEDIC RESEARCH Tay, B. K., Le, A. X., Gould, S. E., Helms, J. A. 1998; 16 (5): 636-642

    Abstract

    A tibial lengthening scheme in the mouse was used to study the molecular and cellular events regulating tissue regeneration during distraction osteogenesis. Here, we report on the surgical technique and frame design and describe the histochemical and molecular aspects of distraction during different phases of treatment. A total of 26 mice were used in this study. The treatment protocol was divided into a latency period of 7 days, a phase of active distraction that lasted 10 days with a distraction rate of 0.42 mm/day, and a maturation phase of 9 days. During latency, the distraction site resembled a stabilized fracture callus on both a histochemical and a molecular level. During active distraction, the gap was characterized by a central fibrous interzone bordered by primary matrix fronts, regenerate bone aligned with the distraction force, parallel columns of vascular sinusoids, and a medullary cavity. Alkaline phosphatase activity was detected in the endosteal and periosteal surfaces of the bone ends. Tartrate resistant acid phosphatase staining revealed that osteoclasts remodeled the bone regenerate as it formed. Collagen type I was expressed in the periosteum and the primary matrix front during distraction, whereas collagen type-II transcripts were localized to discrete regions on the periosteal surfaces, immediately adjacent to the osteotomy ends. Collagen type-II transcripts were not detected in the fibrous interzone. During the maturation phase, cells within the fibrous interzone expressed collagen type I and exhibited abundant alkaline phosphatase activity, suggesting that they had begun to terminally differentiate. Collectively, these data demonstrate the utility of a mouse model to study the molecular and cellular bases for the regeneration and remodeling of tissue.

    View details for Web of Science ID 000076861700017

    View details for PubMedID 9820290

  • MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes CELL Vu, T. H., Shipley, J. M., Bergers, G., Berger, J. E., Helms, J. A., Hanahan, D., Shapiro, S. D., Senior, R. M., WERB, Z. 1998; 93 (3): 411-422

    Abstract

    Homozygous mice with a null mutation in the MMP-9/gelatinase B gene exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive lengthening of the growth plate to about eight times normal. After 3 weeks postnatal, aberrant apoptosis, vascularization, and ossification compensate to remodel the enlarged growth plate and ultimately produce an axial skeleton of normal appearance. Transplantation of wild-type bone marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by gelatinase B-expressing cells of bone marrow origin, designated chondroclasts. Growth plates from gelatinase B-null mice in culture show a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.

    View details for Web of Science ID 000073471500016

    View details for PubMedID 9590175

  • Common molecular pathways in skeletal morphogenesis and repair MORPHOGENESIS: CELLULAR INTERACTIONS Ferguson, C. M., Miclau, T., Hu, D., Alpern, E., Helms, J. A. 1998; 857: 33-42

    Abstract

    The formation of bone is a continual process in vertebrate development, initiated during fetal development and persisting in adulthood in the form of remodeling and repair. The remarkable capacity of skeletal tissues to regenerate has led to the hypothesis that the molecular signaling pathways regulating skeletogenesis are shared during fetal development and adult wound healing. A number of key regulatory pathways that are required for endochondral ossification during fetal development are described, and their reintroduction in fracture repair demonstrated. Secreted proteins such as Sonic and Indian hedgehog exert their effect on pattern formation and chondrogenesis in the appendicular skeleton, partly through regulation of molecules such as bone morphogenic proteins (Bmps) and parathyroid hormone-related peptide (PTHrP). Once chondrocytes have matured and hypertrophied, they undergo apoptosis and are replaced by bone; the transcription factor Cbfal plays a critical role in this process of chondrocyte differentiation and ossification. Analyses of the expression patterns of these genes during fracture healing strongly suggest that they play equivalent roles in adult wound repair. Knowledge acquired through the study of fetal skeletogenesis will undoubtedly contribute to an understanding of fracture repair, and subsequently guide the development of biologically based therapeutic interventions.

    View details for Web of Science ID 000077211500004

    View details for PubMedID 9917830

  • Expression of Indian hedgehog, bone morphogenetic protein 6 and gli during skeletal morphogenesis MECHANISMS OF DEVELOPMENT Iwasaki, M., Le, A. X., Helms, J. A. 1997; 69 (1-2): 197-202

    Abstract

    A complex signaling pathway involving members of the Hedgehog, Bone morphogenetic protein (Bmp) and Gli families regulates early patterning events in fetal skeletogenesis (Hui and Joyner, 1993. A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toes mutation contains an intragenic deletion of the Gli3 gene. Nat. Genet. 3, 241-246; Bitgood and McMahon, 1995. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172, 126-138; Lanske et al., 1996. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273, 663-666; Vortkamp et al., 1996. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273, 613-622). Hedgehog genes encode secreted proteins that mediate patterning and growth through the induction of secondary signals (reviewed in Hammerschmidt et al., 1997. The world according to hedgehog. Trends Genet. 13, 14-21). Two potential targets of Ihh are bmp6 and gli (Johnson et al., 1995. Patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development 121, 4161-4170; Dominguez et al., 1996. Sending and receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus. Science 272, 1621-1625; Marigo et al., 1996. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol. 180, 273-283). We investigated the molecular similarities and differences between fetal and postnatal skeletal development by analyzing the coincident and complimentary expression domains of indian hedgehog (ihh), bmp6 and gli in adjacent sections throughout the process of skeletogenesis. In almost all of the skeletal tissues examined, the expression domains of ihh and bmp6 were adjacent to one another and this region was surrounded by gli-expressing cells. These observations are in keeping with the proposed function of gli as a negative regulator of Ihh signaling and the induction of Bmps by Hedgehog proteins (Roberts et al., 1995. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 121, 3163-3174; Kawakami et al., 1996. BMP signaling during bone pattern determination in the developing limb. Development 122, 3557-3566). By puberty, ihh, bmp6 and gli transcripts were no longer detected in the growth plate, despite the fact that physeal chondrocytes continued to hypertrophy and differentiate. Although bmp6 was expressed, ihh transcripts were not found in primordia of intramembranous bones, nor in cells lining the future articular surfaces. Collectively our findings suggest that ihh participates in, but is not required for chondrocyte hypertrophy.

    View details for Web of Science ID 000071716400015

    View details for PubMedID 9486541

  • Neuronal pentraxin receptor, a novel putative integral membrane pentraxin that interacts with neuronal pentraxin 1 and 2 and taipoxin-associated calcium-binding protein 49 JOURNAL OF BIOLOGICAL CHEMISTRY Dodds, D. C., Omeis, I. A., Cushman, S. J., Helms, J. A., Perin, M. S. 1997; 272 (34): 21488-21494

    Abstract

    We have identified the first putative integral membrane pentraxin and named it neuronal pentraxin receptor (NPR). NPR is enriched by affinity chromatography on columns of a snake venom toxin, taipoxin, and columns of the taipoxin-binding proteins neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and taipoxin-associated calcium-binding protein 49 (TCBP49). The predominant form of NPR contains an putative NH2-terminal transmembrane domain and all forms of NPR are glycosylated. NPR has 49 and 48% amino acid identity to NP1 and NP2, respectively, and NPR message is expressed in neuronal regions that express NP1 and NP2. We suggest that NPR, NP1, NP2, and TCBP49 are involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris.

    View details for Web of Science ID A1997XR78900077

    View details for PubMedID 9261167

  • Sonic hedgehog participates in craniofacial morphogenesis and is down-regulated by teratogenic doses of retinoic acid DEVELOPMENTAL BIOLOGY Helms, J. A., Kim, C. H., Hu, D., Minkoff, R., Thaller, C., Eichele, G. 1997; 187 (1): 25-35

    Abstract

    The face is one of the most intricately patterned structures in human and yet little is known of the mechanisms by which the tissues are instructed to grow, fuse, and differentiate. We undertook a study to determine if the craniofacial primordia used the same molecular cues that mediate growth and patterning in other embryonic tissues such as the neural tube and the limb. Here we provide evidence for the presence of organizer-like tissues in the craniofacial primordia. These candidate organizers express the polarizing signal sonic hedghog (shh) and its putative receptor, patched, as well as fibroblast growth factor 8 and bone morphogeneic protein 2. Shh-expressing epithelial grafts functioned as organizing tissues in a limb bud assay system, where they evoked duplications of the digit pattern. High doses of retinoic acid, which are known to truncate the growth of the frontonasal and maxillary processes and thus produce bilateral clefting of the lip and palate, inhibited the expression of shh and patched but not fgf8, in the craniofacial primordia, and abolished polarizing activity of these tissues. From these studies we conclude that the embryonic face contains signaling centers in the epithelium that participate in craniofacial growth and patterning. In addition, we discuss a novel mechanism whereby retinoids can exert a teratogenic effect on craniofacial morphogenesis independent of its effects on Hox gene expression or neural crest cell migration.

    View details for Web of Science ID A1997XL41900003

    View details for PubMedID 9224671

  • Retinoic acid signaling is required during early chick limb development DEVELOPMENT Helms, J. A., Kim, C. H., Eichele, G., Thaller, C. 1996; 122 (5): 1385-1394

    Abstract

    In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.

    View details for Web of Science ID A1996UM55800005

    View details for PubMedID 8625827

  • EFFECTS OF CHLORHEXIDINE ON HUMAN TASTE PERCEPTION ARCHIVES OF ORAL BIOLOGY Helms, J. A., DELLAFERA, M. A., Mott, A. E., Frank, M. E. 1995; 40 (10): 913-920

    Abstract

    Chlorhexidine gluconate at a dose used to control bacteria in the mouth has a reversible effect on taste perception. Taste-intensity ratings and taste-quality identification for concentration series of sucrose, sodium chloride, citric acid and quinine hydrochloride were obtained from 15 healthy humans. The participants rinsed with 0.12% chlorhexidine for 3 min twice a day. Each individual was tested 3 times: before the 4-day rinse period, 30 min after the final rinse, and 4 days after the rinse period. Chlorhexidine rinses reduced the perceptual intensity of sodium chloride and quinine hydrochloride, not sucrose or citric acid. No effects on taste perception were detected 4 days after the rinse period. The identification of sodium chloride as salty was seriously impaired by chlorhexidine but the identification of quinine hydrochloride as bitter was not affected. Specific sites of action of chlorhexidine on the taste epithelium are not known but its effects on salty taste may be related to its strong positive charge and its effect on bitter taste may be related to its amphiphilicity. Chlorhexidine has promise as a probe of taste transduction, as well as for the management of salty/bitter dysgeusias in humans.

    View details for Web of Science ID A1995TB53700004

    View details for PubMedID 8526801

  • NEURONAL PENTRAXIN, A SECRETED PROTEIN WITH HOMOLOGY TO ACUTE-PHASE PROTEINS OF THE IMMUNE-SYSTEM NEURON SCHLIMGEN, A. K., Helms, J. A., Vogel, H., Perin, M. S. 1995; 14 (3): 519-526

    Abstract

    We have identified, by affinity chromatography, a binding protein for the snake venom toxin taipoxin. The sequence of this 47 kDa protein is unique, is characteristic of a secreted protein, and has homology to the acute phase proteins serum amyloid P protein and C-reactive protein of the pentraxin family. We have named this protein neuronal pentraxin (NP), as Northern analysis and in situ hybridization demonstrate high message levels in neurons of cerebellum, hippocampus, and cerebral cortex. Because NP may be released synaptically and has homology to immune proteins potentially involved in uptake of lipidic, toxic, or other antigenic material, we suggest that NP may be involved in a general uptake of synaptic macromolecules.

    View details for Web of Science ID A1995QP23000005

    View details for PubMedID 7695898

  • CLONING AND ANALYSIS OF A NEW DEVELOPMENTALLY-REGULATED MEMBER OF THE BASIC HELIX-LOOP-HELIX FAMILY MECHANISMS OF DEVELOPMENT Helms, J. A., Kuratani, S., Maxwell, G. D. 1994; 48 (2): 93-108

    Abstract

    We have isolated a basic helix-loop-helix (bHLH) family member from an embryonic chick-brain cDNA library. This 3.86-Kb cDNA, GbHLH1.4, exhibits extensive sequence similarity in the bHLH domain with Drosophila daughterless and the vertebrate cDNAs E12 and HTF4. Outside of the bHLH region the similarity is significantly reduced. GbHLH1.4 recognizes a 4.0-Kb mRNA and in situ hybridization analysis shows that GbHLH1.4 mRNA is widely expressed at early stages of development but becomes progressively restricted as embryogenesis proceeds. At later stages of embryonic development, mRNA transcripts are localized to several structures including the ventricular layers of the spinal cord and brain, the facial primordia, dorsal root ganglia and heart muscle and cardiac valves. Strikingly, GbHLH1.4 expression in chick embryos exhibits significant overlap with that reported for the murine negative HLH regulator, Id.

    View details for Web of Science ID A1994PU06200003

    View details for PubMedID 7873406

  • RELATIONSHIP BETWEEN RETINOIC ACID AND SONIC HEDGEHOG, 2 POLARIZING SIGNALS IN THE CHICK WING BUD DEVELOPMENT Helms, J., Thaller, C., Eichele, G. 1994; 120 (11): 3267-3274

    Abstract

    Local application of all-trans-retinoic acid (RA) to the anterior margin of chick limb buds results in pattern duplications reminescent of those that develop after grafting cells from the zone of polarizing activity (ZPA). RA may act directly by conferring positional information to limb bud cells, or it may act indirectly by creating a polarizing region in the tissue distal to the RA source. Here we demonstrate that tissue distal to an RA-releasing bead acquires polarizing activity in a dose-dependent manner. Treatments with pharmacological (beads soaked in 330 micrograms/ml) and physiological (beads soaked in 10 micrograms/ml) doses of RA are equally capable of inducing digit pattern duplication. Additionally, both treatments induce sonic hedgehog (shh; also known as vertebrate hedgehog-1, vhh-1), a putative ZPA morphogen and Hoxd-11, a gene induced by the polarizing signal. However, tissue transplantation assays reveal that pharmacological, but not physiological, doses create a polarizing region. This differential response could be explained if physiological doses induced less shh than pharmacological doses. However, our in situ hybridization analyses demonstrate that both treatments result in similar amounts of mRNA encoding this candidate ZPA morphogen. We outline a model describing the apparently disparate effects of pharmacologic and physiological doses RA on limb bud tissue.

    View details for Web of Science ID A1994PV97100020

    View details for PubMedID 7720566

Conference Proceedings


  • The origins of species-specific facial morphology: the proof is in the pigeon Helms, J. A., Brugmann, S. A. OXFORD UNIV PRESS INC. 2007: 338-342

    Abstract

    One of the principal objectives of developmental research is to understand morphogenesis and in doing so, gain insights into the genetic basis of variation observed throughout the Animal Kingdom. In this review we take an approach, first popularized by Darwin, to understanding how diversity is created by using the domesticated pigeon as a model organism. Nearly 3000 years of selective breeding has produced an astonishing array of feather patterns, behaviors, skeletal shapes, and body sizes. Cumulatively, these features make the pigeon an exemplar of morphological variation. Our research interests center around exploiting the unique properties of domesticated pigeons to gain critical insights into the molecular and cellular basis for craniofacial variation.

    View details for DOI 10.1093/icb/icm051

    View details for Web of Science ID 000249765100002

    View details for PubMedID 21672843

  • Molecular aspects of healing in stabilized and non-stabilized fractures Le, A. X., Miclau, T., Hu, D., Helms, J. A. JOHN WILEY & SONS INC. 2001: 78-84

    Abstract

    Bone formation is a continuous process that is initiated during fetal development and persists in adults in the form of bone regeneration and remodeling. These latter two aspects of bone formation are clearly influenced by the mechanical environment. In this study we tested the hypothesis that alterations in the mechanical environment regulate the program of mesenchymal cell differentiation, and thus the formation of a cartilage or bony callus, at the site of injury. As a first step in testing this hypothesis we produced stabilized and non-stabilized tibial fractures in a mouse model, then used molecular and cellular methods to examine the stage of healing. Using the "molecular map" of the fracture callus, we divided our analyzes into three phases of fracture healing: the inflammatory or initial phase of healing, the soft callus or intermediate stage, and the hard callus stage. Our results show that indian hedgehog(ihh), which regulates aspects of chondrocyte maturation during fetal and early postnatal skeletogenesis, was expressed earlier in an non-stabilized fracture callus as compared to a stabilized callus. ihh persisted in the non-stabilized fracture whereas its expression was down-regulated in the stabilized bone. IHH exerts its effects on chondrocyte maturation through a feedback loop that may involve bone morphogenetic protein 6 [bmp6; (S. Pathi, J.B. Rutenberg, R.L. Johnson, A. Vortkamp, Developmental Biology 209 (1999) 239-253)] and the transcription factor gli3. bmp6 and gli3 were re-induced in domain adjacent to the ihh-positive cells during the soft and hard callus stages of healing. Thus, stabilizing the fracture, which circumvents or decreases the cartilaginous phase of bone repair, correlates with a decrease in ihh signaling in the fracture callus. Collectively, our results illustrate that the ihh signaling pathway participates in fracture repair, and that the mechanical environment affects the temporal induction of ihh, bmp6 and gli3. These data support the hypothesis that mechanical influences affect mesenchymal cell differentiation to bone.

    View details for Web of Science ID 000168179000011

    View details for PubMedID 11332624

Stanford Medicine Resources: