Bio

Academic Appointments


Administrative Appointments


  • Member, Institute for Stem Cell Biology and Regenerative Medicine (2006 - Present)
  • Member, Stanford Cancer Center (2006 - Present)

Honors & Awards


  • Keio Medical Science Prize, Keio University, Japan (2011)
  • March of Dimes Prize in Developmental Biology, March of Dimes Birth Defects Foundation (2008)
  • Fellow, American Academy of Arts and Sciences (2003)
  • Member, National Academy of Sciences (2002)
  • National Academy of Sciences Award in Molecular Biology, National Academy of Sciences (1998)
  • Outstanding Young Scientist Award, Maryland Academy of Sciences (1997)

Professional Education


  • Ph.D., Stanford University, Biochemistry (1986)
  • B.S., Goshen College, Goshen, Indiana, Natural Sciences (1979)

Research & Scholarship

Current Research and Scholarly Interests


My lab studies the function of Hedgehog proteins and other extracellular signals in morphogenesis (pattern formation) and in injury repair and regeneration (pattern maintenance). We study how the distribution of such signals is regulated in tissues, how cells perceive and respond to distinct concentrations of signals, and how such signaling pathways arose in evolution. We also study the normal roles of such signals in stem-cell physiology and their abnormal roles in the formation and expansion of cancer stem cells.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Itraconazole and Arsenic Trioxide Inhibit Hedgehog Pathway Activation and Tumor Growth Associated with Acquired Resistance to Smoothened Antagonists CANCER CELL Kim, J., Aftab, B. T., Tang, J. Y., Kim, D., Lee, A. H., Rezaee, M., Kim, J., Chen, B., King, E. M., Borodovsky, A., Riggins, G. J., Epstein, E. H., Beachy, P. A., Rudin, C. M. 2013; 23 (1): 23-34

    Abstract

    Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

    View details for DOI 10.1016/j.ccr.2012.11.017

    View details for Web of Science ID 000313759100005

    View details for PubMedID 23291299

  • Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder NATURE Shin, K., Lee, J., Guo, N., Kim, J., Lim, A., Qu, L., Mysorekar, I. U., Beachy, P. A. 2011; 472 (7341): 110-U145

    Abstract

    Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

    View details for DOI 10.1038/nature09851

    View details for Web of Science ID 000289199400047

    View details for PubMedID 21389986

  • Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Monje, M., Mitra, S. S., Freret, M. E., Raveh, T. B., Kim, J., Masek, M., Attema, J. L., Li, G., Haddix, T., Edwards, M. S., Fisher, P. G., Weissman, I. L., Rowitch, D. H., Vogel, H., Wong, A. J., Beachy, P. A. 2011; 108 (11): 4453-4458

    Abstract

    Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

    View details for DOI 10.1073/pnas.1101657108

    View details for Web of Science ID 000288450900040

    View details for PubMedID 21368213

  • Patterning Axon Targeting of Olfactory Receptor Neurons by Coupled Hedgehog Signaling at Two Distinct Steps CELL Chou, Y., Zheng, X., Beachy, P. A., Luo, L. 2010; 142 (6): 954-966

    Abstract

    We present evidence for a coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events.

    View details for DOI 10.1016/j.cell.2010.08.015

    View details for Web of Science ID 000281855000017

    View details for PubMedID 20850015

  • Interactions between Hedgehog proteins and their binding partners come into view GENES & DEVELOPMENT Beachy, P. A., Hymowitz, S. G., Lazarus, R. A., Leahy, D. J., Siebold, C. 2010; 24 (18): 2001-2012

    Abstract

    Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.

    View details for DOI 10.1101/gad.1951710

    View details for Web of Science ID 000281815500003

    View details for PubMedID 20844013

  • Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, J., Lee, J. J., Kim, J., Gardner, D., Beachy, P. A. 2010; 107 (30): 13432-13437

    Abstract

    Aberrant Hedgehog (Hh) pathway activation has been implicated in cancers of diverse tissues and organs, and the tumor growth-inhibiting effects of pathway antagonists in animal models have stimulated efforts to develop pathway antagonists for human therapeutic purposes. These efforts have focused largely on cyclopamine derivatives or other compounds that mimic cyclopamine action in binding to and antagonizing Smoothened, a membrane transductory component. We report here that arsenicals, in contrast, antagonize the Hh pathway by targeting Gli transcriptional effectors; in the short term, arsenic blocks Hh-induced ciliary accumulation of Gli2, the primary activator of Hh-dependent transcription, and with prolonged incubation arsenic reduces steady-state levels of Gli2. Arsenicals active in Hh pathway antagonism include arsenic trioxide (ATO), a curative agent in clinical use for acute promyelocytic leukemia (APL); in our studies, ATO inhibited growth of Hh pathway-driven medulloblastoma allografts derived from Ptch+/-p53-/- mice within a range of serum levels comparable to those achieved in treatment of human APL. Arsenic thus could be tested rapidly as a therapeutic agent in malignant diseases associated with Hh pathway activation and could be particularly useful in such diseases that are inherently resistant or have acquired resistance to cyclopamine mimics.

    View details for DOI 10.1073/pnas.1006822107

    View details for Web of Science ID 000280602800045

    View details for PubMedID 20624968

  • Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth CANCER CELL Kim, J., Tang, J. Y., Gong, R., Kim, J., Lee, J. J., Clemons, K. V., Chong, C. R., Chang, K. S., Fereshteh, M., Gardner, D., Reya, T., Liu, J. O., Epstein, E. H., Stevens, D. A., Beachy, P. A. 2010; 17 (4): 388-399

    Abstract

    In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

    View details for DOI 10.1016/j.ccr.2010.02.027

    View details for Web of Science ID 000276879700010

    View details for PubMedID 20385363

  • Dally-like core protein and its mammalian homologues mediate stimulatory and inhibitory effects on Hedgehog signal response PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Williams, E. H., Pappano, W. N., Saunders, A. M., Kim, M., Leahy, D. J., Beachy, P. A. 2010; 107 (13): 5869-5874

    Abstract

    The distribution and activities of morphogenic signaling proteins such as Hedgehog (Hh) and Wingless (Wg) depend on heparan sulfate proteoglycans (HSPGs). HSPGs consist of a core protein with covalently attached heparan sulfate glycosaminoglycan (GAG) chains. We report that the unmodified core protein of Dally-like (Dlp), an HSPG required for cell-autonomous Hh response in Drosophila embryos, alone suffices to rescue embryonic Hh signaling defects. Membrane tethering but not specifically the glycosylphosphatidylinositol linkage characteristic of glypicans is critical for this cell-autonomous activity. Our studies further suggest divergence of the two Drosophila and six mammalian glypicans into two functional families, an activating family that rescues cell-autonomous Dlp function in Hh response and a family that inhibits Hh response. Thus, in addition to the previously established requirement for HSPG GAG chains in Hh movement, these findings demonstrate a positive cell-autonomous role for a core protein in morphogen response in vivo and suggest the conservation of a network of antagonistic glypican activities in the regulation of Hh response.

    View details for DOI 10.1073/pnas.1001777107

    View details for Web of Science ID 000276159500033

    View details for PubMedID 20231458

  • Genetic and biochemical definition of the Hedgehog receptor GENES & DEVELOPMENT Zheng, X., Mann, R. K., Sever, N., Beachy, P. A. 2010; 24 (1): 57-71

    Abstract

    Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. Here we show that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. We demonstrate that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, we conclude that Ihog and Ptc together constitute the Drosophila Hh receptor.

    View details for DOI 10.1101/gad.1870310

    View details for Web of Science ID 000273249300008

    View details for PubMedID 20048000

  • Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, J., Kato, M., Beachy, P. A. 2009; 106 (51): 21666-21671

    Abstract

    Stimulation by the extracellular Hedgehog (Hh) protein signal has been shown to alter ciliary localization of the mammalian Hh receptor components Smoothened (Smo) and Patched (Ptc), and mutations that disrupt the structure and function of the cilium also disrupt Hh-induced changes in gene expression. But how ciliary events affect gene expression in the nucleus is not known, and to address this question we have characterized the cellular trafficking of Gli2, the principal mediator of Hh-dependent transcriptional activation. From a combination of pharmacological and genetic manipulations we find in resting cells that both Gli2 and Smo appear to shuttle in and out of the cilium, with Gli2 but not Smo requiring intact cytoplasmic microtubules for ciliary entry and both requiring the ciliary retrograde motor, cytoplasmic dynein 2, for ciliary exit. We also find that changes in ciliary and nuclear trafficking of Gli2 are triggered by the Hh-dependent accumulation of activated Smo in the cilium, resulting in a shift from primarily cytoplasmic localization to accumulation at the distal tip of the cilium and within the nucleus. Gli2 thus functions as a dynamic monitor of Smo activity in the cilium and thereby links Hh pathway activation in the cilium to transcriptional activation in the nucleus.

    View details for DOI 10.1073/pnas.0912180106

    View details for Web of Science ID 000272994200038

    View details for PubMedID 19996169

  • Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form GENES & DEVELOPMENT Creanga, A., Glenn, T. D., Mann, R. K., Saunders, A. M., Talbot, W. S., Beachy, P. A. 2012; 26 (12): 1312-1325

    Abstract

    Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action.

    View details for DOI 10.1101/gad.191866.112

    View details for Web of Science ID 000305485300006

    View details for PubMedID 22677548

  • Identification of a cKit(+) Colonic Crypt Base Secretory Cell That Supports Lgr5(+) Stem Cells in Mice GASTROENTEROLOGY Rothenberg, M. E., Nusse, Y., Kalisky, T., Lee, J. J., Dalerba, P., Scheeren, F., Lobo, N., Kulkarni, S., Sim, S., Qian, D., Beachy, P. A., Pasricha, P. J., Quake, S. R., Clarke, M. F. 2012; 142 (5): 1195-?

    Abstract

    Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a ?-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

    View details for DOI 10.1053/j.gastro.2012.02.006

    View details for Web of Science ID 000303113600038

    View details for PubMedID 22333952

  • Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, M., Saunders, A. M., Hamaoka, B. Y., Beachy, P. A., Leahy, D. J. 2011; 108 (32): 13112-13117

    Abstract

    Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-? crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, ?-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

    View details for DOI 10.1073/pnas.1109877108

    View details for Web of Science ID 000293691400035

    View details for PubMedID 21828006

  • Hedgehogs, Flies, Wnts and MYCs: The Time Has Come for Many Things in Medulloblastoma JOURNAL OF CLINICAL ONCOLOGY Monje, M., Beachy, P. A., Fisher, P. G. 2011; 29 (11): 1395-1398

    View details for DOI 10.1200/JCO.2010.34.0547

    View details for Web of Science ID 000289276900016

    View details for PubMedID 21357776

  • Truncating loss-of-function mutations of DISP1 contribute to holoprosencephaly-like microform features in humans HUMAN GENETICS Roessler, E., Ma, Y., Ouspenskaia, M. V., Lacbawan, F., Bendavid, C., Dubourg, C., Beachy, P. A., Muenke, M. 2009; 125 (4): 393-400

    Abstract

    Defective function of the Sonic Hedgehog (SHH) signaling pathway is the most frequent alteration underlying holoprosencephaly (HPE) or its various clinical microforms. We performed an extensive mutational analysis of the entire human DISP1 gene, required for secretion of all hedgehog ligand(s) and which maps to the HPE 10 locus of human chromosome 1q41, as a HPE candidate gene. Here, we describe two independent families with truncating mutations in human DISP1 that resemble the cardinal craniofacial and neuro-developmental features of a recently described microdeletion syndrome that includes this gene; therefore, we suggest that DISP1 function contributes substantially to both of these signs in humans. While these clinical features are consistent with common HPE microforms, especially those linked to defective signaling by Sonic Hedgehog, we have insufficient evidence so far that functionally abnormal DISP1 alleles will commonly contribute to the more severe features of typical HPE.

    View details for DOI 10.1007/s00439-009-0628-7

    View details for Web of Science ID 000265383500004

    View details for PubMedID 19184110

  • Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia NATURE Zhao, C., Chen, A., Jamieson, C. H., Fereshteh, M., Abrahamsson, A., Blum, J., Kwon, H. Y., Kim, J., Chute, J. P., Rizzieri, D., Munchhof, M., VanArsdale, T., Beachy, P. A., Reya, T. 2009; 458 (7239): 776-U117

    Abstract

    Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established, its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the loss of Smoothened (Smo), an essential component of the Hh pathway, impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR-ABL1 oncoprotein. Loss of Smo causes depletion of CML stem cells--the cells that propagate the leukaemia--whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wild-type BCR-ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML might be avoided by targeting this essential stem cell maintenance pathway.

    View details for DOI 10.1038/nature07737

    View details for Web of Science ID 000265193600044

    View details for PubMedID 19169242

  • The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla NATURE McLellan, J. S., Zheng, X., Hauk, G., Ghirlando, R., Beachy, P. A., Leahy, D. J. 2008; 455 (7215): 979-U62

    Abstract

    Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

    View details for DOI 10.1038/nature07358

    View details for Web of Science ID 000260038300053

    View details for PubMedID 18794898

  • Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer BIOORGANIC & MEDICINAL CHEMISTRY Kumar, S. K., Roy, I., Anchoori, R. K., Fazli, S., Maitra, A., Beachy, P. A., Khan, S. R. 2008; 16 (6): 2764-2768

    Abstract

    A promising agent for use in prostate cancer therapy is the Hedgehog (Hh) signaling pathway inhibitor, cyclopamine. This compound, however, has the potential for causing serious side effects in non-tumor tissues. To minimize these bystander toxicities, we have designed and synthesized two novel peptide-cyclopamine conjugates as prostate-specific antigen (PSA)-activated prodrugs for use against prostate cancer. These prodrugs were composed of cyclopamine coupled to one of two peptides (either HSSKLQ or SSKYQ) that can be selectively cleaved by PSA, converting the mature prodrug into an active Hedgehog inhibitor within the malignant cells. Of the two prodrugs, Mu-SSKYQ-Cyclopamine was rapidly hydrolyzed, with a half-life of 3.2h, upon incubation with the PSA enzyme. Thus, modulating cyclopamine at the secondary amine with PSA-cleavable peptides is a promising strategy for developing prodrugs to target prostate cancer.

    View details for DOI 10.1016/j.bmc.2008.01.012

    View details for Web of Science ID 000255127700005

    View details for PubMedID 18249125

  • Disruption of the basal body comprises proteasomal function and perturbs intracellular Wnt response NATURE GENETICS Gerdes, J. M., Liu, Y., Zaghloul, N. A., Leitch, C. C., Lawson, S. S., Kato, M., Beachy, P. A., Beales, P. L., DeMartino, G. N., Fisher, S., Badano, J. L., Katsanis, N. 2007; 39 (11): 1350-1360

    Abstract

    Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of beta-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic beta-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.

    View details for DOI 10.1038/ng.2007.12

    View details for Web of Science ID 000250575900019

    View details for PubMedID 17906624

  • Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice MATRIX BIOLOGY Akiyama, H., Stadler, H. S., Martin, J. F., Ishii, T. M., Beachy, P. A., Nakamura, T., de Crombrugghe, B. 2007; 26 (4): 224-233

    Abstract

    Our previous studies have demonstrated the essential roles of the transcription factor Sox9 in the commitment of mesenchymal cells to a chondrogenic cell lineage and in overt chondrogenesis during limb bud development. However, it remains unknown if Sox9 induces chondrogenesis in mesenchyme ectopically in vivo as a master regulator of chondrogenesis. In this study, we first generated mutant mice in which Sox9 was misexpressed in the limb bud mesenchyme. The mutant mouse embryos exhibited polydactyly in limb buds in association with ectopic expression of Sox5 and Sox6 although markers for the different axes of limb bud development showed a normal pattern of expression. Misexpression of Sox9 stimulated cell proliferation in limb bud mesenchyme, suggesting that Sox9 has a role in recruiting mesenchymal cells to mesenchymal condensation. Second, despite the facts that misexpression of Sonic hedgehog (Shh) induces polydactyly in a number of mutant mice and Shh-null mutants have severely defective cartilage elements in limb buds, misexpression of Sox9 did not restore limb bud phenotypes in Shh-null mutants. Rather, there was no expression of Sox9 in digit I of Hoxa13Hd mutant embryos, and Sox9 partially rescued hypodactyly in Hoxa13Hd mutant embryos. These results provide evidence that Sox9 induces ectopic chondrogenesis in mesenchymal cells and strongly suggest that its expression may be regulated by Hox genes during limb bud development.

    View details for DOI 10.1016/j.matbio.2006.12.002

    View details for Web of Science ID 000246755000003

    View details for PubMedID 17222543

  • Oxysterols pathway in are novel activators of the hedgehog pluripotent mesenchymal cells JOURNAL OF BIOLOGICAL CHEMISTRY Dwyer, J. R., Sever, N., Carlson, M., Nelson, S. F., Beachy, P. A., Parhami, F. 2007; 282 (12): 8959-8968

    Abstract

    Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.

    View details for DOI 10.1074/jbc.M611741200

    View details for Web of Science ID 000245780300042

    View details for PubMedID 17200122

  • Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peacock, C. D., Wang, Q., Gesell, G. S., Corcoran-Schwartz, I. M., Jones, E., Kim, J., Devereux, W. L., Rhodes, J. T., Huff, C. A., Beachy, P. A., Watkins, D. N., Matsui, W. 2007; 104 (10): 4048-4053

    Abstract

    The cancer stem cell hypothesis suggests that malignant growth depends on a subset of tumor cells with stem cell-like properties of self-renewal. Because hedgehog (Hh) signaling regulates progenitor cell fate in normal development and homeostasis, aberrant pathway activation might be involved in the maintenance of such a population in cancer. Indeed, mutational activation of the Hh pathway is associated with medulloblastoma and basal cell carcinoma; pathway activity is also critical for growth of other tumors lacking such mutations, although the mechanism of pathway activation is poorly understood. Here we study the role and mechanism of Hh pathway activation in multiple myeloma (MM), a malignancy with a well defined stem cell compartment. In this model, rare malignant progenitors capable of clonal expansion resemble B cells, whereas the much larger tumor cell population manifests a differentiated plasma cell phenotype that pathologically defines the disease. We show that the subset of MM cells that manifests Hh pathway activity is markedly concentrated within the tumor stem cell compartment. The Hh ligand promotes expansion of MM stem cells without differentiation, whereas the Hh pathway blockade, while having little or no effect on malignant plasma cell growth, markedly inhibits clonal expansion accompanied by terminal differentiation of purified MM stem cells. These data reveal that Hh pathway activation is heterogeneous across the spectrum of MM tumor stem cells and their more differentiated progeny. The potential existence of similar relationships in other adult cancers may have important biologic and clinical implications for the study of aberrant Hh signaling.

    View details for DOI 10.1073/pnas.0611682104

    View details for Web of Science ID 000244972400068

    View details for PubMedID 17360475

  • Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc. Natl. Acad. Sci. U.S.A. Peacock, C.D., Wang, Q., Gesell, G.S., Corcoran-Schwartz, I.M., Jones, E., Kim, J., Devereux, W.L., Rhodes, J.T., Huff, C.A., Beachy, P.A., Watkins, D.N., Matsui, W. 2007; 104: 4048-4053
  • Structure of a heparin-dependent complex of hedgehog and Ihog PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McLellan, J. S., Yao, S., Zheng, X., Geisbrecht, B. V., Ghirlando, R., Beachy, P. A., Leahy, D. J. 2006; 103 (46): 17208-17213

    Abstract

    Hedgehog (Hh) signaling molecules mediate key tissue-patterning events during animal development, and inappropriate activation of Hh signaling in adults has been associated with human cancers. Recently, a conserved family of type I integral membrane proteins required for normal response to the Hh signal was discovered. One member of this family, Ihog (interference hedgehog), functions upstream or at the level of Patched (Ptc), but how Ihog participates in Hh signaling remains unclear. Here, we show that heparin binding induces Ihog dimerization and is required to mediate high-affinity interactions between Ihog and Hh. We also present crystal structures of a Hh-binding fragment of Ihog, both alone and complexed with Hh. Heparin is not well ordered in these structures, but a basic cleft in the first FNIII domain of Ihog (IhogFn1) is shown by mutagenesis to mediate heparin binding. These results establish that Hh directly binds Ihog and provide the first demonstration of a specific role for heparin in Hh responsiveness.

    View details for DOI 10.1073/pnas.0606738103

    View details for Web of Science ID 000242249400028

    View details for PubMedID 17077139

  • Minimizing the risk of reporting false positives in large-scale RNAi screens NATURE METHODS Echeverri, C. J., Beachy, P. A., Baum, B., Boutros, M., Buchholz, F., Chanda, S. K., Downward, J., Ellenberg, J., Fraser, A. G., Hacohen, N., Hahn, W. C., Jackson, A. L., Kiger, A., Linsley, P. S., Lum, L., Ma, Y., Mathey-Prevot, B., Root, D. E., Sabatini, D. M., Taipale, J., Perrimon, N., Bernards, R. 2006; 3 (10): 777-779

    Abstract

    Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

    View details for DOI 10.1038/nmeth1006-777

    View details for Web of Science ID 000240942600008

    View details for PubMedID 16990807

  • Prevalence of off-target effects in Drosophila RNA interference screens NATURE Ma, Y., Creanga, A., Lum, L., Beachy, P. A. 2006; 443 (7109): 359-363

    Abstract

    RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

    View details for DOI 10.1038/nature05179

    View details for Web of Science ID 000240622000051

    View details for PubMedID 16964239

  • A genomewide screen for components of the RNAi pathway in Drosophila cultured cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dorner, S., Lum, L., Kim, M., Paro, R., Beachy, P. A., Green, R. 2006; 103 (32): 11880-11885

    Abstract

    Posttranscriptional silencing by RNAi is initiated by dsRNAs that are processed into siRNAs that ultimately target homologous mRNAs for degradation. We used luciferase reporter constructs and a cultured cell-based assay to perform a genomewide screen for components of the RNAi pathway in Drosophila melanogaster. The screen identified seven genes that affect the RNAi response, five with previously described function (AGO2, Tis11, Hsc70-3, Hsc70-4, and hdc) and two annotated genes (CG17265 and CG10883).

    View details for DOI 10.1073/pnas.0605210103

    View details for Web of Science ID 000239701900013

    View details for PubMedID 16882716

  • Transcriptional activation by extradenticle in the Drosophila visceral mesoderm DEVELOPMENTAL BIOLOGY Stultz, B. G., Jackson, D. G., Mortin, M. A., Yang, X., Beachy, P. A., Hursh, D. A. 2006; 290 (2): 482-494

    Abstract

    decapentaplegic (dpp) is a direct target of Ultrabithorax (Ubx) in parasegment 7 (PS7) of the embryonic visceral mesoderm. We demonstrate that extradenticle (exd) and homothorax (hth) are also required for dpp expression in this location, as well as in PS3, at the site of the developing gastric caecae. A 420 bp element from dpp contains EXD binding sites necessary for expressing a reporter gene in both these locations. Using a specificity swap, we demonstrate that EXD directly activates this element in vivo. Activation does not require Ubx, demonstrating that EXD can activate transcription independently of homeotic proteins. Restoration is restricted to the domains of endogenous dpp expression, despite ubiquitous expression of altered specificity EXD. We demonstrate that nuclear EXD is more extensively phosphorylated than the cytoplasmic form, suggesting that EXD is a target of signal transduction by protein kinases.

    View details for DOI 10.1016/j.ydbio.2005.11.041

    View details for Web of Science ID 000235382500021

    View details for PubMedID 16403493

  • Defective cerebellar response to mitogenic Hedgehog signaling in Down's syndrome mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Roper, R. J., Baxter, L. L., Saran, N. G., Klinedinst, D. K., Beachy, P. A., Reeves, R. H. 2006; 103 (5): 1452-1456

    Abstract

    Trisomy 21 is the cause of Down [corrected] syndrome (DS) which is characterized by a number of phenotypes, including a brain which is small and hypocellular compared to that of euploid individuals. The cerebellum is disproportionately reduced. Ts65Dn mice are trisomic for orthologs of about half of the genes on human chromosome 21 and provide a genetic model for DS. These mice display a number of developmental anomalies analogous to those in DS, including a small cerebellum with a significantly decreased number of both granule and Purkinje cell neurons. Here we trace the origin of the granule cell deficit to precursors in early postnatal development, which show a substantially reduced mitogenic response to Hedgehog protein signaling. Purified cultures of trisomic granule cell precursors show a reduced but dose-dependent response to the Sonic hedgehog protein signal in vitro, demonstrating that this is a cell-autonomous deficit. Systemic treatment of newborn trisomic mice with a small molecule agonist of Hedgehog pathway activity increases mitosis and restores granule cell precursor populations in vivo. These results demonstrate a basis for and a potential therapeutic approach to a fundamental aspect of CNS pathology in DS.

    View details for DOI 10.1073/pnas.0510750103

    View details for Web of Science ID 000235094300054

    View details for PubMedID 16432181

  • Defective cerebellar response to mitogenic Hedgehog signaling in Down syndrome mice. Proc. Natl. Acad. Sci. U.S.A. Roper, R.J., Baxter, L.L., Saran, N.G., Klinedinst, D.K., Beachy, P.A., Reeves, R.H. 2006; 103: 1452-1456
  • The ihog cell-surface proteins bind Hedgehog and mediate pathway activation Cell Yao, S., Lum, L., Beachy, P.A. 2006; 125: 343-357
  • Structure of a heparin-dependent complex of Hedgehog and Ihog. Proc. Natl. Acad. Sci. U.S.A. McLellan, J.S., Yao, S., Zheng, X., Geisbrecht, B.V., Ghirlando, R., Beachy, P.A., Leahy, D.J. 2006; 103: 17208-17213
  • Molecular mechanisms of Sonic hedgehog mutant effects in holoprosencephaly PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maity, T., Fuse, N., Beachy, P. A. 2005; 102 (47): 17026-17031

    Abstract

    Holoprosencephaly (HPE), a human developmental brain defect, usually is also associated with varying degrees of midline facial dysmorphism. Heterozygous mutations in the Sonic hedgehog (SHH) gene are the most common genetic lesions associated with HPE, and loss of Shh function in the mouse produces cyclopia and alobar forebrain development. The N-terminal domain (ShhNp) of Sonic hedgehog protein, generated by cholesterol-dependent autoprocessing and modification at the C terminus and by palmitate addition at the N terminus, is the active ligand in the Shh signal transduction pathway. Here, we analyze seven reported missense mutations (G31R, D88V, Q100H, N115K, W117G, W117R, and E188Q) that alter the N-terminal signaling domain of Shh protein, and show that two of these mutations (Q100H and E188Q), which are questionably linked to HPE, produce no detectable effects on function. The remaining five alterations affect normal processing, Ptc binding, and signaling to varying degrees. These effects include introduction of a recognition site for furin-like proteases by the G31R alteration, resulting in cleavage of 11 amino acid residues from the N terminus of ShhNp and consequent reduced signaling potency. Two other alterations, W117G and W117R, cause temperature-dependent misfolding and retention in the sterol-poor endoplasmic reticulum, thus disrupting cholesterol-dependent autoprocessing.

    View details for DOI 10.1073/pnas.0507848102

    View details for Web of Science ID 000233463200023

    View details for PubMedID 16282375

  • Extensive phosphorylation of Smoothened in Hedgehog pathway activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, C., Williams, E. H., Guo, Y., Lum, L., Beachy, P. A. 2004; 101 (52): 17900-17907

    Abstract

    The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in Drosophila have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes in vivo and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1alpha in Hh pathway activation.

    View details for DOI 10.1073/pnas.0408093101

    View details for Web of Science ID 000226102700006

    View details for PubMedID 15598741

  • Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2 SCIENCE Chen, W., Ren, X. R., Nelson, C. D., Barak, L. S., Chen, J. K., Beachy, P. A., de Sauvage, F., Lefkowitz, R. J. 2004; 306 (5705): 2257-2260

    Abstract

    Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

    View details for DOI 10.1126/science.1104135

    View details for Web of Science ID 000225950000047

    View details for PubMedID 15618519

  • Tissue repair and stem cell renewal in carcinogenesis NATURE Beachy, P. A., Karhadkar, S. S., Berman, D. M. 2004; 432 (7015): 324-331

    Abstract

    Cancer is increasingly being viewed as a stem cell disease, both in its propagation by a minority of cells with stem-cell-like properties and in its possible derivation from normal tissue stem cells. But stem cell activity is tightly controlled, raising the question of how normal regulation might be subverted in carcinogenesis. The long-known association between cancer and chronic tissue injury, and the more recently appreciated roles of Hedgehog and Wnt signalling pathways in tissue regeneration, stem cell renewal and cancer growth together suggest that carcinogenesis proceeds by misappropriating homeostatic mechanisms that govern tissue repair and stem cell self-renewal.

    View details for DOI 10.1038/nature03100

    View details for Web of Science ID 000225161400042

    View details for PubMedID 15549094

  • The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas CANCER RESEARCH Hallahan, A. R., Pritchard, J. I., Hansen, S., Benson, M., Stoeck, J., Hatton, B. A., Russell, T. L., Ellenbogen, R. G., Bernstein, I. D., Beachy, P. A., Olson, J. M. 2004; 64 (21): 7794-7800

    Abstract

    To develop a genetically faithful model of medulloblastoma with increased tumor incidence compared with the current best model we activated the Sonic Hedgehog (Shh) pathway by transgenically expressing a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors (ND2:SmoA1 mice). This resulted in early cerebellar granule cell hyper-proliferation and a 48% incidence of medulloblastoma formation. Gene expression studies showed an increase in the known Shh targets Gli1 and Nmyc that correlated with increasing hyperplasia and tumor formation. Notch2 and the Notch target gene, HES5, were also significantly elevated in Smoothened-induced tumors showing that Shh pathway activation is sufficient to induce Notch pathway signaling. In human medulloblastomas reverse transcription-PCR for Shh and Notch targets revealed activation of both of these pathways in most tumors when compared with normal cerebellum. Notch pathway inhibition with soluble Delta ligand or gamma secretase inhibitors resulted in a marked reduction of viable cell numbers in medulloblastoma cell lines and primary tumor cultures. Treatment of mice with D283 medulloblastoma xenografts with a gamma secretase inhibitor resulted in decreased proliferation and increased apoptosis, confirming that Notch signaling contributes to human medulloblastoma proliferation and survival. Medulloblastomas in ND2:SmoA1 mice and humans have concomitant increase in Shh and Notch pathway activities, both of which contribute to tumor survival.

    View details for Web of Science ID 000224790600024

    View details for PubMedID 15520185

  • Hedgehog signalling in prostate regeneration, neoplasia and metastasis NATURE Karhadkar, S. S., Bova, G. S., Abdallah, N., Dhara, S., Gardner, D., Maitra, A., Isaacs, J. T., Berman, D. M., Beachy, P. A. 2004; 431 (7009): 707-712

    Abstract

    Metastatic cancers adopt certain properties of normal cells in developing or regenerating organs, such as the ability to proliferate and alter tissue organization. We find here that activity of the Hedgehog (Hh) signalling pathway, which has essential roles in developmental patterning, is required for regeneration of prostate epithelium, and that continuous pathway activation transforms prostate progenitor cells and renders them tumorigenic. Elevated pathway activity furthermore distinguishes metastatic from localized prostate cancer, and pathway manipulation can modulate invasiveness and metastasis. Pathway activity is triggered in response to endogenous expression of Hh ligands, and is dependent upon the expression of Smoothened, an essential Hh response component that is not expressed in benign prostate epithelial cells. Monitoring and manipulating Hh pathway activity may thus offer significant improvements in diagnosis and treatment of prostate cancers with metastatic potential.

    View details for DOI 10.1038/nature02962

    View details for Web of Science ID 000224299300045

    View details for PubMedID 15361885

  • Mending and malignancy NATURE Beachy, P. A., Karhadkar, S. S., Berman, D. M. 2004; 431 (7007): 402-402

    View details for DOI 10.1038/431402a

    View details for Web of Science ID 000224000500020

    View details for PubMedID 15385990

  • The Hedgehog response network: Sensors, switches, and routers SCIENCE Lum, L., Beachy, P. A. 2004; 304 (5678): 1755-1759

    Abstract

    The Hedgehog (Hh) signaling pathway is intimately linked to cell growth and differentiation, with normal roles in embryonic pattern formation and adult tissue homeostasis and pathological roles in tumor initiation and growth. Recent advances in our understanding of Hh response have resulted from the identification of new pathway components and new mechanisms of action for old pathway components. The most striking new finding is that signal transmission from membrane to cytoplasm proceeds through recruitment, by the seven-transmembrane protein Smoothened, of an atypical kinesin, which routes pathway activation by interaction with other components of a complex that includes the latent zinc finger transcription factor, Ci.

    View details for Web of Science ID 000222089500031

    View details for PubMedID 15205520

  • Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis DEVELOPMENTAL BIOLOGY Berman, D. M., Desai, N., Wang, X., Karhadkar, S. S., Reynon, M., Abate-Shen, C., Beachy, P. A., Shen, M. M. 2004; 267 (2): 387-398

    Abstract

    Previous studies have demonstrated that the Hedgehog (Hh) signaling pathway plays a critical role in the development and patterning of many endodermally derived tissues. We have investigated the role of Sonic hedgehog (Shh) in formation of the prostate gland by examining the urogenital phenotype of Shh mutant fetuses. Consistent with earlier work reporting an essential role for Shh in prostate induction, we have found that Shh mutant fetuses display abnormal urogenital development and fail to form prostate buds. Unexpectedly, however, we have discovered that this prostate defect could be rescued by three different methods: renal grafting, explant culture in the presence of androgens, and administration of dihydrotestosterone (DHT) to pregnant mice, indicating that the prostate defect in Shh mutants is due to insufficient levels of androgens. Furthermore, we find that the inhibition of Hh pathway signaling by treatment with cyclopamine does not block prostate formation in explant culture, but instead produces morphological defects consistent with a role for Hh signaling in ductal patterning. Taken together, our studies indicate that the initial organogenesis of the prostate proceeds independently of Shh, but that Shh or other Hh ligands may play a role in subsequent events that pattern the prostate.

    View details for DOI 10.1016/j.ydbio.2003.11.018

    View details for Web of Science ID 000220351400009

    View details for PubMedID 15013801

  • Novel lipid modifications of secreted protein signals ANNUAL REVIEW OF BIOCHEMISTRY Mann, R. K., Beachy, P. A. 2004; 73: 891-923

    Abstract

    Secreted signaling proteins function in a diverse array of essential patterning events during metazoan development, ranging from embryonic segmentation in insects to neural tube differentiation in vertebrates. These proteins generally are expressed in a localized manner, and they may elicit distinct concentration-dependent responses in the cells of surrounding tissues and structures, thus functioning as morphogens that specify the pattern of cellular responses by their tissue distribution. Given the importance of signal distribution, it is notable that the Hedgehog (Hh) and Wnt proteins, two of the most important families of such signals, are known to be covalently modified by lipid moieties, the membrane-anchoring properties of which are not consistent with passive models of protein mobilization within tissues. This review focuses on the mechanisms underlying biogenesis of the mature Hh proteins, which are dually modified by cholesteryl and palmitoyl adducts, as well as on the relationship between Hh proteins and the self-splicing proteins (i.e., proteins containing inteins) and the Hh-like proteins of nematodes. We further discuss the cellular mechanisms that have evolved to handle lipidated Hh proteins in the spatial deployment of the signal in developing tissues and the more recent findings that implicate palmitate modification as an important feature of Wnt signaling proteins.

    View details for DOI 10.1146/annurev.biochem.73.011303.073933

    View details for Web of Science ID 000223246400028

    View details for PubMedID 15189162

  • Hedgehog signal transduction via smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2 MOLECULAR CELL Lum, L., Zhang, C., Oh, S., Mann, R. K., von Kessler, D. P., Taipale, J., Weis-Garcia, F., Gong, R. Y., Wang, B. L., Beachy, P. A. 2003; 12 (5): 1261-1274

    Abstract

    The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Here, we present evidence that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo.

    View details for Web of Science ID 000186764700021

    View details for PubMedID 14636583

  • Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours NATURE Berman, D. M., Karhadkar, S. S., Maitra, A., de Oca, R. M., Gerstenblith, M. R., Briggs, K., Parker, A. R., SHIMADA, Y., Eshleman, J. R., Watkins, D. N., Beachy, P. A. 2003; 425 (6960): 846-851

    Abstract

    Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.

    View details for DOI 10.1038/nature01972

    View details for Web of Science ID 000186118500047

    View details for PubMedID 14520411

  • A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis NATURE GENETICS Cooper, M. K., Wassif, C. A., Krakowiak, P. A., Taipale, J., Gong, R. Y., Kelley, R. I., Porter, F. D., Beachy, P. A. 2003; 33 (4): 508-513

    Abstract

    Smith-Lemli-Opitz syndrome (SLOS), desmosterolosis and lathosterolosis are human syndromes caused by defects in the final stages of cholesterol biosynthesis. Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hedgehog (Hh) family of secreted proteins. Here we report that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. We show that decreasing levels of cellular sterols correlate with diminishing responsiveness to the Hh signal. This diminished response occurs at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. We further find that sterol depletion affects the activity of Smoothened (Smo), an essential component of the Hh signal transduction apparatus.

    View details for DOI 10.1038/ng1134

    View details for Web of Science ID 000181928600023

    View details for PubMedID 12652302

  • Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells SCIENCE Lum, L., Yao, S. Q., Mozer, B., Rovescalli, A., Von Kessler, D., Nirenberg, M., Beachy, P. A. 2003; 299 (5615): 2039-2045

    Abstract

    Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

    View details for Web of Science ID 000181834200046

    View details for PubMedID 12663920

  • Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer NATURE Watkins, D. N., Berman, D. M., Burkholder, S. G., Wang, B. L., Beachy, P. A., BAYLIN, S. B. 2003; 422 (6929): 313-317

    Abstract

    Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.

    View details for DOI 10.1038/nature01493

    View details for Web of Science ID 000181637300040

    View details for PubMedID 12629553

  • Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened GENES & DEVELOPMENT Chen, J. K., Taipale, J., Cooper, M. K., Beachy, P. A. 2002; 16 (21): 2743-2748

    Abstract

    The steroidal alkaloid cyclopamine has both teratogenic and antitumor activities arising from its ability to specifically block cellular responses to vertebrate Hedgehog signaling. We show here, using photoaffinity and fluorescent derivatives, that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo.

    View details for DOI 10.1101/gad.1025302

    View details for Web of Science ID 000179027900002

    View details for PubMedID 12414725

  • Small molecule modulation of Smoothened activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. 2002; 99 (22): 14071-14076

    Abstract

    Smoothened (Smo), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Although the cellular mechanisms that regulate Smo function remain unclear, the direct inhibition of Smo by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved. Here we demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle in a manner that antagonizes cyclopamine action. In addition, we have identified four small molecules that directly inhibit Smo activity but are structurally distinct from cyclopamine. Functional and biochemical studies of these compounds provide evidence for the small molecule modulation of Smo through multiple mechanisms and yield insights into the physiological regulation of Smo activity. The mechanistic differences between the Smo antagonists may be useful in the therapeutic manipulation of Hh signaling.

    View details for DOI 10.1073/pnas.182542899

    View details for Web of Science ID 000178967400021

    View details for PubMedID 12391318

  • Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched CELL Ma, Y., Erkner, A., Gong, R. Y., Yao, S. Q., Taipale, J., Basler, K., Beachy, P. A. 2002; 111 (1): 63-75

    Abstract

    The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.

    View details for Web of Science ID 000178461900009

    View details for PubMedID 12372301

  • Differential requirements for Shh in mammary tissue and hair follicle morphogenesis DEVELOPMENTAL BIOLOGY Gallego, M. I., Beachy, P. A., Hennighausen, L., Robinson, G. W. 2002; 249 (1): 131-139

    Abstract

    Sonic Hedgehog (Shh) is a secreted morphogen that directs patterning and cellular differentiation through binding to its receptor Patched (Ptc). It is required for the development of skin-derived organs, such as hair, whiskers, and teeth. The mammary gland is a skin-derived organ that develops mainly during adult life in which Shh is expressed from puberty to lactation. We have investigated the role of Shh in mammary gland morphogenesis and differentiation by two transplantation approaches. Since Shh-null fetuses die at late embryogenesis, we transplanted Shh-null mammary anlagen into cleared fat pads and under the renal capsule of wild type host mice. Pregnancy-mediated functional differentiation of Shh-null mammary epithelium was indistinguishable from wild type transplants, while hair follicles derived from cotransplanted skin only developed in wild type transplants. Transplants of Ihh-null anlagen also developed normally. To assess the molecular consequences of Shh deletion in mammary tissue, we compared mRNA levels of patched 1, a target gene of Hedgehog signaling, in Shh-null and wild type mammary epithelial transplants. No reduction of Ptc1 transcripts was observed in Shh-null mammary tissues. Our results demonstrate that neither Shh nor Ihh is required for mammary gland morphogenesis and functional differentiation, suggesting that the two members of the Hedgehog family may have redundant function in activating the Ptc1 signaling pathway during mammary gland development.

    View details for DOI 10.1006/dbio.2002.0761

    View details for Web of Science ID 000177924600011

    View details for PubMedID 12217324

  • Medulloblastoma growth inhibition by Hedgehog pathway blockade SCIENCE Berman, D. M., Karhadkar, S. S., Hallahan, A. R., Pritchard, J. I., Eberhart, C. G., Watkins, D. N., Chen, J. K., Cooper, M. K., Taipale, J., Olson, J. M., Beachy, P. A. 2002; 297 (5586): 1559-1561

    Abstract

    Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.

    View details for Web of Science ID 000177697300059

    View details for PubMedID 12202832

  • Patched acts catalytically to suppress the activity of Smoothened NATURE Taipale, J., Cooper, M. K., Maiti, T., Beachy, P. A. 2002; 418 (6900): 892-897

    Abstract

    Mutations affecting the transmembrane proteins Patched (Ptc) or Smoothened (Smo) that trigger ligand-independent activity of the Hedgehog (Hh) signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma. Despite extensive genetic studies demonstrating the importance of these receptor components in embryonic patterning and cancer, the mechanism by which Ptc regulates Smo is not understood. Here we report that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, we show that free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; we demonstrate that the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumour suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule.

    View details for DOI 10.1038/nature00989

    View details for Web of Science ID 000177555600042

    View details for PubMedID 12192414

  • Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal SCIENCE Chamoun, Z., Mann, R. K., Nellen, D., von Kessler, D. P., Bellotto, M., Beachy, P. A., Basler, K. 2001; 293 (5537): 2080-2084

    Abstract

    One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.

    View details for DOI 10.1126/science.1064437

    View details for Web of Science ID 000171028700072

    View details for PubMedID 11486055

  • Manifestation of the limb prepattern: Limb development in the absence of sonic hedgehog function DEVELOPMENTAL BIOLOGY Chiang, C., Litingtung, Y., Harris, M. P., Simandl, B. K., Li, Y., Beachy, P. A., Fallon, J. F. 2001; 236 (2): 421-435

    Abstract

    The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.

    View details for Web of Science ID 000170497400013

    View details for PubMedID 11476582

  • The Hedgehog and Wnt signaling pathways in cancer NATURE Taipale, J., Beachy, P. A. 2001; 411 (6835): 349-354

    Abstract

    The Wnt and Hedgehog (Hh) signalling pathways have long been known to direct growth and patterning during embryonic development. Recent evidence also implicates these pathways in the postembryonic regulation of stem-cell number in epithelia such as those of the skin and intestine, which undergo constant renewal. A pathological role for the Wnt and Hh pathways has emerged from studies showing a high frequency of specific human cancers associated with mutations that constitutively activate the transcriptional response of these pathways. This article focuses on Hh and Wnt signal transduction and reviews evidence suggesting that tumorigenesis associated with pathway activation may result from mis-specification of cells towards stem-cell or stem cell-like fates.

    View details for Web of Science ID 000168710000057

    View details for PubMedID 11357142

  • Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 during craniofacial morphogenesis DEVELOPMENTAL DYNAMICS Munoz-Sanjuan, I., Cooper, M. K., Beachy, P. A., Fallon, J. F., Nathans, J. 2001; 220 (3): 238-245

    Abstract

    Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during chicken craniofacial development. cFHF-4 is expressed in the mesenchyme of the frontonasal process, and in the mesenchyme and ectoderm of the mandibular processes. The expression of cFHF-4 and other genes implicated in facial patterning have been analyzed in talpid(2) embryos or in the presence of exogenous retinoic acid. Talpid(2) mutants show abnormal patterns of gene expression, including up-regulation of cFHF-4 in the developing face, which correlate with defects in cartilage formation. By contrast, expression of cFHF-4 in the developing face is strongly downregulated by teratogenic doses of all-trans retinoic acid in a dose-dependent manner. Low levels of retinoic acid that produce distal upper beak truncations do not affect cShh, c-Patched-1, or c-Bmp-2 expression in the face, but downregulate cFHF-4 in the frontonasal process.

    View details for Web of Science ID 000167131400005

    View details for PubMedID 11241832

  • Cholesterol modification of proteins BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS Mann, R. K., Beachy, P. A. 2000; 1529 (1-3): 188-202

    Abstract

    The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning. Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol. Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins. We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues. Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal.

    View details for Web of Science ID 000166141400017

    View details for PubMedID 11111088

  • Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine NATURE Taipale, J., Chen, J. K., Cooper, M. K., Wang, B. L., Mann, R. K., Milenkovic, L., Scott, M. P., Beachy, P. A. 2000; 406 (6799): 1005-1009

    Abstract

    Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

    View details for Web of Science ID 000089020200048

    View details for PubMedID 10984056

  • Genetics of ventral forebrain development and holoprosencephaly CURRENT OPINION IN GENETICS & DEVELOPMENT Muenke, M., Beachy, P. A. 2000; 10 (3): 262-269

    Abstract

    The disease holoprosencephaly is the basis of the most common structural anomaly of the developing forebrain in humans. Numerous teratogens when administered during early gastrulation, have been associated with this condition. Recent studies have characterized molecules expressed in the prechordal plate which are critical for normal brain formation. Perturbation of signaling pathways involving these molecules have been shown to cause holoprosencephaly in humans and other organisms.

    View details for Web of Science ID 000087349400004

    View details for PubMedID 10826992

  • Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb CELL Wang, B. L., Fallon, J. F., Beachy, P. A. 2000; 100 (4): 423-434

    Abstract

    Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. We demonstrate that PKA-dependent processing of vertebrate Gli3 in developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation.

    View details for Web of Science ID 000085446900005

    View details for PubMedID 10693759

  • Nuclear trafficking of cubitus interruptus in the transcriptional regulation of hedgehog target gene expression CELL Chen, C. H., von Kessler, D. P., Park, W. J., Beachy, P. A. 1999; 98 (3): 305-316

    Abstract

    Transcriptional activation of Hedgehog (Hh) target genes requires Cubitus interruptus, a 155 kDa cytoplasmic zinc finger protein (Ci155), which in the absence of Hh signaling is processed to form a nuclear repressor (Ci75). We show that Hh signaling reduces phosphorylation of Ci155, and this reduction in turn appears to decrease processing. Blocking processing with proteasome inhibitors or altered Ci proteins, however, is insufficient for activation of Hh targets. We find that Hh signaling increases the rate of Ci155 nuclear import, resulting in significant nuclear accumulation. Even in the absence of signaling, nuclear accumulation of Ci155 suffices for significant induction of Hh targets, and active nuclear export of Ci155 is an essential mechanism for maintenance of the unstimulated state.

    View details for Web of Science ID 000081950300006

    View details for PubMedID 10458606

  • Essential role for Sonic hedgehog during hair follicle morphogenesis DEVELOPMENTAL BIOLOGY Chiang, C., Swan, R. Z., Grachtchouk, M., Bolinger, M., Ying, L. T., Robertson, E. K., Cooper, M. K., Gaffield, W., Westphal, H., Beachy, P. A., Dlugosz, A. A. 1999; 205 (1): 1-9

    Abstract

    The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.

    View details for Web of Science ID 000078367800001

    View details for PubMedID 9882493

  • Teratogen-mediated inhibition of target tissue response to Shh signaling SCIENCE Cooper, M. K., Porter, J. A., Young, K. E., Beachy, P. A. 1998; 280 (5369): 1603-1607

    Abstract

    Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent teratogens capable of inducing cyclopia and other birth defects. Here, it is shown that these compounds specifically block the Sonic hedgehog (Shh) signaling pathway. These teratogens did not prevent the sterol modification of Shh during autoprocessing but rather inhibited the response of target tissues to Shh, possibly acting through the sterol sensing domain within the Patched protein regulator of Shh response.

    View details for Web of Science ID 000074061200041

    View details for PubMedID 9616123

  • Patterning of the embryonic forebrain CURRENT OPINION IN NEUROBIOLOGY Rubenstein, J. L., Beachy, P. A. 1998; 8 (1): 18-26

    Abstract

    The vertebrate forebrain is derived from the anterior neural plate, where anteroposterior, dorsoventral and local patterning mechanisms specify regional identify. The recent identification of genetic regulators of these processes has opened the way to elucidating how the major forebrain regions (i.e. cerebral cortex, basal ganglia, thalamus, and hypothalamus) are formed, and how molecular lesions in these processes cause human birth defects.

    View details for Web of Science ID 000072773400002

    View details for PubMedID 9568388

  • Mediation of Sonic Hedgehog induced expression of COUP-TFII by a protein phosphatase SCIENCE Krishnan, V., Pereira, F. A., Qiu, Y., Chen, C. H., Beachy, P. A., Tsai, S. Y., Tsai, M. J. 1997; 278 (5345): 1947-1950

    Abstract

    A Sonic hedgehog (Shh) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter that binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway.

    View details for Web of Science ID A1997YL00200035

    View details for PubMedID 9395397

  • Crystal structure of a hedgehog autoprocessing domain: Homology between hedgehog and self-splicing proteins CELL Hall, T. M., Porter, J. A., Young, K. E., Koonin, E. V., Beachy, P. A., Leahy, D. J. 1997; 91 (1): 85-97

    Abstract

    The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.

    View details for Web of Science ID A1997XZ80900011

    View details for PubMedID 9335337

  • Multiple roles of cholesterol in hedgehog protein biogenesis and signaling COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Beachy, P. A., Cooper, M. K., Young, K. E., von Kessler, D. P., Park, W. J., Hall, T. M., Leahy, D. J., Porter, J. A. 1997; 62: 191-204

    View details for Web of Science ID 000073570200024

    View details for PubMedID 9598352

  • Cholesterol modification of hedgehog signaling proteins in animal development SCIENCE Porter, J. A., Young, K. E., Beachy, P. A. 1996; 274 (5285): 255-259

    Abstract

    Hedgehog (Hh) proteins comprise a family of secreted signaling molecules essential for patterning a variety of structures in animal embryogenesis. During biosynthesis, Hh undergoes an autocleavage reaction, mediated by its carboxyl-terminal domain, that produces a lipid-modified amino-terminal fragment responsible for all known Hh signaling activity. Here it is reported that cholesterol is the lipophilic moiety covalently attached to the amino-terminal signaling domain during autoprocessing and that the carboxyl-terminal domain acts as an intramolecular cholesterol transferase. This use of cholesterol to modify embryonic signaling proteins may account for some of the effects of perturbed cholesterol biosynthesis on animal development.

    View details for Web of Science ID A1996VM67100046

    View details for PubMedID 8824192

  • Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function NATURE Chiang, C., Ying, L. T., Lee, E., Young, K. E., Corden, J. L., Westphal, H., Beachy, P. A. 1996; 383 (6599): 407-413

    Abstract

    Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

    View details for Web of Science ID A1996VL46300053

    View details for PubMedID 8837770

  • pH-dependent enchancement of DNA binding by the ultrabithorax homeodomain BIOCHEMISTRY Li, L. K., VONKESSLER, D., Beachy, P. A., Matthews, K. S. 1996; 35 (30): 9832-9839

    Abstract

    Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. We report in the present study that Ubx-HD binding to a single optimal target site displayed significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA was unaffected by pH. Results from studies of chimeric Ubx-Dfd homeodomains showed that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH was greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo.

    View details for Web of Science ID A1996VA02500024

    View details for PubMedID 8703957

  • Hedgehog patterning activity: Role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain CELL Porter, J. A., Ekker, S. C., Park, W. J., VONKESSLER, D. P., Young, K. E., Chen, C. H., Ma, Y., Woods, A. S., Cotter, R. J., Koonin, E. V., Beachy, P. A. 1996; 86 (1): 21-34

    Abstract

    Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

    View details for Web of Science ID A1996UX93400005

    View details for PubMedID 8689684

  • Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Szakmary, A., Huang, S. M., Chang, D. T., Beachy, P. A., Sander, M. 1996; 93 (4): 1607-1612

    Abstract

    Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

    View details for Web of Science ID A1996TW69800048

    View details for PubMedID 8643678

  • A POTENTIAL CATALYTIC SITE REVEALED BY THE 1.7-ANGSTROM CRYSTAL-STRUCTURE OF THE AMINO-TERMINAL SIGNALING DOMAIN OF SONIC HEDGEHOG NATURE Hall, T. M., Porter, J. A., Beachy, P. A., Leahy, D. J. 1995; 378 (6553): 212-216

    Abstract

    Within the past few years, members of the hedgehog (hh) family of secreted signalling proteins have emerged as the primary signals generated by certain embryonic patterning centres. In vertebrate embryos, for example, sonic hedgehog expression in the notochord appears to be responsible for the local and long-range induction of ventral cell types within the neural tube and somites (reviewed in refs 1, 2). Protein products encoded by hh family members are synthesized as precursors that undergo autoprocessing to generate an amino-terminal domain that appears to be responsible for both local and long-range signalling activities, and a carboxy-terminal domain that contains the autoprocessing activity. As part of an effort to understand how hh family members participate in cell-to-cell signalling, we have determined and report here the crystal structure at 1.7 A of the amino-terminal domain of murine Sonic hedgehog (Shh-N). The structure revealed a tetrahedrally coordinated zinc ion that appears to be structurally analogous to the zinc coordination sites of zinc hydrolases, such as thermolysin and carboxypeptidase A. This previously unsuspected catalytic site represents a distinct activity from the autoprocessing activity that resides in the carboxy-terminal domain.

    View details for Web of Science ID A1995TD75900062

    View details for PubMedID 7477329

  • CONTROL OF DROSOPHILA TRACHEAL BRANCHING BY THE NOVEL HOMEODOMAIN GENE UNPLUGGED, A REGULATORY TARGET FOR GENES OF THE BITHORAX COMPLEX DEVELOPMENT CHAING, C., Young, K. E., Beachy, P. A. 1995; 121 (11): 3901-3912

    Abstract

    We have identified a novel Drosophila homeodomain gene, unplugged (unp), whose function is required for formation of the tracheal branches that penetrate the CNS. In unp mutant embryos the segmentally repeated ganglionic branches stall and fail to penetrate the CNS and the segment-specific cerebral branch and associated cerebral anastomosis fail to form. Expression of unp in the founder cells for the cerebral branch within the first tracheal metamere is repressed in posterior segments by Ubx and other bithorax complex genes. This pattern of expression and homeotic gene regulation is reproduced by an unusual 2.6 kb cis-regulatory sequence located downstream of the unp transcription unit. Since the unp protein is localized to the nucleus of tracheal precursor cells as they migrate and extend, unp protein appears to play a regulatory role in neural branching of the tracheae, and the segment-specific aspects of these neural branching patterns appear to be generated by homeotic regulation of unp expression.

    View details for Web of Science ID A1995TF89600038

    View details for PubMedID 8582298

  • PATTERNING ACTIVITIES OF VERTEBRATE HEDGEHOG PROTEINS IN THE DEVELOPING EYE AND BRAIN CURRENT BIOLOGY Ekker, S. C., Ungar, A. R., Greenstein, P., VONKESSLER, D. P., Porter, J. A., MOON, R. T., Beachy, P. A. 1995; 5 (8): 944-955

    Abstract

    The hedgehog (hh) family of secreted signaling proteins is responsible for developmental patterning in a variety of systems, including the neural tube, limbs and somites. Within the neural tube, at the level of the spinal cord, products of the vertebrate gene sonic hedgehog (shh) are proposed to function as a ventral patterning influence, with the capability of inducing floor plate and motor neurons.We report the isolation of tiggy-winkle hedgehog (twhh), a novel member of the zebrafish hh gene family. Both twhh and shh are expressed in the ventral midline of the embryonic zebrafish neural tube and brain, but twhh expression becomes limited to the neural tube, whereas shh is also expressed in the notochord. Both genes are expressed in the developing brain, in domains that include a discrete region in the floor of the diencephalon, located between the sites of the future optic stalks. Using pax-2 and pax-6 as markers of proximo-distal fate within the developing eye, we found that ectopic expression of either hh gene promoted proximal fates and suppressed distal fates. In contrast, proximal fates were lost in cyclops mutant embryos, which lack twhh- and shh-expressing forebrain cells. Both twhh and shh proteins undergo autoproteolytic processing in vivo; a fragment corresponding to the amino-terminal cleavage product was sufficient to carry out all signaling activities associated with twhh in eye and brain development.These findings suggest that secreted signals encoded by members of the hedgehog gene family, emanating from the ventral midline of the neural tube, not only play important roles in dorso-ventral patterning of the brain but also appear to constitute an early patterning activity along the proximo-distal axis of the developing eyes.

    View details for Web of Science ID A1995RN56900024

    View details for PubMedID 7583153

  • PATTERNING OF THE NEURAL ECTODERM OF XENOPUS-LAEVIS BY THE AMINO-TERMINAL PRODUCT OF HEDGEHOG AUTOPROTEOLYTIC CLEAVAGE DEVELOPMENT Lai, C. J., Ekker, S. C., Beachy, P. A., MOON, R. T. 1995; 121 (8): 2349-2360

    Abstract

    The patterns of embryonic expression and the activities of Xenopus members of the hedgehog gene family are suggestive of role in neural induction and patterning. We report that these hedgehog polypeptides undergo autoproteolytic cleavage. Injection into embryos of mRNAs encoding Xenopus banded-hedgehog (X-bhh) or the amino-terminal domain (N) demonstrates that the direct inductive activities of X-bhh are encoded by N. In addition, both N and X-bhh pattern neural tissue by elevating expression of anterior neural genes. Unexpectedly, an internal deletion of X-bhh (delta N-C) was found to block the activity of X-bhh and N in explants and to reduce dorsoanterior structures in embryos. As elevated hedgehog activity increases the expression of anterior neural genes, and as delta N-C reduces dorsoanterior structures, these complementary data support a role for hedgehog in neural induction and anteroposterior patterning.

    View details for Web of Science ID A1995RN70300009

    View details for PubMedID 7671801

  • DISTINCT EXPRESSION AND SHARED ACTIVITIES OF MEMBERS OF THE HEDGEHOG GENE FAMILY OF XENOPUS-LAEVIS DEVELOPMENT Ekker, S. C., McGrew, L. L., Lai, C. J., Lee, J. J., VONKESSLER, D. P., MOON, R. T., Beachy, P. A. 1995; 121 (8): 2337-2347

    Abstract

    The hedgehog family of signaling proteins is associated with a variety of spatial patterning activities in insects and vertebrates. Here we show that new members of this family isolated from Xenopus laevis are expressed embryonically in patterns suggestive of roles in patterning in the ectoderm, nervous system and somites. Banded hedgehog is expressed throughout the neural plate and subsequently in both the nervous system and in the dermatome of somites. Cephalic hedgehog is expressed in anterior ectoderm and endodermal structures, and sonic hedgehog is expressed in patterns which parallel those in other species. Injection of RNAs encoding Xenopus hedgehogs induces ectopic cement gland formation in embryos. Similar to reported activities of noggin and follistatin, Xenopus hedgehogs share a common ability to induce cement glands in animal cap explants. However, hedgehog activities in naive ectoderm appear capable of acting independently of noggin and follistatin since, although all three are induced by activin in animal cap explants, X-hh expression does not induce noggin or follistatin.

    View details for Web of Science ID A1995RN70300008

    View details for PubMedID 7671800

  • INDUCTION OF MIDBRAIN DOPAMINERGIC-NEURONS BY SONIC HEDGEHOG NEURON Hynes, M., Porter, J. A., Chiang, C., Chang, D., TESSIERLAVIGNE, M., Beachy, P. A., Rosenthal, A. 1995; 15 (1): 35-44

    Abstract

    Midbrain dopaminergic neurons, whose loss in adults results in Parkinson's disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinson's disease.

    View details for Web of Science ID A1995RL75600007

    View details for PubMedID 7619528

  • LIMB-PATTERNING ACTIVITY AND RESTRICTED POSTERIOR LOCALIZATION OF THE AMINO-TERMINAL PRODUCT OF SONIC HEDGEHOG CLEAVAGE CURRENT BIOLOGY LOPEZMARTINEZ, A., Chang, D. T., Chiang, C., Porter, J. A., Ros, M. A., Simandl, B. K., Beachy, P. A., Fallon, J. F. 1995; 5 (7): 791-796

    Abstract

    Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH.We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud.Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

    View details for Web of Science ID A1995RL95300022

    View details for PubMedID 7583126

  • FLOOR PLATE AND MOTOR-NEURON INDUCTION BY DIFFERENT CONCENTRATIONS OF THE AMINO-TERMINAL CLEAVAGE PRODUCT OF SONIC HEDGEHOG AUTOPROTEOLYSIS CELL Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A., Jessell, T. M. 1995; 81 (3): 445-455

    Abstract

    The differentiation of floor plate cells and motor neurons can be induced by Sonic hedgehog (SHH), a secreted signaling protein that undergoes autoproteolytic cleavage to generate amino- and carboxy-terminal products. We have found that both floor plate cells and motor neurons are induced by the amino-terminal cleavage product of SHH (SHH-N). The threshold concentration of SHH-N required for motor neuron induction is about 5-fold lower than that required for floor plate induction. Higher concentrations of SHH-N can induce floor plate cells at the expense of motor neuron differentiation. Our results suggest that the induction of floor plate cells and motor neurons by the notochord in vivo is mediated by exposure of neural plate cells to different concentrations of the amino-terminal product of SHH autoproteolytic cleavage.

    View details for Web of Science ID A1995QW89400016

    View details for PubMedID 7736596

  • LONG-RANGE SCLEROTOME INDUCTION BY SONIC HEDGEHOG - DIRECT ROLE OF THE AMINO-TERMINAL CLEAVAGE PRODUCT AND MODULATION BY THE CYCLIC-AMP SIGNALING PATHWAY CELL Fan, C. M., Porter, J. A., Chiang, C., Chang, D. T., Beachy, P. A., TESSIERLAVIGNE, M. 1995; 81 (3): 457-465

    Abstract

    A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.

    View details for Web of Science ID A1995QW89400017

    View details for PubMedID 7736597

  • THE PRODUCT OF HEDGEHOG AUTOPROTEOLYTIC CLEAVAGE ACTIVE IN LOCAL AND LONG-RANGE SIGNALING NATURE Porter, J. A., VONKESSLER, D. P., Ekker, S. C., Young, K. E., Lee, J. J., Moses, K., Beachy, P. A. 1995; 374 (6520): 363-366

    Abstract

    The secreted protein products of the hedgehog (hh) gene family are associated with local and long-range signalling activities that are responsible for developmental patterning in multiple systems, including Drosophila embryonic and larval tissues and vertebrate neural tube, limbs and somites. In a process that is critical for full biological activity, the hedgehog protein (Hh) undergoes autoproteolysis to generate two biochemically distinct products, an 18K amino-terminal fragment, N, and a 25K carboxy-terminal fragment, C (ref. 16); mutations that block autoproteolysis impair Hh function. We have identified the site of autoproteolytic cleavage and find that it is broadly conserved throughout the hedgehog family. Knowing the site of cleavage, we were able to test the function of the N and C cleavage products in Drosophila assays. We show here that the N product is the active species in both local and long-range signalling. Consistent with this, all twelve mapped hedgehog mutations either affected the structure of the N product directly or otherwise blocked the release of N from the Hh precursor as a result of deletion or alteration of sequences in the C domain.

    View details for Web of Science ID A1995QN63000061

    View details for PubMedID 7885476

  • ULTRABITHORAX PROTEIN IS NECESSARY BUT NOT SUFFICIENT FOR FULL ACTIVATION OF DECAPENTAPLEGIC EXPRESSION IN THE VISCERAL MESODERM EMBO JOURNAL Sun, B., Hursh, D. A., Jackson, D., Beachy, P. A. 1995; 14 (3): 520-535

    Abstract

    To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression.

    View details for Web of Science ID A1995QF71800012

    View details for PubMedID 7859741

  • AUTOPROTEOLYSIS IN HEDGEHOG PROTEIN BIOGENESIS SCIENCE Lee, J. J., Ekker, S. C., VONKESSLER, D. P., Porter, J. A., Sun, B. I., Beachy, P. A. 1994; 266 (5190): 1528-1537

    Abstract

    Extracellular signaling proteins encoded by the hedgehog (hh) multigene family are responsible for the patterning of a variety of embryonic structures in vertebrates and invertebrates. The Drosophila hh gene has now been shown to generate two predominant protein species that are derived by an internal autoproteolytic cleavage of a larger precursor. Mutations that reduced the efficiency of autoproteolysis in vitro diminished precursor cleavage in vivo and also impaired the signaling and patterning activities of the HH protein. The two HH protein species exhibited distinctive biochemical properties and tissue distribution, and these differences suggest a mechanism that could account for the long- and short-range signaling activities of HH in vivo.

    View details for Web of Science ID A1994PV01500035

    View details for PubMedID 7985023

  • THE NOVEL HOMEODOMAIN GENE BUTTONLESS SPECIFIES DIFFERENTIATION AND AXONAL GUIDANCE FUNCTIONS OF DROSOPHILA DORSAL MEDIAN CELLS DEVELOPMENT Chiang, C., Patel, N. H., Young, K. E., Beachy, P. A. 1994; 120 (12): 3581-3593

    Abstract

    We have identified a novel homeodomain gene, buttonless (btn), that is specifically expressed in 20 cells of a single type during Drosophila embryonic development. These cells, the dorsal median (DM) cells, are arranged as a single pair within each segment along the dorsal midline of the CNS. Distinctive features of the DM cells include a large cell body and a long thick process extending laterally to the muscle attachment site. In the absence of btn gene function the initial commitment to the DM cell fate is made but differentiation fails to occur and the DM cells are lost. The btn mutation thus specifically eliminates the DM cells, and this genetic ablation in turn reveals a requirement for DM cells as cellular cues for axonal guidance during transverse nerve outgrowth and bifurcation of the median nerve.

    View details for Web of Science ID A1994QB20000022

    View details for PubMedID 7821224

  • PRODUCTS, GENETIC-LINKAGE AND LIMB PATTERNING ACTIVITY OF A MURINE HEDGEHOG GENE DEVELOPMENT Chang, D. T., Lopez, A., VONKESSLER, D. P., Chiang, C., Simandl, B. K., Zhao, R. B., Seldin, M. F., Fallon, J. F., Beachy, P. A. 1994; 120 (11): 3339-3353

    Abstract

    The hedgehog (hh) segmentation gene of Drosophila melanogaster encodes a secreted signaling protein that functions in the patterning of larval and adult structures. Using low stringency hybridization and degenerate PCR primers, we have isolated complete or partial hh-like sequences from a range of invertebrate species including other insects, leech and sea urchin. We have also isolated three mouse and two human DNA fragments encoding distinct hh-like sequences. Our studies have focused upon Hhg-1, a mouse gene encoding a protein with 46% amino acid identity to hh. The Hhg-1 gene, which corresponds to the previously described vhh-1 or sonic class, is expressed in the notochord, ventral neural tube, lung bud, hindgut and posterior margin of the limb bud in developing mouse embryos. By segregation analysis the Hhg-1 gene has been localized to a region in proximal chromosome 5, where two mutations affecting mouse limb development previously have been mapped. In Drosophila embryos, ubiquitous expression of the Hhg-1 gene yields effects upon gene expression and cuticle pattern similar to those observed for the Drosophila hh gene. We also find that cultured quail cells transfected with a Hhg-1 expression construct can induce digit duplications when grafted to anterior or mid-distal but not posterior borders within the developing chick limb; more proximal limb element duplications are induced exclusively by mid-distal grafts. Both in transgenic Drosophila embryos and in transfected quail cells, the Hhg-1 protein product is cleaved to yield two stable fragments from a single larger precursor. The significance of Hhg-1 genetic linkage, patterning activity and proteolytic processing in Drosophila and chick embryos is discussed.

    View details for Web of Science ID A1994PV97100026

    View details for PubMedID 7720571

  • EXPRESSION OF A NOVEL TOLL-LIKE GENE SPANS THE PARASEGMENT BOUNDARY AND CONTRIBUTES TO HEDGEHOG FUNCTION IN THE ADULT EYE OF DROSOPHILA MECHANISMS OF DEVELOPMENT Chiang, C., Beachy, P. A. 1994; 47 (3): 225-239

    Abstract

    Many proteins involved in signal transduction and cell adhesion are characterized by the presence of an extracellular domain with repeated copies of a leucine-rich motif (LRR). Here we report the isolation and characterization of a novel gene, tlr (for Toll-like receptor), which encodes a protein containing multiple LRRs in its presumed extracellular domain, a single transmembrane segment and homology to the cytoplasmic domain of the interleukin 1 receptor in its presumed intracellular domain. The pattern of tlr expression at the extended germ band stage is characterized by 15 transverse stripes in the gnathal and trunk segments, with four patches of expression corresponding to head segments and an additional patch of expression in the presumptive hindgut. The segmentally repeated tlr stripes in the trunk overlap both the wingless and engrailed stripes and thus span the parasegment boundary. The tlr stripes require pair rule gene function for their establishment and later become dependent upon segment-polarity gene function for their maintenance. Segmental modulation of tlr expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. The tlr gene also is prominently expressed in the imaginal discs. In the eye disc, this expression occurs in two stripes at the anterior and posterior margins of the morphogenetic furrow; this expression is consistent with a genetic interaction between a tlr mutation and an eye-specific allele of hedgehog. All of these data combine to suggest a role for tlr in interactions between cells at critical boundaries during development.

    View details for Web of Science ID A1994PJ97200003

    View details for PubMedID 7848870

  • THE DEGREE OF VARIATION IN DNA-SEQUENCE RECOGNITION AMONG 4 DROSOPHILA HOMEOTIC PROTEINS EMBO JOURNAL Ekker, S. C., Jackson, D. G., VONKESSLER, D. P., Sun, B. I., Young, K. E., Beachy, P. A. 1994; 13 (15): 3551-3560

    Abstract

    The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. We compare here the DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity. We further show that amino acid residues within the N-terminal arm are responsible for the sequence specificity differences between the Ultrabithorax and Abdominal-B homeodomains. Similarities and differences at the corresponding positions within the N-terminal arms are conserved in the vertebrate Abdominal-B-like HOM proteins, which play critical roles in limb specifications as well as in regional specification along the anterior-posterior axis. This and other patterns of residue conservation suggest that differential DNA sequence recognition may play a role in HOM protein function in a wide range of organisms.

    View details for Web of Science ID A1994PB90900017

    View details for PubMedID 7914870

  • THE SEGMENT POLARITY GENE HEDGEHOG IS REQUIRED FOR PROGRESSION OF THE MORPHOGENETIC FURROW IN THE DEVELOPING DROSOPHILA EYE CELL Ma, C. Y., Zhou, Y., Beachy, P. A., Moses, K. 1993; 75 (5): 927-938

    Abstract

    Cell-type specification in the Drosophila compound eye begins at the morphogenetic furrow. The furrow sweeps across the developing eye epithelium and is coincident with four classes of cellular events: coordinated changes in cell shape, changes in gene expression, synchronization of the cell cycle, and the specification of a regular array of ommatidial founder cells. The molecular mechanisms that induce these events in the developing eye have hitherto been unknown. We identify here a gene specifically required for furrow progression, hedgehog (hh). We show that hh expression posterior to the morphogenetic furrow is continuously required for its progression. We propose that forward diffusion of hh protein induces anterior cells to enter the furrow.

    View details for Web of Science ID A1993MK96600012

    View details for PubMedID 8252628

  • COOPERATIVE BINDING OF AN ULTRABITHORAX HOMEODOMAIN PROTEIN TO NEARBY AND DISTANT DNA SITES MOLECULAR AND CELLULAR BIOLOGY Beachy, P. A., Varkey, J., Young, K. E., VONKESSLER, D. P., Sun, B. I., Ekker, S. C. 1993; 13 (11): 6941-6956

    Abstract

    Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity.

    View details for Web of Science ID A1993MC84400033

    View details for PubMedID 8105373

  • DIFFERENTIAL DNA-SEQUENCE RECOGNITION IS A DETERMINANT OF SPECIFICITY IN HOMEOTIC GENE-ACTION EMBO JOURNAL Ekker, S. C., VONKESSLER, D. P., Beachy, P. A. 1992; 11 (11): 4059-4072

    Abstract

    The homeotic genes of Drosophila encode transcriptional regulatory proteins that specify distinct segment identities. Previous studies have implicated the homeodomain as a major determinant of biological specificity within these proteins, but have not established the physical basis of this specificity. We show here that the homeodomains encoded by the Ultrabithorax and Deformed homeotic genes bind optimally to distinct DNA sequences and have mapped the determinants responsible for differential recognition. We further show that relative transactivation by these two proteins in a simple in vivo system can differ by nearly two orders of magnitude. Such differences in DNA sequence recognition and target activation provide a biochemical basis for at least part of the biological specificity of homeotic gene action.

    View details for Web of Science ID A1992JT32800028

    View details for PubMedID 1356765

  • SECRETION AND LOCALIZED TRANSCRIPTION SUGGEST A ROLE IN POSITIONAL SIGNALING FOR PRODUCTS OF THE SEGMENTATION GENE HEDGEHOG CELL Lee, J. J., VONKESSLER, D. P., Parks, S., Beachy, P. A. 1992; 71 (1): 33-50

    Abstract

    The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.

    View details for Web of Science ID A1992JQ62300005

    View details for PubMedID 1394430

  • OPTIMAL DNA-SEQUENCE RECOGNITION BY THE ULTRABITHORAX HOMEODOMAIN OF DROSOPHILA EMBO JOURNAL Ekker, S. C., Young, K. E., VONKESSLER, D. P., Beachy, P. A. 1991; 10 (5): 1179-1186

    Abstract

    The 61 amino acid homeodomain is conserved among members of a family of eukaryotic DNA-binding proteins that play regulatory roles in transcription and in development. We have refined a rapid method for determining optimal DNA binding sites and have applied it to a 72 amino acid peptide containing the homeodomain of the Ultrabithorax (Ubx) homeotic gene of Drosophila. The site (5'-TTAATGG-3') is tightly bound (KD approximately 7 x 10(-11) M) by the Ubx homeodomain peptide; the four central TAAT bases of this sequence play a primary role in determining the affinity of binding, with significant secondary contributions deriving from the flanking bases. Although previously defined genomic sites contain multiple TAAT sequences with flanking bases distinct from those in the optimal binding site, we have found a new binding site with seven near-perfect repeats of the optimal sequence; this site is located in the promoter region of decapentaplegic, a probable Ubx regulatory target. The presence of a TAAT motif in the binding sites for most other homeodomain proteins suggests the existence of a conserved mechanism for recognition of this core sequence, with further specificity conferred by interactions with bases flanking this core.

    View details for Web of Science ID A1991FJ82200018

    View details for PubMedID 1673656

  • A MOLECULAR VIEW OF THE ULTRABITHORAX HOMEOTIC GENE OF DROSOPHILA TRENDS IN GENETICS Beachy, P. A. 1990; 6 (2): 46-51

    Abstract

    The pioneering work of E.B. Lewis drew attention early on to the developmental role of Ultrabithorax (Ubx), one of a group of homeotic genes that specify the distinguishing features of Drosophila segments. Recent molecular work sheds light on the genetic complexity discovered by Lewis and demonstrates that Ubx executes its developmental role by producing a family of closely related DNA-binding proteins that can function as transcription factors.

    View details for Web of Science ID A1990CM95100007

    View details for PubMedID 1970912

  • STRUCTURE AND EXPRESSION OF A FAMILY OF ULTRABITHORAX MESSENGER-RNAS GENERATED BY ALTERNATIVE SPLICING AND POLYADENYLATION IN DROSOPHILA GENES & DEVELOPMENT Kornfeld, K., Saint, R. B., Beachy, P. A., Harte, P. J., PEATTIE, D. A., Hogness, D. S. 1989; 3 (2): 243-258

    Abstract

    The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.

    View details for Web of Science ID A1989T437900011

    View details for PubMedID 2565858

  • AN ULTRABITHORAX PROTEIN BINDS SEQUENCES NEAR ITS OWN AND THE ANTENNAPEDIA P1 PROMOTERS CELL Beachy, P. A., Krasnow, M. A., Gavis, E. R., Hogness, D. S. 1988; 55 (6): 1069-1081

    Abstract

    The homeotic gene Ultrabithorax (Ubx), located in the bithorax complex of Drosophila, encodes a family of closely related proteins that direct the developmental fates of posterior thoracic and anterior abdominal metameres. We have purified a member of the Ubx protein family from an overproducing E. coli strain and have shown that it is sequence-specific DNA binding protein. The protein binds tightly to sequences near its own promoter and near the P1 promoter of Antenna-pedia (Antp), a homeotic gene Ubx is known to repress from genetic studies. The binding sites occur in clusters downstream of the transcription start sites, and far upstream at Antp P1. They range in size from 40 to 90 bp, and contain tandem repeats of the trinucleotide TAA or the related hexanucleotide TAA-TCG. These results suggest that the regulatory activities of Ubx are direct and are mediated by binding of Ubx proteins to promoter region sequences.

    View details for Web of Science ID A1988R562400015

    View details for PubMedID 2904838

  • REGULATION AND PRODUCTS OF THE UBX DOMAIN OF THE BITHORAX COMPLEX COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Hogness, D. S., Lipshitz, H. D., Beachy, P. A., PEATTIE, D. A., Saint, R. B., GOLDSCHMIDTCLERMONT, M., Harte, P. J., Gavis, E. R., Helfand, S. L. 1985; 50: 181-194

    View details for Web of Science ID A1985C628800023

    View details for PubMedID 3938361

  • SEGMENTAL DISTRIBUTION OF BITHORAX COMPLEX PROTEINS DURING DROSOPHILA DEVELOPMENT NATURE Beachy, P. A., Helfand, S. L., Hogness, D. S. 1985; 313 (6003): 545-551

    Abstract

    The Ubx and bxd transcription units comprise a single functional domain in the bithorax complex of Drosophila melanogaster. The segmental distributions and nuclear localization of proteins encoded by the Ubx unit have been determined by immunofluorescence staining with antibodies raised against a fusion protein containing Ubx coding sequences. Wild-type and mutant distributions are consistent with a model in which the protein-coding functions of the domain derive from the Ubx unit and are regulated by the bxd unit.

    View details for Web of Science ID A1985ABQ6600040

    View details for PubMedID 3918274

  • MOLECULAR-GENETICS OF THE BITHORAX COMPLEX IN DROSOPHILA-MELANOGASTER SCIENCE Bender, W., Akam, M., KARCH, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B., Hogness, D. S. 1983; 221 (4605): 23-29

    Abstract

    The bithorax complex in Drosophila melanogaster is a cluster of homeotic genes that specify developmental pathways for many of the body segments of the fly. The DNA of the bithorax complex has been isolated, and a region of 195,000 base pairs that covers the left half of the complex is described here. The lesions associated with many of the bithorax complex mutants have been identified, and most are due to DNA rearrangements. Most of the spontaneous mutants have insertions of a particular mobile element named "gypsy." This element affects the functions of sequences removed from the site of insertion. Mutant lesions for a given phenotypic class are distributed over large DNA distances of up to 73,000 base pairs.

    View details for Web of Science ID A1983QV59400006

    View details for PubMedID 17737996

Conference Proceedings


  • Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched Fuse, N., Maiti, T., Wang, B. L., Porter, J. A., Hall, T. M., Leahy, D. J., Beachy, P. A. NATL ACAD SCIENCES. 1999: 10992-10999

    Abstract

    The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.

    View details for Web of Science ID 000082868500012

    View details for PubMedID 10500113

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