Instructor, Medicine - Oncology
Ph.D., Columbia University, Biochemistry and Mol. Biophysics (1995)
Our interests are to understand the mechanism and control of signals that regulate proliferation and differentiation in adult tissue. We are currently focused on studying modulation of the Hedgehog pathway in brain and muscle stem cell compartments during normal homeostasis and in degeneration or disease.
The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry. VIDEO ABSTRACT:
View details for DOI 10.1016/j.neuron.2012.02.009
View details for Web of Science ID 000301998700010
View details for PubMedID 22445340
In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.
View details for DOI 10.1038/nature05246
View details for Web of Science ID 000242018300049
View details for PubMedID 17086203
A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximately 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.
View details for DOI 10.1073/pnas.0505474102
View details for Web of Science ID 000231916300034
View details for PubMedID 16129827
Otx1 is a homeodomain protein required for axon refinement by layer 5 neurons in developing cerebral cortex. Otx1 localizes to the cytoplasm of progenitor cells in the rat ventricular zone, and remains cytoplasmic as neurons migrate and begin to differentiate. Nuclear translocation occurs during the first week of postnatal life, when layer 5 neurons begin pruning their long-distance axonal projections. Deletion analysis reveals that Otx1 is imported actively into cell nuclei, that the N-terminus of Otx1 is necessary for nuclear import, and that a putative nuclear localization sequence within this domain is sufficient to direct nuclear import in a variety of cell lines. In contrast, GFP-Otx1 fusion proteins that contain the N-terminus are retained in the cytoplasm of cortical progenitor cells, mimicking the distribution of Otx1 in vivo. These results suggest that ventricular cells actively sequester Otx1 in the cytoplasm, either by preventing nuclear import or by promoting a balance of export over import signals.
View details for DOI 10.1006/mcne.2001.1076
View details for Web of Science ID 000174845800010
View details for PubMedID 11906214
CDO is a cell-surface protein of the immunoglobulin/fibronectin type III repeat family that positively regulates myogenic differentiation in vitro. To gain a better understanding of the role of cdo during vertebrate development, we carried out an extensive in situ hybridization study to characterize its expression pattern from postimplantation to late stages of mouse embryogenesis and in rat brain from E13 to adult. Our results show a broad pattern of cdo expression that is spatially and temporally restricted during embryogenesis. In the central nervous system (CNS), cdo expression is detected as early as E7.5 and maintained in the dorsal ventricular zones of the brain and spinal cord, becoming increasingly restricted in the adult. High levels of cdo are detected in developing sensory organs, such as the eye and ear. Outside the CNS, cdo is expressed mainly in neural crest and mesodermal derivatives, including skeletal muscle precursors. Overall, the highest levels of cdo expression are seen from E9.0 to E15.5. The temporal onset and restricted expression of cdo suggest that cdo plays a role in the determination and/or differentiation of a number of cell types during embryogenesis.
View details for Web of Science ID 000089062600006
View details for PubMedID 10974670
T cell receptor (TCR) beta variable region genes are assembled in progenitor T cells from germ-line Vbeta, Dbeta, and Jbeta segments via an ordered two-step process in which Dbeta to Jbeta rearrangements occur on both alleles before appendage of a Vbeta to a preexisting DJbeta complex. Direct joining of Vbeta segments to nonrearranged Dbeta or Jbeta segments, while compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCRbeta locus. We have analyzed mechanisms that mediate ordered Vbeta, Dbeta, and Jbeta assembly via an approach in which TCRbeta minilocus recombination substrates were introduced into embryonic stem cells and then analyzed for rearrangement in normal thymocytes by recombinase-activating gene 2-deficient blastocyst complementation. These analyses demonstrated that Vbeta segments are preferentially targeted for rearrangement to Dbeta as opposed to Jbeta segments. In addition, we further demonstrated that Vbeta segments can be appended to nonrearranged endogenous Dbeta segments in which we have eliminated the ability of Dbeta segments to join to Jbeta segments. Our findings are discussed in the context of the mechanisms that regulate the ordered assembly and utilization of V, D, and J segments.
View details for Web of Science ID 000088048400058
View details for PubMedID 10869424
To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.
View details for Web of Science ID 000079906200014
View details for PubMedID 10328944
T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.
View details for Web of Science ID 000077188400011
View details for PubMedID 9850857
We have tested the ability of the T cell receptor beta (TCR-beta) transcriptional enhancer (E beta) to confer transcriptional activation and tissue-specific V(D)J recombination of TCR-beta V, D, and J segments in a transgenic minilocus recombination substrate. We find that the minimal E beta element, as previously shown for a DNA segment that contained the E mu element, promotes a high level of substrate D to J beta rearrangement in both B and T cells, but only promotes V beta to DJ beta rearrangement in T cells. Thus, both the E mu and E beta elements similarly direct V(D)J recombination of this substrate in vivo, supporting a general role for transcriptional enhancers in the normal regulation of this rearrangement process. Surprisingly, however, we found that both the V beta and DJ beta portion of the constructs were transcribed in an enhancer-dependent fashion (conferred by either E mu or E beta) in both B and T lineage cells, including normal precursor B cells propagated in culture. These findings indicate that, at least in some contexts, transcriptional activation, per se, is not sufficient to confer V(D)J recombinational accessibility to a substrate V gene segment.
View details for Web of Science ID A1994NR98900029
View details for PubMedID 8006587
The somatic assembly of antigen receptor genes--known as V(D)J recombination--is a highly regulated process at several different levels. V(D)J rearrangement is limited to stages of early B and T cell development by the lymphoid specific expression of recombinase activity. A common recombinase is apparently responsible for the rearrangement of all antigen receptor loci. Thus, the tissue specific and ordered rearrangement of each antigen receptor locus must depend on controlling their 'accessibility' to the recombinase. Here, we outline recent work that has contributed to the current understanding of mechanisms that control V(D)J rearrangement.
View details for PubMedID 7948958
During the assembly of immunoglobulin and T cell receptor variable region genes from variable (V), diversity (D), and joining (J) segments, the germline-encoded repertoire is further diversified by processes that include the template-independent addition of nucleotides (N regions) at gene segment junctions. Terminal deoxynucleotidyl transferase (TdT)-deficient lymphocytes had no N regions in their variable region genes, which shows that TdT is responsible for N region addition. In addition, certain variable region genes appeared at increased frequency in TdT-deficient thymocytes, which indicates that N region addition also influences repertoire development by alleviating sequence-specific constraints imposed on the joining of particular V, D, and J segments.
View details for Web of Science ID A1993LU58600033
View details for PubMedID 8356451
We have utilized subtractive hybridization to isolate 16 distinct cDNA sequences representing genes expressed in pre-B-cell lines but not myeloma cell or fibroblast lines. These sequences represent RNA transcripts that vary in abundance in pre-B-cell lines from 0.001% to 0.05%. Five of these sequences were not related to any known genes. One was related to but distinct from known myosin regulatory light chain genes and another encoded a protein with lectin domains. Three represented previously identified genes encoding carbonic anhydrase type II, thymosin, and CD2; these genes were not previously known to be specifically expressed in early stages of B-cell development. Other isolated genes corresponded to pre-B-cell-specific or pre-B-cell/B cell-specific genes recently described by others. The isolated cDNA sequences may be divided into two general categories--those representing genes expressed only in the pre-B-cell stage of B-cell development and those expressed in both the pre-B-cell and B-cell stages. The in vivo expression patterns of the identified genes suggest that some function specifically in lymphocytes while others may have roles in additional lineages.
View details for Web of Science ID A1990DR71400036
View details for PubMedID 1696011
The CD3+, IL-2-dependent normal human thymocyte clone, CII, expresses on its surface a CD3-associated gamma/delta TCR. We have further elucidated the structure of this receptor from the nucleotide sequence of cDNA and genomic clones from CII that encode functional TCR-gamma and -delta chains. We find that the CII line expresses a C gamma 2 constant region that is a polymorphic form lacking a copy of an internal exon; the sequence of this constant region accounts for the size of the gamma chain and noncovalent linkage of gamma and delta chains in the CII TCR. The V gamma region used for the CII TCR is identical to the several previously characterized expressed human V gamma segments. Possible implications of this finding are discussed.
View details for Web of Science ID A1988Q919000021
View details for PubMedID 2844954
A novel human T cell receptor (TcR) gene, located 85 kbp upstream to the C alpha coding regions, was isolated using human genomic clones to identify cDNA homologous to messages encoded by this region. The deduced protein sequence of this gene is highly homologous to that of the newly identified constant region found in the murine TcR alpha chain locus. This gene undergoes rearrangements and is expressed at the RNA level in human thymocytes, peripheral T cells and several leukemic T cell lines which have been shown to express the surface gamma-delta heterodimer, suggesting that this gene encodes the human T cell delta chain.
View details for Web of Science ID A1988M795400015
View details for PubMedID 2965024
A study was undertaken to test for the occurrence of DNA rearrangements or amplifications during embryonic development in Xenopus laevis. DNA isolated from testes and liver was digested with four restriction enzymes, separated on agarose gels, transferred to nitrocellulose, and hybridized with over 50 cloned cDNA probes generated from embryonic poly (A)+ RNA. No qualitative or quantitative differences were detectable in the DNA hybridization patterns of testes and liver DNA, suggesting that, at least during liver development, selective amplifications or rearrangements occur rarely if at all. In the course of this investigation a wide range of restriction-site polymorphisms for different genes was observed. While some genes showed little polymorphism among different animals, several genes showed considerable polymorphism, involving changes in several restriction enzyme sites. These complex polymorphisms could be the result of gene rearrangements that occur occasionally during the course of sexual reproduction rather than during development.
View details for Web of Science ID A1985AKF0300003
View details for PubMedID 2991055