Key processes in development, including differential cell proliferation, programmed cell death, migration and cell-cell interactions are recapitulated in a pathologic manner during oncogenesis and metastasis, suggesting an aberrant regulation or 're-activation' of such processes. For example, FGF, Wnt, and Shh signaling pathways are all involved in the genesis of certain human neoplasias. Understanding how developmental processes are normally regulated will be invaluable in deciphering tumor biology and ultimately help to identify new ways to intercept cellular targets that drive tumor cell behavior. My lab identified several new transcription factors (homeobox genes and T-box genes) that appear to play roles in the formation and pattern of the primary embryonic axis and the limb axis in vertebrate embryos. Analyzing the role that such regulators play in different developmental contexts may offer new insights into the sorts of basic processes that they govern in the cell. We are analyzing the normal developmental functions of several regulators with the long term aim of linking steps from initial differences in patterns of gene expression to ultimate differences in tissue morphology and structure in the embryo. In this context, potential roles in oncogenesis and other pathologic conditions are also being considered. 1. Initiation of limb budding: function of the transcription factor T (Brachyury): The initiation of limb bud formation and induction of the apical ectodermal ridge (AER) that directs subsequent limb outgrowth appear to be regulated by a cascade of FGF and Wnt signals. Intermediate target genes in this signal relay are largely unknown. Using retroviral misexpression in chick embryos, we have found that the T-box transcription factor T regulates the formation and maintenance of the AER. T is expressed in the early limb bud (in the mesoderm just underneath the AER) and is regulated by both FGF and Wnt signals. Overexpression causes extension of the AER and additional limb outgrowth, producing extra digits. Mouse embryos mutant for T (-/-) die at E10.5 but do form forelimb buds which are small. These limb buds do not have a normal AER and have very irregular Fgf-8 expression, also consistent with a role for T in normal AER formation. We don't yet know whether T misexpression by itself is sufficient to induce formation of an ectopic limb bud in the flank, as for example, FGF beads or Wnt misexpression can do. Other T-box genes, Tbx5 and Tbx4, regulate both limb identity and limb outgrowth, and also function apparently downstream of Wnt and FGF signals. We are evaluating whether AER formation and outgrowth is regulated by T-Tbx heterodimers and we are generating a T conditional knock out to further analyze T function in limb development. In parallel studies, we are developing chromatin immunoprecipitation assays in embryos to allow direct identification of target promoters which T binds to in vivo. 2. Determination of limb skeletal pattern: early Hoxd functions in SHH pathway: The limb skeleton arises from a bud by progressive branching of skeletal precursors from proximal to distal (ie. shoulder to hand). The 'pattern' of skeletal elements that form from anterior to posterior (thumb to little finger) is regulated by secreted Sonic hedgehog (SHH) signals from the posterior edge of the limb bud. Hoxd genes are thought to be key targets of SHH signaling that regulate the pattern of skeletal components forming in the limb. Their function in molecular terms and the target genes they regulate are still unknown. Using transgenic mouse models, we found that Hoxd12 regulates Shh expression and the Shh pathway as part of a positive feedback loop in the early limb bud which reinforces and amplifies patterning signals at the right sites. We are evaluating the mechanism by which Hoxd genes can alter the Shh pathway. The Gli3 transcriptional repressor is a major mediator of SHH in the developing limb. We have now found that a direct interaction between Gli3 and Hoxd proteins alters the Sonic hedgehog pathway and skeletal patterning during limb development. Antagonism of Gli3 transcriptional repressor function by SHH signaling derepresses SHH target genes in the limb. Gli3 and Hoxd12 interact genetically and physically, and this interaction converts Gli3 from a repressor into an activator of SHH target genes. Several 5'Hoxd genes interact with Gli3, and we propose that the sum of these interactions determines the levels of expression of Gli3-regulated SHH target genes during the later stages of digit patterning, when Shh expression has declined, but 5'Hoxd genes and Gli3 are still highly active. We are planning Hoxd and Gli3 misexpression experiments in the chick to evaluate the effects of varying Hoxd:Gli3 ratios on digit patterns formed, in collaboration with Dr. John Fallon (Univ. Wisconsin, Madison), who has demonstrated late ability of interdigit signals to change digit identities. These interdigit zones are also the late sites of Hoxd and Gli3 repressor expression. 3. Chondrogenic differentiation of limb skeletal elements: late Hoxd functions: Hoxd genes continue to be expressed late at the periphery of chondrogenic condensations that will form the skeletal elements, and then in growth plates of the limb. We developed an inducible transgenic model to selectively analyze later developmental functions of 5' Hoxd genes in condensing and proliferating cartilage and also identify molecular targets using DNA microarray analysis of transgenic embryonic limb buds. Our preliminary results with late Hoxd12 or Hoxd13 misexpression reveal an ability to regulate limb pattern and growth at very late (differentiation) stages of development. When limb mesoderm first condenses to initiate chondrogenesis, prolonging Hoxd expression within the differentiating condensations has a striking effect on long bone precursors and on digit number. Failure to shut off Hoxd expression during early condensation phase prevents the differentiation of the large, proximal long bone condensations into cartilage. Digit number is also decreased, but surpisingly this appears to be indirect; due to the loss of the normal earlier forming proximal condensations. This finding reveals an unexpected and novel function of proximal limb elements in supporting the survival of more distal elements together with AER signals from the distal limb bud. In the absence of the proximal condensations, a survival signal is lost and extensive apoptosis occurs in the distal digit-forming region. We are developing organ-culture strategies to identify the nature of this proximal 'signal' that is disturbed due to Hoxd misexpression. Chromatin immunoprecipitation assays for Hoxd binding in vivo are being developed in parallel with the conditional transgenic approaches and microarray analyses to identify and analyze regulation of Hoxd gene target promoters. 4. Direct evaluation of retinoid morphogen gradients in embryos: There is evidence that retinoids regulate a large number of developmental events, including the positions for limb budding to initiate along the trunk of the embryo, early steps in the establishment of left/right body asymmetry and the regionalization/differentiation of the central nervous system. Retinoic acid may function as a `morphogen' in such processes, but RA gradients have never been directly demonstrated. In collaboration with Dr. Gordon Hager (NCI) we have developed a new, sensitive assay for retinoids using a glucocorticoid receptor-GFP fusion chimera that contains the retinoic acid receptor ligand-binding domain (LBD) in place of the GR LBD. This chimera is cytoplasmic but rapidly translocates to the nucleus when retinoids are present (in 20-30min), allowing for detection of RA levels in situ in live embryos by confocal microscopy. Physiologic RA concentrations (~10-100nM) are sufficient to cause translocation. We plan to introduce this chimera into embryos using several approaches (in zebrafish, chick and mouse) to evaluate retinoid distributions during RA-regulated developmental events and determine whether gradients are present and important for the regulation of these events. We are particularly interested in addressing several questions regarding the role of RA morphogen gradients in the initiation of left/right asymmetry, the establishment of limb position and the formation of limb polarizing regions--the sites of Shh signaling in the limb bud. Because of ease of manipulating gene expression in early zebrafish embryos, we are piloting the technique in zebrafish embryos to examine initiation of L/R asymmetry in collaboration with Dr. Brant Weinstein (NICHD). 5. Origin of the sternum as part of the forelimb: In the course of our analyses of limb development, we have found that the sternum is regulated by SHH signals together with the limb. This may explain the association of both sternal and limb abnormalities with Gorlin's syndrome (basal cell nevus syndrome), and has led us to suspect that the sternum arose during evolution from a proximal part of the forelimb. The sternum arises from two separate prechondrogenic condensations that form adjacent to each forelimb bud and subsequently move together to meet in the ventral midline. The developmental origin becomes understandable if the sternum arose as an adaptation of the shoulder girdle for land locomotion to bring the forelimbs together and to generate a closed rib cage for land breathing. We are using defective retroviral reporters for lineage mapping to determine whether the sternum arises from a common precursor condensation together with the proximal forelimb.
