Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are using combined genetic, genomic, and proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior pattern (thumb to pinky) and the final morphogenesis of distinct digits. Learning how this 3-dimensional structure forms will be generally relevant for understanding how organogenesis is achieved and insights on how growth and morphogenesis are orchestrated will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. 1) Early events downstream of Shh: Our analyses of temporal requirements for Shh signals in mutant mouse limb buds suggests that Shh acts at early stages to specify digits through an indirect signal relay rather than acting as a classical morphogen, and more likely acts to divide the limb field into discrete domains with differing potential to respond to secondary downstream signals, than to specify 'final' distinct digit identities. To determine the initial differences established during early signaling, we will perform single cell transcriptome analysis from normal limb buds at, and shortly after Shh activation to identify expression signatures and characterize immediate-early response zones. This will provide a foundation for subsequent studies using mouse mutants in which early Shh activity is altered. Furthermore, our genetic studies indicate that there are 2 classes of Shh responsive target genes with very different regulatory features: those that respond to a transient signal and become stably expressed, and those that require continuous signaling to maintain their expression. From our analysis, the former class would include targets critical for organizing a basic pattern of limb elements that can form, and the latter would include regulators of growth and survival necessary for the later expansion and morphogenesis of these elements. We are comparing the transcriptomes of control, Shh mutant, and rescued Shh mutant limb buds (enforced cell survival substituting for late function), to begin to characterize the genes in these two dstinct target classes and determine the basis of their differential regulation. Understanding the proliferative and anti-apoptotic roles of Shh in the context of these differentially regulated target classes will provide a reference for deciphering and intercepting Shh roles in cancer as well as normal development. 2) Feedback circuits between Shh and Fgf signaling: Reciprocal positive and negative feedback loops between the mesodermal Shh-expressing and ectodermal Fibroblast growth factor (Fgf)-expressing signaling centers in the limb bud act to both maintain and restrict each other's activity in regulating digit pattern and outgrowth and eventually to terminate activity when limb organogenesis is complete. We are using genetic strategies to manipulate Shh and Fgf levels at different limb bud stages, to begin to unravel the positive and negative regulatory inputs controlling their expression. These results will be incorporated into the analysis of the regulatory networks operating at different stages of limb morphogenesis to arrive at a more complete model of how these circuits are integrated. 3) Gli3-Hox interactions and regulation of morphogenesis: Gli3-Hox protein-protein interactions govern multiple processes in limb morphogenesis, including the rate of proliferation and timing of cell adhesion during formation of progenitor skeletal condensations, and the control of distinct final digit morphologies by late signals from interdigital tissues (webbing) adjacent to each of the digit primordia. We previously identified a highly conserved domain in Gli3 that interacts with Hox factors and also several other key developmental regulators (Smad1, beta-catenin). We will use mass spectrometry to elucidate the range of partners that can modulate Gli3 activity in the limb, and may also compete Hox protein binding, which will be validated using other biochemical and genetic strategies. In parallel, to gain insight into the mechanisms by which Gli3 and Hoxd proteins act antagonistically, we are comparing the normal limb transcriptome with 5'Hoxd, Gli3, and compound mutants, to identify expression changes in potential gene targets. Our results will be compared with known direct transcriptional targets of Hoxd13 and of Gli3 (from available ChIPseq data) and supplemented with ChIPseq in our lab for later limb stages if needed. We have engineered an epitope-tagged Hoxd13 conditional transgene allele for ChIP in collaboration with Steve Vokes (UT Austin), who generated a similarly epitope-tagged Gli3 mouse line used for genomewide ChIP. Identifying late-stage Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. The transcriptional network regulated by Hoxd and Gli3 in the limb will also be analyzed in relation to Shh-pathway targets that form two distinct classes, requiring either transient or sustained signaling for their stable activation. Finally, single cell expression profiling will be used to characterize the digit progenitor regions (digit tips) that are instructed to form phalangeal segments and joints by the interdigit signaling network that is controlled by Gli3-Hox balance. This region behaves as a stem cell pool for the digit skeleton and has some limited regenerative potential even in mammals. Using this combination of approaches, we hope to uncover the regulatory cascade leading to formation of defined digit morphologies with distinct numbers of segments and joints. Gli3 and Hox genes are also aberrantly co-expressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts. 4) Insights on regulatory network from adaptive limb modifications: The basic regulatory network instructing formation of the limb skeleton is largely conserved throughout vertebrates. Uncovering regulatory changes that underlie evolutionary adaptations can illuminate critical network parameters and basis for robustness. Previous work in chick, and in mouse from our lab, have shown that digit morphology (identity) is regulated at late stages by interdigit signals. Our genetic evidence indicates that 5'Hoxd and Gli3 are part of an interdigit signaling center that regulates final digit identity. Elucidating signaling pathway differences between different interdigits will provide new insights on how digit identity is regulated at late stages and potential mechanisms by which Hoxd and Gli3 genes act. We are comparing interdigit expression profiles in species with digit adaptations, to correlate morphogenetic changes with changes in signaling activity, comparing three vertebrates: chick, mouse, and bat (collaborators J. Rasweiler, SUNY; M. Ros, U. Cantabria). Both bats and birds have evolved striking digit adaptations for flight and also have highly adapted hindlimbs including changes in phalanx number and joint formation. Comparative transcriptome analysis (collaboration with R. Agarwala, NCBI) of interdigits and responsive digit condensations of different organisms with very different digit morphologies will provide new insights on how digit identity is regulated and evolutionary adaptation occurs. Together, these studies will also be highly relevant to congenital malformations and regenerative medicine.
