Genome-wide target analysis of Shh-activated transcription network in limb bud

Division Of Basic Sciences - Nci

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Abstract

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. 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 using transcriptome profiling of Shh mutant limb buds to begin to characterize the genes in these two distinct target classes and determine the basis of their differential regulation. Transcriptome profiles of Shh conditional mutant (floxed allele) vs. Shh conditional mutant rescued by a transient Shh exposure (early "trigger" function) and bypassing the need for Shh to support cell survival (Bax/Bak knockout, see also Project ZIA BC 011118) will be compared to identify expression signatures in the transient Shh signaling phase and characterize immediate early response genes. Similar comparisons of Shh null with control embryos will identify all targets, including those dependent on sustained Shh signaling that play important roles in cell survival. 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. Gli2/3 activator (GliA) has been proposed to play a minor role in limb development relative to Gli3 repressor (GliR) inactivation and derepression of HH-regulated targets by Shh, but GliA-dependent targets aside from Gli1 and Ptch1 have not been clearly identified. We also plan to comprehensively identify limb GliA regulated genes by comparing transcriptomes of Gli3KO (GliA present, GliR lost) with ShhKO/Gli3KO (in which both GliA and GliR are absent). This will enable a definitive assessment of the role of GliA in limb patterning. Our recent work examining how posterior digit number is restrained (which is also regulated by Shh) suggests that GliA may play a role in constraining digit expansion inthe posterior limb, possibly by inhibiting expression of Bmp antagonist Grem1, as well as contributing to activation of target Bmps by Shh. This would identify an unappreciated and key role for GliA function in limb digit patterning. 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 have used 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. We have found that short range Shh/ZPA and Fgf/AER ectoderm interactions that modulate AER function and limit posterior digit formation are mediated by Shh induced Bmps, as mentioned above. Bmp signaling to AER induces AER regression and loss of Fgf8 expression. We found that the Shh target Bmps also signal in autocrine fashion to inhibit Shh expression in a negative feedback loop. Together, these counteracting negative loops prevent posterior digit expansion beyond the normal 5 digits. Removing the major Bmp receptor (Bmpr1a) from both AER and Shh producing ZPA abolishes this negative homeostatic mechanism and results in posterior digit polydactyly. Our results on the Bmp driven dual negative feedback loops have been submitted for publication and deposited at Biorxiv (https://doi.org/10.1101/2025.07.17.665402). 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.