Role of Shh in developmental patterning and growth of digit skeleton

Division Of Basic Sciences - Nci

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Abstract

Sonic hedgehog (Shh) acts as a mitogen and cell survival factor in many adult processes during normal tissue renewal and in many types of cancer, but acts as a morphogen in several developmental contexts. How the mitogenic role of Shh is integrated with the morphogenetic role in developmental contexts is still poorly understood. In the limb, Shh regulates both digit number and identity of different digits (A-to-P,thumb to pinky). Shh is thought to act as a morphogen forming a gradient along the limb AP axis, with higher concentrations specifying more posterior digit types. We have determined the time-requirements for Shh function in limb (using a tamoxifen-regulated Cre to remove Shh at different times in mice). To perform this analysis, we generated and characterized a novel conditional Cre recombinase line selectively expressed in early limb mesoderm, neural crest, gut and tailbud. This line provides an excellent tool available to the scientific community to illuminate different temporal roles of key developmental regulators in several important developmental models using mouse mutants, as well as for genetic lineage tracing studies in mice. Our results, deleting Shh function at different time points, are most consistent with a model in which Shh activity is required only very transiently (several hours) to specify the complete complement of 5 different digit types, but is required for a prolonged time (about 2 days) to maintain cell survival and proliferation, enabling 5 normal digits to form. To further test this model for Shh function, we have assessed whether restoring cell survival can rescue normal digit formation in mutant embryos after a transient period of Shh activity that is terminated by Shh gene deletion. To rescue cell survival, the compound mutant for the pro-apoptotic Bcl2 family members Bax/Bak (which play roles in normal interdigital apoptosis) has been introduced to inactivate the intrinsic death pathway. Our results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. In addition, genetic lineage tracing of cells in the limb that have responded to Shh signals at different time points indicates that Shh only signals directly to the very posterior part of the limb bud at the time when all 5 digit progenitors have been specified. This result indicates that Shh acts indirectly in the early limb bud, via a system of relay signals, to specify digits. We developed a genetic assay to test for relay signaling by artificially enforcing Shh-response in the posterior limb bud in embryos lacking all Shh function (Shh KO). This partly rescues anterior digit formation, and strongly suggests the presence of relay signals acting downstream of Shh. Our results are incompatible with Shh acting as a classic morphogen in the limb and suggest that Shh acts as a trigger to activate a relay mechanism. Furthermore, our results indicate that there are 2 classes of Shh responsive target genes, those that respond to a transient signal and become stably expressed, and those that require continuous signaling to maintain expression. We are comparing the transcriptomes of Shh mutant and rescued limb buds to characterize the types of genes in these two differentially regulated target classes (see Project: Genome-wide target analysis of Shh-activated transcription network in limb bud; ZIA BC 0111120). Our results also indicate that both Shh expression and response are highly dynamic, contributing to robustness of Shh activity, and we are characterizing these features using several approaches. Our lineage tracing results of Shh production and response indicate that Shh producing cells arise from a renewing progenitor pool in the proximal limb bud margin, providing an avenue for restoring the Shh signaling center after insults or injury have damaged the center in the limb bud proper. In parallel, we performed single cell transcriptome profiling from normal limb buds to identify expression signatures in the transient Shh signaling phase and characterize immediate early response genes. These studies have revealed a potential renewing progenitor population by trajectory analysis and identified progenitor specific markers. We are following up on these observations using a conditional Cre driven by regulatory regions from one of these progenitor 'markers' to genetically tag and further characterize the progenitor lineage. To further dissect the dynamics of Shh expression and response over time at the single cell level, we are performing single cell transcriptome analyses in early limb buds in which the Shh lineage is tagged by a "reporter" to track Shh descendant cells, enabling us to compare cells actively expressing Shh with descendants that have ceased expression. This approach will also allow us to evaluate the dynamic change in autocrine Shh responsivensss in the Shh lineage over time (see below). Understanding the basis for robustness and renewal capacity in Shh expressing progenitors may be valuable in devising therapeutic intervention for Shh driven cancers. The lineage analysis of Shh production and response also indicate that Shh producing cells are completely refractory to autocrine signaling response, which is a feature shared by many Shh driven tumors in humans (signaling is often paracrine and regulates a supportive tumor niche). Our genetic and transcriptomic studies suggest that nonresponsiveness to Shh signaling is tightly coupled to functional Shh ligand production, but occurs in a cell autonomous manner in the producing cells. We are dissecting the requirements for ligand production in a transgenic mouse model using comparison of the endogenous Shh locus vs Shh cDNA transgene which contains only coding exon sequences to assess the basis for this autonomous nonresponsiveness. Understanding the underlying mechanisms for this strict inhibition of autonomous response will also provide insights on how this inhibition is bypassed in some Shh driven cancers. Using a complementary approach (mentioned above) that will allow us to compare gene expression differences between Shh expressing cells that respond to autocrine signals and those that no longer respond at the single cell level. This comparison will provide mechanistic insights on how selective autocrine nonresponsiveness may be regulated. Our preliminary scRNA results are encouraging that these comparisons are feasible and will be insightful.