The identification of distinct expression

The identification of distinct expression domains in the limb suggested that these domains might have specific biological functions. We explored this possibility by determining enriched biological processes for each category using GO ontology term analysis (WebGestalt) (Zhang et al., 2005). Category 1 was notably enriched for transcriptional regulation and cell proliferation processes (P≤0.000001) (Supplementary material Fig. S2). In contrast, category 2 was notably enriched for cell differentiation, cell adhesion, and ossification processes (P≤0.05), and category 3 was enriched for skeletal development processes (P≤0.05) (Supplementary material Fig. S2). Differential enrichment within these categories suggests that the three distinct SHH-mediated domains in the limb may have unique functions. Our categorization of expression patterns was based on E10.5 limb buds. Because SHH signaling within the limb bud is dynamic, it is possible that the expression of GLI target genes within these domains could also be dynamic. To assess this, we examined the temporal changes in the expression domains of GLI target genes by comparing their expression patterns between E10.5 and E11.5. Most genes expressed in a particular domain at E10.5 remain expressed in a broader version of the same domain at E11.5 (category 1, n=18/24; category 2, n=9/12; category 3, n=8/9) (Fig. 2A–C and Supplementary material Fig. S1A). Taken together, these results indicate that the expression domains of GLI target genes are relatively stable despite the large changes in SHH signaling that occur during this period. In summary, we identified three stable domains of GLI target gene expression within the SHH-responsive region of the limb bud.
Discussion In this study, we have identified a set of genes that by multiple criteria are likely to be directly regulated by GLI proteins in the developing limb bud. These genes are expressed in three domains, which have distinct temporal requirements for SHH signaling (Fig. 7A). The GLI-bound CRMs associated with genes in two of the domains are enriched for additional DNA motifs that are unique and common, representing OG-L002 receptor for potential transcriptional co-regulators (Fig. 7B). The identification of distinct expression domains has greatly improved the understanding of HH patterning in the Drosophila imaginal wing disk and vertebrate neural tube, and we hope that the identification of different domains in this study will facilitate the determination of the gene regulatory networks underlying SHH-mediated limb patterning.
Acknowledgments We thank Martha Vokes for identifying plasmids and designing primers for generating templates for antisense riboprobes. We thank Xia Li for performing the initial analysis of RNA-seq dataset on cyclopamine-treated embryos. We are grateful to Dr. Simone Probst and the other members of Dr. Rolf Zeller's laboratory for teaching us the limb bud culture technique. We thank Dr. Jacqueline Tabler and Simone Giovanetti for providing critical comments on the manuscript. This work was supported by NIHR01HD073151 (to S.A.V and H.J.) and startup funds from the College of Natural Sciences and the Institute for Cellular and Molecular Biology at the University of Texas at Austin (to S.A.V).
Introduction The Hedgehog (Hh) pathway is a signaling cascade that plays a crucial role in embryonic development and tissue homeostasis [1,2]. Although in adults Hh signaling is activated during tissue repairing and wound healing, its aberrant activation has been reported to drive tumorigenesis and tumor progression in several cancers [3]. Germline or somatic mutations of genes encoding Hh pathway components have been identified in human and murine basal cell carcinoma (BCC) and medulloblastoma (MB) [[4], [5], [6]]. Moreover a growing number of reports correlate uncontrolled Hh signaling with multiple cancers such as lung, breast, stomach, pancreas, ovarian carcinoma and hematopoietic malignancies [5,[7], [8], [9], [10]]. Targeting the Hh signaling pathway with small molecule modulators has thus emerged as a profitable anticancer strategy, as also highlighted in recent reviews [[11], [12], [13], [14], [15]]. The largest part of Hh modulators developed so far act as antagonists of the upstream Smoothened receptor (Smo) [[16], [17], [18], [19], [20]], whereas only a few inhibitors of the downstream effectors Glioma-associated oncogene homologue (Gli) proteins have been disclosed, most of which acting by an unclear or indirect mechanism of action [12,[21], [22], [23], [24]]. Two Smo antagonists, namely Vismodegib and Sonidegib, have been approved by the Food and Drug Administration (FDA) in 2012 and 2015, respectively, for the treatment of metastatic and locally advanced BCC, while a number of additional Smo antagonists are currently undergoing clinical evaluation [19,20,25]. However, the emergence of drug-resistance [15,[26], [27], [28], [29]], and the occurrence of aberrant Hh activation downstream of Smo seriously limited the use of these drugs, and raised some concerns on the efficacy of therapeutic approaches that target Smo. Indeed, pharmacological modulation of the final and most powerful effector Gli1 is nowadays considered a more efficient – although challenging – strategy to block Hh signaling in cancer [12]. However, only the Gli antagonist Arsenic trioxide (ATO) has entered clinical evaluation so far [30]. It is therefore widely accepted that novel Hh inhibitors acting through different mechanisms of action than Smo antagonism, or able to overcome drug resistance, are in high demand as therapeutic candidates for the treatment of Hh-dependent tumors.