SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

Abstract

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

TBX3 is essential to establish the posterior boundary of anterior genes and up-regulate posterior genes with HAND2 during onset of limb bud development

“…3B, 3E). Even though transcriptional up-regulation is more difficult to detect than down-regulation (Gamart et al, 2021), upregulation and spatial alterations are detected for several target genes in Tbx3 1/1c forelimb buds (Fig. 3E).…”

“…The TBX3 (this study), HAND2 (Osterwalder et al, 2014), HOXD13 (Sheth et al, 2016), SMAD4 (Gamart et al, 2021) and ß-Catenin ChIP seq-profiles (Sheth et al, unpublished) were used together with the H3K27ac profile were used to map the core regions in both enhancers. The candidate HAND2 binding sites were mapped using the HAND1::TCF3 motif (Jaspar ID MA0092.1) and the TBX3 binding sites using the human TBX3 motif (Jaspar ID MA1566.1) with ap-value cut-off of 0.01.…”

“…In turn, the upregulation and maintenance of posterior Tbx3 expression depends on both Hand2 and SHH signaling (Galli et al, 2010). In the anterior limb bud mesenchyme, Tbx3 expression is activated slightly later and requires SMAD4-mediated BMP signal transduction (Emechebe et al, 2016; Gamart et al, 2021). The anterior TBX3 protein colocalises with the GLI3 protein to primary cilia and interacts with the SUFU/Kif7 complex to stabilize the GLI3 full-length (GLI3FL) protein and its processing to the GLI3R isoform (Emechebe et al, 2016).…”

Recombinant Limb Assay as in Vivo Organoid Model

“…When the ZPA is grafted to the anterior zone of a limb bud, it induces mirror-image digit duplications. This signaling center is characterized by Sonic hedgehog ( Shh ) expression ( Riddle et al, 1993 ; Fujii et al, 2021 ; Gamart et al, 2021 ). Finally, the ventral and dorsal limb bud ectoderms specify DV polarity.…”

Single-cell profiling of penta- and tetradactyl mouse limb buds identifies mesenchymal progenitors controlling digit numbers and identities

“…1,12,[14][15][16][17][18] An integral part of this self-regulatory signaling system is the spatially dynamic transcriptional regulation of Grem1, which preferentially antagonizes SMAD4-mediated BMP signal transduction in the posterior and distal limb bud mesenchyme. 19,20 In Grem1-deficient mouse limb buds, the SHH/GREM1/AER-FGF feedback signaling system is disrupted, which cause mesenchymal apoptosis and formation of only 3 very rudimentary digits. 21,22 The spatial robustness of Grem1 cisregulation is paramount to normal limb and digit development as progressive genetic 4 reduction of its spatial domain and posterior expression bias causes transition from wildtype pentadactyly (5 digits) to stable tetradactyly (4 digits) via intermediate and variable anterior digits fusions.…”