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 use an epitope-tagged Smad4 allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lacking Smad4 in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced in Smad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared between Smad4- and Shh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.

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 downregulation (Gamart et al, 2021), upregulation and spatial alterations are detected for several target genes in Tbx3 D/Dc 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 SMAD4mediated 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).…”