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 TGF{beta} 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.