SMAD4: a multifunctional regulator of limb bud initiation and outgrowth

Abstract

During mouse embryonic development, the spatio-temporal expression of genes is controlled by both interlinked signalling pathways and interactions between transcription factors and their target cis-regulatory modules. To gain global insights into the roles of a trans-acting transcriptional regulator in a specific tissue, the genome-wide profiling of its target regulatory regions and their association with the putative target genes are essential. Therefore, I have combined several types of genome-wide analyses such as ChIP-seq using epitope-tagged transcription factors with ATAC-seq and RNA-seq to study the functions of HAND2 and SMAD4 during heart and limb bud development, respectively. In Hand2-deficient embryos, we observed that cells of the atrioventricular canal do not undergo the endothelial-mesenchymal transition that underlies cardiac cushion development. By combining HAND23xF ChIP-seq and RNA-seq analysis, we have identified the HAND2 gene regulatory network involved in these processes and show that HAND2 is a key regulator of heart valve development. Limb bud outgrowth and patterning are regulated by a self-regulatory feedback signalling system operating between the SHH and FGF signalling pathways that critically depends on the BMP antagonist GREMLIN1. However, the establishment of these signalling feedback loops requires initiation of Gremlin1 expression by high BMP activity. For my PhD research, I have investigated the roles of the BMP signalling pathway during limb bud initiation by studying the functions of the BMP signal transducer SMAD4. By combining genome-wide SMAD43xF ChIP-seq, ATAC-seq and RNA-seq analyses, I am able to show that SMAD4 participates in activation of Gremlin1 expression by interacting with Grem1 coding exon 2 (a putative regulatory region). Furthermore, the identification of the SMAD4 gene regulatory network reveals multiple functions of SMAD4 during the onset of limb bud development. Especially, SMAD4 directly regulates target genes involved in limb bud outgrowth and patterning. Rather unexpected, my analysis reveals that SMAD4 directly regulates cholesterol homeostasis and controls the gradient and activity of the SHH signalling pathway during early limb bud development.