A paradigm to study genetic robustness is the regulation of gene expression by cis-regulatory modules (CRMs) embedded in their genomic landscapes. We have shown that Gremlin1 (Grem1) mediated BMP antagonism is pivotal to mouse organogenesis and its dynamic regulation by inputs from different transcription factors defines a key node in the self-regulatory signaling system that orchestrates limb bud outgrowth and patterning. Several CRMs interact to regulate Grem1 expression and we uncovered the complex cis-regulatory logics and robustness of its regulation in mouse limb buds. While Grem1 transcript levels are regulated in an additive manner, the spatial control of its expression depends on enhancer cooperativity. The latter provides limb bud development with cis-regulatory robustness as any significant spatial changes result in digit fusions and loss. The progression from digit fusions to digit loss due to increasing spatial alterations very reminiscent of that changes that occurred during evolutionary diversification of mammalian limbs. Indeed, the Grem1 cis-regulatory landscape is evolutionary ancient and analysis of enhancer activities and Grem1 expression in limb buds of different vertebrate embryos reveals the underlying cis-regulatory plasticity. Three of the CRMs are evolutionary ancient as they are conserved from basal fishes to mammals and our ongoing studies provide molecular insights into the cis-regulatory alterations underlying the fin-to-limb transition and tetrapod limb skeletal diversification.
Another research line aims to determine the range of target genes for the key transcriptional regulators HAND2 and TBX3 that function in concert with the GLI3 repressor during limb bud initiation. This analysis identifies the target GRNs that function upstream of self-regulatory signaling system in establishment of axes polarities. In addition, we use computational analysis of time-series of the genome-wide ATAC-seq and transcriptome profiles in the paradigm mouse and chicken limb bud models to study the impact of conserved and species-specific chromatin remodeling on target gene expression. In mouse forelimb buds, the temporal dynamics of open chromatin and the expression of the associated target genes are strikingly synchronous, while there is stage-specific divergence during chicken wing bud development. In silico mapping of transcription factor (TF) binding reveals the dynamic interactions of TF with the open chromatin regions and indicates that they function as CRMs regulating the expression of the associated differentially expressed target gene. Using the HAND2 and GLI3 key regulators as paradigm allows identification and in silico modelling of conserved and species-specific target gene interactions in both species despite the fact that ChIP-seq analysis is not possible in chicken embryos. Computational analysis also identifies enhancers with diverged chicken-specific enhancer activities indicative of species-specific traits. This comprehensive computational analysis reveals the impact of genome evolution on the chromatin remodeling and the trans-regulatory hierarchies and gene regulatory circuits during mouse and chicken limb bud development.