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Abstract
In multicellular organisms, cell fates are established through precise patterns of gene expression. This requires a complex set of interactions among cis-regulatory elements and transcription factors. Enhancers, in particular, play a central role in driving cell-type specific gene expression patterns and are capable of activating transcriptional target genes. Both the limb and the mandible are classic model systems for the study of developmental regulation. In mouse, the hindlimb and mandible share similar developmental processes, including specification of the prospective field, induction of tissue outgrowth, maintenance of the outgrowth and patterning tissue along different axes. Many major signaling pathways contribute to the development of both organs, including FGF, SHH, BMP and WNT. Beyond signaling pathways, there is a relatively narrow network of hindlimb-restricted or mandible-restricted regulators, such as PITX1, TBX4, ISL1, DLX5/6 and HAND1/2. PITX1 is a tissue-specific transcription factor, which is encoded by the paired-like homeodomain 1. Strong Pitx1 expression is found in both the hindlimb and mandible mesenchyme. Haploinsufficiency for Pitx1 in mice and humans can result in clubfoot, mandibular hypoplasia and other hindlimb- or jaw-related malformations. However, the hindlimb and mandible PITX1-transcriptional targets that are misregulated in the absence of PITX1 remain largely unknown. In this work, I have applied a combination of comparative genomics, epigenetic profiles and functional analyses to study the regulatory network of PITX1 transcriptional targets that direct mouse hindlimb and mandible development. I performed a genome-wide ChIP-seq analysis to identify the location of cis-regulatory elements bound by PITX1 during embryonic development of the mouse hindlimb and mandible. The transcriptional targets of PITX1 in developing hindlimbs and mandibles are revealed by performing RNA-seq. PITX1-dependent enhancers and target genes of hindlimb and mandible are defined with the combination of sequence conservation, ChIP-seq, RNA-seq and functional analyses. The results support the conclusions that PITX1 can promote chondrogenic and myogenic differentiation in mouse hindlimb through conserved regulatory targets, and modulate both hindlimb and mandible development through the shared regulatory network.