Transcriptional Regulation of Cardio-Pulmonary Development
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Organogenesis is a complex process, disruption of which results in developmental anomalies. In recent years, genetic dissection of the pathways involved in cardiogenesis, have shown a striking similarity in molecular mechanisms across species. One conserved protein is dHAND, a basic helix-loop-helix (bHLH) transcription factor that is required for normal development of the right ventricle, the pharyngeal arches and limb buds. Loss of dHAND leads to apoptosis in the aforementioned tissues and to embryonic lethality at E10.0. A differential display analysis was performed to identify genes dysregulated in dHAND-/- hearts. Characterization of such genes could potentially shed light on the molecular mechanisms involved in the defects seen in dHAND mutants, while also identifying genes required for normal embryonic development. This thesis represents work on two molecules that were identified in this screen. Bnip3, a hypoxia inducible, pro-apoptotic molecule that can induce mitochondrial damage, was upregulated in the dHAND-/- pharyngeal arches and heart, suggesting a role for mitochondrial damage in the observed apoptosis. I have shown that while Apaf-1, a downstream mediator of mitochondrial-induced apoptosis, is required for the apoptosis observed in dHAND-null pharyngeal and aortic arch mesenchyme, cardiomyocyte apoptosis in dHAND mutants is Apaf-1 independent. Rescue of pharyngeal arches revealed that premature closure of the pharyngeal arch arteries likely contributes to the early lethality observed in dHAND-/- embryos. The mouse ortholog of Bcl-2 associated transcription factor (Btf), which was similar to thyroid hormone receptor associated protein 150 (TRAP150), was down regulated in dHAND mutants. TRAPs are a family of transcriptional co-activators that are required for normal cardiac and embryonic development. Mice lacking Btf showed normal cardiac development, however, the animals had hypercellular lungs and died within 24 hours after birth. Analysis of lung ultrastructure and cell specific markers showed presence of immature secretory cells in the proximal airways of the lung and aberrant proximal-distal patterning. The ectopic presence of stem cell-like proximal epithelial cells (Clara cells) in the distal epithelium may explain the hypercellularity observed in btf-null lungs. These results show that Btf is required for normal maturation and patterning of the pulmonary epithelium and survival of the animal.