The Molecular Regulation of MAP3K1 in Eyelid Development

Tissue morphogenesis is a highly organized and evolutionarily conserved process controlled by the temporal and tissue-specific activation of signal transduction pathways. The transient closure and then reopening of eyelid is an essential step for vertebrate ocular surface morphogenesis. In mice, eyelid closure takes place at embryonic day 16 (E16), while the closed eyelids re-open at post-natal day 12-14 (P12-14). The temporary closed eyelid is believed to provide a microenvironment favorable for the immature cornea, lens and retina to develop. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) plays a crucial role in eyelid morphogenesis. Previous studies from our laboratory have shown that MAP3K1 is responsible for the transmission of morphogenetic activin B signals to Jun N-terminal kinases (JNKs), which in turn phosphorylates transcription factor c-JUN to regulate gene expression events involved in eyelid closure. MAP3K1 ablation in mice results in defective embryonic eyelid closure and an obvious eye open at birth (EOB) phenotype. Our previous studies also show that MAP3K1 is highly expressed in the leading edge of the eyelid epithelium just prior to the onset of eyelid closure. The central hypothesis of this dissertation is that activation of Map3k1 gene promoter and expression serves as a critical determinant factor for embryonic eyelid closure. We test this hypothesis by addressing the following pertinent questions. (1) What are the factors responsible for induction of MAP3K1 expression? We tested various eyelid morphogenetic factors on their abilities to induce the Map3k1 promoter activity. Our results show that activation of EGFR by its ligands and by microtubule depolymerizing agents could induce MAP3K1 expression. (2) What are the intracellular signaling pathways that lead to activation of Map3k1 promoter? We established an EGFR/TGFalpha-RhoA/ROCK signaling cascade that led to induction of c-JUN. c-JUN in turn bound to and activated the Map3k1 promoter. (3) Are epigenetic mechanisms involved in the regulation of the Map3k1 promoter? Because the Map3k1 promoter sequence has extremely high CpG content, it could be regulated by epigenetic mechanisms. We showed that both DNA methylation and histone acetylation were involved in regulation of the Map3k1 promoter. (4) What are the consequences of MAP3K1 expression? MAP3K1 is an intracellular protein kinase, involved in the JNK-c-JUN cascade activation. We showed that one of the downstream events of MAP3K1 was transcriptional activation of AP-1 and the induction of AP-1 target gene, such as Pai-1, during eyelid development.