Despite abundant evidence for the centrality of FGF receptor (FGFR) signaling to normal fiber cell differentiation, our understanding of the key downstream target genes that carry out the differentiation process in response to FGFR stimulation remains incomplete. An innovative, combined use of RNA-Seq and systems biology approaches identified Nkx6-1, a homeobox transcription factor, as a potential key mediator of FGFR-induced lens fiber cell differentiation. In support of this argument, Nkx6-1 mRNA is expressed 70x more abundantly in lens fiber cells than in lens epithelial cells. Nkx6-1 is also co-expressed with the fundamental lens transcription factor, Pax6 within the neural tube and pancreas and these two transcription factors coordinately pattern different cell types in these tissues. These discoveries led to the hypothesis that FGF receptor signaling induces the expression of Nkx6-1 to promote secondary fiber cell differentiation, in part by antagonizing PAX6 during the epithelial to fiber cell transition. This hypothesis will be tested by specifically removing Nkx6-1 during lens development and characterizing any resultant defects in lens development. If lens development is compromised in Nkx6-1 deficient lenses, a transcriptome analysis will determine which transcripts are deregulated. Testing the hypothesis will also utilize post-natal lens epithelial explants where effects of FGFR loss on prenatal lens cell survival can be separated the effects of FGFR loss on fiber cell differentiation. Lens explants lacking FGFRs lose the ability to undergo fiber cell differentiation in response to either FGF or vitreous humor. However, the removal of both FGFRs and PTEN restores vitreous humor-induced fiber cell differentiation in lens epithelial explants. The requirement of NKX6-1 for this restoration of vitreous humor-induced fiber cell differentiation in lens explants lacking both FGFRs and PTEN will be tested. It will also be determined if overexpression of Nkx6- 1 restores vitreous humor-induced lens fiber cell differentiation in lens epithelial explants lacking only FGFRs. Several experiments will also test for antagonism between NKX6-1 and PAX6 during lens fiber cell differentiation. The effect of Nkx6-1 loss on the expression pattern of PAX6 during lens development will be determined. The expression of PAX6-regulated transcripts will also be tested in lens epithelial explants that either lack or overexpress NKX6-1 in response to vitreous humor stimulation. ChIP-Seq analysis will determine which gene regulatory sequences are directly bound by NKX6-1 in the lens and combined with the RNA-Seq data to determine the genes which NKX6-1 directly regulates. The resulting data will also be compared with previously published lens ChIP-Seq data from lens to determine which genes share binding sites for both PAX6 and NKX6- 1. These studies will provide the first comprehensive investigation of the role of NKX6-1 during lens development and will both definitively show if NKX6-1 is an essential mediator of FGFR-induced fiber cell differentiation and set the stage for future studies to elucidate the molecular mechanisms NKX6-1 uses to coordinate lens development.
The proposed research is relevant to public health because abnormalities in tissue patterning underlie a significant portion of organ malfunction and birth defects. Nxk6-1 plays a well-recognized role in patterning the nervous system and pancreas where it coordinates with Pax6, and while both of these proteins are expressed in the lens, the role of Nkx6-1 in lens development remains unknown. The proposed research is relevant to the NIH?s mission because uncovering the mechanisms by which NKX6-1 and PAX6 interact to regulate lens development could both provide insights into cataract formation, and also have an impact on developmental disorders of the nervous system and pancreas as well.