Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression

Abstract

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF.

Novel phenotypes and loci identified through clinical genomics approaches to pediatric cataract

“…It has been shown that enriched expression in developing lens tissue can be used as a criterion to evaluate potential function in lens development (Lachke et al 2011, 2012a, b; Anand and Lachke 2016; Anand et al 2015; Agrawal et al 2015; Kasaikina et al 2011; Wolf et al 2013; Manthey et al 2014; Dash et al 2015; Audette et al 2016). Consistent with those data, we find that all six candidates are significantly expressed in mouse lens development, and five exhibit lens-enrichment.…”

Signaling and Gene Regulatory Networks in Mammalian Lens Development

“…The regulatory interaction between Pax6, c-Maf, Prox1, and their crystallin targets is termed as a “feed-forward loop”. Evidence also exists that Pax6, Prox1, and Hsf4 directly control the expression of genes encoding Fgfs and their receptors, Fgfrs [139, 170, 186], and that FGF-regulated mitogen-activated protein kinase (MAPK) Erk1/2 phosphorylates Hsf4 to increase its binding to DNA [187]. Likewise, Pax6 can also be phosphorylated by Erk, p38, or by HIPK2 [188]; however, none of these studies were conducted in the context of lens development.…”

“…Constitutively active Ras signaling can compensate for the loss of Frs2 and Shp2 in cell proliferation and differentiation, whereas PI3K signaling rescues an FGF receptor 2 (Fgfr2) deficiency for cell survival, suggesting that FGF signaling employs a compendium of downstream pathways to regulate lens development [146, 207]. Downstream to Ras, MAP kinases such as Erk phosphorylate and stimulate the activity of AP1 and ETS family transcription factors, which induce the expressions of crystallins and other lens determination genes such as Prox1 and c-Maf [186, 189]. This pathway is also finely tuned by negative regulators including the GTPase activating protein NF1 and the Ras inhibitor Sprouty, as well as by the transcription factor Prox1 that activates the expression of FGF receptors [186, 210–212].…”

“…Downstream to Ras, MAP kinases such as Erk phosphorylate and stimulate the activity of AP1 and ETS family transcription factors, which induce the expressions of crystallins and other lens determination genes such as Prox1 and c-Maf [186, 189]. This pathway is also finely tuned by negative regulators including the GTPase activating protein NF1 and the Ras inhibitor Sprouty, as well as by the transcription factor Prox1 that activates the expression of FGF receptors [186, 210–212]. FGF signaling has been shown to induce the expression of Jag1, the ligand for Notch signaling that suppresses lens epithelial cell differentiation as described above, and Fizzled-6, the receptor for Wnt-planar cell polarity (PCP) signaling that may facilitate the orientation of the lens fibers [213, 214].…”

“…Expression of γA-crystallin requires Sox1 [171, 175] and from E14.5 is further augmented by Hsf4 [170] and the FGF/MAPK pathway [187]. Other regulatory layers are involved as Hsf4 inhibits the expression of Fgf1, Fgf4, Fgf7, and Fgfr1 [170], and Prox1 regulates the expression of Fgfr3, Fgfrl1, and Lctl and its expression is downstream of FGF/MAPK cascade [186]. …”