Pax6 is essential for lens fiber cell differentiation

Abstract

The developing ocular lens provides an excellent model system with which to study the intrinsic and extrinsic cues governing cell differentiation. Although the transcription factors Pax6 and Sox2 have been shown to be essential for lens induction, their later roles during lens fiber differentiation remain largely unknown. Using Cre/loxP mutagenesis, we somatically inactivated Pax6 and Sox2 in the developing mouse lens during differentiation of the secondary lens fibers and explored the regulatory interactions of these two intrinsic factors with the canonical Wnt pathway. Analysis of the Pax6-deficient lenses revealed a requirement for Pax6 in cell cycle exit and differentiation into lens fiber cells. In addition, Pax6 disruption led to apoptosis of lens epithelial cells. We show that Pax6 regulates the Wnt antagonist Sfrp2 in the lens, and that Sox2 expression is upregulated in the Pax6-deficient lenses. However, our study demonstrates that the failure of differentiation following loss of Pax6 is independent of β-catenin signaling or Sox2 activity. This study reveals that Pax6 is pivotal for initiation of the lens fiber differentiation program in the mammalian eye.KEY WORDS: Pax6, Sox2, Lens, Crystallin, Wnt, β-catenin, Mouse Development 136, 2567Development 136, -2578Development 136, (2009 2568Chen et al., 2004;Chen et al., 2006). When the Wnt coreceptor Lrp6 was deleted in mice, aberrant LFCs appeared in the anterior pole of the lens (Stump et al., 2003). Upon inactivation of the canonical Wnt effector β-catenin, LE markers, proliferation and differentiation were disrupted (Cain et al., 2008). These findings suggest a role for Wnt signaling in LE cell fate.Owing to the severe ocular phenotype of Pax6 mutants, the later developmental roles of Pax6 could only be extrapolated from in vitro research. Herein, we introduce the first in vivo loss-of-function model of Pax6 and its presumed transcriptional partner Sox2. We show that the loss of Pax6 prevents LFC differentiation and results in cell death and in an increase in Sox2. However, conditional deletion of Sox2 reveals that it is dispensable for LFC differentiation. Furthermore, overexpression of β-catenin results in a differentiation failure that is similar to, but independent of, that observed following Pax6 loss. These findings place Pax6 upstream in the cascade of events leading to the differentiation of LE into lens fibers in mammals. MATERIALS AND METHODS Mouse linesMouse lines employed in this study were: Pax6 lox (Ashery-Padan et al., 2000), Mlr10 (Zhao et al., 2004), Catnb lox(ex3) (Harada et al., 1999) and BATlacZ (Nakaya et al., 2005) and are described in Fig. S1 in the supplementary material. The Sox2 loxP line (see Fig. 7A) contains two loxP sites inserted around the single exon of the murine Sox2 gene using conventional gene-targeting methods (Joyner, 1995). In the gene-targeting vector, loxP-frt-pMC1neopA-frt, the neo gene is flanked by frt sites. Flp recombinase activity within the B6.SJL-Tg(ACTFLPe)9205Dym/J mouse line (Rodriguez et al.,...

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation

“…Additionally, Sipa1l3 or Epha4 deficiency resulted in an upregulation of the direct Wnt/β-catenin target axin2, which is in agreement with a previously published study that shows axin2 overexpression upon Wnt/β-catenin activation in mouse lenses (Antosova et al, 2013). These observations fit very well to published data showing that cataract formation, smaller lenses and inhibited lens cell differentiation can be induced by β-catenin overexpression (Antosova et al, 2013;Shaham et al, 2009;Martinez et al, 2009). Note that overexpression of β-catenin in LFCs alone is sufficient to induce cataract formation (Antosova et al, 2013).…”

“…Interaction of Epha4 and Sipa1l3 causes proper eye development through β-catenin-independent Wnt signaling In studies previously published by others, it has been shown that overexpression of β-catenin in LFCs results in an inhibition of LFC differentiation and cataract formation (Antosova et al, 2013;Shaham et al, 2009) that is similar to the Sipa1l3 LOF phenotype in the mouse (Greenlees et al, 2015). Gain of β-catenin function in the lens also leads to microphthalmia (Martinez et al, 2009) comparable to the loss of Sipa1l3 phenotype ( Fig.…”

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

“…The transcription factor Pax6 is abundant in the LV and lens epithelium (Fig. 4C), with lower levels present in LFs (Shaham et al, 2009). The highest Pax6 levels are restricted to the anterior cells of the Prox1 cKO lens (Fig.…”

Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation

“…BMP, FGF and Notch signaling pathways regulate lens fiber cell differentiation in conjunction with DNA-binding transcription factors, including FoxE3 (Blixt et al, 2007;Brownell et al, 2000;Medina-Martinez et al, 2005), Gata3 (Maeda et al, 2009), Pax6 (Shaham et al, 2009), Pitx3 (Ho et al, 2009;Medina-Martinez et al, 2009), Prox1 (Duncan et al, 2002;Wigle et al, 1999), Hey1 (Herp2) and Rbpj (Jia et al, 2007;Rowan et al, 2008). Although little is known about links between BMP and FGF signaling and these factors, disruption of Prox1 blocks expression of p27 Kip1 and p57 Kip2 in the posterior part of the lens vesicle, followed by arrested lens fiber cell elongation (Wigle et al, 1999).…”