Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. Here we show that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6Sey/flox embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. Our data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner. KEY WORDS: Pax6, Retinal progenitor, mRx-Cre, Lens induction INTRODUCTIONProper eye development is dependent on the coordinated formation of two main tissues in the eye: the retina and the lens. Vertebrate eye development begins with invagination of the optic vesicle (OV) toward the lens-competent head surface ectoderm (SE). As OV contacts SE, a series of reciprocal inductive signals elicit formation of the lens placode (LP) and subsequent invagination of both LP and OV to form a two-layered optic cup (OC), with retinal pigmented epithelium (RPE) surrounding the retina (reviewed by Fuhrmann, 2010; Chow and Lang, 2001;Ogino et al., 2012). Genetic studies have identified multiple transcription factors and signaling pathways interacting in a complex network orchestrating early eye development (reviewed by Fuhrmann, 2010; Chow and Lang, 2001;Ogino et al., 2012;Xie and Cvekl, 2011). Among the signaling pathways, BMP (Furuta and Hogan, 1998;Rajagopal et al., 2009;Sjödal et al., 2007;Wawersik et al., 1999) and FGF (Faber et al., 2001;Garcia et al., 2011;Gotoh et al., 2004;Pan et al., 2006) were found to be essential for lens induction and coordinated OV-to-OC transition, as severe eye defects are associated with their inactivation. RESEARCH ARTICLEInstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 14420 Prague 4, Czech Republic. At the time the LP is formed, the dorsal region of the OV becomes specified to the retina and is populated with mitotically active retinal progenitor cells (RPCs) (Fuhrmann, 2010;Levine and Green, 2004). Lineage-tracing studies have shown that RPCs a...

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens-specific gene expression in the surface-ectoderm. Supression of canonical Wnt/beta-catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down-regulation of canonical Wnt signaling in the presumptive lens ectoderm.