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.

Genetic studies of the last decades strongly indicated that generation of particular retinal cell types is governed by gene regulatory networks of transcription factors and their target genes. The paired and homeodomain transcription factor Pax6 plays a pivotal role in retinal development as its inactivation in the retinal progenitor cell population leads to abolished differentiation of all retinal cell types. However, until now, only a few transcription factors operating downstream of Pax6 responsible for generation of individual retinal cell types have been identified. In this study, we identified two transcription factors of the Onecut family, Onecut1 and Onecut2, as Pax6 downstream-acting factors. Onecut1 and Onecut2 were previously shown to be expressed in developing horizontal cells, retinal ganglion cells and cone photoreceptors; however, their role in differentiation of these cell types is poorly understood. In this study, we show that the horizontal cell genesis is severely disturbed in Onecut-deficient retinae. In single Onecut1 and Onecut2 mutants, the number of horizontal cells is dramatically reduced while horizontal cells are completely missing in the Onecut1/Onecut2 compound mutant. Analysis of genes involved in the horizontal cell genesis such as Foxn4, Ptf1a, Prox1 and Lim1 showed that although horizontal cells are initially formed, they are not maintained in Onecut-deficient retinae. Taken together, this study suggests the model in which Pax6 regulates the maintenance of horizontal cells through the activation of Onecut1 and Onecut2 transcription factors.

The vertebrate eye is derived from the neuroepithelium, surface ectoderm, and extracellular mesenchyme. The neuroepithelium forms an optic cup in which the spatial separation of three domains is established, namely, the region of multipotent retinal progenitor cells (RPCs), the ciliary margin zone (CMZ)—which possesses both a neurogenic and nonneurogenic potential—and the optic disk (OD), the interface between the optic stalk and the neuroretina. Here, we show by genetic ablation in the developing optic cup that Meis1 and Meis2 homeobox genes function redundantly to maintain the retinal progenitor pool while they simultaneously suppress the expression of genes characteristic of CMZ and OD fates. Furthermore, we demonstrate that Meis transcription factors bind regulatory regions of RPC-, CMZ-, and OD-specific genes, thus providing a mechanistic insight into the Meis-dependent gene regulatory network. Our work uncovers the essential role of Meis1 and Meis2 as regulators of cell fate competence, which organize spatial territories in the vertebrate eye.