Dose-dependent regulation of horizontal cell fate by Onecut family of transcription factors

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of eitherOc1orOc2gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion ofOc1andOc2leads to a complete loss of HCs. Here we study the genetic redundancy betweenOc1andOc2paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

<div><p>The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.</p></div

Chromatin Remodeling Enzyme Snf2h Is Essential for Retinal Cell Proliferation and Photoreceptor Maintenance

Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina

β-catenin stabilization in lens fiber cells results in cataract.

<p>(A) Schematic diagram of delβ-CAT transgenic construct. (B) delβ-CAT transgenic protein is detected in adult mutant lenses (Tg) with anti-Ha-tag antibody. (C) N-terminally deleted β-catenin is detected in transgenic lenses (Tg) with anti-β-catenin antibody. Note that the delβ-cat protein is expressed in higher amount than endogenous β-catenin. (D) qRT-PCR demonstrates upregulated mRNA expression of Axin2 in newborn delβ-CAT lenses (*p<0.05). Ocular phenotype of adult wild-type (E, G) and delβ-CAT (F, H) mice, adult transgenic mice develop cataract, indicated with arrows (F, H). (I-L) Histological sections of adult wild-type (I) and transgenic (J, K, L) eyes and detail view of wild-type (I′) and delβ-cat (J′, K′, L§) lens fiber cell compartment (fc). Scale bars indicate (I, J, K, L) 500 µm and (I′, J′, K′, L′) 200 µm.</p

Expression of cell cycle markers persists in the fiber cell compartment of αA-CLEF lenses.

<p>(A) Cyclin D1 and (E) cyclin D2 expression is detected mainly in the equatorial region and transitional zone of wild-type lenses at E13.5. However, in αA-CLEF lenses, cyclin D1 (B) and cyclin D2 (F) reactivity is detected in the fiber cell compartment. (J) p27^Kip1 and (N) p57^Kip2 expression is unaltered in E13.5 αA-CLEF lenses compared to wild-type lenses (I, M). At E16.5, (D) cyclin D1, (H) cyclin D2, (I) p27^Kip1 and (P) p57^Kip2 expression is inappropriately maintained in the fiber compartment in the central part of αA-CLEF lenses compared to wild-type lenses (C, G, K, O), where the highest levels of expression are normally observed at the transitional zone (indicated with green arrowheads) at E16.5. Scale bars indicate 50 µm. Abbreviations: fc, fiber cell compartment.</p

Ectopic Wnt/β-catenin signaling activation affects fiber cell nuclei localization and expression of lens regulatory proteins.

<p>Cryosections of E16.5 wild-type (A, C, E, G, I, K, O) and αA-CLEF (B, D, F, H, J, L, N, P) embryos stained with hematoxylin and eosin (A, B), DAPI (C, D), for lens epithelial cell marker Pax6 (I, J), its target Foxe3 (M, N), for early differentiation marker Prox1 (O, P) and for fiber cell differentiation markers Sox1 (G, H) and c-Maf (K, L). (E, F) CLEF transgenic protein is detected with anti-Lef1 antibody nuclei of fiber cells from transitional zone to fiber cell compartment. Fiber–cell-nuclei are detected throughout the fiber cell compartment in αA-CLEF lenses (B, D), and the expression of Pax6 (F), Sox1 (H) and c-Maf (L) is stronger in the fiber cell compartment of αA-CLEF lenses (indicated with red arrows) compared to wild-type (E, G, K). Scale bars indicate 50 µm.</p

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of <i>Pax6</i>

<div><p>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). <i>Pax6</i> represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of <i>Pax6</i> during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of <i>Pax6</i> 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 <i>Meis1</i> and <i>Meis2</i> 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 <i>Pax6</i>. We further show, using <i>in vivo</i> 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, <i>Pax6</i>, during lens induction.</p></div

Downregulation of γ-crystallin protein and mRNA in adult αA-CLEF lenses.

<p>(A, C) Western blot analysis shows less γ-crystallin in total (A) and soluble (C) protein extract of adult αA-CLEF lenses compared to wild-type. (B, D) Quantification of band density of total (B) and soluble (D) lens protein extract western blot analysis. (E) Quantitative RT-PCR expression analysis of γ-crystallins in adult wild-type and αA-CLEF lenses. mRNA expression of γA-, γC-, γD- and γEF-crystallin is significantly lower in adult αA-CLEF lenses. (F) Quantitative RT-PCR expression analysis of γ-crystallins in E16.5 wild-type and αA-CLEF lenses. mRNA expression of γA-, γB-, γC-, γD- and γEF-crystallin is significantly lower already in E16.5 αA-CLEF lenses (**p<0.01).</p

The phenotypic consequences of Meis1 and Meis2 deficiency.

<p>(<b>A-E</b>) At E12.5, external eyes of whole-mount <i>Meis1</i>^<i>-/-</i>, <i>Le-Cre;Meis2</i>^<i>f/f</i>, <i>Le-Cre;Meis1</i>^<i>+/-</i><i>;Meis2</i>^<i>f/f</i> mutant appear comparable to control eye, whereas the eye of <i>Le-Cre;Meis1</i>^<i>-/-</i><i>;Meis2</i>^<i>f/f</i> double mutant has abnormal shape. The insets show high magnification of eye region (boxed). (<b>F-O</b>) Hematoxylin-eosin stained parrafin sections show histology of control or mutant E10.5 and E12.5 eyes. (<b>F-H</b>, <b>K-M</b>) Formation of lens placode is followed by invagination of surface ectodem, formation of lens pit (LPi) and subsequent formation of lens in control, <i>Meis1</i>^<i>-/-</i> and <i>Le-Cre;Meis2</i>^<i>f/f</i> embryos. (<b>I</b>, <b>N</b>) One active <i>Meis1</i> allele in <i>Le-Cre;Meis1</i>^<i>-/+</i><i>; Meis2</i>^<i>f/f</i> embryos is sufficient for lens placode and lens formation. (<b>J</b>, <b>O</b>) In <i>Le-Cre;Meis1</i>^<i>-/-</i><i>;Meis2</i>^<i>f/f</i> embryos, deficient for both Meis1 and Meis2, lens development is arrested in pre-placodal stage (arrowheads). * Artefact, le-lens, nr-neural retina.</p

Current model of transcriptional regulatory network operating during mammalian lens induction.

<p>Direct interactions are indicated with solid lines, whereas dashed lines show possible direct interactions inferred from gain- and loss-of-function studies.</p