Conservation and Diversification of an Ancestral Chordate Gene Regulatory Network for Dorsoventral Patterning

Formation of a dorsoventral axis is a key event in the early development of most animal embryos. It is well established that bone morphogenetic proteins (Bmps) and Wnts are key mediators of dorsoventral patterning in vertebrates. In the cephalochordate amphioxus, genes encoding Bmps and transcription factors downstream of Bmp signaling such as Vent are expressed in patterns reminiscent of those of their vertebrate orthologues. However, the key question is whether the conservation of expression patterns of network constituents implies conservation of functional network interactions, and if so, how an increased functional complexity can evolve. Using heterologous systems, namely by reporter gene assays in mammalian cell lines and by transgenesis in medaka fish, we have compared the gene regulatory network implicated in dorsoventral patterning of the basal chordate amphioxus and vertebrates. We found that Bmp but not canonical Wnt signaling regulates promoters of genes encoding homeodomain proteins AmphiVent1 and AmphiVent2. Furthermore, AmphiVent1 and AmphiVent2 promoters appear to be correctly regulated in the context of a vertebrate embryo. Finally, we show that AmphiVent1 is able to directly repress promoters of AmphiGoosecoid and AmphiChordin genes. Repression of genes encoding dorsal-specific signaling molecule Chordin and transcription factor Goosecoid by Xenopus and zebrafish Vent genes represents a key regulatory interaction during vertebrate axis formation. Our data indicate high evolutionary conservation of a core Bmp-triggered gene regulatory network for dorsoventral patterning in chordates and suggest that co-option of the canonical Wnt signaling pathway for dorsoventral patterning in vertebrates represents one of the innovations through which an increased morphological complexity of vertebrate embryo is achieved

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development

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

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

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

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

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

Characterization of SIMO wild-type and mutant enhancer by reporter gene assays in chick and zebrafish.

<p>(<b>A</b>, <b>B</b>) Schematic view of reporter constructs used for <i>in ovo</i> electroporation of chick embryos. Reporter constructs carry wild-type or mutant mouse SIMO element upstream of <i>hsp68</i> minimal promoter and β-galactosidase open reading frame. In mutant SIMO Meis binding sites were abolished by introduction of specific single-point mutations changing Meis recognition sequence TGACAG/A into TcACAG/A. (<b>C–F</b>) Whole-mount view or histological sections through the eye of β-galactosidase–stained chick embryos of stage HH21-22 electroporated either with (<b>C, E</b>) wild-type or with (<b>D, F</b>) mutant SIMO fragment. Positive X-gal staining correlates with the activity of reporter constructs. Wild-type SIMO fragment supports reporter construct expression in lens but not the mutant SIMO fragment. (<b>G, H</b>) Schematic view of reporter constructs used for transgenesis in zebrafish. Reporter constructs carry wild-type or mutant zebrafish SIMO element upstream of zebrafish <i>gata2a</i> minimal promoter and EGFP open reading frame. In mutant zebrafish SIMO Meis binding sites were abolished by introduction of specific single-point mutations changing Meis recognition sequence TGACAG/A into TcACAG/A. In order to control for transgenesis efficiency <i>in vivo</i> the reporter genes contain a second cassette composed of a cardiac actin promoter driving the expression of a red fluorescent protein (DsRed). EGFP and DsRed transcriptional units are separated by an insulator. (<b>I-L</b>) Wild-type SIMO enhancer activity is detected at 48 hpf (n = 160, 68% EGFP of DsRed positive), <b>(I, J)</b>, but not for the mutant construct (n = 36, 89% EGFP negative of DsRed positive) (<b>K, L</b>). LE—lens, NR—neural retina.</p

Genetic analysis of SIMO deletion <i>in vivo</i>.

<p>(<b>A</b>) Scheme of wild-type <i>Pax6</i> locus and alleles carrying EE [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006441#pgen.1006441.ref017" target="_blank">17</a>] or SIMO deletion (this study). EE is indicated with red oval and SIMO with yellow oval. (<b>B</b>) Phenotypic consequences of SIMO deletion in <i>Pax6</i>^{<i>eSIMOdel710/Sey</i>} compound heterozygote mice. Whole-mount view of E13.5 embryos of the indicated genotype with eye in the inset (top panel). Histological sections through the eye demonstrating the absence of lens at E13.5 (middle panel) and arrested development prior to lens pit stage at E11.0 in <i>Pax6</i> ^{<i>SIMOdel710/Sey</i>} embryos. nr—neural retina, le-lens.</p