Xenopus cadherin-6 regulates growth and epithelial development of the retina

Cadherins are crucial for tissue cohesion, separation of cell layers and cell migration during embryogenesis. To investigate the role of classical type II Xcadherin-6 (Xcad-6), we performed loss-of-function studies by morpholino oligonucleotide injections. This resulted in severe eye defects which could be rescued with murine cadherin-6. In the absence of Xcadherin-6, morphological alterations and a decrease in cell proliferation were observed with eye cup formation. Eye field transplantations of Xcadherin-6 depleted donors yielded grafts that failed to form a proper neuroepithelium in a wildtype environment. At later developmental stages Xcadherin-6 deficient eyes showed lamination defects in the outer neural retina, a reduced thickness of the ganglion cell layer (GCL) and a fragmented retina pigment epithelium (RPE). Thus, Xcadherin-6 is essential early in eye development for structural organization and growth of the neuroepithelium before it differentiates into neural retina and RPE.     

Directionality of heart looping: effects of Pitx2c misexpression on flectin asymmetry and midline structures

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating head formation in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of beta-catenin. Thus, the IGF pathway is required for head formation during embryogenesis.     

Mutations in laminin alpha 1 result in complex, lens-independent ocular phenotypes in zebrafish

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.     

Ectopic lens induction in fish in response to the murine homeobox gene Six3

Recent findings show an unexpected conservation of genes involved in vertebrate and insect eye development. The Drosophila homeobox gene sine oculis is crucial for eye development. Its murine homologue, Six3 is expressed in the anterior neural plate, a region which is involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. Our findings suggest a conserved function for Six3 in metazoan eye development.     

Independent requirements for Hedgehog signaling by both the anterior heart field and neural crest cells for outflow tract development

Cardiac outflow tract (OFT) septation is crucial to the formation of the aortic and pulmonary arteries. Defects in the formation of the OFT can result in serious congenital heart defects. Two cell populations, the anterior heart field (AHF) and cardiac neural crest cells (CNCCs), are crucial for OFT development and septation. In this study, we use a series of tissue-specific genetic manipulations to define the crucial role of the Hedgehog pathway in these two fields of cells during OFT development. These data indicate that endodermally-produced SHH ligand is crucial for several distinct processes, all of which are required for normal OFT septation. First, SHH is required for CNCCs to survive and populate the OFT cushions. Second, SHH mediates signaling to myocardial cells derived from the AHF to complete septation after cushion formation. Finally, endodermal SHH signaling is required in an autocrine manner for the survival of the pharyngeal endoderm, which probably produces a secondary signal required for AHF survival and for OFT lengthening. Disruption of any of these steps can result in a single OFT phenotype.     

Neural crest derivatives in ocular development: Discerning the eye of the storm

Neural crest cells (NCCs) are vertebrate‐specific transient, multipotent, migratory stem cells that play a crucial role in many aspects of embryonic development. These cells emerge from the dorsal neural tube and subsequently migrate to different regions of the body, contributing to the formation of diverse cell lineages and structures, including much of the peripheral nervous system, craniofacial skeleton, smooth muscle, skin pigmentation, and multiple ocular and periocular structures. Indeed, abnormalities in neural crest development cause craniofacial defects and ocular anomalies, such as Axenfeld‐Rieger syndrome and primary congenital glaucoma. Thus, understanding the molecular regulation of neural crest development is important to enhance our knowledge of the basis for congenital eye diseases, reflecting the contributions of these progenitors to multiple cell lineages. Particularly, understanding the underpinnings of neural crest formation will help to discern the complexities of eye development, as these NCCs are involved in every aspect of this process. In this review, we summarize the role of ocular NCCs in eye development, particularly focusing on congenital eye diseases associated with anterior segment defects and the interplay between three prominent molecules, PITX2, CYP1B1, and retinoic acid, which act in concert to specify a population of neural crest‐derived mesenchymal progenitors for migration and differentiation, to give rise to distinct anterior segment tissues. We also describe recent findings implicating this stem cell population in ocular coloboma formation, and introduce recent evidence suggesting the involvement of NCCs in optic fissure closure and vascular development. Birth Defects Research (Part C) 105:87–95, 2015. © 2015 Wiley Periodicals, Inc.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.     

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in Xenopus tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus.     

Requirement for Dlgh-1 in Planar Cell Polarity and Skeletogenesis during Vertebrate Development

The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg) plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP). Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.     

Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse.

Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis.     

Restoring eye size in Astyanax mexicanus blind cavefish embryos through modulation of the Shh and Fgf8 forebrain organising centres

The cavefish morph of the Mexican tetra (Astyanax mexicanus) is blind at adult stage, although an eye that includes a retina and a lens develops during embryogenesis. There are, however, two major defects in cavefish eye development. One is lens apoptosis, a phenomenon that is indirectly linked to the expansion of ventral midline sonic hedgehog (Shh) expression during gastrulation and that induces eye degeneration. The other is the lack of the ventral quadrant of the retina. Here, we show that such ventralisation is not extended to the entire forebrain because fibroblast growth factor 8 (Fgf8), which is expressed in the forebrain rostral signalling centre, is activated 2 hours earlier in cavefish embryos than in their surface fish counterparts, in response to stronger Shh signalling in cavefish. We also show that neural plate patterning and morphogenesis are modified in cavefish, as assessed by Lhx2 and Lhx9 expression. Inhibition of Fgf receptor signalling in cavefish with SU5402 during gastrulation/early neurulation mimics the typical surface fish phenotype for both Shh and Lhx2/9 gene expression. Fate-mapping experiments show that posterior medial cells of the anterior neural plate, which lack Lhx2 expression in cavefish, contribute to the ventral quadrant of the retina in surface fish, whereas they contribute to the hypothalamus in cavefish. Furthermore, when Lhx2 expression is rescued in cavefish after SU5402 treatment, the ventral quadrant of the retina is also rescued. We propose that increased Shh signalling in cavefish causes earlier Fgf8 expression, a crucial heterochrony that is responsible for Lhx2 expression and retina morphogenesis defect.     

Feature-based attention in the frontal eye field and area V4 during visual search

When we search for a target in a crowded visual scene, we often use the distinguishing features of the target, such as color or shape, to guide our attention and eye movements. To investigate the neural mechanisms of feature-based attention, we simultaneously recorded neural responses in the frontal eye field (FEF) and area V4 while monkeys performed a visual search task. The responses of cells in both areas were modulated by feature attention, independent of spatial attention, and the magnitude of response enhancement was inversely correlated with the number of saccades needed to find the target. However, an analysis of the latency of sensory and attentional influences on responses suggested that V4 provides bottom-up sensory information about stimulus features, whereas the FEF provides a top-down attentional bias toward target features that modulates sensory processing in V4 and that could be used to guide the eyes to a searched-for target.     

Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon

Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.     

Drosophila nemo promotes eye specification directed by the retinal determination gene network

Drosophila nemo (nmo) is the founding member of the Nemo-like kinase (Nlk) family of serine-threonine kinases. Previous work has characterized nmo's role in planar cell polarity during ommatidial patterning. Here we examine an earlier role for nmo in eye formation through interactions with the retinal determination gene network (RDGN). nmo is dynamically expressed in second and third instar eye imaginal discs, suggesting additional roles in patterning of the eyes, ocelli, and antennae. We utilized genetic approaches to investigate Nmo's role in determining eye fate. nmo genetically interacts with the retinal determination factors Eyeless (Ey), Eyes Absent (Eya), and Dachshund (Dac). Loss of nmo rescues ey and eya mutant phenotypes, and heterozygosity for eya modifies the nmo eye phenotype. Reducing nmo also rescues small-eye defects induced by misexpression of ey and eya in early eye development. nmo can potentiate RDGN-mediated eye formation in ectopic eye induction assays. Moreover, elevated Nmo alone can respecify presumptive head cells to an eye fate by inducing ectopic expression of dac and eya. Together, our genetic analyses reveal that nmo promotes normal and ectopic eye development directed by the RDGN.     

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation

TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-β signaling. Depletion of RNF12 dramatically reduced TGF-β/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-β family signaling.     

Dpp and Hh signaling in the Drosophila embryonic eye field.

We have analyzed the function of the Decapentaplegic (Dpp) and Hedgehog (Hh) signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. As reported previously, Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). We discuss the similarities between vertebrate systems and Drosophila with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling.