Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure.
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ABSTRACT:
SUBMITTER: Brown JD
PROVIDER: S-EPMC2631080 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of childhood blindness. The molecular and cellular mechanisms that facilitate optic fissure fusion remain elusive. We have profiled global gene expression during optic fissure morphogenesis by transcriptome analysis of tissue dissected from the margins of the zebrafish optic fissure and the opposing dorsal retina before (32 hours post fertilisation, hpf), during (48 hpf) and after (56 hpf) optic fissure fusion. Differential expression analysis between optic fissure and dorsal retinal tissue resulted in the detection of several known and novel developmental genes. The expression of selected genes was validated by qRT-PCR analysis and localisation investigated using in situ hybridisation. We discuss significantly overrepresented functional ontology categories in the context of optic fissure morphogenesis and highlight interesting transcripts from hierarchical clustering for subsequent analysis. We have identified netrin1a (ntn1a) as highly differentially expressed across optic fissure fusion, with a resultant ocular coloboma phenotype following morpholino antisense translation-blocking knockdown and downstream disruption of atoh7 expression. To support the identification of candidate genes in human studies, we have generated an online open-access resource for fast and simple quantitative querying of the gene expression data. Our study represents the first comprehensive analysis of the zebrafish optic fissure transcriptome and provides a valuable resource to facilitate our understanding of the complex aetiology of ocular coloboma.
| S-EPMC6367446 | biostudies-literature
Project description:Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We show that the mouse Zfp503/Nlz2 gene is dynamically regulated in developing mouse eyes and that loss of its expression results in uveal coloboma. KO embryos exhibit altered expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1 and Vax2, resulting in reduced melanin pigmentation in the presumptive retinal pigment epithelium and its differentiation into a neural-retina-like lineage. From a large cohort of patients with exome sequencing records, only one patient with clinical diagnosis of retinal degeneration was identified carrying a pPresumed loss-of-function variants in the human ortholog, ZNF503. Although no disease-causing association was demonstrated, because of the retinal degeneration rather than of uveal coloboma phenotype, are rare and may be associated with retinal degeneration, rather than a uveal coloboma phenotype, suggesting it is tempting to speculate that a pleiotropic role of ZNF503 might have a pleiotropic role in ocular development and physiology
2022-11-16 | GSE180641 | GEO
Project description:PurposeMutations in the CYP1B1 gene are the most commonly identified genetic causes of primary infantile-onset glaucoma. Despite this disease association, the role of CYP1B1 in eye development and its in vivo substrate remain unknown. In the present study, we used zebrafish to elucidate the mechanism by which cyp1b1 regulates eye development.MethodsZebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Cyp1b1 and retinoic acid (RA) levels were genetically (morpholino oligonucleotide antisense and mRNA) and pharmacologically manipulated to examine gene function.ResultsUsing zebrafish, we observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency. Decreased Cyp1b1 resulted in the premature breakdown of laminin in the ventral fissure and altered subsequent neural crest migration into the anterior segment. In contrast, cyp1b1 overexpression inhibited cell survival in the ventral ocular fissure and prevented fissure closure via an RA-independent pathway. Cyp1b1 overexpression also inhibited the ocular expression of vsx2, pax6a, and pax6b and increased the extraocular expression of shha. Importantly, embryos injected with human wild-type but not mutant CYP1B1 mRNA also showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species.ConclusionsCyp1b1 regulation of ocular fissure closure indirectly affects neural crest migration and development through an RA-independent pathway. These studies provide insight into the role of Cyp1b1 in eye development and further elucidate the pathogenesis of primary infantile-onset glaucoma.
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Project description:SoxC transcription factors play critical roles in many developmental processes, including neurogenesis, cardiac formation, and skeletal differentiation. In vitro and in vivo loss-of-function studies have suggested that SoxC genes are required for oculogenesis; however the mechanism was poorly understood. Here, we have explored the function of the SoxC factor Sox4 during zebrafish eye development. We show that sox4a and sox4b are expressed in the forebrain and periocular mesenchyme adjacent to the optic stalk during early eye development. Knockdown of sox4 in zebrafish resulted in coloboma, a structural malformation of the eye that is a significant cause of pediatric visual impairment in humans, in which the choroid fissure fails to close. Sox4 morphants displayed altered proximo-distal patterning of the optic vesicle, including expanded pax2 expression in the optic stalk, as well as ectopic cell proliferation in the retina. We show that the abnormal ocular morphogenesis observed in Sox4-deficient zebrafish is caused by elevated Hedgehog (Hh) signaling, and this is due to increased expression of the Hh pathway ligand Indian Hedgehog b (ihhb). Consistent with these results, coloboma in sox4 morphants could be rescued by pharmacological treatment with the Hh inhibitor cyclopamine, or by co-knockdown of ihhb. Conversely, overexpression of sox4 reduced Hh signaling and ihhb expression, resulting in cyclopia. Finally, we demonstrate that sox4 and sox11 have overlapping, but not completely redundant, functions in regulating ocular morphogenesis. Taken together, our data demonstrate that Sox4 is required to limit the extent of Hh signaling during eye development, and suggest that mutations in SoxC factors could contribute to the development of coloboma.
| S-EPMC4339509 | biostudies-literature
Project description:A key embryonic process that occurs early in ocular development is optic fissure closure (OFC). This fusion process closes the ventral optic fissure and completes the circumferential continuity of the 3-dimensional eye. It is defined by the coming together and fusion of opposing neuroepithelia along the entire proximal-distal axis of the ventral optic cup, involving future neural retina, retinal pigment epithelium (RPE), optic nerve, ciliary body, and iris. Once these have occurred, cells within the fused seam differentiate into components of the functioning visual system. Correct development and progression of OFC, and the continued integrity of the fused margin along this axis, are important for the overall structure of the eye. Failure of OFC results in ocular coloboma-a significant cause of childhood visual impairment that can be associated with several complex ocular phenotypes including microphthalmia and anterior segment dysgenesis. Despite a large number of genes identified, the exact pathways that definitively mediate fusion have not yet been found, reflecting both the biological complexity and genetic heterogeneity of the process. This review will highlight how recent developmental studies have become focused specifically on the epithelial fusion aspects of OFC, applying a range of model organisms (spanning fish, avian, and mammalian species) and utilizing emerging high-resolution live-imaging technologies, transgenic fluorescent models, and unbiased transcriptomic analyses of segmentally-dissected fissure tissue. Key aspects of the fusion process are discussed, including basement membrane dynamics, unique cell behaviors, and the identities and fates of the cells that mediate fusion. These will be set in the context of what is now known, and how these point the way to new avenues of research.
| S-EPMC7829581 | biostudies-literature
Project description:Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of childhood blindness. The molecular and cellular mechanisms that facilitate optic fissure fusion remain elusive. We have profiled global gene expression during optic fissure morphogenesis by transcriptome analysis of tissue dissected from the margins of the zebrafish optic fissure and the opposing dorsal retina before (32 hours post fertilisation, hpf), during (48 hpf) and after (56 hpf) optic fissure fusion. Differential expression analysis between optic fissure and dorsal retinal tissue resulted in the detection of several known and novel developmental genes. The expression of selected genes was validated by qRT-PCR analysis and localisation investigated using in situ hybridisation. We discuss significantly overrepresented functional ontology categories in the context of optic fissure morphogenesis and highlight interesting transcripts from hierarchical clustering for subsequent analysis. We have identified netrin1a (ntn1a) as highly differentially expressed across optic fissure fusion, with a resultant ocular coloboma phenotype following morpholino antisense translation-blocking knockdown and downstream disruption of atoh7 expression. To support the identification of candidate genes in human studies, we have generated an online open-access resource for fast and simple quantitative querying of the gene expression data. Our study represents the first comprehensive analysis of the zebrafish optic fissure transcriptome and provides a valuable resource to facilitate our understanding of the complex aetiology of ocular coloboma.
2020-10-22 | GSE159822 | GEO
Project description:During vertebrate eye morphogenesis, a transient fissure forms at its inferior part, known as the optic fissure. This will gradually close, giving rise to a healthy, spherical optic cup. Failure of the optic fissure to close gives rise to an ocular disorder known as coloboma. During this developmental process, Foxg1 is expressed in the optic neuroepithelium, with highest levels of expression in the nasal optic stalk. Foxg1-/- mutant mice have microphthalmic eyes with a large ventral coloboma. We found Wnt8b expression upregulated in the Foxg1-/- optic stalk and hypothesized that, similar to what is observed in telencephalic development, Foxg1 directs development of the optic neuroepithelium through transcriptional suppression of Wnt8b To test this, we generated Foxg1-/-;Wnt8b-/- double mutants of either sex and found that the morphology of the optic cup and stalk and the closure of the optic fissure were substantially rescued in these embryos. This rescue correlates with restored Pax2 expression in the anterior tip of the optic fissure. In addition, although we do not find evidence implicating altered proliferation in the rescue, we observe a significant increase in apoptotic cell density in Foxg1-/-;Wnt8b-/- double mutants compared with the Foxg1-/- single mutant. Upregulation of Wnt/β-catenin target molecules in the optic cup and stalk may underlie the molecular and morphological defects in the Foxg1-/- mutant. Our results show that proper optic fissure closure relies on Wnt8b suppression by Foxg1 in the nasal optic stalk to maintain balanced apoptosis and Pax2 expression in the nasal and temporal edges of the fissure.SIGNIFICANCE STATEMENT Coloboma is an ocular disorder that may result in a loss of visual acuity and accounts for ∼10% of childhood blindness. It results from errors in the sealing of the optic fissure (OF), a transient structure at the bottom of the eye. Here, we investigate the colobomatous phenotype of the Foxg1-/- mutant mouse. We identify upregulated expression of Wnt8b in the optic stalk of Foxg1-/- mutants before OF closure initiates. Foxg1-/-;Wnt8b-/- double mutants show a substantial rescue of the Foxg1-/- coloboma phenotype, which correlates with a rescue in molecular and cellular defects of Foxg1-/- mutants. Our results unravel a new role of Foxg1 in promoting OF closure providing additional knowledge about the molecules and cellular mechanisms underlying coloboma formation.
| S-EPMC5559767 | biostudies-literature
Project description:The different stages of the optic fissure can be clearly visualized by making sagittal sections through the mouse eye during early development which represent the optic fissure at open (E10.5), closing (E11.5) and fused (E12.5) states. Laser capture microdissection (LCM) was employed to dissect tissue from the margins of the optic fissure consisting of the outer (presumptive RPE) and inner (presumptive neurosensory retina) layers of the retina. An approximately square-shaped block of optic fissure (50 x 50 mm) was dissected from each side of the fissure. Two rounds of linear amplification were performed on RNA isolated from each of the samples prior to microarray hybridization. Expression data were gathered in biological triplicate at E10.5, E11.5 and duplicate at E12.5, each array representing pooled optic fissure tissue from three embryos from a single litter. Expression signals were ascertained from 45,101 probe sets and normalized across arrays. Experiment Overall Design: Differential gene expression over the course of three days in laser capture microdissected tissue from wild type mouse embryos.
2008-10-26 | E-GEOD-13103 | biostudies-arrayexpress
Project description:Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37 highly selected individuals with severe ocular anomalies plus variable defects associated with BOFS for mutations or deletions in TFAP2A. We identified one individual with a de novo TFAP2A four amino acid deletion, a second individual with two non-synonymous variations in an alternative splice isoform TFAP2A2, and a sibling-pair with a paternally inherited whole gene deletion with variable phenotypic expression. Second, we determined that TFAP2A is expressed in the lens, neural retina, nasal process, and epithelial lining of the oral cavity and palatal shelves of human and mouse embryos--sites consistent with the phenotype observed in patients with BOFS. Third, we used zebrafish to examine how partial abrogation of the fish ortholog of TFAP2A affects the penetrance and expressivity of ocular phenotypes due to mutations in genes encoding bmp4 or tcf7l1a. In both cases, we observed synthetic, enhanced ocular phenotypes including coloboma and anophthalmia when tfap2a is knocked down in embryos with bmp4 or tcf7l1a mutations. These results reveal that mutations in TFAP2A are associated with a wide range of eye phenotypes and that hypomorphic tfap2a mutations can increase the risk of developmental defects arising from mutations at other loci.
| S-EPMC3083835 | biostudies-literature
Project description:Exome sequence analysis of affected individuals from two families with autosomal-dominant inheritance of coloboma identified two different cosegregating heterozygous nonsense mutations (c.370C>T [p.Arg124*] and c. 1066G>T [p.Glu356*]) in YAP1. The phenotypes of the affected families differed in that one included no extraocular features and the other manifested with highly variable multisystem involvement, including hearing loss, intellectual disability, hematuria, and orofacial clefting. A combined LOD score of 4.2 was obtained for the association between YAP1 loss-of-function mutations and the phenotype in these families. YAP1 encodes an effector of the HIPPO-pathway-induced growth response, and whole-mount in situ hybridization in mouse embryos has shown that Yap1 is strongly expressed in the eye, brain, and fusing facial processes. RT-PCR showed that an alternative transcription start site (TSS) in intron 1 of YAP1 and Yap1 is widely used in human and mouse development, respectively. Transcripts from the alternative TSS are predicted to initiate at codon Met179 relative to the canonical transcript (RefSeq NM_001130145). In these alternative transcripts, the c.370C>T mutation in family 1305 is within the 5' UTR and cannot result in nonsense-mediated decay (NMD). The c. 1066G>T mutation in family 132 should result in NMD in transcripts from either TSS. Amelioration of the phenotype by the alternative transcripts provides a plausible explanation for the phenotypic differences between the families.
| S-EPMC3928658 | biostudies-literature