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.
Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five ...families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1. These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2, a close homolog of MAB21L1, cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral ...anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.
Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust ...spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that
-1 (
) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that
is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.
Tissue fusion is a critical process that is repeated in multiple contexts during embryonic development and shares common attributes to processes such as wound healing and metastasis. Ocular coloboma ...is a developmental eye disorder that presents as a physical gap in the ventral eye, and is a major cause of childhood blindness. Coloboma results from fusion failure between opposing ventral retinal epithelia, but there are major knowledge gaps in our understanding of this process at the molecular and cell behavioural levels. Here we catalogue the expression of cell adhesion proteins: N-cadherin, E-cadherin, R-cadherin, ZO-1, and the EMT transcriptional activator and cadherin regulator SNAI2, in the developing chicken embryonic eye. We find that fusion pioneer cells at the edges of the fusing optic fissure have unique and dynamic expression profiles for N-cad, E-cad and ZO-1, and that these are temporally preceded by expression of SNAI2. This highlights the unique properties of these cells and indicates that regulation of cell adhesion factors may be a critical process in optic fissure closure.
Ocular coloboma (OC) is a failure of complete optic fissure closure during embryonic development and presents as a tissue defect along the proximal-distal axis of the ventral eye. It is classed as ...part of the clinical spectrum of structural eye malformations with microphthalmia and anophthalmia, collectively abbreviated to MAC. Despite deliberate attempts to identify causative variants in MAC, many patients remain without a genetic diagnosis. To reveal potential candidate genes, we utilised transcriptomes experimentally generated from embryonic eye tissues derived from humans, mice, zebrafish, and chicken at stages coincident with optic fissure closure. Our in-silico analyses found 10 genes with optic fissure-specific enriched expression:
,
,
,
,
,
,
,
,
, and
. In situ hybridization revealed that all 10 genes were broadly expressed ventrally in the developing eye but that only
and
were expressed in cells at the edges of the optic fissure margin. Of these conserved optic fissure genes,
,
, and
have not previously been associated with human MAC cases. Targeted genetic manipulation in zebrafish embryos using CRISPR/Cas9 caused the developmental MAC phenotype for
and
. We analysed available whole genome sequencing datasets from MAC patients and identified a range of variants with plausible causality. In combination, our data suggest that expression of genes involved in ventral eye development is conserved across a range of vertebrate species and that
,
, and
are candidate loci that warrant further functional analysis in the context of MAC and should be considered for sequencing in cohorts of patients with structural eye malformations.
We report heterozygous, loss-of-function SOX2 mutations in three unrelated individuals with Anophthalmia-Esophageal-Genital (AEG) syndrome. One previously reported case Rogers, R.C. (1988) Unknown ...cases. Proceedings of the Greenwood Genetic Center. 7, 57. has a 2.7 Mb deletion encompassing SOX2 and associated with a cryptic translocation t(3;7)(q28;p21.3). The deletion and translocation breakpoints on chromosome 3q are >8.6 Mb apart and both chromosome rearrangements have occurred de novo. Another published case Petrackova et al. (2004) Association of oesophageal atresia, anophthalmia and renal duplex. Eur. J. Pediatr., 163, 333–334. has a de novo nonsense mutation, Q55X. A previously unreported case with severe bilateral microphthalmia and oesophageal atresia has a de novo missense mutation, R74P, that alters a highly evolutionarily conserved residue within the high mobility group domain, which is critical for DNA-binding of SOX2. In a yeast one-hybrid assay, this mutation abolishes Sox2-induced activation of the chick delta-crystallin DC5 enhancer. Four other reported AEG syndrome cases were extensively screened and do not have detectable SOX2 mutations. Two of these cases have unilateral eye malformations. SOX2 mutations are known to cause severe bilateral eye malformations but this is the first report implicating loss of function mutations in this transcription factor in oesophageal malformations. SOX2 is expressed in the developing foregut in mouse and zebrafish embryos and an apparently normal pattern of expression is maintained in Shh−/− mouse embryos, suggesting either that Sox2 acts upstream of Shh or functions in a different pathway. Three-dimensional reconstructions of the major morphological events in the developing foregut and eye from Carnegie Stages 12 and 13 human embryos are presented and compared with the data from model organisms. SOX2, with NMYC and CHD7, is now the third transcriptional regulator known to be critical for normal oesophageal development in humans.
Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing ...reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families ...currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked
(eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the
linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to
. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male
embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between
and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in
, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the
-refined critical interval. We conclude that the refined
critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered
regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in
, but further work is required to determine the precise causative allele and its genetic mechanism.
Goniodysgenesis is a developmental abnormality of the anterior chamber of the eye. It is generally considered to be congenital in dogs (
), and has been associated with glaucoma and blindness. ...Goniodysgenesis and early-onset glaucoma initially emerged in Border Collies in Australia in the late 1990s and have subsequently been found in this breed in Europe and the USA. The objective of the present study was to determine the genetic basis of goniodysgenesis in Border Collies. Clinical diagnosis was based on results of examinations by veterinary ophthalmologists of affected and unaffected dogs from eleven different countries. Genotyping using the Illumina high density canine single nucleotide variant genotyping chip was used to identify a candidate genetic region. There was a highly significant peak of association over chromosome 17, with a
-value of 2 × 10
Expression profiles and evolutionary conservation of candidate genes were assessed using public databases. Whole genome sequences of three dogs with glaucoma, three severely affected by goniodysgenesis and three unaffected dogs identified a missense variant in the olfactomedin like 3 (
) gene in all six affected animals. This was homozygous for the risk allele in all nine cases with glaucoma and 12 of 14 other severely affected animals. Of 67 reportedly unaffected animals, only one was homozygous for this variant (offspring of parents both with goniodysgenesis who were also homozygous for the variant). Analysis of pedigree information was consistent with an autosomal recessive mode of inheritance for severe goniodysgenesis (potentially leading to glaucoma) in this breed. The identification of a candidate genetic region and putative causative variant will aid breeders to reduce the frequency of goniodysgenesis and the risk of glaucoma in the Border Collie population.
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