Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities-an analysis of CRISPR genome-edited mouse embryos.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid gene-targeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesicle-like tissues or histological anophthalmia. Genotyping of neck tissue cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.

Profiling retinal biochemistry in the MPDZ mutant retinal dysplasia and degeneration chick: a model of human RP and LCA.

PURPOSE: Raman microscopy, a rapid nondestructive technique that profiles the composition of biological samples, was used to characterize retinal biochemistry in the retinal dysplasia and degeneration (rdd) and wild-type (wt) chick retina during retinogenesis and at hatching. METHODS: Embryonic day (E)13 and posthatch day (P)1 rdd and wt retinal cross-sections (n = 3 of each line at each age) were profiled using 633 helium-neon laser excitation. The biochemical composition was determined using computational analysis of the Raman spectra. In parallel histology, TUNEL and glial fibrillary acidic protein (GFAP) immunostaining were used to visualize retinal dysfunction. RESULTS: Principal component (PC) analysis of the Raman spectra identified 50 major biochemical profiles, but only PCs that made significant contributions to variation within rdd and wt retina were mapped. These significant PCs were shown to arise from DNA, various fatty acids, melanin, and a number of proteins. Distinct patterns of GFAP immunostaining and a larger population of TUNEL-positive nuclei were observed in the rdd versus wt retina. CONCLUSIONS: This study has demonstrated that Raman microscopy can discriminate between major retinal biomolecules, thus providing an unbiased account of how their composition varies due to the impact of the MPDZ null mutation in the rdd chick relative to expression in the normal wt retina.

A nonautonomous role for retinal frizzled-5 in regulating hyaloid vitreous vasculature development.

PURPOSE: Frizzled-5 (Fzd5) is expressed in the developing retina of multiple species and appears to play species-specific roles during eye development. The present study analyzed the effects of tissue-specific deletion of Fzd5 on mammalian eye development. METHODS: To generate Fzd5 conditional knockout (CKO) mice, Fzd5(+/-) mice carrying the Six3-Cre transgene were crossed with Fzd5(LoxP/LoxP) mice. To determine which cell lineages in the eye had Cre recombinase activity, Six3-Cre transgenic mice were crossed with ROSA-26 reporter mice, and lacZ activity was assayed. Histologic analysis, immunofluorescence, and TUNEL labeling were performed from embryonic day (E)12.5 to postnatal stages to analyze vascularization, cell proliferation, retinal organization, and apoptosis. RESULTS: On conditional disruption of Fzd5 specifically in the retina, but not in vitreous hyaloid vasculature (VHV), an abnormal accumulation consisting of pericytes and endothelial cells was observed in the vitreous as early as E12.5. The abundant retrolental cells persisted into postnatal stages and appeared as a pigmented intravitreal mass. In Fzd5 CKO mice there was failure of normal apoptosis of the VHV, and cells in the persistent VHV were maintained in the cell cycle up to postnatal day 23. Moreover, morphogenesis of the retina adjacent to the vasculature was disrupted, leading to retinal folds, detachment, and abnormal lamination. This phenotype is similar to that of human eye disease persistent hyperplastic primary vitreous (PHPV). CONCLUSIONS: Selective loss of Fzd5 in the retina results in PHPV and retinal defects through an apparently cell-nonautonomous effect, revealing a potential requirement for retina-derived signals in regulating the development of the VHV.

Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance.

BACKGROUND: The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey) and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. RESULTS: Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection. CONCLUSION: Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6+/- heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm.

Disgenesias papilares y de retina: incluyendo un caso de papila contráctil y uno de enfermedad de Norrie / Disc and retina disgenesia: including a contractil papilla and 1 of Norrie`s disease

Se presentan 21 ojos con anomalías congénitas de papila y retina, detallando un caso de disgenesia papilar total o síndrome de Morning Glory, de tipo contráctil que evolucionó hacia una PVR masiva, y un caso de displasia retinal o Enfermedad de Norrie típicamente bilateral. Se discute la embriogénesis de estas anomalías, concluyendo que la displasia retinal ocurre en la sexta semana de gestación y que la disgenesia papilar total probablemente se sitúe en la cuarta semana gestacional, produciéndose por falta de obliteración de la copa óptica