RNA-seq of wild-type human iPSC-derived retinal organoids at different developmental stages
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE326554 | GEO | 2026/04/05
REPOSITORIES: GEO
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2022-11-16 | GSE180641 | GEO
Project description:Anophthalmia, microphthalmia and coloboma (AMC) comprise a spectrum of developmental eye disorders, accounting for approximately 20% of childhood visual impairment. While non-coding regulatory sequences are increasingly recognised as contributing to disease burden, characterising their impact on gene function and phenotype remains challenging. Furthermore, little is known of the nature and extent of their contribution to AMC phenotypes. We identified two families with variants in or near MAB21L2. The first proband, with microphthalmia and coloboma, had a likely pathogenic missense variant (c.338G>C; p.[Trp113Ser]), segregating within the family. The second individual, presenting with microphthalmia, had an ~113.5kb homozygous deletion 19.38kb upstream of MAB21L2. Modelling the deletion led to transient small lens and coloboma in zebrafish, and microphthalmia and coloboma in Xenopus tropicalis. Conservation analysis identified 15 non-genic conserved elements (CE) within the deleted region, while ChIP-seq data from mouse embryonic stem cells demonstrated one of these (CE14) binds OTX2, a protein with an established role in eye development. Targeted disruption of CE14 in Xenopus tropicalis recapitulated an ocular coloboma phenotype, supporting its role in eye development. Together, our data provides insights into regulatory mechanisms underlying eye development, and highlights the importance of non-coding sequences as a source of genetic diagnoses in AMC.
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Project description:Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have limited the use of these models for more high throughput applications such as disease modelling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrates the development of a novel, highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal/forebrain cell fate specification.
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Project description:The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson’s chorioretinal atrophy and congenital retinal coloboma.
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Project description:PAX6 is essential for eye and forebrain development but how PAX6 instructs retinal versus neuroectoderm specification remains unknown. We found that the paired-less PAX6, PAX6D, is uniquely expressed in retinal cells during human eye development and along human embryonic stem cell (hESC) differentiation to retinal cells. HESCs with deletion of PAX6D failed to enter retinal differentiation. Induced expression of PAX6D but not PAX6A under the PAX6-null background restored the retinal differentiation capacity. ChIP-Seq, confirmed by functional assays, revealed a set of retinal genes and neural genes that are targets of PAX6D, including WNT8B. Inhibition of WNTs restored the retinal differentiation capacity of neuroepithelia with PAX6D knockout whereas activation of WNTs blocks retinal differentiation even when PAX6D is induced. Thus, PAX6D specifies neuroepithelia to retinal cells, partly via regulation of WNTs.
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Project description:PAX6 is essential for eye and forebrain development but how PAX6 instructs retinal versus neuroectoderm specification remains unknown. We found that the paired-less PAX6, PAX6D, is uniquely expressed in retinal cells during human eye development and along human embryonic stem cell (hESC) differentiation to retinal cells. HESCs with deletion of PAX6D failed to enter retinal differentiation. Induced expression of PAX6D but not PAX6A under the PAX6-null background restored the retinal differentiation capacity. ChIP-Seq, confirmed by functional assays, revealed a set of retinal genes and neural genes that are targets of PAX6D, including WNT8B. Inhibition of WNTs restored the retinal differentiation capacity of neuroepithelia with PAX6D knockout whereas activation of WNTs blocks retinal differentiation even when PAX6D is induced. Thus, PAX6D specifies neuroepithelia to retinal cells, partly via regulation of WNTs.
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