Six3 in a small population of progenitors at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.
ABSTRACT: Neuroretina and retinal pigment epithelium (RPE) are differentiated from the progenitors in optic vesicles, but it is unclear when and how the two lineages are segregated. Manipulation of chick embryos reveals that the early anteroventral optic vesicle is crucial for neuroretinal development, but the molecular mechanism is unclear. Homeodomain transcription factor Six3 is required for neuroretinal specification and is dispensable for RPE formation, but the cell fates of Six3-deficient progenitors and the origins of remnant RPE are unknown. Here, we performed lineage tracing of Six3-Cre positive cells in wild-type and Six3-deficient mouse embryos. Six3-Cre positive progenies were found in a population of progenitors in the anteroventral optic pits/vesicles starting at E8.5, and were found in neuroretina, optic stalk, ventral forebrain, but not RPE, at E10.5. Six3-deletion in the small population of progenitors at E8.5 was sufficient to cause rostral expansion of Wnt8b and drastic reduction of Fgf8/MAPK signaling, ablating neuroretinal specification without affecting RPE. Lineage tracing revealed Six3-deficient progenitors at E8.5 were eventually lost and the remnant RPE was derived from Six3-Cre negative cells. Thus, Six3 in a small population of progenitors expressing Six3-Cre at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.
Project description:Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell-based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis-related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional-mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.
Project description:Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.
Project description:Gene regulation of multipotent neuroretinal progenitors is partially understood. Through characterizing Six3 and Six6 double knockout retinas (DKOs), we demonstrate Six3 and Six6 are jointly required for the maintenance of multipotent neuroretinal progenitors. Phenotypes in DKOs were not found in either Six3 nulls or Six6 nulls. At the far periphery, ciliary margin (CM) markers Otx1 and Cdon together with Wnt3a and Fzd1 were ectopically upregulated, whereas neuroretinal progenitor markers Sox2, Notch1, and Otx2 were absent or reduced. At the mid periphery, multi-lineage differentiation was defective. The gene set jointly regulated by Six3 and Six6 significantly overlapped with the gene networks regulated by WNT3A, CTNNB1, POU4F2, or SOX2. Stimulation of Wnt/?-catenin signaling by either Wnt-3a or a GS3K? inhibitor promoted CM progenitors at the cost of neuroretinal identity at the periphery of eyecups. Therefore, Six3 and Six6 together directly or indirectly suppress Wnt/?-catenin signaling but promote retinogenic factors for the maintenance of multipotent neuroretinal progenitors.
Project description:Fibroblast growth factor (FGF) signaling is an essential regulator of lens epithelial cell proliferation and survival, as well as lens fiber cell differentiation. However, the identities of these FGF factors, their source tissue and the genes that regulate their synthesis are unknown. We have found that Chx10-Cre;Lhx2lox/lox mice, which selectively lack Lhx2 expression in neuroretina from E10.5, showed an early arrest in lens fiber development along with severe microphthalmia. These mutant animals showed reduced expression of multiple neuroretina-expressed FGFs and canonical FGF-regulated genes in neuroretina. When FGF expression was genetically restored in Lhx2-deficient neuroretina of Chx10-Cre;Lhx2lox/lox mice, we observed a partial but nonetheless substantial rescue of the defects in lens cell proliferation, survival and fiber differentiation. These data demonstrate that neuroretinal expression of Lhx2 and neuroretina-derived FGF factors are crucial for lens fiber development in vivo.
Project description:Colobomata, persistent optic fissures, frequently cause congenital blindness. Here, we focused on optic fissure fusion using in vivo time-lapse imaging in zebrafish. We identified the fusion initiating cells, which we termed "pioneer cells." Based on morphology, localization, and downregulation of the neuroretinal (NR) precursor marker rx2, these cells could be considered as retinal pigment epithelial (RPE) progenitors. Notably, pioneer cells regain rx2 expression and integrate into the NR after fusion, indicating that they do not belong to the pool of RPE progenitors, supported by the lack of RPE marker expression in pioneer cells. They establish the first cellular contact between the margins in the proximal fissure region and separate the hyaloid artery and vein. After initiation, the fusion site is progressing distally, increasing the distance between the hyaloid artery and vein. A timed BMP (Bone Morphogenetic Protein) induction, resulting in coloboma, did not alter the morphology of the fissure margins, but it did affect the expression of NR and RPE markers within the margins. In addition, it resulted in a persisting basal lamina and persisting remnants of periocular mesenchyme and hyaloid vasculature within the fissure, supporting the necessity of BMP antagonism within the fissure margins. The hampered fissure fusion had severe effects on the vasculature of the eye.
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 Tead-responsive 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 dosage-dependent 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.
Project description:Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.
Project description:During early brain development, the organisation of neural progenitors into a neuroepithelial sheet maintains tissue integrity during growth. Neuroepithelial cohesion and patterning is essential for orderly proliferation and neural fate specification. Neuroepithelia are regionalised by the expression of transcription factors and signalling molecules, resulting in the formation of distinct developmental, and ultimately functional, domains.We have discovered that the Six3/6 family orthologue Optix is an essential regulator of neuroepithelial maintenance and patterning in the Drosophila brain. Six3 and Six6 are required for mammalian eye and forebrain development, and mutations in humans are associated with severe eye and brain malformation. In Drosophila, Optix is expressed in a sharply defined region of the larval optic lobe, and its expression is reciprocal to that of the transcription factor Vsx1. Optix gain- and loss-of-function affects neuroepithelial adhesion, integrity and polarity. We find restricted cell lineage boundaries that correspond to transcription factor expression domains.We propose that the optic lobe is compartmentalised by expression of Optix and Vsx1. Our findings provide insight into the spatial patterning of a complex region of the brain, and suggest an evolutionarily conserved principle of visual system development.
Project description:Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that the reactivation of anterior neural transcription factor OTX2, eye field transcription factor RX1 and SIX3 in iPSCs is a feature of pluripotency acquisition and predetermines the potential of RPE differentiation.
Project description:The hemispheric, bi-layered optic cup forms from an oval optic vesicle during early vertebrate eye development through major morphological transformations. The overall basal surface, facing the developing lens, is increasing, while, at the same time, the space basally occupied by individual cells is decreasing. This cannot be explained by the classical view of eye development. Using zebrafish (Danio rerio) as a model, we show that the lens-averted epithelium functions as a reservoir that contributes to the growing neuroretina through epithelial flow around the distal rims of the optic cup. We propose that this flow couples morphogenesis and retinal determination. Our 4D data indicate that future stem cells flow from their origin in the lens-averted domain of the optic vesicle to their destination in the ciliary marginal zone. BMP-mediated inhibition of the flow results in ectopic neuroretina in the RPE domain. Ultimately the ventral fissure fails to close resulting in coloboma.