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.

Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective

“…Several genes have been attributed to neuroretinal specification as well as the proliferative and multipotent ability of retinal progenitor cells, including Vsx2 (also known as Chx10), Pax6, Lhx2, Rax, Six3, and Six6 [71][72][73][74][75][76][77][78][79]. Many of the genes are also implicated in retinal abnormalities; for instance, Pax6 mutations can lead to foveal hypoplasia, while Rax mutations can cause microphthalmia leading to retinal dysplasia [80,81].…”

Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation

“…Conditional Six3 inactivation using Rx-Cre ablates NR specification but expands RPE following a rostral expansion of Wnt8b ( Liu et al, 2010 ). Six3 in a small population of progenitors at E8.5 is required for NR specification through suppressing Wnt8b, maintaining FGF/ERK signaling, and cell survival ( Liu and Cvekl, 2017 ). Six6, a paralog of Six3, is expressed in optic vesicles starting at E8.5–9.0, a later stage compared to Six3, and is largely co-localized with Six3 in the progenitors in optic vesicles and optic cups ( Jean et al, 1999 ; Liu and Cvekl, 2017 ).…”

“…Six3 in a small population of progenitors at E8.5 is required for NR specification through suppressing Wnt8b, maintaining FGF/ERK signaling, and cell survival ( Liu and Cvekl, 2017 ). Six6, a paralog of Six3, is expressed in optic vesicles starting at E8.5–9.0, a later stage compared to Six3, and is largely co-localized with Six3 in the progenitors in optic vesicles and optic cups ( Jean et al, 1999 ; Liu and Cvekl, 2017 ). Neither Six3 inactivation at E9.5–10.5 nor Six6 deletion ( Li et al, 2002 ) overtly affect NR specification, but Six3 inactivation at E9.5 together with Six6 deletion ablates NR specification, indicating genetic interactions between Six3 and Six6 in retinal development ( Liu and Cvekl, 2017 ).…”

A distal enhancer that directs Otx2 expression in the retinal pigment epithelium and neuroretina

“…37,40,41 Briefly, embryos or eyes were fixed for 10 minutes (1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP40 in PBS), rinsed with PBS, and then stained in an X-gal solution (1 mg/mL X-gal, 0.5 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 ) at 30 C overnight. 37,40,41 Briefly, embryos or eyes were fixed for 10 minutes (1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP40 in PBS), rinsed with PBS, and then stained in an X-gal solution (1 mg/mL X-gal, 0.5 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 ) at 30 C overnight.…”

“…X-gal staining of transgenic embryos and eyes was performed using a method described previously. 37,40,41 Briefly, embryos or eyes were fixed for 10 minutes (1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP40 in PBS), rinsed with PBS, and then stained in an X-gal solution (1 mg/mL X-gal, 0.5 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 ) at 30 C overnight. Stained samples were fixed in 4% paraldehyde at room temperature for 1 hour and then rinsed in PBS.…”