Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate
Wei Liu, … , Milan Jamrich, Guillermo Oliver
Wei Liu, … , Milan Jamrich, Guillermo Oliver
Published September 20, 2010
Citation Information: J Clin Invest. 2010;120(10):3568-3577. https://doi.org/10.1172/JCI43219.
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Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate

Abstract

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.

Authors

Wei Liu, Oleg Lagutin, Eric Swindell, Milan Jamrich, Guillermo Oliver

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Figure 6

Six3 binds to Wnt8b regulatory elements in vivo.

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Six3 binds to Wnt8b regulatory elements in vivo. (A) DNA sequence co...
(A) DNA sequence comparison of the Wnt8b genomic loci of mouse, human, zebrafish, chick, and fugu fish revealed 2 conserved regions located outside the exons: the promoter and a 3′ flanking fragment. (B) DNA fragments containing potential Six3-binding motifs for both areas were subjected to EMSA analysis. Addition of GST-Six3 protein caused a band shift of the radiolabeled probes, and addition of the Six3 antibody supershifted (red rectangle) those bands. Cold probes (in ×400 excess) were used to compete with the band shift. (C–E) Forced Six3 expression in the presumptive forebrain territory of E8.5 mouse embryos repressed Wnt8b expression. The anterior boundaries of Wnt8b expression in control (C) and in the transgenic embryo (D) are indicated by arrowheads. Expression of the transgene is monitored by YFP fluorescence (E). (F) Chromatin prepared from the head (Six3 positive) or trunk (Six3 negative) of 8- to 15-somite staged wild-type embryos was used for ChIP using either Six3 antibody or normal rabbit IgG and quantified by SYBR qPCR. The y axis indicates the value ratio between the Six3 and the corresponding IgG immunoprecipitated samples. Six3 was highly enriched in the promoter area and moderately enriched in the 3′ flanking area. (G) Working model of NR specification. Normally, the eye field (EF) territory will be derived from the anterior neural plate (ANP). Later on, the NR and RPE will be derived from this region, but while RPE specification requires active Wnt/β-catenin signaling, the specification of the NR fate requires its repression by Six3. Scale bar: 100 μm.