Proper differentiation of photoreceptors and amacrine cells depends on a regulatory loop between NeuroD and Six6

Abstract

SUMMARYTimely generation of distinct neural cell types in appropriate numbers is fundamental for the generation of a functional retina. In vertebrates, the transcription factor Six6 is initially expressed in multipotent retina progenitors and then becomes restricted to differentiated retinal ganglion and amacrine cells. How Six6 expression in the retina is controlled and what are its precise functions are still unclear. To address this issue, we used bioinformatic searches and transgenic approaches in medaka fish (Oryzias latipes) to characterise highly conserved regulatory enhancers responsible for Six6 expression. One of the enhancers drove gene expression in the differentiating and adult retina. A search for transcription factor binding sites, together with luciferase, ChIP assays and gain-of-function studies, indicated that NeuroD, a bHLH transcription factor, directly binds an 'E-box' sequence present in this enhancer and specifically regulates Six6 expression in the retina. NeuroD-induced Six6 overexpression in medaka embryos promoted unorganized retinal progenitor proliferation and, most notably, impaired photoreceptor differentiation, with no apparent changes in other retinal cell types. Conversely, Six6 gain-and loss-of-function changed NeuroD expression levels and altered the expression of the photoreceptor differentiation marker Rhodopsin. In addition, knockdown of Six6 interfered with amacrine cell generation. Together, these results indicate that Six6 and NeuroD control the expression of each other and their functions coordinate amacrine cell generation and photoreceptor terminal differentiation.

MiR-204 is responsible for inherited retinal dystrophy associated with ocular coloboma

“…Despite its pervasive role in many aspects of ocular differentiation, miR-204 has not previously been reported to be significantly expressed in photoreceptor cells (3,11,25,26,28) or to have an effect on photoreceptor function. Here, we provide for the first time to our knowledge RNA ISH data for miR-204 in the human retina, which also reveals its expression in photoreceptors (Fig.…”

“…As a negative control, a double-DIG-labeled LNA scrambled microRNA probe, included in the above kit, was hybridized in parallel at the same concentration and optimal hybridization temperature. Whole-mount RNA ISHs were performed, photographed, and sectioned (25-μm vibratome sections), as described by Conte et al (25). Digoxigenin-labeled anti-sense and sense riboprobes for olCrx, olPax6, olOtx2, and olRhodopsin were used (25).…”

“…Whole-mount RNA ISHs were performed, photographed, and sectioned (25-μm vibratome sections), as described by Conte et al (25). Digoxigenin-labeled anti-sense and sense riboprobes for olCrx, olPax6, olOtx2, and olRhodopsin were used (25). Medaka embryos were cryostat-sectioned (12-μm), and immuno-labeling was performed as previously described (25), using mouse monoclonal antibodies to Rhodopsin (1:2,000; Sigma) and Zpr-1 (1:2,000; ZIRC) and the rabbit polyclonal antibody to Prox1 (1:200; Chemicon).…”

“…Digoxigenin-labeled anti-sense and sense riboprobes for olCrx, olPax6, olOtx2, and olRhodopsin were used (25). Medaka embryos were cryostat-sectioned (12-μm), and immuno-labeling was performed as previously described (25), using mouse monoclonal antibodies to Rhodopsin (1:2,000; Sigma) and Zpr-1 (1:2,000; ZIRC) and the rabbit polyclonal antibody to Prox1 (1:200; Chemicon). Alexa-488-conjugated goat α-mouse and goat α-rabbit (1:1,000; Invitrogen) secondary antibodies were used.…”

“…To test the possible contribution of miR-204 in photoreceptor homeostasis, we therefore interfered with miR-204 processing and activity in medaka fish, using a multiblocking morpholino (Mo), as previously described (9,25), and assessed the consequences in the differentiated retina. Comparison of the morphology of the differentiated retina of control and Mo-miR-204-injected embryos revealed that morphant retinas displayed significant alteration in photoreceptor marker staining, which was more evident in ventral regions (Fig.…”