Proper differentiation of photoreceptors and amacrine cells depends on a regulatory loop between NeuroD and Six6.
Timely 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.
Conte
Ivan
I
Instituto Cajal, CSIC and CIBER de Enfermedades Raras, Madrid, Spain.
Marco-Ferreres
Raquel
R
Beccari
Leonardo
L
Cisneros
Elsa
E
Ruiz
José María
JM
Tabanera
Noemí
N
Bovolenta
Paola
P
D025042:Amacrine Cells / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D015870:Gene Expression; D009474:Neurons / Q000378:metabolism; D009990:Oryzias / Q000235:genetics / Q000378:metabolism; D010786:Photoreceptor Cells; D012160:Retina / Q000166:cytology / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D012243:Rhodopsin; D014157:Transcription Factors