Onecut1 and Onecut2 redundantly regulate early retinal cell fates during development

Abstract

Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general.transcription factors | neural development | retinal development | cell fate determination | gene regulation

Photoreceptor cell fate specification in vertebrates

“…It was also shown that Onecut1 and Otx2 synergize to activate Thrb2 expression, suggesting that the combinatorial action of these two factors is necessary for the acquisition of cone potential or cone fate commitment. In mice that lack Onecut1 and its similarly expressed paralog Onecut2, there is an initial lack of Rxrγ-expressing cones, but the number of cones is only modestly reduced by the end of development (Sapkota et al, 2014). In addition, nearly all cones in these mutants express high levels of S-opsin and few cones express M-opsin, similar to Thrb2 mutant mice.…”

Two transcription factors, Pou4f2 and Isl1, are sufficient to specify the retinal ganglion cell fate

“…Immunofluorescence staining followed a protocol we have described previously (15,66,67). Briefly, after fixation with 4% paraformaldehyde for 30 min and three washings with PBS (pH7.4) plus 0.1% Tween 20 (PBST), embryos (E14.5) or eyes (P0 and older) were embedded in Optimum Cutting Temperature (OCT) compound.…”

“…For whole-mount staining, the same general procedure was followed, except that the incubation time and washing time were considerably longer. Sources of all antibodies and their dilutions also have been published previously (15,66,67). Counting of positive cells was performed as described previously (66), and the significance of difference (P < 0.1) between various genotypes and the control was determined by Student's t test.…”

“…In situ hybridization followed a protocol described previously (15,66). The tissues were fixed in 4% (wt/vol) paraformaldehyde overnight, embedded in OCT, and sectioned at 16 μm.…”

“…Total RNA isolation from retinal tissues and reverse transcription reaction were carried out as described previously (66). cDNA equivalent to 40 ng of total RNA then was used as template for each PCR.…”