Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

Abstract

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.

iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

“…We cloned Dam-f-GFP using promoters that included ubiquitin (Mosimann et al, 2011), heat shock (Blechinger et al, 2002) and Rx2 (Reinhardt et al, 2015) in plasmids carrying transgenesis markers such as Cmlc2:GFP or RFP. Surprisingly, none of the Dam-f-GFP constructs showed GFP expression, whereas the unfused GFP construct did (Fig.…”

“…Although Rx2 was provided as mRNA and was therefore expressed in the whole embryo, a careful inspection of the regions of Rx2 enrichment revealed many players known to be involved in retinal development, including Six3 (Loosli et al, 1998), Otx2 (Zuber et al, 2003), Pax6 (Loosli et al, 1998) and Sox2 (Reinhardt et al, 2015). Interestingly, we also found enrichment of Rx2 on its own proximal upstream locus (…”

“…Immunostainings were performed on 16 μm cryosections as previously described (Inoue and Wittbrodt, 2011) using the primary antibodies rabbit anti-OlRx2 (1:250; Reinhardt et al, 2015) and chicken anti-GFP (1:500, A10262, Thermo Fisher Scientific), and the secondary antibodies goat anti-rabbit IgG Alexa Fluor 647 (1:1000, A-21245, Thermo Fisher Scientific) and goat anti-chicken IgY Alexa Fluor 488 (1:1000, A-11039, Thermo Fisher Scientific).…”

The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages

“…It has been reported that Müller glia cells can function as late-stage retinal progenitors of the photoreceptor lineage, with their cell bodies occasionally migrating to the horizontal cell layer to divide asymmetrically, generating one photoreceptor and one Müller glial cell [12]. To assess the existence of such Müller glial cell divisions, we carried out immunolabelling with Rx2, which allows labelling cell bodies of photoreceptor as well as Müller glial precursors of the INL (Fig 2F and 2G [30]). Some Rx2 immunoreactive cells could be indeed observed with their cell bodies located sub-apically, between the outer limiting membrane and the horizontal cell layer.…”

Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development

“…In a cancer cell line, MID1 marks Fu by ubiquitination for subsequent cleavage leading to the cytosolic retention of GLI3 (31) and thus blocks Hh signaling. In the eye, gli3 was shown to interact with other transcription factors to define retinal stem cells and gli3 may cooperate with pax6 during eye morphogenesis (42,43). Thus, a feedback loop may exist, which regulates a balance between mid1 expression with medium levels of Shh that induce mid1 and lower levels that suppress mid1 expression.…”