Regulation of Brn3b by Dlx1 and Dlx2 is required for retinal ganglion cell differentiation in the vertebrate retina

Abstract

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo. We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.

Neurogenesis and Specification of Retinal Ganglion Cells

“…Dlx1 and Dlx2 were also identified as transcriptional activators of Pou4f2 expression ( Figure 2) supported by in utero retinal electroporation of Dlx2 and siRNA-mediated knockdown of Dlx2 in primary embryonic retinal cultures [119]. Furthermore, dlx1/dlx2/pou4f2 triple knockout mice show defects that are stronger than the combined effect of pou4f2-KO and dlx1/dlx2 −/− , with an almost complete loss of RGCs and a marked increase in amacrine cells in the ganglion cell layer (GCL) [120]. Therefore, the regulation of Pou4f2 by Dlx1 and Dlx2 is required for RGC differentiation in the vertebrate retina [120].…”

“…Moreover, in vitro re-aggregation experiments showed that this miRNA function is cell-autonomous [135]. In the next RGC differentiation step, Pou4f2 (Brn-3b) is necessary for the terminal differentiation and survival of most RGCs [120]. This TF seems to be down regulated by miR-23a and miR-374 [137].…”

De novo genesis of retinal ganglion cells by targeted expression of Klf4 in vivo

“…Atoh7 and Eya2, both of which were upregulated upon Klf4 overexpression, have been described as necessary for Brn3b expression (Gao et al, 2014;Wang et al, 2001) and the latter together with Isl1 participates in essential steps of ganglion cell differentiation, such as specification, maturation, axonal projection and survival (Gan et al, 1999;Mu et al, 2008;Qiu et al, 2008;Wang et al, 2000;Wu et al, 2015). Recently, Dlx1 and Dlx2 were also described as crucial for the differentiation of RGCs downstream of Atoh7, both in parallel and cooperating with Brn3b (Zhang et al, 2017). Neither of those transcription factors were upregulated in response to Klf4 overexpression.…”

De novogenesis of retinal ganglion cells by targeted expression of KLF4in vivo

“…Atoh7 and Eya2, both of which were upregulated upon Klf4 overexpression, were described as necessary for Brn3b expression (Wang et al, 2001; Gao et al, 2014) and the latter together with Isl1 participate in essential steps of ganglion cell differentiation, such as specification, maturation, axonal projection and survival (Gan et al, 1999; Wang et al, 2000; Mu et al, 2008; Qiu et al, 2008; Wu et al, 2015). Recently, Dlx1 and Dlx2 were also described as critical for the differentiation of RGCs downstream of Atoh7 and both in parallel as well as cooperating with Brn3b (Zhang et al, 2017). Neither of those transcription factors were upregulated in response to Klf4 overexpression.…”