Epitope‐tagging Math5 and Pou4f2: New tools to study retinal ganglion cell development in the mouse

Abstract

Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5HA and Pou4f2HA alleles. We demonstrate that the tagged alleles recapitulated the wild-type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co-staining, we found that Math5 and Pou4f2 were transiently co-expressed in newly-born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope-tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development.

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

“…Consistent with this notion, most Pou4f2/Isl1-expressing cells in the NBL (>80%) were Atoh7 + , as indicated by colabeling of Pou4f2 and HA in the Atoh7 tTA/HA ;tetO-P&I retina (Fig. S1 E-G) in which Atoh7 expression was marked by an HA tag (10). However, many Atoh7-expressing cells did not express Pou4f2, indicating a lapse between the expression of tTA from the Atoh7 promoter and the activation of the tetO-P&I transgene.…”

“…S1D). Because Atoh7 is active in a larger population of cells than Pou4f2 and Isl1 in the NBL of wild-type retinas (10), the increased number of Pou4f2-and Isl1-expressing cells indicated that tTA from the Atoh7 tTA allele activated the tetO-P&I transgene and thus produced Pou4f2 and Isl1 in RPCs that normally express Atoh7. Consistent with this notion, most Pou4f2/Isl1-expressing cells in the NBL (>80%) were Atoh7 + , as indicated by colabeling of Pou4f2 and HA in the Atoh7 tTA/HA ;tetO-P&I retina (Fig.…”

“…Both are downstream of Atoh7 in the gene-regulatory network of RGC development and have essentially identical retinal expression patterns at the early stages of development (22)(23)(24)(25). More importantly, they are transiently coexpressed with Atoh7 in newly formed RGCs and are the earliest known transcription factors expressed specifically in developing RGCs (10,17). Thus, the initiation of expression of these two factors appears to coincide with RGC fate commitment.…”

“…Many such transcription factors have been identified by loss-and gain-of-function analyses, but these studies often fail to reveal the specific roles these factors play in the development of the cell types with which they are involved (7)(8)(9). RPCs are heterogeneous, as has been demonstrated by the nonuniform expression of many RPC genes (10)(11)(12)(13). RPCs expressing specific genes, particularly those encoding transcription factors, although still multipotent, tend to be biased for certain retinal cell types.…”

Onecut1 Is Essential for Horizontal Cell Genesis and Retinal Integrity

“…The floxed Oc1 allele (Oc1 flox ) and the Six3-cre transgenic line have been described previously (Furuta et al, 2000;Zhang et al, 2009). The Ptf1a Ϫ (Ptf1a cre ) mice (Fujitani et al, 2006) were obtained from MMRRC-UNC (Stock number: 000435-UNC). All lines were maintained in C57BL/6ϫ129 mixed background.…”