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Proc Biol Sci
2016 Mar 16;2831826:20152978. doi: 10.1098/rspb.2015.2978.
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Substituting mouse transcription factor Pou4f2 with a sea urchin orthologue restores retinal ganglion cell development.
Mao CA
,
Agca C
,
Mocko-Strand JA
,
Wang J
,
Ullrich-Lüter E
,
Pan P
,
Wang SW
,
Arnone MI
,
Frishman LJ
,
Klein WH
.
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Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures.
Figure 1. Sequence alignment of SpPou4f1/2 and Pou4f and generation of the Pou4f2SpPou4f1/2 KI allele. (a) Generation of Pou4f2SpPou4f1/2 KI allele, FRT indicates FLP recombinase sites to remove the Neo selection cassette (brown box) by a Rosa26-FLPeR mouse line. SpPou4f1/2 cDNA sequences (green box) were fused in frame to a HA-epitope tag (yellow box). The dark purple thick lines indicate the site of recombination into a construct carrying the Pou4f2 coding region and upstream regulatory regon. TK (orange box) indicates the TK cassette used for negative selection. The BamHI site was used for Southern genotyping, p1 and p2 are PCR primers to detect wild-type mouse Pou4f2 allele, and p3 and p4 are primers to detect knock in SpPou4f1/2 allele. (b) Phylogeny analysis of SpPou4f1/2 and Pou4f genes from other organisms. The tree was constructed with MEGA software v. 5.2. using neighbour-joining method with 1000 bootstrap repeats. Ci, Ciona intestinalis; Dr, Danio rerio; Mm, Mus musculus; Sp, Strogylocentrotus purpuratus; Xt, Xenopus tropicalis.
Figure 3. Normal RGC differentiation programme in Pou4f2SpPou4f1/2/Z retinas. TUNEL assay shows lesser cell death in retinas of Pou4f2Z/+ (a), Pou4f2SpPou4f1/2/Z (b) compared with Pou4f2Z/Z (c). SpPou4f1/2 activates Pou4f2 downstream RGC genes Pou4f1 and Tbr2 in the absence of Pou4f2 (d–i). Immunostaining of retina from E15 embryos with anti-Pou4f1/Brn3a (d–f) and anti-Tbr2 (g–i).
Figure 4. ERG recordings of Pou4f2SpPou4f1/2/SpPou4f1/2 retinas. (a) Scotopic full-field flash ERG responses recorded from one mouse in each of the three genotypes. From left to right, Pou4f2+/+ (+/+), Pou4f2SpPou4f1/2/SpPou4f1/2 (SP/SP), Pou4f2−/− (−/−). Stimulus strength increases from bottom to top. Arrows in the right column indicated missing STRs in this animal. (b–d) Stimulus versus ERG amplitude plots measured for the three genotypes. Pou4f2+/+(+/+; n = 4), Pou4f2SpPou4f1/2/SpPou4f1/2 (Sp/Sp; n = 4), Pou4f2−/− (−/−; n = 4). (b) pSTR (box) and b-wave amplitudes. (c) nSTR amplitudes and (d) a-wave amplitudes. The nSTR amplitudes saturated around −4.1 log sc cd-s m−2, and then a larger negative wave of unknown origin emerged. The error bars are standard errors.
Figure 5. Co-expression analysis of SpPou4f1/2, SpPax6 and SpOpsin4 in S. purpuratus tube feet. (a) Schematic view of a sea urchin tube feet displaying the morphology of tube feet stalk (tfs), disc (tfd) and rosette (ros); the nervous system (green) is represented by tube feet nerve (tfn), ganglion (ga) and disc ring nerve (drn). Sp-Opsin4 positive photoreceptor cells (red) are located at the rim of the disc and in a depression of the skeleton (ske) at the base of the tube feet. (b,c,e,f,g,h) Z-stack projection of tube feet disc. (b) Bottom view (seen from the stalk), (c,e,f,g,h) top view, showing SpPou4f1/2 and SpPax6 mRNA expression and Sp-Opsin4 protein in disc photoreceptors. The whole tube feet disc is shown in (b) while (c), (e–g) display only a quadrant of it. SpPou4f1/2 mRNA is shown in yellow/green (b) or green (c,f–h), SpPax6 mRNA in red (b) or purple (c), SpOpsin4 protein in red (c,e,g and h). (b) SpPou4f1/2 mRNA expression associated with skeletal rosettes, SpPax6 mRNA in stalk and disc. (b′ and b″) are details of (b) as indicated. (c) Expression of SpPou4f1/2 corresponds to neuronal tissue and axonal projection area of photoreceptors. SpPax6 expression in the area between skeletal rosette elements correlates with localization of photoreceptor cell bodies and SpOpsin4 protein in the apical part of the photoreceptor cells. (d) Three-dimensional reconstruction of serial TEM sections clarifies cell position and identity in SpPou4f1/2, SpPax6 and SpOpsin4 expression regions. Skeletal rosette (grey) with associated nerve tissue (yellow) and two photoreceptors (purple and blue). (e–g) Different overlays showing coexpression of SpPou4f1/2 mRNA and SpOpsin4 protein in the apical region of photoreceptors. (h) A twofold zoom of the area indicated by the rectangle in (e–g) showing a single photoreceptor (arrow shows nucleus).
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