XB-ART-53973Neural Dev. September 1, 2017; 12 (1): 16.
Prdm13 forms a feedback loop with Ptf1a and is required for glycinergic amacrine cell genesis in the Xenopus Retina.
Amacrine interneurons that modulate synaptic plasticity between bipolar and ganglion cells constitute the most diverse cell type in the retina. Most are inhibitory neurons using either GABA or glycine as neurotransmitters. Although several transcription factors involved in amacrine cell fate determination have been identified, mechanisms underlying amacrine cell subtype specification remain to be further understood. The Prdm13 histone methyltransferase encoding gene is a target of the transcription factor Ptf1a, an essential regulator of inhibitory neuron cell fate in the retina. Here, we have deepened our knowledge on its interaction with Ptf1a and investigated its role in amacrine cell subtype determination in the developing Xenopus retina. We performed prdm13 gain and loss of function in Xenopus and assessed the impact on retinal cell fate determination using RT-qPCR, in situ hybridization and immunohistochemistry. We found that prdm13 in the amphibian Xenopus is expressed in few retinal progenitors and in about 40% of mature amacrine cells, predominantly in glycinergic ones. Clonal analysis in the retina reveals that prdm13 overexpression favours amacrine cell fate determination, with a bias towards glycinergic cells. Conversely, knockdown of prdm13 specifically inhibits glycinergic amacrine cell genesis. We also showed that, as in the neural tube, prdm13 is subjected to a negative autoregulation in the retina. Our data suggest that this is likely due to its ability to repress the expression of its inducer, ptf1a. Our results demonstrate that Prdm13, downstream of Ptf1a, acts as an important regulator of glycinergic amacrine subtype specification in the Xenopus retina. We also reveal that Prdm13 regulates ptf1a expression through a negative feedback loop.
PubMed ID: 28863786
PMC ID: PMC5580440
Article link: Neural Dev.
Genes referenced: gad1.1 gad1.2 neurog2 prdm13 ptf1a rbpj slc17a7 slc6a9
Morpholinos referenced: ptf1a MO3
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|Fig. 1. Prdm13 expression in the Xenopus retina. a Whole-mount in situ analysis of prdm13 expression during embryogenesis, shown as lateral views of the embryo heads or as transversal retinal sections at the indicated stages. At stage 42, the in situ hybridization has been performed on retinal section. The brown colour is the retinal pigment epithelium. Dotted lines delineate the ciliary marginal zone (CMZ). b Stage 28/30 sections following prdm13 in situ hybridization (red) and EdU incorporation assay (green). Below are enlargements of areas delineated with dotted lines. White arrows point to prdm13 + /Edu− cells while yellow arrows point to double labelled cells. c Schematic of a CMZ in the periphery of a Xenopus tadpole retina. d Stage 42 retinal section following prdm13 in situ hybridization (dark blue) and EdU incorporation assay (green). Since EdU labels cells that are in the S-phase during the 3-h EdU pulse, not all proliferative cells are labelled, in particular the slowly cycling stem cells. Panel on the right is an enlargement of the area delineated with dotted lines. Arrows point to double labelled cells. e Prdm13 fluorescent in situ hybridization (red) on stage 41 retinal section, counterstained with Hoechst to visualize nuclei (blue). Panel on the right is an enlargement of the white square. The white and yellow arrows point to prdm13 positive cells localized in the ganglion cell layer (GCL), and the outer part of the inner nuclear layer (INL), respectively. Dotted lines delineate the three nuclear layers. f Pie chart showing the distribution of prdm13 + cells among the ganglion cell layer (GCL, 1.3 ± 0.5%), the inner part of INL (87.8 ± 1.3%) and the outer part of the INL (10.9 ± 1.3%). Data are presented as mean ± SEM, n = 20 sections. g Quantification of the percentage of prdm13 positive cells among amacrine cells (defined by their localization in the inner part of the INL). Data are presented as mean ± SEM. Number of analysed sections is indicated in the bar. NR: Neural Retina, CMZ: ciliary marginal zone; RPE: Retinal Pigmented Epithelium, GCL: Ganglion Cell Layer, INL: Inner Nuclear Layer, ONL: Outer Nuclear layer. Scale bar represents 200 μm (whole mount), 100 μm (sections)|
|Fig. 2. Prdm13 is expressed in glycinergic- and GABAergic-amacrine cells. a Double in situ hybridization with prdm13 (red) and gad1 (green) probes on stage 39/40 retinal section. bIn situ hybridization with prdm13 probe (red) coupled with anti-Glycine immunostaining (green) on stage 41/42 retinal section. Arrows point to double labelled cells. Dotted lines separate the GCL and the INL. c Quantification of the percentages of GABA (gad1 +), Glycine (Gly) and Calretininin (Cal) amacrine cells among prdm13 + cells in the INL and in the GCL. Data are presented as mean ± SEM. Number of analysed sections is indicated in each bar. GCL: Ganglion Cell Layer, INL: Inner Nuclear Layer. Scale bar represents 25 μm|
|Fig. 3. Prdm13 overexpression promotes amacrine cells with a bias toward a glycinergic cell fate. a Illustration of the lipofection technique. i DNA is injected in the eye fields (green) of stage 18 embryos (frontal view). ii Retinas (green) are then sectioned (dashed line) at stage 41. iii Schema of a retinal section showing a clone of transfected cells (green) in the different retinal layers. iv Picture of a retinal section area (square in c) showing transfected cells (green). Nuclei are counterstained with Hoechst (blue). b Proportion of different retinal cell types in stage 41 prdm13 lipofected and control embryos. The table indicates the absolute numbers of counted cells for each cell type. c Double-immunostaining with anti-GFP and anti-Glycine or anti-GABA antibodies on retinal sections of prdm13 lipofected embryos. Arrows point to GFP positive cells that are Glycine or GABA-positive. d,e Quantification of GABA-positive and Glycine-positive cells among total GFP+ cells (d) or among GFP+ cells in the inner part of the INL where amacrine cells reside (e). Number of analysed retinas is indicated in each bar. GC: ganglion cells; AM: amacrine cells; BI: bipolar cells; HO: horizontal cells; PR: photoreceptor cells; MU: Müller cells. Values are given as mean ± SEM. * p-value <0,05; ** p-value <0,01; *** p-value <0,001 (Mann-Whitney test). Scale bar represents 50 μm|
|Fig. 4. Prdm13 loss of function leads to a decrease in glycinergic but not GABAergic-amacrine cells. a Whole-mount in situ hybridization analysis of prdm13, glyt1, gad1 and vglut1 expression on stage 39/40 embryos injected with prdm13-M0 or control-MO. Lateral views of the head and transversal sections of the retinas are shown. The number of analysed embryos and the percentage of embryos with represented phenotypes are indicated in each panel. b Stage 39/40 sections following GABA or Glycine-immunostaining on control-MO and prdm13-MO injected embryos. Arrows point to Glycine-positive cells. c Quantification of the average number of GABA- or Glycine-positive cells per section. Number of analysed sections is indicated in each bar. Data are presented as mean ± SEM. p < 0.001 (***) (Mann-Whitney test). Scale bars represent 200 μm (whole mount), 100 μm (sections)|
|Fig. 5. Prdm13 expression in the retina upon Ptf1a gain and loss of function. a Double fluorescent in situ hybridization with prdm13 (red) and ptf1a (green) probes on wild type retina at stage 35/36 and 40. Right panels are enlargement of central or peripheral retina (white squares). Arrows show double labelled cells. b Quantification of the percentage of ptf1a + cells among the prdm13 + cell population (top graph) and the percentage of prdm13 + cells among the ptf1a + cell population (bottom graph) at different stages of retinogenesis. Data are presented as mean ± SEM. Number of analysed sections is indicated in each bar. c Analysis of gad1, prdm13 and glyt1 expression on stage 41 retinal transversal sections following whole mount in situ hybridization on embryos injected with ptf1a-MO, control-MO, GFP mRNA (control) or ptf1a-GR mRNA. Dexamethasone was added at stage 21/22 to activate the Ptf1a-GR fusion protein. Scale bar represents 100 μm|
|Fig. 6. Prdm13 expression is deregulated upon Ptf1a gain of function. Whole-mount in situ hybridization analysis of prdm13 and gad1 expression in ptf1a-GR mRNA injected embryos treated with dexamethasone (Dex) and analysed at the indicated stages. Shown are lateral views of the embryos (a), of the head at higher magnification (b), and transversal sections of the retinas (c). The number of analysed embryos and percentage of embryos with represented phenotype are indicated in each panel. Scale bars represent 400 μm (a, b) or 100 μm (c)|
|Fig. 7. Prdm13 negatively regulates Ptf1a in a feedback loop. a RT-qPCR analysis of prdm13 and ptf1a expression in animal cap explants isolated from embryos injected with ptf1a-GR, mprdm13, lacZ mRNA and morpholinos as indicated, and collected when sibling embryos reach stage 26. Expression levels in non-injected caps have been set to 1. Shown are representative results of one out of two independent experiments. Data are presented as means of technical triplicates ± SD. b RT-qPCR analysis of ptf1a, prdm13 and neurog2 expression in stage 39/40 dissected eyes from control-MO or prdm13-MO injected embryos. Expression level in control caps has been set to 1. The graph represents a pool of 3 to 4 experiments. Data are presented as mean ± SEM. p < 0.05 (*) (Mann-Whitney test). c Drawing illustrating the interactions between ptf1a and prdm13 suggested by our results. As in the neural tube , we found that Ptf1a positively regulates prdm13 expression. It has previously been shown that Ptf1a binds, along with an E protein and Rbpj (PTF1-J complex), to a conserved 2.3 kb sequence located 13.4 kb 5′ to the ptf1a coding region and regulates its own transcription . We showed here that Prdm13 negatively regulates its own expression through a negative retro-control of ptf1a expression. The underlying mechanism remains to be investigated. Our results also do not exclude the possibility that Prdm13 could in addition directly repress its own expression|
|slc17a7 (solute carrier family 17 (vesicular glutamate transporter), member 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.|