Mazurier N et al. (2014), Ascl1 as a novel player in the Ptf1a transcript...

XB-ART-48702

PLoS One 2014 Mar 18;93:e92113. doi: 10.1371/journal.pone.0092113.

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Ascl1 as a novel player in the Ptf1a transcriptional network for GABAergic cell specification in the retina.

Mazurier N , Parain K , Parlier D , Pretto S , Hamdache J , Vernier P , Locker M , Bellefroid E , Perron M .

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In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.

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Species referenced: Xenopus laevis
Genes referenced: ascl1 atoh7 dpysl3 gad1.2 hand2 myc neurod1 neurog2 phox2a ptf1a slc17a7 th tubb2b
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	Figure 2. Ascl1 overexpression in the retina favours GABAergic cell genesis.Cell fate analysis following overexpression of the indicated construct by either mRNA injection in one blastomere at the four-cell stage and dex treatment at stage 16 (A, B; analysis at stage 38) or in vivo lipofection at the neurula stage (C, D; analysis at stage 41). In both cases, GFP was used as a tracer to visualize injected/transfected cells. Note that Ascl1, Neurog2 and Atoh7 lipofections all result in an increased percentage of cells in the ganglion cell layer but only Ascl1-overexpressing cells are biased towards a GABAergic destiny. Total number of analyzed retinas and counted cells per condition is indicated in each bar. Values are given as mean +/– s.e.m. p<0,001 (*), p<0,01 (), p<0,05 (*) (Student’s t-test). (E) shows a typical section of stage 41 retinas lipofected with GFP plus Ascl1 and immunostained with anti-GABA (red) and anti-GFP (green) antibodies. Panels on the right are higher magnifications of the dotted square delineated region. The dotted line indicates the inner plexiform layer, with the ganglion cell layer on the left and the inner nuclear layer on the right. White arrow points to a transfected GABA-positive cell within the ganglion cell layer. L: Lens. Scale bar represents 300 μm (heads) or 50 μm (sections).
	Figure 3. Comparison of GABAergic and glutamatergic inducing activities of five different bHLH factors.Whole mount in situ hybridization analyses of tubb2b, VGlut1 or gad1 expression on stage 24 embryos injected with the indicated mRNAs in one blastomere at the two-cell stage. (A) Arrows indicate ectopic expression in the epidermis (anterior on the left, dorsal side up). All the tested bHLH factors exhibit neurogenic activity, as inferred from tubb2b ectopic expression. However, only Ascl1 specifically induces gad1+ neurons without inducing VGlut1+ ones. (B, C) Quantification of embryos displaying VGlut1 (B) or gad1 (C) ectopic expression. Ascl1 interferes with Ptf1a-, Neurog2-, NeuroD1- and Atoh7-dependent production of VGlut1+ neurons, while none of these factors affect Ascl1 GABAergic inducing activity. (D) Quantification of embryos with weak or strong ectopic gad1 staining following mRNA injection of Ascl1 alone or together with Neurog2. Note that Neurog2 enhances Ascl1 GABAergic inducing activity. Total number of analyzed embryos per condition is indicated in each bar. Error bars represent 95% confidence intervals. p<0,001 (*), p<0,01 (), p<0,05 (*) (binomial test). Scale bar represents 500 μm.
	Figure 4. Ascl1 overexpression together with Neurog2 still promotes production of GABAergic cells in the retina.(A) Stage 41 retinal sections immunostained with anti-GABA (red) and anti-GFP (green) antibodies. Embryos were injected with the indicated mRNAs in one blastomere at the four-cell stage and treated with dexamethasone at stage 16. Panels on the right are higher magnifications of the dotted square delineated region. The dotted line indicates the inner plexiform layer. Arrowheads point to GFP/GABA-positive cells within the ganglion cell layer. (B) Quantification of GABAergic cell proportion among GFP+ cells in the ganglion cell layer, showing that Ascl1 GABAergic inducing activity is not affected by Neurog2 misexpression. Total number of analyzed retinas and counted cells per condition is indicated in each bar. p<0,05 (*) (Student’s t-test). Values are given +/– s.e.m. L: Lens. Scale bar represents 50 μm.
	Figure 5. Comparison of the catecholaminergic inducing activities of five different bHLH factors.(A) Whole mount in situ hybridization analysis of Tyrosine Hydroxylase (TH) expression on stage 24 embryos injected with the indicated mRNAs in one blastomere at the two-cell stage (anterior on the left, dorsal side up). Arrowheads indicate the position of previously described TH-positive antero-ventral neurons [53], while arrows point to ectopic TH staining. Note that, among all tested bHLH factors, only Ascl1 induces ectopic TH expression. Sibling embryos were also hybridized with the tubb2b probe as a positive control (see Figure 3A). (B) Quantification of embryos displaying ectopic TH+ neurons, showing that Neurog2 and Ptf1a significantly reduce the catecholaminergic inducing activity of Ascl1. Total number of analyzed embryos per condition is indicated in each bar. Error bars represent 95% confidence intervals. p<0,05 (*) (binomial test). Scale bar represents 500 μm.
	Figure 6. Mapping of Ascl1 and Neurog2 domains accounting for their functional specificity in neuronal subtype specification.(A) bHLH protein sequences of the Myc-tagged inducible Ascl1:Neurog2 chimeric constructs used in B and C. Ascl1 and Neurog2 sequences are depicted in red and black, respectively. Shown on the right and illustrated in (C) is the ability of the different proteins to induce dpysl3, VGlut1, gad1 or TH. Percentages of embryos with ectopic expression are indicated. (B, C) Embryos were injected in one blastomere at the four-cell stage with the indicated wild-type or chimeric mRNA and dexamethasone was added at stage 13. (B) Western-blot using an anti-Myc antibody showing that each chimeric protein has been produced. Detection of GAPDH serves as a loading control. (C) Whole mount in situ hybridization analyses of dpysl3, VGlut1, gad1 or TH expression on stage 22 or 28 injected embryos (anterior on the right, dorsal side up). Altogether, these domain-swapping experiments show that the GABAergic inducing activity of Ascl1 resides in its basic domain, while the glutamatergic inducing activity of Neurog2 is imparted by its HLH domain. In each panel the number of analysed embryos is indicated. Scale bar represents 250 μm.
	Figure 7. Retinal GABAergic inducing activity of Ascl1 resides in its basic domain.(A) Stage 41 retinal sections immunostained with anti-GABA (red) and anti-GFP (green) antibodies. Embryos were lipofected with Ascl1 or the inducible ABN chimeric construct (encoding a Neurog2 protein with the basic domain of AsclI). Dexamethasone was added immediately after lipofection at stage 18. Panels on the right are higher magnifications of the dotted square delineated region. The dotted line indicates the inner plexiform layer. Arrowheads indicate GABA+/GFP+ cells within the ganglion cell layer. (B-C) Quantification of GFP+ cells and GABA+/GFP+ in the ganglion cell layer showing that similarly to Ascl1, ABN increases the percentage of cells in the ganglion cell layer but also biases these cells towards a GABAergic destiny. Total number of analyzed retinas and counted cells per condition is indicated in each bar. Values are given as mean +/– s.e.m. p<0,01 (*), p<0,05 () (Student’s t-test). L: Lens. Scale bar represents 50 μm.
	Figure 8. Distinct transcriptional networks sustain Ascl1 GABAergic and catecholaminergic inducing activities.Whole mount in situ hybridization analyses of TH or gad1 expression on stage 24 embryos following injection of mRNA and/or morpholinos (Mo), as indicated, in one blastomere at the two-cell stage. (A, C) Arrows point to ectopic TH or gad1 expression (anterior on the left, dorsal side up). Sibling embryos were also hybridized with the tubb2b probe as a positive control (data not shown). (B, D) Quantification of embryos displaying ectopic TH or gad1 expression. Note that Phox2a and Hand2 knockdown significantly reduces Ascl1 ability to induce TH expression but does not impair its GABAergic inducing activity (B), which specifically depends on Ptf1a (D). Total number of analyzed embryos per condition is indicated in each bar. Error bars represent 95% confidence intervals. P<0,001 (*), p<0,01 () (binomial test). Scale bar represents 500 μm.
	Figure 9. Ptf1a is a direct transcriptional target of Ascl1.(A) Whole mount in situ hybridization analyses of Ptf1a expression on stage 22 embryos injected with Ascl1 or Neurog2 mRNA in one blastomere at the two-cell stage. Note that Ascl1 is able to activate Ptf1a expression in the epidermis (arrow). Sibling embryos were also hybridized with the tubb2b probe as a positive control (data not shown). (B) Quantification of embryos displaying Ptf1a ectopic expression. Total number of analysed embryos per condition is indicated in each bar. Error bars represent 95% confidence intervals. P<0,001 (***) (binomial test). (C, D) RT-qPCR analyses of tubb2b and Ptf1a expression in embryos injected with Ascl1-GR at the two-cell stage and treated with dexamethasone (dex) and/or cycloheximide (CHX) from stage 19 to stage 21. Note that Ptf1a up-regulation upon dex treatment is maintained in the presence of CHX. Scale bar represents 500 μm.
	Figure 10. Comparison of Ascl1 and Ptf1a expression patterns during retinogenesis.(A, B) Whole mount in situ hybridization analysis of Ascl1 and Ptf1a expression during retinogenesis. Shown are representative pictures of the observed labelling in the head region (A; lateral views, anterior on the left) and on retinal cross sections (B; dorsal side up). Ascl1 and Ptf1a are expressed in overlapping domains of the neural retina at stage 30 and in the ciliary marginal zone (CMZ, delineated by doted line) from stage 35 onwards. (C) Double fluorescent in situ hybridization against Ptf1a (green) and Ascl1 (red) performed on stage 40 retinal sections. Ascl1 staining in the CMZ is delineated by a white doted line. Panel on the right shows a magnification of the dorsal CMZ. Neither Ascl1 nor Ptf1a are detected in zone 1 (stem cell compartment). In zone 2 (early progenitors), only Ascl1 is expressed. The expression patterns of both genes overlap in zone 3 (late progenitors; yellow doted line). In zone 4 (postmitotic retinoblasts), Ascl1 expression vanishes while that of Ptf1a persists. OP: Optic vesicle, L: Lens. Scale bar represents 300 μm (A) or 50 μm (B, C).
	Figure 11. Ptf1a overexpression rescues Ascl1 knockdown in the retina.(A) Whole mount in situ hybridization analysis of gad 1 expression at stage 37. Embryos were injected with the indicated mRNAs and/or morpholinos (Mo) in two blastomeres at the two-cell stage and treated with dexamethasone at stage 22. Shown are representative pictures of the observed labelling in the head region (lateral views, anterior on the left) and on retinal sections (dorsal side up). (B) Quantification of gad1 staining intensity in the eye of injected embryos. Results are presented as percentage increase/decrease relative to the average intensity found in controls. Total number of analyzed embryos per condition is indicated in each bar. Values are given as mean +/– s.e.m. p<0,001 (***) (Student’s t-test); N.S. : Non significant; L: Lens. Scale bar represents 300 μm (heads) or 50 μm (sections).
	Figure 12. Summary of Ascl1 genetic network in neuronal subtype specification.Green, red and blue arrows point to neuronal subtypes induced by Neurog2 and Ascl1. Identified downstream components required for their respective activities are indicated. Grey arrows illustrate epistatic relationships between Neurog2 and Ascl1, as suggested by our results. Protein domains in red confer to Ascl1 and Neurog2 their specific GABAergic/catecholaminergic and glutamatergic activities, respectively (see text for more details).
	Figure 1. Ascl1 is required for retinal GABAergic cell genesis.Whole mount in situ hybridization on stage 35 embryos following Ascl1 or control morpholino (Mo) injection in one blastomere at the two-cell stage. Shown for each indicated probe are representative pictures of the observed labelling in the head region (lateral views, anterior on the left) and on retinal cross sections (dorsal side up). The expression of the pan-neuronal marker Bru (A, B) is not affected by Ascl1 knockdown while gad1 and Ptf1a stainings are severely reduced (C-F). VGlut1 expression (G, H) is unequally affected in the different cell layers: decreased in the photoreceptor layer (white arrow) and extended in the inner nuclear layer (white arrowhead). L: Lens. Scale bar represents 300 μm (heads) or 50 μm (sections).

References [+] :

Adler, Have we achieved a unified model of photoreceptor cell fate specification in vertebrates? 2008, Pubmed, Xenbase