Jaurena MB et al. (2015), Zic1 controls placode progenitor formation non-...

XB-ART-53288

Nat Commun 2015 Jun 23;6:7476. doi: 10.1038/ncomms8476.

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Zic1 controls placode progenitor formation non-cell autonomously by regulating retinoic acid production and transport.

Jaurena MB , Juraver-Geslin H , Devotta A , Saint-Jeannet JP .

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All cranial placode progenitors arise from a common precursor field anterior to the neural plate, the pre-placodal region (PPR). We showed that transcription factor Zic1, expressed at the anterior neural plate, is necessary and sufficient to promote placode fate. Here we reveal the non-cell autonomous activity of Zic1 and implicate retinoic acid (RA) signalling as a key player in cranial placode progenitor specification. In a screen for genes activated by Zic1, we identify several factors involved in RA metabolism and function. Among them we show that retinaldehyde dehydrogenase 2 (RALDH2) and lipocalin-type prostaglandin D2 synthase (LPGDS), which, respectively, regulate the synthesis and transport of RA, directly participate in the establishment of the PPR. We propose that RALDH2 and LPGDS induction by Zic1 at the anterior neural plate allows for the localized production and transport of RA, which in turn activates a cranial placode developmental programme in neighbouring cells.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 eya1 foxi4 hnf1b odc1 ptgds rab40b six1 zic1

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	Figure 2. LPGDS is required for placode development(a) Increasing amounts of LPGDS-MO 10 ng (+), 100 ng (++), and 1000 ng (+++) blocks translation directed by LPGDS mRNA in an in vitro coupled transcription/translation reaction. The position of markers of known molecular weight (kDa) is indicated. (b) In situ hybridization for pan-placodal and (c) placode specific genes in control and LPGDS-MO injected embryos (frontal views, dorsal to top). Arrowheads indicate reduced expression on the injected side. (d) Quantification of the results. Four independent experiments were performed. The number of embryos analyzed (n) is indicated on the top of each bar. (e) Amino acid sequences alignment showing the conserved cysteine residue (Cyst65), center of LPGDS enzymatic activity. (f) Foxi1c and Six1 expression domains are rescued in LPGDS-MO-injected embryos by co-injection of either WT or C65A mouse LPGDS mRNA. Frontal views, dorsal to top; injected side is indicated by the lineage tracer (Red-Gal). (g) Quantification of the rescue experiment. Three independent experiments were performed. The number of embryos analyzed (n) is indicated on the top of each bar. LPGDS-MO vs. LPGDS-MO+WT or LPGDS-MO+C65A mRNA injected embryos (p<0.001, Fisher exact test.); LPGDS-MO+WT mRNA vs. LPGDS-MO+C65A mRNA injected embryos show no significant differences. Scale bars, 200 μm.
	Figure 3. Retinoic acid affects PPR formation in a dose dependent manner(a) Treatment of intact embryos at stage 11 with increasing doses of RA (0.01 μM, 0.1 μM, 1 μM and 10 μM) disrupts Fox1ic and Six1 expression at the PPR. DMSO was used as a control. Frontal views, dorsal to top. (b) Quantification of the results. Four independent experiments were performed. The number of embryos analyzed for each condition (n) is indicated on the top of each bar. Scale bars, 200 μm.
	Figure 4. Retinoic acid signaling regulates placode formation(a) Treatment at stage 11 of LPGDS-depleted embryos (LPGDS-MO) with 0.1 μM RA or with the RA receptor agonist, TTNPB, restored completely or partially Foxi1c and Six1 expression on the injected side. Frontal views, dorsal to top. Injected side is indicated by the lineage tracer (Red-Gal). Double arrowheads indicate Foxi1c or Six1 expansion on the control side. (b) Quantification of the rescue experiment. Four independent experiments were performed. The number of embryos analyzed for each condition (n) is indicated on the top of each bar. DMSO vs. RA or TTNPB treated embryos (p< 0.001, Fisher exact test) RA vs. TTNPB treated embryos show no significant differences. (c) Fold induction of RALDH2 from the microarray data. (d) By in situ hybridization RALDH2 is detected in the trunk mesoderm (dorsal view, anterior to right) and at the anterior neural plate (frontal view; arrowheads). (e) RALDH2 expression is lost in Zic1-depleted embryos. The graph is a quantification of the results. Three independent experiments were performed. The number of embryos analyzed (n) is indicated on the top of each bar. (f) Foxi1c expression is reduced in RALDH2-MO injected embryos. The graph is a quantification of the results. Three independent experiments were performed. The number of embryos analyzed (n) is indicated on the top of each bar. (e–f) Arrowheads indicate reduced expression on the injected side. Scale bars, 200 μm.
	Figure 5. Zic1 regulates placode formation non-cell autonomously(a) Double in situ hybridization for Zic1/Foxi1c, Foxi1c/LPGDS and Foxi1c/RALDH2 showing that Zic1, LPGDS and RALDH2 are expressed at a distance from the PPR (Foxi1c-expressing cells). Frontal views, dorsal to top. Scale bars, 200 μm. (b) Animal explants dissected from embryos injected in one blastomere at the 2-cell stage with Zic1GR and GFP mRNAs were cultured for 8 hours in dexamethasone and analyzed by In situ hybridization. (c) In situ hybridization (ISH) for Foxi1c, Six1, LPGDS and RALDH2 on sections of animal explants derived from embryos injected with Zic1GR and GFP mRNA in one blastomere at the 2-cell stage (left panels). In each case the Zic1GR-expressing cells (GFP-positive) are shown (middle panels). Merge of fluorescence and in situ hybridization images (right panels). Three independent experiments were performed with similar results for each probe as pictured (Foxi1c, n=14; Six1, n=13; LPGDS, n=13 and RALDH2, n=11). Scale bars, 100 μm.
	Figure 6. Zic1 regulates placode fate independently of canonical RA receptors(a) Animal explants dissected from embryos injected in one blastomere at the 2-cell stage with Zic1GR mRNA and cultured for 8 hours in dexamethasone, with or without the pharmacological inhibitors Disulfiram (100 μM), Citral (100 μM) or AGN193109 (10 μM). (b) RT-PCR analysis of Six1 and Eya1 expression in animal explants expressing Zic1GR treated with Disulfiram or Citral. Odc (Ornithine decarboxylase) is shown as a loading control. Controls are uninjected (Uninj) and GR mRNA injected (GR) animal explants. Similar results were obtained in four independent experiments for each inhibitor. The position of markers of known size is indicated (bp). (c) RT-PCR analysis of Six1 and Eya1 expression in Zic1GR injected animal explants treated with the pan-RAR antagonist, AGN193109. Controls are uninjected (Uninj), GR mRNA injected (GR) and Zic1GR mRNA injected treated with DMSO (+DMSO) animal explants. Similar results were obtained in six independent experiments. The position of markers of known size is indicated (bp). (d) AGN193109 treatment blocks Hnf1b expression in the posterior hindbrain (100% of the embryos; n=94), while in DMSO-treated embryos Hnf1b expression is similar to that of control embryos (100% of the embryos; n=52 and n=37, respectively). Three independent experiments were performed. Stage 13 embryos, dorsal views, anterior to top. Scale bars, 200 μm.
	Figure 7. Model for the regulation of PPR formation by Zic1 and RA signalingZic1 controls RA signaling at the anterior neural plate through the activation of RALDH2 to produce RA, and LPGDS to transport RA extracellularly. As a consequence RA induces the expression of Six1, Eya1 and Foxi1c in neighboring cells (PPR).

References [+] :

Ahrens, Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. 2005, Pubmed, Xenbase