Plouhinec JL et al. (2014), Pax3 and Zic1 trigger the early neural crest ge...

XB-ART-48413

Dev Biol 2014 Feb 15;3862:461-72. doi: 10.1016/j.ydbio.2013.12.010.

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Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers.

Plouhinec JL , Roche DD , Pegoraro C , Figueiredo AL , Maczkowiak F , Brunet LJ , Milet C , Vert JP , Pollet N , Harland RM , Monsoro-Burq AH .

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Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

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Species referenced: Xenopus laevis
Genes referenced: acod1lb acta1 angpt4 anos1 axin2 bnip3 btc c3 capn8.1 col18a1 cyp26c1 dact1 dynll1 elavl3 ets1 fgfr4 fkbp9 foxd3 fst fzd10 gbx2.1 gbx2.2 glipr2 greb1l guk1 hapln3 hoxa2 hsp90aa1.1 irf1 irx1 irx2 irx3 lmx1b.1 mafb meis3 mfap2 mmp28 msx1 msx2 myo10.2 myo1d nipal2 nrp1 olig4 pax3 pcdh8l pdgfra peli1 pfkfb4 phyhdlb plekhn1 pnhd prtg pts rab11fip4l rara ror2 sdhb snai1 snai2 sox10 sox9 tcf7 tenm4 tfap2a tfap2b tpbg tuba1cl.3 twist1 wls wnt1 wnt11b wnt8a zfp36l2 zic1

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GSE53678: NCBI
GSE53679: NCBI

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	Fig. 1. Identification of the premigratory neural crest transcriptome signature during neurulation (A) Several types of early embryo explants were dissected for microarray analysis: the animal cap ectoderm, cut at blastula stage 9 and allowed to develop until stage 14 in vitro (AC14), the early neural plate at stage 12 (NP12), the lateral neural border at stage 14 (NB14), the premigratory cranial neural crest and its overlying ectoderm at stage 18 (NB18), and the anterior neural fold at stage 18 (ANF18). Expression level thresholding, differential analysis, and clustering defined a group of 83 genes enriched in neural border samples. (B) Outline of the experimental strategy used to identify the neural crest transcriptome signature.
	Fig. 2. Developmental expression of a sample of novel genes belonging to the neural border/crest signature Pax3 and snail2 serve as markers for the lateral neural border and the premigratory neural crest respectively. Novel markers, identified in the neural border/neural crest signature, are shown at the most significant stages. (A-M,O-T) Dorsal views, with anterior to the bottom and posterior to the top. (N) Side view.
	Fig. 3. Pax3 and Pax3/Zic1 immediate early target genes in the neural crest signature (A) Experimental strategy used to identify Pax3 and Pax3/Zic1 immediate early target genes. (B) List of direct targets belonging to the neural crest signature. (C) Independent validation of a subset of targets by quantitative PCR. Embryos were injected as indicated, either with Pax3GR mRNA, or Zic1GR mRNA, or a combination of both. The animal cap ectoderm was treated with cycloheximide and dexamethasone as described in the text. (D) In vivo validation of the novel Pax3/Zic1 target genes using by either Pax3 or Zic1 knockdown at stage 17. Abbreviations: Log FC, log 2 fold change.
	Fig. 4. Identification of Pax3 and Zic1 binding sites on Snail1/2 promoters in vitro (A) Induction of snail2 between stage 11.5 and 14 is strongly reduced by Pax3 knock-down, but only mildly affected by Zic1 knock-down. (B) The increase in snail1 transcription between stage 11.5 and 14 is blocked by both Pax3 and Zic1 knock-down. (C) Location of the ECRs studied here (yellow boxes), containing Pax3 or Zic1 putative binding site (B.S.), in the genomic sequence upstream of snail1 and snail2 transcription start site (TSS). (D) Pax3 binds specifically to the motif identified in the snail2 promoter ECR: the electrophoretic mobility of a radiolabeled oligonucleotide containing the putative Pax3 B.S. (Pax3BS) is shifted in the presence of Pax3-transfected cell extract, but not with the non-specific (N.S) control (i.e. GFP-transfected cell extract). Intensity of the shift is reduced when the Pax3 binding site is mutated (mut. Pax3BS). In the presence of a specific anti-Pax3 antibody, we detect a mobility supershift, indicating that the Pax3 protein is indeed responsible for the observed shift. (E) Zic1 binds specifically to the motif identified in the snail1 ECR: the electrophoretic mobility shift detected in the presence of Zic1-transfected cell extract can be competed by increasing doses of non-labeled oligonucleotide (Zic1BS), but only weakly by the mutated oligonucleotide (mut. Zic1BS).
	Fig. 5. Identification of Pax3 binding sites on Pax3 promoters in vitro (A) Pax3, but not Zic1 alone, can trigger synthesis of the endogenous Pax3 transcript (primers in 3'UTR, not amplifying the inducible form) either in absence of the translation inhibitor cycloheximide (induction is 5-fold compared to uninjected animal caps) or in the presence of cycloheximide (induction is 31-fold, compared to cycloheximide-treated uninjected animal caps). Endogenous pax3 expression level in a stage 11 whole embryo is set at a relative value of 1 unit. After co-injection of Pax3 and Zic1, endogenous pax3 was activated as an immediate-early target as well (77-fold compared to uninjected animal caps; and, in presence of cycloheximide, 16-fold when compared to cycloheximide-treated uninjected animal caps). (B) Location of the ECR containing Pax3 putative binding site in the genomic sequence upstream of the Pax3 TSS. (C) Pax3 binds specifically to the motif identified in the pax3 promoter ECR: an electrophoretic mobility shift of the radiolabeled Pax3 binding site (Pax3BS) oligonucleotide probe is detected in the presence of Pax3-transfected cell extract, but not with the GFP-transfected cell extract. Intensity of the shift is reduced when the Pax3 binding site is mutated (mut. Pax3BS). In the presence of a specific anti-Pax3 antibody, we detect a supershift, indicating that the Pax3 protein is indeed responsible for the observed shift.
	Fig. 6. Model of the Pax3/Zic1-linked neural crest gene regulatory network This model summarizes the targets of Pax3 and of Pax3 combined to Zic1, validated in this study. Red arrows indicate Pax3 targets, blue arrows indicate Zic1 targets. Bold arrows indicate that regulation by Pax3 or by Zic1 was confirmed by RTqPCR and/or in vivo.
	greb1l (growth regulation by estrogen in breast cancer-like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterior view, dorsal up.
	greb1l (growth regulation by estrogen in breast cancer-like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	pnhd (pinhead) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterior view, dorsal up.
	pnhd (pinhead) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	tcf7 (transcription factor 7 (T-cell specific, HMG-box) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterior view, dorsal up.
	tcf7 (transcription factor 7 (T-cell specific, HMG-box) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	hapln3 (hyaluronan and proteoglycan link protein 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	hapln3 (hyaluronan and proteoglycan link protein 3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.
	anos1 (anosmin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	anos1 (anosmin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
	mfap2 (microfibrillar-associated protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	mfap2 (microfibrillar-associated protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.
	mmp28 (matrix metallopeptidase 28) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	mmp28 (matrix metallopeptidase 28) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.
	tenm4 (teneurin transmembrane protein 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	tenm4 (teneurin transmembrane protein 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.

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Alarcón, A dual requirement for Iroquois genes during Xenopus kidney development. 2008, Pubmed, Xenbase