Kaufmann LT and Niehrs C (2011), Gadd45a and Gadd45g regulate neural development...

XB-ART-43784

Mech Dev 2011 Jan 01;1287-10:401-11. doi: 10.1016/j.mod.2011.08.002.

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Gadd45a and Gadd45g regulate neural development and exit from pluripotency in Xenopus.

Kaufmann LT , Niehrs C .

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Gadd45 genes encode a small family of multifunctional stress response proteins, mediating cell proliferation, apoptosis, DNA repair and DNA demethylation. Their role during embryonic development is incompletely understood. Here we identified Xenopus Gadd45b, compared Gadd45a, Gadd45b and Gadd45g expression during Xenopus embryogenesis, and characterized their gain and loss of function phenotypes. Gadd45a and Gadd45g act redundantly and double Morpholino knock down leads to pleiotropic phenotypes, including shortened axes, head defects and misgastrulation. In contrast, Gadd45b, which is expressed at very low levels, shows little effect upon knock down or overexpression. Gadd45ag double Morphants show reduced neural cell proliferation and downregulation of pan-neural and neural crest markers. In contrast, Gadd45ag Morphants display increased expression of multipotency marker genes including Xenopus oct4 homologs as well as gastrula markers, while mesodermal markers are downregulated. The results indicate that Gadd45ag are required for early embryonic cells to exit pluripotency and enter differentiation.

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Species referenced: Xenopus laevis
Genes referenced: fgf8 gadd45a gadd45b gadd45g gal.2 myf5 pou5f3 sox10 sox3 tbxt
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	Fig. 1. Sequence comparison of Gadd45 genes. (A) Multiple alignment of predicted full-length amino acid sequences of Gadd45b from X. laevis (xla), X. tropicalis (xtr), human (h), mouse (m) and zebrafish (z). Conserved residues in at least five sequences or in at least three sequences are highlighted in black and gray, respectively. (B) Phylogenetic tree of vertebrate Gadd45a, Gadd45b and Gadd45g genes based on the average distance using % identity (ClustalW)
	Fig. 2. Differential expression of Gadd45a, Gadd45b and Gadd45g during Xenopus embryogenesis. (A–C) qPCR analyses showing the mean concentration normalized to GAPDH of Xenopus Gadd45a (A), Gadd45b (B) and Gadd45g (C). (D) Quantification of relative transcript levels of Xenopus Gadd45a, Gadd45b and Gadd45g in tadpole stage (st. 32) embryos. (E–S) Whole mount in situ hybridization expression analyses. (E–H) Gadd45a expression in lateral view of whole (E) and sagitally halved (F) gastrula stage (st. 10.5) embryos, dorsal is right. (G) Anterior and (H) dorsal view of late neurula stage (st. 18) embryo. (I and J) Gadd45b expression in anterior view of stage 18 embryo using antisense (I) and sense control probe (J). (K and L) Gadd45g expression in anterior view (K) and dorsal view (L) of stage 18 embryo. (M–P) Expression analysis of stage 18 embryos cut transversally with the probes indicated. (Q–S) Lateral view of tadpole stage (st. 32) embryos hybridized with probes for Gadd45a (Q), Gadd45b (R) and Gadd45g (S). The cement glands are outlined by dashed lines in (I–K). Key: (ba) branchial arches, (bp) blastopore, (cg) cement gland, (ey) eye, (fb) forebrain, (hb) hindbrain, (ht) heart field, (mes) mesoderm, (mb) midbrain, (nf) neural fold, (np) primary neuron precursors, (nt) neural tube, (pd) proctodeum, (pn) pronephros, (so) somites, (tg) trigeminal placode.
	Fig. 3. Gadd45a, Gadd45b and Gadd45g overexpression and knock down in Xenopus embryos cause pleiotropic phenotypes. (A–H) Phenotypes of stage 41 embryos resulting from Gadd45 mRNA injection (A–D) or antisense MO knock down (E–H). The topmost tadpoles represent the least affected specimen of the respective group, the lower two show the representative phenotypes. Embryos were injected with 5 ng of lacZ (A), Gadd45a (B), Gadd45b (C) or 1 ng of Gadd45g (D) mRNA, or the respective MO (E–H), as described in Section 4. (I) Quantification of the phenotypes shown in A–H caused by Gadd45 overexpression or knock down. (J) qPCR analysis of Gadd45 expression in neurulae (stage 18) injected with Gadd45 single or double MOs as described in Section 4. Key: (co) Control, (G45) Gadd45, (g.d.) gastrulation defect, (wt) wild type.
	Fig. 4. Gadd45a–Gadd45g double knock down is rescued by hGadd45a and hGadd45g mRNAs. (A–D) Phenotypes of stage 41 embryos coinjected with the indicated combinations of mRNA (left) and Morpholinos (MO, top). (E) Quantification of the phenotypes shown in A–D. (F–I) Phospho-Histone 3 immunostaining (blue nuclei) of neurula embryos (anterior view) injected unilaterally with lineage tracer (red β-gal staining) with the indicated combinations of mRNA (left) and MO (top). Insets, enlarged boxed areas. (J) Quantification of p-Histone 3 positive cells on injected and uninjected sides. Data represent the mean ± SD of 16 embryos per sample. (K) Cell cycle inhibitor qPCR expression analysis in double Gadd45ag Morphant neurulae and rescue by mRNA coinjection. Key: (ag) Gadd45a MO + Gadd45g MO, (co) control MO, (G45) Gadd45, (g.d.) gastrulation defect, (hG45ag) hGadd45a + hGadd45g mRNA, (pH3+) p-Histone 3 positive, (wt) wild type.
	Fig. 5. Gadd45a and Gadd45g act redundantly to inhibit pluripotency and promote cell differentiation. (A) qPCR expression analysis of neurula (stage 18) embryos injected with the indicated agents analyzing early expressed developmental genes which are repressed by Gadd45ag MO and rescued by hGadd45ag mRNA coinjection. (B–E) sox10 expression in neurula embryos (stage 18, anterior view) unilaterally injected with the indicated combinations of mRNA (left) and MO (top) and lineage tracer (blue β-gal staining). Note reduced sox10 expression in neural crest (nc) after Gadd45ag MO injection (arrowhead in B). Coinjection of hGadd45a and hGadd45g mRNA rescues this repression (asterisk in D). (F–H) qPCR expression analyses of the indicated genes at neurula stage (st. 18) injected with hGadd45ag mRNA and Gadd45ag MO. (F) Pluripotency- and early germ layer specific markers upregulated by Gadd45ag MO. (G) Terminal differentiation markers unaffected by Gadd45ag overexpression or knock down. (H) FGF related genes upregulated by Gadd45ag MO. (I) qPCR expression analysis of Xbra in stage 11 animal caps after induction with FGF8 mRNA, coinjected with the indicated agents (see Section 4). Key: (G45) Gadd45, XFD (dominant negative FGF receptor).
	gadd45a (growth arrest and DNA-damage-inducible, alpha) gene expression in Xenopus laevis, NF stage 18 embryo, assayed via in situ hybridization, dorsal view, anterior up.
	gadd45a (growth arrest and DNA-damage-inducible, alpha) gene expression in Xenopus laevis, NF stage 32 embryo, assayed via in situ hybridization, lateral view, dorsal up.
	gadd45b (growth arrest and DNA-damage-inducible, beta ) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 32 lateral view, anterior left, dorsal up.
	gadd45g (growth arrest and DNA-damage-inducible, alpha) gene expression in Xenopus laevis, NF stage 18, dorso-anterior view ( left) and dorsal view (right)., via in situ hybridization, lateral view, dorsal up.
	gadd45g (growth arrest and DNA-damage-inducible, alpha) gene expression in Xenopus laevis, NF stage 32, lateral view, via in situ hybridization, lateral view, dorsal up.
	gadd45a (growth arrest and DNA-damage-inducible, alpha) gene expression in Xenopus laevis, NF stage 18 embryo, via in situ hybridization, anterior view, dorsal up.