Janesick A et al. (2018), RARγ is required for mesodermal gene expression...

XB-ART-55309

Development 2018 Sep 17;14518:. doi: 10.1242/dev.147769.

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RARγ is required for mesodermal gene expression prior to gastrulation in Xenopus.

Janesick A , Tang W , Shioda T , Blumberg B .

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The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 btg2 cyp26a1 dact1 dhh ednra fgf8 foxa4 foxi1 fstl1 fzd10 fzd4 gbx2 gdf3 hapln3 hnf1b hoxa1 hoxa2 hoxa5 hoxb1 hoxb6 hoxd4 jag1 kcp kremen2 mamdc2 myod1 ncam1 nkx6-2 nodal pax6 rarg skida1 tbxt tgif2 tubb2b twsg1 wnt5b wnt8a zeb2 zic1 znf503 znf703
GO keywords: somite specification [+]
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???displayArticle.gses??? GSE119124: NCBI

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	Fig 1 RARÎ³1 is required for the expression of mesoderm markers. (A-D) Embryos were microinjected unilaterally at the 2-cell or 4-cell stage with 3.3â€…ng RARÎ³1.S+3.3â€…ng RARÎ³1.L/S MOs (Fig. S3). Injected side is to the right of the dashed line, and is indicated by the magenta Î²-gal lineage tracer. WISH shows that RARÎ³1 MOs result in the loss of T, fgf8, wnt8 and gdf3. (E-H) Embryos were microinjected unilaterally at the 2-cell or 4-cell stage with 6.6â€…ng RARÎ±2 MOs (Janesick et al., 2013). Injected side is to the right of the dashed line, and is indicated by the magenta Î²-gal lineage tracer. RARÎ±2 MOs did not affect T, wnt8 or gdf3 expression, but did produce loss of N-tubulin as previously published (Janesick et al., 2013). All embryos, except in H, are shown in vegetal view at stage 10.5/11. H is shown in dorsal view, with anterior on the bottom at stage 14. Fractions represent the portion of embryos displaying the phenotype. NC, no change.
	Fig 2 Results of RNA-seq data analysis. (A) GO analysis of differentially expressed genes after TTNPB treatment. GO terms shown to be statistically over-represented (P=0.001) in 1â€…ÂµM TTNPB-treated embryos (q<0.05, FPKMVEH>0.1) conducted in DAVID (Huang da et al., 2009a, b). Gene symbols from Xenopus were used as input, and GO terms were derived from human (for better annotation). Enrichment score is measured by Fisher's exact statistics by the DAVID server. *zic1 is a downregulated gene, and zeb2, tgif2 are <1.5-fold activated by TTNPB. (B) TTNPB-induced genes selected for further analysis in mesoderm development. Activation by TTNPB (represented as log2FPKM) is mapped for both .L and .S subgenomes (Matsuda et al., 2015; Session et al., 2016). kielin and btg2 are not found in .S and .L, respectively; hapln.S is not detected by RNA-seq.
	Fig 3 RAR-selective agonist TTNPB modulates expression of genes identified by RNA-seq. (A-P) WISH from embryos treated at stage 6/7 with 1â€…Î¼M TTNPB or control vehicle (0.1% ethanol). (A-D,I-L) Control expression of znf703, znf503, kremen2, mamdc2, skida1, nkx6-2, dhh and cyp26a1. (E-H,M-P) TTNPB expands expression of these genes. All embryos are shown in vegetal view at stage 10.5/11 with dorsal lip at the top. N, number of embryos scored in the experiment; 100% of embryos displayed the phenotype shown.
	Fig 4 RARÎ³1 acts as a transcriptional activator in rescuing znf703 expression in RARÎ³1 MO embryos. (A-H) Embryos injected unilaterally at the 2- or 4-cell stage. Injected side is to the right of the dashed line, and is indicated by the magenta Î²-gal lineage tracer. (A,E) 3.3â€…ng RARÎ³1.S+3.3â€…ng RARÎ³1.L/S MOs diminish znf703 expression. (B,F) VP16-RARÎ³1 mRNA (0.2 ng) rescues and expands znf703 expression beyond its usual boundary. (C,D,G,H) WT RARÎ³1 mRNA (0.5 ng) partially rescues znf703 (C,G) and 2â€…ng DN-RARÎ³1 mRNA does not rescue znf703 at all, and the severity of the knockdown is increased (D,H). Embryos are shown at stage 10.5/11 in vegetal (A-D) or dorsal (E-H) view. Fractions represent the portion of embryos displaying the phenotype.
	Fig 5 RARÎ³1 MO inhibits cell adhesion in animal caps. (A-C) Embryos were injected bilaterally at the 2-cell stage with 3.3â€…ng RARÎ³1.S+3.3â€…ng RARÎ³1.L/S MOs. Animal caps were harvested at stage 9 and incubated in control vehicle or defined doses of activin (0.8, 4, 20 and 100â€…ng/ml) overnight. (A,B) RARÎ³1 MOs result in animal cap dissociation (note the large number of single cells) (B), compared with control MO (A) in all treatment conditions. This effect was replicated in three separate experiments. mRNA was harvested 8â€…h after treatment from the third and final animal cap experiment and evaluated as shown in C. (C) ncam1 expression was significantly reduced in RARÎ³1 MO animal caps compared with control MO, despite constant housekeeping (histone H4) expression. The upper y-axis represents ncam1 expression 2âˆ’Î”Ct values normalized to histone H4. The bottom y-axis shows raw Ct values of histone H4.
	Fig 6 RARÎ³1 knockdown yields a complex phenotype on myod mRNA and Myod protein. Embryos were injected unilaterally at 2- or 4-cell stage. (A) Injection of 3.3â€…ng RARÎ³1.S+3.3â€…ng RARÎ³1.L/S MOs lead to reduced Myod mRNA expression (10/14 embryos, dorsal view). (B) RARÎ³1 MOs cause loss of PSM expression of Myod protein and ectopic expression in the trunk of coronal sections of stage 26 embryos (4/4 embryos). (C) RARÎ³1 MOs cause loss and disorganization of mature somite marker 12/101 in coronal sections of stage 26 embryos (4/4 embryos).
	Supplemental Figure S1. RARÎ³1 is required for the expression of mesoderm markers. Embryos were microinjected unilaterally at the 2-cell or 4-cell stage with 10ng RARÎ³.L/S MO (see Fig S3). Injected side is to the right of the dotted line, and is indicated by the turquoise Î²-gal lineage tracer. RARÎ³1 MO results in the loss of T (Brachyury) and Foxa4.
	Supplemental Figure S2. Whole mount in situ hybridization showing the expression of RarÎ³,Aldh1a2, and Cyp26a1 at stage 10.5/11, vegetal view, dorsal lip at the top.
	Supplemental Figure S3. Specificity of RARÎ³1 MO phenotype. Two different morpholinos were designed to target Xenopus laevis RarÎ³1. (Top) Mapping of MOs to the .S and .L subgenomes (Session, 2016). MO#1 matches nearly perfectly to both .S and .L, whereas MO #2 matches only .S, and will not likely to bind .L. (Bottom) Embryos were microinjected unilat-erally at the 2-cell or 4-cell stage with 6.6 ng of either RarÎ³1.L/S MO (#1) or RarÎ³1.S (#2). Injected side is to the right of the dotted line, and is indicated by the magenta Î²-gal lineage tracer. RARÎ³1 MO #1 and MO#2 give the same knockdown phenotype on T and Znf703. Fractions represent the portion of embryos displaying the phenotype.
	Supplemental Figure S5. VP16-RarÎ³1 mRNA expands Znf703 while DN-RarÎ³1 mRNA diminishes Znf703. (A-E) Embryos were injected unilaterally at 2- or 4-cell stage. Injected side is to the right of the dotted line, and is indicated by magenta.Î²-gal lineage tracer. (A, B, E) 0.2 ng VP 16- RarÎ³1 mRNA expands Znf703 dorsally and animally. (C, D) 2 ng DN-RarÎ³1 mRNA diminishes Znf703. Embryos are shown at stage 10.5/11 in vegetal (A, C), dorsal (B, D) or animal (E) view.
	znf503 (zinc finger protein 503) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.
	dhh (desert hedgehog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.
	mamdc2 (MAM domain containing 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.