Sridharan J , Haremaki T , Jin Y , Teegala S , Weinstein DC .

R01 GM061671 NIGMS NIH HHS , R01-GM61671 NIGMS NIH HHS

Xla Wt + mab21l3 (fig.3.b)
Xla Wt + mab21l3 MO (fig.5.b)
Xla Wt + mab21l3 MO (fig.5.b)
Xla Wt + mab21l3 MO (fig.5.d)

	Fig. 1. Xmab21l3, a novel Mab-21 family protein, is a target of Xema, (A) Xema misexpression upregulates Tx1 in animal caps. RT-PCR analysis of Tx1 expression in animal caps derived from Xema RNA-injected embryos. 25 cycles of PCR were used for detection of Tx1. (B) Xema knockdown induces mesoderm and inhibits expression of Tx1. 33 cycles of PCR were used for detection of Tx1. (C) Tx1/Xmab21l3 encodes a Mab-21 family protein domain (Xmab21l3 aa 5856). (D) The Mab21l3 proteins are more similar to each other than to other Mab-21 family proteins. Dendrogram highlighting the percentage similarity between Xmab21l3 and other Mab-21 family proteins in H. sapiens, M. musculus, X. laevis, D. melanogaster, C. elegans. The dendrogram was generated using ClustalW (http://www.genome.jp/tools/clustalw/) and visualized by NJpolt software (http://pbil.univ-lyon1.fr/software/njplot.html). Branch lengths are proportional to distance. Numbers indicate GI.
	Fig. 2. Spatiotemporal expression of Xmab21l3. (A) Xmab21l3 expression is preceded by the onset of Xema expression. RT-PCR analysis of embryos collected at the indicated stages. The Spemann organizer marker chordin is first detected at stage 9 and increases dramatically at the initiation of gastrulation at stage 10. (B) RT-PCR analysis of embryos collected at the indicated stages. (C) RT-PCR of Xmab21l3 in explants of gastrula stage embryos. Xmab21l3 is expressed predominantly in the animal cap (AC) explants and to a lesser extent in ventral marginal zone (VMZ) explants of stage 10 embryos. DMZ, dorsal marginal zone. (D, D′) Xmab21l3 is expressed in the animal pole ectoderm of gastrula stage embryos. Animal and lateral views of whole mount in situ hybridization analysis of gastrula stage embryos. (E′) Xmab21l3 is excluded from the dorsal neural tube (arrowheads). (E) Dorsal view of stage 20 embryos with anterior to the left. (E′) Posterior view of stage 20 embryos with anterior to the top. (E′) Transverse cross-section of the dorsal neural tube. Dorsal is to the top.
	Fig. 3. Xmab21l3 misexpression promotes dorsalization. (A) Overexpression of Xmab21l3 in embryos causes head defects and ectopic tail-like lateral protrusions. Lateral (top) and dorsal (bottom) views of stage 35 embryos injected with 1 ng of Xmab21l3 RNA in the animal pole of both blastomeres at the 2-cell stage. (B) Xmab21l3-induced lateral protrusions are positive for a somite-specific antigen. Whole mount immunohistochemistry of Xmab21l3 RNA-injected tadpoles with the 12/101 antibody (Kintner and Brockes, 1984). Arrows in (A) indicate protrusions; arrowheads in (B) indicate secondary axes. (C) Xmab21l3 overexpression promotes induction of dorsal markers chordin and goosecoid by Activin. RT-PCR analysis of uninjected and Xmab21l3 RNA-injected animal cap explants cultured until stage 10.5 in the absence or presence of a low dose of Activin (5 ng/ml). (D) Xmab21l3 overexpression induces neural markers sox2 and sox3 in competent ectoderm. RT-PCR analysis of animal cap explants from Xmab21l3 RNA-injected embryos cultured until stage 18. Xmab21l3 (l) and Xmab21l3 (h) indicate 0.5 ng/embryo and 1 ng/embryo doses, respectively, of injected Xmab21l3 RNA.
	Fig. 4. Xmab21l3 does not directly repress transcription of a heterologous promoter. (A) Gal4 constructs used in heterologous promoter repression assays. The constructs were designed for expression of the Gal4 DNA binding domain (147aa) alone or fused to full length Xmab21l3 (168) (Gal4-Xmab21l3), the Engrailed repressor domain (299aa) (Gal4-En) or the VP16 activation domain (41090aa) (Gal4-VP16). (B) Expression of a Gal4-Xmab21l3 fusion protein has little or no effect on Luciferase reporter gene expression. Gal4-En blocks reporter expression completely and Gal4-VP16 strongly upregulates reporter expression. Graph measures expression in RLU (Relative luciferase units). Error bars denote s.d.
	Fig. 5. Loss of function of Xmab21l3 causes anterior defects and lethality. (A) Xmab21l3MO blocks translation of a 3′Myc-tagged Xmab21l3 protein in a dose-dependent manner. Western blot analysis was used to detect Myc-tagged Xmab21l3 protein. (B) Loss of Xmab21l3 affects eye development and axis formation. Low doses of Xmab21l3 MO (2.5 ng) leads to a loss of eyes and/or mild axis curvature in a subset of embryos (middle). Higher doses of Xmab21l3 MO (6.4 ng) give rise to pronounced head defects and shortened body axes (right). Embryos in left panel were injected with 20 ng of scrambled (control) MO. (C) Coinjection of 1 ng RNA encoding a Xmab21l3MO-insensitive silent mutant (Xmab21l3SMT) decreases the percentage of embryos with severe phenotypes. (D) High levels of Xmab21l3MO (250 ng) cause lethality in 95% of gastrula stage embryos. Survival can be rescued in a dose-dependent manner by injection of Xmab21l3SMT. (C, D) Injection of a 5 base-pair mismatch (5MM) control morpholino does not give rise to developmental defects or lethality. Representative experiments shown in C, D; n ⩾ 15 for each condition.
	Fig. 6. Xmab21l3 is required for dorsal fates. (A) Xmab21l3MO injection inhibits induction of chordin and goosecoid, but not Xbrachyury, by Activin. Injection of RNA encoding the Xmab21l3MO-insensitive silent mutant Xmab21l3SMT RNA rescues this effect. (B) A 5 base pair-mismatch Xmab21l3MO (5MM) morpholino does not inhibit mesodermal marker expression by Activin.
	Fig. 7. Analysis of effects of Xmab21l3 on BMP and FGF/ERK signaling. (A) Injection of BMP2 RNA inhibits neuralization by Xmab21l3. Graphs show relative expression, assayed by RT-PCR, of the neural markers sox2 and sox3 in animal caps normalized to expression in uninjected explants. (B) Xmab21l3 misexpression inhibits expression of the BMP-responsive gene sizzled (szl), and induces expression of the dorsal marker fgf4. (C) Xmab21l3 misexpression inhibits expression of a Vent2-Luciferase reporter fusion protein; this construct includes a mutation in a TCF binding site that renders it insensitive to Wnt activation (Hikasa et al., 2010). (D) Xmab21l3 activates ERK1/2 signaling in animal cap explants. Western blot analysis using an antibody against di-phosphorylated ERK1/2 demonstrates that ERK1/2 is activated in response to treatment with FGF or following injection of Xmab21l3 RNA. The FGFR1 inhibitor SU5402 blocks ERK activation by FGF, Xmab21l3 and Xmab21l3SMT. Error bars denote s.d.
	Mab-21-like 3 (mab21l3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, animal view.
	Mab-21-like 3 (mab21l3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, lateral view, animal hemisphere up.
	Mab-21-like 3 (mab21l3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, posterior view, dorsal up.
	Mab-21-like 3 (mab21l3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.

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