Baldessari D , Badaloni A , Longhi R , Zappavigna V , Consalez GG .

E.0740 Telethon, TI_E.0740 Telethon

	Figure 1. Expression of Mab21l2 gene and protein in Xenopus laevis development. A) Double whole mount in situ hybridization for Xmab21l2 (blue) and Xkrox20 (brown). At the end of neurulation (dorsal view of a st. 19 embryo is shown) Xmab21l2 expression (arrows) is restricted to the eye primordium (bottom arrowhead) and midbrain (top arrowhead). Xkrox20 labels the third and fifth rhombomeres (arrows). B) Frontal view of a stage 24 embryo. Xmab21l2 expression (blue, arrows) is restricted to the eye (left, solid arrow) and midbrain (right, solid arrow), clearly posterior to the forebrain marker Emx2 expression domain (brown, empty arrow). C–E: Sections obtained from late tadpole embryos were wholemount-hybridized with Xmab21l2. Three transverse sections, from anterior to posterior, show Xmab21l2 expression in the retina and lens (Ey), in the branchial arches (BA), midbrain (arrow in C), hindbrain (arrow in D), and in the dorsal neural tube, (SC in E). F: Western blot analysis of MAB21l2 protein expression in Xenopus oocytes (stage 0) and Xenopus embryos (stages 3–23). ME: day 12 mouse embryo (positive control). Protein concentrations were quantitated through a Bradford assay and loaded on gel in equal amounts. The experiment shows maternal expression of MAB21l2 (41 kD, arrow), the levels of which decrease around blastula stage to resume zygotically thereafter. In particular, stage 11 gastrulae are positive for the MAB21l2 protein.
	Figure 2. Isolated overexpression of Xmab21l2 produces partially dorsalized embryos. A, C, E: control-injected embryos; B, D, F: Xmab21l2-injected embryos. A–D: tailbud-tadpole stages; E, F: gastrula stage. In B: external appearance of Xmab21l2-injected embryos. D: horizontal section of an Xmab21l2-injected section stained with orange-G/aniline blue. Note enlarged notocord (nc). F: expanded and enhanced Chordin expression in Xmab21l2-injected gastrulae (blue signal).
	Figure 3. Xmab21l2 rescues dorsal structures in BMP4 injected embryos. (A) As previously described, injection of 0.6 ng of BMP4 mRNA gave rise to ventralized embryos. The most severely ventralized phenotype of BMP4 injection, called bauchstück, was scored with a dorso-anterior index (DAI) of 0 (top), whereas normally dorsalized embryos were assigned a DAI of 5 (bottom). (B) Embryos were injected with 0.6 ng BMP4 (n = 111), with 0.6 ng BMP4 plus 0.8 ng Mab21l2 (n = 83), or with 0.6 ng BMP4 plus 1.6 ng Mab21l2 (n = 101). In each group, we scored the percentage of complete ventralization (DAI = 0, grey bars) and normal dorsal axis formation (DAI = 5, black bars) in embryos that completed development. Intermediate classes are omitted in the plot. Coinjection of embryos with Mab21l2 significantly increased the percentage of correctly dosalized embryos (see text for details). Statistical analysis was conducted using the Chi square algorithm (1 df). *: p = 0.0005; **: p = 0.0087.
	Figure 4. Xmab21l2 restores the normal expression of Xvent2 and Chordin in BMP4-injected embryos. Chordin (A–C) and Xvent2 (D–E) expression were analyzed by whole mount in situ hybridization. Embryos are shown in a vegetal view, dorsal to the top, ventral to the bottom. (A, D) Wild type expression of Chordin (A) and Xvent2 (D) in stage 10.5 gastrulae; (B, E) embryos injected with 1.2 ng of BMP4 mRNA alone showed reduced Chordin expression (B) (22/25) and expanded Xvent2 expression (E) (16/25); (C, F) embryos co-injected with 1.2 ng of BMP4 and 1.6 ng Xmab21l2 mRNA showed a rescue of wild type Chordin (C) (17/36) and Xvent2 expression (F) (24/29). (G) Percentage of embryos exhibiting wild type (black bars) or reduced (grey bars) Chordin gene expression in embryos injected with 1.2 ng BMP4 alone, and in embryos coinjected with 1.6 ng Mab21l2. Coinjection of Mab21l2 significantly increased the number of embryos exhibiting a wild type Chordin expression pattern; *: p = 0.004. (H) Percentage of embryos exhibiting wild type (black bars) or expanded (grey bars) Xvent2 gene expression in embryos injected with 1.2 ng BMP4 and in embryos coinjected with 1.6 ng Mab21l2. Again, Mab21l2 significantly increased the number of embryos exhibiting a wild type Xvent2 expression pattern; **: p = 0.00044. Statistical analysis was conducted using the Chi square algorithm (1 df).
	Figure 5. Mab21l2 interacts with Smad1 in vitro and in vivo. (A) Lanes 1, 2: immunoblotting of total lysates from P19 cells, mock- and Smad1-transfected, respectively. P19 cells do not express detectable levels of endogenous SMAD1. Lanes 3–8: Affinity chromatography (pull-down) experiment. High-stringency eluates from untransfected P19 were separated by SDS-PAGE and transfered onto nitrocellulose filters. The blots were immunolabeled using an anti-SMAD1 antibody. Lanes 3–6: the in vitro interaction is not strictly dependent on activation of BMP signaling: the pull-down experiment was performed with 20 μl (3, 4) and 40 μl of P19 cell lysates (5, 6). Lysates came from P19 cells treated (4, 6) or untreated (3, 5) with BMP4. Lanes 7, 8: an His-ZZ-MAB21L2 protein (HisMab) synthesized in E. coli (lane 8) was coupled to a sepharose-Ig resin and incubated with cell lysates of P19 cells treated with BMP4. As a negative control, an in-vitro synthesized His-ZZ incubated with the same P19 lysates fails to pull down a 53 kD band. Arrows: SMAD1 (53 kD). (B) direct interaction between SMAD1 and MAB21L2; no direct interaction between SMAD4 and MAB21L2. An His-ZZ-MAB21L2 protein (+Mab) synthesized in E. coli (lane 2) was coupled to a sepharose-Ig resin and incubated with in vitro-translated SMAD1 (lanes 2), SMAD4 (lanes 4), and SMAD1 + SMAD4 (lanes 6). Negative controls were represented by His-ZZ-coupled resins (-Mab) incubated with the same in vitro-synthesized proteins (lanes 3, 5, 7). Lanes 1 and 8 contain in vitro-synthesized SMAD1 and SMAD4, respectively. (C) BMP4-dependent in vivo co-immunoprecipitation of flag-SMAD1 and myc-MAB21L2 in P19 cells. Cells were mock-transfected, transfected with flag-SMAD1 and/or with myc-MAB21L2, as indicated, and either treated with BMP4 or left untreated. In lanes 3–7, cell lysates were immunoprecipitated with a monoclonal anti-flag antibody. Cell lysates in lanes 1, 2 and immunoprecipitates in lanes 3–7 were gel fractionated and transfered to nitrocellulose filters. Blots were immunolabeled with an anti myc antibody. Arrow (42 kD) points to a band in lanes 2, 3) corresponding to myc-MAB21L2. (D) in vivo co-immunoprecipitation of flag-SMAD1 and myc-MAB21L2 in stage 11 Xenopus embryos, facilitated by BMP4 overexpression. Embryos were water-injected, injected with flag-Smad1 and/or with myc-Mab21l2 RNA, as indicated, and coinjected with BMP4 where indicated. In lanes 3–7, cell lysates were immunoprecipitated with a monoclonal anti-flag antibody. Embryo lysates in lanes 1, 2 and immunoprecipitates in lanes 3–6 were gel fractionated and transfered to nitrocellulase filters. Blots were immunolabeled with an anti myc antibody. Arrow (42 kD) points to a band in lanes 2–4) corresponding to myc-MAB21L2.
	Figure 6. When targeted to a heterologous promoter, Mab21l2 behaves as a strong transcriptional repressor. (A) An in vitro-translated chimeric protein containing the GAL4 DNA binding domain fused to the N-terminus of MAB21L2 was used in an electrophoretic mobility shift assay. An end-labeled ds-DNA containing the GAL4 nucleotide binding site (5XUAS) was incubated in native conditions with in vitro translated GAL4-MAB21L2 (G-M) and separated electrophoretically by nondenaturing PAGE. As negative controls, we used free oligonucleotides (F) and rabbit reticulocyte lysates (RRL). As a positive control we used a previously tested GAL4 fusion protein, GAL4-D9NT (G-D9) [40]. The arrow indicates a bandshift specific for GAL4-MAB21L2 and absent in negative controls. u: unbound. (B) 5XUAS-luc transcription is clearly downregulated in cells transfected with GAL4-MAB21L2 (G-M) vs. untransfected COS7 cells or same cells transfected with GAL4 DBD, G-D) alone (*: p = 0.0019). No downregulation of 5XUAS-luc activity was observed in COS7 cells transfected with a fusion of GAL4 and the N-terminal domain of the HOXB3 protein, which possesses no transcriptional activation or repression activity [24] (ns: not significant). Statistical analysis was conducted using the T test method (two tails).
	mab21l2 (mab-21-like 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, anterior view, dorsal up. (note: white arrow head indicates expression of emx2 in forebrain/telencephalon).

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