Goto T and Shibuya H (2023), maea affects head formation through ß-catenin d...

XB-ART-59499

Dev Growth Differ 2023 Jan 01;651:29-36. doi: 10.1111/dgd.12828.

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maea affects head formation through ß-catenin degradation during early Xenopus laevis development.

Goto T , Shibuya H .

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Canonical Wnt signalling plays important roles in early embryogenesis, such as axis formation due to its activation and head formation due to its inhibition. ß-catenin protein stability is a key factor in canonical Wnt signalling. Several E3 ubiquitin ligases contribute to ß-catenin degradation through the ubiquitin/proteasome system. We characterised an E3 ubiquitin ligase gene, Xenopus laevis macrophage erythroblast attacher (maea), during early development. maea transcripts were ubiquitously detected in early embryos. The expression levels of the Wnt target genes nodal homolog 3, gene 1 (nodal3.1), and siamois homeodomain 1 (sia1), which were induced by injection with ß-catenin mRNA, were reduced by maea.S mRNA co-injection. maea.S overexpression at the anterior dorsal region enlarged head structures, whereas Maea knockdown interfered with head formation in Xenopus embryos. Maea.S decreased and ubiquitinated ß-catenin protein. ß-catenin-4KRs protein, which mutated the four lysine (K) residues known as ubiquitinated sites to arginine (R) residues, was also ubiquitinated and degraded by Maea.S. These findings suggest that Maea contributes to β-catenin degradation by ubiquitination of unknown lysine residues in early Xenopus development.

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Species referenced: Xenopus laevis
Genes referenced: hbg1 maea myc nodal nodal3.1 odc1 sia1 sla
GO keywords: beta-catenin binding [+]
???displayArticle.antibodies??? Nkx6-1 Ab1
???displayArticle.morpholinos??? maea MO1

Phenotypes: Xla Wt + ctnnb1 (Fig. 3CD) [+]

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	Figure 1. maea expression patterns in Xenopus laevis. (a) RT-PCR analysis. Temporal expression patterns of maea transcripts. U indicates unfertilized eggs. The numbers indicate the developmental stages. (b) Whole-mount in situ hybridization results for maea. Stages and views are indicated at the bottom of the images. Anti-sense RNA probes of maea were used on the left panels, whereas sense RNA probes of maea were used on the right panels. Arrowheads indicate the blastopore. Arrows indicate the eye. (c) RT-PCR analysis. Spatial expression patterns of maea transcripts. Dissections were performed at the indicated developmental stages. Abbreviations: D, dorsal; V, ventral; Ec, ectoderm; Me, mesoderm; En, endoderm; H, head
	Figure 2. Ã-catenin degradation and ubiquitination by Maea.S. Plasmids of the indicated constructs were transfected. Indicated antibodies were used for western blotting (WB) and immunoprecipitation (IP). (a) WB of ectopically expressed Xenopus Ã-catenin protein in HEK 293âT cells. Transfected maea.S-FLAG plasmid amounts are indicated (0, 0.5, 1.5, and 5 Î¼g per dish). Anti-GAPDH antibody was used as a loading control. (b) WB of lysates of Xenopus animal cap cells from embryos (st. 9) injected with MYC-Ã-catenin mRNA (50âpg/blastomere) or co-injected with maea.S-FLAG mRNA (500âpg/blastomere) into four animal blastomeres of 8-cell embryos. (c) Cycloheximide chase assay. maea.S-FLAG plasmid was transfected at 24âh after MYC-Ã-catenin plasmid transfection in HEK 293âT cells. From 5 h after the transfection of maea.S-FLAG plasmid, cells were treated with cycloheximide (CHX) (20âmg/ml) during the indicated hours (h). (d) Interaction between ectopically expressed Xenopus maea.S and Ã-catenin in HEK 293âT cells. At 24âh after transfection, immunoprecipitation assay was examined. (e) WB of immunoprecipitates of ubiquitinated Ã-catenin protein treated with MG-132 (10 Î¼M, 4 h) 24âh after transfection in HEK293T cells. (f) RT-PCR analysis of the animal caps from embryos injected with maea.S mRNA (1000âpg/blastomere). odc1 expression was used as an internal control. (g) RT-PCR analysis of the animal caps from embryos injected with Ã-catenin mRNA (50âpg/blastomere).
	Figure 3. Effects of Ã-catenin on head and secondary axis formation by maea.S. (a) RT-PCR analysis of the dorsal or ventral sectors from embryos (st. 10) injected with maea.S mRNA. D, dorsal sectors. V, ventral sectors. (b) Phenotypes of embryos when injected with maea.S mRNA into animal dorsal blastomeres at the 8-cell stage. The appearance rate of embryos with enlarged head structures was 68.1% (n = 47). Central panels indicate enlarged images of the cement gland region. (c) Phenotypes of embryos (st. 30) when injected with Ã-catenin mRNA or co-injected with maea.S mRNA into ventral blastomeres at the 4-cell stage. Each injected embryo had no secondary axis, a partial secondary axis without a head structure, or a complete secondary axis with a head structure. Views are indicated at the bottom of the images. (d) The appearance rates of phenotypes in Figure 3c. No secondary axis (light gray); partial secondary axis (dark gray); complete secondary axis (black). Injected mRNA amounts are indicated at the bottom of the graph. Lane 1: n = 50, no secondary axis (22.0%), partial secondary axis (22.0%), and complete secondary axis (56.0%). Lane 2: n = 55, no secondary axis (25.5%), partial secondary axis (43.6%), and complete secondary axis (30.9%). Lane 3: n = 59, no secondary axis (59.3%) and partial secondary axis (40.7%). Lane 4: n = 34, no secondary axis (23.5%) and partial secondary axis (76.5%). Lane 5: n = 35, no secondary axis (57.1%) and partial secondary axis (42.9%). Lane 6: n = 40, no secondary axis (70.0%) and partial secondary axis (30.0%). (e) RT-PCR analysis of the ventral sectors from embryos injected with Ã-catenin mRNA (50âpg/blastomere) or co-injected with maea.S mRNA (500âpg/blastomere). The un-injected dorsal sectors were used as a positive control. (f) WB analysis. The indicated mRNAs and MOs were injected, and FLAG-tagged hemoglobin subunit gamma 1, hbg1-FLAG mRNA was co-injected as loading control. (g) RT-PCR analysis of the dorsal or ventral sectors from embryos injected with Control-MO (40âng/blastomere), maea-MO (40âng/blastomere), or co-injected with 5-mis-maea.S mRNA (500âpg/blastomere). (h) Phenotypes of embryos when injected with maea-MO or co-injected with 5-mis-maea mRNA into animal dorsal blastomeres at the 8-cell stage. Each injected embryo was categorised as having normal eyes, small eyes, or no eyes. The centre panel shows a representative example of the weak phenotype of embryos rescued by co-injection of 5-mis-maea mRNA. The bottom panel shows a representative example of the severe phenotype of embryos injected with maea-MO. (i) The appearance rates of phenotypes in Figure 3h. Normal eyes (light gray); small eyes (dark gray); no eyes (black). Injected mRNAs and MO amounts are indicated at the bottom of the graph. Lane 1: n = 59, normal eyes (96.6%) and small eyes (3.4%). Lane 2: n = 55, small eyes (83.6%) and no eyes (16.4%). Lane 3: n = 56, small eyes (21.4%) and no eyes (78.6%). Lane 4: n = 54, normal eyes (1.9%), small eyes (70.4%), and no eyes (27.7%). Lane 5: n = 51, normal eyes (19.6%) and small eyes (80.4).
	Figure 4. Inhibition effects of maea.S on the Ã-catenin-4KRs construct. (a) WB of ectopically expressed Ã-catenin-4KRs in HEK 293âT cells. Indicated plasmids were transfected, and indicated antibodies were used. (b) Interaction between ectopically expressed Maea.S and Ã-catenin-4KRs in HEK 293âT cells. (c) WB of ubiquitinated Ã-catenin-4KRs treated as described in Figure 2e. (d) The appearance rates of embryo phenotypes when injected with Ã-catenin-4KRs mRNA or co-injected with maea.S mRNA into ventral blastomeres at the 4-cell stage (see Figure 3c,d). No secondary axis (light gray); partial secondary axis (dark gray); complete secondary axis (black). Injected mRNA amounts are indicated at the bottom of the graph. Lane 1: n = 85, no secondary axis (14.1%), partial secondary axis (23.5%), and complete secondary axis (62.4%). Lane 2: n = 82, no secondary axis (36.6%), partial secondary axis (32.9%), and complete secondary axis (30.5%). Lane 3: n = 83, no secondary axis (57.8%), partial secondary axis (38.6%), and complete secondary axis (3.6%). Lane 4: n = 80, no secondary l axis (33.8%), partial secondary axis (63.8%), and complete secondary axis (2.4%). Lane 5: n = 81, no secondary axis (61.7%) and partial secondary axis (38.3%). Lane 6: n = 80, no secondary axis (73.8%) and partial secondary axis (26.3%). (e) RT-PCR analysis of the ventral sectors of embryos injected with Ã-catenin-4KRs mRNA (50âpg/blastomere) or co-injected with maea.S mRNA (500âpg/blastomere). The un-injected dorsal sectors were used as the positive control.
	Supplemental Figure 1. Phenotypes of the injected embryos. The appearance rate (%) of phenotype and the number of the examined embryos (n) are indicated. (A) Phenotypes of embryos injected dorsally with maea.S mRNA-injected (500 pg/blastomere) (st. 28). No significant phenotype was observed, 100% (n=58). (B) The injection of a large amount of maea.S mRNA (2 ng/blastomere) led blastomereâs death before late blastula stage (st. 9.5), 100% (n=38). (C) Phenotypes of embryos injected dorsally with maea-MO-injected (40 ng/blastomere) (st. 28). No significant phenotype was observed, 100% (n=56).
	maea (macrophage erythroblast attacher) gene expression in X. laevis embryo, NF stage 9, as assayed via in situ hybridization, animal view.
	maea (macrophage erythroblast attacher) gene expression in a X. laevis embryo, NF stage 10.5, as assayed via in situ hybridization, vegetal view, dorsal up.
	maea (macrophage erythroblast attacher) gene expression in X. laevis embryo, NF stage 13, as assayed via in situ hybridization, dorsal view, anterior left.
	maea (macrophage erythroblast attacher) gene expression in X. ;aevis embryo, NF stage 23, as assayed via insitu hybridization, lateral view, anterior left, dorsal up.