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PMesogenin1 and 2 function directly downstream of Xtbx6 in Xenopus somitogenesis and myogenesis.
T-box transcription factor tbx6 and basic-helix-loop-helix transcription factor pMesogenin1 are reported to be involved in paraxial mesodermal differentiation. To clarify the relationship between these genes in Xenopus laevis, we isolated pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2 appeared to be transcriptional activators and were induced by a hormone-inducible version of Xtbx6 without secondary protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential T-box binding sites with which Xtbx6 protein was shown to interact, and a reporter gene construct containing these sites was activated by Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6 and pMesogenin1 and 2 knockdowns caused similar phenotypic abnormalities including somite malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis including that of body wall muscle in Xenopus laevis.
Figure 3. The pMesogenin2 5 prime regulatory region drives expression of the EGFP reporter gene in PSM. A: Schematic diagrams of reporter constructs used in transgenesis. In pMsgn2-(-1501)-EGFP, the pMesognein2 5 prime regulatory region-EGFP cassette is flanked at both ends by two I-Sce I recognition sites. In pMsgn2-(-1501)-EGFP-p3U, the pMesogenin2 3 prime UTR is inserted between EGFP and the poly(A) signal (pA). In pMsgn2-(-1501+)-EGFP, the pMesogenin1 5 prime regulatory region is inserted between pMesogenin2 5 prime regulatory region and the I-Sce I recognition site. B: X. laevis pMesogenin1 and X. tropicalis pMesgenin1 5 prime regulatory regions compared using mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) with the X. laevis sequence as the baseline. The downward bracket indicates the region added in pMsgn2-(-1501+)-EGFP. C-E: Whole-mount in situ hybridization for pMesogenin2 or EGFP. C: The endogenous pMesogenin2 expression at stage 27. D: In transgenic embryos with pMsgn2-(-1501)-EGFP, EGFP expression was observed in PSM, somites, and the head region. E: In transgenic embryos with pMsgn2-(1501+)-EGFP, EGFP expression was observed in PSM and somites, but was reduced in the head. F-H: Transverse sections at the positions indicated in C and D.
Figure 5. Knockdown of Xtbx6, or pMesogenin1 and 2. A: Schematic diagram of partial genomic structure of the Xtbx6 gene. Bent line displays intron1. B: RT-PCR analysis of Xtbx6 exint-MO- or 5mis-MO-injected embryos (50 ng each). Arrow indicates the position of the long splicing variant mRNAs. Genome, Xtbx6 genomic DNA. ODC was used as the loading control. C: Xtbx6 exint-MO (25 ng) and beta -gal mRNA (200 pg) were co-injected into the VMZ and DLMZ at the 4-cell stage. D-G: Expression patterns of pMesogenin1 (D, E) and pMesogenin2 (F, G) in Xtbx6 exint-MO-injected embryos at stage 20, revealed by whole-mount in situ hybridization. D, F: Representative embryo injected with Xtbx6 exint-MO. E, G: Representative embryo rescued by co-injecting Xtbx6 mRNA (2 pg) with Xtbx6 exint-MO. H: pMsgn MO designed to bind to the translational start regions of both pMesogenin1 and 2. I: pMsgn MO suppressed the luciferase activity of pMsgn2-Luc. J: pMsgn MO (50 ng) or pMesogenin1-EnR (100 pg) and/or pMesogenin2-EnR (100 pg) was injected with beta -gal mRNA (200 pg) into the VMZ at the 4-cell stage. K, L: The expression pattern of Xtbx6 in pMsgn MO- or dominat-negative pMesogenins-injected embryos at stage 18, revealed by whole-mount in situ hybridization.
Figure 6. Knockdowns of Xtbx6 or pMesogenin1 and 2 cause somite malformation and ventral body wall muscle hypoplasia. Representative embryos injected with Xtbx6 5mis-MO (25 ng) (A, B), Xtbx6 exint-MO (25 ng) (C-H), pMsgn MO (50 ng) (I, J), or pMesogenin1-EnR and pMesogenin2-EnR (100 pg each) (K, L). In A-L, the uninjected sides are on the left (A, C, E, G, I, K) and injected sides on the right (B, D, F, H, J, L). In some cases, images are flipped horizontally to facilitate comparison. A-D, I-L: Immunohistochemistry with the 12/101 antibody at stage 41. E-H: Analysis of expression patterns of XmyoD (E, F) and Xmyf-5 (G, H) at stage 32 by whole-mount in situ hybridization. Arrows indicate the expression of Xmyf-5 in hypaxial muscle cells. M, N: The expression of NCAM was not detectably changed in Xtbx6 exint-MO (50 ng)-injected embryos at stage 30, revealed by 4d antibody staining.
Figure 7. pMesogenin1 and 2 is involved in myogenesis initiated by Xtbx6. A: RT-PCR analysis of muscle marker gene expressions in the animal cap explants injected with pMesogenin1 (200 pg) or pMesogenin2 (200 pg) mRNA. B-E: Animal cap explants were immunostained with the 12/101 antibody at stage 24. B: Uninjected explants. C-E: Doses of injected mRNAs or MOs were Xtbx6 mRNA (50 pg), noggin mRNA (100 pg), pMsgn MO (100 ng), and Co MO (100 ng). Note that muscle differentiation was inhibited by pMsgn MO. F: Regulatory hierarchy in somitogenesis and myogenesis involving Xtbx6, Wnt/ beta -catenin signaling, pMesogenin1 and 2. Solid arrows indicate known controls and the dashed arrow, the deduced control; for details see Discussion section.