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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. |
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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. |
