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First published online 18 May 2005
doi: 10.1242/dev.01865

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Xema, a foxi-class gene expressed in the gastrula stage Xenopus ectoderm, is required for the suppression of mesendoderm

¹ Graduate training program in Mechanisms of Disease and Therapy, Mount Sinai School of Medicine, New York, NY 10029, USA
² Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (23K): [in a new window]   Fig. 1. Identification of the Xenopus Foxi-class gene Xema (GenBank accession number DQ026065). (A) Activin inhibits A07 (Xema) expression. RT-PCR analysis of animal cap explants dissected at late blastula stages and cultured until midgastrula stages. Activin (0.5 ng/ml) was added to stage 9 animal caps, as listed. (B) Putative Xema amino acid sequence. The forkhead box (Fox) DNA-binding domain is indicated.

View larger version (34K): [in a new window]   Fig. 2. Expression of Xema during early development. (A) RT-PCR analysis of Xema temporal expression. The `-RT' lane contains all reagents except for reverse transcriptase and was used as a negative control. Ornithine decarboxylase (ODC) was used as a loading control (Bassez et al., 1990). Chordin expression is initiated zygotically and was used as a staging control. (B) Whole-mount in situ hybridization of early gastrula (stage 10+), midgastrula (stage 11), and early neurula (stage 15) stage embryos, using antisense Xema and Xenopus brachyury (Xbra) probes. Xema expression is seen as a blue stain throughout the animal pole of gastrula-stage albino embryos; Xema expression is excluded from the marginal zone (denoted by arrows at stage 11) and vegetal pole. Expression of the panmesodermal marker Xbra is found only in the marginal zone of gastrula stage embryos and was used as a control. Xema is expressed in the vental ectoderm (epidermis) of early neurula stage embryos (ventral view); expression is excluded from the neural plate (dorsal view). (C) RT-PCR analysis of Xema expression in early gastrula stage explants. EF1- was used as a loading control (Krieg et al., 1989). Xbra is a panmesodermal marker at this stage (Smith et al., 1991), chordin is a dorsal endomesodermal marker (Sasai et al., 1994), and Xwnt8 is a ventrolateral marker (Christian et al., 1991; Smith and Harland, 1991).

View larger version (52K): [in a new window]   Fig. 3. Ectopic Xema inhibits mesendoderm induction. (A) Xema misexpression disrupts embryonic development. Lateral views of stage 35 embryos, anterior is to the right. (B) Whole-mount immunohistochemistry of tailbud stage embryos using the somite-specific antibody 12/101; lateral views, anterior is to the right. Embryos were stained with Red Gal as a substrate prior to immunohistochemistry. (C) RT-PCR analysis of dorsal (DMZ) and ventral (VMZ) marginal zone explants from embryos injected with Xema RNA, harvested immediately after dissection at early gastrula stages. (D) Whole-mount in situ hybridization of midgastrula stage embryos, using an antisense Xenopus brachyury probe; vegetal pole views are shown. Embryos were stained with Red Gal as a substrate prior to in situ hybridization. The embryos shown at the top of the figure were co-injected with lacZ RNA and Xema RNA in two dorsal or ventral blastomeres, at the four-cell stage, as listed; note the presence of ß-galactosidase activity (red, arrows) in the gap in Xenopus brachyury expression (blue). The embryos shown at the bottom of the figure were injected with lacZ RNA in the same regions, as indicated; note the overlap (arrows) between Xenopus brachyury expression and ß-galactosidase activity. (E) Inhibition of Activin-mediated mesendoderm induction by Xema. RT-PCR analysis of animal cap explants dissected at late blastula stages and cultured until midgastrula stages. Xema RNA was injected into the animal pole region of both blastomeres at the two-cell stage. Activin (0.5 ng/ml) was added to stage 9 animal caps, as listed. (F) Inhibition of Fgf-mediated mesoderm induction by Xema. bFgf (10 ng/ml) was added to stage 9 animal caps, as listed. For all experiments in this figure, 1 ng of Xema RNA, and/or 100 pg lacZ RNA was injected, as listed.

View larger version (66K): [in a new window]   Fig. 4. Activity of Xema activator and repressor constructs. (A) VP-Xema-mediated inhibition of mesoderm formation. RT-PCR analysis of animal caps dissected at late blastula stages and cultured until midgastrula stages. VP-Xema RNA (1 ng) was injected at early cleavage stages, as indicated. Activin (0.5 ng/ml) was added to stage 9 animal caps, as indicated. (B) Expression of EnR-Xema induces mesoderm. RT-PCR analysis of animal caps dissected at late blastula stages and cultured until midgastrula stages. EnR-Xema RNA (1 ng) was injected at early cleavage stages, as indicated. (C) Expression of EnR-Xema induces ectopic structures. Whole-mount immunohistochemistry of tailbud stage embryos using the somite-specific antibody 12/101; lateral views, anterior is to the right. Embryos were stained with X-Gal as a substrate prior to immunohistochemistry. The embryo on the right was co-injected with 100 pg lacZ and 1 ng EnR-Xema RNA in the animal pole at the two-cell stage; note the absence of somite staining (red) in the secondary structure containing the ß-galactosidase activity (blue). The embryo on the left was injected with 100pg lacZ RNA in the animal pole at the two-cell stage and was not probed with the somite-specific antibody.

View larger version (59K): [in a new window]   Fig. 5. Knockdown of Xema protein induces mesoderm formation. (A) Xema morpholinos block the translation of Xema RNA in vitro. Morpholino oligonucleotides (500 ng each) and Xema RNA (1 ng) were simultaneously added to a rabbit reticulocyte lysate mix (Promega), in the presence of [35S]methionine. (B) The effect of Xema morpholinos on exogenous Xema protein levels. Western blot analysis of whole-cell lysates extracted from embryos injected with 1 ng Xema-Myc RNA; 10 ng MO-1, 10 ng MM-1, and/or 20 ng MO-2 were injected, as listed. (C) Mesoderm and endoderm are induced by injection of Xema MO-1 (10 ng), but not by Xema MM-1 (10 ng). RT-PCR analysis of animal cap explants from embryos injected with Xema morpholinos in the animal pole at early cleavage stages, dissected at blastula stages and harvested during midgastrula stages. (D) Xema MO-1 (10 ng) and MO-2 (20 ng) potently synergize to induce mesendoderm. (E) Co-expression of Xema MO-1 and MO-2 disrupts normal development. (Left column) Lower panels show embryos co-injected with 5 ng of MO-1 and 10 ng of MO-2 in the animal pole of early cleavage stage embryos. The top embryo is uninjected. Second embryo from top is a dorsal view; all other views are lateral; anterior is to right. Note anterior pigmented ectopic lateral structures. (Right column) Lower panels show embryos injected with 5 ng of MO-1, 10 ng MO-2 and 166 pg of lacZ RNA in the animal pole of early cleavage embryos. The top embryo is injected with 166 pg of lacZ RNA only. Note the presence of ß-galactosidase staining (blue), primarily in the ectopic structures. All views are lateral; anterior is to right. (F) Rescue of Xema morpholino-mediated mesendoderm formation by injection of 1 ng Myc-Xema RNA; 10 ng MO-1 and 20 ng MO-2 were injected, as listed.

View larger version (41K): [in a new window]   Fig. 6. Fgf signaling is required for mesoderm induction by either EnR-Xema or Xema morpholinos. (A) EnR-Xema activity is inhibited by co-expression of a truncated Fgf receptor (XFD), but is not inhibited by expression of a truncated activin receptor (TAR). RT-PCR analysis of animal cap explants dissected at blastula stages and harvested during midgastrula stages. EnR-Xema RNA (1 ng), alone or with either 4 ng XFD or 4 ng TAR, was injected at early cleavage stages, as indicated. Activin (0.5 ng/ml) or Fgf (10 ng/ml) was added to stage 9 animal caps, as indicated. (B) XFD inhibits Xema morpholino-mediated Xbra and Xwnt8 expression. TAR did not inhibit Xbra or Xwnt8 expression in nine out of 12 independent trials. Neither TAR nor XFD inhibit dorsal endomesodermal (chordin) or endodermal (Sox17ß) marker induction by Xema morpholino injection; 4 ng TAR, 4 ng XFD, 10 ng MO-1 and 20 ng MO-2 were injected at early cleavage stages, as indicated.