Zohn IE and Brivanlou AH (2001), Expression cloning of Xenopus Os4, an evolution...

XB-ART-7834

Dev Biol 2001 Nov 01;2391:118-31. doi: 10.1006/dbio.2001.0420.

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Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Zohn IE , Brivanlou AH .

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Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.

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Species referenced: Xenopus laevis
Genes referenced: actc1 acvr2b ag1 bmp2 chrd col2a1 ctdsp2 eomes foxh1 gsc hoxb9 inhbb krt12.4 ldb1 lhx1 ncam1 nodal3.2 odc1 sia1 smad10 smad2 smad4 smad7 tbxt twist1 wnt8a
???displayArticle.antibodies??? Ncam1 Ab2 Notochord Ab1 Somite Ab3

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Phenotypes: Xla Wt + ctdsp2 (Fig.1.a, c) [+]

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	FIG. 1. (A) Characterization of the secondary axis induced by ventral expression of XOs4. (a) XOs4 induces a secondary dorsal axis when injected into the ventral–marginal zone of four- to eight-cell stage embryos. (b) Transverse section of embryo probed with a neural-specific antibody (6F-11). (c) Transverse section of an embryo probed with somite-specific (12/101) antibodies. (B) Nucleotide and predicted amino acid sequence of XOs4. Conserved domain is underlined in red. Conserved phosphatase motif is boxed in blue.
	FIG. 2. (A) Expression of XOs4 gene during early Xenopus embryogenesis. Expression of XOs4 was detected by RT-PCR. ODC is a ubiquitously expressed gene and represents a loading control. XOs4 is expressed maternally and expression continues through tadpole stage. The cDNA clone encoding XOs4 was used in the last lane as a positive control. (B) XOs4 is ubiquitously expressed at gastrula stage. Stage-101 embryos were dissected as diagrammed into dorsal marginal zone (D), ventral–marginal zone (V), animal pole (AN), and vegetal pole (VG) explants and subjected to RT-PCR analysis using primers that amplify XOs4 and various markers to control for proper dissection of explants.
	FIG. 3. (A) Spatial expression pattern of XOs4 in Xenopus embryos. (a) Vegetal view of a Stage-101 embryo; dorsal is up; arrows mark dorsal blastopore lip. (b) Dorsal view of a Stage-15 embryo; anterior is to the left. (c) Transverse section of a Stage-28 embryo demonstrating staining in dorsal neural tube and neural crest. (d) Lateral view of a Stage-32 embryo; anterior is to the left. np, neural plate; epi, epidermis; nc, neural crest; nt, neural tube; nc, notochord; ey, eye; ov, otic vessicle; ba, branchial arches; pn, pronephros. (B) Intracellular localization of XOs4. (b) Ectopically expressed Flag-tagged XOs4 is localized in Stage-101 Xenopus embryos to the cytoplasm and the plasma membrane and exhibits staining characteristic of nuclear localization. (a) FLAG-ldb1 is localized to the nucleus and exhibits characteristic staining. (d) XOs4 is localized to the cytoplasm and nucleus when expressed in Cos-1 cells. (c) pCMV-FLAG-DPC4 coexpressed with an activated Activin receptor (pCMV-ActRIB(T-D), FLAG-Smad4, is localized to the nucleus in Cos-1 cells.
	FIG. 4. (A) XOs4 induces a secondary axis distinct from that induced by activation of FGF or Wnt pathways but similar to activation of Activin or inhibition of BMP pathways. Uninjected (2) or RNA encoding v-Ras (25 pg, b), Wnt-8 (20 pg, c), Smad7 (100 pg, d), Smad2 (1 ng, e), or XOs4 (100 pg, f) was injected into the ventral marginal zone of eight-cell stage embryos. Embryos were allowed to develop until tadpole stage where the induced secondary axis was observed. (B, C) Lineage-tracing analysis reveals that XOs4 does not remain in the endoderm but participates in the formation of the secondary axis. One D-teir ventral–vegetal cell (B) of a 32-cell stage embryo was coinjected with RNA encoding bGal (C-a and C-e) or combined with RNA encoding Wnt8 (C-b and C-f), Smad7 (C-c and C-g), or XOs4 (C-d and C-h). (D) XOs4 is not able to induce the Wnt-responsive markers Xnr3 and Sia. The indicated amounts of RNA were injected into the animal pole regions of two-cell stage embryos and ectodermal explants were processed for RT-PCR analysis as described in Materials and Methods.
	FIG. 5. (A) XOs4 dorsalizes ventral–marginal zone explants. Fourcell stage embryos were injected in the marginal zone region in all four blastomeres with RNA (1 ng/embryo) encoding XOs4 or Smad2. Ventral or dorsal marginal zone explants were dissected at Stage 101 and cultured until sibling control embryos reached tailbud stage (Stage 28). (B) XOs4 does not inhibit BMP signaling. Uninjected animal caps or animal caps expressing XOs4 (1 ng) were dissociated at late blastula stage and cultured in the presence or absence of BMP (10 ng/ml). At the end of neurulation, cells were reaggregated and cultured until control embryos reached late neurula stage (Stage 15) and processed for RT-PCR analysis for molecular markers for neural and epidermal tissue.
	FIG. 6. XOs4 induces dorsal and lateral mesoderm in animal cap explants. (A) XOs4-expressing (1 ng) animal caps exhibit a partially elongated morphology indicated an inductive event. (a) Uninjected animal caps. (b) XOs4-injected animal caps at Stage 17. (B) XOs4- expressing animal caps express markers of posterior neural, neural crest, and dorsal and lateral mesoderm. Embryos were injected at the two-cell stage with the indicated RNAs. Animal caps explants were assayed by RT-PCR for expression of the indicated molecular markers at Stage 28.
	FIG. 7. XOs4 induces expression of mesoderm markers in animal cap explants by late gastrula stages. (A) RNA encoding Smad2 (1 ng) or XOs4 (1 ng) was injected into the animal pole region of two-cell stage embryos. Animal cap explants expressing Smad2 or XOs4 were assayed by RT-PCR for expression of the indicated mesoderm markers at early gastrula stage (10.5) (A) or late gastrula stage (12) (B).
	FIG. 8. (A) XOs4 does not require activity of the Activin/Nodal/ Vg1 signal-transduction pathway upstream of FAST1 for induction of mesodermal derivatives. Embryos were injected with RNA (1 ng/embryo) encoding either tAR or Fast-EnR in the absence or presence of XOs4. Animal cap explants were dissected at blastula stage, incubated in the presence or absence of Activin as indicated, and cultured until control embryos reached tailbud stages (Stage 28). Animal cap explants were then processed for RT-PCR analysis for expression of HoxB9, which is strongly induced by both Activin and XOs4. (B) Like Smad2, XOs4 requires the FGF pathway for expression of mesoderm markers at tadpole stages. Embryos were injected with the indicated RNAs and animal cap explants were assayed by RT-PCR for HoxB9 expression at tadpole stage. XFR (1.5 ng/embryo), dominant-inhibitiory Ras(17N), Smad2, and XOs4 (1 ng/embryo). Basic FGF (FGF) was added to a final concentration of 50 mg/ml.
	ctdsp2 (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
	ctdsp2 (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, dorsal up, anterior left.