Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
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 ventralmesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.
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 lateralmesoderm 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 lateralmesoderm. 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, anteriorleft.
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, anteriorleft.
