August 1, 2006;
Xenopus POU factors of subclass V inhibit activin/nodal signaling during gastrulation.
Three POU factors of subclass V, Oct-25
are expressed in Xenopus oocytes and early embryos. We here demonstrate that vegetal overexpression of Oct-25
or mammalian Oct-3/4 suppresses mesendoderm
formation in Xenopus embryos. Oct-25
are shown to inhibit activin/nodal
and FGF signaling pathways. Loss of Oct-25
function results in elevated transcription of mesendodermal marker genes and ectopic formation of endoderm
in the equatorial region of gastrula stage
embryos. Within the ectoderm
promotes neural fate by upregulating neuroectodermal genes, such as Xsox2
, which prevent differentiation of neural progenitors into neurons. We also show that mouse Oct-3/4 and Xenopus Oct-25
behave as functional homologues. We conclude that Xenopus Oct proteins are required to control the levels of embryonic signaling pathways, thereby ensuring the correct specification of germ layers.
[+] show captions
Fig. 1. Vegetally overexpressed Oct-25, Oct-60, Oct-91 or mOct-3/4 blocks mesendoderm formation. (A) An uninjected control embryo at stage 11.5 showing normal gastrulation. (B–E) Embryos injected with Oct-25 (B), Oct-60 (C), Oct-91 (D) and mOct-3/4 (E) show the same failure of blastopore formation and lack of gastrulation. (F) A control embryo at stage 10.5 shows normal expression of Xbra. (G–J) Embryos injected with Oct-25 (G), Oct-60 (H), Oct-91 (I) and mOct-3/4 (J) show nearly no or severely reduced expression of Xbra. (K) A control embryo at stage 10.5 shows normal expression of Xsox17α. (L–O) Embryos injected with Oct-25 (L), Oct-60 (M), Oct-91 (N) and mOct-3/4 (O) show nearly no or severely reduced expression of Xsox17α. All embryos above are shown in vegetal view. (P) Real-time RT-PCR shows that Oct-25, Oct-60 or mOct-3/4 injections at the vegetal pole significantly inhibit transcription of mesendodermal genes.
Fig. 5. Functional knockdown of Oct-25 or Oct-60 expands the mesendodermal territory towards the upper equatorial region. (A) Immunoblotting using an Oct-25 antibody shows that Oct25MO specifically inhibits protein expression of Oct-25 in embryos. Lane 1, uninjected whole embryos at stage 10; lanes 2 and 3, embryos injected with 10 and 15 ng of ctrlMO; lanes 4 and 5, embryos injected with 10 and 15 ng of Oct25MO. Arrowhead indicates the band corresponding to Oct-25. (B) Immunoblotting using an antibody against the myc-tag shows that Oct60MOa1 specifically inhibits Oct-60MT protein expression. Lane 1, uninjected control animal caps at stage 10; lane 2, caps injected with 400 pg Oct-60MT RNA alone; lanes 3 and 4, caps co-injected with 400 pg Oct-60MT RNA and 20 or 40 ng of Oct60MOa1; lane 5, caps co-injected with Oct-60MT RNA and 40 ng of ctrlMO. (C) Posterior view of an embryo injected with ctrlMO showing normal development with fully closed blastopore at the end of gastrulation. (D–F) Embryos injected with Oct25MO (D), Oct60MOs (E), or a mixture of them (F) show similar defect in blastopore closure. (G) Whole mount in situ hybridization (vegetal view, lateral view, and tissue section) shows normal expression pattern for Xbra in embryos injected with ctrlMO. (H) In embryos injected with Oct25MO and Oct60MOs, the Xbra expression domain expands significantly towards the equatorial region. (I) Whole mount in situ hybridization (vegetal view, lateral view, and tissue section) shows normal expression pattern for Xsox17α in embryos injected with ctrlMO. (J) In embryos co-injected with Oct25MO and Oct60MOs, expression is enhanced and shifted towards the equatorial region. (K) Real-time RT-PCR shows significant upregulation of mesodermal genes and ectopic expression of endodermal genes in equatorial explants excised from embryos injected with Oct25MO and Oct60MOs. (L) Real-time RT-PCR shows downregulation of the neuroectodermal gene, Xsox2, in response to Oct-25 knockdown.
Fig. 2. Overexpression of Oct-25 promotes expression of early neuroectodermal genes. Real-time RT-PCR shows upregulation of Xsox2, Xsox3, Geminin and Zic1 at stage 10 in animal caps overexpressing Oct-25.
Fig. 3. Oct-25 and Oct-60 inhibit the activities of bFGF and activin A in animal caps. (A) Naïve animal caps at stage 20 develop into atypical epidermis. (B) Animal caps from embryos injected with Oct-25 RNA. (C) Animal caps from embryos injected with Oct-60 RNA. (D) Animal caps treated with bFGF at 500 ng/ml show mild elongation. (E and F) Animal caps injected with Oct-25 (E) or Oct-60 (F) and treated with the same dose of bFGF show no elongation. (G) Animal caps treated with activin A at 10 ng/ml show characteristic elongation. (H and I) Animal caps injected with Oct-25 (H) or Oct-60 (I) and treated with the same dose of activin A show no elongation. (J) Real-time RT-PCR demonstrates that Oct-25 and Oct-60 dramatically inhibit tissue differentiation induced by bFGF. (K) Real-time RT-PCR demonstrates that Oct-25 and Oct-60 block tissue differentiation induced by activin A.
Fig. 4. Oct-25 inhibits the activities of Xnrs in embryos. (A) An uninjected control embryo at stage 32. (B, D and F) Injection of Xnr2 (B), Xnr4 (D) or Xnr5 (F) into two ventral vegetal cells at 8-cell stage causes a dorsalization effect. (C, E and G) Co-injection of Oct-25 with Xnr2 (C), Xnr4 (E) or Xnr5 (G) represses the dorsalization effect.
Fig. 6. Functional knockdown of Oct-25 or Oct-60 can be rescued by mOct-3/4. (A) A tailbud embryo injected with ctrlMO shows normal development. (B and C) Embryos injected with Oct25MO (B) or Oct60MOs (C) show similar phenotypic defects. (D) Embryos injected with a mixture of Oct25MO and Oct60MOs reveal even a stronger phenotype. (E–H) Co-injection of Oct-25 RNA (E and G) or Oct-60 RNA (F and H) rescues the Oct-25MO (E and H) or the Oct-60MOs (F and G) phenotypes, respectively. (I and J) Co-injection of mOct-3/4 RNA rescues defects caused by Oct25MO (I) or Oct60MOs (J).