Witta SE et al. (1995), XIPOU 2, a noggin-inducible gene, has direct ne...

XB-ART-20031

Development 1995 Mar 01;1213:721-30.

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XIPOU 2, a noggin-inducible gene, has direct neuralizing activity.

Witta SE , Agarwal VR , Sato SM .

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XIPOU 2, a member of the class III POU domain family, is expressed initially in Spemann's organizer, and later, in discrete regions of the developing nervous system in Xenopus laevis. XIPOU 2 may act downstream from initial neural induction events, since it is activated by the neural inducer, noggin. To determine if XIPOU 2 participates in the early events of neurogenesis, synthetic mRNA was microinjected into specific blastomeres of the 32-cell stage embryo. Misexpression of XIPOU 2 in the epidermis causes a direct switch in cell fate from an epidermal to a neuronal phenotype. In the absence of mesoderm induction, XIPOU 2 has the ability to induce a neuronal phenotype in uncommitted ectoderm. These data demonstrate the potential of XIPOU 2 to act as a master regulator of neurogenesis.

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Species referenced: Xenopus laevis
Genes referenced: fst gsc lhx1 mrc1 ncam1 nog pou3f4

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	Fig. 1. Nucleotide and deducted amino acid sequence of the open reading frame of XlPOU 2. The position of the starting methionine and stop codon are designated by symbols M and * respectively. An arrowhead shows the PstI site. An open arrow indicates the end of the truncated construct, tXlPOU 2. The introduced EcoRI site at position 780 bp in the mutant form of XlPOU 2 ORF is indicated. The sequence of the PCR primers used to generate the DNA internal standard are underlined by dashed lines.
	Fig. 2. The expression pattern of XlPOU 2 during development determined using whole-mount in situ hybridization and sections of stained embryos. (A). View of the vegetal pole of an embryo at stage 10. The arrow indicates expression in the dorsal lip. (B). Vegetal view of an embryo at stage 10.25 showing staining in the involuting mesoderm. The white line indicates the plane of section in C. (C) A cryostat section of the dorsal region of an embryo at stage 10.25 after whole-mount in situ hybridization showing the expression of XlPOU 2 in the involuting dorsal mesoderm. The nuclear staining in the endoderm is indicated by the white lines. (D) Lateral view of an embryo at stage 12. XlPOU 2 is expressed in both the dorsal mesoderm and neuroectoderm. (E) A cryostat sagittal section of a stage 12 embryo, after wholemount in situ hybridization, showing the expression of XlPOU 2 in the dorsal mesoderm, the overlying neuroectoderm and weak expression in the ventral mesoderm. (F) Lateral view of an embryo at stage 16. XlPOU 2 is expressed in the entire neural folds. (G) Lateral view of an embryo at stage 20. The arrow indicates strong expression in the midbrain-hindbrain boundary. (H) Tailbud embryo, stage 27. Lateral view showing expression in subdivisions of the brain and in the pronephros. (I) A dorsal view of the same embryo depicted in H showing expression in portions of the hindbrain, midbrain and forebrain. A, anterior; ar, archenteron; bl, blastocoel; dl, dorsal lip; dm; dorsal mesoderm; en, endoderm; ec, ectoderm; fb, forebrain; hb, hindbrain; mb, midbrain; ne, neuroectoderm; nt, neural folds; P, posterior, pn, pronephros; vm, ventral mesoderm; yp, yolk plug.
	Fig. 3. The expression of XlPOU 2 during development. (A) RNA was extracted from embryos at the indicated stages of development and assayed for the expression of XlPOU 2 by RT-PCR. (B) The data from A was quantified by using a scanning densitometer. An internal standard was used to calculate the copy number of RNA/mg total RNA at each stage (see Materials and Methods). (C) RNA was extracted from dissected pieces of embryos at stage 10.25, and assayed for the expression of XlPOU 2 by RT-PCR. (D) The quantitation of data shown in C. The ubiquitous marker, EF1-a serves as a control.
	Fig. 4. Ectopic b-tubulin expression. (A) A transverse cryostat section from a late neurula stage embryo that had been probed first for b-tubulin expression using the whole-mount in situ hybridization procedure (blue reaction product), and then sectioned and immunostained with anti-12/101 (red reaction product). This embryo had been injected with 200 pg of XlPOU 2 mRNA into an A4 blastomere. The ectopic b-tubulin expression is indicated by the open arrows. (B) A late neurula stage embryo (experimental control, injected with 200 pg tXlPOU 2 mRNA) that had undergone the whole-mount in situ hybridization procedure using the b-tubulin probe (blue reaction product) followed by wholemount imunostaining with anti-12/101 (brown reaction product). No ectopic b- tubulin was ever detected in these embryos. (C) A transverse cryostat section from a tailbud stage embryo that had been probed for b-tubulin expression using the whole-mount in situ hybridization procedure (blue reaction product). This embryo had been injected with 200 pg of XlPOU 2 mRNA into an A4 blastomere. The ectopic neural expression is indicated by an arrow. e, endoderm; hb, hindbrain; nc, notochord; nt, neural tube; s, somites
	Fig. 5. XlPOU 2 induces b-tubulin and NCAM protein expression in animal cap explants. In A-C, animal cap explants were grown until the equivalent of stage 30 and then probed for b-tubulin expression by the whole-mount in situ hybridization procedure, followed by whole-mount immunostaining with anti-12/101. (A) Explants prepared from embryos in which one A4 blastomere had been injected with 200 pg XlPOU 2 mRNA. The b-tubulin expression is the dark blue reaction product and is indicated by the open arrows in A. (B) A cryostat section of one of the animal caps in A. Bar, 50 mm. (C) Explants prepared from experimental control embryos in which one A4 blastomere had been injected with 200 pg tXlPOU 2 mRNA. In E-F and H-I, animal cap explants were cultured, and then subjected to whole-mount immunostaining with an NCAM antibody, whose reaction product is dark blue. (D) Stage 10.5 sibling control showing no NCAM protein expression. (E) Explant cultured until the equivalent of stage 10.5 showing NCAM protein. Explants were prepared from embryos in which both pairs of A1 and A4 blastomeres had injected with XlPOU 2 mRNA. (F) Explant cultured until the equivalent of stage 10.5 showing no NCAM expression. Explants were prepared from embryos in which both pairs of A1 and A4 blastomeres had been injected with 200 pg of the control mRNA, tXlPOU 2 mRNA. (G) Stage 25 sibling control showing the expected pattern of NCAM protein expression in the central nervous system and peripheral nerves. (H) Explant cultured until the equivalent of stage 25 showing NCAM protein expression. White arrows indicate two of the many axons in this explant. Explants were prepared as in E. (I) Explant cultured until the equivalent of stage 25 showing no NCAM protein expression. Explants were prepared as in F.
	Fig. 6. Absence of mesoderm induction in animal cap explants from embryos injected with XlPOU 2 mRNA. (A) Northern blot analyses using cDNA probes for Xlim-1 and Gsc, 5 mg of total mRNA loaded per lane. Lane a, RNA from animal cap explants, prepared from embryos injected with 200 pg XlPOU 2 mRNA, aged to stage 10.5. Lane b, RNA from animal cap explants prepared from the sibling control embryos. Lane c, RNA prepared from whole embryos at stage 10.5; these embryos had been injected with XlPOU2 mRNA. Lane d, RNA prepared from the sibling control embryos. (B) The ethidium-stained gel from A showing that equal amounts of RNA had been loaded in each lane.
	Fig. 7. The induction of XlPOU 2 by noggin, but not follistatin or activin. RNA was extracted from 24 animal cap explants that had been dissected at stage 8, and cultured until the equivalent of stage 10.5, under varying conditions. (A) The expression of XlPOU 2, follistatin, gsc, and EF1-a was assayed by RT-PCR. In lanes 1 and 2, explants were prepared from embryos that had been injected at the 2- cell stage with 2 ng of XFS-319 mRNA or b-galactosidase mRNA, respectively. In lane 3, explants were prepared from uninjected embryos. In lanes 4 and 5, stage 8 explants were incubated in conditioned medium with, or without noggin, respectively. In lanes 6 and 7, explants were incubated in 100 pM activin/0.5´ MMR, or in 0.5´ MMR, respectively. The marker, gsc, served as a positive control for mesoderm induction by activin. (B) The data from A was quantified using a scanning densitometer. An internal XlPOU 2 DNA standard was used to quantify the induction of XlPOU 2 by noggin (see Materials and Methods).
	Fig. 8. Noggin and follistatin are not induced by XlPOU 2. Stage 8 explants were prepared from injected embryos (from the same experimental group shown in Fig. 6D-F), and cultured until stage 10.5. Total RNA was extracted, and the expression of NCAM, noggin, follistatin, and EF1-a was assayed by RT-PCR. The follistatin mRNA that is present in all samples is probably maternal mRNA.