Ault KT et al. (1996), A novel homeobox gene PV.1 mediates induction o...

XB-ART-18058

Proc Natl Acad Sci U S A 1996 Jun 25;9313:6415-20. doi: 10.1073/pnas.93.13.6415.

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A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.

Ault KT , Dirksen ML , Jamrich M .

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The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a bmp4 evx1 gsc tbxt ventx1 ventx1.2

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	FIG. 1. Identification of a ventral-specific homeobox gene. (A) PV.1 homeodomain sequence compared with the murine Emxl. Dots indicate identities. (B) Temporal expression pattern of Xenopus PV.1. Northern blot analysis of Xenopus total RNA isolated from embryos at indicated stages. Equal amounts of RNA were loaded in each lane. The blot was probed with a fragment of PVl. cDNA; a single 1.5-kb transcript was detected. PV.1 expression is first detected at stage 10, peaks at stage 11, slowly decreases and is not detectable by stage 30. (C and D) Spatial expression pattern of PV. 1. Transcripts were detected by whole mount in situ hybridization of albino embryos using digoxigenin-labeled PV.1 antisense probe. (C) Vegetal view of stage 11 embryos showing the ventral marginal zone expression of PV.1. Arrow marks the position of the organizer. (D) Lateral view of stage 14 embryos (anterior is to the left and dorsal on top). PV1 expression is evident throughout the ventral and posterior regions, whereas the dorso-anterior region is negative.
	FIG. 2. Induction of PV.1 by BMP4. (A) Northern blot analysis of total RNA isolated from embryos injected with 1.0 ng of BMP4 mRNA and from uninjected control embryos. Total RNA was isolated from stages 11, 14, and 26 embryos. Five micrograms of total RNA was loaded on each lane. Hybridization of the blot with PV.1 probe revealed that PV.1 is strongly induced by overexpression of BMP4. (B and C) In situ hybridization of PV.1 to animal caps from uninjected embryos (B) or to animal caps from embryos overexpressing BMP4 (C). Animal caps were dissected at stage 9 and cultured until stage 12. (D) In situ hybridization of PV.1 to a stage 14 embryo injected with BMP4 (right) and an uninjected stage 14 embryo (left).
	FIG. 3. Misexpression of PV.1 in dorsal mesoderm ventroposteriorizes intact embryos and rescues embryos partially dorsalized by LiCl treatment. (A) Lateral view of stage 36 embryos dorsally injected with 1 ng of PV.1 mRNA. PV.1 injected embryos appear extremely ventroposteriorized. (B) Control embryos dorsally injected with 1 ng of B-galactosidase mRNA. (C) Lateral view of stage 36 embryos ventrally injected at the 4-cell stage with 1.5 ng of PV.1 mRNA and treated at the 32-cell stage with 0.3 M LiCl. (D) Control embryos treated with 0.3 M LiCl alone. Ventral injection of PV.1 mRNA rescues embryos partially dorsalized by LiCl.
	FIG. 4. (A-D) PV.1 inhibits elongation of animal caps in response to activin. Two-cell stage embryos were injected in the animal pole with 1.5 ng of mRNA encoding PVI or f3-galctosidase. Animal caps were excised from stage 8 injected embryos and were treated with 100 pM activin A or a control solution and cultured for two days. Caps excised from B-galactosidase-injected embryos and treated with activin A (A) show significant elongation relative to untreated controls (B). Elongation in response to activin is inhibited in caps overexpressing P1K (C). No elongation is observed in caps overexpressing PV1I and not treated with activin (D). (E) Overexpression of PV.l in animal caps inhibits induction of gsc and C-actin and enhances Xhox3 expression in response to activin. Animal caps overexpressing PV1 and control caps were treated with activin A or a control solution. Caps were cultured until siblings reached stage 11. Total RNA was isolated and assayed for expression of gsc, C-actin, and Xhox3 by RT-PCR. Expression levels of Xbra were measured as an indicator of generic mesoderm induction by activin. Histone H4 was used to normalize between samples. Template from which RT was omitted (indicated as NRT in the figure) was used to confirm absence of contaminating genomic DNA sequences. Relative to activin-induced control caps, caps overexpressing PV.1 show significant reduction in gsc and C-actin expression levels and enhancement of Xhox3 in response to activin. Caps overexpressing PV.1 but not induced with activin A express low levels of Xbra and Xhox3 relative to uninduced control caps.
	FIG. 5. PV.1 counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Embryos were ventrally injected at the 4-cell stage with a mixture of mRNAs encoding the DNR (2 ng) and PV.1 (1.5 ng) (A) or with mRNA encoding the DNR alone (2 ng) (B). Embryos were cultured until stage 42 and were scored according to the DAI. Embryos receiving injections of mRNA encoding the DNR alone display DAIs ranging from 6-8 (n = 30), whereas embryos injected with mRNA encoding the DNR plus PV.1 develop normally (DAI = 5; n = 28).

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Bailey, Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. 1976, Pubmed