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???displayArticle.abstract??? Bone morphogenetic protein 4 (BMP4), a member of the TGF beta superfamily, has been implicated in the dorsoventral specification of both mesoderm and ectoderm. High levels of BMP4 signaling appear to specify ventral lineages, while lower levels are causally associated with the development of dorsal lineages. We have previously identified a homeobox-containing transcription factor (PV. 1) which is a likely mediator of the ventralizing effects of BMP4 in the mesoderm. Here we provide evidence that PV.1 also functions downstream of BMP4 in the patterning of ectoderm, specifying epidermal and suppressing neural gene expression. PV.1 is expressed in the prospective neuroectoderm at the time of ectodermal fate determination. BMP4 and xSmad1 (a downstream effector of BMP4) induce PV.1 in uncommitted ectoderm and the dominant negative form of the BMP4 receptor (DN-BR) blocks PV.1 expression. In animal pole explants PV.1 counteracts the neuralizing effects of chordin and the DN-BR and restores them to their original epidermal fate. To address the physiological significance of these observations we employed an animal cap transplantation system and demonstrated that overexpression of PV.1 in the prospective neuroectoderm specifically blocks neurogenesis in intact embryos. Thus, PV.1 plays an important role in the ventralization of both mesoderm and ectoderm. We have previously shown that PV.1 is also preferentially expressed in the ventral endoderm, suggesting that this transcription factor may be involved in the ventralization of all three germ layers.
FIG. 1. Comparison of the coding regions of PV.1 (upper line) and Xvent1 (Gawantka et al., 1995; lower line). Amino acids are in the
single-letter IUPAC code. Colons indicate identities. Asterisks represent stop codons. The homeodomain is underlined.
FIG. 2. PV.1 is expressed in the ectoderm of gastrula stage Xenopus
embryos. Transcripts were detected by whole-mount in situ hybridization of albino embryos using digoxigenin-labeled PV.1 antisense probe. Lateral viewof a stage 11 embryo. Dorsal is left and ventral is right. Animal pole ectoderm is on top. PV.1 expression is evident in the ectoderm in a ventral to dorsal gradient.
FIG. 3. Induction of PV.1 by BMP4 and XMad1. RT-PCR analysis. Animal caps injected with mRNA encoding BMP4 (1 ng), xSmad1 (2 ng), the DN-BR (1 ng), or b-galactosidase (2 ng) were cultured until siblings reached stage 11. Total RNA was isolated and assayed for expression of PV.1 by RT-PCR. EF-1a was used to normalize between samples. Template from which RT was omitted (indicated as NRT in the figure) was used to confirm the absence of contaminating genomic DNA sequences. Overexpression of BMP4 or xSMad1 in animal caps induces PV.1, whereas ectopic expression of the DN-BR strongly inhibits PV.1 expression.
FIG. 4. PV.1 rescues embryos dorsalized by ventral overexpression of chordin. (A) Lateral view of stage 36 embryos dorsally injected
with 1 ng of chordin mRNA plus 1 ng of PV.1 mRNA. (B) Control embryos coinjected with 1 ng each of mRNA encoding chordin and bgalactosidase.
Embryos receiving injections of mRNA encoding chordin plus b-galactosidase display DAIs ranging from 6 to 9 (n  27),
whereas embryos injected with mRNA encoding chordin plus PV.1 develop normally (DAI  5; n  32).
FIG. 5. PV.1 inhibits neuralization in response to chordin and the DN-BR. Two-cell stage embryos were injected in the animal pole with
1.0 ng each of mRNA encoding chordin and PV.1 or chordin and b-galactosidase. Animal caps were excised from stage 8 injected embryos
and cultured until siblings reached stage 28. Caps excised from chordin plus b-galactosidase-injected embryos (A) show extensive cement
gland formation relative to controls (C). Cement gland formation in response to chordin is inhibited in caps coexpressing PV.1 (B). (D)
Coexpression of PV.1 inhibits induction of NCAM and NF-M and enhances XK-81 expression in response to chordin or the DN-BR.
Injected animal caps were cultured until siblings reached stage 28. Total RNA was isolated and assayed for expression of NCAM, NF-M,
or XK81 by RT-PCR. Histone H4 was used to normalize between samples. Caps coexpressing chordin or the DN-BR with PV.1 show
significant reduction in NCAM and NF-M expression levels and enhancement of XK-81 expression.
FIG. 6. PV.1 inhibits neurogenesis in vivo. Two-cell stage donor embryos were injected in the animal hemisphere with 2 ng of PV.1 or
control b-galactosidase RNA . ACs were dissected from the injected embryos at stage 8 and transplanted to the region of the prospective
neuroectoderm of recipient stage 10 embryos. Transplant embryos were allowed to develop to stage 30. Some PV.1- or b-galactosidaseinjected
embryos were left intact and served as no transplantation controls (0TP). Embryos were then harvested for whole-mount immunostaining
and photography (A) or RT-PCR analysis (B). Embryos receiving transplants expressing PV.1 (lower right panel) lack a significant
dorsoanterior structure, while those receiving b-galactosidase-expressing transplants (lower left panel) develop normally. Immunostaining
of embryos with 12/101 antibodies reveals somites in PV.1 transplant embryos (lower right, n  8/10), while embryos globally expressing
PV.1 (upper right) show no somite development (n  10/10). (B) Some transplant embryos were cultured until stage 12 or 30. Total RNA
was isolated and assayed for expression of actin, gsc, chordin (chd), and NCAM by RT-PCR. EF1 was used to normalize between samples.
PV.1 transplant embryos show reduced NCAM expression and normal levels of actin, chd, and gsc relative to controls (b-galactosidase
transplant embryos). Embryos expressing PV.1 and not transplanted display reduced levels of NCAM as well as actin, gsc, and chordin.
