Hwang YS et al. (2002), Antimorphic PV.1 causes secondary axis by induc...

XB-ART-7332

Biochem Biophys Res Commun 2002 Apr 12;2924:1081-6. doi: 10.1006/bbrc.2002.6740.

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Antimorphic PV.1 causes secondary axis by inducing ectopic organizer.

Hwang YS , Seo JJ , Cha SW , Lee HS , Lee SY , Roh DH , Kung Hf HF , Kim J , Ja Park M .

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Xenopus homeobox gene, PV.1 ventralizes activin-induced dorsal mesoderm and inhibits neuralization of ectoderm in animal cap when overexpressed. Here we generated PV.1/engrailed fusion construct (N-PV1-EnR) to perform loss-of-function study for this transcription factor. N-PV1-EnR showed an extremely antimorphic effect, causing a partial secondary embryonic axis when expressed at ventral marginal zone of blastula. In ventral marginal zone cells, this chimeric protein induced organizer genes and suppressed ventral markers mimicking those effects reported for dominant negative BMP-4 receptor (DNBR). Moreover, N-PV1-EnR rescued the ventralized embryos caused by the ectopic dorsal expression of PV.1 but not by that of Xvent-2. These results suggested that PV.1 functions at downstream of BMP-4 as a ventralizing effector which acts separately from Xvent-2 and the dominant negative effect gained by this specific mutant is applicable for the further studies of BMP-4 downstream pathway.

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Species referenced: Xenopus
Genes referenced: bmp4 chrd evx1 ncam1 not ventx1 ventx2 ventx2.2

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	FIG. 1. Schematic diagram of constructs for antimorphic PV.1. To make chimeric PV.1 protein we generated three constructs by fusing Drosophila Engrailed repressor domain to the C-terminal end of PV.1 whole peptide (1; PV1-EnR) or to the C-terminus-deleted form of PV.1 (2; NH-PV1EnR) or to the N-terminus alone(3; N-PV1- EnR). Engrailed repressor domain is dotted box. Homeodomain is gray box.
	FIG. 2. Antimorphic PV.1 can induce secondary embryonic axis. Embryos at 4-cell stage were microinjected with indicated mRNA dorsally (A, C, E, G, I, and J) or ventrally (B, D, F, and H; injection site is depicted in small diagram at upper right corner) and allowed to grow until stage 29–33. The amount of injected mRNA is given in Table 1 (all below are also presented in Table 1). The rightmost embryos in each picture are injected with higher dose. -galactosidase injected control (J) embryos were normal. Ventral injection of N-PV1-EnR resulted in secondary axis formation (52, 89%; H; arrows indicate secondary axes) while dorsal injection of N-PV1-EnR did not (G). PV1-EnR, NH-PV1-EnR still has ventralizing effect when dorsally injected (C, E). Embryos injected with BMP-4 mRNA showed typical acephaly or microcephaly (arrowheads indicate shrunken anterior structure; I).
	FIG. 3. Specificity of PV1-N-EnR with respect to ventralizing gene, Xvent-2. 4-cell stage embryos were microinjected dorsally with PV.1 (0.25 ng/blastomere; showed in parentheses) or mixture of PV.1 (0.25 ng) and N-PV1-EnR (0.5 ng) or Xvent-2 (0.2 ng) or mixture of Xvent-2 (0.2 ng) and N-PV1-EnR (0.5 ng). Embryos injected with PV.1 mRNA showed acephaly or microcephaly revealing extreme ventralization of dorsal structure as shown (A; n 25; mean DAI 1.3; also given in Table 2) with low(0–3) grade of DAI. When N-PV1- EnR mRNA was coinjected with PV.1 mRNA, this phenotypic change was rescued (B; n 27; mean DAI 4.9). When Xvent-2 mRNA alone (0.2 ng) was dorsally injected, all embryos were extremely ventralized (C; n 28; mean DAI 1.23). This ventralized phenotype induced by injection of Xvent-2 (0.2 ng) mRNA was not rescued at all when co-injected with N-PV1-EnR (0.5 ng) mRNA (D; n 25; mean DAI 1.3; also given in Table 2).
	FIG. 4. Antimorphic PV.1 induces organizer genes and suppresses ventral specific markers. Embryos were either noninjected (VMZ; DMZ; whole embryo, WE), or injected ventrally at 4-cell stage with mRNAs encoding N-PV1-EnR(0.5 ng/blastomere), PV.1 (0.25), DNBR (0.5)DNBR (0.5). Ventral and dorsal marginal zone explants of some embryos were dissected at stage10–10.5 followed by culture. At stage 12 or stage 25, tissue explants were harvested for RT-PCR analysis. Total RNA was isolated and assayed for expression of Xnot, goosecoid, chordin, folistatin, GATA-2, Xhox3, NCAM, and globin by RT-PCR. EF1 was used to normalize the quantity of samples. One-twentieth of extracted RNA pool from each group was gathered and used for no-RT control.