Biochem Biophys Res Commun
April 12, 2002;
Antimorphic PV.1 causes secondary axis by inducing ectopic organizer.
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
. 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
Biochem Biophys Res Commun
[+] show captions
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
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.