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The Xvent family of homeobox transcription factors is essential for the establishment of the dorsal-ventral body axis during Xenopus embryogenesis. In contrast to Xvent-2B and other members of the Xvent-2 subfamily, Xvent-1B is not a direct response gene of bone morphogenetic protein-4 signaling. Xvent-1B is activated by Xvent-2, but CHX experiments revealed the requirement of additional factors. In this study, we report on the cooperative effect of Xvent-2 and the zinc finger transcription factor GATA-2 on the promoter of the Xvent-1B gene. We show that GATA-2 is a direct target gene of bone morphogenetic protein-4 and that GATA-2 interacts with Xvent-2 to activate transcription of Xvent-1B. Both transcription factors bind to distinct elements within the Xvent-1B promoter, and GATA-2 physically interacts with the C-terminal domain of Xvent-2. Promoter/reporter studies in Xenopus embryos revealed that full activation of Xvent-1B requires both Xvent-2 and GATA-2. Moreover, the two factors are sufficient to direct transcription of Xvent-1B in the presence of CHX at the ventral side of the embryo. The failure of both factors to activate Xvent-1B on the dorsal side suggests the existence of a dorsal inhibitor. This inhibitor is likely a component of the dorsal Wnt signaling pathway because nuclear translocation of beta-catenin before midblastula transition results in a suppression of Xvent-1B transcription.
FIG. 1. GATA-2 is activated directly by BMP-4 and activates
Xvent-1B. Xenopus embryos were injected with BMP-4 RNA (A) or with
GATA-2 RNA (B) into both dorsal blastomeres at the four-cell stage.
Embryos were treated with or without CHX at stage 7.0. Total RNA
isolated from stage 10.5 embryos was subjected to RT-PCR to evaluate
GATA-2 (A) or Xvent-1B (B) transcripts. Histone H4 transcripts were
used as internal controls. CâE, Xvent-1B transcripts visualized by
whole mount in situ hybridization in wild type embryos (C) and in
embryos injected previously at the four-cell stage dorsally with 250 pg
of GATA-2 RNA (D) or with 250 pg of Xvent-2 RNA (E). Embryos are
viewed from the vegetal pole, with the dorsal side at the top.
FIG. 2. GATA-2 rescues the effects of Xvent-2 P(40). The wild
type Xvent-1B promoter fragment (249/52) fused to the luciferase
(Luc) reporter gene was coinjected into both ventral blastomeres of
Xenopus embryos at the four-cell stage with 400 pg of dominant-negative
Xvent-2 RNA (Xvent-2 P(40)), 400 pg of tBR RNA, 50 pg of GATA-2
RNA, or a combination of these RNAs, as indicated. Luciferase activity
was measured at stage 11.
FIG. 3. GATA-2 interacts with the C-terminal domain of
Xvent-2. A, GATA-2 was labeled with [35S]methionine by in vitro transcription/translation
and incubated with GST or GST-Xvent-2. After
washing, the reactions were analyzed by 10% SDS-PAGE. B, extracts
from Xenopus embryos containing Myc-tagged (MT) Xvent-2 were incubated
with GST or GST-GATA-2. Bound Xvent-2 was detected by Western
blot analysis using a Myc antibody. C, Xvent-2 including the mutants
Xvent-2 C, Xvent-2 N, and Xvent-2 P(40) were labeled with
[
35S]methionine and incubated with GST or GST-GATA-2. Reactions
were analyzed by 10% SDS-PAGE.
FIG. 4. GATA-2 and Xvent-2 bind to
the Xvent-1B promoter. A, schematic
summary of results from electrophoretic
mobility shift assay with 32P-labeled fragments
of the Xvent-1B promoter and
Xvent-2, Xvent-2 P(40), or GATA-2 proteins
(as indicated). indicates retardation
of the DNA fragment; indicates no
retardation. BâD, DNase I footprinting
analysis of GATA-2 (B) or Xvent-2 protein
(C) to the 32P-labeled 249/13 or of
Xvent-2 to the 32P-labeled 249/164
Xvent-1B promoter fragment (D). G/A indicates
guanine and adenine residues
from chemical sequencing reactions; triangles
show increasing amounts of protein
(from 40 to 160 ng). Protected regions
are indicated by vertical lines, and
GATA-2 and Xvent-2 target motifs are
shown in boxes.
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FIG. 5. Effects of Xvent-2 and GATA-2 binding sites within the Xvent-1B promoter. A, the wild type Xvent-1B promoter and indicated
deletions or mutations of this fragment (see inset; marks the GATA-2 and # the Xvent-2 binding sites; the arrow denotes a GATA-2 target
sequence, and mutations are indicated by asterisks) were cloned in front of the luciferase (Luc) reporter and either injected alone or coinjected with
400 pg of Xvent-2 RNA or 50 pg of GATA-2 RNA into both dorsal blastomeres of four-cell stage embryos. Luciferase activity was measured at stage
11. B, pGL3 constructs of the wild type (WT) or an Xvent-1B promoter mutated in the GATA-2 binding site (Gm) were coinjected with 50 pg of
GATA-2 and/or 200 pg of Xvent-2 RNA into both dorsal blastomeres of Xenopus embryos at the four-cell stage. Embryos were collected at stage 11
for luciferase reporter assay.
FIG. 6. Direct activation of Xvent-1B
by Xvent-2 and GATA-2. Xenopus embryos
were injected with (A) 400 pg of
Xvent-2 RNA or Xvent-2 and 50 pg of
GATA-2 RNA or (B) Xvent-2, GATA-2,
and 500 pg of BMP-4 RNA at the four-cell
stage as indicated. At stage 7, half of the
embryos were treated with CHX. Total
RNA was extracted when control embryos
had reached stage 10.5 and subjected to
RT-PCR to evaluate Xvent-1B transcripts.
Histone H4 transcripts were determined
as an internal controls.
FIG. 7. Inhibition of Xvent-1B by
dorsal Wnt signaling. Microinjection of
200 pg of Xvent-2 and 50 pg of GATA-2
RNA into both dorsal blastomeres of Xenopus
embryos at the four-cell stage is
shown. Embryos were treated with 300
mM lithium chloride at stage 5 (LiCl St. 5)
and/or 25 g/ml CHX at stage 7 and cultured
until control embryos reached stage
10.5. cDNA synthesis was performed by
using total RNA. Xvent-1B transcripts
were detected using the Roche LightCycler
system and quantified in relation to
ornithine decarboxylase.
FIG. 8. Effects of pre- and post-MBT Wnt signaling on Xvent-1B
promoter activity. A, Xenopus embryos were injected at the four-cell
stage into both ventral blastomeres with 200 pg of Xvent-2/50 pg of
GATA-2 and/or 500 pg of -catenin RNA together with 20 pg of the wild
type (WT) –249/52 or a 249/Tm/52 Xvent-1B (see inset; the arrow
denotes a LEF/TCF target sequence, and mutations are indicated by
asterisks). B, 20 pg of –249/52, 249/Tm/52, or –249/Gm/52
Xvent-1B promoter pGL3 DNA was injected with or without 350 pg of
CMV-Xwnt-8 DNA into both dorsal blastomeres of four-cell stage embryos.
Luciferase (Luc) activity was measured at stage 11.
