Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dev Growth Differ
2009 Oct 01;518:699-706. doi: 10.1111/j.1440-169X.2009.01129.x.
Show Gene links
Show Anatomy links
Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development.
Laurent A
,
Masse J
,
Deschamps S
,
Burel A
,
Omilli F
,
Richard-Parpaillon L
,
Pellerin I
.
???displayArticle.abstract??? ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N-cam expression patterns, whereas krox-20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos-7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.
Fig. 1. Expression patterns of ZFPIP (h) and Pbx1 ( ) in Xenopus.
Quantitative reverse transcription–polymerase chain reaction
(qRT–PCR) assays were carried out in order to follow ZFPIP and
Pbx1 mRNAs dynamics during Xenopus laevis development (stage
1–33). For both genes, relative expression was primarily low,
started to increase at stage 17 and kept on augmenting until stage
33 (a). Western blot analysis revealed overlapping expression of
ZFPIP and Pbx1b proteins from stage 33–46. Proliferating Cell
Nuclear antigen (PCNA) was used as an internal control (b).
Fig. 2. Interaction of xZFPIPp and
mZFPIPp with Pbx1 in vitro. Xenopus
laevis (Xl) peptide was compared to
Mus musculus (Mm) ZFPIPp. The
xZFPIPp peptide contained 660 AA
and corresponded to the Pbx1
binding domain of the mouse ZFPIP
protein (mZFPIPp) identified from the
two hybrid screens. The identical
or equivalent amino-acids between
xZFPIPp and mZFPIPp proteins are
indicated respectively by ‘‘:’’ and ‘‘.’’
(a). GST pull-down assays were
carried out using bacterially
expressed Pbx1-GST and in vitro
translated xZFPIPp and mZFPIPp.
The mouse protein corresponded to
the band at approximately 32 kDa,
whereas the xenopus protein band
was about 35 kDa. While neither
xZFPIPp nor mZFPIPp was retained
by control GST proteins, they both
interacted with Pbx1-GST (b).
Fig. 3. Injection of a dominant
negative form of ZFPIP (ZFPIPp)
perturbed neural markers. The xZFPIPp
transcripts were injected in one
blastomere of two cell-stage embryos.
The correct injection of xZFPIPp
mRNAs were checked by whole mount
hybridization using xZFPIPp as
riboprobes (a). The correct translation
of the protein was checked by Western
blots carried out with a-ZFPIP
antibodies on extracts prepared from
injected and non-injected stage 22
embryos (b). Whole mount hybridizations
of late neurula embryos using
N-cam, en2 or krox-20 riboprobes
demonstrated an altered expression of
N-cam and en2 expression in injected
blastomeres. The side injected with
xZFPIPp capped mRNAs and the
non-injected side are visualized by
b-galactosidase staining and are
indicated respectively by * and ** (c).
Fig. 4. Co-expression of ZFPIPp-
FLAG or N-ZFPIP-FLAG and Pbx1b-HA
in Cos-7 cells. Immunocytochemistry
assays were carried out using a-HA and
a-FLAG antibodies on cells expressing
independently or co-expressing ZFPIP
proteins (ZFPIPp-FLAG or N-ZFPIPFLAG)
and Pbx1b-HA. ZFPIPp-FLAG,
N-ZFPIP-FLAG and Pbx1b-HAproteins
were observed, respectively, in the
nucleus and in the cytoplasm when they
were expressed separately. In contrast,
co-expression in the same cell type
of ZFPIPp or N-ZFPIP and Pbx1b,
led to cytoplasmic and perinuclear
localization of ZFPIP â„ Pbx1 complexes.
Fig. 5. ZFPIP is contained within a syntenic region. Using
AutoGRAPH software (see methods), ZFPIP region in Homo
sapiens (chromosome 9), Mus musculus (chromosome 4) and
Canis familiaris (chromosome 11) were compared. The ZFPIP
gene belongs to a syntenic genomic region, which has not been
rearranged in the dog, mouse and human lineages since their
last common ancestor. ZFPIP is contiguous to another zing
finger-containing gene referred to as KLF4 (AC: NC000070.5).
Arrows indicate the location of the ZFPIP gene on the different
genomes (from left to right: murin, human and canine genomes).
The colored lines connecting orthologous genes show the
conservation of gene order within conserved segments. Black
lines between colored segments represent breakpoints.
