GIPC (GAIP interacting protein, C terminus) is a PDZ-domain-
containing protein initially identified by virtue of its interaction with
RGS-GAIP (regulator of G protein signaling-GTPase activating
protein for G?i) (De Vries et al., 1998b). The list of identified
binding partners for GIPC includes numerous membrane proteins
(Katoh, 2002), such as the transmembrane semaphorin M-SemF
(Wang et al., 1999), neuropilin 1 (Cai and Reed, 1999), the Wnt
receptor frizzled 3 (Tan et al., 2001), the ?1-adrenergic receptor (Hu
et al., 2003), the TGF?-type III receptor (Blobe et al., 2001),
melanosomal membrane protein gp75 (tyrosinase-related protein 1)
(Liu et al., 2001), the tyrosine kinase receptor TrkA (Lou et al.,
2001), intergrin ? subunits (El Mourabit et al., 2002; Tani and
Mercurio, 2001), insulin-like growth factor 1 receptor (IGF1R)
(Booth et al., 2002; Ligensa et al., 2001), human lutropin receptor
(Hirakawa et al., 2003), myosin VI (Dance et al., 2004; Hasson,
2003), dopamine D2 and D3 receptors (Jeanneteau et al., 2004a;
Jeanneteau et al., 2004b), a myeloid cell-surface marker CD93
(Bohlson et al., 2005) and human papillomavirus type 18 E6 protein
(Favre-Bonvin et al., 2005). Although the list of GIPC-binding
partners is long, the endogenous function of GIPC and the
physiological significance of these associations are much less
studied. Loss-of-function studies published so far have been
primarily in ex vivo or in vitro systems, such as Xenopusectodermal
explants (Tan et al., 2001) and cultured cell lines (Favre-Bonvin et
al., 2005; Hirakawa et al., 2003); the in vivo role of GIPC in
embryonic development has not yet been examined.
Previously, we identified a Xenopus homolog of GIPC, kermit
(Tan et al., 2001), that is 72% identical to mammalian GIPC (De
Vries et al., 1998b). Knockdown of kermit using antisense
morpholino oligonucleotides blocked neural crest induction by
Xenopus frizzled 3 in ectodermal (animal cap) explants (Tan et al.,
2001), but did not inhibit neural crest formation in whole embryos.
We reasoned that this negative result may indicate the presence of a
compensating or redundant activity in whole embryos that is absent
in animal cap explants. We therefore identified a second kermitgene
(67% amino acid identity with kermit 1) in Xenopus early embryos
to study the redundancy between kermitand kermit2. Unexpectedly,
we discovered a novel function for kermit 2 in IGF signaling that
does not overlap with kermit 1.
Kermit 2 is identical to XGIPC, identified in a yeast two hybrid
screen for IGF1 receptor binding proteins in Xenopus oocytes
(Booth et al., 2002). XGIPC/kermit 2 binds to the cytoplasmic
domain of the XenopusIGF1R and this interaction appears to require
the PDZ domain of XGIPC. Overexpression of C-terminal
truncation mutants of XGIPC that retain the PDZ domain blocks
insulin-induced Xenopus MAP kinase activation and oocyte
maturation. Human GIPC was also identified as a binding partner
for human IGF1R (Ligensa et al., 2001).
IGF signaling has been recently implicated in neural induction
in Xenopus and zebrafish embryos (Eivers et al., 2004; Pera et al.,
2003; Pera et al., 2001; Richard-Parpaillon et al., 2002). Ectopic
expression of IGF in dorsal cells leads to the induction of ectopic
eyes and eye expansion and, when expressed in ventral cells,
induces secondary head-like structures (Pera et al., 2001; Richard-
Parpaillon et al., 2002). Inhibition of IGF signaling by either
dominant-negative IGF1R or IGF1R depletion reduces head
structures (Pera et al., 2001; Richard-Parpaillon et al., 2002). As
kermit 2/XGIPC physically interacts with XIGF1R, we examined
the involvement of kermit 2/XGIPC in IGF signaling in Xenopus
We report here that kermit2/XGIPC is expressed throughout
Xenopus early embryonic development and is localized to the
anterior region during neurula stage in a pattern highly similar to
XIGF1R (Richard-Parpaillon et al., 2002). Knockdown of kermit 2
leads to embryos with reduced anterior structures, specifically
reduction in the presumptive eye region. Furthermore, depletion
of kermit 2 and expression of dominant-negative XIGF1R
Kermit 2/XGIPC, an IGF1 receptor interacting protein, is
required for IGF signaling in Xenopus eye development
Jinling Wu1, Michael O’Donnell2, Aaron D. Gitler1and Peter S. Klein1,2,*
GIPC is a PDZ-domain-containing protein identified in vertebrate and invertebrate organisms through its interaction with a variety
of binding partners including many membrane proteins. Despite the multiple reports identifying GIPC, its endogenous function and
the physiological significance of these interactions are much less studied. We have previously identified the Xenopus GIPC homolog
kermit as a frizzled 3 interacting protein that is required for frizzled 3 induction of neural crest in ectodermal explants. We
identified a second Xenopus GIPC homolog, named kermit 2 (also recently described as an IGF receptor interacting protein and
named XGIPC). Despite its high amino acid similarity with kermit, kermit 2/XGIPC has a distinct function in Xenopus embryos. Loss-
of-function analysis indicates that kermit 2/XGIPC is specifically required for Xenopus eye development. Kermit 2/XGIPC functions
downstream of IGF in eye formation and is required for maintaining IGF-induced AKT activation. A constitutively active PI3 kinase
partially rescues the Kermit 2/XGIPC loss-of-function phenotype. Our results provide the first in vivo loss of function analysis of GIPC
in embryonic development and also indicate that kermit 2/XGIPC is a novel component of the IGF pathway, potentially functioning
through modulation of the IGF1 receptor.
KEY WORDS: Kermit 2, GIPC, Insulin like growth factor (IGF), IGF1 receptor (IGF1R), Eye development, Xenopus
Development 133, 3651-3660 (2006) doi:10.1242/dev.02547
1Cell and Molecular Biology Graduate Group, University of Pennsylvania School of
Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA 19104,
USA. 2Department of Medicine (Hematology-Oncology), University of Pennsylvania
School of Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia,
PA 19104, USA.
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 18 July 2006
synergistically inhibit eye development. Kermit 2 is required for
IGF1 induced eye formation in whole embryos and for the induction
of eye molecular markers in ectodermal explants. Finally, we present
evidence that kermit 2 is required to maintain IGF/PI3 kinase-
dependent activation of AKT. These results indicate that kermit
2/XGIPC is required for IGF signaling in Xenopuseye development
likely through its interaction with the IGF1 receptor.
MATERIALS AND METHODS
Cloning of kermit2, plasmid constructs and in vitro transcription
kermit2 was identified through a Blast search of the Xenopus EST database
using the kermit sequence (Tan et al., 2001). The GenBank accession
number for kermit2 is BC089139. kermit2 5?UTR sequence was identified
from a stage 10 Xenopus 5?RACE cDNA library. Capped mRNAs were
synthesized by in vitro transcription of plasmids encoding XIGF1R
(Richard-Parpaillon et al., 2002), DN-IGFR (Pera et al., 2001), XIGF1
(Pera et al., 2001), p110* (Carballada et al., 2001), kermit 2/pCS2, kermit
2-GFP/pCS2 and 5?UTR-kermit 2-GFP/pCS2 (described below) using SP6
mMessage mMachine kits (Ambion). Kermit 2/pCS2 was cloned by
RT-PCR amplifying stage 1 Xenopus RNA using primers (forward,
AGGGATCCATGCCTCTGGGATTGCGCGTA; reverse, AGTCTAGAT-
TAAAAGCGTCCTTGTTTAGC) with engineered BamHI and XbaI sites
and cloned directionally into pCS2+. Kermit 2-GFP/pCS2 was generated
by PCR using kermit 2/pCS2 as the template and cloned into
BamHI/EcoRI sites of pCS2-EGFP. 5?UTR-kermit 2-GFP/pCS2 was
cloned by PCR amplifying using primers (upstream, CGGGATC-
TTCAAAGCGTCCTTGTTTAGCATC) with engineered BamHI and
EcoRI sites and cloned directionally into pCS2-EGFP.
RNA isolation and RT-PCR methods are described elsewhere (Yang et al.,
2002). Primers for EF-1? (Yang et al., 2002), ODC (Yang et al., 2002),
Otx2 (Cox and Hemmati-Brivanlou, 1995), En2 (Hemmati-Brivanlou et
al., 1994), muscle actin (Hemmati-Brivanlou and Melton, 1994), XAG
(Blitz and Cho, 1995) and Xrx (Mathers et al., 1997) have been described
previously. Primers were designed to detect kermit2 (upstream, ATGC-
CTCTGGGATTGCGCGTAAAG; downstream, TTTAACTTTGTCA-
ATCGTGCTCTC), HoxD1 (upstream, CACTTCTTGCGGGGATGTTT;
downstream, AGAGTCCTGTAGCTCAGCTG), Pax6 (by N. Hirsch and
W. Harris; upstream, AGTGTCCTCATTCACATCG; downstream, AG-
TACTGAGACATGTCAGG) and Vent1 (upstream, GCATCTCCTTG-
GCATATTTGG; downstream, TTCCCTTCAGCATGGTTCAAC).
Lineage tracing and in situ hybridization
Embryos were co-injected with RNA encoding ?-galactosidase with a
nuclear localization motif and control morpholino or kermit 2 morpholino
(5?-AGAGGCATCTTTCTTTCAGCGAAGG-3?). ?-Galactosidase activity
was visualized in embryos with Red-Gal (Research organics). Whole-mount
in situ hybridization was performed as described (Deardorff et al., 1998).
Antisense probes detected chordin (Sasai et al., 1994), gooscoid (Blumberg
et al., 1991), Bf1 (Papalopulu and Kintner, 1996), Otx2 (Lamb et al., 1993),
Pax6 (Hirsch and Harris, 1997) and Xrx (Mathers et al., 1997). For sense
probes, kermit 2/pCS2 was linearized by NotI and transcribed by Sp6
polymerase. For antisense probes, kermit 2/pCS2 was linearized by BamHI
and transcribed by T7 polymerase.
Embryos, microinjection, immunoprecipitation and
Eggs were obtained from Xenopus females, in vitro fertilized and
microinjected as described (Deardorff et al., 1998). Each injected blastomere
received 10 nl of RNA or morpholino. For unilateral injections, morpholino
and/or mRNA were injected into one dorsal blastomere at the four-cell stage.
Animal pole explants assays were performed as described previously
(Deardorff et al., 2001). For immunoprecipitation, Xenopus embryos were
injected in the animal pole at the one-cell stage, cultured to stage 10 and
lysed in embryo lysis buffer [20 mM Tris (pH 7.5), 140 mM NaCl, 10%
glycerol, 1 mM DTT, 2 mM sodium vanadate, 25 mM NaF, 1% Nonidet P-
40 and protease inhibitor cocktail for mammalian cells (Sigma)]. Anti-GFP
monoclonal antibody (1 ?l) (Roche, 1 814 460) was incubated with 250 ?l
of cleared lysate overnight at 4°C, collected on 30 ?l protein G agarose
beads (Invitrogen, 15920-010) for 1 hour, washed three times with PBS, and
analyzed by western blot using IGF1R antibody (Cell Signaling, 3022).
AKT, P-AKT, MAPK, P-MAPK and ?-tubulin antibodies are from Sigma
(p2482), Cell Signaling (9271), Cell Signaling (9102), Sigma (M9692) and
BD Biosciences (556321), respectively.
TUNEL assay and phosphorylated histone H3 staining
Phosphorylated histone H3 staining was performed as described (Bellmeyer
et al., 2003). TUNEL staining protocol was from www.xenbase.org
Expression of kermit2/XGIPC in Xenopus embryos
To characterize the temporal pattern of kermit2 expression, we
extracted RNA from multiple stages of embryogenesis and
performed RT-PCR. kermit2 was detected maternally (four-cell
embryos) and throughout early embryonic development (Fig. 1A).
kermit2 is ubiquitously expressed in early and mid-neurula stage
embryos (Fig. 1B and data not shown). As neurulation progresses,
kermit2 expression is restricted to the anterior region (Fig. 1B,D),
including the cement gland and neural plate border, adjacent to
and/or overlapping the presumptive eye region (Fig. 1D). The
expression pattern of kermit2is very similar to XIGF1Rat this stage
(Richard-Parpaillon et al., 2002). By tailbud stages, kermit2
expression becomes further restricted, with strong expression in the
cement gland, otic vesicles (Fig. 1E), pronephros, branchial arches
(Fig. 1F), and cranial nerves V, VII and IX (data not shown).
Kermit2/XGIPC is required for eye development
To study the endogenous function of kermit 2/XGIPC in Xenopus
embryos, we depleted kermit 2 using morpholino antisense
oligonucleotides. The kermit 2 morpholino blocked translation of
kermit 2-GFPmRNA containing the endogenous 5?UTR but had no
effect on kermit 2-GFP mRNA lacking the 5?UTR (Fig. 2A).
Unilateral injection (into one dorsal blastomere at the four-cell stage)
of kermit 2 morpholino strongly inhibited anterior development,
especially eye development, in a dose-dependent manner (Fig. 2B;
data not shown). Although there was a distribution of phenotypes,
60% (n=38) of embryos receiving 40 ng of kermit 2 morpholino
developed without any detectable eyes on the injected side and 32%
had small eyes (Fig. 2B). Co-injection of kermit2-GFP mRNA
lacking the morpholino target sequence restored eye formation in
72% (n=43) of kermit2-depleted embryos, and of these, 28%
appeared completely normal (Fig. 2B). kermit2-GFP mRNA alone
did not cause apparent developmental defects. In addition, bilateral
injection of kermit 2 morpholino caused absent or miniscule eyes in
83% (n=73) of embryos, and this effect was reversed in 60% (n=85)
of embryos co-injected with the kermit2-GFP rescuing mRNA,
although bilateral injections were also associated with more severe
trunk defects that were not completely rescued by kermit2-GFP
(data not shown). These results suggest that the reduced eye
formation phenotype is specifically due to loss of kermit 2.
To characterize further the kermit 2 loss-of-function phenotype,
we analyzed molecular markers at various stages by in situ
hybridization. Kermit 2 depletion disrupted anterior development,
which could indicate direct inhibition of anterior neural development
or could alternatively indicate mild ventralization. To examine
whether kermit 2 plays a role in dorsal development, we examined
the expression of the dorsal organizer genes chordinand goosecoid.
Kermit 2 or control morpholinos were injected into two dorsal
animal blastomeres of four-cell/eight-cell embryos, and chordin or
Development 133 (18)
goosecoid expression was assessed at the early gastrula stage (Fig.
3A-D). Expression of chordin and goosecoid was not affected by
depletion of kermit 2, suggesting that loss of kermit 2 does not
disrupt early dorsal specification. We then examined the expression
of the anterior neural markers Bf1 (a forebrain marker), Otx2
(expressed in the forebrain, eyes and anterior midbrain) and Pax6(a
forebrain and eye marker) at the neurula stage (stage 20). To our
surprise, unilateral injection of kermit 2 morpholino into
dorsoanimal blastomeres did not obviously affect Bf1 expression
(80% no change, n=45, Fig. 3F). Furthermore Pax6 and Otx2
expression were reduced only in the presumptive eye domain (Pax6,
93% eye reduction, n=75; Otx2, 81% eye reduction, n=32, Fig.
3H,J). Pax6 expression in the spinal cord was not affected.
Consistent with an apparently eye-specific phenotype, the
expression of the eye-specific markers Xrx (85% reduction, n=32,
Fig. 3L) and Xath5 (data not shown) was also strongly reduced by
kermit 2 depletion. This eye-specific phenotype is probably due to
loss of kermit 2, as co-injection of kermit2 mRNA lacking the
5?UTR partially recovered Xrx expression in 73% (n=26, Fig. 3S,
compared with 3R and 3L) of kermit 2-depleted embryos. We also
analyzed the expression of Xrx and Pax6 at earlier stages of
development (early neurula stage, stage 14) and found that depletion
of kermit 2 did not have an obvious effect on Xrx (74% no change,
n=49, Fig. 3N) or Pax6 (84% no change, n=50, Fig. 3P) expression
at this stage, suggesting that kermit 2 is required for the
maintenance, but not the initiation, of eye formation.
Interaction between kermit2/XGIPC and XIGF1R in
Kermit 2, despite its high amino acid similarity with Kermit, does
not bind to the Kermit interaction partner, frizzled 3 (unpublished
data). However, kermit 2/XGIPC was identified as an XIGF1R-
Kermit 2/XGIPC in IGF-dependent eye development
Fig. 1. Temporal and spatial pattern of kermit2 expression. (A) RT-
PCR analysis of kermit2 temporal expression. Total RNA was isolated
from embryos at different stages and analyzed by RT-PCR. –RT, negative
control without reverse transcriptase. ODC was used as a loading
control. (B) RT-PCR analysis of kermit2 spatial expression. Stage 16
embryos were dissected into dorsal, ventral, anterior and posterior
pieces, as shown in the diagram. Stage 20 embryos were dissected into
anterior and posterior pieces. EF1? was used as a loading control.
kermit2 is equally expressed in anterior, posterior, dorsal and ventral
regions at mid-neurula stage (stage16), but is localized to the anterior
region by stage 20. Anterior marker Otx2, posterior dorsal marker
HoxD1, and posterior ventral marker Vent-1 were used as positive
controls. (C-F) In situ hybridization of neurula and tadpole stage
embryos. (C,D) Stage 20, anterior view, dorsal towards the top. (C)
Sense control; (D) antisense probe. At stage 20, kermit2 is expressed in
the cement gland and neural plate border, which is adjacent to and/or
overlapping the presumptive eye region. (E,F) Antisense probe. By
tailbud stages, kermit2 expression becomes further restricted, strongly
expressed in the cement gland and otic vesicles (ov: arrow) at stage 26
(E), and is also expressed in the pronephros and branchial arches in
stage 33 tadpoles (F).
Fig. 2. Kermit 2 loss-of-function phenotype. (A) A kermit 2-directed
antisense morpholino blocks translation of kermit 2-GFP mRNA (5?UTR-
kermit 2-GFP, which contains the morpholino target sequence in the
5?UTR), but not kermit 2-GFP mRNA lacking the 5?UTR. Kermit 2-GFP
mRNA (1 ng) with or without the 5?UTR was injected into one-cell
embryos together with kermit 2 morpholino (K2M; 20 ng). Embryos
were harvested at stage 10 and analyzed by western blot with GFP
antibodies. ?-Tubulin was used as a loading control. (B) Unilateral
injection of kermit 2 morpholino (40 ng) into one dorsal blastomere at
the four-cell stage completely blocked eye development in 60% of
embryos (lower left panel, n=38) and reduced eye formation in an
additional 32% (not shown). Co-expression of kermit2-GFP mRNA,
which lacked the morpholino target sequence, restored eye formation
in 72% of kermit2-depleted embryos (lower right panel, n=43).
kermit2-GFP mRNA alone did not cause apparent embryonic defects
(upper right panel).
binding protein by Booth et al. from a yeast two-hybrid screen
(Booth et al., 2002). To study the role of kermit 2/XGIPC in the IGF
signaling pathway, we confirmed that kermit 2/XGIPC interacts with
the IGF1R in Xenopus embryos. Full-length XIGF1R co-
immunoprecipitated with GFP-tagged kermit 2 expressed in
gastrula-stage embryos (Fig. 4A), suggesting that kermit 2/XGIPC
physically interacts with the XIGF1R in Xenopus.
Inhibition of IGF signaling by a dominant-negative XIGF1R
(DN-IGFR) reduces the size of anterior structures, including eyes,
in Xenopus embryos (Pera et al., 2001). To test for a functional
interaction between kermit 2 and IGF1R, we used doses of the
kermit 2 morpholino and DN-IGFR mRNA that alone only mildly
reduce eye formation (Fig. 4B). When co-injected, kermit 2
morpholino and DN-IGFR led to embryos with strongly reduced
eyes (Fig. 4B), suggesting a synergistic effect between the two.
Furthermore, although these levels of kermit 2 morpholino or DN-
IGFR alone minimally affected Pax6 expression (72.5%, n=24;
100%, n=27, respectively), co-injection strongly reduced Pax6
expression in the presumptive eye in 82% (n=22) of the embryos
(Fig. 4C). Taken together, these data indicate that kermit 2/XGIPC
could be involved in the IGF pathway, potentially functioning at the
Kermit 2/XGIPC is required for IGF1 induced eye
In order to test the requirement of kermit 2/XGIPC for IGF
signaling, we examined whether depletion of kermit 2 inhibits IGF
function in embryos. Dorsal overexpression of IGF1 mRNA has
been shown to induce ectopic eyes and overgrowth of endogenous
eyes (Pera et al., 2001; Richard-Parpaillon et al., 2002). We found
similarly that dorsal injection of XIGF1 mRNA caused expanded
eyes in 64% (n=22) of the embryos. Depletion of kermit 2
dramatically inhibited the XIGF1 induced eye phenotype and led to
embryos (73%, n=20) with small eyes or no eyes (Fig. 5A). Kermit
2 depletion alone reduced eye formation (similar to Fig. 2). Dorsal
injection of IGF1 mRNA also expanded cement gland formation in
embryos, but this phenotype was not affected by kermit 2 depletion.
Kermit 2 is also required for IGF induced expression of eye
markers in animal cap explants. As reported (Pera et al., 2001;
Richard-Parpaillon et al., 2002), expression of IGF1alone in animal
caps induces anterior neural markers (Fig. 5B, lane 4), including
XAG (a cement gland marker), Otx2 (a marker for the forebrain and
anterior midbrain), Pax6 (a marker for eyes and the forebrain) and
Xrx (an eye marker), but not the midbrain-hindbrain boundary
marker En2 or the mesodermal marker muscle actin. Kermit 2
Development 133 (18)
Fig. 3. Depletion of kermit 2/XGIPC
specifically inhibits Xenopus eye
development. (A-D) Depletion of kermit 2
does not affect the expression of dorsal
mesoderm markers chordin and goosecoid.
Embryos are viewed from the vegetal side and
dorsal is towards the top. Control morpholino
or kermit 2 morpholino (40 ng) was injected
into two dorsal animal blastomeres of four-cell
embryos. Embryos were fixed at the gastrula
stage (stage 10) and whole-mount in situ
hybridization was performed for chordin (A,B)
and goosecoid (C,D). (E-L) Depletion of kermit
2 specifically reduces marker gene expression
within the presumptive eye field in stage 20
embryos. Embryos are viewed from the
anterior side with dorsal towards the top.
Control or kermit 2 morpholino was injected
into one dorsal animal blastomere of four-
cell/eight-cell embryos with 500 pg of mRNA
for nuclear ?-galactosidase. Embryos were
fixed at stage 20 and ?-galactosidase activity
was measured in situ (red) followed by whole-
mount in situ hybridization for Bf1 (E,F), Otx2
(G,H), Pax6 (I,J) and Xrx (K,L). Bf1 expression
was not affected by depletion of kermit 2 (F).
Expression of Otx2 (H) and Pax6 (J) were
reduced only within the presumptive eye-
forming region (red arrowheads) in embryos
injected with kermit 2 morpholino. The
expression of the eye marker Xrx was also
inhibited (L red arrowhead). (M-P) Depletion of
kermit 2 does not reduce Xrx (N) or Pax6 (P)
expression in early neurula stage embryos
(stage 14). (Q-S) Kermit 2 mRNA restored Xrx
expression in kermit 2-depleted embryos. Red
arrowhead in R indicates strongly reduced Xrx
expression within presumptive eye domain in
kermit 2-depleted embryo; red arrow in S
indicates recovered Xrx expression in embryo
co-injected with kermit 2 morpholino and
kermit 2 mRNA.
depletion strongly reduced the induction of Xrx, mildly reduced
Pax6 induction, and did not affect the induction of the anterior neural
markers XAG and Otx2(lane 6). Co-injection of control morpholino
did not affect any of the IGF1-induced markers (lane 5). These
results suggest that kermit 2/XGIPC is required for IGF1-induced
eye formation in Xenopus.
Kermit 2/XGIPC is required for long-term
activation of AKT by IGF1
As shown above, kermit 2 is required for IGF induced embryonic
phenotypes, but whether it is required for the activation of
downstream IGF signaling is still unknown. The two main
intracellular pathways activated by IGF/IGFR are the PI3 kinase/
AKT pathway and the MAP kinase pathway (Oldham and Hafen,
2003). We first examined the requirement of kermit 2/XGIPC for
IGF signaling in Xenopusoocytes. Stage VI oocytes have low levels
of background activation of AKT or MAP kinase, but respond
robustly to exogenous IGF1. We injected control morpholino or
kermit 2 morpholino into oocytes and cultured these oocytes for 48
hours to deplete endogenous kermit 2. We then treated these oocytes
with IGF1 protein for 30 minutes or overnight (18 hours). IGF1
induced phosphorylation of AKT (Ser 473) and MAPK (e.g. Thr183
and Tyr185 in ERK2) at sites associated with activation as
documented previously (Alessi et al., 1996; Payne et al., 1991;
Stephens et al., 1998; Stokoe et al., 1997; Yung et al., 1997). Kermit
2 depletion blocked phosphorylation/activation of AKT induced by
long-term exposure to IGF1, but not by short-term exposure (30
minutes) (Fig. 6A). MAP kinase phosphorylation induced by IGF1
protein was not affected by depletion of kermit 2 at any time points
(Fig. 6A). The control morpholino did not affect IGF1-induced AKT
or MAP kinase phosphorylation/activation. These data suggest that
kermit 2/XGIPC is required to maintain activation of AKT induced
by IGF1, but is not required for the initiation of signaling.
This analysis was extended to ectodermal (animal cap) explants.
Compared with oocytes, animal caps have high levels of endogenous
AKT and MAP kinase phosphorylation/activation. We first tested
the effect of kermit 2 morpholino on the endogenous
phosphorylation of AKT and MAP kinase. Control morpholino or
kermit 2 morpholino was injected into fertilized eggs. Animal caps
were dissected at the late blastula stage (stage 9) and cultured for 2
hours (stage 10/10.5) or overnight (stage 20). Similar to the result
in oocytes, kermit 2 depletion inhibited AKT phosphorylation/
activation after overnight incubation, but not after 2 hours
incubation; kermit 2 morpholino had no effect on MAP kinase
phosphorylation (Fig. 6B). To test whether kermit 2 is required for
AKT phosphorylation/activation by exogenous IGF1, embryos were
injected with IGF1 mRNA with or without kermit 2 morpholino,
animal caps were explanted at the blastula stage and cultured until
late neurula stage (stage 20). We found that kermit 2 depletion
blocked phosphorylation/activation of AKT by overexpressed IGF1
Kermit 2/XGIPC in IGF-dependent eye development
Fig. 4. Interaction between kermit2/XGIPC
and XIGF1R in eye development. (A) Co-
immunoprecipitation of kermit 2 and full-length
XIGF1R. Embryos were injected at the one-cell
stage with mRNAs encoding XIGF1R (2 ng) and
GFP-tagged kermit 2 (1 ng), and cultured until the
gastrula stage. XIGF1R/kermit 2 complexes were
immunoprecipitated from embryo lysates with
anti-GFP antibody and XIGF1R was visualized by
western blotting. Mouse IgG was used as a
negative control. (B) Kermit 2 morpholino and
DN-IGFR synergistically inhibit eye formation in
embryos. Kermit 2 morpholino (20 ng), DN-IGFR
mRNA (500 pg), or both, were injected into two
dorsal animal blastomeres of four-cell/eight-cell
embryos. Embryos were cultured until tadpole
stages to score phenotypes. The percentage of
embryos with either strong or mild reduction in
eyes is tabulated in the panel on the right side.
(C) Kermit 2 morpholino and DN-IGFR
synergistically reduce expression of Pax6 in
presumptive eye domain (arrowhead).
Microinjections were performed as in Fig. 2B.
Nuclear ?-galactosidase mRNA was co-injected as
a lineage tracer. Embryos were fixed at stage 20
and ?-galactosidase activity was measured in situ
(red) followed by whole-mount in situ
hybridization for Pax6.
(Fig. 6C, compare lane 5 with lane 4). (Kermit 2 morpholino did not
interfere with translation of injected mRNAs, as GFP mRNA co-
injected with IGF1 mRNA was expressed at equal levels with or
without kermit 2 morpholino co-injection.) Taken together, the
results in oocytes and animal cap explants suggest that kermit
2/XGIPC is required for maintaining IGF induced AKT activation,
but not for MAPK activation.
Activated PI3K partially rescues the kermit
2/XGIPC loss-of-function phenotype
As kermit 2 depletion
phosphorylation/activation induced by IGF1, we examined the
physiological relevance of this inhibition by rescuing the kermit 2
loss-of-function phenotype using a constitutively active p110
subunit (p110*) of phosphatidylinositol-3? kinase (PI3 kinase)
strongly inhibits the AKT
Development 133 (18)
Fig. 5. Kermit 2/XGIPC is required for IGF1 induced eye formation
in whole embryos and in animal cap explants. (A) Kermit 2 is
required for IGF1 induced eye formation in Xenopus embryos. XIGF1
mRNA (1 ng), kermit 2 morpholino (40 ng), or both, were injected into
one dorsal animal blastomere of four-cell/eight-cell embryos. IGF1
injection alone leads to embryos with expanded eyes and cement
glands and co-injection of kermit 2 morpholino specifically inhibits the
IGF1-induced eye phenotype. (B) Kermit 2 is required for IGF1-induced
eye marker expression in animal cap explants. XIGF1 mRNA alone (2
ng), or with control morpholino (CM) or kermit 2 morpholino (K2M)
was injected into four animal blastomeres of four-cell/eight-cell
embryos. Animal caps were explanted from stage 9 blastulae, cultured
until stage 20, and then harvested for RT-PCR. St. 20 WE indicates the
whole embryo control; uninjected represents animal caps from
uninjected embryos; –RT, without reverse transcriptase; EF1? is the
loading control. Kermit 2 morpholino specifically reduces XIGF1-
induced expression of the presumptive eye markers Xrx and Pax6.
Fig. 6. Kermit 2/XGIPC is required to maintain IGF1 induced
phosphorylation of AKT in oocytes and animal cap explants.
(A) Kermit 2 is required for maintaining IGF1-induced AKT
phosphorylation in oocytes. Oocytes were injected with 40 ng of
control morpholino (CM) or kermit 2 morpholino (K2M), cultured for
48 hours and then treated with recombinant human IGF1 protein (18
ng/ml) for 30 minutes or overnight. Kermit 2 is required for IGF1-
induced AKT phosphorylation/activation after overnight treatment, but
not for short-term phosphorylation of AKT or MAPK. Total AKT or total
MAPK was used as the loading control. (B) Effects of kermit 2
morpholino on the endogenous activation of AKT and MAPK. Control
morpholino or kermit 2 morpholino was injected into four animal
blastomeres of four-cell/eight-cell embryos. Animal caps were explanted
at stage 9 and harvested after 2 hours or overnight. Kermit 2
morpholino reduces endogenous AKT phosphorylation after overnight
incubation but not after 2 hours incubation and does not affect MAPK
phosphorylation at either time point. (C) Kermit 2 is required for IGF1-
induced AKT phosphorylation in stage 20 animal cap explants. 500 pg
of XIGF1 mRNA and 400 pg of GFP mRNA were injected into one-cell
embryos and 40 ng of kermit 2 morpholino was injected into four
animal blastomeres of four-cell/eight-cell embryos. Animal caps were
dissected at stage 9 and harvested at stage 20. GFP was used as the
(Carballada et al., 2001), a lipid kinase upstream of AKT and
downstream of IGF1R. Expression of p110* partially rescues the
kermit 2 depletion phenotype (Fig. 7B). In a typical experiment,
96% (n=24) of the embryos injected dorsally with the kermit 2
morpholino alone had either miniscule or no apparent eyes, whereas
only 26% (n=20) of embryos co-injected with p110* showed this
phenotype, and 74% developed small but normal-appearing eyes.
Dorsal expression of p110* itself did not cause apparent
developmental defects in embryos. p110* was confirmed to be active
as it induced strong AKT phosphorylation in gastrula-stage animal
caps (Fig. 7A). In addition to the morphological rescue, p110* also
partially recovers the expression of Xrx, an eye marker, in kermit 2-
depleted embryos. Compared with control embryos, most kermit 2-
depleted embryos had strongly reduced Xrx expression (63%, n=39;
Fig. 7D, middle panel) at the neurula stage (stage 19), whereas the
majority of embryos co-injected with p110* showed only a mild
reduction of Xrxexpression (73%, n=53; Fig. 7D, right panel). These
rescue results suggest that the kermit 2 loss-of-function phenotype
is at least partially due to the inhibition of the PI3 kinase/AKT
Kermit 2/XGIPC is required for cell survival, but
not for cell proliferation in Xenopus
The IGF pathway affects multiple cellular processes, including cell
survival and cell proliferation. We examined whether kermit
2/XGIPC is involved in these processes. We injected control
morpholino or kermit 2 morpholino into the right side of embryos
(leaving the left side as the control) and followed apoptosis by
TUNEL analysis. Seventy-two percent (n=61) of kermit 2-depleted
embryos demonstrate a substantial increase in the number of
TUNEL-positive nuclei on the injected side compared with the
control side (Fig. 8B). This effect was dose dependent (data not
shown) and no difference was detected with control morpholino at
equivalent doses (Fig. 8A). Expression of kermit2 mRNA without
the 5?UTR significantly reduced TUNEL-positive nuclei in 83%
(n=66) of kermit 2-depleted embryos (Fig. 8C), which indicates that
the elevated cell death was specifically due to loss of kermit 2.
Expression of the same amount of n?-gal mRNA did not reduce
apoptosis in kermit 2-depleted embryos (data not shown). We also
assessed whether kermit 2 regulates cell proliferation. Embryos
unilaterally injected with kermit 2morpholino were immunostained
Kermit 2/XGIPC in IGF-dependent eye development
Fig. 7. Activation of PI3K partially
rescues eye development in kermit
2/XGIPC-depleted embryos. (A) p110*,
a constitutively active subunit of PI3 kinase,
induces AKT phosphorylation/activation in
stage 10 animal cap explants. mRNA
encoding p110* was injected into
fertilized eggs and animal caps were
explanted at the gastrula stage and
analyzed by western blot with antibodies
recognizing AKT phosphorylated at serine-
473 (upper panel) or general anti-AKT
antibodies (lower panel). (B) p110*
partially rescues eye development in kermit
2-depleted embryos. Kermit 2 morpholino
(40 ng), 3 ng of p110* mRNA, or both,
were injected into two dorsal animal
blastomeres of four-cell/eight-cell embryos.
No apparent eyes are present in embryos
injected with the kermit 2 morpholino,
while small eyes are present in over 70%
of embryos co-injected with p110* mRNA
and morpholino. p110* injection alone
does not affect embryo development.
(C) The percentage of embryos with
normal, small or absent/miniscule eyes is
summarized. (D) p110* partially recovers
the expression of Xrx in kermit 2-depleted
embryos. One dorsal-animal blastomere of
four-cell/eight-cell embryos was injected
with kermit 2 morpholino with or without
p110* mRNA (right side, indicated by GFP
lineage tracer) and harvested at stage 20.
Whole-mount in situ hybridization was
performed for Xrx. Embryos are viewed
from the anterior side with dorsal towards
with an antibody against phosphorylated histone H3, which
specifically recognizes mitotic chromosomes. We did not observe an
obvious change in the number of mitotic cells on the injected side
compared with the control side in early neurula stage (stage 14) and
late neurula stage (stage 22) embryos (Fig. 8E,F). These results
indicate that kermit 2/XGIPC is required for cell survival, but do not
support a requirement for kermit 2 in proliferation in Xenopus
embryos. As kermit 2 is involved in IGF signaling, we further tested
whether inhibition of the IGF pathway increases cell death in
Xenopus embryos. Indeed, DN-IGFR, like kermit 2 depletion,
markedly increased the number of TUNEL-positive nuclei (76%,
n=29; Fig. 8D) when expressed in embryos. Activation of the
MAPK branch of IGF signaling leads to transcriptional responses
associated with cell proliferation, whereas activation of the
PI3K/AKT branch regulates the activity of BCL2 family members
and anti-apoptotic responses (O’Connor, 2003; Oldham and Hafen,
2003). Therefore, these data are consistent with a requirement for
kermit 2 in IGF-induced AKT activation. Finally, to determine
whether the kermit 2 depletion phenotype is due to apoptosis of cells
within the eye-field, we examined whether inhibition of apoptosis
restores eye formation in kermit 2-depleted embryos. Kermit 2
morpholino injection alone strongly reduced Pax6expression in the
presumptive eye in 75% (n=24, Fig. 8H) of embryos. Co-expression
of bcl2 rescued this phenotype and restored Pax6 expression in the
presumptive eye in almost 72% (n=25, Fig. 8I) of embryos, as well
as restoring eye development to normal or near normal morphology
in a majority of sibling embryos allowed to develop to later stages
(data not shown). This result suggests that the kermit 2 phenotype is
at least partially due to elevated cell death in the presumptive eye
We report here the identification of the second Xenopus GIPC
homolog, kermit 2. Despite its high amino acid similarity with the
first Xenopus GIPC homolog Kermit, kermit 2 has a different
function in Xenopus embryos. Loss-of-function and molecular
marker analyses indicate that kermit 2 is specifically required for
Xenopus eye development. We have confirmed the physical
interaction of kermit 2/XGIPC with XIGF1R in Xenopusand further
established that kermit 2 is required for IGF-dependent eye
development. In addition, kermit 2 is required for maintaining IGF
induced AKT phosphorylation/activation and a constitutively active
PI3 kinase partially rescues kermit 2 loss of function. These results
provide the first demonstration of the in vivo role of GIPC in
embryonic development and also show that kermit 2/XGIPC is a
novel component of the IGF pathway, potentially acting through the
modulation of IGF1R function.
kermit 2/XGIPC and IGF signaling in Xenopus
Inhibition of IGF signaling in Xenopusby either dominant-negative
IGF receptor or IGF receptor depletion disrupts anterior neural
development broadly (Pera et al., 2001; Richard-Parpaillon et al.,
2002), yet loss of kermit 2/XGIPC specifically inhibits eye
formation without apparent effect on other aspects of anterior neural
development. At least two mechanisms could explain the more-
restricted requirement for kermit 2/XGPIC. First, depletion of kermit
2 blocks the activation of AKT, but not of MAPK. It is likely that the
IGF/PI3K/AKT branch is specifically involved in eye formation,
while the IGF/MAPK branch regulates anterior neural development
more broadly. Indeed, IGF inhibits SMAD1 activity and induces
neural differentiation via MAPK (Pera et al., 2003; Kuroda et al.,
2005). Second, eye development could have a distinct temporal
requirement for IGF signaling. For example, the specification of
anterior neural tissue in general may only require an early exposure
to IGF, whereas proper eye formation may require continual
activation of IGF signaling. As kermit 2/XGIPC is required for the
maintenance, but not the initiation of IGF signaling, depletion of
kermit 2/XGIPC may inhibit eye formation only at late neurula
stages, without disrupting general neural specification.
We also found that depletion of kermit 2 dramatically increases
cell death in embryos, but does not reduce cell proliferation. This
result is consistent with the data that kermit 2 depletion blocks
IGF induced AKT activation, not MAPK activation. However, the
Development 133 (18)
Fig. 8. Kermit 2/XGIPC is required for cell survival, but
not for cell proliferation in Xenopus. (A-F) Control
morpholino (A), kermit 2 morpholino alone (B,E,F), kermit 2
morpholino with 3 ng of kermit 2 mRNA (C) or 1 ng of DN-
IGFR (D) was injected into the right side dorsal animal
blastomere of four-cell/eight-cell embryos. The left side
serves as a control. TUNEL staining and phosphorylated
histone H3 staining were performed on neurula stage
embryos. Embryos are viewed from the dorsal side, anterior
towards the top. Compared with the control side (left side),
the side injected with kermit 2 morpholino (right side) shows
a substantial increase in the number of TUNEL-positive nuclei
(B), without apparent change in the number of mitotic cells
(E,F). The increased apoptosis in kermit 2-depleted embryos
can be rescued by co-expression of kermit 2 mRNA lacking
the 5?UTR (C). DN-IGFR also leads to elevated cell death on
the injected side, as shown in D. (G-I) Co-injection of Bcl2
mRNA recovers Pax6 eye expression in kermit 2-depleted
embryos. Microinjections were performed as above and n?-
gal was used as the lineage tracer. Embryos were fixed at
stage 20 and ?-galactosidase activity was measured in situ
(red) followed by whole-mount in situ hybridization for Pax6.
Red arrowhead in H indicates strongly reduced eye region in
kermit 2 morpholino-injected embryo and red arrow in I
indicates recovered eye expression domain by Bcl2 mRNA.
increased cell death in kermit 2-depleted embryos is not restricted
to the presumptive eye field. Thus, the extent to which increased
cell death contributes to the eye phenotype remains to be
Regulation of the IGF1 receptor by kermit 2/XGIPC
As kermit 2/XGIPC functions downstream of the IGF ligand and
upstream of PI3 kinase and physically associates with the XIGF1R,
kermit 2 probably regulates IGF signaling through modulation of the
receptor. In principle, kermit 2 could regulate the stability, activity
or subcellular localization of the receptor. However, kermit 2
depletion did not obviously reduce the level of XIGF1R under
conditions that reduced AKT activation (data not shown), arguing
against kermit 2 regulation of IGF1R stability. Furthermore, kermit
2 depletion did not significantly affect the level of IGF-induced
MAPK phosphorylation, arguing against a role for kermit 2 in
regulating overall stability or activity of the IGF1R. Kermit 2
depletion also did not interfere with the early (30 minute)
phosphorylation of AKT in response to IGF, suggesting that
coupling to downstream signaling, at least initially, is intact.
Alternatively, mammalian GIPC is involved in the regulation of
endocytic trafficking, and kermit 2/XGIPC may similarly regulate
the subcellular localization of the IGF receptor. A function for GIPC
in endocytic trafficking was first proposed based on its localization
to endocytic vesicles (Dance et al., 2004; De Vries et al., 1998a; De
Vries et al., 1998b; Jeanneteau et al., 2004a; Lou et al., 2002; Lou
et al., 2001). For example, GIPC is enriched at clathrin-rich
invaginations and the endocytic compartments found between
microvilli in proximal tubule kidney cells (Lou et al., 2002). A role
for GIPC in endocytosis is also supported by functional studies in
cultured cells (Hirakawa et al., 2003; Jeanneteau et al., 2004a).
Hirakawa et al. used RNAi to knockdown GIPC in 293 cells and
found that GIPC is partially responsible for maintaining a relatively
constant level of the human lutropin receptor (LHR) at the cell
surface during ligand-induced internalization and for recycling of
the ligand (CG) (Hirakawa et al., 2003). In a similar manner, kermit
2 could be required for the recycling of the IGF1R to the plasma
membrane after ligand-dependent internalization. A failure to
recycle internalized IGF1R could explain why depletion of kermit 2
reduces AKT phosphorylation after prolonged exposure to IGF, but
not after short-term exposure. A defect in recycling of IGF1R would
not be expected to disrupt MAP kinase phosphorylation, as MAP
kinase can be activated by receptors that have been internalized in
association with ?-arrestin and component kinases of the MAPK
cascade (DeFea et al., 2000; Lefkowitz and Shenoy, 2005; Lin et al.,
1998; Luttrell et al., 2001; Tohgo et al., 2003), while AKT and PI3
kinase have not been reported to be activated by internalized
receptors within endosomes. However, we have so far been unable
to demonstrate an effect of kermit 2 depletion on IGF1R trafficking,
and this will remain a focus of future studies.
In summary, we have shown that kermit 2/XGIPC is required for
eye development in Xenopusembryos. Kermit 2/XGIPC physically
and functionally interacts with the IGF1R and is required for IGF
signaling in anterior neural development specifically in eye
formation. Expression of molecular markers of eye development is
induced but not maintained in kermit 2-depleted embryos, and
phosphorylation of AKT is similarly induced but not maintained
after prolonged exposure to IGF when kermit 2 is depleted. Eye
development can be partially rescued in kermit 2-depleted embryos
by an active PI3 kinase. Based on these observations, we propose
that kermit 2/XGIPC, through the modulation of IGF1R, mediates
endogenous IGF signaling in Xenopus eye formation.
We thank Eddy M. De Robertis, Laurent Richard-Parpaillon, Patrick Lemaire,
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Pierre Saint-Jeannet for plasmids and reagents. We thank Dan Kessler, Jean-
Pierre Saint-Jeannet, Mary Mullins, Jonathan Epstein, Tom Kadesch and Jing
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