DEVELOPMENTAL DYNAMICS 236:214 –225, 2007
RESEARCH ARTICLE
Mouse Rab11-FIP4 Regulates Proliferation and
Differentiation of Retinal Progenitors in a
Rab11-Independent Manner
Akihiko Muto, Yutaka Aoki, and Sumiko Watanabe*
We identified Rab11-family interacting protein 4 (Rab11-FIP4) as a gene strongly expressed in the
developing mouse retina. The major transcript encoding a full-length protein, mRab11-FIP4A, was
expressed predominantly in neural tissues; whereas an alternative transcript encoding an N-terminally
truncated form of the protein, mRab11-FIP4B, was expressed ubiquitously as a minor form. Gain-offunction of mRab11-FIP4A in retina promoted cell cycle exit and increased subpopulations of retinal cells
localized in the inner nuclear layer, such as bipolar cells and Müller glia. Reversal of the phenotype was
observed in the loss-of-function experiment. Furthermore, Shh signaling was suggested to be involved in
these functions. Analysis using truncation mutants revealed the essential role of the N-terminal region
containing a conserved EF-hand motif for the retinal phenotypes induced by the expression of mRab11FIP4A, whereas binding to Rab11 was dispensable, suggesting the involvement of a novel Rab11independent mechanism for mRab11-FIP4A action in the regulation of retinal development. Developmental
Dynamics 236:214 –225, 2007. © 2006 Wiley-Liss, Inc.
Key words: Rab11-FIP4; retina; mouse; proliferation; differentiation
Accepted 4 October 2006
INTRODUCTION
The vertebrate neural retina contains
six major types of neurons and a single type of glial cell; and these cells
are organized into three nuclear layers: the outer nuclear layer (ONL),
consisting of cone and rod photoreceptors; the inner nuclear layer (INL),
consisting of three types of interneurons, i.e., bipolar, amacrine, and horizontal cells, as well as the Müller glial
cells; and the ganglion cell layer
(GCL), consisting of retinal ganglion
cells (RGC) and a small number of
displaced amacrine cells. Although all
of these types of retinal cells are
thought to be derived from a single
population of retinal progenitor cells
(Perron and Harris, 2000; Ahmad et
al., 2004), the generation of each type
occurs at distinct stages during eye
development and its timing is strictly
regulated. To identify genes regulated
for expression in a retinal developmental stage-specific manner, we conducted differential display analysis
using cDNA prepared from various
stages of mouse eyes as templates. As
a result, we found that a gene named
rab11-family interacting protein 4
(Rab11-FIP4) was expressed with a
unique pattern in the developing retina.
Rab11-FIP4 was originally cloned
as a gene named KIAA1821 in a human cDNA sequencing project (Nagase et al., 2001) and recently identified as a member of the Rab11-family
interacting protein (Rab11-FIPs) family (Wallace et al., 2002a). Rab11 is a
member of the Rab family of small
GTPases and is known to regulate diverse pathways of vesicle trafficking
including protein recycling and intracellular protein transport (Zerial and
McBride, 2001) as well as cytokinesis
(Riggs et al., 2003). At least six members of Rab11-FIPs, which all share a
highly conserved short motif named
Rab11-binding domain (RBD) at the
C-termini of the proteins, have been
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Grant sponsor: RIKEN Center for Developmental Biology, Kobe, Japan.
*Correspondence to: Sumiko Watanabe, Department of Molecular and Developmental Biology, Institute of Medical Science,
University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639, Japan. E-mail: sumiko@ims.u-tokyo.ac.jp
DOI 10.1002/dvdy.21009
Published online 3 November 2006 in Wiley InterScience (www.interscience.wiley.com).
© 2006 Wiley-Liss, Inc.
mRab11-FIP4 REGULATES RETINAL DEVELOPMENT 215
so far been identified (Hales et al.,
2001; Prekeris et al., 2001; Lindsay et
al., 2002; Wallace et al., 2002b), and
these molecules are thought to play
important roles in the regulation of
the vesicle trafficking by Rab11 as
downstream effectors (Cullis et al.,
2002; Meyers and Prekeris, 2002).
Rab11-FIP4 and a closely related protein Rab11-FIP3 are categorized into
class II subfamily of Rab11-FIPs by
the presence of an N-terminal EFhand motif as well as by a similarity to
the Drosophila protein Nuclear Fallout (Nuf), which was identified as a
gene required for cellularization
(Rothwell et al., 1998; Riggs et al.,
2003). In vitro studies failed to reveal
the involvement of Rab11-FIP4 in the
Rab11-mediated intracellular transport of transferrin (Wallace et al.,
2002b) or its receptor (Hickson et al.,
2003), whereas it was demonstrated
that Rab11-FIP4 in cooperation with
Rab11-FIP3 was required for cytokinesis (Fielding et al., 2005). However,
these studies were performed in vitro
using human cell lines and the physiological function of Rab11-FIP4 during the vertebrate development is still
unclear. We recently identified a zebrafish orthologue of Rab11-FIP4
(zRab11-FIP4) as a gene specifically
expressed in the neural tissues, including the developing retina, and
found that the amino acid sequence as
well as protein motifs were highly conserved between human and zebrafish
(Muto et al., 2006). In more detailed
analysis, we showed that zRab11FIP4 plays an important role in regulating the proliferation and differentiation of the retinal progenitors during
development. Also, functional interaction between zRab11-FIP4 and shh
was indicated. These observations,
therefore, have prompted us to examine whether these biological functions
of Rab11-FIP4 are conserved in other
vertebrates.
Here, we report the identification
and characterization of mouse Rab11FIP4 (mRab11-FIP4). In this study,
we found mRab11-FIP4A, a longer
form of mRab11-FIP4, to be involved
in retinal development. mRab11FIP4A was predominantly expressed
in the developing neural tissues, and
gain- and loss-of-function analyses using a retinal explant system indicated
that this molecule played roles in reg-
Fig. 1. Structure and expression pattern of Rab11-FIP4. A: Schematic representation of protein
structures of Rab11-FIP4 homologues. The identity at the amino acid level and the positions of
conserved motifs are indicated. The mouse Rab11-FIP4B–specific 5⬘-region is indicated by the
hatched box. B: Northern blot analysis of mRab11-FIP4 in the developing eye and brain was
performed by using a probe recognizing both A- and B-forms. Ten micrograms of total RNA
prepared from the indicated tissues was applied in each lane. The sizes of RNA markers are shown
at the right of the panel. C: Expression of A- and B-forms of mRab11-FIP4 as well as mRab11-FIP3
in various tissues of postnatal day (P) 1 mice (lower) and adult mice (upper) was examined by
semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Glyceraldehyde-3phosphate dehydrogenase (G3PDH) was used as a control in both “B” and “C.”
ulating the exit of retinal progenitor
cells from the cell cycle and their subsequent differentiation into INL cells.
We also found that Rab11 could not
mimic the phenotype observed by the
overexpression
of
mRab11-FIP4.
These results suggest that, although
Rab11-FIP4 has been structurally and
functionally conserved among vertebrates, the detailed mechanism underlying its action between zebrafish
and mouse Rab11-FIP4 appears to be
different.
RESULTS
Identification and Cloning of
Mouse DD76 cDNA
To identify genes the transcriptional
level of which is regulated during eye
development, we performed differential display using cDNAs prepared
from RNAs obtained from mouse eyes
at various stages of development. We
sequenced 91 clones that had unique
expression patterns during retinal development and focused on a fragment
of 221 bp, which we named DD76. Homology search against the Celera and
public DNA databases showed that
the sequence of this DD76 fragment
corresponded to an intron region of a
putative mouse gene similar to that of
human (h) Rab11-FIP4. We isolated
the full-length cDNA by reverse transcriptase-polymerase chain reaction
(RT-PCR) in combination with 5⬘rapid amplification of cDNA ends
(RACE) and found it to encode a putative protein of 635 amino acids with
91% identity to the amino acid sequence of hRab11-FIP4 (Fig. 1A). A
motif search identified a single EFhand motif and the RBD in regions
proximal to the N- and C-termini, respectively. A coiled-coil structure and
two leucine-zipper like motifs were
identified within the C-terminal half.
All these characteristic features are
conserved in human and zebrafish
Rab11-FIP4 (Muto et al., 2006). Moreover, a search of the genomic databases mapped the DD76 gene to the
mouse chromosome 11B5 region,
which is syntenic to a locus encompassing the hRab11-FIP4 gene on human chromosome 17q11.2 (data not
shown). All these results strongly indicate that the DD76 gene is a mouse
homologue of hRab11-FIP4; therefore
hereafter, we refer to it as mouse
Rab11-FIP4 (mRab11-FIP4).
By the 5⬘-RACE method, we found
an alternative transcript, which was
predicted to carry 185 bp of a unique
5⬘-sequence in place of the N-terminal
region containing the EF-hand motif.
A putative initiation codon was found
in its unique 5⬘-sequence. We desig-
216 MUTO ET AL.
nated this N-terminal–truncated form
as Rab11-FIP4B, and the full-length
one as Rab11-FIP4A (Fig. 1A).
Rab11-FIP4 Is Expressed
Predominantly in the Neural
Tissues
By Northern blot analysis using a
1.5-kb cDNA fragment from a region
shared by Rab11-FIP4 A- and B-forms
as a probe, we found a single 7-kb
band in samples derived from the eyes
and brain of a postnatal day (P) 1
mouse. The bands were surmised to
contain both A- and B-form transcripts because of a slight difference in
their mobility (Fig. 1B). As development proceeded, the amount of
mRab11-FIP4 mRNA in the eyes
gradually increased up to the end of
the embryonic period (data not
shown); then, after postnatal day 1
(P1), it decreased toward the adult
stage (Fig. 1B). In contrast, that in the
brain did not change significantly
(Fig. 1B). We then examined the expression pattern of mRab11-FIP4 in a
variety of P1 and adult mouse tissues
by RT-PCR using primers specific for
each form of transcript. The A-form in
both P1 and adult was predominantly
expressed in neural tissues such as
eye and brain, whereas the B-form
transcript was detected in a broad
range of tissues, except the heart and
muscle (Fig. 1C).
Next, the spatial and temporal expression pattern of Rab11-FIP4 in developing mouse embryos was examined by whole-mount in situ
hybridization. Since we failed to detect A and B transcripts separately by
using specific probes, probably due to
the short length of the probes (data
not shown), we adopted the same
probe used for the Northern blot analysis, which hybridized with both Aand B-forms, for the in situ hybridization. At embryonic day (E) 8.5, the
mRab11-FIP4 gene was clearly expressed in the region along the midline (Fig. 2A). On the next day (E9.5),
the expression level became much
higher; and the transcript was detected predominantly in the central
nervous system (CNS), including the
entire brain, neural tube, and otic vesicles. At this stage, the optic vesicle
started to evaginate, and mRab11FIP4 was strongly expressed in this
region (Fig. 2B). In addition to these
neural tissues, the branchial arches
also expressed Rab11-FIP4 at this
stage. At E10.5, the expression pattern was essentially the same as that
in E9.5 embryos; weak expression in
fore- and hindlimbs was additionally
observed (Fig. 2C).
To analyze in more detail the expression pattern of mRab11-FIP4 in
the developing mouse eye, we performed in situ hybridization on tissue
sections. At E10.5, expression was
found in the retinal pigment epithelium (RPE) but not in the neural retina (Fig. 2D); that in the RPE gradually declined and disappeared by
E13.5 (Fig. 2E–G). The expression of
mRab11-FIP4 in the neural retina
was first detected at E11.5 in a small
number of cells in the central area of
the retina (Fig. 2E), and a large number of mRab11-FIP4 – expressing cells
were observed by E12.5 in the GCL
(Fig. 2F). The expression in the GCL
expanded peripherally at later stages,
and finally was detected throughout
the retina by E16.5 (Fig. 2H). On the
other hand, mRab11-FIP4 started to
be expressed in the neuroblastic layer
(NBL) around E13.5, and its expression there increased by E16.5 (Fig.
2H). Rab11-FIP4 continued to be expressed in the GCL after birth; however, at later stages, the expression in
the NBL gradually disappeared from
the outer side (Fig. 2J,K) concomitant
with differentiation of photoreceptors,
and finally disappeared from the adult
retina (Fig. 2L).
In the brain at E16.5, expression of
mRab11-FIP4 was observed in the
cortical plate (Fig. 2M), as well as in
the olfactory epithelium and in some
peripheral nerve ganglia (Fig. 2N,O).
In addition, otic and nasal capsules
expressed mRab11-FIP4 mRNA (Fig.
2O).
In contrast, the closely related gene
mRab11-FIP3 (GenBank accession no.
AB093257) was expressed ubiquitously in P1 and adult mice when examined by RT-PCR (Fig. 1C). During
development, mRab11-FIP3 was expressed throughout the retina in all
stages for all the times we examined,
although the expression level was relatively higher in RGC (later than
E13.5) and INL (later than P5; Fig.
2P–V). It is, therefore, indicated that
the predominant expression in neural
tissues is a
mRab11-FIP4.
pattern
unique
to
mRab11-FIP4 Regulates the
Fate Decision of Retinal Cell
To examine the function of mRab11FIP4 for retinal development, we
overexpressed either the A- or B-form
of mRab11-FIP4 in retinal explants by
using a retrovirus vector containing
enhanced green fluorescent protein
(EGFP) -driven by the internal ribosomal entry site (IRES; Ouchi et al.,
2005). Retinal explants prepared from
E17.5 mouse eyes were infected with
the retrovirus and cultured for 2
weeks. We first examined the localization of virus-infected cells by examining frozen sections of the explants.
When the control virus was used for
the infection, more than 80% of the
EGFP-positive (virus-infected) cells
were localized in the ONL, and the
rest of them were found in the INL
(Fig. 3A,B). On the other hand, by
overexpressing mRab11-FIP4A, we
found the proportion of EGFP-positive
cells localized in the INL to be significantly increased (Fig. 3A,B). This
phenotype was not observed by overexpression of the B-form. We then examined differentiation of the virus-infected cells by immunostaining
sections with antibodies against various marker proteins of the subpopulations of retinal cells. Antibodies used
were anti-rhodopsin (rod photoreceptors), -HuC/D (RGC and amacrine
cells), -PKC (bipolar cells), -glutamine
synthetase (GS; Müller glia), and -cyclin D3 (Müller glia) antibodies. The
relative proportions of Müller glia, bipolar cells, and amacrine cells were
increased by the expression of
mRab11-FIP4A (Fig. 3D,E). We observed neither the mislocalization of
photoreceptors (Fig. 3D) nor the enhanced cell death in the outer region
of NBL or ONL at any of the stages
examined (Fig. 3F).
Analysis of the localization of cells
at various time points revealed that
the increase in the number of cells
expressing mRab11-FIP4 in the INL
was first recognized at day 5 of the
culture period (Fig. 3G). When we
used a retinal explant prepared from
an E15.5 embryo, which was 2 days
younger than the retina used in the
experiment just described, an increase
mRab11-FIP4 REGULATES RETINAL DEVELOPMENT 217
in the proportion of cells located in the
INL was observed from day 7, indicating that the onset of abnormal sublocalization of the mRab11-FIP4A– expressing cells corresponded to P3 of
the developmental stage. The decline
in the expression of the endogenous
mRab11-FIP4 in the outer region of
the NBL between P1 and P5 (Fig. 2I,J)
thereby suggests that a persistent expression of mRab11-FIP4A during
this period may lead retinal progenitors to differentiate into cells in the
INL.
We next examined the effects of
down-regulation of the expression of
mRab11-FIP4 by using small hairpin
RNAs (shRNA) directed against both
(ABi) or either (Ai and Bi) of the two
forms of mRab11-FIP4 mRNAs (Fig.
4B). For this loss-of-function study, we
used the retrovirus vector pSSCG
(Yamamichi et al., 2005), which contains the U6 promoter to express
shRNA and the EGFP gene driven by
the CMV promoter. We examined the
efficiencies and specificities of these
shRNAs in 293T cells and confirmed
that all of shRNAs acted as expected
(Fig. 4B). Then, retinal explants prepared from E17.5 mice were infected
with the virus encoding these shRNA.
Fig. 2. Expression of class II Rab11-FIPs in the
developing mouse. A–O: The expression of
mRab11-FIP4 in the developing mouse.
A–C: Results of whole-mount in situ hybridization of mouse embryos at embryonic day (E) 8.5
(A), E9.5 (B), and E10.5 (C). D–L: Sections subjected to in situ hybridization for detection of
mRab11-FIP4 mRNA. Transverse sections of
embryonic heads at E10.5 (D), E11.5 (E), E12.5
(F), E13.5 (G), and E16.5 (H), and retinal sections prepared from postnatal mice at postnatal
day (P) 1 (I), P5 (J), P10 (K), and adult (L) were
examined by in situ hybridization. M–O: Expression of mRab11-FIP4 in areas around the forebrain (M), nose (N), and the inner ear (O) at
E16.5 was analyzed by using transverse sections. P–V: Expression of mRab11-FIP3 in the
developing retina. Expression pattern of
mRab11-FIP3 was assessed using cryosections from embryonic mice (P–R) and postnatal
retina (S–V). al, allantois; ba, branchial arch; co,
cochlea; cp, cortical plate; di, diencephalon; fl,
forelimb bud; gcl, retinal ganglion cell layer; h,
heart; hl, hindlimb bud; inl, inner nuclear layer;
iz, intermediate zone; le, lens; nbl, neuroblastic
layer; nc, nasal cavity; oe, olfactory epithelium;
onl, outer nuclear layer; op, optic vesicle; ot,
otic vesicle; pe, retinal pigment epithelium; sc,
spinal cord; st, striatum; tg, trigeminal ganglion;
vg, vagal ganglion; vz, ventricular zone. Scale
bars ⫽ 400 ␮m in A–C, 100 ␮m in D–V.
Fig. 2.
When both forms of mRab11-FIP4
were simultaneously knocked down by
expressing ABi in the explants, the
Fig. 3.
Fig. 4.
Fig. 3. Overexpression of mRab11-FIP4A leads
to an increase in the population of inner nuclear
layer (INL) cells at the expense of outer nuclear
layer (ONL) cells. A: Retinal explants prepared
from embryonic day (E) 17.5 mouse embryos
were infected with retroviruses encoding
mRab11-FIP4A, mRab11-FIP4B, or mRab11FIP4A⌬RBD, and enhanced green fluorescent
protein (EGFP) -positive virus-infected cells
were examined by immunohistochemistry in
sections prepared from 14-day cultures. DAPI
(4⬘,6-diamidine-2-phenylidole-dihydrochloride)
was used to visualize nuclei. B: Proportions of
EGFP-positive cells in each layer to total EGFPpositive cells in the sections. C: 293T cells were
transfected with pMX-mRab11-FIP4 (A, B or
⌬RBD) -internal ribosomal entry site (IRES)
-EGFP plasmid and the expression of each
form of mRab11-FIP4 was examined by Western blotting using an anti–mRab11-FIP4 antibody. EGFP was used as a control. D: Differentiation into each retinal cell type was
examined immunohistochemically. Markers
used are rhodopsin for rod photoreceptors,
PKC for bipolar cells, HuC/D for retinal ganglion
cells (RGC) and amacrine cells, and glutamine
synthetase (GS) and cyclin D3 for Müller glia.
E: Proportions of marker-positive cells among
total EGFP-positive cell population. F: Cell
death in the virus-infected retina prepared from
E17.5 mice examined by TUNEL (terminal
deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) assay at
the indicated days. G: Effect of mRab11-FIP4A
expression on subretinal localization of retinal
cells examined at different developmental stages.
Retinal explants prepared from E17.5 and
E15.5 embryos were infected with control or
pMXc-mRab11-FIP4A virus and analyzed for
the localization of the EGFP-positive cells at the
indicated times. Corresponding stages of the in
vivo development are indicated between the
two panels. Error bars indicate SEM. *P ⬍ 0.01;
**P ⬍ 0.05.
Fig. 4. mRab11-FIP4A is required for the differentiation of cell types in the inner nuclear layer
(INL). A: Schematic showing regions in
mRab11-FIP4 targeted by small hairpin RNAs
(shRNA). B: Specificity of shRNAs analyzed in
293T cells. 293T cells were cotransfected with
mRab11-FIP4A or mRab11-FIP4B and a vector
for the expression of shRNA, and the expression of mRab11-FIP4A and B was examined by
Western blotting using total cell lysates (upper
panel). The CBB staining pattern of the membrane is shown in the lower panel. C,D: Effect of
down-regulation of the endogenous mRab11FIP4A and/or mRab11-FIP4B on retinal development. C: Retinal explants prepared from embryonic day (E) 17.5 embryos were infected with
retroviruses encoding the indicated shRNAs,
and subretinal localization was examined in
sections prepared from 14-day cultures. D: The
proportion of EGFP-positive cells in each layer
to total EGFP-positive cells was then determined. Error bars indicate SEM. *P ⬍ 0.01.
mRab11-FIP4 REGULATES RETINAL DEVELOPMENT 219
proportion of EGFP-positive cells in
the INL was significantly decreased,
and most of them were localized in the
ONL (Fig. 4C,D). Subsequent analysis
using Ai or Bi revealed that only the
Ai had the effect of decreasing the
number of INL-located cells. This phenotype is the reverse of that found
when mRab11-FIP4A was overexpressed, which suggests that mRab11FIP4A plays an important role in regulation of differentiation into retinal
subpopulations localized in the INL
and ONL.
mRab11-FIP4A Promotes
Exit From the Cell Cycle
The determination of cell fate is
closely related to the timing of exit
from the cell cycle. Therefore, next we
analyzed the effect of knockdown or
overexpression of mRab11-FIP4A on
retinal cell proliferation by examining
the expression of Ki67, which is a nuclear antigen expressed in the proliferating cells (Gerdes et al., 1983; Fig.
5B). In the retinal explants infected
with either one of the control viruses,
approximately 25–27% of the EGFPpositive cells expressed Ki67 at day 4
of the culture period (Fig. 5). We found
that the population of proliferating
cells was decreased when mRab11FIP4A was overexpressed, whereas it
was increased when mRab11-FIP4A
was knocked down, indicating that
mRab11-FIP4A promoted the retinal
progenitors to exit from the cell cycle.
This result may account for the observation that the number of EGFP-positive cells was lower in the Rab11FIP4A– expressing retina than that in
control at day 14, despite equivalent
numbers of infected cells at day 3 (Fig.
3A,G). As to the mechanism responsible for this phenomenon, we examined
whether down-regulation of mRab11FIP4 would affect the expression of
p27Kip1 or not and found that its expression was partially suppressed in
the ABi shRNA-expressing cells (Fig.
5C,D).
mRab11-FIP4A Regulates the
Expression of Genes
Involved in Retinal
Development
We showed here that mRab11-FIP4A
influenced both proliferation and dif-
ferentiation of developing retinal
cells. To gain insight into the mechanism of mRab11-FIP4A– dependent
regulation of retinal development,
cells ectopically expressing mRab11FIP4A were sorted as retrovirus-infected (EGFP-positive) cells from retinal explants by flow cytometry and
the gene expression patterns in the
isolated cells were compared with
those in cells sorted from the control
virus-infected explants. Retinal explants prepared from E17.5 embryos
were infected with either control or
mRab11-FIP4A virus and trypsinized
to prepare single cell suspension at
day5, when the effect of mRab11FIP4A expression first appeared (Fig.
3G). Proportions of EGFP-positive
cells were approximately 24%, 8%,
and 22% in control, mRab11-FIP4A–
expressing, and mRab11-FIP4B– expressing retinal explants, respectively
(Fig. 6A). After staining with propidium iodide (PI), a fluorescent reagent specifically staining dead cells,
virus-infected (EGFP-positive) and
living (PI-staining negative) cells
were enriched by cell sorting using
FACSAria (BD Bioscience). In each
sample, 4 ⫻ 105 cells expressing
EGFP at a high level were isolated
from 1 ⫻ 107 dissociated cells (corresponding to 11 retinal explants) and
more than 90% of isolated cells were
indeed EGFP-positive (data not
shown). cDNA prepared from the isolated cells was subjected to RT-PCR,
in which the amount of cDNA was
equalized first by the amount of RNA
used and then compensated by the expression level of glyceraldehyde-3phosphate dehydrogenase (G3PDH;
Fig. 6B).
We first examined the growth-related genes, including cyclinD1,
p27Kip1, and p57Kip2 (Fig. 6B). As
expected, the expression level of cyclin
D1 was decreased and those of
p27Kip1 and p57Kip2 were increased
by ectopic expression of mRab11FIP4A. The expression of p27Kip1
was apparently, but only slightly, upregulated, probably because this gene
is already expressed in more then 80%
of control cells (Fig. 5D). Consistently,
in mRab11-FIP4A– expressing cells,
the expression of Pax6 and Rx, which
are known to be expressed in the retinal progenitors (Ahmad et al., 2004),
was decreased. Moreover, as expected
from the effects of mRab11-FIP4A on
retinal cell differentiation, Müller
glial markers, GS, and Vimentin (Hojo
et al., 2000), were up-regulated,
whereas the expression of rhodopsin
was down-regulated (Fig. 6B).
When we assessed the expression of
Wnt signaling-related genes, including
several ligands (Wnt), receptors (Mfz),
and soluble antagonists (Sfrp), the
expression of Wnt-5a and -5b, Mfz-3,
and Sfrp-3 were significantly enhanced
in mRab11-FIP4A– expressing cells,
whereas Mfz-4 and Sfrp-2 were detected in the cells at a comparable level
to those in control cells. While Wnt signaling is known to regulate proliferation of retinal progenitors during embryonic stages, genes related to Wnt
signaling were shown to be expressed
even in the perinatal and adult retina,
where most cells have ceased proliferating, with patterns unique to each gene
(Liu et al., 2003): i.e., Wnt-5a and -5b,
Mfz-3, and Sfrp-3 were expressed only
in the INL but not in ONL, whereas
Mfz-4 was expressed in both INL and
ONL. Therefore, given the results obtained from the immunohistochemical
analysis, the expression patterns of the
Wnt-related genes we observed could be
due to an increased proportion of INL
cells induced by mRab11-FIP4A expression.
We recently demonstrated that
Rab11-FIP4A regulated the retinal development by means of the activation of
Sonic Hedgehog (Shh) signaling in zebrafish (Muto et al., 2006). To evaluate
whether the expression of mRab11FIP4A affected on Shh signaling in the
mouse retina, we next examined the expression level of Shh as well as its target
genes, including Gli family of transcription factors (gli1 to 3) and patched-1
(ptc1). We found that the expression of
ptc1 was decreased in cells expressing
mRab11-FIP4A (Fig. 6B), thereby indicating that Shh signaling was likely to be
impaired in the cells expressing mRab11FIP4A. In addition, while the expression
levels of shh, gli1, and gli2 were not affected, the gli3 was significantly up-regulated when the mRab11-FIP4A was ectopically expressed (Fig. 6B). Gli3 is
known to be a negative regulator of Shh
signaling (Lipinski et al., 2006), suggesting that mRab11-FIP4A may regulate
Shh function negatively by means of the
induction of Gli3. The expression patterns of these genes were not affected by
220 MUTO ET AL.
expressing mRab11-FIP4B, further indicating the importance of the N-terminal
domain on the retinal development (Fig.
6B).
Interaction With Rab11 Is
Dispensable for the
Regulation of the Retinal
Development by mRab11FIP4
We next examined whether the RBD
found in the C-terminal end of Rab11FIP4 was functional to interact with
Rab11 or not. Fusion proteins of the
glutathione-S transferase (GST) protein fused to either the N-terminal
375 amino acid (a.a.; GST-4N) or Cterminal 260 a.a. (GST-4C) of
mRab11-FIP4 (Fig. 7A) were purified,
and the interaction of these proteins
with Rab11 was examined by conducting a pull-down assay (Fig. 7B).
EGFP-fused human Rab11 protein
(EGFP-hRab11), but not control
EGFP, was coprecipitated with the
GST-4C protein. In contrast, neither
the GST-4N protein (Fig. 7B) nor GST
(data not shown) was coprecipitated
with EGFP-hRab11. The binding of
the GST-4C to EGFP-hRab11 was
completely abolished by deleting a Cterminal 30-a.a. region encompassing
RBD from GST-4C (GST-4C⌬RBD),
indicating that the RBD was indeed
essential for the interaction of Rab11FIP4 with Rab11. This result is consistent with the previously reported
observations on other Rab11-binding
proteins (Prekeris et al., 2001).
To examine whether the binding with
the Rab11 plays a role in the biological
effects of mRab11-FIP4, we examined
the effects of overexpression of a C-terminally truncated mutant of mRab11FIP4A, mRab11-FIP4A⌬RBD, on the
retinal explants by means of retrovirusmediated gene expression. We found
that the expression of mRab11FIP4A⌬RBD also increased the proportion of INL-located cells, in fact to a
degree rather higher than that achieved
by the full-length A-form (Fig. 3A,B),
suggesting that the binding with Rab11
is dispensable for the function of
mRab11-FIP4 in retinal development.
It should be noted that the C-terminally
truncated protein (⌬RBD) was able to
be expressed stably at the level comparable to those of the A- and B-forms in
mammalian cells, including 293T (Fig.
3C) and NIH3T3 cells (data not shown),
despite the partial degradation of the
GST-fusion protein with the same truncation (GST4C-⌬RBD) in Escherichia
coli cells (Fig. 7B).
To further address this notion, we
examined the role of Rab11 activity in
retinal development by using wildtype (Rab11 WT), dominant-negative
(Rab11 S25N), and constitutively active (Rab11 Q70L) Rab11. As already
reported (Wallace et al., 2002b),
mRab11-FIP4A was localized in the
perinuclear region when expressed in
a cell line; and its expression there
was enhanced by coexpressing it with
either Rab11 WT or Rab11 Q70L (Fig.
7C). When coexpressed with Rab11
S25N, mRab11-FIP4A was diffusely
distributed in the cytoplasm, suggesting that Rab11-FIP4A forms a complex only with the active form of
Rab11. Although we found that the
proportion of cells was increased in
ONL when wild-type Rab11 was expressed, similar effects were also observed by the expression of either the
constitutively active or dominant-negative mutant of Rab11 (Fig. 7D,E),
suggesting that the activity of Rab11
had no effect on the sublayer localization of retinal cells. In addition, the
expression of either Rab11 or any of
its mutants had no effect on proliferation of retinal progenitors or localization of the retinal cells (Fig. 7F,G). All
these results, therefore, suggest that
mRab11-FIP4 may play a role in retinal development in a Rab11-independent manner.
DISCUSSION
We identified mRab11-FIP4A as a
gene predominantly expressed in neural tissues, including the retinal differentiating progenitors and RGC. By
gain- and loss-of-function experiments
using retinal explants, the involvement of mRab11-FIP4A for cell cycle
exit of retinal progenitors through the
up-regulation of p27Kip1 as well as
down-regulation of cyclinD1 and the
fate decision to subpopulations localized in the INL, such as bipolar cells
and Müller glia, was revealed. Analysis of the birth date, or the onset of the
differentiation, of various retinal subpopulations has indicated that the
late-born cells differentiate from pro-
genitors that have undergone longer
periods of proliferation (Young, 1985;
Livesey and Cepko, 2001). The bipolar
and Müller glia cells are the latestborn retinal cells and, therefore,
would be expected to be decreased in
number when the progenitors exit the
cell cycle earlier. However, several
Fig. 5. mRab11-FIP4A promotes cell cycle exit
of the retinal cells. A: Effect of overexpression
(left column) and down-regulation (right column) of mRab11-FIP4A on the proliferation of
retinal progenitors was analyzed in terms of
Ki-67 expression. Retinal explants prepared
from embryonic day (E) 17.5 embryos were infected with the indicated retroviruses, and proliferating cells in 4-day cultures were identified
immunohistochemically by using anti–Ki-67 antibody. B: Proportions of Ki-67–positive cells to
total enhanced green fluorescent protein
(EGFP) -positive cell population are shown.
C: Effect of mRab11-FIP4 on the expression of
p27Kip1 in 4-day cultures was examined immunohistochemically by using anti-p27Kip1 antibody. D: Proportions of p27Kip1-positive cells
to total EGFP-positive cell population are
shown. Error bars indicate SEM. *P ⬍ 0.01,
**P ⬍ 0.05.
Fig. 7. Neither binding to Rab11 nor Rab11
activity is required for the effect of mRab11FIP4A on the sublayer localization of retinal
cells. A: Schematic representation of the glutathione-S transferase (GST) -Rab11-FIP4 –fusion
proteins used for pull-down assay. B: Pulldown assay. Whole protein extracts prepared
from PLAT-E cells expressing either wild-type
enhanced green fluorescent protein (EGFP)
-Rab11 or EGFP were incubated with the purified GST-mRab11-FIP4 –fusion proteins and
then affinity-purified by using glutathione–
Sepharose beads. EGFP-Rab11 and EGFP coprecipitated with the GST-fusion proteins were
analyzed by Western blotting using an anti-GFP
antibody (upper panel). The GST-fusion proteins were visualized by CBB staining after having been transferred onto the membrane (lower
panel). The asterisk indicates degradation
products of GST-4C and GST-4C⌬RBD proteins. C: Myc-tagged mRab11-FIP4A was expressed solely (control, a– c) or coexpressed
with wild-type (WT, d–f), constitutively active
(Q70L, g–i), or dominant-negative (S25N, j–l)
Rab11 fused to EGFP in COS7 cells, and their
localization was examined by immunostaining
with anti-myc (red) and anti-EGFP (green) antibodies. Nuclei were visualized with DAPI (4⬘,6diamidine-2-phenylidole-dihydrochloride).
D–G: Rab11 mutants fused to EGFP, i.e., wildtype (WT), dominant-negative (S25N), and constitutively active (Q70L) Rab11, were expressed
in retinal explants prepared from E17.5 embryos. D–G: Then, subretinal localization (D,E)
and proliferation (F,G) of virus-infected cells
were examined immunohistochemically by using frozen sections prepared from 2-week (D)
and 4-day (F) cultures. DAPI was used to visualize nuclei. Error bars indicate SEM. *P ⬍ 0.01.
Fig. 5.
Fig. 6. Effects of mRab11-FIP4A expression
on the expression patterns of genes involved in
the retinal development. A: Schematic diagram
of cell sorting to isolate retrovirus-infected cells
from retinal explants. Retrovirus-infected retinal
explants prepared from embryonic day (E) 17.5
embryos were trypsinized at day 5 to prepare
single cell suspension and a population of cells
expressing enhanced green fluorescent protein
(EGFP) at high level was sorted by flow cytometry using FACSAria (BD Bioscience). Proportions of EGFP-positive cells in total retinal cells
were indicated. A total of 4 ⫻ 105 EGFP-positive cells were sorted from each sample. Total
RNA was prepared from the sorted cells, and
the gene expression pattern was examined by
reverse transcriptase-polymerase chain reaction (RT-PCR). B: Expression of genes involved
in the retinal development was analyzed by the
semiquantitative RT-PCR. The amount of cDNA
used was normalized by the expression of
glyceraldehydes-3-phosphate dehydrogenase
(G3PDH).
Fig. 7.
222 MUTO ET AL.
lines of evidence suggest that cell cycle regulation and histogenesis of the
retinal cells are not simply coordinated. In the developing mouse retinal progenitor cells, the cell cycle is
regulated positively by cyclin D1 and
negatively by the cdk inhibitors
p27Kip1 and p57Kip2 (Dyer, 2003).
Cyclin D1-deficient mice showed small
eyes with significantly impaired proliferation of retinal progenitors; however, their eye function was normal,
suggesting that the histogenesis was
unaffected and the neural network of
the retinal cells was properly formed
in the absence of cyclin D1 (Sicinski et
al., 1995). Differentiation of all major
types of retinal cells was also observed
in mice lacking either one of the above
cdk inhibitors (Levine et al., 1995;
Dyer and Cepko, 2000, 2001). On the
other hand, the misexpression of homeodomain and basic helix–loop– helix transcription factors in the retinal
explant promotes differentiation of
certain subsets of retinal cells without
affecting their cell proliferation or survival (Hatakeyama et al., 2001). On
the basis of these observations, we
surmised that mRab11-FIP4A regulates the retinal cell fate by the mechanism distinct from that for cell cycle
exit.
The single EF-hand motif in the Nterminal domain and the C-terminal
RBD are highly similar among orthologues of Rab11-FIP4, suggesting that
this gene has been evolutionally conserved. However, our observation revealed that interaction with Rab11
through the C-terminal RBD was dispensable for the retinal phenotypes
induced. There are several reports on
Rab11-FIPs to analyze the mechanism of the interaction with Rab11 by
introducing point mutations in their
conserved RBD (Prekeris et al., 2001;
Meyers and Prekeris, 2002; Fielding
et al., 2005). Although here we used
the C-terminal truncation mutant of
mRab11-FIP4A mRab11-FIP4A⌬RBD,
a point mutant of mRab11-FIP4 lacking
ability to bind to Rab11 could allow us
to examine the role of Rab11 in the retinal development in more detail.
In contrast, the N-terminal domain
containing the EF-hand motif was required for mRab11-FIP4A to induce
phenotypes when expressed in the retinal explant. The N-terminal EF-hand
motif is a Ca2⫹-binding motif found
in many Ca2⫹-binding proteins; and
in many cases, it functions in pairs to
induce a conformational change in the
protein in a Ca2⫹-dependent manner.
However, some proteins possess an
odd number of this motif, and a single
unpaired EF-hand is thought to contribute to the interaction with other
molecules (Lewit-Bentley and Rety,
2000), suggesting that the function of
mRab11-FIP4A in retinal development may be mediated by some molecule(s) interacting with the N-terminal domain.
The EF-hand motif was also found
in the N-terminal region of Rab11FIP3 (Meyers and Prekeris, 2002;
Wallace et al., 2002a). Despite
mRab11-FIP3 expression in the retina
throughout development, we demonstrated that retinal development was
perturbed when the expression of
mRab11-FIP4A alone was modified.
Although we could not exclude the
possibility that mRab11-FIP3 plays a
role in retinal development, mRab11FIP4A is likely to have a specific function, which cannot be complemented
by mRab11-FIP4B or mRab11-FIP3.
There are two possible mechanisms to
explain the specificity between Rab11FIP3 and Rab11-FIP4. One is the interaction with distinct proteins
through the EF-hand. Specificity of
the protein binding through the EFhand is in part determined by the primary amino acid sequence within and
around this motif (Bhattacharya et
al., 2004). The N-terminal sequence
surrounding the EF-hand in Rab11FIP4 is well conserved among species
but distinct from that found in Rab11FIP3, probably mediating the specific
protein interaction. The other possible
mechanism is differential affinities of
Rab11-FIP3 and Rab11-FIP4 for
small GTPases. Rab11-FIP3 and
Rab11-FIP4 in humans are also
known as arfophilin1 and arfophilin2,
respectively, and have been shown to
interact with Arf5 and Arf6 through a
binding site, which was located at the
C-terminal regions but distinct from
that for Rab11 (Fielding et al., 2005).
Biochemical analysis indicated that
Rab11-FIP3 bound to Rab11 with an
affinity greater than that for Arf6,
whereas Rab11-FIP4 was preferentially interacted with Arf6 rather than
Rab11 (Fielding et al., 2005), suggesting that Arf6 may be involved in the
retinal function of Rab11-FIP4. Although Arf6 was shown to be involved
at least in the neurite outgrowth in
the chick retina (Albertinazzi et al.,
2003), further analysis is required to
uncover the role of Arf6 in the retinal
development regulated by mRab11FIP4.
Recent studies indicated that mammalian class II Rab11-FIPs (FIP3 and
FIP4) and Drosophila Nuf were shown
to regulate the formation of the cleavage furrow in cytokinesis in mammalian cell lines (Fielding et al., 2005;
Wilson et al., 2005) and in cellularization in Drosophila embryos (Rothwell
et al., 1998; Riggs et al., 2003), respectively. In addition, effects of Rab11FIP4 expression on the morphology of
recycling endosome observed in HeLa
cells was reproduced by the expression of Nuf (Hickson et al., 2003).
These observations suggested that
Nuf was not only structurally but also
functionally related to the class II
Rab11-FIPs. However, Nuf contains
neither N-terminal EF-hand motif nor
Arf-binding domain (Hickson et al.,
2003; Wilson et al., 2005), and the
functional analysis of Rab11-FIP4 in
some previous works has been performed using an N-terminally truncated form of the protein (Wallace et
al., 2002b; Hickson et al., 2003), suggesting that the role of mRab11FIP4A we observed in the retinal development was mediated by the
mechanism distinct from that previously reported.
In this study, we identified two alternative forms of mRab11-FIP4, Aand B-forms, expressed in developing
mouse embryos. We found that
zRab11-FIP4 was also expressed as
two forms of transcripts with similarities in their structures and expression patterns (Muto et al., 2006). In
addition, the loss-of-function experiments using a morpholino antisense
oligo demonstrated that zRab11FIP4A was required for cell cycle exit
and differentiation of the retinal progenitors. These results suggest that
Rab11-FIP4 has been conserved structurally and functionally between
mouse and zebrafish. As in the zebrafish, mouse Rab11-FIP4 may play
roles in the differentiation of RGC;
however, we could not examine this
possibility because of technical difficulty.
mRab11-FIP4 REGULATES RETINAL DEVELOPMENT 223
zRab11-FIP4A has been implicated
to interact functionally with Shh signaling (Muto et al., 2006). Shh was
earlier shown to define the timing of
the cell cycle exit by regulating the
expression of cyclin D1 and p57Kip2
(Shkumatava and Neumann, 2005)
and to promote the differentiation of
RGC and photoreceptors (Stenkamp
et al., 2002; Shkumatava et al., 2004)
in the zebrafish retina. We found that
all these events were impaired in the
zRab11-FIP4A knockdown embryos
(morphants) and that the delayed cell
cycle exit found in the morphant retina was recovered by activating the
Shh signaling (Muto et al., 2006), suggesting that zRab11-FIP4A positively
interacts with Shh signaling in the
developing zebrafish retina. Shh has
also been shown to play important
roles in mouse retinal development,
but the roles are different from those
in the zebrafish retina; i.e., in mouse
retina, Shh promotes the proliferation
of retinal progenitors (Jensen and
Wallace, 1997), represses RGC production (Wang et al., 2005), and accelerates rod photoreceptor differentiation (Levine et al., 1997). Our results
indicated that mRab11-FIP4A affected negatively some of these
events; and, therefore, a mechanism
similar to that of zRab11-FIP4A and
Shh cannot be applied for mouse
Rab11-FIP4. In an effort to clarify the
role of mRab11-FIP4A on the retinal
development, we obtained some evidence suggesting the negative regulation of Shh signaling by the expression of mRab11-FIP4A. An analysis of
the expression patterns of genes related to Shh signaling showed that the
expression of ptc1, which is a major
target of Shh signaling, was down-regulated in the retinal cells expressing
mRab11-FIP4A. We also found that
only gli3 among three gli genes was
up-regulated by ectopic expression of
mRab11-FIP4A. These expression
patterns were similar to those observed in neurosphere derived from
neocortex of Shh mutant mice (Palma
and Ruiz i Altaba, 2004), suggesting
that mRab11-FIP4A regulated the expression of these genes by repressing
Shh signaling. Alternatively, the ectopic expression of gli3 might repress
Shh signaling in mRab11-FIP4A– expressing cells, as Gli3 was indicated to
be a negative regulator of Shh signal-
ing in vivo by means of the analysis
using mutant mice lacking Gli3 activity (Meyer and Roelink, 2003; Lipinski
et al., 2006). The role of Gli3 in the
context of mRab11-FIP4A function remains elusive.
Several lines of evidence have demonstrated that Shh signaling is also
regulated by vesicle trafficking at
multiple steps, including at the level
of the subcellular localization of the
Shh receptors Patched and Smoothened (Incardona et al., 2000; Martin et
al., 2001) and by the signaling pathway downstream of these receptors
(Eggenschwiler et al., 2006). One of
the molecules recently identified as a
negative regulator of Shh signaling is
Rab23, another member of Rab GTPase (Eggenschwiler et al., 2001). Recent studies demonstrated that Rab23
repressed Shh signaling by regulating
the activities Gli2 and Gli3 proteins
(Eggenschwiler et al., 2006). Rab23 is
expressed in eyes (Marcos et al.,
2003), and Rab23 mutant mice,
known as open brain (opb), were reported to have poorly developed eyes,
suggesting that Rab23 is involved in
the retinal development by regulating
Shh signaling. Rab11-FIP4 was demonstrated to interact with multiple
small GTPases (Hickson et al., 2003);
it is, therefore, tempting to speculate
that Rab11-FIP4 plays a role in the
retinal development by regulating
membrane trafficking system through
interaction with other small GTPases
such as Rab23. Further analysis is required to elucidate the exact mechanism of Rab11-FIP4 in retinal development and the interaction of this
protein with Shh signaling.
EXPERIMENTAL
PROCEDURES
Differential Display and
cDNA Cloning of Mouse
Rab11-FIP4
For differential display analysis, firststrand cDNA was synthesized from
2.5 ␮g of total RNA prepared from
whole eyes by using GT15V primer (5⬘GTTTTTTTTTTTTTTT(A/G/C)-3⬘)
and Superscript II reverse transcriptase (Invitrogen). DD-PCR was performed using a combination of the
GT15V primer and an arbitrary 10mer primer. Then, 30 cycles of PCR
reaction (95°C for 0.5 min, 40°C for 2
min, and 72°C for 1 min) were performed, and the products were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel and
visualized by staining with SYBR
green. Differentially displayed bands
were extracted from the gels, amplified under the same PCR conditions,
and subcloned into the pGEM-T easy
(Promega) or pCR2.1 vector (Invitrogen). Nucleotide sequences of the subcloned fragments were determined,
and database searching was performed by using the BLAST algorithm. Based on the partial sequence
found in Celera Discovery System, we
cloned a cDNA encoding the full
length of a putative mouse Rab11FIP4 protein by RT-PCR in combination with the 5⬘-RACE method using
cDNA prepared from P1 mouse retina
as a template.
Plasmid Construction
Plasmids to express GST-fused
mRab11-FIP4 in E. coli cells were constructed by subcloning PCR-amplified
partial fragments of mRab11-FIP4 into
pGEX6P-2 (Amersham). Retrovirus
vectors, pMXc-IRES-EGFP (Morita et
al., 2000; Tabata et al., 2004; Ouchi et
al., 2005) and pSSCG (Yamamichi et
al., 2005) were used for the expression
of genes and shRNAs, respectively.
cDNAs for wild-type, dominant-negative (S25N), and constitutively active
(Q70L) Rab11 (Savina et al., 2002),
which were kindly donated by Dr. M. I.
Colombo of the Universidad Nacional
de Cuyo-CONICET, were subcloned
into pMX-IRES-EGFP. For shRNA expression, double-stranded oligonucleotides covering shRNA sequences were
first subcloned into BbsI and EcoRI
sites of a pU6 vector, and then the U6
promoter-shRNA cassettes were further subcloned into pSSCG vector by
using BamHI and EcoRI sites.
Animals, Cell Culture, and
Transfection
Normal ICR, FVB, and C57BL6 mice
were purchased from SLC Japan. All
experiments on animals were approved by the Animal Care Committee
of the Institute of Medical Science of
the University of Tokyo.
COS7, 293T, or PLAT-E cells
224 MUTO ET AL.
(Morita et al., 2000), the last being a
derivative of HEK293 cells, were
grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal
calf serum. For PLAT-E cells, the medium was supplemented with 1 ␮g/ml
puromycin and 10 ␮g/ml brastcidine.
Exponentially growing cells (4 ⫻ 106
cells) were transfected with 2 ␮g of
plasmid by using Fugene6 (Roche Diagnostics GmbH) according to the
manufacturer’s instructions. The effect of shRNA was examined in 293T
cells expressing either the A- or Bform of mRab11-FIP4. The expression
of mRab11-FIP4 was examined by
Western blotting using rabbit polyclonal anti–mRab11-FIP4 antibody
prepared in our laboratory.
Preparation of retinal explants and
retrovirus infection was performed as
described previously (Ouchi et al.,
2005). To quantify the distribution of
retrovirus-infected cells, at least 10
sections from two independent retinas
were used and EGFP-positive cells on
these sections were counted under the
fluorescence microscope.
Cell Sorting and RT-PCR
Retinal explants infected with retrovirus were cultured for 5 days and dissociated by incubating in 0.25% trypsin/phosphate buffered saline (PBS)
at 37°C for 10 min. After removing
aggregated cells by filtration, virusinfected (EGFP-positive) retinal cells
in the dissociated single cell suspension were sorted using FACSAria
(Becton Dickinson). Gating parameters by forward and side scatters were
set to remove remaining dead or aggregated cells, and dead cells were
also removed as cells stained with PI.
Equal numbers of EGFP-positive cells
were isolated from all samples, and
total RNA extracted from the sorted
cells by a standard AGPC method was
used to prepare cDNA. All primers
used for the following RT-PCR were
designed from cDNA sequences in
Genbank.
Pull-Down Assay
GST and GST-fusion proteins were affinity purified from E. coli (BL21DA3
strain) lysates, which were prepared
in E. coli lysis buffer (PBS containing
1% Triton X-100 and 1 mM phenylm-
ethyl sulfonyl fluoride [PMSF]), by the
binding of the proteins to glutathione–
Sepharose 4B beads (Amersham).
Mammalian cell lysates containing
EGFP-Rab11 or EGFP (control) protein were prepared from PLAT-E cells
transfected with either pEGFP or
pEGFP-Rab11wt in 0.5 ml of a lysis
buffer (50 mM Hepes, pH 7.5, containing 150 mM NaCl, 5 mM MgCl2, 0.5%
Triton X-100, 1 mM PMSF, 2 ␮g/ml
leupeptin, and 1 ␮g/ml NaVO3). After
removal of debris by ultracentrifugation, supernatants were stored at
⫺80°C until used. Then, the lysate supernatants were thawed and incubated at 4°C for 6 hr with GST or
either one of the GST-Rab11-FIP4
proteins bound to the glutathione–
Sepharose 4B beads. These samples
were extensively washed, after which
the bound proteins were subjected to
SDS-PAGE. Precipitated EGFP and
EGFP-Rab11wt proteins were detected by Western blotting using an
anti-EGFP antibody (Clontech).
In Situ Hybridization and
Immunohistochemistry
For in situ hybridization, a digoxigenin (DIG)-labeled RNA probe was prepared from cDNA template subcloned
into the pGEM-T easy vector (Promega) by using a DIG RNA labeling
kit (Roche Diagnostics GmbH). For
whole-mount in situ hybridization,
mouse embryos (E8.5 to E10.5) were
fixed in 4% paraformaldehyde (PFA)/
PBS for overnight at 4°C. These embryos were dehydrated and rehydrated by passage through a series of
diluted methanol, bleached with 6%
hydrogen peroxide, treated with Proteinase K (10 ␮g/ml) for an appropriate time at room temperature, and
then fixed with 4% PFA/0.2% glutaraldehyde solution. Next, the samples
were hybridized with DIG-labeled
RNA probe for overnight at 70°C. On
the next day, the samples were extensively washed; and in situ hybridization and immunohistochemistry for
the tissue sections were performed, as
described elsewhere (Muto et al.,
2006). Antibodies used in this study
were anti-rhodopsin (4D2, 1:200;
kindly provided gift from Dr. Molday),
anti-Hu C/D (1:1,000; Molecular
Probes, Inc.), anti-PKC (1:20; Oncogene Research Product), anti-glu-
tamine synthetase (GS, 1:500; Chemicon International), anti-cyclin D3
(1:500, Santa Cruz Biotechnology),
anti-p27Kip1 (clone 57, 1:50; BD
Transduction Laboratories), anti-Ki67
(1:200, BD Biosciences), and anti-GFP
(1:5,000; Clontech).
ACKNOWLEDGMENTS
We thank Drs. M.I. Colombo and R.S.
Molday for providing Rab11 cDNAs
and anti-rhodopsin antibody 4D2, respectively. We thank Drs. H. Sagara
and R. Kurita for helpful comments
and discussion and also A. Usui for
technical support.
REFERENCES
Ahmad I, Das AV, James J, Bhattachary S,
Zhao X. 2004. Neural stem cells in the
mammalian eye: types and regulation.
Semin Cell Dev Biol 15:53–62.
Albertinazzi C, Za L, Paris S, de Curtis I.
2003. ADP-ribosylation factor 6 and a
functional PIX/p95-APP1 complex are
required for Rac1B-mediated neurite
outgrowth. Mol Biol Cell 14:1295–1307.
Bhattacharya S, Bunick CG, Chazin WJ.
2004. Target selectivity in EF-hand calcium binding proteins. Biochim Biophys
Acta 1742:69 –79.
Cullis DN, Philip B, Baleja JD, Feig LA.
2002. Rab11-FIP2, an adaptor protein
connecting cellular components involved
in internalization and recycling of epidermal growth factor receptors. J Biol
Chem 277:49158 –49166.
Dyer MA. 2003. Regulation of proliferation, cell fate specification and differentiation by the homeodomain proteins
Prox1, Six3, and Chx10 in the developing
retina. Cell Cycle 2:350 –357.
Dyer MA, Cepko CL. 2000. p57(Kip2) regulates progenitor cell proliferation and
amacrine interneuron development in
the mouse retina. Development 127:
3593–3605.
Dyer MA, Cepko CL. 2001. p27Kip1 and
p57Kip2 regulate proliferation in distinct retinal progenitor cell populations.
J Neurosci 21:4259 –4271.
Eggenschwiler JT, Espinoza E, Anderson
KV. 2001. Rab23 is an essential negative
regulator of the mouse Sonic hedgehog
signalling pathway. Nature 412:194 –
198.
Eggenschwiler JT, Bulgakov OV, Qin J, Li
T, Anderson KV. 2006. Mouse Rab23 regulates Hedgehog signaling from Smoothened to Gli proteins. Dev Biol 290:1–12.
Fielding AB, Schonteich E, Matheson J,
Wilson G, Yu X, Hickson GR, Srivastava
S, Baldwin SA, Prekeris R, Gould GW.
2005. Rab11-FIP3 and FIP4 interact
with Arf6 and the exocyst to control
membrane traffic in cytokinesis. EMBO
J 24:3389 –3399.
mRab11-FIP4 REGULATES RETINAL DEVELOPMENT 225
Gerdes J, Schwab U, Lemke H, Stein H.
1983. Production of a mouse monoclonal
antibody reactive with a human nuclear
antigen associated with cell proliferation. Int J Cancer 31:13–20.
Hales CM, Griner R, Hobdy-Henderson
KC, Dorn MC, Hardy D, Kumar R, Navarre J, Chan EK, Lapierre LA, Goldenring JR. 2001. Identification and characterization of a family of Rab11interacting proteins. J Biol Chem 276:
39067–39075.
Hatakeyama J, Tomita K, Inoue T,
Kageyama R. 2001. Roles of homeobox
and bHLH genes in specification of a retinal cell type. Development 128:1313–
1322.
Hickson GR, Matheson J, Riggs B, Maier
VH, Fielding AB, Prekeris R, Sullivan
W, Barr FA, Gould GW. 2003. Arfophilins are dual Arf/Rab 11 binding proteins
that regulate recycling endosome distribution and are related to Drosophila nuclear fallout. Mol Biol Cell 14:2908 –
2920.
Hojo M, Ohtsuka T, Hashimoto N, Gradwohl G, Guillemot F, Kageyama R. 2000.
Glial cell fate specification modulated by
the bHLH gene Hes5 in mouse retina.
Development 127:2515–2522.
Incardona JP, Lee JH, Robertson CP, Enga
K, Kapur RP, Roelink H. 2000. Receptormediated endocytosis of soluble and
membrane-tethered Sonic hedgehog by
Patched-1. Proc Natl Acad Sci U S A
97:12044 –12049.
Jensen AM, Wallace VA. 1997. Expression
of Sonic hedgehog and its putative role
as a precursor cell mitogen in the developing mouse retina. Development 124:
363–371.
Levine EM, Close J, Fero M, Ostrovsky A,
Reh TA. 1995. p27(Kip1) regulates cell
cycle withdrawal of late multipotent progenitor cells in the mammalian retina.
Dev Biol 219:299 –314.
Levine EM, Roelink H, Turner J, Reh TA.
1997. Sonic hedgehog promotes rod photoreceptor differentiation in mammalian
retinal cells in vitro. J Neurosci 17:6277–
6288.
Lewit-Bentley A, Rety S. 2000. EF-hand
calcium-binding proteins. Curr Opin
Struct Biol 10:637–643.
Lindsay AJ, Hendrick AG, Cantalupo G,
Senic-Matuglia F, Goud B, Bucci C, McCaffrey MW. 2002. Rab coupling protein
(RCP), a novel Rab4 and Rab11 effector
protein. J Biol Chem 277:12190 –12199.
Lipinski RJ, Gipp JJ, Zhang J, Doles JD,
Bushman W. 2006. Unique and complimentary activities of the Gli transcription factors in Hedgehog signaling. Exp
Cell Res 312:1925–1938.
Liu H, Mohamed O, Dufort D, Wallace VA.
2003. Characterization of Wnt signaling
components and activation of the Wnt
canonical pathway in the murine retina.
Dev Dyn 227:323–334.
Livesey FJ, Cepko CL. 2001. Vertebrate
neural cell-fate determination: lessons
from the retina. Nat Rev Neurosci 2:109 –
118.
Marcos I, Borrego S, Antinolo G. 2003. Molecular cloning and characterization of
human RAB23, a member of the group of
Rab GTPases. Int J Mol Med 12:983–
987.
Martin V, Carrillo G, Torroja C, Guerrero
I. 2001. The sterol-sensing domain of
Patched protein seems to control
Smoothened activity through Patched
vesicular trafficking. Curr Biol 11:601–
607.
Meyer NP, Roelink H. 2003. The aminoterminal region of Gli3 antagonizes the
Shh response and acts in dorsoventral
fate specification in the developing spinal cord. Dev Biol 257:343–355.
Meyers JM, Prekeris R. 2002. Formation of
mutually exclusive Rab11 complexes
with members of the family of Rab11interacting proteins regulates Rab11 endocytic targeting and function. J Biol
Chem 277:49003–49010.
Morita S, Kojima T, Kitamura T. 2000.
Plat-E: an efficient and stable system for
transient packaging of retroviruses.
Gene Ther 7:1063–1066.
Muto A, Arai K, Watanabe S. 2006. Rab11FIP4 is predominantly expressed in neural tissues and involved in proliferation
as well as in differentiation during zebrafish retinal development. Dev Biol
292:90 –102.
Nagase T, Nakayama M, Nakajima D,
Kikuno R, Ohara O. 2001. Prediction of
the coding sequences of unidentified human genes. XX. The complete sequences
of 100 new cDNA clones from brain
which code for large proteins in vitro.
DNA Res 8:85–95.
Ouchi Y, Tabata Y, Arai K, Watanabe S.
2005. Negative regulation of retinal-neurite extension by beta-catenin signaling
pathway. J Cell Sci 118:4473–4483.
Palma V, Ruiz i Altaba A. 2004. HedgehogGLI signaling regulates the behavior of
cells with stem cell properties in the developing neocortex. Development 131:
337–345.
Perron M, Harris WA. 2000. Retinal stem
cells in vertebrates. Bioessays 22:685–
688.
Prekeris R, Davies JM, Scheller RH. 2001.
Identification of a novel Rab11/25 binding domain present in Eferin and Rip
proteins. J Biol Chem 276:38966 –38970.
Riggs B, Rothwell W, Mische S, Hickson
GR, Matheson J, Hays TS, Gould GW,
Sullivan W. 2003. Actin cytoskeleton remodeling during early Drosophila furrow
formation requires recycling endosomal
components Nuclear-fallout and Rab11.
J Cell Biol 163:143–154.
Rothwell WF, Fogarty P, Field CM, Sullivan W. 1998. Nuclear-fallout, a Drosophila protein that cycles from the cytoplasm to the centrosomes, regulates
cortical microfilament organization. Development 125:1295–1303.
Savina A, Vidal M, Colombo MI. 2002. The
exosome pathway in K562 cells is regu-
lated by Rab11. J Cell Sci 115:2505–
2515.
Shkumatava A, Fischer S, Muller F,
Strahle U, Neumann CJ. 2004. Sonic
hedgehog, secreted by amacrine cells,
acts as a short-range signal to direct differentiation and lamination in the zebrafish retina. Development 131:3849 –
3858.
Shkumatava A, Neumann CJ. 2005. Shh
directs cell-cycle exit by activating
p57Kip2 in the zebrafish retina. EMBO
Rep 6:563–569.
Sicinski P, Donaher JL, Parker SB, Li T,
Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA. 1995.
Cyclin D1 provides a link between development and oncogenesis in the retina
and breast. Cell 82:621–630.
Stenkamp DL, Frey RA, Mallory DE,
Shupe EE. 2002. Embryonic retinal gene
expression in sonic-you mutant zebrafish. Dev Dyn 225:344 –350.
Tabata Y, Ouchi Y, Kamiya H, Manabe T,
Arai K, Watanabe S. 2004. Specification
of the retinal fate of mouse embryonic
stem cells by ectopic expression of Rx/
rax, a homeobox gene. Mol Cell Biol 24:
4513–4521.
Wallace DM, Lindsay AJ, Hendrick AG,
McCaffrey MW. 2002a. The novel Rab11FIP/Rip/RCP family of proteins displays
extensive homo- and hetero-interacting
abilities. Biochem Biophys Res Commun
292:909 –915.
Wallace DM, Lindsay AJ, Hendrick AG,
McCaffrey MW. 2002b. Rab11-FIP4 interacts with Rab11 in a GTP-dependent
manner and its overexpression condenses the Rab11 positive compartment
in HeLa cells. Biochem Biophys Res
Commun 299:770 –779.
Wang Y, Dakubo GD, Thurig S, Mazerolle
CJ, Wallace VA. 2005. Retinal ganglion
cell-derived sonic hedgehog locally controls proliferation and the timing of RGC
development in the embryonic mouse
retina. Development 132:5103–5113.
Wilson GM, Fielding AB, Simon GC, Yu X,
Andrews PD, Hames RS, Frey AM, Peden AA, Gould GW, Prekeris R. 2005.
The FIP3-Rab11 protein complex regulates recycling endosome targeting to the
cleavage furrow during late cytokinesis.
Mol Biol Cell 16:849 –860.
Yamamichi N, Yamamichi-Nishina M, Mizutani T, Watanabe H, Minoguchi S,
Kobayashi N, Kimura S, Ito T, Yahagi N,
Ichinose M, Omata M, Iba H. 2005. The
Brm gene suppressed at the post-transcriptional level in various human cell
lines is inducible by transient HDAC inhibitor treatment, which exhibits antioncogenic potential. Oncogene 24:5471–
5481.
Young RW. 1985. Cell differentiation in the
retina of the mouse. Anat Rec 212:199 –
205.
Zerial M, McBride H. 2001. Rab proteins as
membrane organizers. Nat Rev Mol Cell
Biol 2:107–117.