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First published online 31 January 2007
doi: 10.1242/dev.02776
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1 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235,
USA.
2 Department of Medicine, Vanderbilt University Medical School, Nashville, TN
37232, USA.
3 Nephrology Division, Harvard Medical School, Massachusetts General Hospital,
Charlestown, MA 02129, USA.
* Author for correspondence (e-mail: lilianna.solnica-krezel{at}vanderbilt.edu)
Accepted 6 December 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12/13 or Plexin B1. The fruit fly
homolog, RhoGEF2, interacts with heterotrimeric G protein subunits to activate
Rho, associates with microtubules, and is required during gastrulation for
cell shape changes that mediate epithelial folding. Here, we report functional
characterization of a zebrafish homolog of ARHGEF11 that is expressed
ubiquitously at blastula and gastrula stages and is enriched in neural tissues
and the pronephros during later embryogenesis. Similar to its human homolog,
zebrafish Arhgef11 stimulated actin stress fiber formation in cultured cells,
whereas overexpression in the embryo of either the zebrafish or human protein
impaired gastrulation movements. Loss-of-function experiments utilizing a
chromosomal deletion that encompasses the arhgef11 locus, and
antisense morpholino oligonucleotides designed to block either translation or
splicing, produced embryos with ventrally-curved axes and a number of other
phenotypes associated with ciliated epithelia. Arhgef11-deficient embryos
often exhibited altered expression of laterality markers, enlarged brain
ventricles, kidney cysts, and an excess number of otoliths in the otic
vesicles. Although cilia formed and were motile in these embryos, polarized
distribution of F-actin and Na+/K+-ATPase in the
pronephric ducts was disturbed. Our studies in zebrafish embryos have
identified new, essential roles for this RhoGEF in ciliated epithelia during
vertebrate development.
Key words: Pronephros, Left-right asymmetry, Otoliths, PDZ-RhoGEF, Arhgef11, Cell polarity, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Rossman et al., 2005). Most of
these RhoGEFs possess both Dbl-homology (DH) and Pleckstrin-homology (PH)
domains in tandem. These domains often interact specifically with the target
GTPase(s) and constitute the functional nucleotide exchange subunit
(Cerione and Zheng, 1996). In
addition to the DH and PH domains, many RhoGEFs possess domains that modulate
their functions and connect Rho to a variety of signaling pathways. The
regulator of G protein-coupled signaling (RGS) domains link G protein-coupled
signaling to Rho by binding the alpha subunits of activated heterotrimeric G
proteins to RhoGEFs. The family of RGS-domain-containing RhoGEFs includes
ARHGEF1 (p115RhoGEF), ARHGEF12 (Leukemia Associated RhoGEF, LARG) and ARHGEF11
(KIAA0380, PDZ-RhoGEF) (Fukuhara et al.,
2001). Both ARHGEF11 and ARHGEF12 also contain a PDZ
(PSD-95/DLG/ZO1) domain, which has been shown to interact with the C-terminus
of the Semaphorin receptor, Plexin B1
(Swiercz et al., 2002).
Through this interaction, Semaphorin 4D stimulation of Plexin B1 activates Rho
signaling pathways and influences axon guidance
(Aurandt et al., 2002;
Perrot et al., 2002;
Swiercz et al., 2002). These
studies demonstrate that activation of Rho via ARHGEF11 and ARHGEF12 can be
modulated through both the PDZ and RGS domains.
Thus far, human ARHGEF11 and its closely related family member, ARHGEF12,
have been studied primarily in cell culture, where they activate Rho and
promote reorganization of the actin cytoskeleton in response to stimulation by
heterotrimeric G protein subunits G12/13
(Fukuhara et al., 1999). In
addition to the work illuminating the interactions and roles of the conserved
domains of ARHGEF11, further studies have delineated other regions of the
protein that may be important for its function. One such region is the
C-terminus, which may interact with p-21 activated kinase 4 (PAK4), or homo-
or heterodimerize with the C-terminus of another ARHGEF11 or ARHGEF12 molecule
(Barac et al., 2004;
Chikumi et al., 2004).
Moreover, a small region of ARHGEF11 between the RGS and DH domains has been
shown to interact with the actin cytoskeleton
(Banerjee and Wedegaertner,
2004). These reported interactions and functions were determined
using yeast and mammalian cell culture systems, where both ARHGEF11 and
ARHGEF12 exhibit very similar activities. Interestingly, there is evidence
from mouse studies suggesting that the expression profiles of these two
proteins are somewhat different, with ARHGEF11 detected predominantly in
neural tissues and ARHGEF12 found in both neural and non-neural tissues
(Kuner et al., 2002).
Therefore, although ARHGEF11 and ARHGEF12 seem to have mostly redundant
activities in cultured cells, these functions may be modulated differently and
in a tissue-specific manner.
Most of our knowledge of in vivo roles for ARHGEF11 has come from work
conducted with the fruit fly homolog, RhoGEF2, which contains all the major
domains and displays significant sequence similarity with ARHGEF11. RhoGEF2
was first identified in a screen for Rho signaling pathway components, and was
further shown to control cell shape changes during gastrulation
(Barrett et al., 1997;
Hacker and Perrimon, 1998).
This RhoGEF also associates with microtubules via the plus-end-binding
protein, EB1, and regulates actomyosin contraction in the epithelia of the
developing embryo (Rogers et al.,
2004; Padash Barmchi et al.,
2005). Additionally, apical distribution of RhoGEF2 and other Rho
activators together with the basolateral distribution of Rho inhibitors are
required to modulate actin via Rho during cell invagination and lumen
formation for proper development of the spiracle in the fruit fly
(Simoes et al., 2006).
Studies of these vertebrate and invertebrate PDZ-domain-containing RhoGEFs suggest they may act similarly to regulate the cytoskeleton by modulating Rho in response to G protein-coupled signaling, possibly while associating with actin and/or microtubules. Despite all the information garnered from cultured cells and the D. melanogaster system, the roles of ARHGEF11 in developing and adult vertebrates remain to be elucidated.
Here we use the zebrafish model, which is particularly amenable to in vivo analysis, to assess the functions of a zebrafish homolog of human ARHGEF11 during vertebrate development. Employing several loss-of-function approaches, we have identified new and unanticipated roles for this vertebrate RhoGEF in processes involving ciliated epithelia, including establishment of left-right asymmetry, formation of otoliths in the otic vesicle, and development of the pronephros.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Embryos were obtained from natural matings and staged according to morphology
(Kimmel et al., 1995).
Cloning and sequencing of arhgef11
Positional cloning of the trilobite locus revealed
arhgef11 nearby on chromosome 7
(Jessen et al., 2002). Using
5' and 3' SMART RACE (BD Biosciences), partial UTR and the entire
coding sequence of arhgef11 were determined. Total RNA was isolated
from 2- to 8-cell or 48-hour WT embryos using TRIzol Reagent (Invitrogen), and
used to produce cDNA via the Superscript First-Strand Synthesis System
(Invitrogen). Using primers to the 5'
(5'-CGGAATTCCGTCATGAATGTCCGACACC-3' or
5'-CGGAATTCCACTACACTAATAACCGGACAAACC-3') and 3'
(5'-GCTCTAGAAGCGTCAGAAACCAGTGGAC-3') UTR, both the `early' and
`late' splice variants of arhgef11 were PCR-amplified from cDNA using
Platinum Pfx DNA Polymerase (Invitrogen). The complete coding sequence was
amplified and cloned into the EcoRI-XbaI sites of the pCS2+
vector (Rupp et al., 1994),
then verified by sequencing. N-terminal-tagged arhgef11 constructs
were made by subcloning into myc-pCS2+.
RT-PCR was performed by isolating RNA and producing cDNA as described above from embryos at the indicated stages. The resultant cDNAs were used as templates to amplify small regions of the gene encompassing the alternatively-spliced exons.
The construct encoding the dominant-negative form of Arhgef11,
dhph, was generated so as to lack the DH and PH domains by
using the myc-tagged full-length `early' construct as a template to amplify
the protein-encoding regions directly 5' and 3' of the DH-PH
tandem, while inserting an XhoI site in its place with the following
primers: 5'-CGCTCGAGTAATATGTGCGGCTCCACTG-3' and
5'-CGCTCGAGTCTGCCGATCAGTCAGAAGG-3'. This site was then used to
ligate the fragments together without the DH and PH domains.
The arhgef11 full-length gene sequence reported in this paper has been deposited in GenBank under the accession number AY295347.
In situ hybridization
Sense and antisense probes for arhgef11 were made with
digoxigenin-labeled NTPs (Roche) using pCS2-arhgef11 constructs
linearized with NotI or EcoRI as templates for RNA synthesis
with SP6 or T7 RNA polymerases, respectively. Antisense probes for
southpaw (spaw) (Long et
al., 2003), pitx2
(Tsukui et al., 1999),
cardiac myosin light chain 2 (cmlc2; myl7 -
Zebrafish Information Network) (Yelon et
al., 1999) and preproinsulin (ins)
(Milewski et al., 1998) were
prepared as described previously. Embryos were fixed at the stages indicated
and processed essentially as previously described
(Thisse and Thisse, 1998).
Some of the stained embryos were embedded in a solution of 1.2% agarose, 30%
sucrose and cryosectioned using a Leica CM1900.
RNA and morpholino injections
Capped sense RNA encoding Arhgef11 was synthesized with SP6 RNA polymerase
(Ambion mMessage mMachine system) after linearization of the
pCS2-arhgef11 construct with NotI. RNA was purified using
G-50 Sephadex Quick Spin Columns (Roche) and diluted with tissue culture grade
distilled water. Microinjections into 1- to 4-cell embryos were performed as
described previously (Marlow et al.,
1998).
A translation-blocking morpholino oligonucleotide (MO), MOAUG (Gene-Tools, LLC), targeted to the 5'-UTR (5'-GACGGAGGTTTGTCCGGTTATTAGT-3') and a splice-blocking MO, MOSPL (Open Biosystems), targeted to the exon 10-intron 10 boundary (5'-GGATACACTCACCTCCACGTCTCCT-3') were used. MOs were diluted and microinjected as described above.
Stress fiber formation assay
HEK293 cells were grown to confluence in DMEM supplemented with 10% FBS,
1x penicillin/streptomycin and 2 mM GlutaMAX (Gibco), then plated onto
poly-D-lysine (Sigma) -coated coverslips in 6-well plates at
4x105 cells per well. After reaching 50-70% confluence, cells
were transfected with myc-pCS2 containing arhgef11 or
dhph, or pCS2-GFP as a control, using TransIT-LT1
Transfection Reagent (Mirus). After 24 hours, cells were serum-starved for 4
hours. As a positive control, 10 nM thrombin (a gift from P. E. Bock,
Vanderbilt University Medical School, Nashville, TN) was then added for 20
minutes. After fixation with 4% paraformaldehyde (PFA) in PBS, cells were
immunostained using AlexaFluor546-phalloidin (Molecular Probes) and Rabbit
anti-c-myc (Research Diagnostics) with Cy2-anti-Rabbit (Jackson ImmunoResearch
Laboratories) antibodies, then visualized by confocal microscopy (Zeiss LSM510
META). The GFP construct used as transfection control was made by subcloning
GFP coding sequence from the pEGFP-N1 vector (Clontech) into pCS2+.
Antibody production
A portion of the `early' arhgef11 construct encoding amino acids
509-736 was amplified and cloned into a pET30a+ vector (Novagen). The
construct was expressed and purified essentially as described previously
(Panizzi et al., 2006). The
fragment was dialyzed into PBS, then sent to ProSci for rabbit polyclonal
antibody production.
Western blotting
Zebrafish embryos were manually dechorionated and deyolked before lyzing.
Laemmli SDS reducing sample buffer
(Laemmli, 1970) was added to
the lyzate before loading onto 4-15% Tris-HCl Ready Gels (BioRad) and
electrophoresis in Tris-glycine-SDS buffer
(Laemmli, 1970). Proteins were
then transferred to Immobilon-P membrane (Millipore) in Tris-glycine buffer
and the membranes processed essentially as described previously
(Iwamoto et al., 2006), using
the primary antibodies indicated.
Immunohistochemistry
Embryos were grown to the indicated stages and processed essentially as
described previously (Topczewska et al.,
2001), using CY2-, CY5- or CY3-conjugated secondary antibodies
(Jackson ImmunoResearch Laboratories). Where indicated,
AlexaFluor546-phalloidin was added with secondary antibody to visualize
F-actin. Embryos were analyzed by confocal microscopy (Zeiss LSM510 META).
Antibody staining on cryosections was performed essentially as described above, except that Tween 20 was omitted and that PBS containing 5% evaporated milk, 5% goat serum was used for blocking. For Na+/K+-ATPase detection, fixation was performed overnight at -20°C in Dent's fixative (80% methanol, 20% DMSO).
In addition to anti-zRG, the following primary antibodies were used:
6F (anti-Na+/K+-ATPase; Developmental Studies
Hybridoma Bank), anti-aPKC
(Santa Cruz Biotechnology), anti-ZO1 (Zymed
Laboratories), anti-
-tubulin and anti-acetylated tubulin (Sigma).
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4 µm sections were obtained, and then processed with
hematoxylin and eosin (BBC Biochemical) according to the manufacturer's
protocol.
Live imaging of cilia
Movies were captured and processed as described previously
(Kramer-Zucker et al.,
2005).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Sequence analysis by BLAST (Tatusova and
Madden, 1999) revealed that this gene encodes a protein of 1419
amino acids that shares 40% identity and a further 13% similarity with human
ARHGEF11 (Nagase et al., 1997;
Fukuhara et al., 1999), and
bears even higher similarity within each of the conserved domains
(Fig. 1A). In addition, the
zebrafish protein has significant similarity to RhoGEF2 from D.
melanogaster (Barrett et al.,
1997; Hacker and Perrimon,
1998), particularly within each of the conserved domains
(Fig. 1A). Based on our
analysis of the zebrafish genomic DNA sequence, we predict arhgef11
comprises more than 40 small exons (data not shown), much like its human
counterpart (Hubbard et al.,
2005). Interestingly, there is also conserved synteny between the
region of zebrafish chromosome 7 containing arhgef11 and
vangl2/trilobite and the region of human chromosome 1 that contains
their human homologs (Fig. 1K).
The amino acid sequence similarity coupled with the syntenic relationship
support the notion that arhgef11 is the ortholog of human
ARHGEF11, and the encoded proteins are likely to share many of the
same functions. To study the gene in the developing zebrafish embryo, we cloned two arhgef11 isoforms using cDNA synthesized from the RNA of embryos at 2 days post-fertilization (dpf). Both cloned forms of arhgef11 contain the complete coding sequence for all four of the conserved domains, whereas small exons outside these regions are alternatively-spliced (Fig. 1A, pink bars). Further analysis by RTPCR on embryos at stages from 1 hour post-fertilization (hpf) to 2 dpf demonstrated that these two isoforms are expressed dynamically during early embryogenesis (Fig. 1B). Sequence analysis of the first isoform revealed that it lacks the exon encoding amino acids 1232-1257 of the predicted full-length transcript, situated near the C-terminus just after the PH domain (Fig. 1A). Furthermore, RT-PCR amplification of a small fragment using primers flanking this alternatively-spliced exon showed that this form persists from early cleavage stages through to 2 dpf, and we henceforth refer to it as the `early' form (Fig. 1B). In addition, we identified another site of alternative splicing between the RGS and DH domains at the exon encoding amino acids 556-566 of the predicted full-length transcript (Fig. 1A). The form lacking this exon was only detected after 1 dpf (Fig. 1B), and is hereafter referred to as the `late' form. Although these seem to be the most highly represented forms of arhgef11 during early development, RT-PCR experiments indicated that the full-length form is also present (data not shown).
Arhgef11 induces actin stress fiber formation in cultured cells
We next assayed for functional similarities between the human and zebrafish
forms of ARHGEF11 in cell culture. After serum starvation, cultured HEK293
cells transfected with human ARHGEF11 develop actin stress fibers as a result
of RhoA stimulation (Rumenapp et al.,
1999). We predicted that zebrafish Arhgef11 acts similarly owing
to the conservation of crucial amino acids within the DH domain required for
Rho specificity (see Fig. S1 in the supplementary material)
(Oleksy et al., 2006).
Accordingly, we found that after transfection of HEK293 cells with plasmid
containing myc-arhgef11 (early form), the protein was distributed
throughout the cell with modest enrichment near the cell membrane
(Fig. 1H' and data not
shown), and the cells developed actin stress fibers following serum starvation
(Fig. 1H). Cells transfected
with a GFP-containing plasmid (Fig.
1F) did not form actin stress fibers, serving as both a
transfection and negative control. As a positive control, GFP-transfected
cells were incubated with thrombin (Fig.
1G), a potent stimulator of actin stress fiber formation
(Murphy et al., 2001).
In addition to the phenotypic similarities observed after overexpression of human or zebrafish Arhgef11 in cell culture, the effects they had on the developing zebrafish embryo were virtually indistinguishable. When either form was overexpressed by microinjection of synthetic RNAs, blastula stage embryos accumulated a large mass of cells within the blastoderm and failed to properly complete gastrulation movements (data not shown).
Spatiotemporal expression pattern of Arhgef11 RNA and protein during zebrafish development
To further understand the roles of this RhoGEF during vertebrate
development, we used digoxigenin-labeled antisense probes to determine the
localization of RNA encoding Arhgef11 by in situ hybridization in zebrafish
embryos at different developmental stages. Transcripts were detected in each
cell at early cleavage stages, less than 1 hpf and before zygotic
transcription begins (Kane and Kimmel,
1993), indicating a maternal contribution of arhgef11
(Fig. 1C). Embryos undergoing
gastrulation movements, at just over 8 hpf, also expressed arhgef11
ubiquitously (Fig. 1D). By 24
hpf, the transcripts became localized largely to the head, central nervous
system and pronephric ducts (Fig.
1E,E'). No signal was detected when a digoxigenin-labeled
sense probe for arhgef11 was used (data not shown).
To gain insight into the function of Arhgef11 protein, we synthesized a
peptide encoding amino acids 509-736, which are located between the RGS and DH
domains, and developed a rabbit polyclonal antibody (-zRG). We first
tested the ability of this antibody to detect the myc-tagged full-length
protein by western blotting. A band of the predicted molecular weight
(157 kDa) was detected in embryo extracts using either whole serum or
affinity-purified antibody, but not with preimmune serum
(Fig. 1J and data not shown).
In extracts of embryos microinjected with synthetic RNA encoding a myc-tagged
Arhgef11, the 157 kDa band detected by -zRG was more intense and
was also detected by polyclonal antibodies for c-myc
(Fig. 1J). We next tested the
ability of the whole serum and the affinity-purified antibody to detect
endogenous protein via whole-mount immunostaining of the zebrafish embryos. By
this method, we were unable to detect significant amounts of Arhgef11 in cells
of the early gastrula (6 hpf) or during early segmentation (13 hpf) (data not
shown). However, at 24 hpf, fluorescent immunostaining was remarkably similar
to the RNA expression data, revealing enrichment of Arhgef11 in the anterior
region of the embryos and in cells lining the pronephric ducts
(Fig. 6A and data not
shown).
Loss-of-function studies uncover novel and essential developmental roles for Arhgef11
To assess the function of Arhgef11 in the developing zebrafish embryo, we
employed four different loss-of-function strategies. First, we utilized an
antisense morpholino oligonucleotide, MOAUG, designed to block
translation by binding to 25 bases of the 5' UTR just upstream of the
start codon (Nasevicius and Ekker,
2000). Whereas this type of interference does not affect
maternally-deposited protein, it does inhibit production of new protein from
both maternally-contributed and zygotically-produced RNA, making it a powerful
tool for studying the effects of near-total loss of function during early
stages of development. In fact, microinjection of 2-20 ng of MOAUG
per embryo led to marked dosedependent developmental defects. The ability of
MOAUG to block the translation of endogenous Arhgef11 was tested by
western blotting analysis on extracts from embryos microinjected with a
moderate dose (4 ng) of the oligonucleotide. When compared with that of
uninjected WT control embryo extracts, MOAUG-injection yielded a
dramatic reduction in Arhgef11 protein levels after 13 hpf, but produced no
change in levels of an unidentified protein also recognized by the antibody
(Fig. 2A). Interestingly, at
least 85% of embryos injected with MOAUG exhibited an abnormal
ventrally-curved body shape and enlarged brain ventricles at 32 hpf
(Fig. 2C), as compared with
uninjected WT siblings (Fig.
2B). After 80 hpf, MOAUG-injected embryos developed
pericardial edema and severe distension of the pronephros
(Fig. 6I). Most of these
embryos did not survive past 4 dpf of development, suggesting that Arhgef11
function is crucial during these early developmental stages.
Since rescue attempts by co-injection with full-length RNA were impeded by the aforementioned gain-of-function phenotypes during gastrulation, we used a second MO to confirm that the observed morphant defects were due to loss of Arhgef11 function. This morpholino, MOSPL, was designed to bind to the exon 10-intron 10 boundary and block splicing of the zygotically-synthesized arhgef11 transcript (Fig. 2G). RT-PCR and sequencing analysis of RNA isolated from embryos injected with 5 ng MOSPL confirmed that the resulting arhgef11 RNA encodes a protein truncated just before the RGS domain as a result of a frameshift and premature stop codon after exclusion of exon 10 (Fig. 2H and data not shown). Moreover, the resulting embryos displayed phenotypes remarkably similar to those observed in embryos microinjected with MOAUG, including the ventral body curvature (Fig. 2F). Whereas Arhgef11 levels at 13 hpf were modestly reduced in embryos injected with MOSPL alone, co-injection of both MOs led to near-complete absence of this protein (Fig. 2I).
In a parallel approach to study Arhgef11 loss-of-function, we employed a
gamma-irradiation-induced deletion mutation, vu7, encompassing
arhgef11, vangl2/trilobite and at least two other nearby genes
(J.R.P., J.R.J. and L.S.-K., unpublished). Homozygous vu7/vu7 mutant
embryos, recognized by their shortened bodies resulting from loss of the
vangl2/trilobite gene (Jessen et
al., 2002), also showed a marked decrease in Arhgef11 protein
levels at 13 hpf (Fig. 2A).
Similar to the arhgef11 morphants, these embryos had slight ventral
body curvature, enlarged brain ventricles at 32 hpf, and developed pericardial
edema and distension of the pronephros by 2 dpf
(Fig. 2E and data not shown).
However, owing to the deletion of genes other than arhgef11 and
vangl2/trilobite, these mutant embryos also exhibited additional
phenotypes, including brain degeneration after 2 dpf, which we attribute to
loss of an unidentified gene within the deletion (J. A. Clanton, J.R.P. and
L.S.-K., unpublished). Additionally, as vu7/vu7 mutant embryos are
produced by heterozygous matings, maternally-contributed Arhgef11 protein and
RNA account for a milder reduction of protein levels, and therefore milder
phenotypes, than those observed in arhgef11 morphants
(Fig. 2A;
Fig. 3E,J;
Fig. 5C).
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DHPH)
previously shown to be required for Rho activation
(Fig. 1A,J). Notably, a similar
construct has been reported to block G protein-coupled activation of RhoA in
mammalian cell culture (Rumenapp et al.,
1999). This construct presumably acts as a competitive inhibitor
of the endogenous protein through its binding to upstream components and
inability to activate Rho. Owing to the nature of this interference technique,
it could also disrupt signaling of other PDZ- and RGS-domaincontaining RhoGEFs
such as Arhgef1 and Arhgef12. Expression of the DHPH construct in
HEK293 cells revealed that it neither localized to the cell membrane like its
WT counterpart, nor induced formation of actin stress fibers
(Fig. 1I,I'). Embryos
injected with 350-700 pg of synthetic RNA encoding DHPH exhibited
dose-dependent developmental defects including a widened anterior body region,
pericardial edema and frequent cardia bifida
(Fig. 2D and data not shown).
These defects could be suppressed by co-injection of the full-length construct
(data not shown). Taken together, the data obtained from all these approaches
indicate that Arhgef11 plays an essential role during zebrafish
embryogenesis.
Arhgef11 function is important for establishment of left-right asymmetry
The curved body axis and pronephric cysts observed in Arhgef11 morphant
embryos were similar to phenotypes observed in inversin and
polaris (ift88 - Zebrafish Information Network) morphant and
mutant embryos, which display defects in left-right asymmetry and other
cilia-mediated processes (Otto et al.,
2003; Bisgrove et al.,
2005; Kramer-Zucker et al.,
2005). Left-right asymmetry is marked by sided gene expression
that ultimately affects the orientation of many organs including the heart,
pancreas, liver and certain regions of the brain
(Levin, 2005). Kupffer's
vesicle, a small epithelial structure comprising cells with motile cilia that
create a directional fluid flow, has been shown to play a key role in the
early steps of establishing the left-right axis
(Essner et al., 2005;
Kramer-Zucker et al., 2005).
Given these phenotypic similarities, we examined the effect of loss of
Arhgef11 function on left-right asymmetry.
First, we analyzed WT embryos injected with MOAUG,
MOSPL, or the dhph construct, and vu7/vu7
mutants using in situ hybridization with antisense probes for the
asymmetrically-expressed genes spaw and pitx2
(Tsukui et al., 1999;
Long et al., 2003).
spaw RNA, encoding a Nodal-related protein, was detected in the left
lateral plate mesoderm (LPM) at 19 hpf in most uninjected WT embryos,
whereas this expression was somewhat randomized or absent in vu7/vu7
embryos and in WT injected with MOAUG, MOSPL or
dhph (Fig.
3A-E; Table 1).
Expression of pitx2 RNA, encoding a Bicoidrelated transcription
factor, was also disrupted in the LPM of Arhgef11-deficient embryos at 21 hpf
(Fig. 3E-I;
Table 1). Whereas most
uninjected WT embryos had normal left-sided expression, embryos in which
Arhgef11 function was disturbed exhibited not only left-sided but also
bilateral, right-sided, and absent expression in the LPM. By contrast, the
expression of pitx2 in Rohon Beard cells was not affected in any of
these loss-of-function experiments (arrowheads in
Fig. 3F-I).
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) to visualize the heart and ins
(Milewski et al., 1998) to
mark the position of the pancreas. In both cases, we observed randomized
placement of the two organs in all of the above Arhgef11 loss-of-function
scenarios. Whereas uninjected WT control embryos had heart fields localized on
the left side, embryos deficient in Arhgef11 had randomized positioning of
heart fields (Fig. 3J-M;
Table 1). Moreover, in
DHPH-overexpressing embryos, many had two heart fields bilaterally
located (data not shown).
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dhph construct, the
pancreas was observed on the right side in a smaller percentage of the
embryos, with many exhibiting medially-localized, or left-sided positioning.
Additionally, in some DHPH-overexpressing embryos, there were two
separate bilateral fields of RNA expression (data not shown). Most
vu7/vu7 mutants failed to survive to 53 hpf, and therefore expression
was not assayed in these embryos.
Given the left-right phenotypes observed in Arhgef11-deficient embryos, we
assayed for disruption of structures that influence asymmetry. It has been
proposed that proper midline development is important for establishing normal
left-right asymmetries (Danos and Yost,
1996). In arhgef11 morphants, the notochord and neural
tube formed and the neural tube was patterned properly
(Fig. 4A,B; see Fig. S3 in the
supplementary material). However, these assays cannot completely exclude the
possibility that a more subtle midline defect is present and contributes to
the left-right defects in these embryos. In addition, endoderm and dorsal
forerunners cells, which ultimately form Kupffer's vesicle
(Cooper and D'Amico, 1996),
appeared normal in morphants (see Fig. S2 in the supplementary material).
Furthermore, the morphology of Kupffer's vesicle was comparable to that of
uninjected WT control embryos and comprised ciliated cells
(Fig. 4A-D). Cilia of
MOSPL-injected and vu7/vu7 embryos were of normal length;
however, cilia were significantly shortened after MOAUG-injection
(Fig. 4E).
Arhgef11 is necessary for formation of the normal number of ear otoliths
Since ciliated cells have multiple developmental roles in addition to
affecting left-right asymmetry, we examined the effects on other processes
that require ciliated epithelia in embryos with reduced Arhgef11 function.
Ciliated cells of the developing ear are essential for proper formation of
mineral-rich structures, called otoliths, which are important for its sensory
functions (Riley et al., 1997;
Popper and Lu, 2000). In
mutants with defective cilia, such as oval, which harbors a mutation
in the polaris gene, an excess number of otoliths is observed
(Tsujikawa and Malicki, 2004).
Likewise, whereas 98% (n=342) of WT control embryos had two otoliths
and 2% had three, we observed three otoliths in 43% (n=252) of
MOAUG-injected embryos, with the remaining 43% and 14% having two
or one otolith, respectively (Fig.
5A-C). Similarly, 49% (n=92) of embryos injected with
MOSPL had two otoliths and 51% had three. Among vu7/vu7
mutants, 33% (n=99) had three otoliths and 67% had two. Finally, we
also observed three otoliths in a small number (6%, n=66) of
DHPH-overexpressing embryos, with 83% having two otoliths and the
remaining 11% one otolith.
Proper development of the pronephros requires Arhgef11
We next examined the developing pronephros, which includes pronephric ducts
comprising both mono- and multi-ciliated epithelial cells. In many mutants
with abnormal cilia, fluid-filled cysts develop within these ducts
(Otto et al., 2003;
Sun et al., 2004;
Kramer-Zucker et al., 2005).
As mentioned above, both protein and RNA encoding Arhgef11 were found in the
pronephric ducts of uninjected WT embryos at 24 hpf
(Fig. 1E',
Fig. 6A). Notably, Arhgef11
protein was enriched in apical regions of these cells, whereas it was reduced
or absent in embryos injected with MOAUG and in vu7/vu7
mutants (Fig. 6A,A' and
data not shown). We also observed considerable distention of the pronephric
ducts in live morphants at 24 hpf and later. Indeed, cross-sections of
morphant embryos after 54 hpf showed the presence of cysts within the
pronephric ducts (Fig. 6E-G),
and these cysts became morphologically apparent by 80 hpf
(Fig. 6H,I). However,
anti-acetylated tubulin staining revealed that cilia were still present in
Arhgef11 morphants (Fig.
6C,C'). In addition, we monitored the ability of these cilia
to beat in morphant pronephric ducts using highspeed video imaging. Cilia
within cystic morphant ducts appeared to beat at rates similar to, or slightly
faster than, those in uninjected control embryos (see Movies 1, 2 in the
supplementary material). Additionally, as in WT embryos, both mono- and
multi-ciliated cells were observed in the pronephric ducts of Arhgef11
morphants.
|
|
; Prkci - Zebrafish Information Network) were localized to
the apical side of the pronephric ducts in uninjected WT and morphant embryos
(Fig. 7C,D). Localization of
the microtubule-organizing center, as visualized by gamma-tubulin antibody,
was also apically-localized in both control and morphant embryos
(Fig. 7E,F).
|
), we hypothesized that Arhgef11-deficient embryos would have
a disrupted actin organization. We visualized the distribution of filamentous
actin in the pronephric ducts using phalloidin. As in control embryos,
morphants showed an enrichment of actin at the apical side of these cells, but
it appeared slightly less organized (Fig.
6B,B'; Fig.
7A,B). Notably, Na+/K+-ATPase, an ion
channel localized baso-laterally in the pronephric ducts of uninjected WT
embryos (Drummond et al.,
1998) (Fig. 7G),
was reduced and frequently absent from the basal regions of the cells in
morphant embryos (Fig. 7H).
Taken together, these experiments indicate that interference with
arhgef11 function does not completely eradicate the apical-basal
polarity of pronephric duct cells, but causes mislocalization of certain
proteins. Thus, we propose that such defective intracellular protein
distribution in pronephric ducts could underlie their abnormal function in
Arhgef11-deficient embryos. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The large number of exons in arhgef11 in zebrafish and mammals
provides ample opportunity for alternative splicing events to produce
different transcripts and modulate the function of the resulting protein. We
have detected two alternative splice forms of arhgef11 expressed
during early fish development. Interestingly, the alternatively-spliced exon
encoding amino acids 556-566 of the zebrafish protein corresponds to an exon
of the same length encoding human ARHGEF11 that was also reported to undergo
an alternative splicing event (Hart et
al., 2000), and 9 of the 11 amino acids are identical.
Conservation of this alternative splicing and the location of this exon near a
region that mediates the interaction of ARHGEF11 with actin
(Banerjee and Wedegaertner,
2004), suggest that splicing events may be important for
regulating this interaction. As mentioned above, the C-terminus of the human
homolog associates with p-21 activated kinase 4
(Barac et al., 2004), and is
important for homo- and heterodimerization with ARHGEF11 and ARHGEF12
molecules, respectively (Chikumi et al.,
2004). We predict that modification through alternative splicing
of the exon encoding amino acids 1232-1257 could affect this or other
important interactions.
|
; Rumenapp et al.,
1999). One would predict expression of this form to yield a
somewhat weaker defect than the MOs, as the endogenous full-length form is not
eliminated by this method and may still achieve some level of Rho activation.
Indeed, we did observe defects in left-right asymmetry and otolith formation,
similar to those detected in morphants. The major differences we noted between
these and morpholino-injected embryos were two expression domains of
cmlc2 and ins, cardia bifida, and the absence of a ventral
curve. Since cardia bifida was not observed in the morphants or the
vu7/vu7 mutants, we attribute this phenotype to interference with the
function of a similar RhoGEF, such as Arhgef12, which interacts with many of
the same molecules as ARHGEF11 via its PDZ and RGS domains
(Fukuhara et al., 2001;
Swiercz et al., 2002).
Interestingly, previous work using the chick embryo showed defects in
left-right asymmetry and cardia bifida after inhibition of Rho kinase, a
downstream effector of Rho (Wei et al.,
2001). Inhibition of Rho kinase or Rho in zebrafish impairs
gastrulation movements (Marlow et al.,
2002; Zhu et al.,
2006) and causes cardia bifida
(Matsui et al., 2005),
consistent with involvement of RhoGEFs in midline convergence of heart
precursors.
Since all the laterality markers we examined were affected to some degree
by loss of Arhgef11 activity, we conclude that it functions upstream of these
genes in an early step to establish left-right asymmetry, possibly by
regulating the ciliated cells that constitute Kupffer's vesicle. The fact that
loss of function also leads to defects in other processes involving ciliated
epithelia, such as the formation of otoliths in the otic vesicles and proper
development of the pronephric ducts, supports the notion that Arhgef11 plays
an important role in the cells of these structures. This is an unexpected
finding because we anticipated defects similar to those in the D.
melanogaster RhoGEF2 mutants, where loss of function disrupted
gastrulation cell movements and epithelial folding
(Barrett et al., 1997;
Hacker and Perrimon, 1998;
Nikolaidou and Barrett, 2004;
Padash Barmchi et al., 2005).
Many recent reports point to cilia as important components in an increasing
number of processes, but their formation and ability to beat appear largely
unaffected in Arghef11-deficient embryos. In addition to the aforementioned
roles, these structures are also implicated in Hedgehog signaling and growth
control in mammalian embryos (Corbit et
al., 2005; Huangfu and
Anderson, 2005; Schneider et
al., 2005). By contrast, our examination of Hedgehog-dependent
cell types and gene expression in embryos after Arhgef11 loss-of-function did
not reveal any defects (see Fig. S3 in the supplementary material). However,
we cannot completely rule out a role for zebrafish Arhgef11 in cilia formation
and function because a reduction in cilia length correlates with our strongest
loss-of-function phenotypes.
In the case of the pronephric ducts, these tubular structures contain
ciliated epithelial cells. The apical side of the cells faces the lumen and
the cell junctions are important for maintaining its structural integrity and
to serve as a selectively-permeable barrier to water and solutes. Examination
of the cilia and apical-basal polarity of the renal epithelial cells did not
reveal any obvious defects in localization of most polarized proteins, with
the exception of Na+/K+-ATPase, the basal expression of
which was lost in morphants. Indeed, the distribution of this protein was
often disrupted in other zebrafish kidney mutants where cysts developed
(Drummond et al., 1998).
Whether this mislocalization is directly or indirectly due to Arhgef11
loss-of-function remains to be determined, but given its known functions, the
mechanism is likely to be through modulation of the cytoskeleton. Accordingly,
our data also hint that apical actin structures are slightly misorganized in
morphants. Since Na+/K+-ATPase is important for
maintaining the ionic gradient and the osmotic balance in these cells
(Drummond, 2000;
Rajasekaran et al., 2005),
incorrect positioning in morphants, as a result of cytoskeletal disruption,
could account for impaired pronephric function. Interestingly, recent work
with RhoGEF2 demonstrated that this protein stimulates Rho and, subsequently,
actin remodeling in apical regions of epithelial cells within the fruit fly
embryo so as to modulate invagination and lumen maintenance of the tubular
structures called spiracles (Simoes et
al., 2006). It is plausible that the zebrafish homolog functions
similarly to maintain the epithelial structures affected in our experiments.
Since this study has not exhaustively examined the cilia of the pronephric
ducts, the possibility remains that some other aspect of the cilia are
affected in Arhgef11-deficient embryos, such as coordination of beating and
anchoring of the basal body, thereby leading to cyst formation.
Although the exact mechanism by which Arhgef11 affects ciliated epithelia
remains unclear, its mere involvement opens up a wide range of possible
pathways that could also play a role here. For the pronephric ducts, this
seems to be a highly specialized RhoGEF, where its expression is
transcriptionally-regulated and its localization is enriched to the apical
side of the cell. With its multiple functional domains and the many
interactions identified in other systems, this RhoGEF could prove a very
interesting protein, possibly linking Plexins or G protein-coupled signaling
to cytoskeletal modification of ciliated and other highly polarized cells in
vivo. Interestingly, RhoGEF2 is enriched on the apical surface of epithelial
cells during development in D. melanogaster
(Padash Barmchi et al., 2005).
We hypothesize that there may be some similar roles for Arhgef11 and its
invertebrate homolog, but as ciliated epithelia are unique to vertebrates, so
might be these new functions that we have uncovered for Arhgef11.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/921/DC1
|
ACKNOWLEDGMENTS |
|---|
|
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