Department of Cell Biology and Anatomy, University of Tokyo, Graduate School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 Japan
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Abstract |
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KIF3A is a classical member of the kinesin
superfamily proteins (KIFs), ubiquitously expressed although predominantly in neural tissues, and which
forms a heterotrimeric KIF3 complex with KIF3B or
KIF3C and an associated protein, KAP3. To elucidate
the function of the kif3A gene in vivo, we made kif3A
knockout mice. kif3A/
embryos displayed severe developmental abnormalities characterized by neural
tube degeneration and mesodermal and caudal dysgenesis and died during the midgestational period at ~10.5
dpc (days post coitum), possibly resulting from cardiovascular insufficiency. Whole mount in situ hybridization of Pax6 revealed a normal pattern while staining
by sonic hedgehog (shh) and Brachyury (T) exhibited abnormal patterns in the anterior-posterior (A-P) direction at both mesencephalic and thoracic levels.
These results suggest that KIF3A might be involved in
mesodermal patterning and in turn neurogenesis.
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Introduction |
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Furthermore, homozygotes for kif3A showed randomization of laterality in heart looping. In contrast to wild-type embryos where almost all have a D-loop heart (situs solitas), ~40% of homozygous embryos exhibited cardiac L-loops (situs inversus). Moreover, their pericardial space was filled with effusion resulting from/in circulatory insufficiency. Scanning electron microscopy (SEM) and video-enhanced contrast DIC and fluorescent images (VEC-DIC/FL) microscopy revealed the absence of motile monocilia on mutant nodal pit cells, which could not generate leftward nodal flow of extraembryonic fluid and are considered to be responsible for initial determination of the left-right (L-R) asymmetry. These results collectively suggest the important role of the KIF3A protein in determination of embryonic body planning, particularly for the laterality.
INTRACELLULAR trafficking is one of the fundamental
mechanisms for maintaining cellular organization by
distributing the molecules properly to the lieu where
they are required. In the case of neurons, this transportation system is highly differentiated and is termed axonal
transport (Grafstein and Forman, 1980; Hirokawa, 1993) in accordance with their highly polarized structure. To
carry out these tasks, three major kinds of cytoskeleton-associated motor proteins are recognized today: kinesin
superfamily proteins (KIFs)1 (Aizawa et al., 1992
; Hirokawa, 1996
, 1998
), dynein superfamily proteins (Gibbons et al., 1994
; Tanaka et al., 1995
; Gibbons, 1996
), and
unconventional myosins (Fath and Burgess, 1994
; Wang et
al., 1996
). All of these motor proteins exert their force by hydrolysis of ATP, although the track for the former two is
microtubules (MTs) while the latter one slides along the
microfilaments composed of actin. Among them, KIFs
comprise several dozens of molecules that take part in various processes including axonal transport (Kondo et al.,
1994
; Okada et al., 1995
; Yamazaki et al., 1995
), intracellular traffic such as intraciliary-flagellar transport (IFT; Kozminski et al., 1995
; Morris and Scholey, 1997
; Cole et al.,
1998
), and cell division (Boleti et al., 1996
).
KIF3A protein is one of the authentic murine KIFs discovered by our PCR screening (Aizawa et al., 1992) that
takes on heterotrimers with KIF3B (Kondo et al., 1994
;
Yamazaki et al., 1995
) or KIF3C (Muresan et al., 1998
;
Yang and Goldstein, 1998
) and an associated molecule
KAP3 (Yamazaki et al., 1995
, 1996
), which are in toto called KIF3 complex. Both KIF3A/B have ATPase activity at their NH2-terminal head region followed by a
coiled-coil stalk region assumed to be responsible for
dimerization and a COOH-terminal tail domain. Previous
biochemical studies (Kondo et al., 1994
) have revealed
that KIF3A is expressed ubiquitously but predominantly in brain, testis, and adrenal medulla. Furthermore, it has
been biochemically revealed that only KIF3A can exist in
a soluble form in the absence of its counterpart (Yamazaki
et al., 1995
) and can form heterodimers with either KIF3B
or KIF3C (Yamazaki et al., 1995
; Muresan et al., 1998
;
Yang and Goldstein, 1998
), whereas KIF3B aggregates
under physiological conditions in the absence of KIF3A in
the baculovirus expression system and does not dimerize
with KIF3C. Moreover, developmental expression of the
KIF3A protein precedes that of KIF3B. Recently, we have
made kif3B knockout mice (Nonaka et al., 1998
) and revealed that KIF3B is essential in early developmental
stages, especially for left-right (L-R) determination. From
another standpoint, KIF3 has diverse orthologues among
species such as sea urchin kinesin II (Cole et al., 1993
),
Chlamydomonas FLA10 (Walther et al., 1994
), C. elegans
Osm3 (Shakir et al., 1993
), and a fish photoreceptor KIF3 antigen (Beech et al., 1996
). Considering all the aforementioned properties of KIF3A, this molecule proper must
therefore have essential and regulatory roles in various animals, and it is anticipated that more severe phenotype
shall be observed in the kif3A gene-targeting mice.
To pave the way into the investigation of murine KIF3A
in situ, we disrupted the murine kif3A gene by homologous recombination. The resulting kif3A/
mice displayed severe developmental defects at the mid-gestational stage and died before 10.5 dpc (days post coitum).
The defects were mainly localized to the neural tube,
heart, brachial arches, and lower truncal mesoderm, leading to highly underdeveloped posterior structures. Surprisingly, the cardiac looping was randomized, i.e., ~40% of
all homozygote embryos exhibited a L-loop pattern (situs
inversus). Because it has been predicted that the node plays an important role in L-R determination, we examined the node of kif3A
/
and compared it with that
of wild-type embryos. Since Chlamydomonas orthologue
FLA10 has been reported to be localized in the motile flagella as a motor for the IFT (Kozminski et al., 1995
; Cole et al., 1998
; Rosenbaum et al., 1999
), we also focused our
attention on the monocilia of the embryonic node. In the
wild-type embryos, the monocilia of nodal pit cells are motile and generate a leftward flow of extraembryonic fluid
(nodal flow). However, formation of cilia on the surface of
nodal pit cells was severely affected and no flow was recorded in kif3A
/
homozygous embryos. These data suggest the possibility that KIF3A molecules are involved in
the initial determination of the L-R asymmetry by contributing to formation of cilia, which generates leftward flow
of the extraembryonic fluid. In addition, the lack of nodal
cilia might bring about abnormal distribution of mesoderm-inducing activity such as sonic hedgehog (shh), and
homozygous embryos displayed disturbed mesodermal structures.
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Materials and Methods |
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Generation of the Targeting Vector
A 20-kb genomic fragment containing full-length kif3A was selected from
a murine genomic library of embryonic stem (ES) cell line J1 (Tanaka et al.,
1998) and probed using a mouse cDNA fragment encoding the P-loop
exon of kif3A (Kondo et al., 1994
). The two independent phage clones obtained were cloned into pBS (Stratagene) vector. These clones were subsequently subcloned into pBS and mapped by a combination of several restriction enzymes and partial sequencing. We applied the conventional
positive-negative selection (Yagi et al., 1993
) together with the poly(A)
trap strategy. As shown in Fig. 1 A, we applied a 1-kb-long HindIII-SalI
fragment as a 5'-homologous arm and a 5-kb-long fragment as a 3'-homologous arm. Then poly(A)-less pGKneo positive selection cassette was flanked by these arms and cloned into a modified multiple cloning site in
the DT-A cassette B vector (GIBCO BRL). Finally, this construct was
prepared on a large scale with a QIAGEN miniprep kit. This plasmid was
linearized by NotI and an appropriate concentration of vector (25 µg/ml)
was prepared for the subsequent transfection into ES cells.
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Transfection and Screening of Recombinant ES Clones
We have performed the electroporation of targeting vector to J1 cell line
basically according to Harada et al. (1994) with a slight modification in
G418 concentration. After electroporation, the transfected cells were selected in the presence of G418 (GIBCO BRL) at a concentration of 175 µg/ml. About 10 d after electroporation, the growing positive selected colonies of ES cells were picked up in duplicated 96-well plates, one for cell
stock and the other for genomic DNA preparation. Screening of recombinants was performed by genomic Southern blotting with 32P-radiolabeled
external probe (generated by PCR ~0.4 kb) as indicated in Fig. 1. Then
the integrity of candidate clones was further confirmed with an internal
and neo probes (data not shown). Three independent recombinants out of
~450 ES colonies were obtained by this poly(A) trap targeting strategy.
These recombinant clones were injected into the blastocysts recovered
from superovulated C57BL/6N line and raised chimeric mice from two independent cell lines. We identified chimeric mice by contribution of
agouti coat color. Male chimeras were bred with C57BL/6J to obtain heterozygous offspring, from the mating of which we could obtain kif3A null mutants.
PCR Genotyping
The generated mice were routinely probed by PCR for the identification
of their genotype as described previously (Tanaka et al., 1998). In brief, a
small portion of mouse tail was incubated in a tail lysis buffer (10 mM
Tris-HCl, 25 mM EDTA, 1% SDS, and 75 mM NaCl, pH 8.0) supplemented with 100 µg/ml proteinase K (Merck). The Eppendorf tubes containing these samples were shaken in an air incubator maintained at 55°C
overnight. Phenol-ethanol extraction and ethanol precipitation were performed before the PCR reaction. About a hundredth amount of each purified DNA was routinely used for PCR. The following two pairs of
primer sets were used for genotyping offsprings: (a) neo primers, neoF
(1620-1649) 5'-TGG GCA CAA CAG ACA ATC GG-3', neoR, (1841-
1820) 5'-ACT TCG CCC AAT AGC AGC CAG-3'; (2) internal primers,
kif3A-F4, (346-369) 5'-TGT TCC ATA TAG CCC AGG ATA CCC-3',
kif3A-B1, (545-525) 5'-GAT GGT CCC TGA AAA TGG TGC-3'.
To determine the embryonal genotype, we collected amniotic membranes. They were dissolved in aqua destillata supplemented with 100 µg/ ml proteinase K under shaking at 55°C for 1 h. A small amount (2 µl) of the lysate was used for PCR amplification.
Western Blotting of the Embryos
The whole embryos ranging from 9.5 to 10 dpc were killed and minced by
using a pair of ophthalmological scissors in PBS supplemented with protease inhibitor cocktails (5 mM PMSF, 10 ng/ml leupeptin, pepstatin, benzamidine, and 100 mM DTT; Wako Pure Chemicals Co.) on ice. By using
a Potter's homogenizer, small pieces of embryos were completely destroyed. The resultant homogenate was centrifuged at 15,000 rpm by using
a Beckman TL-100 for 30 min and the supernatant was collected. The protein concentration of each sample was adjusted to 10 µg/ml and used for
standard Western blotting procedure as described previously (Tanaka et al.,
1998). Here we used the following antibodies to ascertain the expression
level of KIF3A and KIF3B proteins in the mutant mice: both monoclonal
and polyclonal anti-KIF3A antibodies (Kondo et al., 1994
), and polyclonal anti-KIF3B antibody (Yamazaki et al., 1995
).
Preparation of Embryos for Light Microscopy
The embryos dissected from the pregnant mice were processed for the
light microscopic observation by the following method. As soon as the
embryos were extirpated from the uterus, a small portion of extraembryonic membranes was retained for the determination of genotype by PCR
and the embryos proper were soaked into Bouin's fixative (75% pycric
acid, 5% glacial acetic acid, and 25% formaldehyde; Wako Pure Chemicals Co.). After fixing embryos for 2 h at room temperature (RT), the
samples were dehydrated by a graded series of ethanol solutions, followed
by substitution of xylene. Finally, the whole mount embryos were placed
in melted Paraplast (Oxford Labware) at 65°C for ~90 min with two
changes of this embedding material. The samples were embedded in fresh
Paraplast after complete penetration of the samples by the embedding
material. The blocks were cut by using a rotatory microtome (HM355;
Carl Zeiss) into 7-µm-thick serial sections, then the sections were
mounted onto glass slides, which were in turn deparaffinized and stained
with hematoxylin and eosin according to Kaufman (1995) with slight modifications. These sections were observed and photographed by a Nikon
Optiphot-2 microscope.
Preparation of Embryos for Scanning Electron Microscopy
The dissected embryos were fixed by using half Karnovsky solution (2%
paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer,
pH 7.2) for 2 h at RT. Then we soaked these embryos into 10% sucrose
solution buffered by 0.1 M cacodylate for 20 min, followed by postfixation
with 1% osmium tetraoxide in cacodylate buffer on ice for 15 min. After
washing the embryos extensively with aqua destillata, dehydration with a
graded series of ethanol solutions was performed. After completing the
dehydration process, the media was substituted with isoamyl acetate and
the embryos were left to stand in this solution at RT for over 30 min. They were then prepared for scanning electron microscopy by critical point
freeze-dry procedure as described previously (Nonaka et al., 1998). In
brief, they were soaked into liquid carbon dioxide under high pressure
(JCPD-5; JEOL). Then the chamber was gradually heated to the critical
point, where the embryos could be dried without being destroyed. The
samples obtained were surface-coated using a gold spattering device
(JSM-5200; JEOL) under the optimal conditions for 10 min. The samples
were viewed under a JEOL scanning electron microscope (JEM2000) at
100 kV and photographed by a Polaroid.
Transmission Electron Microscopy of the Nodal Monocilia
Embryos at 7.5 dpc were dissected out from the uterus and were fixed by
the method of Mizuhira and Futaesaku (1972) to obtain good preservation
of axonemal proteins. Then the samples were processed and observed
routinely as described previously (Takeda et al., 1995
).
Whole Mount In Situ Hybridization
Whole mount in situ hybridization of the mouse embryos other than lefty-2
was carried out principally according to Conlon and Rossant (1992) with
several modifications. In brief, the embryos were dissected in sterilized
RNase-free PBS and fixed with 4% paraformaldehyde not equilibrated
with NaOH at 4°C. After incubation for 2 h, they were washed twice with
PBT (PBS supplemented with 0.1% Tween 20), followed by bleaching
with PBT and 30% hydrogen peroxide. They were then treated with proteinase K (10 µg/ml; Merck) for several minutes at RT. Embryos were further fixed with 0.2% glutaraldehyde containing 4% paraformaldehyde,
prehybridized, and then hybridized with appropriate markers (overnight,
70°C). On the following day, the sample embryos were washed with buffers and treated with RNase to remove unbound or nonspecific binding probes. After that, alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche Diagnostics) was applied and incubated overnight at 4°C.
On the next day, the embryos were washed with TBST (Tris-buffered saline containing 0.2% Tween 20) with medium changes every 1 h. The
washing process continued into the following day when the final AP color
reaction was performed. The reaction was carried out according to the
standard method (NBT and BCIP; Roche Diagnostics) and the color reaction was stopped whenever the localization of markers became clear and
distinct. We have performed a series of dehydration/rehydration processes
(Hogan et al., 1994
) to darken the color of the AP reaction precipitate
products. The embryos were then soaked in a glycerol/TBST solution to
make the body transparent for easier identification of staining at a three
dimensional level.
For staining the embryos by using the lefty-2 probe, we followed our
previous method (Nonaka et al., 1998).
Nodal Cilia Motility Assay
Nodal cilia can be observed during the nodal formation and bulging at 7.5 dpc (Nonaka et al., 1998). The lower halves of the embryos dissected from
the uterus were eliminated by using a pair of electrolytically sharpened
tungsten needles. The half-egg like structure was embedded into a small
hole of silicon-rubber membrane (thickness ~300 µm) attached onto the
surface of a glass slide. Another silicon membrane having a hole at its center (diameter ~5 mm) was overlaid onto it. We filled the hole with motility solution (50% heat-inactivated rat serum, 49% DMEM and 1% 1 mM sodium pyruvate) and sealed it by simply placing a coverslip on it. Fluorescent beads (Fluosheres, carboxylate-modified microspheres; Molecular
Probes) with a diameter of ~220 nm were added to the assay solution to
~5% for visualization of flow generated by the motile cilia. The preparations were viewed under VEC-DIC/FL microscopy (Olympus Inc.) and a
series of motility assay was videotaped simultaneously. The embryos under investigation were genotyped after observation.
Immunocytochemistry of the Nodal Cilia and the Embryo Section
Embryos were fixed with 4% paraformaldehyde and processed by a standard cryosectioning method. The blocks were sectioned with a Leica cryomicrotome, and extended on the surface of glass slides. The samples were
permeabilized with 0.1% Triton X-100 and blocked with 5% skim milk
followed by a standard immunocytochemical procedure as described elsewhere (Nonaka et al., 1998). For the staining by anti-axonemal dynein antibody, we generally followed the method of Umeda et al. (1995)
with
slight modifications. The stained preparations were examined under a
confocal laser scanning microscope (Bio-Rad).
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Results |
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Targeting Strategy and Confirmation of Homologous Recombination
Initially, we tried to obtain homologous recombinants by using a standard positive-negative selection vector and screened ~800 ES cell colonies. However, we could not successfully obtain any recombinant clones, which led us to adopt the poly (A) less pGK-neo cassette with an A-T rich/pausing signal (Fig. 1 A). Under this condition, three independent clones out of ~450 colonies were isolated, all of which have been used for blastocyst injection. As shown in Fig. 1 B, wild-type embryos displayed a single 3.2-kb fragment when digested with EcoRI, while an additional 2.1-kb band was observed in heterozygotes by genomic Southern blotting. Another restriction enzyme (XbaI) being diagnostic for targeting events, also verified successful homologous recombination (data not shown). Genotypes were also identified by PCR where primer pairs explicitly detect the targeted events. As represented in Fig. 1 D, almost all littermates of heterozygous mothers intercrossed with male heterozygotes revealed proper separation of genotype according to Mendel's law. We made a necropsy of 20 pregnant heterozygotes expected to have homozygote offsprings during 7.5-11.5 dpc, which revealed that no homozygotes were observed after 10.5 dpc (Fig. 1 D, Table I), suggesting the midgestational lethality of homozygous embryos. Western blotting analysis of all littermates revealed a complete absence of KIF3A protein from homozygous mice. Interestingly, KIF3B protein, being a counterpart in the KIF3 complex, was expressed at a normal level (Fig. 1 C, lane 3).
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Severe Developmental Abnormalities in kif3A/
Mice
kif3A homozygotes were generally smaller compared with heterozygous and wild-type littermates at 9.5 dpc. Furthermore, no living embryos were obtained when cesarean section of pregnant mice was carried out after 11 dpc (Table I). At 9.5 dpc, the homozygotes displayed severe malformation characterized by swelling and degeneration of the neural tube (Fig. 2 and see Fig. 5, B and D) mimicking the phenotype of hydrocephalus. Furthermore, extreme distension of the pericardium due to pericardial effusion was noted. A regular array of somites as seen in wild-type embryos (see Fig. 5 A) could not be observed in homozygote embryos (see Fig. 5 C), simply showing degenerated bulk in the lower truncal part and abnormal somitegenesis, i.e., sirenomelia. In most cases, embryonic turning was not completed at 9.5 dpc, suggesting growth retardation/inhibition of the homozygotes. Staggering of the neural tube was prominent at this incomplete turning region. These phenotypes are suggestive of disturbed mesodermal development resulting in failure of neural tube induction. In addition, the severity of the phenotype observed at 9.5 dpc was almost correlated with the expression level of the KIF3A molecule, which revealed relatively strong signal intensity in the neural tube (see Fig. 4 B, Nt) and the heart (idem, Cor).
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Moreover, we encountered abnormal heart looping. In
wild-type cases (Fig. 3, A and D), almost 100% of embryos
displayed a D-loop pattern while a few atypical phenotype
also existed. On the contrary, in homozygotes, ~40% of
the embryos revealed a L-loop type heart (Fig. 3, C and F).
In some cases (~20%), incomplete cardiac looping, i.e., a
tuba rectae, was observed. These results suggested that
randomization of the heart looping occurred in kif3A homozygous mice. To address this point more precisely, we scrutinized these embryos with lefty-2 RNA probe, to see
whether it involves abnormal lefty-2 expression, as lefty-2
product is considered to be one of the earliest left-right determinant in early murine development (Meno et al., 1997,
1998
). As has been observed in our previous report for
kif3B
/
(Nonaka et al., 1998
), the expression of lefty-2
was bilateral or downregulated in kif3A
/
embryos (see
Fig. 6 B). In some cases, the expression was extended to
the contralateral paraxial mesoderm (Fig. 6, the right two) while normal pattern still existed (Fig. 6 B, the left-most
embryo). On the contrary, almost all wild-type embryos
and heterozygotes displayed normal left-side restricted
pattern (Fig. 6 A). Regarding a cardiogenesis, hypoplasia
of the myocardium was another striking feature of kif3A
homozygous embryos. A sagittal section of the heart wall
indicated fairly underdeveloped myocardium (Fig. 6 E)
compared with normal embryos (Fig. 6 F), which might be
a cause of circulatory insufficiency in utero.
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No Cilia Were Formed in the Node of 7.5 dpc
kif3A/
Embryos
The node is normally formed at 7.5 dpc and is essential for
further induction of mesodermal anlage and in turn for
neural tube formation. As Chlamydomonas orthologues of
the KIF3 complex have been reported to engage in IFT
(Kozminski et al., 1995; Cole et al., 1998
; Rosenbaum et
al., 1999
), we focused on the ciliated mesodermal cells
which normally accommodate monocilia (Sulik et al.,
1994
). As shown in Fig. 7, A and B, the nodal region forms a small dimple at the base of the dorsal part of embryo
where the cell density is quite high. Since the cells constituting the node are smaller than surrounding endodermal
cells at this developmental stage, this region could readily
be discriminated from other parts of the embryonic body.
At a glance, the wild-type/heterozygous cells normally exhibited cilia ranging from 2 to 4 µm in length and having
pinpoint-like ends (Fig. 7 C). However, in the case of
kif3A
/
mice (Fig. 7 D), the surface structure displayed
quite striking differences. There were almost no mature
cilia, although there existed some very short rudimentary
processes. This phenotype was invariably observed among
homozygous mouse littermates, suggesting the possible
role of the KIF3A molecule in constructing and maintaining the monocilia structure.
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The KIF3A-null Mutant Embryos Were Defective of the Leftward Extraembryonic Nodal Flow as Revealed by Fluorescent Beads
To decipher the degree of dysfunction of nodal pit cell
cilia in kif3A/
embryos, we have carried out a motility
assay. An embryonic body harboring a node was placed
under the VEC-DIC/FL microscope and fluorescent beads
were added to the solution surrounding the embryos. By
using this method, we can simultaneously observe both ciliary movement and nodal flow. In wild-type and heterozygous embryos, distinct flow generated by the rotating cilia
was recorded and this flow was directed leftward with reference to the presumed body A-P axis (Fig. 8 A). This regular flow was especially evident in the posterior part of the
node. On the contrary, in KIF3A-deficient embryos, we
could identify no such a regular flow, while rather Brownian movement of beads was observed (Fig. 8 B). This result clearly indicates that the cilia of nodal pit cells is involved in gradient formation within the nodal groove,
implying some important roles of the KIF3A molecule in
the determination of early body planning.
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We have carried out immunostaining of nodal cilia to
determine whether KIF3A molecules are really installed
in the motile cilia. Cryosection of the node stained with
anti--tubulin (DM1A) indicates the presence of cilia
containing MT cytoskeleton (Fig. 8, B and D). Double immunostaining of the same embryo with an anti-KIF3A antibody revealed the localization of KIF3A molecule within
the cilia (Fig. 9 A), suggesting that the KIF3A molecule might be engaged in the construction and maintenance of
the ciliary structure. These results are almost the same as
those obtained from the KIF3B-deficient embryos (Nonaka et al., 1998
), strongly suggesting that KIF3A is really
a counterpart of KIF3B in vivo. Collectively, KIF3A molecules may be essential for ciliary formation, determining
the subsequent developmental cascades.
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Moreover, staining with an anti-outer axonemal dynein
antibody (AD2; Yoshida et al., 1989) and anti-axonemal
dynein intermediate chain (IC140; Yang and Sale, 1998
)
also revealed the presence of this molecule within the
monocilia (Fig. 9, C and E). Because the nodal area is
composed of several optical sections by confocal laser
scanning microscopy while that for cilia is made of a few sections, the background fluorescence in the nodal area
seems to be higher. However, as there are no positive
staining of cilia in the negative control panel (Fig. 9 G), we
could state that the localization of ciliary axonemal dynein
is specific. We could also identify both inner and outer arm
dynein localizing on the surface of doublet MTs with in the
monocilia (arrows for inner arm dynein and for outer arm
dynein, Fig. 9, I and J) by a conventional electron microscopy. These results collectively imply that axonemal dynein is a candidate cargo of KIF3A that may in turn be engaged in the movement of monocilia.
Shh Expression Was Somewhat Reduced in Mutant Rostral Portion
As both development of the mesodermal structure and the
determination of L-R asymmetry were highly affected in
the kif3A/
mice, we also examined the expression pattern of some developmental markers for delineating the
role of the KIF3A molecule in this process. Pax6 (Jordan
et al., 1992
) expression in 9.5 dpc embryos did not exhibit
any apparent changes in the eyes except for slightly reduced expression in the forebrain (Fig. 10, A and B).
Brachyury (T, see review by Willison, 1990
) is a mesodermal marker that is normally expressed in the midline
structures with a strong accent at the tail bud region. At
8.5 dpc, its expression patterns in both embryos (wild-type;
Fig. 10 C, homozygote; Fig. 10 D) were very similar, but
midline staining corresponding to the notochord was
fainter in the case of homozygous embryos. Of noteworthiness, another midline mesodermal marker, sonic hedgehog (shh; Conlon et al., 1995
), exhibited the most dramatic
change in homozygote embryos even at early developmental stage (7.5 dpc, Fig. 10 G). Normally, this marker is expressed in the node, notochord and subsequently in the
floor plate. Furthermore, the ventral half of the mesencephalon also expresses it resulting from induction by the
underlying floor plate at this region (Ekker et al., 1995
). In
the case of homozygous embryos (Fig. 10, E and F, lower
embryos), the expression of shh at the thoracic notochord
was significantly reduced and the staining per se became
staggered to a certain extent. Moreover, the expression in
the mesencephalon was completely absent in homozygous
mice (Fig. 10 E, lower embryo) while abundant expression
could be encountered in wild-type embryos (Fig. 10 E, upper embryo). This absence of shh expression in brain
structures might result in abnormal development of the
neural tube, leading to the hydrocephalus-like phenotype
and neural tube hypoplasia. Actually, exencephaly (Fig. 3
C) was relatively frequently observed in our kif3A null
mutants under the genetic background with lesser contribution of C57BL/6J, suggesting the possibility that some modifier loci of this phenotype are present in the 129/Sv
strain. In addition, reduced expression of shh in the rostral
region together with reduced T expression in the truncal
part collectively suggest the influence of the KIF3A molecule in the formation of A-P axis possibly through transporting inductive signals or their receptors.
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Discussion |
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In summary, this is one of the first reports describing the
general function of the KIF3 complex in vivo. The null
mutant embryos displayed smaller body size and early developmental failure characterized by mesodermal hypoplasia and degeneration followed by the development of
neural tube defects. Furthermore, determination of L-R
asymmetry was randomized, exhibiting the phenotype represented by L-loop formation of the cardiac tube (situs inversus). This phenotype was presumably due to defective
nodal cilia as identified in our previous kif3B mutant
(Nonaka et al., 1998). These results reinforce our biochemical data that KIF3A is a counterpart of KIF3B
(Yamazaki et al., 1995
). Another new idea on the function of KIF3A is that it is involved in mesodermal formation,
which then regulates the development of neural tissues.
Here we should like to discuss the possible role of the
KIF3A molecule with special reference to (a) the early developmental events and (b) the determination of laterality.
KIF3A and Early Developmental Sequella
KIF3A is reported to be already expressed in ES cells and
precedes that of KIF3B. Considering the biochemical data
that KIF3A could remain soluble in the absence of its
counterpart (Yamazaki et al., 1995), the earlier death of
kif3A
/
embryos than that of kif3B homozygous embryos
(no surviving embryos after 11.5 dpc; Nonaka et al., 1998
)
is quite reasonable. Although the macroscopic phenotype
was not so evident until 8.5 dpc in kif3A null mutants, microscopical changes in kif3A homozygotes were even evident early in the node formation stage (7.5 dpc). The node
is generally believed to be responsible for the induction of
further midline mesodermal structure. The absence of cilia on the surface of nodal pit cells in kif3A null mutants and
the normal distribution of KIF3A molecules within these
cilia in wild-type embryos strongly suggest that the nodal
cilia are to some extent constructed and maintained with
the aid of KIF3A molecules. Although a previous report
suggested the presence of axonemal outer arm dynein in
primary cilia (9 + 0 structure, two basal bodies; Umeda et al.,
1995
) of mammalian sperm and lung ciliated epithelium, and it might be responsible for actual motility, our data
presented here and in a previous study (Nonaka et al.,
1998
) indicated KIF3A and KIF3B are essential for transporting these ciliary components. Axonemal outer arm dynein per se is probably transported down within the cilia as
a transporting complex for the following two reasons.
On the first line, our immunocytochemical staining clearly demonstrated the colocalization of KIF3A and outer axonemal dynein within monocilia (Fig. 9, C and E). Secondarily, mutations in a putative axonemal dynein heavy chain
(lrd), being abundantly expressed in these ciliated nodal
cells (Supp et al., 1997
), de facto resulted in situs inversus.
These results are in good agreement with the case of
Chlamydomonas FLA10, where large IFT complexes are
transported by this KIF3 orthologue and depletion of this motor protein resulted in the absence of normal motile flagella (Cole et al., 1998
; Rosenbaum et al., 1999
).
Then by what mechanism are the early developmental
events disorganized, giving rise to multisystem failure? Although there is no direct evidence interfacing the function
of KIF3A and early inductive events, it is supposable that
the defective nodal cilia could not properly distribute
some presumably soluble substances to their proper destinations. Alternatively, intracellular transport of some
kinds of receptors to proper direction might be affected. Especially, the abnormal expression pattern of Brachyury
(T) and sonic hedgehog (shh) could partly explain the hypoplasia of midline structure and degeneration of posterior body structure. The generally accepted idea holds that
shh is secreted as a complex form bound to cholesterol
(Cooper et al., 1998). Considering the size of this complex
and cilia, it is reasonable that the lack of cilia resulted in
disturbance of orthotopic shh expression in kif3A
/
homozygote mice. In addition, spontaneous mutation of shh,
cyclopia (Chiang et al., 1996
), and targeted disruption of
shh (Lanske et al., 1996
) partly share the phenotypes observed in this kif3A null mutant. These ideas collectively
imply the relationship between the KIF3 molecule and
transport of the Shh molecule.
From another standpoint, as neural induction depends
on the mesoderm, abnormal neural tube formation could
be attributed to this general failure of anterior development. Indeed, the lack of shh staining at the ventral half of
the mesencephalon implies that the neural phenotype is
partly secondary to mesodermal abnormality. In fact, expression of shh at 7.5 dpc in homozygotes is already more
altered than that of wild-type embryo, displaying very
faint trace amount with no nodal accentuation. However,
considering that neuronal morphogenesis also involves
cellular movement and assortment by themselves, the neural phenotype might be explained independently by genuine neuronal failure brought about by kif3A disruption. In
addition, as some reports suggested the presence of cilia
on the neuron (Vigh-Teichmann et al., 1980), the neural phenotype may partially be attributable to the lack of neuronal cilia.
Ciliary Movement in Node: Implications for Left-Right Asymmetry
As has been discussed in the previous section, KIF3A
might transport materials required for the cilia assembly
and maintenance, so that the absence of KIF3A molecules
results in ciliary disorganization, leading to several developmental defects. One of the most remarkable phenotypes
in these mutant mice was randomization of L-R asymmetry. As we have previously reported in kif3B null mutant
mice (Nonaka et al., 1998), these null mutant mice exhibited randomized heart loop formation. Importantly, failure of cardiac looping to occur in neither direction was
also encountered in kif3A null mutant embryos (~20%).
Then what mechanism is responsible for left-right determination? Our experiment by using fluorescent beads
clearly indicated that the nodal cilia propelled certain
kinds of substances in the extraembryonic fluid in the vicinity of the node from right to left. A concentration gradient that reaches the threshold for switching on lefty expression is probably formed and it might further destine
the L-R asymmetry. This conclusion is further supported
by our recent unpublished work showing randomness of left-right asymmetry occurs in mutant whose nodal pit cells
harbor primary cilia, but they are immotile (Okada, Y., S. Nonaka, and N. Hirokawa, unpublished data). As a candidate of determinants, we expect that N-Shh protein might
be involved since the cholesterol-conjugated form of N-Shh
(Cooper et al., 1998) could not diffuse out a significant distance from a source of secretion and be concentrated to
where it should be localized. There is no direct evidence
for the relationship between lefty and shh, but it is now
generally accepted that shh expression in chick is restricted to the left side before that of lefty-2 (Levin et al.,
1995
). Furthermore, although the expression of shh is bilateral in mammals, it is most probable that the leftward flow of the extraembryonic fluid generated by nodal cilia
produce the concentration gradient of secreted morphogens including Shh which are upstream of the L-R determinants such as lefty-1, -2. The absence of this flow in
kif3A null mutant mice (Fig. 8 B) might result in disturbance of the correct topology of determinants.
As a next step in delineating the total function of KIF3 complex in vivo, conditional gene targeting is now under way. Furthermore, we could also examine the genetic interaction between these molecules by making double knockout mice for KIF3A/B. From another standpoint, by using the cell biological disciplinary, now we are also conducting experiments to unveil the function of KIF3 complex in mesodermal induction and neural development.
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Footnotes |
---|
Address correspondence to Nobutaka Hirokawa, Department of Cell Biology and Anatomy, University of Tokyo, Graduate School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 Japan. Tel.: 81-3-3812-2111, ext. 3326. Fax: 81-3-5802-8646. E-mail: hirokawa{at}m.u-tokyo.ac.jp
Received for publication 14 January 1999 and in revised form 24 March 1999.
We should like to express our gratitude to Dr. Toshimichi Yoshida (Mie
University, Japan) for the anti-axonemal outer arm dynein antibody
(AD2); Dr. Winfield Sale (Emory University, Georgia) for the anti-dynein
intermediate chain antibody (IC140); Dr. Hiroshi Hamada (Osaka University, Japan) for the lefty-2 and shh probe; Drs. Yumiko Saga (National
Institute of Health Science, Japan) and Bernhard G. Herrmann (Max-Planck Institut für Entwicklungsbiologie, Tübingen, Germany) for the
Brachyury probe; Dr. Peter Gruss (Max-Planck Institut für Biophysikalische Chemie, Göttingen, Germany) for the Pax6 probe. We appreciate Dr. Mika Karasawa (Chiba University, Japan) for providing us with a
good protocol for whole mount in situ hybridization. We are also grateful
to Drs. Yoshimitsu Kanai and Akihiro Harada, Ms. Chunjie Zhao, and
Ms. Noriko Homma for their assistance in transgenic technology, and to
Mrs. Haruyo Fukuda, Ms. Hiromi Sato, and Mr. Nobuhisa Ono-uchi for
their technical and secretarial assistance.
This work was supported by a grant for Center of Excellence (COE) from the Ministry of Education, Science, Sports and Culture of Japan to Nobutaka Hirokawa.
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Abbreviations used in this paper |
---|
AP, alkaline phosphatase; dpc, days post coitum; ES, embryonic stem; IFT, intraciliary-flagellar transport; KIF, kinesin superfamily protein; MT, microtubule; RT, room temperature; VEC-DIC/FL, simultaneous observation of video-enhanced contrast DIC and fluorescent images.
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References |
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