Targeted deletion of the novel cytoplasmic dynein mD2LIC disrupts the embryonic organiser, formation of the body axes and specification of ventral cell fates
Amer Ahmed Rana1,*,**,
Juan Pedro Martinez Barbera1,
,
Tristan A. Rodriguez1,
,
Denise Lynch1,
,
Elizabeth Hirst1,
James C. Smith2,* and
Rosa S. P. Beddington1,¶
1 Division of Mammalian Development, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Division of Developmental Biology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
**
Author for correspondence (e-mail:
a.rana{at}gurdon.cam.ac.uk)
Accepted 23 July 2004
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SUMMARY
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Dyneins have been implicated in left-right axis determination during
embryonic development and in a variety of human genetic syndromes. In this
paper, we study the recently discovered mouse dynein 2 light intermediate
chain (mD2LIC), which is believed to be involved in retrograde
intraflagella transport and which, like left-right dynein, is
expressed in the node of the mouse embryo. Cells of the ventral node of mouse
embryos lacking mD2LIC have an altered morphology and lack monocilia,
and expression of Foxa2 and Shh in this structure is reduced
or completely absent. At later stages, consistent with the absence of nodal
cilia, mD2LIC is required for the establishment of the left-right
axis and for normal expression of Nodal, and the ventral neural tube
fails to express Shh, Foxa2 and Ebaf. mD2LIC also functions
indirectly in the survival of anterior definitive endoderm and in the
maintenance of the anterior neural ridge, probably through maintenance of
Foxa2/Hnf3ß expression. Together, our results indicate
that mD2LIC is required to maintain or establish ventral cell fates
and for correct signalling by the organiser and midline, and they identify the
first embryonic function of a vertebrate cytoplasmic dynein.
Key words: D2LIC, Axis formation, Handedness, Mouse embryo, Intra-flagellar transport, Lefty2, Hnf3ß
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Introduction
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Dynein is a large multi-subunit protein complex consisting of two heavy
chains complexed to intermediate, light intermediate and/or light chains
(Harrison and King, 2000
). The
dynein family has been divided into two subfamilies: the axonemal and
cytoplasmic dyneins. Axonemal dyneins function as structural elements in cilia
and flagella, and provide the force responsible for ciliary motion. By
contrast, cytoplasmic dyneins are motor proteins involved in the intracellular
transport of cargo along microtubules and in retrograde intraflagellar
transport during the assembly of cilia
(Perrone et al., 2003
).
The dyneins have been implicated in many cellular and developmental
processes. For example, the gene responsible for the spontaneous classical
mouse mutation inversus viscerum (iv) is an axonemal heavy
chain dynein named left-right dynein (previously lrd;
Dnahc11 Mouse Genome Informatics)
(Supp et al., 1997
).
Dnahc11 is expressed in the node of the mouse embryo and is necessary
for the motility of cilia in the node. It is required for the establishment of
the left-right body axis and has been implicated in Kartagener's and other
immotile cilia syndromes (Supp et al.,
1997
). In addition, dyneins have been associated with severe
clinical abnormalities, such as heterotaxia and isomerism (including
polysplenia or asplenia), and with single organ inversions, such as
dextrocardia (Casey and Hackett,
2000
), as well as in human genetic disease syndromes including
spinal bulbar muscular atrophy and spinal muscular atrophy
(Hafezparast et al.,
2003
).
The importance of intra-flagellar transport in embryonic development has
been emphasised by analysis of wimple (previously Wim; Ift172 Mouse
Genome Informatics) and flexo (previously Fxo; Tg737Rpw Mouse Genome
Informatics) (Huangfu et al.,
2003
), which, together with the kinesin Kif3a
(Marszalek et al., 1999
;
Takeda et al., 1999
), are
involved in anterograde intra-flagellar transport. Mouse mutants lacking
Wim or Fxo fail to specify ventral cell fates, probably
because Shh signalling is disrupted downstream of the patched receptor
(Huangfu et al., 2003
).
Together, these observations emphasise that the study of dyneins is of
great cellular, developmental and medical interest. Recently, on the basis of
its amino acid sequence and its ability to interact with cytoplasmic dynein 2
heavy chain (DHC2), Grissom and colleagues
(Grissom et al., 2002
) have
classified dynein 2 light intermediate chain (D2LIC) as a novel member of the
dynein family of proteins. Consistent with this proposal, XBX-1, the
Caenorhabditis elegans homologue of D2LIC, proves to be required for
retrograde intraflagellar transport
(Schafer et al., 2003
). We
previously identified mouse D2LIC (mD2LIC; 4933404O11Rik
Mouse Genome Informatics) as a gene that is expressed in the node of
the developing embryo (Sousa-Nunes et al.,
2003
), and in this paper we investigate its function during
development. Our work shows that mD2LIC is needed to maintain or
establish ventral cell fates, for monocilium formation in the ventral node,
and for correct signalling by the organiser and midline. Our experiments
define the first embryonic function for a vertebrate cytoplasmic dynein.
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Materials and methods
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In situ hybridisation
In situ hybridisation of mD2LIC was performed as described
(Wilkinson and Nieto, 1993
)
using probes derived from EST clone AL024282. Probes specific for other genes
are described in the text. The lefty probe
(Meno et al., 1997
) detects
both Leftb (previously lefty1) and Ebaf (previously
lefty2) transcripts.
Northern blot analysis
Total RNA was isolated from adult tissues and from 11.5 dpc whole embryos
and placenta as described (Chomczynski and
Sacchi, 1987
). A probe was prepared from a full-length
mD2LIC cDNA. Hybridisation was performed as described
(Martinez-Barbera et al.,
1997
).
Scanning electron microscopy
Scanning electron microscopy was performed as described
(Sulik et al., 1994
), with 15
minutes uranyl acetate treatment.
TUNEL staining
TUNEL staining was performed as described
(Barbera et al., 2002
).
Gene targeting
Genomic clones were isolated from a 129/Olac genomic library (Stratagene)
using an mD2LIC full-length cDNA to generate a probe. A 2.75 kb
region of the endogenous genomic locus was replaced by homologous
recombination in E14TG2A ES cells with a 2 kb loxP flanked PGK
neomycin (PGKneo) cassette using a diphtheria toxin A
(DTA) cassette for negative selection
(Fig. 3). Correctly targeted
cells from five independent cell lines were used to generate chimeric animals
that were subsequently mated to produce heterozygous individuals. Only
heterozygotes derived from the same cell line were intercrossed. The
phenotypes of homozygous individuals derived from each of the five cell lines
were similar, indicating that the embryonic defects observed in this paper are
consequences of the targeting event.

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Fig. 3. Targeted deletion of mD2LIC. (A) The targeting strategy involved
removing 2.75 kb of the endogenous mD2LIC locus. This contains 500 bp
upstream of the predicted transcriptional start site together with the first
two exons, which include the translation start site and half the P-loop
domain. The targeting vector incorporates both positive (neomycin resistance)
and negative (diphtheria toxin A) selection, and comprises a 5.5 kb 5'
homology arm, a 2 kb loxP flanked PGKneo cassette (which
replaces the targeted region) and a 2.3 kb 3' homology arm. During the
targeting event, an exogenous HindIII restriction site is introduced
into the locus just downstream of the PGKneo cassette. (B) Use of a
3' probe in Southern blot analyses of transfected ES cell genomic DNA
digested with HindIII reveals a 13 kb band corresponding to the
wild-type locus and a 6 kb band representing a correctly targeted locus. (C)
Northern blot analysis of littermates from heterozygous crosses demonstrates
loss of the mD2LIC transcript in homozygous mutant individuals and a
decrease in heterozygous animals. (D) Primers P1 and P2 (indicated in A),
together with a pair of primers designed to detect neo, distinguish
between targeted and non-targeted alleles when genotyping. (E) Classification
of mD2LIC/ mutants into three groups
according to the extent of embryonic turning at 9.5 dpc
(Table 1). Class I mutants
(bottom, 61%; n=39), which exhibit the most severe phenotype, fail to
initiate embryonic turning. Three examples are shown here. Class II (middle,
21%) mutants start but do not complete embryonic turning, and Class III (top,
18%) mutants complete turning but always display an open neural tube in the
region of the head (arrowhead). Other defects include reversal of heart
looping (Class II, white arrowhead), ballooning of the pericardial sac,
anterior truncations (Class III, white arrowhead) and defects in truck and
tail development.
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Genotyping of wild-type and mutant mD2LIC alleles
Individuals were genotyped using the polymerase chain reaction (PCR) or by
Southern blot analysis. In the PCR strategy, a 450 bp product specific to the
wild-type locus was amplified using the primers P1
(5'-GCCCAACATTGTTTCAGCTTCC-3') and P2
(5'-TGACAGCGAGGTACTACTGCT-3'), and a 350 bp neomycin-specific PCR
product was amplified using the primers 5'-CAAGATGGATTGCACGCAGG-3'
and 5'-CGGCAGGAGCAAGGTGAGAT-3'. Southern blotting was performed as
described using a 700 bp probe that lies externally to the targeted region and
recognises 13 kb (wild-type locus) and 6 kb (targeted locus) genomic fragments
following digestion with HindIII (see
Fig. 3).
 |
Results
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Sequence and expression pattern of mD2LIC
In contrast to Grissom and colleagues
(Grissom et al., 2002
), we
find that mD2LIC protein is predicted to contain 351 amino acids, with a
putative P-loop (nucleotide binding domain) near its N terminus
(Fig. 1). We believe that this
discrepancy has arisen because Grissom and colleagues
(Grissom et al., 2002
) made
use of an EST sequence (mouse locus AK008822, BAB25915; M. musculus
Genome Sequencing Consortium) that contains a frame shift relative to the
sequence presented in our study; when translated, this results in a 209 amino
acid protein that lacks the P-loop domain. Our translation of the
mD2LIC cDNA (Sousa-Nunes et al.,
2003
) yields a protein that closely resembles the 350 amino acid
human D2LIC, such that there is 87% identity and 92% amino acid charge matches
between the two proteins.

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Fig. 1. cDNA sequence and translation of mD2LIC. The cDNA comprises 1390
bp and codes for a 351 amino acid protein predicted to contain a P-loop
(nucleotide binding domain) near its N terminus (red box). This sequence
differs from the previously published mD2LIC
(Grissom et al., 2002 ), but
the predicted protein resembles closely the rat and human gene products. We
suspect that the previously published sequence contains a frame-shift near the
5' end of the cDNA, resulting in a predicted protein of only 209 amino
acids that lacks the P loop.
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Expression of mD2LIC in the late streak to early somite stage
mouse embryo is restricted to the monociliated cells of the ventral node
(Sousa-Nunes et al., 2003
)
(Fig. 2A-E). Northern blot
analysis reveals a single transcript of 1.4 kb, and in the adult strongest
expression of mD2LIC occurs in kidney and brain
(Fig. 2F). The initial
expression pattern of mD2LIC resembles that of the axonemal
left-right dynein (lrd), suggesting that mD2LIC,
like lrd, is involved in establishment of the embryonic axis
(Supp et al., 1999
).

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Fig. 2. Expression pattern of mD2LIC. Transcription of mD2LIC is
not detected by in situ hybridisation before the onset of gastrulation (A),
but from late streak (B) to headfold stages (C,D) transcription is observed in
the node (black arrowheads). Sections reveal that this expression is
restricted to cells of the ventral node (E). Northern blot analysis reveals a
single transcript of 1.4 kb, and in the adult expression is highest in brain
and kidney (E).
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Targeted mutation of mD2LIC
To analyse the function of mD2LIC, we generated a targeted
mutation in embryonic stem cells. The mutation results in loss of both
transcriptional and translational start sites and is likely to represent a
null allele (Fig. 3A-D).
mD2LIC/ mutants die before 11.5 days post
coitum (dpc) (Table 1) and
gross phenotypic effects are detectable from 8.5 dpc, with defects in
notochord and floorplate formation and a reduction in definitive endoderm.
These are followed by anterior truncations of the forebrain, defects in the
ventral body wall and in closure of the neural tube, and either an arrest of
embryonic turning and heart looping or a randomisation in their direction
(Fig. 3E;
Table 2). The severity of this
phenotype varies from embryo to embryo, and we have defined three phenotypic
classes (Fig. 3E;
Table 2). Class I, the most
severe, was observed most frequently, and our analysis therefore concentrates
on such embryos unless stated otherwise.
The gross phenotype at 8.5 dpc and later is presaged at 7.5 dpc by a
disruption of cilium formation in the ventral node, where expression of
mD2LIC is highest. Although scanning electron microscopy revealed
that the gross morphology of the node is normal in null mutants
(Fig. 4A,C,E), the single
cilium that is normally carried by ventral node cells was absent in four out
of 12 null mutant embryos. A further eight embryos formed only stunted
cilium-like structures that were restricted to the posterior region of the
node (Fig. 4B,D,F-I). Cells of
the notochordal plate lacked cilia completely, suggesting that the stunted
cilium-like structures visible in the node of some embryos are later lost,
perhaps through reabsorption. We note that cells in the node and notochordal
plate also appear flatter than their wild-type counterparts
(Fig. 4B,D,F-I).

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Fig. 4. Scanning electron microscopy reveals defects in monocilium formation in
mD2LIC/ mouse embryos. (A) The distal tip of
a wild-type embryo at 7.5-8.0 dpc (1-2 somite stage) viewed at low power.
(C,E) The gross morphology of the node of homozygous mutant embryos is normal.
Conditions and magnification are identical in A and C, and E is at twice the
magnification. (B,G) Higher-power view of a wild-type embryo reveals rounded
cells bearing monocilia (arrowheads). (D,H,F,I) Ventral node cells in
mD2LIC/ embryos are flatter than their
wild-type counterparts and they lack normal monocilia. In some cases stunted
structures are formed in the place of monocilia (arrows). G-I are at the same
magnification. White boxes in B,D,F indicate regions of the node shown at
higher magnification in G-I respectively. (F) Cells in the anterior region of
the node and notochordal plate have the most extreme phenotype.
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Gene expression patterns in mD2LIC/ mutant embryos
The abnormal appearance of the node suggests that these cells might be
incorrectly specified. To address this point, we examined the expression
patterns of Fgf8, Shh, T and Foxa2 (previously Hnf3ß)
(Ang and Rossant, 1994
;
Crossley and Martin, 1995
;
Echelard et al., 1993
;
Wilkinson et al., 1990
). At
the late streak stage, expression of Fgf8 is normal in null mutant
embryos (Fig. 5A,B), while
expression of both Shh (n=5/5;
Fig. 5C-E) and T
(n=5/5; data not shown) is slightly reduced. Most significantly,
expression of Foxa2 in the region of the node was severely reduced in
mD2LIC/ embryos (n=7/9), and in
some was almost undetectable (n=2/9)
(Fig. 5F-J).

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Fig. 5. Analysis of the mD2LIC phenotype at gastrula stages. (A,B)
Expression of Fgf8 is unaffected by loss of mD2LIC function.
(A) Lateral view of wild-type embryo; (B) Lateral view of
mD2LIC/ embryo. (C-E) Shh
expression is reduced in the node and axial mesoderm of mD2LIC-null
mutants. (C) Wild-type embryo showing expression of Shh in the node
and in axial structures. (D,E) Reduction of Shh expression
(arrowheads) in mD2LIC/ embryos. (F-J)
Reduction in Foxa2 expression in mD2LIC null mutant embryos. (F)
Wild-type embryo showing expression of Foxa2 in the node and axial
mesendoderm. (G,H) Expression of Foxa2 is severely reduced (G) or
absent (H) in mD2LIC-null mutant embryos. (I,J) Foxa2
expression is later reduced in anterior definitive endoderm in
mD2LIC/ embryos. (I) Wild-type embryo. (J)
mD2LIC/ embryo. Arrowheads mark anterior
definitive endoderm. (K-P) TUNEL analysis of
mD2LIC/ embryos reveals no elevation of cell
death in the node or its derivatives but apoptosis does occur in anterior
definitive endoderm. (K-M) Little apoptosis is observed in the embryonic
region of wild-type embryos at 7.5 dpc (K, lateral view; L, anterior view; M,
distal view). In mD2LIC/ embryos, no
apoptosis is observed in the node or its derivatives (P) but substantial cell
death occurs in the anterior definitive endoderm (arrowheads, N, lateral; O,
anterior).
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Gene expression patterns in derivatives of the node are also disrupted.
T (Fig. 6A,B) and
Shh (data not shown) are both expressed in a discontinuous manner in
the region of the notochord, while Foxa2 is completely absent here
and in the floor plate of null mutant embryos, and its expression in foregut
endoderm is markedly reduced (Fig.
6E,F). Shh transcripts are also absent from the floor
plate and endoderm of mD2LIC/ embryos
(Fig. 6C,D). The downregulation
of these genes is unlikely to be due to cell death; TUNEL staining revealed no
abnormal levels of apoptosis in the node or its derivatives at any of the
stages examined (Fig. 5K-M).
Consistent with the downregulation of T, Shh and Foxa2 in
the midline of mD2LIC/ embryos, histological
analysis revealed that loss of mD2LIC impairs differentiation of the notochord
(see Table 3).

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Fig. 6. Analysis of mD2LIC/ mutants at 8.5-9.0
dpc. (A,B) Expression of T is discontinuous and distorted in the notochord of
embryos lacking mD2LIC. (A) Wild-type embryo; (B)
mD2LIC/ embryo. (C,D) Expression of
Shh is also discontinuous in null mutant embryos (data not shown) and
is absent in the ventral neural tube as seen in histological sections (black
arrowheads; sections shown here are from the head region). (C) Wild-type
embryo; (D) mD2LIC/ embryo. (E,F)
mD2LIC-null embryos lack expression of Foxa2 in notochord and ventral
neural tube and expression is greatly reduced in definitive embryonic
endoderm. (E) Wild-type embryo (ventral view); (F) mutant embryo (ventral
view); Foxa2 expression is lacking in notochord and ventral neural
tube (arrowhead), and there is a reduction of expression in definitive
endoderm (arrow). (G-Q) Defective establishment and maintenance of the
left-right axis in mD2LIC/ mutants.
Wild-type (G, distal view) asymmetric expression of nodal around the node is
not observed in null mutants (H, distal view). At the early somite stage,
left-lateral plate specific expression of nodal (I, posterior view) is either
expressed bilaterally (J, ventral view), on the right (K, dorsal view, white
arrowheads; black arrowhead indicates expression in the node), on the left or
absent (data not shown). Expression of Leftb in the left side of the
ventral neural tube (L, anterior view, arrowhead; broken white line indicates
the anterior edge of the head folds) is absent in null mutants (M, dorsal
view, arrowhead) and Ebaf expression is randomised (in this case
showing bilateral expression). Left lateral plate Pitx2 specific
expression (N, ventral view) is randomised in null mutants, appearing
bilaterally in class I and II mutants (O,P, ventral view, arrowheads) or on
the left (Q, left-hand embryo, ventral view, arrowhead) or on the right (Q,
right-hand embryo, ventral view, arrow) in class III mutants. White lines
indicate the boundary of the headfolds. (R,S) Lack of Fgf8 expression
in the anterior neural ridge (ANR) of
mD2LIC/ embryos. (R) Wild-type embryo
(ventral view). Arrowhead shows expression of Fgf8 in the ANR. (S)
mD2LIC/ mutant embryo (ventral view).
Fgf8 expression does not occur the in ANR (arrowhead). (T-Y) TUNEL
staining reveals altered apoptotic profiles during neural tube closure. Normal
apoptosis in the region of the hindbrain (arrowheads; T, lateral view; U,
dorsal view of trunk; V, dorsal view of head) is absent in mutant (arrowhead
in W, lateral view; X,Y, dorsal view). In addition, ectopic cell death is
observed along the dorsal midline in mutants (X, dorsal view of trunk,
arrowhead), but does not occur in wild-type littermates (U). Cell death is
also observed in the cephalic mesoderm surrounding the notochord in the region
of the head (arrowheads, Y).
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Disruption of signalling by the node and axial mesoderm in mD2LIC/ mutant embryos
The signalling properties of the node and axial mesendoderm in
mD2LIC/ mutant embryos, and the
establishment of the left-right axis, were further investigated by examining
the expression of Nodal, Leftb, Ebaf and Pitx2
(Collignon et al., 1996
;
Kitamura et al., 1999
;
Meno et al., 1997
).
Nodal is the earliest known asymmetric marker of the left-right axis.
In wild-type embryos expression first occurs symmetrically around the
periphery of the node, but it then becomes asymmetrical, with higher levels
and a broader domain of expression on the left-hand side. nodal is
then also expressed in the left-hand lateral plate. At late streak stages
nodal expression was reduced in three
mD2LIC/ embryos, was absent in one, and, in
contrast to its normal expression pattern, was expressed symmetrically around
the node in eight (Fig. 6G,H
and data not shown). At later stages (2-5 somite stages), left-sided
(n=2/9), right-sided (n=1/9) and bilateral (n=5/9)
expression patterns were observed in the lateral plate, and in one embryo
(n=1/9) no expression was detectable. We note that the frequency of
bilateral expression is similar to the frequency of the more severe Class I
and II mutants (Table 2).
Expression of Leftb was undetectable in all null embryos
(n=5/5), while Ebaf expression occurred bilaterally in three
out of five embryos, was expressed on the right-hand side in another, and was
absent in a fifth (Fig. 6L,M). Similar results were observed with Pitx2
(Fig. 6N-Q). Together, these
results indicate that left-right asymmetry is not generated correctly in
mD2LIC/ embryos, perhaps owing to the
impairment of ciliogenesis and subsequent disruption of Nodal signalling,
together with impaired development of the midline (see Discussion).
Anterior truncations and neural tube closure defects in mD2LIC/ mutants
The later phenotypes observed in mD2LIC null embryos, such as
anterior truncations and defects in neural tube closure, are likely to be
consequences of the earlier defects. For example, expression of Foxa2
in the anterior definitive endoderm of
mD2LIC/ mutants is greatly reduced
(Fig. 5I,J), and this is
accompanied by extensive cell death in this tissue
(Fig. 5K-P). mD2LIC is
not normally expressed in the anterior definitive endoderm, suggesting that
these cells are deprived of a survival signal that is normally produced by the
node or its derivatives. During normal development, the anterior definitive
endoderm maintains an Fgf8 signalling centre in the anterior neural
ridge, which in turn is required for correct anterior development
(Martinez Barbera et al.,
2000
; Shimamura and
Rubenstein, 1997
). Fgf8 expression in the anterior neural
ridge is significantly reduced in mD2LIC/
mutants (Fig. 6R,S), presumably
because of defects in the anterior definitive endoderm, and the decrease in
Fgf8 will lead to defects in anterior development
(Fig. 7).

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Fig. 7. A model for the function of mD2LIC in the establishment of the
body axes. (A) In wild-type embryos, mD2LIC is required for the
formation of cilia in the node and for the correct morphology of ventral node
cells (mottled grey circle). It is also necessary for the normal expression of
Foxa2, Shh and T, and for the asymmetric expression of
Nodal (dark blue), which leads to the induction of Nodal,
Ebaf and Pitx2 in the left-hand-side of the embryo. The
notochord (mottled rectangle below the node), also expresses Foxa2,
Shh and T, while the adjacent ventral neural tube (light blue)
expresses Foxa2 and Shh and the nodal signalling antagonist
Leftb. These structures constitute the midline, which is thought to
act as a barrier to maintain left-right character in the developing embryo.
The anterior definitive endoderm (ADE, yellow) receives survival signals from
node derivatives, including the axial mesendoderm and ventral neural tube, and
the ADE in turn is thought to maintain an Fgf8-expressing signalling
centre in the anterior neural ridge (ANR, purple). This is required for
maintenance of the forebrain and anterior identity. (B) In
mD2LIC/ mutants ventral node cells do not
form cilia and are flatter than their wild-type counterparts (solid grey
circle). Expression of Foxa2, Shh and T is severely reduced
or absent (depicted as faded text) and expression of Nodal is usually
symmetrical. The compromised signalling properties of the organiser result in
reduced expression of Shh, T and Foxa2 in the midline and
consequently the absence of Foxa2, Shh and Leftb from the
ventral neural tube. The bilaterally symmetrical expression of Nodal
and the presumed loss of the midline barrier cause the nodal signalling
pathway, normally active only in the left-hand side of the embryo, to be
active on both sides. The defective axial mesendoderm does not emit survival
signals to the ADE, and the Fgf8 signalling centre in the ANR is
lost.
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Defects in neural tube closure are also likely to be an indirect
consequence of loss of mD2LIC. Neural tube closure involves
specification of the floor plate in response to signals derived from the
notochord (Chen and Behringer,
1995
; Huangfu et al.,
2003
; Wallingford and Harland,
2002
) and modelling of the dorsal neural tube through apoptosis
(Martinez Barbera et al.,
2000
); the process also requires mechanical support from adjacent
cephalic mesenchyme (Weil et al.,
1997
). All these steps are likely to be affected in
mD2LIC/ embryos. Perhaps most significantly,
reduced expression of Shh will prevent the notochord from acting as a
normal signalling centre, thereby interfering with formation of the floorplate
and with expression of Foxa2, Shh and Ebaf. In addition,
however, TUNEL analysis at 9.0-9.5 dpc reveals that
mD2LIC/ embryos exhibit reduced levels of
cell death in the hindbrain and also that there is ectopic cell death in the
cephalic mesenchyme (Fig.
6T-Y). The elevated levels of apoptosis in the latter tissue may
deprive the closing neural tube of its required mechanical support.
These and other aspects of the mD2LIC/
phenotype are summarised in Fig.
7 and discussed below.
 |
Discussion
|
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The first detectable defect in mouse embryos lacking mD2LIC is a failure of
cilium formation in the node (Fig.
4). The normal assembly of cilia involves two types of
intraflagellar transport. During anterograde transport, protein particles are
translocated to the tip of the growing cilium; this process requires the
kinesins Kif3a and Kif3b in a heterotrimeric complex with kinesin-associated
protein 3 (Cole et al., 1998
;
Morris and Scholey, 1997
). The
particles are conveyed back to the base of the cilium by retrograde transport,
and as we discuss below, it is this process that requires cytoplasmic dyneins
such as mD2LIC.
Evidence implicating cytoplasmic dyneins in retrograde transport comes from
work in Chlamydomonas and Caenorhabditis elegans.
Chlamydomonas lacking either dynein 2 heavy chain (DHC2) or dynein light
chain (LC8) fail to undergo normal ciliogenesis, and any cilium-like
structures that do form are reabsorbed
(Pazour et al., 1999
;
Pazour et al., 1998
). mD2LIC
can associate with DHC2 (Grissom et al.,
2002
; Perrone et al.,
2003
), suggesting that this cytoplasmic dynein is also involved in
retrograde transport. Consistent with this idea, ciliogenesis is impaired in
C. elegans lacking XBX-1, a homologue of D2LIC, and as in the
Chlamydomonas mutants, those cilia that do form subsequently
disappear, perhaps through reabsorption
(Schafer et al., 2003
).
Together, these results suggest that loss of mD2LIC in the mouse node is
directly responsible for the observed defects in cilium formation.
How does defective ciliogenesis lead to the mD2LIC/ phenotype?
As discussed below, several aspects of the mD2LIC mutant phenotype
may be due directly to the loss of cilia and a disruption of nodal flow. Other
defects, however, such as the early downregulation of genes such as
Foxa2 and Shh, may have a more indirect aetiology. There
may, for example, be a general impairment of cellular function: D2LIC is
present in the Golgi apparatus and centrosomes
(Grissom et al., 2002
), and
its loss may cause defects in protein maturation, in the cytoskeleton or in
cell polarity.
Other elements of the mD2LIC/ phenotype
may result from a reduction in Hedgehog activity. The more severely affected
mD2LIC/ embryos resemble those deficient in
Hedgehog signalling, such as the Smo/ single
mutant and
Shh//Ihh/
double mutants (Zhang et al.,
2001
). mD2LIC/ individuals also
resemble embryos lacking nt, rotatin or Sil, as well as
chimeric and conditional Foxa2/ embryos
(Dufort et al., 1998
;
Faisst et al., 2002
;
Hallonet et al., 2002
;
Izraeli et al., 1999
;
Melloy et al., 1998
). Like
mD2LIC/ embryos, many of these mutants lack
midline expression of Foxa2, one consequence of which would be a
downregulation of Shh (Filosa et
al., 1997
).
It is possible that some of the later elements of the
mD2LIC/ phenotype are also consequences of
defects in the Hedgehog signal transduction pathway. The phenotypes of our
least severe `Class III' embryos resemble those of embryos lacking the
intra-flagellar transport proteins Kif3a, Wim, Polaris and Fxo, in all of
which the neural tube fails to close in the region of the head
(Huangfu et al., 2003
;
Takeda et al., 1999
). This is
a characteristic of embryos that lack Shh
(Huangfu et al., 2003
) and it
is possible that mD2LIC/ individuals, such
as Wim/,
Polaris/ and
Fxo/ embryos, are defective in Shh signal
transduction as well as in Shh expression.
Finally, and as mentioned above, the lack of nodal cilia in
mD2LIC/ mice would be expected to interfere
with nodal flow, and therefore with specification of the left-right axis. In
the nodal flow hypothesis (Nonaka et al.,
1998
), cilia have been suggested to cause the unidirectional flow
of a morphogen that thereby accumulates on just one side of the node and
activates gene expression in an asymmetric fashion. More complicated models
involve two populations of node monocilia, in which one population generates a
fluid flow and the other senses and transduces it, thereby leading to an
asymmetric calcium signal at the left-hand-side of the node
(McGrath et al., 2003
;
Tabin and Vogan, 2003
).
The absence of nodal cilia would disrupt asymmetric gene expression and
thereby set in train the series of events that cause the defects we observe in
mD2LIC/ embryos. These events are described
in Fig. 7, and include the
decreased or symmetrical expression of genes such as Nodal, as well
as the downregulation of T, Foxa2 and Shh. They culminate in
the requirement for mD2LIC in the survival of the anterior definitive
endoderm and thereby for maintenance of the anterior neural ridge and for
normal anterior development. Models involving maintenance of the anterior
neural ridge by anterior definitive endoderm have been proposed previously
(Camus et al., 2000
;
Hallonet et al., 2002
),
although this is the first time that defects in axial mesendoderm have been
shown to lead to cell death in the anterior definitive endoderm.
Regulation of mD2LIC
Recent work has demonstrated that expression of mD2LIC is
downregulated in Rfx3-deficient mouse embryos, and that these embryos too show
defects in cilium development and left-right axis specification
(Bonnafe et al., 2004
).
Expression of mD2LIC is not completely absent in
Rfx3/ individuals, however, and the
phenotype of such mice is not as severe as that of our mD2LIC
mutants, suggesting that other proteins also regulate mD2LIC
expression.
 |
ACKNOWLEDGMENTS
|
---|
This work was supported by the Medical Research Council and is dedicated to
Rosa Beddington much loved and missed. The authors thank members of
the Divisions of Mammalian Development, Developmental Biology and
Developmental Neurobiology, especially Simon Bullock, Jonathan Cooke, Sally
Dunwoodie, Alex Gould, Shankar Srinivas and Derek Stemple, for helpful
discussions. We are also grateful to Melanie Clements for technical assistance
and to the staff of the Dunkin Green building for looking after the mice.
 |
Footnotes
|
---|
* Present address: Wellcome Trust/Cancer Research UK Gurdon Institute and
Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge,
CB2 1QR, UK 
Present address: Neural Development Unit, Institute of Child Health, 30
Guilford Street, London, WC1N 1EH, UK 
Present address: MRC Clinical Sciences Centre, Faculty of Medicine,
Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London,
W12 0NN, UK 
Present address: Developmental Biology Program, Victor Chang Cardiac
Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia 
¶ Deceased 18 May 2001 
 |
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