Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: hueco{at}uclink4.berkeley.edu)
Accepted 22 August 2002
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SUMMARY |
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Key words: Dishevelled, Strabismus, Planar cell polarity, Convergent extension, Neural tube defect, Neurulation, Midline, Floorplate, Craniorachischisis, Looptail, Xenopus
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INTRODUCTION |
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Mammalian neural tube defects (NTDs) stem from a failure of one or more of
these morphogenetic processes (Harris and
Juriloff, 1999), and recent genetic experiments in mice have
identified individual genes that control specifically the many individual
behaviors that comprise neurulation. For example, loss of p190RhoGAP inhibits
neural fold closure by disrupting apical constriction of neuroepithelial cells
(Brouns et al., 2000
). Mutation
of ephrin A5 does not affect the elevation or apposition of neural folds, but
instead precludes their normal fusion at the dorsal midline
(Holmberg et al., 2000
). Genes
controlling extrinsic factors in neurulation have also been identified, such
as AP-2, which is expressed in the non-neural ectoderm but is essential for
tube closure (Zhang et al.,
1996
).
One morphogenetic process that has not been mechanistically related to NTDs
is convergent extension, in which a tissue narrows in one axis and elongates
in a perpendicular axis (reviewed by
Wallingford et al., 2002).
Though it has been described during neurulation in a variety of vertebrates,
the contribution of convergent extension to tube closure has been difficult to
assess without tools for uncoupling it from the many other morphogenetic
processes involved (Burnside and Jacobson,
1968
; Elul and Keller,
2000
; Jacobson and Gordon,
1976
; Keller et al.,
1992
; Lawson et al.,
2001
; Schoenwolf and Alvarez,
1989
; van Straaten et al.,
1996
). Indeed, defects in axial elongation have been postulated to
contribute to NTD in the classical mouse mutant looptail
(Kibar et al., 2001
;
Smith and Stein, 1962
;
Wilson and Wyatt, 1992
),
though other studies have challenged that view
(Gerrelli and Copp, 1997
;
Murdoch et al., 2001
).
Convergent extension is driven by the polarized rearrangement of cells
within the tissue (Elul and Keller,
2000; Keller et al.,
2000
; Shih and Keller,
1992
), and Xenopus Dishevelled (Xdsh) controls this
polarity via a vertebrate cognate of the planar cell polarity (PCP) cascade
(Tada and Smith, 2000
;
Wallingford et al., 2000
). In
our previous work, we showed that disruption of Xdsh signaling results in a
failure of both neural convergent extension and neural tube closure
(Wallingford and Harland,
2001
).
These results suggest the possibility that PCP-mediated convergent
extension is directly required for neural tube closure. But without a
mechanistic analysis, it remains equally possible that Xdsh controls other
important movements during neurulation. Indeed, each of the many processes
comprising neurulation requires coordinated cell polarity
(Fig. 1), and PCP signaling
components have been implicated in establishing both planar and apicobasal
cell polarity in a wide variety of tissues and animals
(Bellaiche et al., 2001;
Marsden and DeSimone, 2001
;
Sun et al., 2001
;
Theisen et al., 1994
;
Wiggan and Hamel, 2002
).
In light of the previous work, two questions need to be addressed. First,
which processes of neurulation require Xdsh signaling? Second, does convergent
extension directly contribute to neural tube closure? In this paper, we show
that Xdsh signaling is dispensable for the elevation, medial movement and
fusion of the neural folds, but time-lapse analysis revealed a strict
correlation between convergent extension and neural tube closure. The results
here demonstrate that midline convergent extension is a crucial component of
normal vertebrate neurulation, narrowing the distance between the neural
folds, allowing them to meet and fuse. In light of the severe NTDs of
Dishevelled mutant mice (Hamblet et al.,
2002), our results may provide important insights into the
pathology of specific human neural tube defects.
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MATERIALS AND METHODS |
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For microinjections, embryos were placed in a solution of 2.5% Ficoll in 1/3xMMR, and injected using forceps and an Oxford universal micromanipulator, then reared in Ficoll + 1/3xMMR to stage 9 then washed and reared in 1/3xMMR alone. Xdd1 and Xdsh-D2 were injected at 1 ng per blastomere. XStbm was injected at 1.25 ng per blastomere.
Embryo culture, solutions, in vitro transcription, and in situ
hybridization were as previously described; some embryos were dehydrated and
cleared (Sive et al.,
2000).
Constructs used were: Xdsh-PDZ/D2 (Xdsh-D2)
(Rothbächer et al.,
2000
), Xdd1 (Sokol,
1996
), XStbm (Darken et al.,
2002
), Xash-3 (Zimmerman et
al., 1993
), Xpax-3 (Bang et
al., 1999
), Xnetrin (de la
Torre et al., 1997
), Sox-2
(Mizuseki et al., 1998
) and
Xfd12 (Solter et al.,
1999
).
Time-lapse microscopy and morphometric analysis
For time-lapse, embryos were devitellinated with forceps and placed in
wells of modeling clay in 1/3xMMR. Images were collected on a Zeiss SV6
stereoscope using a Zeiss Axiocam. Images were collected every 2 minutes, and
embryos were manipulated manually with a hairloop to maintain the proper
viewing angle. Time-lapse stacks were assembled and viewed in NIH Image. LWR
(length/width ratio) measurements (Fig.
5) were made at various stages by tracing embryos in NIH Image and
calculating major and minor axes of the best-fitting ellipse of the trace.
Rates of neural tube closure (Fig.
4) were estimated by tracing the edges of the neural folds in
time-lapse movies and calculating the minor axis of the best-fitting ellipse
of that trace. To confirm that this estimation was accurate, the distance
between the apices of the neural folds from fixed specimens was measured
directly from confocal sections at stages 16 and 17. Rates of fold apposition
obtained from the fixed material paralleled that obtained from the time-lapse
(not shown).
|
|
Confocal microscopy
Embryos were fixed in MEMFA overnight, washed in PBS, dehydrated overnight
in methanol at 4°C, and rehydrated in PBS + 0.1% Tween-20. Embryos were
then bisected transversely using forceps and a fragment of razor blade. These
`half-mounts' were then redehydrated in an ethanol series and cleared for
confocal viewing in 2:1 benzyl benzoate:benzyl alcohol. Images were collected
on an inverted Zeiss 410 confocal microscope using an HQ long-pass 580
emission filter (Chroma).
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RESULTS |
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Mutant Xdsh does not inhibit neural fold elevation
Confocal optical sections revealed the subtle nascent neural folds of stage
16 embryos (Fig. 3A, arrows).
As neurulation proceeded, the folds became more pronounced and moved medially
(Fig. 3D). In embryos
expressing Xdd1, subtle neural folds were also apparent by stage 16, but these
folds were displaced laterally when compared with controls
(Fig. 3B,C). As in controls,
neural folds in Xdd1-expressing embryos were more pronounced at stage 17
(Fig. 3E,F).
|
In many Xdd1-expressing embryos, the midline tissues appeared somewhat disorganized at stage 16 with less clearly defined boundaries between notochord, somites and neural plate when compared with controls. At stage 17, the tissues were more distinct (Fig. 3B,C). In some embryos, the open neural tube was associated with a severely broadened notochord (Fig. 3F). However, in other embryos, open neural tubes were associated with a normal notochord, suggesting that the defect in neural tube closure lies within the neural tissue (Fig. 3E).
Xdsh function is not required for medial movement of the neural
folds
To better understand the nature of the neural tube defects elicited by
mutant Xdsh, we made time-lapse movies of neurulating embryos (see movies at
http://dev.biologists.org/supplemental).
At the doses used here, expression of Xdd1 inhibits neural tube closure in
approximately 50% of embryos. We refer to Xdd1-injected embryos which will
eventually exhibit an open neural tube defect as `open-NT' embryos, and
embryos which will eventually close their neural tube as `closed-NT' embryos.
Xdd1 closed-NT embryos serve as a useful control group for identifying aspects
of morphogenesis which are associated with successful versus failed NT
closure.
In control uninjected embryos, neural folds were evident by stage 16 and the folds moved steadily toward the midline, meeting and fusing at stage 20 (Movie 1 at http://dev.biologists.org/supplemental; Fig. 4A, part a', 4E). The folds of closed-NT embryos were also evident at stage 16 and like the controls, closed-NT embryos displayed a steady movement of the neural folds toward the midline followed by fold fusion (Fig. 4B, part b', 4E). There was often a brief delay in the timing of neural tube closure in closed-NT embryos (Movie 1 at http://dev.biologists.org/supplemental; Fig. 4E).
Surprisingly, Xdd1-injected open-NT embryos also formed pronounced neural folds at stage 16 and these folds moved steadily toward the dorsal midline as neurulation proceeded (Movie 1 at http://dev.biologists.org/supplemental; Fig. 4C,D,F). Similar results were obtained from movies of embryos expressing Xdsh-D2 (Movie 2 at http://dev.biologists.org/supplemental). The distance traveled by the folds of open-NT embryos between stage 16 and 20 was comparable with the distance traveled in the same period by folds of closed-NT or control embryos (Fig. 4E,F), suggesting that Xdsh function is not required for the movement of neural folds toward the midline. In fact, the rate at which neural folds approached one another during mid- and late-neurulation was marginally faster in the open-NT embryos than in wild-type or closed-NT embryos (Fig. 4G).
The crucial difference observed between open-NT embryos and control embryos was the initial distance separating the forming folds at stage 16. The width between the nascent neural folds was considerably greater in open-NT embryos than in control embryos (Fig. 4A-D), and throughout neurulation, folds of open-NT embryos remained substantially farther apart than folds of control embryos (Fig. 4F).
Finally, the dorsal epidermis has been implicated in generating force for the apposition of the neural folds. Our time-lapse analysis revealed no defect in the medial movement of the dorsal epidermis in Xdd1-injected embryos. Dorsal superficial cells can be seen actively and robustly moving medially in both control embryos (Movie 3 at http://dev.biologists.org/supplemental) and open-NT embryos (Movie 4 at http://dev.biologists.org/supplemental).
Failure of neural tube closure accompanies failure of convergent
extension
In our time-lapse movies of neurulation, we noticed a strong correlation
between the degree of axis elongation and the severity of the neural tube
defect. For example, Xdd1 closed-NT embryos elongated substantially between
stage 16 and stage 20 (Fig. 4B, part
b'), while embryos with severely open NTs elongated
negligibly during the same period (Fig. 4D,
part d'). Interestingly, embryos with smaller regions of
open neural tube elongated to an intermediate degree
(Fig. 4C, part c').
Because there is no increase in mass in early amphibian development,
elongation of the AP axis is directly linked to mediolateral narrowing. We
therefore quantified overall embryonic convergent extension by measuring the
length-to-width ratio (LWR) of embryos, and this analysis confirmed a
correlation between LWR and neural tube closure
(Fig. 4H).
To further investigate this correlation, we made time-lapse movies of wild-type and Xdd1-injected embryos from early gastrulation until the end of neurulation and compiled the LWR data from several movies (Movie 5 at http://dev.biologists.org/supplemental; Fig. 5). In normal embryos, a very dramatic increase in LWR was observed at the onset of neurulation (Fig. 5B). Control embryos then underwent steady convergent extension, increasing their LWR consistently throughout the neurula stages (Fig. 5B).
In Xdd1-injected embryos which would eventually close their neural tubes, a
very similar pattern was observed except that by mid- to late-neurula stages,
a mild defect in the overall elongation of the axis became apparent in
closed-NT embryos (Fig. 5B).
This mild defect in elongation probably accounts for the delay in closure
observed in these embryos (Movies 1, 5 at
http://dev.biologists.org/supplemental).
A delay in tube closure is also observed in mice lacking one copy of the PCP
gene Strabismus (Copp et al.,
1994).
In Xdd1-injected open-NT embryos, a severe deficiency in LWR was apparent by the early neurula stages (Movie 5 at http://dev.biologists.org/supplemental; Fig. 5C). This defect became progressively more severe during neurulation. Xdd1-injected open-NT embryos failed to elongate steadily, and periods with no elongation were often observed at mid-neurulation and again at the end of neurulation. Similar defects in axis elongation were observed in movies of Xdsh-D2 expressing embryos (Movie 2 at http://dev.biologists.org/supplemental).
Finally, we observed an overall acceleration in the rate of elongation during neurula stages in control embryos (Fig. 5D). Closed-NT embryos also displayed this continuing increase in rate of elongation, while open-NT embryos did not (Fig. 5D).
Neural tube closure requires Xdsh function in the midline, but not in
the lateral/dorsal neural tube
The neural plate is patterned along its mediolateral axis, and as a result
of neurulation this mediolateral pattern becomes the dorsoventral pattern
within the neural tube; the medial neural plate becomes the ventral neural
tube, while the lateral neural plate becomes the dorsal neural tube
(Bronner-Fraser and Fraser,
1997). To define the regions of the neural plate in which Xdsh is
required for tube closure, we performed targeted injections of Xdsh mutants
(Fig. 6A,B). At the 16-cell
stage, injection into medial animal blastomeres targets expression to the
ventral/medial neural tissue and to the notochord, while injection into the
lateral animal blastomeres targets expression to the dorsolateral neural
tissue and to the dorsal epidermis (Hirose
and Jacobson, 1979
).
|
Because Xdsh-D2 is fused to GFP, the location of cells expressing the construct can be followed. To confirm the proper targeting of injections, embryos were examined at the early neural plate stage. In medially injected embryos, GFP-positive cells were present in a single stripe in the dorsal midline (Fig. 6C). In laterally injected embryos, two distinct domains of GFP-positive cells were observed at the lateral borders of the neural plate, where the neural folds are forming (Fig. 6D).
Open neural tubes were only observed in medially injected embryos (Fig. 6E) where GFP-positive cells could be seen at the floor of the open neural tube (Fig. 6e'). In laterally injected embryos, neural tubes were closed (Fig. 6F) and GFP-positive cells could be observed in the dorsal neural tube and epidermis (Fig. 6f'). Like Xdsh-D2, Xdd1 was also found to elicit neural tube closure defects when targeted medially, but not when targeted laterally (not shown). Consistent with our time-lapse data, this result indicates that Xdsh function is not required in the forming or fusing neural folds or in the medially moving dorsal epidermis. These data suggest that for neural tube closure, Xdsh function and convergent extension are required only in the midline.
Defective midline neural convergent extension is evident at gastrula
stages in Xdd1-injected embryos
To further characterize the convergent extension defects in the neural
midline of Xdd1-injected embryos, we examined the morphology of the
ventral/medial neural tissue with molecular markers. During gastrula stages,
Xfd12 is expressed in ventral/medial neural precursors
(Solter et al., 1999). During
early gastrulation, robust convergent extension reorganizes the neural plate
(Keller et al., 1992
), and
these movements are very closely paralleled by the expression pattern of Xfd12
(Fetka et al., 2000
). At early
gastrula, both control and Xdd1-injected embryos express similar levels of
Xfd12 in a broad, dorsal arc. No difference in the Xfd12 expression pattern
was observed between control and Xdd1-injected embryos at this stage
(Fig. 7A,B). By
mid-gastrulation, the Xfd12 expression pattern in control embryos had
undergone dramatic convergent extension
(Fig. 7C). However, in
Xdd1-expressing embryos Xfd12 still marked a short, wide arc reminiscent of
its earlier expression pattern (Fig.
7D), indicating a failure of convergent extension in the
midline.
|
We examined the medial/ventral limit of the neural plate at neurula stages by in situ hybridization for Xnetrin, which marked a thin line along the length of the neuraxis of control embryos at mid- and late neurula stages (Fig. 7E,G). In Xdd1-injected embryos, the Xnetrin staining pattern was extremely broad and significantly shortened (Fig. 7F,H). In many cases, regions in the center of the Xnetrin domain failed to stain (Fig. 7F, green arrow), suggesting some disruption of ventral neural cell fates in these regions. This disruption was confirmed by expression of the midline marker Sonic Hedgehog (SHH; Fig. 7I). In Xdd1-injected embryos, SHH expression was abnormally broad, and in some embryos SHH expression was lost in certain regions (Fig. 7J, green arrow).
Although failure of floorplate convergent extension always accompanied the open-NT phenotype, disorganization of floorplate marker expression was inconsistently observed in open-NT embryos (Fig. 7, green arrows). Likewise, the expanded floorplate did not necessarily occupy the entire open-NT region (Fig. 7, red arrows).
Xdd1 inhibits the elongation and narrowing of the neural plate at all
dorsoventral levels
For normal neural tube closure, Xdsh signaling is not required in
dorsolateral neural tissue (Fig.
6). However, convergent extension does occur in the lateral
regions of the neural plate (Keller et
al., 1992), and we examined the requirement for Xdsh in convergent
extension movements of these regions of the neural plate.
We examined morphology of the lateral/dorsal neural plate by in situ hybridization to Xpax3. At mid-neurula stages, Xpax3 marks two elongate domains in the lateral neural plate (Fig. 8A). In Xdd1-injected embryos, each of these domains was shorter and wider and the two were separated by a considerable distance (Fig. 8B), consistent with the widened floorplate (Fig. 7).
|
We then examined the effect of Xdd1 on the intermediate neural plate. In normal embryos at the late neurula stages, Xash3 is expressed in two elongate stripes at the borders between the presumptive alar and basal regions on either side of the neural plate (Fig. 8C). Each Xash3 expression domain was foreshortened in Xdd1-injected embryos, and like the Xpax3 domains these Xash3 domains were more widely spaced (Fig. 8D).
Finally, we examined the shape of the entire neural plate at the onset of neurulation (stage 14) as revealed by the pan-neural marker Sox-2 (Fig. 8E). Embryos injected with Xdd1 uniformly stain positive for Sox2, indicating that Xdd1 does not affect neural induction. In Xdd1-injected embryos, the Sox2 expression domain was shorter and much broader than in uninjected embryos (Fig. 8F). Together with the Xfd12 data (above, Fig. 6), this result indicates that defective convergent extension of the neural tissue prior to the onset of neurulation results in an abnormally broad neural plate and subsequently in abnormally wide spacing of the neural folds.
Overexpression of wild-type Xenopus Stbm phenocopies the effects of
mutant Xdsh expression
The similarity between the Xdd1 and the Xdsh-D2 phenotypes (compare Movie 1
with Movie 2 at
http://dev.biologists.org/supplemental)
indicate that the NT defects result from disruption of PCP signaling and not
the canonical Wnt pathway (see Wallingford
and Harland, 2001). To explore this idea further, we made use of
the fact that overexpression of wild-type components of the PCP pathway
specifically disrupt cell polarity but do not affect Wnt signaling
(Krasnow and Adler, 1994
).
Indeed, overexpression of wild-type Xenopus Stbm disrupts
mediolateral cell polarity, convergent extension and NT closure in
Xenopus (Darken et al.,
2002
; Goto and Keller,
2002
).
Like the Xdd1 phenotype, XStbm-induced NT defects (Fig. 9A,B) were accompanied by defective convergent extension of the midline during gastrula stages, as indicated by the abnormally broad expression domain of Xfd12 (Fig. 9C,D). In addition, XStbm disrupted convergent extension throughout the neural plate, as evidenced by expression patterns of XPax3, Xash-3 and Sox2 (Fig. 9E-J).
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DISCUSSION |
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In this report, time-lapse analysis, cross-sections, and targeted injections revealed that Xdsh mutants do not disrupt elevation, medial movement or fusion of neural folds, and do not disrupt the medial movement of the epidermis (Figs 2, 3, 4, 9; Movies 1, 3, 4 at http://dev.biologists.org/supplemental). Although we have not ruled out a role for Xdsh signaling in establishing apicobasal polarity in the neural epithelium, the results presented here do demonstrate that mutant Xdsh specifically disrupts neural convergent extension movements and does not affect other polarized processes of neurulation.
Convergent extension movements in the neural plate and tube have been
documented in a variety of animals, particularly amphibians and chicks
(Burnside and Jacobson, 1968;
Jacobson and Gordon, 1976
;
Keller et al., 1992
;
Lawson et al., 2001
;
Schoenwolf and Alvarez, 1989
;
van Straaten et al., 1996
).
However, without tools for uncoupling the various morphogenetic process, very
limited experimental analysis of its role in tube closure has been possible.
For example, failure of NT closure is correlated with reduced neuraxis
elongation in UV-irradiated chick embryos, but other aspects of neurulation
were not described (Jacobson,
1984
). The specificity of Xdsh mutants as inhibitors of convergent
extension movements in the neural plate allows us to test directly the
contribution of this morphogenetic movement to neural tube closure. While
convergent extension of both dorsal and ventral neural tissue was inhibited by
mutant Xdsh (Fig. 8A-E),
targeted injections reveal that only midline convergent extension is required
for tube closure (Fig. 6).
Convergent extension contributes directly to neural tube closure by
narrowing the distance between the nascent neural folds
What biomechanical roles does convergent extension play in neural tube
closure? It has been shown that proper signaling from underlying mesoderm is
required for the fusion of neural folds in Xenopus
(Poznanski et al., 1997), so
it is possible that the NT defect arises from disruption of involution due to
defective convergent extension in the mesoderm. However, this possibility is
unlikely, as Xdd1 open-NT embryos successfully complete gastrulation (not
shown). Moreover, disruption of convergent extension by targeted injection of
mutant Xdsh into the mesoderm has little effect on tube closure
(Wallingford and Harland,
2001
).
Another possibility is that elongation of the neuroepithelium may be
involved directly in generating force for neural fold apposition. It has been
proposed that anteroposterior elongation of the neural plate results in
transverse buckling of the neuroepithelium, promoting the elevation of the
folds (Jacobson, 1978).
Interestingly, detailed analysis of the rate of elongation in Xenopus
revealed that elongation occurs episodically, with the rate fluctuating even
as it accelerates overall (not shown). Similar episodic elongation is observed
during chick and newt neurulation
(Jacobson, 1978
;
van Straaten et al., 1996
).
Medially directed force generated by amphibian neural folds has also been
demonstrated to be episodic (Selman,
1958
), suggesting that it may arise in part from the episodic
convergence and extension.
It has also been suggested that convergent extension may compensate for the
anteroposterior shortening of the neuroepithelium, which results from apical
constriction (Jacobson and Gordon,
1976; Schoenwolf and Alvarez,
1989
). The constriction of the apical surfaces of neuroepithelial
cells is a crucial component of neural fold elevation, and this constriction
is radial; buckling the neural plate in all directions. Indeed, computer
modeling predicts that apical wedging in the absence of neural plate
elongation will produce a cup or bowl, rather than a tube
(Jacobson and Gordon, 1976
).
Our movies bear out this prediction (see, in particular, Movie 4 at
http://dev.biologists.org/supplemental).
Such a cup-shape appears to mechanically disallow the neural folds reaching
one another and fusing.
Finally, our morphometric and molecular marker data both indicate that the crucial difference between open-NT embryos and control embryos was the width of the early neural plate (Figs 7, 8) and the distance between the forming neural folds (Fig. 4). In addition, our targeted injections demonstrate a requirement for convergent extension in the midline. We conclude that the mechanism which advances the elevated neural folds produces only a finite amount of medial movement. In order for tube closure to be successful, midline convergent extension is required to decrease the width between the folds, reducing the distance folds must travel in order to meet and fuse (Fig. 10).
|
Convergent extension and mammalian caudal neural tube closure
The similarity between the phenotypes of Xenopus and mouse embryos
in which PCP signals are disrupted is striking and suggests a conservation not
only of molecular controls but also of morphogenetic mechanisms. As is the
case in Xenopus, disruption of Dishevelled or Stbm signaling in mice
results in severe defects in both elongation of the AP axis and neural tube
closure (Hamblet et al., 2002;
Kibar et al., 2001
;
Murdoch et al., 2001
). Several
other results also suggest that convergent extension movements may be crucial
to mammalian spinal neurulation. For example, a spontaneous mutation that
results in severe spinal NTD in the rat also elicits a severe foreshortening
of the AP axis (Layton and Smith,
1977
). Likewise, mice lacking retinaldehyde dehydrogenase 2
display specific defects in spinal NT closure and fail to elongate the
posterior region of the embryo
(Niederreither et al., 1999
).
Indeed, a connection between defective axial elongation and defective neural
tube closure has long been suggested for the looptail mouse, which
lacks PCP signaling (Kibar et al.,
2001
; Smith and Stein,
1962
; Wilson and Wyatt,
1992
). It will now be very interesting to assess directly the
convergent extension of the neural plate at early stages in the
looptail and Dishevelled mutant mice.
Convergent extension, PCP signaling, and human neural tube
defects
Neural tube defects are among the most common and most debilitating human
birth defects, affecting close to 1 in 1000 pregnancies
(Botto et al., 1999;
Manning et al., 2000
). While
folate supplements have dramatically reduced the frequency of neural tube
defects, studies indicate an important genetic component to their etiology
(George and Speer, 2000
;
Harris, 2001
). It is therefore
crucial that we understand all of the genetic and embryological mechanisms
that govern normal and abnormal neural tube closure in vertebrates.
Craniorachischisis (CRS), in which the entire caudal NT fails to close,
accounts for 10% of human NTDs
(Kirillova et al., 2000
;
Nakatsu et al., 2000
).
Strikingly, human embryos with CRS consistently display shortened
anteroposterior axes and widened mediolateral axes; in particular, the neural
arches of the vertebrae in these embryos are displaced laterally
(Kirillova et al., 2000
;
Marin-Padilla, 1966
). In
addition to defective convergent extension and failed neural tube closure,
human CRS is associated with bifurcated notochords, disorganized expression of
SHH in the floorplate and bulging of the neural plate midline
(Kirillova et al., 2000
;
Marin-Padilla, 1966
;
Saraga-Babic et al., 1993
).
All of these abnormalities are observed in frog or mouse embryos in which PCP
function is disrupted (this study) (Greene
et al., 1998
; Wallingford and
Harland, 2001
). While the etiology of CRS remains poorly
understood, these data suggest that PCP signaling and convergent extension may
provide an effective starting point for directed studies into the molecular
and biomechanical underpinnings of this neural tube defect.
PCP signaling and floorplate specification
Previous studies have suggested that in embryos with disrupted PCP
signaling, the open-NT phenotype results from an abnormally wide floorplate
caused by a failure to restrict floorplate cell fates
(Murdoch et al., 2001). Our
data are not consistent with that hypothesis, but rather indicate that the
widening of the floorplate, like the widening of all levels of the neural
plate and tube, results from a persistent defect in convergent extension
beginning during gastrulation and continuing throughout neurulation (Figs
5,6,7,8,9,10).
Nonetheless, the disorganization of SHH and Xnetrin expression and the
indistinct tissue boundaries at the midline of some Xdd1-injected
Xenopus embryos (Figs
3,
7) are consistent with the
disruptions of midline structures and cell fates observed in looptail
mice (Greene et al., 1998;
Murdoch et al., 2001
). It is
possible that the defects in floorplate patterning reflect a secondary effect
on cell fate resulting from improper morphogenetic movements. For example,
convergent extension movements enable short-range signaling events that are
crucial to the differentiation of posterior notochord and somite
(Domingo and Keller, 1995
). A
similar mechanism may be required in the floorplate, as the posterior, medial
neural tissue of open-NT embryos often lack Xnetrin expression
(Fig. 7F, red arrow). However,
it has also been suggested that PCP signals influence floorplate cell fates
directly (Murdoch et al.,
2001
). Interestingly, PCP signals interact with the Notch pathway
to modulate cell fate in Drosophila
(Cooper and Bray, 1999
;
Fanto and Mlodzik, 1999
), and
the Notch pathway has been implicated in controlling cell fates in the
vertebrate midline (Appel et al.,
1999
). Together, the data from mice and from frogs indicate that
PCP signaling may play a dual role, integrating cell fate and morphogenetic
cell movement in the vertebrate midline.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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