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First published online 1 November 2006
doi: 10.1242/dev.02678
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Research Report |
1 Department of Physiology, Development and Neuroscience, University of
Cambridge, Downing Street, Cambridge CB2 3DY, UK.
2 Division of Engineering and Applied Sciences, Harvard University, Pierce Hall,
29 Oxford Street, Cambridge, MA 02138, USA.
* Author for correspondence (e-mail: rja46{at}cam.ac.uk)
Accepted 4 October 2006
SUMMARY
Mechanisms for shaping and folding sheets of cells during development are poorly understood. An example is the complex reorganisation of the forebrain neural plate during neurulation, which must fold a sheet into a tube while evaginating two eyes from a single contiguous domain within the neural plate. We, for the first time, track these cell rearrangements to show that forebrain morphogenesis differs significantly from prior hypotheses. We postulate a new model for forebrain neurulation and demonstrate how mutations affecting two signalling pathways can generate cyclopic phenotypes by disrupting normal cell movements or introducing new erroneous behaviours.
Key words: Zebrafish, Time-lapse microscopy, Neurulation, Cyclopia, Quantitative analyses
INTRODUCTION
Forebrain neurulation and common neural-tube defects associated with it,
such as cyclopia (Gripp et al.,
2000
), are best studied dynamically so that normal movements and
deviations from them can be directly measured. We have developed new methods
to address this class of problem and in particular the early morphogenesis of
the eye, using the transparent zebrafish as a model. Fate mapping studies in
teleost fish, mouse and chick have shown that a significant anterior-posterior
rearrangement of tissue takes place to relocate the hypothalamic anlagen
(ventral diencephalon) from a position posterior to the eye field to one
anterior and ventral to it (Woo and
Fraser, 1995; Varga et al.,
1999; Hirose et al.,
2004; Inoue et al.,
2000; Lawson,
1999; Cobos et al.,
2001; Dale et al.,
1999). This diencephalic rearrangement has been interpreted as
splitting the early continuous eye field, with models favouring either a
physical separation by a posterior to anterior movement of the future
hypothalamus through the plane of the neural plate
(Varga et al., 1999;
Hirose et al., 2004), or a
local induction of medial diencephalic tissue growth to bisect the eye field
(Li et al., 1997). Reduced
Nodal (Hatta et al., 1991;
Hatta et al., 1994;
Schier et al., 1996;
Rebagliati et al., 1998;
Sampath et al., 1998), Wnt11
(Heisenberg et al., 1996;
Heisenberg and Nüsslein-Volhard,
1997; Heisenberg et al.,
2000) or Sonic Hedgehog (Chiang
et al., 1996) signalling results in cyclopia, a reduced
interocular distance or eye fusion, it is presumed by altering forebrain
patterning or morphogenesis. We have directly addressed this problem by
producing time-lapse confocal movies of wild-type, cyclops
(cyc) morphant and homozygous silberblicktx226
(slb) mutant zebrafish embryos. We labelled all cell nuclei with
green fluorescent protein (GFP) to visualise and track their movements. In
individual embryos, we tracked the paths of hundreds of cells contributing to
the forebrain regions of eye, optic stalk, telencephalon, hypothalamus
(ventral diencephalon) and dorsal diencephalon to generate high-resolution
dynamic fate maps. Movements were followed from mid-gastrula (8 hours post
fertilisation, hpf) in the anterior neural plate until the 18-somite stage (15
hpf) when forebrain domains are resolved by morphogenesis and cell fates could
be assigned. This enables high-resolution visualisation and quantitative
analysis of forebrain morphogenesis for the first time.
MATERIALS AND METHODS
Fish lines and genetics
Wild-type (AB) embryos were obtained from zebrafish (Danio rerio)
lines raised at 28.5°C, maintained as described by Westerfield
(Westerfield, 2000). Mutant
embryos from homozygous slb carriers were a kind gift of Masazumi
Tada (University College London, London, UK). Embryos were staged according to
standard indicators (Kimmel et al.,
1995).
RNA and morpholino injections
pCS2-nucGFP2 (a kind gift of Betsy Pownall, University of York, York, UK)
was linearised by NotI, and capped mRNA transcripts were made using
the MEGAscript SP6 in vitro transcription kit (Ambion). Embryos were injected
at the one-cell stage with 80-100 pg of pCS2-nucGPF2 mRNA. cyc
morphants were co-injected with 3 ng of cyc morpholino, as previously
described (Karlen and Rebagliati,
2001).
Time-lapse imaging
Suitable embryos were dechorionated and mounted in 0.30% low
gelling-temperature agarose (type VII, Sigma Aldrich) containing 0.017%
tricaine (Sigma Aldrich) anaesthetic solution
(Westerfield, 2000) in
custom-built chambers (Concha and Adams,
1998) maintained at 28.5°C. Minimal volumes of embryo medium,
containing 0.003% tricaine solution, were used to overlay embedded
embryos.
Time-lapse imaging was performed using inverted laser scanning confocal microscopes: Zeiss LSM 510 and Leica Microsystems TSC-SP1-MP using long working-distance 20x/0.5 NA water immersion objectives. Image stacks of 50-60 sections of 2.8-3.2 µm spacing were recorded at 2-minute intervals for periods of 20 hours or more. Embryos were morphologically evaluated upon completion of imaging; any displaying abnormal development were excluded.
Cell tracking and data analysis
Manual and automated (G.B.B. and R.J.A., unpublished) cell tracking was
performed using custom software written in Interactive Data Language (IDL, ITT
Visual Information Solutions). Embryo shape was determined by detecting the
embryos surface from its fluorescent signal. A blanket was fitted to this
surface and the normals from this used to estimate the centroid of the embryo.
Cell locations were expressed relative to this surface, giving a measure of
radial depth within the embryo. Measures of the distances between forebrain
regions were taken along the curved surface of the embryo, within a plane
parallel to the anterior-posterior axis, passing through the centroid. These
distances were also converted to relative angles by taking the angular
displacement subtended by lines connecting the two locations to the centroid
of the embryo. Angular measures were considered less prone to error when
comparing measures in living and fixed embryos. Tissue deformation, expressed
as contraction and expansion, was measured by calculating local velocity
gradients (G.B.B., L.M. and R.J.A., unpublished). For each cell, gradients
were calculated with respect to a fixed Cartesian system by looking at the
movements of cells and their immediate neighbours. This velocity gradient was
used to compute the principal direction and associated principal deformation
rates by solving an eigenvalue problem. Cell velocities and positions were
corrected for the effects of curvature of the embryo surface in these
calculations.
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Tracking cells in wild-type animals confirms that hypothalamic precursors
originate from a region medial and posterior to the eye field in the anterior
neural plate (Fig. 1A)
(Varga et al., 1999). The eye
field begins as a broad bi-lobed domain bordered anteriorly by precursors of
the telencephalon (Fig. 1A,
Fig. 2A) and posteriorly by
optic stalk. More peripherally there are dorsal diencephalon precursors
(Fig. 1A,C) (S.J.E., G.B.B. and
R.J.A., unpublished). Optic stalk precursors also originate more anteriorly
among eye and telencephalic precursors
(Fig. 1A).
Studies in the anterior trunk showed that neurulation in the zebrafish
proceeds by folding of the neural plate about the midline to form a transient
wedge of cells - the keel - in a mechanism analogous to primary neurulation in
higher vertebrates (Papan and
Campos-Ortega, 1994; Lowery
and Sive, 2004). We show that neurulation within the forebrain is
more complex, precluding neurulation through a simple keel or tube. Before
neurulation, the most anterior neural plate begins posterior contraction
towards a focus at the anterior tip of the hypothalamic anlagen
(Fig. 1A,
Fig. 3A,D,J,M). The onset of
neurulation is detected and defined by a ventral movement of the hypothalamic
anlagen, at the focus of contraction, to initiate formation of the anterior
limit of the neural keel (Fig.
1B,F, Fig. 3A,D,G,
and see Table S1 and Fig. S1 in the supplementary material). This keel
subsequently subducts and extends beneath the eye field, without splitting it
(Fig. 1C,G), and moves forward
to emerge finally anterior to the telencephalic field
(Fig. 1D,H,
Fig. 3J,M). This
posterior-to-anterior rearrangement maps to the dorso-ventral axis of the
anterior neural tube (Fig.
3J,M). This movement in the epithelium results in a shear relative
to the adjacent lateral tissue. Consequently, posterior eye cells are drawn
deep and anteriorwards to begin eye folding
(Fig. 1B-D,F-H,
Fig. 3G). Coincidently,
telencephalon and the anterior-lateral eye field fold towards the midline,
which positions them above medial and posterior eye cells
(Fig. 1B,F). This creates a
neuropore within the dorsal seam of the neural tube near the site of
subduction (Fig. 1C,D,G,H),
which closes when the lateral edges meet at the midline
(Fig. 1I-K,M-O). This folding
initiates eye-vesicle evagination by forming an out-pocketing of tissue
(Fig. 1I,M). Neurulation of the
dorsal forebrain far more closely resembles movements observed in higher
vertebrates that involve folding within the neural plate rather than the use
of a neural keel.
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Evagination of the eyes proceeds by a combination of two principal movements. First, the dorsal diencephalon sweeps inwards and forwards, displacing eye and future optic-stalk tissue forwards and then out into the emerging optic vesicles (Fig. 1I-P, Fig. 3P). Second, anterior eye tissue at the midline displaces laterally from above into the eye vesicles as the telencephalon seals the neuropore to occupy the dorsal neural tube (Fig. 1J-K,N-O). This latter movement is predominantly coincident with the segregation of intermingled left and right eye cells into their respective eyes (see Fig. S2I-P in the supplementary material). This description of forebrain morphogenesis is summarised in Fig. 2.
Given this new model of neural-plate folding in the forebrain, we
re-address how neurulation is disrupted in cyclopic mutants. In the
cyc mutant, a loss of Ndr2 signalling causes abnormal patterning and
morphogenesis of mesoderm and forebrain
(Rebagliati et al., 1998;
Sampath et al., 1998). After
neurulation, a portion of the eye field remains medial as a band of eye tissue
spanning the front of the brain between two partial eye cups
(Macdonald et al., 1995;
Fulwiler et al., 1997). The
size and location of the presumptive eye field is normal in cyclops
mutant and morphant embryos; however, hypothalamic tissue is not induced
(Fig. 4D, see Fig. S1 in the
supplementary material) (Varga et al.,
1999). Tracking cells in cyc morphants, we show that the
focus of neural-plate contraction (Fig.
3E), and the initiation of neural keel formation, locate within
the eye field itself (Fig.
3H,N, Fig. 4E,J,
see Table S1 and Fig. S1 in the supplementary material); this is far more
anterior to the usual initiation in wild type, which corresponds to the
anterior tip of the hypothalamus (Fig.
1B,F). This abnormal focus of contraction causes medial eye-field
cells to be brought erroneously anterior and ventral within the neural tube
(Fig. 3H,Q,
Fig. 4F,L). Morphants
additionally show reduced movements of the keel beneath the remaining eye and
telencephalic fields: the keel never emerges anterior to them
(Fig. 3K,N,
Fig. 4F).
A consequence of attenuated subduction movements is that posterior eye
tissue does not move forward within the neural plate
(Fig. 3K,
Fig. 4E,F,P-R). Convergence of
telencephalic and anterior eye fields towards the midline is similar to the
wild-type (Fig. 4E,F,J,L,P,T),
but is not sufficient to resolve the eye-field cells within the keel into
separate vesicles (Fig.
4Q,R,V,X). This shows that cyclopia in cyclops results in
incorporation of eye tissue into an inappropriate location within the medial
neural keel. We speculate that this precludes its resolution into the eye
vesicles by any of the morphogenetic mechanisms observed so far in wild-type
animals. This represents a dissociation of the mechanisms of patterning and
morphogenesis. These defects override potential mechanisms intrinsic to the
eye field that usually result in eye separation, such as the motile capacity
of eye-field cells (Rembold et al.,
2006).
A second mutant, slb, which encodes Wnt11, shows incomplete
eye-field separation (Heisenberg et al.,
2000). This gene has been associated with reduced movements of
convergence and extension, as well as aberrant migration of the mesoderm
(Heisenberg and Nüsslein-Volhard,
1997; Hammerschmidt et al.,
1996; Solnica-Krezel et al.,
1996; Ulrich et al.,
2003). Effects of Wnt11 on eye morphogenesis could be caused
indirectly by disrupting mesodermal induction of neural patterning
(Heisenberg and Nüsslein-Volhard,
1997; Marlow et al.,
1998), or directly from Wnt11 expression in lateral neurectoderm,
posterior to the presumptive forebrain
(Heisenberg et al., 2000). We
investigated eye-field morphogenesis in homozygous slb mutants.
Forebrain territories, although patterned normally, are broadened and
shortened (Fig. 3F,
Fig. 4A, see Fig. S1 in the
supplementary material) (Heisenberg et
al., 1996), possibly because of defective convergence and
extension. The focus of early contraction is less clearly resolved
(Fig. 3F). However, the
initiation of keel formation is at the anterior boundary of the hypothalamus,
as observed in wild type (Fig.
3I,L,O, Fig. 4B,I,
see Table S1 and Fig. S1 in the supplementary material). Anteriorward movement
of the neural keel, however, is much reduced
(Fig. 3L,O,
Fig. 4C,K), as in
cyclops (Fig. 3K,N),
and the hypothalamus never emerges anterior to the telencephalon
(Fig. 3L,O,
Fig. 4C). Instead, the
hypothalamus, optic stalk and posterior eye field are displaced progressively
deeper within the keel (Fig.
3I, Fig. 4I,K).
Along with a severely reduced convergence of the lateral neural plate, this
slows folding of the lateral eye towards the midline
(Fig. 4C). Compared with the
extensive movement of lateral-posterior eye-field cells fated to optic stalk
in wild type (Fig. 1D,H,I-P,
Fig. 3P), much reduced
convergent and forward movement of the slb eye field results in
medial-posterior eye-field cells remaining medial
(Fig. 3R,
Fig. 4M-O,S,U,W). We postulate
that this defect in movement is a major cause of the reduced resolution of
bilateral eye fates in this class of mutant, constituting a second, temporally
and spatially distinct, defect of forebrain morphogenesis. Analyses of these
two mutants reveal two of the intrinsic steps that comprise the complex
morphogenetic program of forebrain development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4613/DC1
ACKNOWLEDGMENTS
We thank Bill Harris and members of the Adams group for discussion and critical reading of this manuscript; and many colleagues for providing reagents. We especially thank Masazumi Tada and Nina Buchan for providing silberblick mutant embryos. A Research Studentship from the BBSRC supported S.J.E. A MRC Senior Research Fellowship and Research Grant to R.J.A. supported this work.
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