Department of Genetics, University of Technology, Dresden, c/o Max Planck Institute for Molecular Cell Biology and Genetics, Germany
* Author for correspondence (e-mail: brand{at}mpi-cbg.de)
Accepted 13 May 2005
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SUMMARY |
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Key words: Midbrain-hindbrain boundary, Isthmic organizer, Lineage restriction, Neuromeres, segments, Zebrafish, Mesencephalon, Metencephalon, otx2, gbx1, fgf8
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Introduction |
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Work in recent years has revealed that hindbrain neuromeres are
evolutionarily conserved units of gene expression, differentiation and cell
behavior (Keynes et al., 1990;
Puelles, 2001
;
Cooke and Moens, 2002
;
Moens and Prince, 2002
;
Pasini and Wilkinson, 2002
).
In terms of their behavior, cells are free to mix within a given neuromere,
but not across the boundary into the neighboring compartment. This important
phenomenon, termed lineage restriction, was discovered in Drosophila
wing development (Garcia-Bellido et al.,
1973
; Crick and Lawrence,
1975
). In the vertebrate brain, lineage restriction acts during
the formation of hindbrain compartments, the rhombomeres
(Fraser et al., 1990
). Here,
cells acquire distinct adhesive properties that prevent them from mixing
between rhombomeres (Mellitzer et al.,
1999
; Xu et al.,
1999
).
In the Drosophila wing, the anteroposterior compartment boundary
coincides with the position of an organizer, a localized group of cells that
controls neighboring cell fate by secreting diffusible signaling proteins. To
ensure proper tissue formation and differentiation, the position of such
potent organizing cells has to be tightly controlled
(Dahmann and Basler, 1999).
Organizers also serve important functions during vertebrate brain development
(Irvine and Rauskolb, 2001
).
The organizer situated at the junction of the midbrain and anterior hindbrain
termed midbrain-hindbrain boundary (mhb) organizer or isthmic
organizer serves as a paradigm for organizer activity in the forming
nervous system (Rhinn and Brand,
2001
; Wurst and Bally-Cuif,
2001
; Raible and Brand,
2004
). The mhb organizer forms at the interface between the
expression domains of two transcription factors in the neural plate an
anterior otx and a posterior gbx domain
(Rhinn et al., 2005
).
otx and gbx expression domains initially overlap, but
subsequently sort out and form a sharp interface
(Hidalgo-Sanchez et al., 1999
;
Garda et al., 2001
;
Rhinn et al., 2003
). Cells on
the posterior side of this interface start to express the signaling protein
Fgf8 as the key molecule exerting organizer function
(Crossley et al., 1996
;
Reifers et al., 1998
). It has
been proposed that cells that might cross this boundary readjust their gene
expression profile via mutual repression
(Jungbluth et al., 2001
;
Wurst and Bally-Cuif,
2001
).
Although lineage restriction in the mhb region has been addressed in
previous studies, we still do not know how tightly cell movement is controlled
in this brain area. No lineage restriction between the chick mesencephalon and
metencephalon was detected using a clonal analysis approach
(Jungbluth et al., 2001),
while other studies using broader labeling techniques or tissue grafting argue
in favor of a cell movement restriction across the mhb
(Millet et al., 1996
;
Alexandre and Wassef, 2003
;
Louvi et al., 2003
). A recent
study strongly suggests lineage restriction between the mouse midbrain and
rhombomere one (Zervas et al.,
2004
). To determine whether the midbrain-hindbrain boundary is a
compartment boundary in the developing vertebrate brain, we analyzed
morphological changes, gene expression patterns and cell behavior during the
formation of the mhb region in zebrafish with single-cell resolution. To this
end, we imaged the developing mhb region in a GFP transgenic line that marks
all nuclei. Using a novel combination of antibody staining and continuous
single cell tracking, we present strong evidence for the existence of a
lineage restriction boundary between the mesencephalon and metencephalon in
the zebrafish. Single cell injection and clonal analysis indicate that this
boundary is established as early as late gastrulation. We argue that lineage
restriction constrains the organizing cell population at the mhb to ensure
proper patterning and differentiation of the mhb region.
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Materials and methods |
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Staining of living embryos
Embryos were stained with the vital dye BODIPY-ceramide (Molecular
Probes/Invitrogen) (Cooper et al.,
1999), mounted for imaging
(Concha and Adams, 1998
;
Langenberg et al., 2003
) and
optically sectioned on a Leica confocal microscope.
Analysis of gene expression
Standard methods for whole-mount RNA in situ hybridization were used, with
laboratory modifications as described elsewhere
(Reifers et al., 1998). DIG
probes were developed with Fast Red substrate (Sigma) to yield a fluorescent
signal. Embryos were moved to 70% glycerol, mounted and imaged on a Zeiss
confocal system with 488/543 nm excitation. The 488 nm excitation gave a
sufficient signal to visualize the tissue background. Probes for the following
genes were used: otx2 (Mori et
al., 1994
), gbx2
(Rhinn et al., 2003
),
wnt1 (Kelly et al., 1993
)
and fgf8 (Reifers et al.,
1998
).
Immunohistochemistry
The Orthodenticle/Otx antibody was a kind gift from Antonio Simeone.
Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS)
overnight at 4°C, washed and incubated in methanol for at least 30 minutes
at 20°C. Embryos were then digested with 0.0025% Trypsin in PBT
(PBS + 0.8% Triton) for 5 minutes on ice, postfixed for 30 minutes with 4%
PFA, washed and blocked for 2 hours in PBT with 10% heat-inactivated normal
goat serum (NGS) and 1% DMSO. Antibody incubations were as follows: overnight
in anti-Otx antibody (1:3000) in PBT + 1% NGS; secondary antibody (Jackson
ImmunoResearch TRITC coupled anti-goat 1:200) for 2 hours at room temperature.
Embryos were postfixed, moved to 70% glycerol, mounted and imaged on a Zeiss
confocal system with 488/543 nm excitation.
RNA injection
RNA injections were carried out as described by Reim
(Reim and Brand, 2002). Fifty
picograms GFP RNA per embryo was sufficient to strongly label donor cells.
Transplantation
Cell transplantations were essentially carried out as described
(Westerfield, 1994). Host
embryos carrying transplanted cells were imaged on an Olympus BX61 microscope
with a Spot RT Slider camera and Metamorph acquisition software.
Iontophoretic single cell injection
Iontophoretic cell labeling was performed as described by Fraser
(Fraser, 1996). Embryos were
photographed on the above described setup.
Nuclei tracking and plotting
Histone H2A.F/Z:GFP transgenic fish were mounted in 1.5% low melting point
agarose in an imaging chamber (Concha and
Adams, 1998) and imaged for up to 12 hours with 1.5 µm
z- and 3- to 4-minute time resolution on a Nikon/BioRad two-photon
confocal system. Image stacks were imported into NIH image, converted to
single tiff files, renamed with FileBuddy (SkyTag Software) and imported as 4D
stacks into the NIH Image4D version (modified NIH Image by Richard Adams).
Nuclei were manually tracked and their positions taken down in Excel files.
Calibration and plotting was performed with self-written routines in MatLab
(The Mathworks) or with Excel.
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Results |
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To analyze the behavior of individual cells during the folding process, we
transplanted cells expressing cytosolic GFP from injected donor embryos into
unlabeled wild-type hosts at the onset of gastrulation (6 hpf) and imaged the
developing mhb region by confocal time-lapse microscopy between the
five-somite stage (11.5 hpf) and 30 hpf
(Fig. 1D-F; see Movie 1 in the
supplementary material). Cells stretch from the apical (ventricular) to the
basolateral surface of the neuroepithelium, forming a pseudo-stratified
epithelium (Schmitz et al.,
1993) (Fig. 1E,
double arrow and see Movie 1 in the supplementary material), while nuclei
constantly cycle between the two sides. Upon division, cells round up at the
ventricular side (Kimmel et al.,
1994
; Papan and Campos-Ortega,
1994
) (Fig. 2G-I and Movies 1, 2 in the supplementary material). We traced groups of cells
divided by the morphological boundary back to separate cells or cell groups at
the beginning of the time-lapse (Fig.
1D-F, pseudo-colored cells and see Movie 1 in the supplementary
material). Between these groups, a gap of unlabeled cells developed over time
(Fig. 1, arrowheads in E,
asterisks in E,F). Cell movement between the groups appeared very restricted.
These data show that the developing midbrain and anterior hindbrain in the
zebrafish go through distinct morphological movements that lead to a folded
anteroposterior axis. The position of the late fold can be traced back to a
small indentation in the prospective mhb region. We took the behavior of
groups of cells on either side of the boundary as the first indication of
restricted cell mixing between the midbrain and anterior hindbrain.
|
In total, we evaluated three independent movies, covering development of the mhb region between the 5- and 26-somite stages (Table 1 and Movie 2 in the supplementary material). After imaging, embryos were immediately fixed, stained for Otx protein, and optically sectioned on a confocal microscope (Fig. 2A,C). By comparing the last image stack of the time-lapse (Fig. 2B) with the antibody staining, we were able to assign a molecular status (Otx-positive or Otx-negative) to the nuclei at the end of the time-lapse (Fig. 2B,C). Nuclei close to the boundary of Otx expression were then backtracked through the time-lapse (Fig. 2D,E) and their position (xy center and z-level) was noted in intervals of about 1 hour. Using this approach, we assigned a molecular status to and followed nearly all cells at the boundary throughout the whole time-lapse (Table 1). We observed virtually no cell death as a result of photodamage, even under almost continuous scanning. Owing to the high temporal resolution, we were able to track cells through divisions, when nuclei temporarily left their respective groups to divide at the midline (Fig. 2G-I). Because of the working distance of the objective and signal quality issues, the analysis was restricted to a focal depth of 80 µm, corresponding to the dorsal three quarters of the neural tube at the mhb.
|
By contrast, nuclei of arbitrarily defined boundaries in the midbrain or cerebellum did not sort out into coherent groups (Fig. 3E,F). Arbitrary boundaries were picked within the midbrain or anterior hindbrain at locations that do not to our knowledge correspond to any gene expression or morphological boundaries. At these arbitrary boundaries, we consistently found violation of the artificial boundary by a large proportion of the tracked nuclei (Fig. 3E,F). We also examined artificial boundaries shifted just one cell row towards posterior or anterior from the detected boundary, and found no lineage restriction at this sharply defined interface (Fig. 3C,D shows an anterior shift as an example). This demonstrates that the movement of cells forming the mhb is specific to this boundary, and that the observed lineage restriction is not due to a general behavior of cells in this region of the neural keel and tube.
|
In summary, these data demonstrate clearly that Otx-positive and Otx-negative cells at the mhb of the 1-day old zebrafish embryo derive from cells that were spatially separated for the last 12 hours of development, arguing that lineage restriction is established and maintained from at least the five-somite stage onwards.
Single cell lineage analysis by iontophoretic injection
Having found that two lineage restricted cell populations are established
from early somitogenesis stages onwards, we wanted to determine the onset of
lineage restriction between the midbrain and hindbrain. Because large-scale
morphogenetic movements make imaging and continuous tracking throughout
gastrulation stages less precise, we decided to test the early mhb for lineage
restriction by labeling individual cells and analyzing their distribution at
later developmental stages.
In the zebrafish, the expression domains of the transcription factors
otx2 and gbx1 (the functional homolog of gbx2 in
the mouse) become mutually exclusive towards the end of gastrulation, at the
80% epiboly stage (Rhinn et al.,
2003). We therefore expected this period to be important for cell
behavior at the otx2/gbx1 interface, and labeled single
cells by iontophoretic injection of a fluorescent dye at successive
gastrulation stages (Fig. 4).
In addition, we transplanted single cells from GFP-injected donors to
wild-type unlabeled hosts at the shield stage (onset of gastrulation). Ability
of the clonal descendants to cross the mhb was determined at 24 hpf
(Fig. 4) and 36 hpf (data not
shown).
Upon labeling or transplanting at the beginning of gastrulation (shield
stage to 60% epiboly), about one quarter of clones had descendant cells on
both sides of the boundary at 24 hpf and 36 hpf, in agreement with earlier
fate-mapping studies (Woo and Fraser,
1995). The proportion of two-sided clones decreased significantly
when cells were injected during later gastrulation stages (80-90% epiboly and
tailbud to one-somite stage, Fig.
4B-E), with no clear two-sided clones after labeling at the
tailbud stage (summary Fig.
4F). These findings argue for the establishment of the lineage
restriction boundary between the prospective midbrain and hindbrain during
late gastrulation stages, when the expression domains of otx2 and
gbx1 become mutually exclusive.
Gene expression domains at the midbrain-hindbrain boundary
During hindbrain segmentation, a number of genes are expressed in a
segmental manner, i.e. they respect the lineage restriction boundaries between
rhombomeres as expression borders. Our data show that Otx2 protein expression
as a marker for the extent of the midbrain and the lineage restriction
boundary coincide. To characterize the expression domains of further known
regulatory genes with respect to the identified lineage restriction boundary
at the mhb, we analyzed gene expression by fluorescent in situ hybridization
and subsequent confocal microscopic imaging.
|
These data show that the lineage restriction boundary coincides with the
expression borders of several important regulatory genes in mhb development.
Of particular importance, the cell population expressing fgf8 as the
key organizer gene (Crossley et al.,
1996; Lee et al.,
1997
; Reifers et al.,
1998
; Martinez et al.,
1999
), appears to respect the lineage boundary at its anterior
end.
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Discussion |
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The detailed analysis of the movement of hundreds of individual nuclei (summarized in Table 1) shows that two lineage restricted cell populations are established at least from the five-somite stage onwards. By comparing the final nuclei position with an anti-Otx staining (Fig. 2), we were able to assign a status to the tracked nuclei, demonstrating that the lineage restriction boundary lies between an Otx-positive and an Otx-negative cell population (Figs 2, 3) and thus between mesencephalic and metencephalic cells. Because the molecular status of the tracked cells was determined only at the end of the time-lapse analysis, it remains a formal possibility that the cells could switch their molecular status during the time period analyzed. However, we consider this possibility unlikely, because Otx-expressing and Otx non-expressing cells form, also at earlier stages, a sharp interface (Fig. 2).
The expression domains of important regulatory genes in the mhb genetic
cascade, among them otx2, have been studied in several vertebrate
model organisms (Rhinn and Brand,
2001; Wurst and Bally-Cuif,
2001
; Raible and Brand,
2004
). After a phase of small overlap between the mesencephalic
and metencephalic expression domains, mesencephalic genes become restricted to
the otx-positive domain, while metencephalic genes become confined to
the gbx-positive domain. In agreement with these data, we have shown
that several mhb genes share a common expression border that coincides with
the lineage restriction boundary and follows the changes in morphology during
mhb development (Figs 1,
5). Interestingly, as in the
chick (Millet et al., 1996
),
the otx expression border, and thus the lineage restriction boundary,
does not correlate with the morphological indentation in the mhb area, but is
situated slightly anterior to it (Fig.
2J-L and see Movie 2 in the supplementary material).
|
|
To determine the onset of the lineage restriction mechanism, we injected
single cells at three developmental stages: at the beginning, middle and end
of the gastrulation period (Fig.
4). This comprises the time window during which the expression
domains of otx2 and gbx1, whose interface correlates with
the position of the mhb, become mutually exclusive. Our results obtained from
this approach suggest that lineage restriction is established already by the
end of gastrulation, at around 80% epiboly
(Fig. 4). Setting up lineage
restriction at this early time point may be important to prevent mixing
between two cell populations that start to express secreted patterning
molecules shortly thereafter: mesencephalic, otx2 and
wnt1-expressing cells; and metencephalic, gbx and
fgf8-expressing cells
(Hidalgo-Sanchez et al., 1999;
Rhinn et al., 2003
). The
fgf8-positive cells are thought to constitute the organizer in the
mhb region (Crossley et al.,
1996
; Lee et al.,
1997
; Reifers et al.,
1998
; Martinez et al.,
1999
). We argue that it is of high importance for the developing
embryo to limit fgf8 expression to a defined compartment in the brain
to ensure proper patterning and differentiation of the mhb region.
Our detailed analysis of cell movement further shows that nuclei can move a significant distance away from the Otx expression boundary (Fig. 1, Fig. 3G,H, see Movie 1 in the supplementary material). This principal capability of cells to move within the neuroepithelium further underscores the importance of restricting movement across the mesencephalon-metencephalon (mes-met) interface: cells expressing the secreted organizer molecule Fgf8 would probably move far into the midbrain in the absence of a cell-sorting mechanism and cause disorganization of mhb development. The putative mechanism that establishes and maintains the lineage restriction boundary between the mesencephalon and metencephalon will need to both restrict mixing across the mes-met interface and to allow mixing within the groups of cells on either side. We consider a mechanism based on differential adhesion between mesencephalic and metencephalic cells as the most likely possibility.
Lineage restriction at the midbrain-hindbrain boundary has been addressed
in other vertebrate model systems. A recent study in mouse
(Zervas et al., 2004)
postulates lineage restriction boundaries in the mhb region, one of them
situated at the dorsal mes-met interface. In this study, a mouse strain
carrying a tamoxifen-inducible Cre recombinase, controlled by a Wnt1 promoter,
was crossed to a lacZ driver line. After induction, the vast majority
of cells was detected in the mesencephalon. However, independent of the time
of labeling, some lacZ-positive cells were found within the
cerebellum. This may be due to the previously described early activation of
Wnt1 on both sides of the mhb (Bally-Cuif
et al., 1995
) or due to a certain leakiness of the lineage
restriction mechanism (Birgbauer and
Fraser, 1994
). Several studies in chick embryos suggest lineage
restriction between the mesencephalon and metencephalon
(Millet et al., 1996
;
Alexandre and Wassef, 2003
;
Louvi et al., 2003
). In one
study, one or two founder cells were labeled, and in this study no lineage
restriction was observed (Jungbluth et
al., 2001
). In neither of these studies has cell movement in the
midbrain-hindbrain boundary area been followed directly. By examining the
movement of individual cells within the mhb region of the zebrafish, we have
detected only two cells (out of 551) within a few cell diameters of the
boundary that were clearly located in the neural tube, and that did not
respect the lineage restriction boundary
(Table 1). These two
Otx-negative cells were traced backwards and derived from the same founder
cell within the future Otx-positive domain at the start of the time-lapse.
Possible explanations for this exception to the rule are as follows. (1) We
may have wrongly assigned these cells or mistracked them repeatedly. However,
given our overall high accuracy of tracking, we consider this unlikely. (2)
Initially lineage restricted cells may move across during later
differentiation, e.g. by neuronal migration in the mantle layer, as reported
in the chick hindbrain (Wingate and
Lumsden, 1996
). (3) The restriction mechanism may be leaky, as
suggested for rhombomere boundaries
(Birgbauer and Fraser, 1994
).
Escapers would need to readjust their gene expression to adopt the fate of the
target tissue. Studies carried out in chick and mouse embryos
(Alexandre and Wassef, 2003
;
Louvi et al., 2003
) suggest
that roof plate cells may escape a lineage restriction mechanism. In our
study, very dorsally located cells also seemed to violate the lineage
restriction boundary (Table 1). However, all of these cells either left the neuroepithelium (18/22) during the
imaged time period or moved very fast and over long distances (4/22),
classifying them as putative neural crest cells. Therefore, we argue that
there is no contribution of midbrain cells to anterior hindbrain structures
and vice versa in the zebrafish and that, although we did not address the
molecular status of these cells, lineage restriction probably extends to roof
plate structures.
Our results are consistent with a study of clonal dispersion after single
cell injections in the brain of another fish species, Medaka (Oryzias
latipes) (Hirose et al.,
2004). Data derived from 150 single cell injections were fitted
onto a model of a developing Medaka embryo. Although the position of cells
relative to putative lineage restriction boundaries or genetic markers was not
followed in this study, reduced mixing between all examined brain regions at
the transition from developmental stage 16+ to 17 was observed, which
corresponds approximately to the time we observe onset of restriction at the
tailbud stage in zebrafish.
In summary, we suggest that the midbrain-hindbrain boundary separates two
neuromeres in the developing zebrafish brain, raising the possibility that the
neuromeric organization of the vertebrate brain extends to this part of the
neural tube. Further studies will show whether the anterior neural tube is
compartmentalized in general, similar to the rhombencephalon. A picture
emerges where cell populations secreting organizing molecules are flanked by
neuromere boundaries. The reverse conclusion can apparently not be drawn, as
several rhombomere boundaries and the diencephalon-mesencephalon border are
not (yet) known to be associated with organizers. Studies with cellular
resolution of the type reported here may help to determine the relationship
between organizing cell populations and lineage restriction boundaries, a link
discovered and well studied in the fly
(Crick and Lawrence, 1975;
Dahmann and Basler, 1999
), but
poorly characterized in vertebrate brain development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/14/3209/DC1
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexandre, P. and Wassef, M. (2003). The
isthmic organizer links anteroposterior and dorsoventral patterning in the
mid/hindbrain by generating roof plate structures.
Development 130,5331
-5338.
Bally-Cuif, L., Cholley, B. and Wassef, M. (1995). Involvement of Wnt-1 in the formation of the mes/metencephalic boundary. Mech. Dev. 53, 23-34.[CrossRef][Medline]
Birgbauer, E. and Fraser, S. E. (1994).
Violation of cell lineage restriction compartments in the chick hindbrain.
Development 120,1347
-1356.
Brand, M. and Granato, M. (2002). Keeping and raising zebrafish. In Zebrafish: A Practical Approach (ed. C. Nüsslein-Volhard and R. Dahm). Oxford: Oxford University Press.
Concha, M. L. and Adams, R. J. (1998). Oriented
cell divisions and cellular morphogenesis in the zebrafish gastrula and
neurula: a time-lapse analysis. Development
125,983
-994.
Cooke, J. E. and Moens, C. B. (2002). Boundary formation in the hindbrain: Eph only it were simple. Trends Neurosci. 25,260 -267.[CrossRef][Medline]
Cooper, M. S., D'Amico, L. A. and Henry, C. A. (1999). Analyzing morphogenetic cell behaviors in vitally stained zebrafish embryos. Methods Mol. Biol. 122,185 -204.[Medline]
Crick, F. H. C. and Lawrence, P. A. (1975). Compartments and polyclones in insect development. Science 189,340 -347.[Medline]
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380,66 -68.[CrossRef][Medline]
Dahmann, C. and Basler, K. (1999). Compartment boundaries: at the edge of development. Trends Genet. 15,320 -326.[CrossRef][Medline]
Fraser, S. E. (1996). Iontophoretic dye labeling of embryonic cells. Methods Cell Biol. 51,147 -160.[Medline]
Fraser, S., Keynes, R. and Lumsden, A. (1990). Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions. Nature 344,431 -435.[CrossRef][Medline]
Garcia-Bellido, A., Ripoll, P. and Morata, G. (1973). Developmental compartmentalisation of the wing disk of Drosophila. Nat. New Biol. 245,251 -253.[Medline]
Garda, A. L., Echevarria, D. and Martinez, S. (2001). Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101,111 -118.[CrossRef][Medline]
Hidalgo-Sanchez, M., Millet, S., Simeone, A. and Alvarado-Mallart, R. M. (1999). Comparative analysis of Otx2, Gbx2, Pax2, Fgf8 and Wnt1 gene expressions during the formation of the chick midbrain/hindbrain domain. Mech. Dev. 81,175 -178.[CrossRef][Medline]
Hirose, Y., Varga, Z. M., Kondoh, H. and Furutani-Seiki, M.
(2004). Single cell lineage and regionalization of cell
populations during Medaka neurulation. Development
131,2553
-2563.
Irvine, K. D. and Rauskolb, C. (2001). Boundaries in development: formation and function. Annu. Rev. Cell Dev. Biol. 17,189 -214.[CrossRef][Medline]
Jungbluth, S., Larsen, C., Wizenmann, A. and Lumsden, A. (2001). Cell mixing between the embryonic midbrain and hindbrain. Curr. Biol. 11,204 -207.[CrossRef][Medline]
Kelly, G. M., Lai, C. J. and Moon, R. T. (1993). Expression of wnt10a in the central nervous system of developing zebrafish. Dev. Biol. 158,113 -121.[CrossRef][Medline]
Keynes, R., Cook, G., Davies, J., Lumsden, A., Norris, W. and Stern, C. (1990). Segmentation and the development of the vertebrate nervous system. J. Physiol. (Paris) 84, 27-32.[Medline]
Kimmel, C. B., Warga, R. M. and Kane, D. A.
(1994). Cell cycles and clonal strings during formation of the
zebrafish central nervous system. Development
120,265
-276.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Langenberg, T., Brand, M. and Cooper, M. S. (2003). Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants. Dev. Dyn. 228,464 -474.[CrossRef][Medline]
Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A.
P. (1997). Evidence that FGF8 signalling from the
midbrain-hindbrain junction regulates growth and polarity in the developing
midbrain. Development
124,959
-969.
Louvi, A., Alexandre, P., Metin, C., Wurst, W. and Wassef,
M. (2003). The isthmic neuroepithelium is essential for
cerebellar midline fusion. Development
130,5319
-5330.
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. and
Martin, G. R. (1999). FGF8 induces formation of an
ectopic isthmic organizer and isthmocerebellar development via a
repressive effect on Otx2 expression.
Development 126,1189
-1200.
Mellitzer, G., Xu, Q. and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. Nature 400, 77-81.[CrossRef][Medline]
Millet, S., Bloch-Gallego, E., Simeone, A. and Alvarado-Mallart,
R. M. (1996). The caudal limit of Otx2 gene
expression as a marker of the midbrain/hindbrain boundary: a study using in
situ hybridisation and chick/quail homotopic grafts.
Development 122,3785
-3797.
Moens, C. B. and Prince, V. E. (2002). Constructing the hindbrain: insights from the zebrafish. Dev. Dyn. 224,1 -17.[CrossRef][Medline]
Mori, H., Miyazaki, Y., Morita, T., Nitta, H. and Mishina, M. (1994). Different spatio-temporal expressions of three otx homeoprotein transcripts during zebrafish embryogenesis. Brain Res. Mol. Brain Res. 27,221 -231.[Medline]
Papan, C. and Campos-Ortega, J. A. (1994). On the formation of the neural keel and neural tube in the zebrafish Danio (Brachydanio) rerio. Roux's Arch. Dev. Biol. 203,178 -186.[CrossRef]
Pasini, A. and Wilkinson, D. G. (2002). Stabilizing the regionalisation of the developing vertebrate central nervous system. BioEssays 24,427 -438.[CrossRef][Medline]
Pauls, S., Geldmacher-Voss, B. and Campos-Ortega, J. A. (2001). A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development. Dev. Genes Evol. 211,603 -610.[CrossRef][Medline]
Puelles, L. (2001). Brain segmentation and forebrain development in amniotes. Brain Res. Bull. 55,695 -710.[CrossRef][Medline]
Raible, F. and Brand, M. (2004). Divide et impera the midbrain-hindbrain boundary and its organizer. Trends Neurosci. 27,727 -734.[CrossRef][Medline]
Reifers, F., Böhli, H., Walsh, E. C., Crossley, P. H.,
Stainier, D. Y. R. and Brand, M. (1998). Fgf8
is mutated in zebrafish acerebellar mutants and is required for
maintenance of midbrain-hindbrain boundary development and somitogenesis.
Development 125,2381
-2395.
Reim, G. and Brand, M. (2002). Spiel-ohne-grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development. Development 129,917 -933.[Medline]
Rhinn, M. and Brand, M. (2001). The midbrainhindbrain boundary organizer. Curr. Opin. Neurobiol. 11,34 -42.[CrossRef][Medline]
Rhinn, M., Lun, K., Amores, A., Yan, Y. L., Postlethwait, J. H. and Brand, M. (2003). Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling. Mech. Dev. 120,919 -936.[CrossRef][Medline]
Rhinn, M., Lun, K., Luz, M., Werner, M. and Brand, M.
(2005). Positioning of the midbrain-hindbrain boundary organizer
through global posteriorization of the neuroectoderm mediated by Wnt8
signaling. Development
132,1261
-1272.
Schmitz, B., Papan, C. and Campos-Ortega, J. A. (1993). Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio. Roux's Arch. Dev. Biol. 202,250 -259.[CrossRef]
Vaage, S. (1969). The segmentation of the primitive neural tube in chick embryos (Gallus domesticus). Ergebnisse der Anatomie und Entwicklungsgeschichte 41, 3-87.[Medline]
Westerfield, M. (1994). The Zebrafish Book. 2.1 edn. Oregon: University of Oregon Press.
Wingate, R. J. and Lumsden, A. (1996).
Persistence of rhombomeric organisation in the postsegmental hindbrain.
Development 122,2143
-2152.
Woo, K. and Fraser, S. E. (1995). Order and
coherence in the fate map of the zebrafish nervous system.
Development 121,2595
-2609.
Wurst, W. and Bally-Cuif, L. (2001). Neural plate patterning: upstream and downstream of the isthmic organizer. Nat. Rev. Neurosci. 2,99 -108.[CrossRef][Medline]
Xu, Q., Mellitzer, G., Robinson, V. and Wilkinson, D. G. (1999). In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature 399,267 -271.[CrossRef][Medline]
Zervas, M., Millet, S., Ahn, S. and Joyner, A. L. (2004). Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1. Neuron 43,345 -357.[CrossRef][Medline]
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