1 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,
MA 02142, USA
2 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA
02139-4307 USA
* Author for correspondence (e-mail: sive{at}wi.mit.edu)
Accepted 15 February 2005
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SUMMARY |
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Key words: Brain ventricle formation, Brain structure, Circulation, Morphogenesis, Zebrafish, Lumen inflation, snakehead (snk), nagie oko (nok), Neural tube, Epithelial polarity, MAGUK family, Na+K+ ATPase, atp1a1a.1
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Introduction |
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The brain ventricles are cavities lying deep within the brain, which
contain cerebrospinal fluid (CSF) and form a circulatory system in the brain
(Cushing, 1914;
Milhorat et al., 1971
;
Pollay and Curl, 1967
). This
system is believed to have essential roles in brain function, including waste
removal, nutrition, protection and pressure equilibration
(Novak et al., 2000
). Recent
evidence suggests that CSF directly regulates neuronal proliferation in the
embryonic brain and is part of a non-synaptic communication system in the
adult (Miyan et al., 2003
;
Owen-Lynch et al., 2003
;
Skinner and Caraty, 2002
). In
addition, certain neurons send processes into the ventricular space,
suggesting that their activity may be connected with regulating CSF
homeostasis (Vigh and Vigh-Teichmann,
1998
). CSF contains hormones, proteoglycans and ions, and its
composition varies between ventricles and over time, suggesting a changing
function for the ventricles during development
(Alonso et al., 1998
;
Skinner and Caraty, 2002
).
Abnormalities in brain ventricle structure can lead to hydrocephaly, one of
the most common birth defects (McAllister
and Chovan, 1998
; Rekate,
1997
), and abnormal brain ventricle size and development have been
correlated with mental health disorders such as autism and schizophrenia
(Hardan et al., 2001
;
Kurokawa et al., 2000
).
While the adult brain ventricles have a complex shape, the embryonic brain
begins as a simple tube, the lumen of which forms the brain ventricles. During
and after neurulation, the anterior neural tube dilates in three specific
locations to form the future forebrain, midbrain and hindbrain ventricles
(also called brain vesicles). This dilation pattern is highly conserved in all
vertebrates. Elegant studies in chick embryos have shown that intraluminal
pressure resulting from the accumulation of CSF inside the brain ventricles is
necessary for normal brain ventricle expansion and cell proliferation
(Desmond, 1985;
Desmond and Levitan, 2002
),
and levels of proteoglycans such as chondroitin sulfate affect this process
(Alonso et al., 1998
;
Alonso et al., 1999
). However,
the molecular mechanisms underlying brain ventricle formation are almost
completely unknown. This has been due, in part, to lack of a genetic model in
which early brain ventricle development could be observed.
We have considered whether the zebrafish is a good model for analyzing
brain morphogenesis. One issue is whether the zebrafish neural tube forms by a
similar mechanism to the amniote neural tube, as teleost neurulation involves
formation of a solid neural keel, whereas amniote and amphibian neurulation
involves rolling of the neuroepithelium into a tube. Our evaluation of the
primary literature clearly indicates that teleost, amniote and amphibian
neurulation occur via fundamentally similar topological mechanisms, supporting
the use of zebrafish as a model for brain morphogenesis
(Lowery and Sive, 2004). We
therefore initiated a project to analyze brain ventricle formation using the
zebrafish as a model. Brain ventricle mutants have been identified in several
mutagenesis screens (Guo et al.,
1999
; Jiang et al.,
1996
; Schier et al.,
1996
); however, most have not been studied further.
In this study, we characterize normal brain ventricle formation in the zebrafish, and examine in detail the phenotypes of two severe brain ventricle mutants, nagie oko and snakehead. Our data define a series of steps necessary for brain ventricle development and demonstrate the utility of the zebrafish as a system for in-depth analysis of this process.
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Materials and methods |
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Lines used were: Tübingen Long Fin, sihtc300B
(Sehnert et al., 2002),
snkto273a (Jiang et
al., 1996
), nokwi83
(Wiellette et al., 2004
),
snkto273a; nokwi83, slhm291
(Yuan and Joseph, 2004
).
Double mutant snkto273a; nokwi83 were
constructed using standard genetic techniques. To genotype double mutant
embryos, PCR analysis of nok and morphology analysis for snk
was used. After sorting snk mutant embryos phenotypically, the heads
from each individual were removed, fixed in 2% paraformaldehyde, 1%
glutaraldehyde, and processed for sectioning as described below. The remaining
body was digested with proteinase K (1 mg/ml) in lysis buffer (10 mmol/l Tris
pH 8, 1 mmol/l EDTA, 0.3% Tween-20, 0.3% NP40) and used for PCR genotyping for
the nok locus. Because nokwi83 has a retroviral
insertion in the nok gene, mutant individuals could be identified by
PCR. Primers used: nokf 5'-GGTGAGCTGCCACTTTTCGGACA-3', nokr
5'-TAGCGACCCGTCACATAACA-3', retroviral-specific primer
5'-CCATGCCTTGCAAAATGGCGTTACTTAAGC-3' (MWG Biotech). To identify
the wild-type allele, nokf and nokr primers were used. To identify the
nokwi83 allele, nokf and insertion primers were used.
Brain ventricle imaging
Embryos were anesthetized in 0.1 mg/ml Tricaine (Sigma) dissolved in embryo
medium (Westerfield, 1995)
prior to injection and imaging. The hindbrain ventricle was micro-injected
with 2-10 nl dextran conjugated to Rhodamine or Texas Red (5% in 0.2 mol/l
KCl, Sigma), the fluorescent molecule then diffused through the brain
cavities, and micrographs were taken with light and fluorescent illumination
within 10 minutes of injection. These two images were superimposed in
Photoshop 6 (Adobe). Injected embryos survive and develop normally.
Comparisons of injected and non-injected brains show ventricle size is not
perturbed by injection.
Live confocal imaging
Bodipy ceramide (Fl C5, Molecular Probes) was dissolved in DMSO to a stock
concentration of 5 mmol/l. Embryos were soaked in 50 nmol/l bodipy ceramide
solution overnight in the dark. The embryos were then washed, dechorionated
and placed in wells in 1% agarose for confocal microscopy. Confocal imaging
was performed using a Zeiss LSM510 laser-scanning microscope, using standard
confocal imaging techniques (Cooper et
al., 1999). Confocal images were analyzed using LSM software
(Zeiss) and Photoshop 6.0 (Adobe).
Histology
Embryos were fixed in 2% paraformaldehyde, 1% glutaraldehyde in PBS
overnight at 4°C, then washed in PBS, dechorionated, dehydrated and
embedded in plastic according to the manufacturer's instructions (JB-4 Plus
Embedding Kit, Polysciences). Sections 5-8 µm were cut on Leica RM2065
microtome and stained with hematoxylin and eosin using standard staining
methods (Polysciences).
Immunohistochemistry
For labeling with anti-phosphohistone H3 antibody, anti-Nagie oko antibody
and phalloidin-Texas Red, embryos were fixed in 4% paraformaldehyde for 2
hours at room temperature, then rinsed in PBS and dechorionated. For labeling
with anti-ß-catenin polyclonal antibody, anti-occludin polyclonal
antibody and anti-alpha Na+K+ ATPase a6F antibody,
dechorionated embryos were fixed in Dent's fixative (80% methanol, 20% DMSO)
for 2 hours at room temperature, then rinsed in PBS
(Dent et al., 1989). Blocking
was done for 4 hours at room temperature in 0.5% Triton X, 4% normal goat
serum, in phosphate buffer. Whole-mount immunostaining was carried out using
anti-phosphohistone H3 rabbit polyclonal antibody (Upstate Biotechnology,
1:800), anti-ß-catenin rabbit polyclonal antibody (Zymed Laboratories,
1:50), anti-occludin rabbit polyclonal antibody (Zymed Laboratories, 1:50),
anti-Nagie oko rabbit polyclonal antibody
(Wei and Malicki, 2002
)
(1:500), and mouse monoclonal antibody alpha6F, raised against chicken alpha1
subunit of Na+K+ ATPase
(Takeyasu et al., 1988
)
(1:100), which was obtained from the Developmental Studies Hybridoma Bank.
Goat anti-rabbit IgG Alexa Fluor 488 (Molecular Probes, 1:500) and goat
anti-mouse Alexa Fluor 488 (Molecular Probes, 1:500) were used as secondary
antibodies. Phalloidin conjugated to Texas Red (Sigma, 1:1000) was used to
label actin filaments. Brains were flat-mounted in glycerol and imaged with a
confocal microscope. For transverse sections, brains were embedded in 4% low
melting agarose and sectioned with a vibratome (200 µm sections) before
confocal imaging.
For cell proliferation quantification, phosphohistone H3-labeled cells in each z-series of the midbrain-hindbrain boundary and hindbrain regions were counted and averaged. Average z-series areas of the regions were measured using Scion Image software (Scion Corporation), and by determining the approximate area occupied by each cell, total cell number and the percentage of labeled cells in each region were calculated. To determine the statistical difference among different regions at the same time point, statistical analyses were performed using a paired sample t-test with SPSS 13.0 for Windows (SPSS). Cell death quantification was performed similarly, using an ANOVA test for comparison of multiple groups. P<0.05 was considered significant.
Cell death labeling
DNA fragmentation during apoptosis was detected by the TUNEL method, using
`ApopTag' kit (Chemicon). Embryos were fixed in 4% paraformaldehyde in PBS for
2 hours, then rinsed in PBS and dechorionated. Embryos were dehydrated to 100%
ethanol, stored at 20°C overnight, then rehydrated in PBS. Embryos
were further permeabilized by incubation in proteinase K (5 µg/ml) for 5
minutes, then rinsed in PBS. TdT labeling was followed per manufacturer's
instructions. Anti-DIG-AP (Gibco, 1:100) was used to detect the DIG labeled
ends. Brains were flat-mounted in glycerol and imaged.
Inhibition of cell proliferation
Cell proliferation was inhibited by treating embryos with 100 µg/ml
aphidicolin (Sigma) in 1% DMSO from 15 hpf until 24 hpf. This treatment
significantly slows, although does not stop, cell proliferation. Previous
studies have indicated that in zebrafish it is not possible to completely
inhibit cell proliferation at the stages observed without severe cell death,
which interferes with brain ventricle development
(Ikegami et al., 1997).
Reduction in cell proliferation was measured using an antibody to
phosphorylated histone H3 as described above.
Detection of snakehead mutation
Total RNA was extracted from mutant embryos and wild-type siblings using
Trizol reagent (Invitrogen), followed by chloroform extraction and isopropanol
precipitation. cDNA synthesis was performed with Super Script II Reverse
Transcriptase (Invitrogen) and random hexamers. PCR was then performed using
five sets of previously published primers, which amplify the coding region of
atp1a1a.1 (Shu et al.,
2003). RT-PCR products were used for sequencing analysis,
performed by Northwoods DNA, Inc. (Solway MN). Sequencing data was analyzed
using the BLAST program
(http://www.ncbi.nlm.nih.gov/BLAST/),
and the cDNA sequence of atp1a1a.1 was obtained from the GenBank
database (NM_131686). Seven single-nucleotide changes in the
snakehead cDNA were found, but only one, within primer set 2, changed
the amino acid sequence.
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Results |
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Circulation is not required for initial brain ventricle opening, but is required for later expansion
The demonstration that brain ventricle opening occurred before the onset of
heartbeat contrasts with previous speculations that circulation is required
for initial brain ventricle development
(Schier et al., 1996). In
order to further explore this point, we analyzed this process in silent
heart (sih) mutants. The sih gene encodes a
cardiac-specific troponin, and while the heart forms normally, heartbeat and
circulation never occur (Sehnert et al.,
2002
). Silent heart mutants formed brain ventricles
indistinguishable from wild type by 24 hpf
(Fig. 2A,B). This continued
through 27 hpf (Fig. 2C,D).
Later during development, by 36 hpf, when wild-type ventricles had expanded
their volume significantly (Fig.
2E), sih mutants showed a smaller ventricle height (and
therefore volume) relative to wild-type embryos
(Fig. 2F). These data confirm
that initial steps in brain ventricle formation are independent of heartbeat
and circulation, but that a later step contributing to ventricle expansion
does require circulation. We subsequently focused our attention on the initial
opening of the brain ventricles, before 24 hpf.
|
|
We also asked whether cell death was correlated with ventricle opening (Fig. 3H-J). Using TUNEL staining in whole-mount embryos, no patterns of localized cell death were apparent from 17 to 24 hpf (Fig. 3H,I and data not shown). Quantification of cell death demonstrated that, while more death occurred at 17 hpf than at 21 hpf, no brain region examined displayed a significant difference in amount of cell death relative to other regions (Fig. 3J) (n=10, P=0.575 at 17 hpf, P=0.368 at 21 hpf).
These results suggest that regulated cell proliferation is necessary for initial brain ventricle formation, although other processes are crucial. Localized cell death does not appear to regulate initial brain ventricle formation.
The snakehead mutant is allelic to small heart and corresponds to a point mutation in Na+K+ ATPase atp1a1a.1
In order to identify brain ventricle mutants, we performed a `shelf' screen
of ethylnitrosourea (ENU) and insertional zebrafish mutants
(Amsterdam et al., 2004;
Jiang et al., 1996
;
Schier et al., 1996
). We
focused on mutants that were previously suggested to have a ventricle
phenotype, although none of their brain phenotypes have been studied further.
From this screen, we have identified 33 mutants with a brain ventricle
phenotype, with the important criterion that these show healthy neural tissue,
with no obvious necrosis (not shown).
One of the most severe ventricle phenotypes is seen in the
snakehead (snk) mutant. snkt0273a was
derived from a large-scale chemical screen and is therefore presumed to be
caused by a point mutation (Jiang et al.,
1996). In addition to a lack of brain ventricles, snk
embryos have heart defects, delayed body pigmentation and no touch response
(Schier et al., 1996
). We
noticed that the snk phenotype appeared identical to that of the
small heart (slh) mutant, which was isolated in an ENU
screen (Yuan and Joseph,
2004
). We therefore performed complementation analysis between
snk and slh and determined that they do not complement, and
are likely to be different alleles of the same locus. In a cross of
snk and slh heterozygotes, 77% showed a wild-type phenotype
and 23% a mutant phenotype (78 embryos in total). slh has been cloned
and encodes a Na+K+ ATPase, Atp1a1a.1 (previously named
1B1). The heart and mind (had) mutant also has a
mutation in the atp1a1a.1 gene
(Shu et al., 2003
).
We asked whether the snakehead phenotype was due to a mutation in
atp1a1a.1 by comparing cDNA sequences from the wild-type and
snakeheadto273a mutants. This analysis revealed that the
to273a allele of snakehead contained a G to A mutation at
position 812 in the atp1a1a.1 coding sequence, which resulted in an
amino acid change from glycine to aspartate at position 271 in the amino acid
sequence (Fig. 4 and data not
shown). This mutation was in the M2-M3 cytoplasmic loop, which is necessary
for catalytic activity and may play a role in ion pumping action
(Kaplan, 2002). Additional
mutations in this loop have been shown to alter the kinetic properties of the
protein in other systems (Kaplan,
2002
), and thus we predict that the glycine to aspartate mutation
in the snakeheadto273a mutant substantially reduces or
eliminates Na+K+ ATPase atp1a1a.1 function.
|
snakehead and nagie oko mutants fail to form brain ventricles by different mechanisms
Another mutant with a severe brain ventricle phenotype is nagie
oko (nok), which encodes a MAGUK family kinase required for
epithelial cell polarity in the zebrafish eye and gut
(Wei and Malicki, 2002;
Horne-Badovinac et al., 2003
).
We analyzed a retroviral insertion allele of nagie oko, isolated in
our laboratory, and this hypomorphic allele showed absence of brain
ventricles, but did not show the eye phenotype of the ENU-derived nok
allele, suggesting these may be separable functions, or that there is a
quantitatively different requirement for Nok in different tissues
(Wei and Malicki, 2002
;
Wiellette et al., 2004
).
By light microscopy of living embryos, brain ventricles appeared completely absent from both mutants in either dorsal views (Fig. 5A-C) or in lateral views (Fig. 5D-F). However, unlike nok mutants, the entire snk embryo displayed a characteristic refractivity. In particular, in dorsal view, the outline of the neural tube was not visible, even though a neural tube was present (see below). The refractivity of the neural tube thus could not be distinguished from that of surrounding tissues, unlike the case in wild-type embryos. In order to ask whether absence of ventricles in both mutants reflected disruption of a similar process, we further analyzed the brain morphology of the mutants.
|
These data show that both snk and nok have an early ventricle phenotype, by 20 hpf, before the onset of heartbeat. Significantly, the `no ventricle' phenotypes of snk and nok mutants were quite different, which indicates that the mechanisms by which these genes affect brain ventricle morphogenesis are distinct.
nagie oko mutants retain epithelial polarity but lose epithelial integrity in the brain
Previous analyses of nok mutants have concluded that, within the
brain, epithelial polarity is normal, whereas in the retina, epithelial
polarity is abnormal and correlated with retinal disorganization
(Wei and Malicki, 2002).
However, the brain epithelium has not been extensively examined, and we
therefore analyzed nok epithelial organization in more detail. We
first examined transverse brain sections at 17 hpf, when the neural tube is
normally straight and ventricle morphogenesis has not yet begun. Sections of
wild-type embryos showed a clear midline, with nuclei lined up on either side
(Fig. 6A), while nok
mutant sections showed disorganized nuclear position and no continuous midline
(Fig. 6B). However, in most
sections of nok mutants extending from forebrain to hindbrain, there
were small, intermittent regions with a clear midline (not shown).
|
In summary, we showed that in the future brain the nok neuroepithelium was highly disorganized, and that while it displayed proper apical/basal organization, junctional actin belts did not form cohesively, and there was no clear or continuous midline.
Epistasis analysis of snakehead/atp1a1a.1 and nagie oko
As both snk and nok mutants fail to form ventricles, we
performed epistasis analysis to determine whether the atp1a1a.1 and
nok genes function in the same or separate genetic pathways
(Fig. 7). To create
double-mutant embryos, heterozygote carriers were mated and their progeny
raised. Double mutants were identified by morphology and PCR. Light microscopy
and histology showed that the double mutant snk;nok had a composite
of both mutant phenotypes (Fig.
7D,H,L,P). In the double mutant, the altered refractivity and lack
of extracellular spaces of the snk phenotype was present
(Fig. 7C,G,K,O). However, the
narrower brain tube, disorganized epithelium, and lack of hinge-points
characteristic of the nok phenotype was also present
(Fig. 7B,F,J,N and
Fig. 7D,H,L,P).
|
|
In summary, the composite double-mutant phenotype and correct localization of a6F staining and Nok protein in nok and snk mutants, respectively, suggest that nagie oko and atp1a1a.1 function in separate pathways.
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Discussion |
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Our data show that initial stages of ventricle formation are not dependent on circulation. Why do many mutants with brain ventricle defects also display heart or circulation defects? One possibility is that some of these mutants show a `late' ventricle phenotype and reflect a deficit in circulation-dependent ventricle expansion. Another possibility is that genes required for heart and brain ventricle morphogenesis are shared and the phenotype of a particular mutant is therefore pleiotropic. In support of this, both nagie oko and snakehead mutants show heart defects.
The role of cell proliferation in brain ventricle development
The lowest level of cell proliferation we observed along the A/P axis of
the brain epithelium was at the MHB, and this deficit may be one of the
reasons that the MHB does not open into a ventricular space. How might cell
proliferation contribute to normal ventricle development? One possibility is
that a critical mass of cells is necessary for ventricle morphogenesis, while
a second is that cells must be actively cycling to respond to signals leading
to cell movement or shape changes. A third possibility is that cell
proliferation is required for lumen inflation, rather than for ventricle
morphogenesis. However, the smaller, but normally shaped, ventricles observed
after inhibition of DNA replication did not resemble the defects seen in
nok or snk mutants, and neither of these mutants showed
differences in cell proliferation (or cell death) relative to wild-type
embryos (not shown). We therefore hypothesize that cell proliferation defines
an independent step during ventricle formation. We cannot rule out that
embryos in which cell proliferation had been inhibited were generally
disrupted; however, other regions of such embryos appeared normal, including
somite number and shape (not shown). Overproliferation of the neuroepithelium
also suppresses ventricle opening, as shown in the mindbomb and
curlyfry mutants (Bingham et al.,
2003; Song et al.,
2004
), and the connection between these data and the requirement
for cell proliferation is not clear.
Previous reports have indicated that in mice blocking cell death by caspase
gene ablation causes an overgrowth of the brain tissue, with obscured or
obstructed brain ventricles (Kuida et al.,
1996). Conversely, mutations that cause too much cell death in the
brain lead to a reduction in brain tissue and overexpansion of the brain
ventricles (Keino et al.,
1994
). Our data do not suggest an early role for regulated cell
death in initial brain ventricle development, and it is likely that these
phenotypes reflect late outcomes of perturbing cell death.
The nagie oko phenotype indicates a requirement for epithelial integrity in ventricle formation
The nagie oko gene encodes a MAGUK family scaffolding protein that
localizes to junctions at the apical surface of epithelia and regulates
epithelial polarity (Wei and Malicki,
2002). MAGUK proteins probably function in the assembly of protein
complexes that control the formation or maintenance of cell junctions, and the
Nok homolog in other organisms (Stardust in Drosophila,
PALS1 in mammals) is part of the Crumbs protein complex, one of the key
regulators of epithelial junction formation
(Bachmann et al., 2001
;
Hong et al., 2001
;
Hurd et al., 2003
;
Knust and Bossinger, 2002
;
Muller and Bossinger, 2003
;
Tepass, 2002
).
Brain ventricle morphogenesis may require a cohesive epithelium, and nok mutants may fail to undergo morphogenesis because the brain neuroepithelium lacks normal epithelial junctions and therefore lacks this cohesiveness. Additionally, in nok mutants, there is no continuous midline, at which opposing epithelial surfaces would normally separate. The nok phenotype may therefore arise because the neuroepithelium is glued shut by cells straddling the midline. In support of this, nok mutants often show short stretches of a clear midline, and perhaps correlated with this, sometimes show very small ventricular openings.
Atp1a1a.1 plays a role in lumen inflation
The mechanism by which the brain ventricles initially inflate is not known.
The Na+K+ ATPase Atp1a1a.1 protein is likely to be
necessary to create an osmotic gradient that would drive movement of water
into the closed ventricles after their morphogenesis
(Blanco and Mercer, 1998;
Speake et al., 2001
;
Therien and Blostein, 2000
).
We therefore hypothesize that Atp1a1a.1 functions to direct initial brain
ventricle lumen inflation. The sequential opening of each ventricle may
reflect sequential activation of this ion pump, thus blowing up the ventricles
like a balloon. Alternately, Atp1a1a.1 function may be continuous along the
length of the brain, and another mechanism may regulate where initial lumen
inflation occurs. Interestingly, although the Na+K+
ATPase family is large, other members cannot compensate for the loss of
embryonic Atp1a1a.1 function. This is consistent with previous results showing
that similar alpha subunits of Na+K+ ATPase do not show
the same expression patterns and cannot substitute for each other during
zebrafish heart development or ear development
(Canfield et al., 2002
;
Shu et al., 2003
;
Blasiole et al., 2003
).
In the older brain, the choroid plexuses are the main source of CSF
secretion, although the brain ependymal cells also contribute
(Wright, 1978;
Brown et al., 2004
;
Bruni, 1998
).
Na+K+ ATPases localize to the ventricular surface of
secretory brain epithelia in adult mammals and amphibia and are necessary for
the secretion of CSF (Masuzawa et al.,
1984
; Saito and Wright,
1983
). Thus, this gene family may be used at different times of
development to initiate, and later maintain, ventricular fluid secretion. We
hypothesize that earlier during development, when the brain ventricles are
initially forming and before the choroid plexuses have formed, the ependymal
cells lining the ventricles are the source of CSF, through the action of the
Atp1a1a.1 protein.
Zebrafish as a model for vertebrate brain ventricle development
In some respects, formation of the zebrafish neural tube appears different
from that of frog, as in the brain region the zebrafish neural tube is
initially straight whereas in the frog Xenopus the presumptive brain
undergoes some morphogenesis prior to neural tube closure. However, the
number, position and shape of the initial brain ventricles are essentially
identical in all vertebrates. We have previously compared processes of trunk
neural tube formation between teleosts and other vertebrates, and have
concluded that these are very similar
(Lowery and Sive, 2004) (see
Introduction). These considerations suggest that zebrafish ventricle formation
is likely to be fundamentally the same as that of other vertebrates. We are
beginning to analyze the phenotypes of other zebrafish ventricle mutants, and
this is likely to uncover additional genetic mechanisms regulating brain
ventricle formation. Of particular interest is the relationship between genes
that regulate neural patterning and the positioning and shaping of the
ventricles, the interaction of nok with other genes that regulate
epithelial polarity, and the mechanism by which atp1a1a.1 and other
genes regulate lumen inflation.
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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/132/9/2057/DC1
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