- Research article
- Open Access
Nodal signaling is required for closure of the anterior neural tube in zebrafish
© Aquilina-Beck et al; licensee BioMed Central Ltd. 2007
- Received: 07 March 2007
- Accepted: 08 November 2007
- Published: 08 November 2007
Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish.
We find that the neural tube in the presumptive forebrain fails to close in zebrafish Nodal signaling mutants. For instance, the cells that will give rise to the pineal organ fail to move from the lateral edges of the neural plate to the midline of the diencephalon. The open neural tube in Nodal signaling mutants may be due in part to reduced function of N-cadherin, a cell adhesion molecule expressed in the neural tube and required for neural tube closure. N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling. Further, N-cadherin mutants and morphants have a pineal phenotype similar to that of mutants with deficiencies in the Nodal pathway. Overexpression of an activated form of the TGFβ Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling. We find that overexpression of Taram-A* also corrects their open neural tube defect. This suggests that, as in mammals, the mesoderm and endoderm have an important role in regulating closure of the anterior neural tube of zebrafish.
This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain.
- Neural Tube
- Neural Tube Defect
- Neural Plate
- Nodal Signaling
- Pineal Organ
Nodals are secreted signaling proteins in the TGFβ superfamily that have many established roles in vertebrate development. The absence of Nodal signaling in mice and zebrafish results in loss of the gastrula organizer, an almost complete failure in the development of mesodermal and endodermal tissue, and severe defects in the cellular movements of gastrulation [1, 2]. Over expression and partial loss-of-function studies indicate that Nodal signaling not only induces mesoderm, but also acts in a concentration dependent fashion to subdivide the mesoderm into different tissue types . Further, Nodal signaling in the left lateral plate mesoderm has a conserved role in regulating left-right asymmetry of visceral organs such as the heart and lungs .
Nodal signals also have extensive roles during neural development. Nodal proteins cooperate with several other signaling proteins, including Bone Morphogenetic Protein (BMP), Fibroblast Growth Factor (FGF), and Wingless Integrated (Wnt) to regulate anterior-posterior patterning of the neurectoderm [5–11]. Nodal signals are also important for patterning the ventral neural tube: lack of the Nodal signal Cyclops (Cyc) in zebrafish results in a complete absence of ventral brain and a severe reduction of the floor plate cells in the ventral spinal cord [12–15]. Because Nodal signaling mutants lack the Sonic hedgehog (Shh) expressing cells of the ventral brain and prechordal plate, they often have holoprosencephaly, or a failure in the forebrain to bifurcate into two hemispheres . In zebrafish, Nodal signals also regulate laterality in the dorsal forebrain. Several genes in the Nodal signaling pathway are expressed on the left side of the developing pineal organ, where they influence left-right asymmetry of the habenula nuclei and the pineal complex, which consists of a medial pineal organ and a left-sided parapineal [17–21].
Zebrafish have three Nodal signals, Cyc, Squint (Sqt), and Southpaw (Spaw), which all function through a receptor complex containing the membrane associated protein One-eyed pinhead (Oep). Here, we demonstrate that Sqt and Oep are important for closure of the anterior neural tube. During development, pineal precursors originate in two domains at the lateral edges of the neural plate [22–27]. As development proceeds, these precursors converge towards the midline of the dorsal diencephalon, where they ultimately fuse to form a single pineal organ. Thus, the position of the pineal precursors serves as a sensitive measure of neural tube closure. We find that the pineal precursors often fail to converge to the midline in sqt mutants, resulting in a pineal anlage that is elongated laterally or divided into two domains. In maternal zygotic oep (MZoep) mutants, which lack both maternally derived and zygotically expressed oep mRNA, the pineal precursors remain in two widely separated domains. The cell adhesion molecule N-cadherin (N-cad) is required for closure of the neural tube in zebrafish [28, 29]. Our data suggests that Nodal signaling may influence neural tube closure by regulating N-cad. N-cad protein localization to the cell membrane was reduced in MZoep mutants, and the structure of the neural tube was severely disrupted. We found that cell autonomous rescue of mesendoderm (a tissue that gives rise to both mesodermal and endodermal cell types) in MZoep mutants corrected their neural tube defect (NTD). This suggests that the role of Nodal signaling is to induce mesoderm formation, and mesoderm in turn regulates neural tube closure. This role of mesoderm may be conserved, as mesoderm also has a key role in rostral neural tube closure in mice . NTDs such as spina bifida and exencephaly occur in approximately one in one thousand human births, but the genetic causes of these disorders are still poorly understood . This work raises the possibility that deficiencies in the Nodal signaling pathway could underlie defects in closure of the rostral neural tube in humans.
Nodal signaling is required for dorsal convergence of pineal precursors
Embryos that lack Nodal signaling or N-cad expression have defects in the convergence of pineal precursors
sqtcz35/+ X sqtcz35/+
To more fully define the role of Nodal signaling in the movement of pineal precursors, we examined pineal morphology in other Nodal signaling mutants. The phenotype of mutants that lack Cyc, another zebrafish Nodal signal, includes a partial loss of mesendodermal tissue, lack of ventral brain and floor plate, and randomized laterality of the pineal complex and habenula nuclei [14, 17, 19, 33]. In contrast to sqt, the pineal precursors in 1 dpf cyc mutants always formed a single pineal anlage with a round morphology, suggesting that the pineal precursors had successfully moved to the midline of the brain (Table 1, Figure 1M).
Cyc and Sqt signal through a common receptor complex that contains the protein Oep, a member of the EGF-CFC family . Maternally derived oep mRNA is loaded into the oocyte and then later transcribed from the zygotic genome . Mutants that lack only zygotic oep (Zoep) have an overall phenotype that is very similar to cyc mutants. For instance, both Zoep and cyc mutants lack ventral brain but have a fairly normal notochord, while the most severe sqt mutants lack both notochord and ventral brain [10, 36–39]. Consistent with the other similarities between cyc and Zoep, we found that Zoep mutants also had a single, round pineal anlage (Table 1, Figure 1N).
In MZoep embryos, which lack both maternal and zygotic oep mRNA, the ventral neural tube, endoderm, and most mesoderm fail to develop [34, 40]. Despite these severe defects, anterior-posterior patterning of the brain is fairly normal and the pineal precursors are specified [17, 19, 34]. However, we found that pineal development was severely affected in MZoep fish: the pineal precursors were found in two widely spaced domains, or in rare cases, in a single, elongated domain (Table 1, Figure 1O).
Together, this suggests that Sqt signaling through a receptor complex containing Oep is important for the convergent movements of the pineal precursors. The fact that the pineal phenotype of MZoep mutants is much more severe than most sqt mutants suggests that loss of zygotically expressed sqt is in part compensated for by one of the other zebrafish Nodal signals or by Sqt translated from maternal mRNA. This is not unexpected, as there is partial redundancy between the three zebrafish Nodal signals in other aspects of their functions, such as patterning the endoderm and mesoderm .
The Nodal signaling mutants sqt and MZoephave an open neural tube
During development, the flat neural plate epithelium folds or rolls to form the neural tube, driving convergence of the pineal precursors to the midline of the brain. This suggests that the elongated/divided pineal phenotypes are due to a failure in neurulation. To test this, we took advantage of the variable pineal phenotype in sqt mutants. If this hypothesis is correct, then the neural tube should be open only in sqt mutants with an elongated or divided pineal morphology.
Correlation between pineal and neural tube phenotypes in Nodal signaling mutants
Neural tube morphology
sqtcz35/+ X sqtcz35/+
The neural tube defect in MZoep mutants was further characterized through examination of ephrina4a (epha4a) expression in the hindbrain. In WT embryos, epha4a was expressed in three stripes that correspond to rhombomeres 1, 3 and 5 (Figure 3K, M). In some MZoep embryos epha4a was expressed in a similar pattern to WT embryos, except that the rhombomeres were somewhat misshapen and slightly wider along the anterior posterior axis (Figure 3L). In approximately half of the MZoep embryos, the hindbrain was severely disrupted, including partial fusion of rhombomeres 3 and 5 and apparent separation of the left and right sides of the neural tube (Figure 3N).
Transverse sections through rhombomere 5 and the diencephalon further demonstrated the extent of the neural tube phenotype in MZoep. The neural tube at both axial levels of WT embryos was symmetric with a small lumen at the dorsal side (Figure 3O, Q). The MZoep neural tube was shortened along the dorsal-ventral axis and the midline was not apparent (Figure 3P, R). As we have previously reported, the brain of MZoep is often twisted, and the pineal precursors were often located on one side of the developing brain (Figure 3R) .
Pineal precursors fail to converge normally in embryos that lack N-cad
N-cad expression is altered in the MZoepneural tube
Expression of N-cad protein can be first detected at low levels around the onset of gastrulation. Levels increase and are quite high by the end of epiboly . Consistent with this, we find that N-cad levels were low at 60% epiboly, and no differences could be found between WT and MZoep (data not shown). In contrast, at 95–100% epiboly, just before neurulation begins, MZoep embryos had significantly reduced levels of N-cad protein (5A, B). In particular the characteristic N-cad staining at the cell membrane was clearly apparent in the WT embryo, but nearly absent in the mutant tissue.
The decreased levels of N-cad localization at the membrane were also present at the six somite stage, when the anterior neural tube is just closing to form a neural rod. In WT embryos, N-cad expression was strong and almost evenly distributed in the head region (Figure 5C, E). In MZoep embryos, N-cad staining was reduced in most regions except in clusters of cells in deeper (ventral) regions of the tissue (Figure 5D, F). Closer examination of the tissue sections revealed that N-cad expression in the WT tissue was confined mainly to the membranes of cells (Figure 5C'). In contrast, labeling in the plasma membrane of MZoep embryos was much reduced in most cells, similar to the phenotype at 100% epiboly (Figure 5D', F). N-cad expression in cells located in the clusters with strong N-cad expression was either inside the cell and on the cell membrane, or mainly inside the cell (Figure 5D'). This suggests that Nodal signaling during early development could directly or indirectly regulate the expression levels and/or intracellular localization of N-cad.
The cellular organization of the neural tube is perturbed in MZoepmutants
If the neural tube defect in MZoep mutants is in part due to the abnormal expression of N-cad, then MZoep embryos should display similar cellular phenotypes to those found in n-cad mutants. These include decreased cell adhesion and failure of the dorsal/lateral cells to lengthen and intercalate to form a single cell layer . To determine whether MZoep mutants displayed similar cellular phenotypes, we injected one cell stage embryos with mRNA encoding membrane bound GFP (mGFP) and assayed neural tube morphology at several points during neurulation.
In MZoep embryos, convergence of cells towards the dorsal side occurs normally, but involution of these cells to form the mesendoderm is severely disrupted . As a result, the precursors to the neural tube accumulate at the dorsal margin. Cross sections through this region revealed cells that appeared similar to those in WT embryos, although the tissue was significantly thicker (Figure 6D). Although the presumptive neural tube of MZoep mutants changed morphology and became "rounder" as the embryos developed to the five and ten somite stages, the boundaries of the neural tube remained indistinct and no midline could be identified (Figure 6E, F).
At 24 hpf, dorsal views of WT embryos revealed their highly ordered neuroepithelium and a distinct midline (Figure 6G). In contrast, the developing neural tube of MZoep embryos was crooked (Figure 6H), consistent with the undulating pattern of wnt1 staining in sqt mutants (Figure 3G). Further, there continued to be no apparent order to the arrangement of cells (Figure 6H). As was previously found in our cross sections (Figure 3P, R), no midline could be discerned (Figure 6H).
The morphology of cells within the neural tube was characterized by injection of mGFP DNA, which labels only a subset of cells and makes it easier to trace cell shape . The WT neural tube contained many elongated cells contacting the midline of the developing brain (Figure 6I)[28, 29, 46, 47]. Elongated cells were also present in the anterior neural tube of MZoep mutants (Figure 6J). However, there were also regions where the cells had a round morphology (Figure 6J'). The phenotypes present in the MZoep neural tube are consistent with the presence of a cell adhesion defect that inhibits formation of an ordered epithelium.
Injection of N-cad mRNA corrects the NTD in a small percentage of MZoepfish
If lack of N-cad expression and decreased cell adhesion is an important cause of the NTD in Nodal signaling mutants, then overexpression of N-cad protein might be able to correct the NTD. To test this hypothesis, we overexpressed N-cad by injecting mRNA encoding Xenopus N-cad into embryos at the one cell stage. In WT embryos, these injections caused an overexpression phenotype similar to that described by Bitzur and colleagues, suggesting that the Xenopus protein was active in zebrafish (Additional file 1) .
Injection of N-cad mRNA does not correct the NTD in MZoep mutants
Dose of N-cad mRNA (pg)
Overexpression of Taram-A* rescues mesendoderm formation and neural tube closure in MZoepmutants
Expression of Tarama* rescues mesoderm formation and neural tube closure
Non-injected MZoep embryos and those injected with mGFP mRNA alone expressed neither mesodermal marker, consistent with their severe lack of mesendodermal tissue (Figure 8E–E", Table 4). In contrast, 68% of embryos injected with Taram-A* mRNA had significant expression of ctsl1b, col2a1a, or both (Figure 8F–F", H-H", Table 4). Taram-A* is also capable of inducing a second axis, and as a consequence, the organization of the notochord cells was often abnormal (Figure 8H).
All of the embryos with a closed neural tube expressed ctsl1b, demonstrating that hatching gland cells were present (Figure 8F–F", Table 4). The hatching glands are derived from the anterior-most mesendodermal precursors, suggesting that anterior mesoderm is important for anterior neurulation. Most rescued embryos also expressed col2a1a in a group of cells that had the characteristic "stack of pennies" morphology of the notochord (Figure 8F–F", Table 4). Thus, more posterior mesoderm may also aid in closure of the anterior neural tube.
Although all embryos with a closed neural tube had rescued mesendoderm, the converse was not true. Many embryos with an elongated or divided pineal had a substantial number of notochord and hatching gland cells (Figure 8H–H", Table 4). This observation suggests that the timing or spatial aspect of mesendoderm formation may be important, which should inform future studies of neural tube closure.
In this study, we establish a new role for Nodal signaling in closure of the anterior neural tube in zebrafish. Further, we begin to identify important factors that may contribute to the neurulation defects in fish with deficiencies in the Nodal pathway. First, the cell adhesion molecule N-cad, which is required for zebrafish neurulation, is severely reduced at the cell membrane in MZoep embryos during the stages that the neural plate is folding into a neural rod. Further, we find that cell autonomously rescuing mesoderm and endoderm formation in MZoep mutants also rescues the neurulation defect. This suggests a model in which activation of the Nodal signaling pathway is required within the cells of the mesendoderm. Interactions between the mesendoderm and the overlying neural epithelium then promote neural tube closure, in part through regulating N-cad levels at the cell membrane. The NTD in zebrafish sqt and MZoep mutants is similar to the human birth defect exencephaly, in which the rostral neural tube fails to close, ultimately resulting in anencephaly, degeneration of the developing brain, and death. N-cad and Nodals are thought to play similar roles in patterning all vertebrates, including humans [52–54]. Thus, these studies may give important insights into the causes of human NTD.
Nodal signaling regulates closure of the anterior neural tube
The process of neurulation in zebrafish starts with the formation of the neural plate during gastrulation. The neural plate then "infolds" to form a neural keel and then a neural rod with no lumen [46, 47]. The neural rod subsequently cavitates to form the neural tube . During this process, the original two cell layers of the neural plate intercalate to form the single cell layer of the neural tube .
Recent work demonstrates that Nodal signaling is not required for neurulation of the trunk spinal cord, as this region of the neural tube is closed in MZoep mutants and in WT embryos overexpressing the Nodal antagonist Lefty . In contrast, we find that Nodal signaling is essential for normal closure of the rostral neural tube. As assayed by pineal anlage morphology, which we have found to be a sensitive indicator of anterior neural tube closure, all MZoep mutants have an open rostral neural tube. Together, these studies demonstrate that Nodal signaling is required for an anterior-specific mechanism of neurulation.
Our data suggests that Nodal signaling is likely required during very early embryogenesis. This is illustrated by a comparison between Zoep and MZoep mutants. Although the former has a closed neural tube, and the latter an open neural tube, they differ only in the presence and absence, respectively, of maternally supplied oep mRNA. Thus, maternal oep mRNA in Zoep mutants is sufficient for normal neural tube closure, suggesting that Nodal signaling before or shortly after the Midblastula transition is important for neurulation in the forebrain. The phenotype of MZoep mutants is consistent with such an early role. In these mutants, the two domains of pineal precursors were typically located very laterally, suggesting that little infolding of the anterior neural plate had occurred.
Mesoderm and anterior neurulation
Nodal signaling has multiple and complex roles during vertebrate development. We have just begun to understand how lack of Nodal signaling could result in an open neural tube. In mice, proliferation of the anterior mesenchyme helps drive shape changes in the cranial neural plate that are required for closure . Our data suggests that mesoderm also has a critical role in neurulation in the rostral zebrafish neural tube. MZoep mutants have only a very small amount of mesoderm in the most caudal part of their tail . Rescue of mesendoderm formation in MZoep mutants via overexpression of Taram-A* in turn rescued the NTD in a large number of embryos. Expression of Taram-A* cell autonomously drives cells to become mesendoderm [12, 48–51]. Therefore, it is probable that Nodal signaling induces mesendoderm, and mesendoderm consequently promotes anterior neurulation. One possibility is that the mesendoderm influences the neural tube closure directly through the secretion of signaling molecules or through inducing shape changes in the neural plate. However, the mesoderm could also be acting in part or completely through other intermediate tissues. For instance, at the doses of Taram-A* used in our study, the ventral brain should also have been significantly rescued .
Role of N-cad in closure of the forebrain neural tube
Our data suggests that lack of Nodal signaling ultimately decreases the expression of N-cad or its localization to the plasma membrane, as N-cad protein at the membrane is severely reduced in MZoep mutants. The ordered arrangement of cells within the neural tube and the ability of the neural tube cells to form an elongated shape are also perturbed. These phenotypes are consistent with a cell adhesion defect [28, 29]. Further support for a connection between Nodal signaling and N-cad comes from our rescue experiments. In a small number of cases, we were able to induce closure of the MZoep neural tube solely through N-cad overexpression.
As yet, we do not understand why injection of N-cad mRNA failed to rescue neurulation in the majority of MZoep fish. One possibility is that the Xenopus protein is not fully functional in zebrafish. Alternatively, N-cad mis-regulation may not the only cause of neurulation failure in MZoep fish. The neural tube defects in MZoep mutants are somewhat different and more severe than those found in fish that lack N-cad. For instance, the hindbrain neural tube in n-cad mutants has a very characteristic "T" shape that reflects the fact that the basal (ventral) neural tube forms fairly normally, while the alar (lateral/dorsal) neural tube fails to fold [28, 29]. In contrast, the hindbrain neural tube of the MZoep mutants was severely disrupted along the whole dorsal-ventral axis. In the most severely affected MZoep mutants, the hindbrain neural tube appeared to have split along the midline. This suggests that Nodal signaling may regulate proteins in addition to N-cad, possibly even other proteins in the cadherin family. As an additional complication, there may be a very narrow range of N-cad protein that promotes neural tube closure. Too little, and the neural tube remains open, as in the zebrafish n-cad mutants and morphants. Overexpression or expression in the wrong place causes morphological defects (this study and ).
In addition, it may be difficult to rescue the MZoep NTDs using mRNA. We found that N-cad mRNA levels are comparable between MZoep mutants WT fish (data not shown). This suggests that the defect is primarily in the post-transcriptional regulation of N-cad. Cadherin-mediated adhesion is subject to many types of post-transcriptional regulation, including modulation by the Wnt signaling pathway and entry into recycling and degradation pathways [57, 58]. Future studies will be needed to define how lack of Nodal signaling effects all of the biochemical mechanisms that control N-cad levels at the cell membrane.
Role of N-cad in closure of the forebrain neural tube
N-cad is expressed strongly in the developing neural tube of many vertebrates, including zebrafish [29, 45, 56, 59, 60]. In Xenopus, downregulation of E-cadherin and upregulation of N-cad in the neural epithelium is critical for its separation from the non-neural epithelium . While N-cad knockout mice have a closed neural tube, the neural tube expresses other cadherins, leading to the suggestion that a related protein(s) is sufficient to mediate the cell adhesion required for folding . In zebrafish, loss of N-cad causes several NTDs that include altered cell adhesion and an open neural tube in the mid-diencephalon, midbrain, and hindbrain [29, 63]. Recently, Hong and Brewster (2006) have demonstrated that N-cad is important for both the infolding of the cells at the lateral edges of the neural plate and for the radial intercalation of these cells to form a monolayer.
Here, we add to the role of N-cad in neurulation by demonstrating that it is also important for neural tube closure in the anterior diencephalon, as convergence of the pineal precursors is often incomplete in pac and glo mutants and n-cad morphants. The variability in the pineal phenotype was unexpected, because other defects in pac and glo mutants are completely penetrant [29, 63]. One possibility is that the alleles we are using are not null mutations, and residual N-cad activity is sufficient to mediate closure in some embryos. The pacp79emcfallele causes an Isoleucine-to-Serine substitution in a conserved amino acid in the fifth cadherin extracellular (EC5) domain . The EC5 domain is not essential for C-cadherin's function in cell-cell adhesion . Thus, there may be some residual N-cad activity in pac79emcfmutants. In contrast, the glom117 allele causes a Trytophan-to-Glycine substitution in the second amino acid in the EC1 domain . This Tryptophan residue is essential for cell-cell adhesion, strongly suggesting that glom117 is a null allele [66–68]. Thus, the different degrees of N-cad depletion could account for why the percentage of embryos with an elongated/divided pineal is higher in glom117 than pac79emcfmutants, but is unlikely to explain why the pineal phenotype is incompletely penetrant even in glom117 embryos. Closure of the anterior region of the mouse neural tube is sensitive to genetic background . Thus, another possibility is that the anterior border of the NTD varies in N-cad depleted embryos due to the fact that they are not isogenic.
Loss of ventral neural tube does not correlate with anterior NTDs
In mice and chick, the ventral neural tube forms the medial hinge point, a key site of bending in the neural plate, and also produces a signal (Sonic Hedgehog, Shh) that regulates bending at more lateral hinge points [14, 16, 30, 69–72]. However, although Zoep and cyc mutants lack ventral brain and floor plate, we find that their anterior neural tube is closed. Thus, ventral brain is not essential for rostral neural tube closure in zebrafish. One possibility is that ventral neural tube does not play a role in closure of the zebrafish anterior neural tube. However, another possibility is that the rostral neural tube closes using an alternative pathway when the ventral neural tube is absent. The spinal cord neural tube is closed in mice mutants that lack floor plate, but the mechanism of closure is different [73–78]. In normal mice, the upper spinal cord neural tube folds by bending at the median hinge point. Ybot-Gonzalez et al. have demonstrated that in the absence of Shh signaling and formation of the medial hinge point, bending at two dorsal lateral hinge points occurs instead and is sufficient to close the neural tube .
Nodal signaling in neural tube in other vertebrates
Although the Nodal signaling pathway has several well-defined and conserved functions in neural development, the role of Nodal signaling in neural tube closure remains largely unexplored. We have been unable to find any known links between Nodal and NTD in humans. However, there are some indications that Nodal signaling has a conserved role in vertebrate neurulation. Zic transcription factors act as negative regulators of nodal gene expression in mice and Xenopus [9, 79]. Specifically, in mice, Zic3 acts upstream of Nodal signals in regulating left-right asymmetry of visceral organs. Interestingly, many aspects of the zic2 and zic3 mutant phenotypes are similar to those found in zebrafish Nodal signaling mutants. Holoprosencephaly, or a failure in the hemispheres of the forebrain to separate , is the cause of the cyclopia in cyc, sqt, and oep mutants. Mutations in zic2 in mice and humans also cause holoprosencephaly [80, 81]. Most importantly for this study, both zic3 and zic2 mutant mice also have neurulation defects. Lack of Zic2 causes a delay in neurulation, spina bifida, and at a lower rate, exencephaly . zic3 mutants have a variable defect that can include an open anterior neural tube (exencephaly and anencephaly) as well as neurulation defects in more posterior regions of the embryo . It is tempting to speculate that the role of Zic2 and Zic3 in neurulation is a reflection of their role in regulation of Nodal signaling.
Failures in neural tube closure are some of the most common birth defects in humans . Here we have demonstrated that Nodal signaling has an essential role in the closure of the anterior neural tube. This study and those of others demonstrate that zebrafish has great potential to reveal new genes involved in neurulation, and thus new potential causes of human NTDs.
Zebrafish stocks were maintained at 28.5°C and a 14:10 hour light:dark cycle using standard methods . WT (Oregon AB) and mutant embryos were produced from natural matings. Mutant alleles used included sqtcz35 , cycm294 , oepm134 , pacp79emcf, glom117 , and flh:eGFP1 (flh:eGFP1). Adult oepm134/oepm134 fish were generated by rescuing homozygous mutants to viability by injection of oep mRNA at the 1–2 cell stage [19, 34], and used to produce MZoep embryos.
Whole mount in situ hybridization
Whole mount RNA in situ hybridization was carried out using established methods [19, 83]. Antisense probes included otx5 , wnt1 [42, 43], cone-rod homeobox (crx) , transducin α-subunits (rod, gnat1; cone, gnat2) , rod opsin (rho) , flh , epha4a, (formerly named receptor tyrosine kinase 1), ctsl1b (formerly named hgg1) , col2a1a , and n-cad .
Standard fluorescent immunocytochemical methods were used for the detection of N-cad protein in 12 μm zebrafish tissue sections. The N-cad antibody was an affinity purified, rabbit polyclonal antibody prepared against zebrafish N-cad extracellular domain one . Tissue sections were incubated overnight at 4°C in the N-cad antibody (6 μg/ml), followed by an anti-rabbit secondary antibody conjugated with Cy3 (1: 100 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA), for 2 hours at room temperature. The sections were washed in PBS, and mounted with 90% glycerol in PBS containing 1 mM CaCl2.
Procedures for whole mount immunohistochemistry were described in detail previously . The primary antibody zpr1 (Zebrafish International Resource Center, University of Oregon, Eugene, OR) was used at a 1:1000 dilution. A biotinylated secondary antibody (anti-mouse IgG from Vector Laboratories, Burlingame, CA) was used at 1:200. Visualization of the reaction was achieved by using a DAB kit (Vector Laboratories).
Embryos were processed as described above for whole mount in situ hybridization and then post-fixed overnight in 4% paraformaldehyde in PBS, pH 7.4, equilibrated in 30% sucrose in DEPC PBS for twenty minutes, and transferred to 100% glucose. The embryos were decapitated to allow for easier orientation, and the heads were dipped in Tissue-Tek® O.C.T. compound and positioned for transverse sections in a Pelco flat embedding chamber. The block was allowed to solidify on dry ice and then sectioned at -20°C on a Leica CM3050S cryostat.
A translation blocking n-cad specific antisense morpholino oligonucleotide (n-cad MO 5'-TCTGTATAAAGAAACCGATAGAGTT-3'), and a standard control MO (5'-CCTCTTACCTCAGTTACAATTTATA-3', gifts from James Marrs (Indiana University) who purchased the oligonucleotides form Gene Tools, Philomath, OR, were used in the experiments. The n-cad MO, dissolved in Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6) to a concentration of 100 μM, was injected into 1–4 cell stage embryos (1–2 nl/embryo). Injection of the n-cad MO at this concentration was previously shown to phenocopy pac mutants , while embryos injected with the control MO were morphologically indistinguishable from uninjected WT embryos .
mRNA and DNA injections
mRNA was synthesized in vivo using the mMessage Machine Kit (Ambion). To express GFP and N-cad throughout the embryo, single blastomeres of one cell stage embryos were injected with 100 pg of mGFP mRNA of the indicated amount of N-cad mRNA using a Harvard Apparatus PLI-90 nitrogen picoinjector. mGFP was expressed in a subset of cells by injecting one cell stage embryos with 100 pg of pCS2+mGFP.
To express Taram-A* in a subset of cells within the embryo, single blastomeres of 16-cell stage embryos were co-injected with a mixture of 0.15–2.4 pg of Taram-A* mRNA and 100 pg mGFP mRNA. Control embryos were injected with mGFP mRNA alone or left uninjected.
Fluorescent and bright field images were taken on an Axiocam camera mounted on a Zeiss Axioplan 2 Imaging microscope or a SPOT camera mounted on an Olympus BX51 epifluorescence microscope. Confocal optical sections were obtained using a Leica True Confocal Scanner.
We would like to thank Drs. Marnie Halpern and Ron Conlon for their valuable suggestions on the manuscript, Drs. Pamela Raymond, Laure Bally-Cuif, Joshua Gamse, and Dr. Barry Gumbiner for providing the cDNAs of photoreceptor-specific genes, neural tube genes, mGFP, and N-cad, respectively, and Dr. James Marrs for the n-cad MO. Onkar Dhande, Bindu Manyam, and Derek Laux provided valuable technical assistance. Supported in part by research grant No. 5-FY02-259 from the March of Dimes Birth Defect Foundation (to J.O.L.), an Edward Mallinckrodt, Jr. Foundation grant (to J.O.L.), and NIH EY13879 (to Q.L.).
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