Mesoderm is required for coordinated cell movements within zebrafish neural plate in vivo
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Morphogenesis of the zebrafish neural tube requires the coordinated movement of many cells in both time and space. A good example of this is the movement of the cells in the zebrafish neural plate as they converge towards the dorsal midline before internalizing to form a neural keel. How these cells are regulated to ensure that they move together as a coherent tissue is unknown. Previous work in other systems has suggested that the underlying mesoderm may play a role in this process but this has not been shown directly in vivo.
Here we analyze the roles of subjacent mesoderm in the coordination of neural cell movements during convergence of the zebrafish neural plate and neural keel formation. Live imaging demonstrates that the normal highly coordinated movements of neural plate cells are lost in the absence of underlying mesoderm and the movements of internalization and neural tube formation are severely disrupted. Despite this, neuroepithelial polarity develops in the abnormal neural primordium but the resulting tissue architecture is very disorganized.
We show that the movements of cells in the zebrafish neural plate are highly coordinated during the convergence and internalization movements of neurulation. Our results demonstrate that the underlying mesoderm is required for these coordinated cell movements in the zebrafish neural plate in vivo.
KeywordsZebrafish neurulation Morphogenesis Mesoderm
Anti-acetylated tubulin antibody
Analysis of variance
Animals (Scientific Procedures) Act 1986
Atypical protein kinase C
Cell division inhibitor
Glial fibrillary acidic protein
Histone H2B/red fluorescent protein fusion
Hours post fertilization
Maternal-zygotic one-eyed pinhead
Green fluorescent protein/polarity protein partitioning defective 3 fusion
Planar cell polarity
Phospho-histone 3 marker
Region of interest
Standard error of the mean
Transforming growth factor beta
Zonula occludens 1.
Morphogenesis of the vertebrate neural tube from the neural plate is a fundamental early step in building the brain and spinal cord. This complex process is likely to be coordinated by a combination of mechanisms both intrinsic and extrinsic to the neural tissue itself. One important intrinsic mechanism is the non-canonical Wnt/planar cell polarity (PCP) pathway that regulates the movements of convergent extension to shape the neural plate. This pathway is thought to act through cell-cell interactions within the neural plate itself and appears to be a prerequisite for efficient neural tube closure and morphogenesis in all vertebrates (reviewed by Ueno and Greene ). However, embryological and genetic approaches have also suggested that adjacent tissues such as the mesoderm can directly influence neural tube morphogenesis [2, 3, 4, 5]. Mutations affecting proliferation in mouse cephalic mesoderm suggest that this tissue is critical for the correct shape and closure of anterior neural tube structures  (reviewed by Copp et al. ). In addition, experiments using amphibian explant cultures suggest that persistent interactions with the underlying mesoderm are required for the cell elongation, cell protrusive and cell intercalation events present during neural plate morphogenesis in vitro[7, 8]. However, the degree to which such tissue interactions influence cell behavior has not been analyzed in vivo.
The zebrafish embryo provides a good model to study tissue interaction in vivo because of its superior optical qualities. A common feature in teleost and other vertebrate embryos is that the neural plate lies on a subjacent layer of mesoderm and the first steps in the process of neurulation involve the convergence of the neural plate towards the dorsal midline [9, 10]. The later stages of neurulation in teleost embryos are different to other vertebrates in that the neural tube is not formed by folding an epithelial neural plate, rather the teleost neural tube is built by generating a lumen at the center of a solid neural rod primordium (reviewed by Lowery and Sive  and Clarke ). The solid neural rod is built by the orchestrated actions of large numbers of cells from both sides of the neural plate that converge towards the dorsal midline where they become internalized. The mechanism of neural internalization in the teleost is a poorly understood process but it results in a structure known as the neural keel, which then condenses into a solid neural rod. Subsequently, the neural rod cavitates to form a neural tube with a single central lumen surrounded by neuroepithelium with clear apicobasal polarity. At a cellular level, neural tube architecture is achieved by a combination of behaviors including cell intercalation, midline-crossing divisions and polarized cell behavior [10, 13, 14, 15, 16, 17, 18]. A possible role for mesoderm in zebrafish neurulation is suggested by the anterior brain defects in maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack Nodal signaling and anterior mesoderm , but a detailed analysis of neural morphogenesis in these mutants is lacking.
To better understand the roles of mesoderm in neural tube morphogenesis we have taken a live imaging approach to analyze neural cell movements in normal embryos and embryos lacking mesoderm. We found that at early stages of zebrafish neurulation the underlying mesoderm is required for the coordinated movements of neural plate cells. In the absence of mesoderm a neural primordium does develop at the dorsal midline but its tissue architecture is severely disorganized.
MZoep embryos have severe defects in neural tube morphogenesis
The mesoderm, but not Nodal signaling, is required for zebrafish neurulation
To test the efficacy of our SB-431542 treatment, we examined the expression of the Nodal downstream genes lefty1 and pitx2. We found the wild-type expression of both lefty1 and pitx2 (Figure 4H) was lost in embryos treated with SB-431542 (Figure 4H) at either the one-cell stage or just prior to neurulation (70 to 80% epiboly).
To test the cell autonomy of neural plate cell behavior in the MZoep embryos we carried out cell transplantation experiments. MZoep cells transplanted into a wild-type background were not only able to incorporate into the wild-type neural plate with a normal elongated morphology, but also undergo midline-crossing divisions at the same time as their wild-type peers (Figure 4I) and generate a pair of daughter cells of mirror-symmetric morphology (Figure 4I’). In complementary experiments, although wild-type cells transplanted into MZoep background followed the abnormal movements of mutant neural primordium, wild-type cells were able to divide and generate a pair of mirror-image sister cells within this disrupted environment (Figure 4J,J’). These results suggest that Nodal signaling is not required during neurulation for the normal cell behaviors leading to neural tube formation.
Movements of the neural plate with and without underlying mesoderm
The development of polarity in MZoep neural plate
During zebrafish neurulation neural plate cells first converge towards the dorsal midline, then internalize to form the neural keel, which subsequently transforms via a solid rod primordium to become the neural tube. In this work we show that underlying mesoderm is required for the coordinated and persistent movements of the neural plate cells during convergence and keel formation.
The loss of mesoderm has dramatic effects on the directionality and coordination of cell movements within the neural plate. Although the initial structure of the neural plate at 10 hpf appears normal, cells are unable to converge normally towards the midline and generate a neural keel. Despite moving in the general direction of the midline, cell velocity is reduced and they are unable to maintain persistent directionality. In comparison to wild-type cells, the nuclei in the neural plates of mesodermless embryos are unable to maintain their relative positions within the depth of the neural plate. We do not know the mechanism by which mesoderm influences the neural plate cells. One possibility is the presence of the underlying mesoderm provides a physical barrier to constrain the space in which neural plate cells can move, that is, it forms the floor of a thin corridor that could restrict the movement of the neural plate cells to the mediolateral axis. Alternatively there may be an active coupling of the mesoderm and the neural plate such that neural plate convergence is at least partially driven by the convergence movements of the mesoderm. Our time-lapse observations show that the movements of neural plate cells and mesoderm cells are indeed very closely coupled during convergence towards the midline (Figure 6B). Although they are in close proximity, there is probably no physical contact between cells of the neural plate and mesoderm because there is an intervening basal lamina. The basal lamina will be enriched with extracellular matrix (ECM) proteins such as laminin and fibronectin and this raises the possibility that they might have a potential role in coordinating the movements of mesoderm and neural plate. ECM has been proposed to play a central role in a variety of processes that could be relevant to this interaction, including cell movements and tissue rearrangements [31, 32, 33, 34]. In frogs, initial gastrulation movements are marked by the involution and subsequent migration of the mesoderm under the ectodermal blastocoel roof towards the animal pole . A number of studies have shown that mesoderm migration depends on a well-developed fibronectin matrix, which is deposited by the overlying blastocoel roof [36, 37, 38, 39, 40]. Interestingly, blocking experiments against fibronectin-fibronectin assembly results in loss of tissue apposition between mesoderm and blastocoel roof and altered tissue dynamics during blastopore closure . In the future it will be important to address whether ECM components contribute to neural plate movements and to the coupling of mesoderm to neural plate in the fish. The role of the ECM could be quite complex as the ECM itself may well be remodeled during this phase of morphogenesis [38, 39] and indeed the ECM may move along with the moving cells and tissues [41, 42].
Since the development of cell polarity is likely to be a critical factor in neural tube formation in the zebrafish [10, 13, 14, 17, 18] we asked whether loss of mesoderm had a major influence on the development of polarity in the neural cells. One measure of polarity in this developing epithelium would be the development of cell-cell junctions. Thus, using ZO-1 immunoreactivity as an assay of polarity we found its expression is initially detected in the neural plate at approximately the same time in MZoep and wild-type cells but that its accumulation into distinct puncta is delayed by approximately 30 minutes in MZoep. After this short delay polarization continues with the same schedule as wild-type, although the distribution of ZO-1 puncta is more scattered in the MZoep tissue. It is possible that the 30-minute delay represents a genuine delay in the cellular mechanisms that drive polarization, but it may also be possible that the more random movements of the MZoep cells will lead to cell-cell contacts between neighboring cells becoming more transient and this could destabilize distinct cell-cell junctions. Since we have previously shown by ectopic transplantation strategies that the dorsal mesoderm is not required for neural polarization and lumen formation , we favor the view that mesoderm is more important in directing neural cell movements than initiating neural cell polarization. It is possible that mesoderm acts to coordinate the orientation of neural polarity and that this could influence how the neural cells move.
One further possibility is that the defective cell movements in MZoep result not from a lack of mesoderm but rather from an unfavorable interaction of the neural plate with the underlying yolk, which is a tissue that it would not normally be exposed to. This possibility suggests that loss of mesoderm simply allows the neural plate to interact with the yolk rather than removing a positive influence of mesoderm. While we cannot rule out this possibility, we feel the tightly coupled movements of mesoderm and neural plate that our analysis reveals, plus the previously published work suggesting mesoderm is required for neural plate movements in Xenopus explants [7, 8], all point to a positive influence of mesoderm on the neural plate.
At present the motive forces driving the neural plate movements of convergence and internalization in the fish are almost completely unknown. In addition to potential influences for the mesoderm, other non-neural tissues could also be involved (reviewed by Gordon  and Colas and Schoenwolf ). In chicken embryos for instance, in vitro explant experiments suggest that the adjacent non-neural ectoderm provides a ‘pushing force’ required to shape neural fold formation and encourage dorsal closure of the neural tube [45, 46]. More recently, functional studies and live tissue analyses in amphibian embryos indicate that pulling forces generated by the deep layer of non-neural ectoderm are required to complete neural tube closure in Xenopus and this partially depends on the cell adhesion molecule E-cadherin and the ECM receptor integrin-β1 .
Finally our observations show that the location and orientation of cell divisions that usually take place across the midline of the neural rod are severely disrupted by loss of mesoderm. The regulation of these morphogenetically powerful divisions [13, 14] has recently been shown to be under the control of the non-canonical Wnt receptor Frizzled 7  and the polarity protein Scribble . Although these divisions are disrupted in the absence of mesoderm, this is not the primary cause of the neural tube defects in these embryos because, unlike the other morphogenetic mutants [13, 14, 15, 16], inhibiting these divisions in the mesodermless embryos does not rescue neural tube morphogenesis. The primary cause of the mesodermless neural phenotype is thus established before and is independent of the midline divisions. In contrast, apart from misregulation of the oriented divisions, in the vangl2, fz7 and scrib mutants any other cellular or molecular defects related to neural tube development must be relatively minor because their neural tube defects are lost when these divisions are blocked [13, 14, 15, 16].
We show that the movements of cells in the zebrafish neural plate are highly coordinated with the movements of the underlying mesoderm during the convergence and internalization movements of neurulation. Our analyses of mesodermless embryos demonstrate that the underlying mesoderm is required for the coordinated convergence movements of the zebrafish neural plate cells in vivo.
All procedures were approved by the College Research Ethics Committee at King’s College London (London, UK) and covered by the Home Office Animals (Scientific Procedures) Act 1986 (ASPA) project licence.
Zebrafish embryos were collected and staged using standard protocols  and provided by the King’s College London fish facility. The following zebrafish alleles were used: wild-type TL, oeptz 257 , cyc m294 , ntl 160 , syut 4  and tg(HUC-GFP) . MZoep mutant embryos were generated by crossing oeptz 257 adult zebrafish previously rescued by injection of oep mRNA at one-cell stage . For time-lapse confocal movies tg(H2A-GFP)  was used. Embryos were grown at 28.5°C and staged according to morphology  and age hpf. mRNA and morpholino injections PCS2+ vectors carrying cDNA fragment encoding for Pard3-GFP (kind gift of Dr Alexander Reugels, University of Cologne, Cologne, Germany), membrane-GFP , membrane-RFP  and histone H2B/red fluorescent protein fusion (H2B-RFP)  were used in this study. Capped RNAs were transcribed using SP6 and T7 RNA polymerase using the mMESSAGE mMACHINE Kit (Ambion, Austin, TX, USA) and 180 pg Pard3-GFP, 120 pg of membrane-GFP, 150 pg of membrane-RFP and 150 pg of H2B-RFP mRNA were injected into wild-type or MZoep embryos. For ubiquitous expression, mRNA was injected at the one-cell stage. For rescue experiments, MZoep embryos were injected with 8 to 10 pg of Taram-A* alone or co-injected with mGFP mRNA in one of the marginal blastomeres at 16- or 32-cell stage . Morpholino oligonucleotides (MOs; Gene Tools, Philomath, OR, USA) were dissolved in water to a concentration of 4 mM and stored at −20°C. All MOs were injected at one-cell stage. To generate MZoep/spt double-mutant embryos, a combination of two previously characterized MOs  against the spt gene were injected into MZoep embryos: spt1-MO 5′-AGCCTGCATTATTTAGCCTTCTCTA-3′ (0.4 pmoles/embryo) and spt2-MO 5′-GATGTCCTCTAAAAGAAAATGTCAG-3′ (0.4 pmoles/embryo). These concentrations of morpholinos have previously been shown to mimic the phenotype of the spadetail mutant .
In situ hybridization
Antisense RNA probes were synthesized with digoxigenin RNA labelling kit (Roche, Basel, Switzerland) using plasmid-containing cDNA for left1, pitx2, krox-20 and foxc1a. Embryos were fixed and stained at appropriate stages. To confirm that neural tube morphology was rescued when Taram-A*-expressing cells became mesoderm, we assessed the expression of the cephalic mesoderm marker, foxc1a.
For whole mount and cryosection immunostaining, embryos were fixed in 4% paraformaldehyde (PFA) at 4°C overnight at different stages (between 11 to 24 hpf). Embryos were blocked in 10% normal goat serum (Sigma-Aldrich, St Louis, MO, USA) for 2 hours at room temperature. The following primary antibodies were used in this study: mouse-anti-ZO-1 (339111; Zymed Laboratories, South San Francisco, CA, USA) at 1:300; rabbit-anti-aPKC (C-20; Santa Cruz Biotechnology, Dallas, TX, USA) at 1:500; rabbit-anti-GFAP (Z0334; DakoCytomation, Glostrup, Denmark) at 1:500; mouse-anti-MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) at 1:50; mouse-anti-acetylated-tubulin (T6793; Sigma-Aldrich); and rabbit-anti-phospho-histone H3 (Upstate Biotechnology, Lake Placid, NY, USA) at 1:200, diluted in 2.5% normal goat serum (Sigma-Aldrich). For secondary antibodies, anti-rabbit and anti-mouse Alexa 488, Alexa 568 and Alexa 633 (Molecular Probes, Eugene, OR, USA) were used at 1:800 in 2.5% normal goat serum. Sections were cut every 14 to 16 μm on a Leica 5100 or Cryo-Star HM 560 MV Micron microtomes.
To block Nodal signaling, wild-type embryos were treated with SB-431542 inhibitor . SB-431542 (4-(4-(1,3-benzodioxol-5-yl)-5-(2-pyrindinyl)-1H-imidazol-2-yl)benzamide; Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) to make a 100 mM stock and stored at −20°C. For drug treatment, approximately 30 embryos at two- to four- cell stage or before neurula stage (70% epiboly, 7 to 8 hpf) were put in a small dish containing 100 μM of SB-431542 dissolved in embryo medium and raised at 28.5°C.
Cell division inhibition
To inhibit cell division, wild-type and MZoep embryos were treated with a combination of 150 μM aphidicolin (Biomol, Hamburg, Germany) and 50 mM hydroxyurea (Sigma-Aldrich) dissolved in 4% DMSO. Inhibition of cell division was performed at tail-bud stages and quantification of PH3+ cells was done for equivalent tissue volumes (360 μm in length × 130 μm in width × 90 μm in depth).
Wild-type and MZoep mutant embryos were used as host and donor embryos. To identify donor cells from host cells, donor embryos were previously injected with either membrane-GFP or membrane-RFP. Between sphere and dome stage (4.0 to 4.3 hpf), dechorionated embryos were transferred into an agarose chamber and about 30 donor cells were transplanted into the prospective hindbrain region of a host embryo.
For time-lapse confocal analysis, embryos were manually dechorionated at tail-bud stage (10 to 11 hpf) and mounted in 1.2% low melting point agarose (Sigma-Aldrich) in embryo medium (E3). For neural plate mesoderm tracking analysis, embryos were imaged in transverse view at the level of the first or second somites. Confocal images were taken 5 to 8 μm apart, using a 40× long working distance water immersion objective in an environment chamber at 28.5°C. Z-stacks were collected at 5-minute intervals, usually starting between 10 to 11 hpf and continuing through to 18 to 20 hpf.
Cell movement analysis
where chdθ equals the length traveled by a cell (the chord) and 2r equals the diameter of the projected circle. For each experimental condition, six to ten embryos from independent experiments were analyzed. To test for significance between mean values, Student’s t-test was applied between wild-type and MZoep embryos. A probability of 0.05 or less was accepted as statistically significant. For each condition, the standard error of the mean (SEM) was calculated. Analysis and graphical representations were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and MATLAB (R14b; MathWorks, Cambridge, UK) statistical programs.
Quantifying the appearance of ZO-1 puncta
For analysis of the appearance of ZO-1 puncta in wild-type and MZoep embryos, the following steps were taken to ensure that staining and imaging across specimens was as consistent as possible. 1) Immunohistochemistry: at the time of fixation, wild-type and MZoep embryos were staged under the dissecting microscope according to the number of somites. Embryos were fixed in 4% PFA from the same aliquot and fixed overnight at 4°C for the same time period. Embryos were then washed in fresh PBS, dehydrated into methanol and put at −20°C to permeabilize the embryos. When all embryos of all stages had been placed at −20°C for at least 1 hour, the normal immunohistochemistry protocol was followed with all tubes of embryos being treated identically. For all stages, the same aliquot of antibody was used at the same dilution. 2) Imaging: after washing of the secondary antibody, all embryos were mounted for dorsal view imaging through the hindbrain. Confocal setting including laser power, gain, offset, pinhole and averaging were set at the beginning of imaging and left unchanged throughout. All embryos were imaged on the same day and the same z-stack parameters were used for each embryo (25 z-slices at 3 μm intervals), starting from the dorsal most part of the neural primordium. 3) Data analysis: a histogram of all pixel intensities (256 gray levels) was derived from a large region of interest (ROI) (100 μm × 100 μm) in each of three different z-levels per specimen. z-levels at comparable dorsoventral depths were chosen for the analysis across embryos to minimize the change in intensity that occurs with z-depth. Care was taken to avoid sampling any signal from the polarized enveloping layer and from the ventral most z-levels because in wild-type embryos these polarize earlier than the rest of the tissue and are not present in MZoep mutants due to the loss of ventral midline-specified cells . For each time point, two to eight mutant or wild-type embryos were analyzed. The average maximum background pixel intensity was calculated from maximum intensity measurements at five locations in each chosen z-slice. Background pixel intensity was sampled from a smaller ROI (10 μm × 10 μm), avoiding obvious real signal. Pixels below this cut-off value were regarded as background and any pixels above were counted as signal. Finally, as the sampled number of pixels was the same for every measurement, the average number of pixels above background was calculated for wild-type or MZoep embryos at each stage.
The authors thank Masa Tada and all members of the Clarke laboratory, London, UK for extensive and helpful discussion of this work. This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) to JC and Fondecyt 11110106 to CA.
Additional file 2: Movie S2: Time-lapse movie taken in the transverse plane through the left-hand side of the neural plate and underlying mesoderm at the level of anterior spinal cord (approximate area of rectangle in first frame of Movie S1). The midline of this wild-type embryo is indicated with an arrow. The nuclei of neural plate cells (yellow dots) and subjacent mesoderm cells (red dots) are seen to move in a closely coordinated way. Nuclei of the overlying enveloping layer (blue dots) are seen to remain stationary. (MOV 1 MB)
Additional file 3: Movie S3: Time-lapse movie taken in the transverse plane through the left-hand side of the neural plate at the level of the hindbrain (approximate area of rectangle in first frame of Movie S1). The midline of this MZoep embryo is indicated with an arrow. In contrast to normal embryos, 12 nuclei (blue dots) that initially lie at the superficial surface of the neural plate are seen to distribute themselves throughout the deeper layers of the neural plate in subsequent frames. Nuclei that appear only in later frames at the superficial surface are marked with red and green dots. The depth of the neural plate is also seen to enlarge as the movie progresses. (MOV 825 KB)
- 3.Takaya H: On the types of neural tissue developed in connection with mesodermal tissues. Ann Zool Jap. 1956, 4: 287-292.Google Scholar
- 9.Jessen JR, Topczewski J, Bingham S, Sepich DS, Marlow F, Chandrasekhar A, Solnica-Krezel L: Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. Nat Cell Biol. 2002, 8: 610-615.Google Scholar
- 11.Lowery LA, Sive H: Strategies of vertebrate neurulation and a reevaluation of teleost neural tube formation. Development. 2004, 2057–2067: 132-Google Scholar
- 15.Quesada-Hernandez E, Caneparo L, Schneider S, Winkler S, Liebling M, Fraser SE, Heisenberg CP: Stereotypical cell division orientation controls neural rod midline formation in zebrafish. Curr Biol. 1966–1972, 2010: 20-Google Scholar
- 22.Imman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS: SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002, 62: 65-74. 10.1124/mol.62.1.65.CrossRefGoogle Scholar
- 27.Thisse C, Thisse B: Antivin, a novel and divergent member of the TGFbeta superfamily regulates anteroposterior endoderm patterning in zebrafish. Mech Dev. 1999, 126: 229-240.Google Scholar
- 38.Davidson LA, Keller R, DeSimone DW: Assembly and remodeling of the fibrillar fibronectin extracellular matrix during gastrulation and neurulation in Xenopus laevis. Dev Biol. 2004, 231: 888-895.Google Scholar
- 48.Westerfield M: The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). 4th edition. 2000, Eugene, OR: University of Oregon PressGoogle Scholar
- 51.Park HC, Kim CH, Bae YK, Yeo SY, Kim SH, Hong SK, Shin J, Yoo KW, Hibi M, Hirano T, Miki N, Chitnis AB, Huh TL: Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. Dev Biol. 2000, 227: 279-293. 10.1006/dbio.2000.9898.CrossRefPubMedGoogle Scholar
- 54.Carreira-Barbosa F, Kajita M, Morel V, Wada H, Okamoto H, Martinez Arias A, Fujita Y, Wilson SW, Tada M: Flamingo regulates epiboly and convergence/extension movements through cell cohesive and signalling functions during zebrafish gastrulation. Development. 2009, 136: 383-392. 10.1242/dev.026542.PubMedCentralCrossRefPubMedGoogle Scholar
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