Primary neurulation is the process by which the neural tube, the central nervous system precursor, is formed from the neural plate. Incomplete neural tube closure occurs frequently, yet underlying causes remain poorly understood. Developmental studies in amniotes and amphibians have identified hingepoint and neural fold formation as key morphogenetic events and hallmarks of primary neurulation, the disruption of which causes neural tube defects. In contrast, the mode of neurulation in teleosts has remained highly debated. Teleosts are thought to have evolved a unique mode of neurulation, whereby the neural plate infolds in absence of hingepoints and neural folds, at least in the hindbrain/trunk where it has been studied. Using high-resolution imaging and time-lapse microscopy, we show here the presence of these morphological landmarks in the zebrafish anterior neural plate. These results reveal similarities between neurulation in teleosts and other vertebrates and hence the suitability of zebrafish to understand human neurulation.

BackgroundShaping of the neural tube, the precursor of the brain and spinal cord, involves narrowing and elongation of the neural tissue, concomitantly with other morphogenetic changes that contribue to this process. In zebrafish, medial displacement of neural cells (neural convergence or NC), which drives the infolding and narrowing of the neural ectoderm, is mediated by polarized migration and cell elongation towards the dorsal midline. Failure to undergo proper NC results in severe neural tube defects, yet the molecular underpinnings of this process remain poorly understood.ResultsWe investigated here the role of the microtubule (MT) cytoskeleton in mediating NC in zebrafish embryos using the MT destabilizing and hyperstabilizing drugs nocodazole and paclitaxel respectively. We found that MTs undergo major changes in organization and stability during neurulation and are required for the timely completion of NC by promoting cell elongation and polarity. We next examined the role of Microtubule-associated protein 1B (Map1b), previously shown to promote MT dynamicity in axons. map1b is expressed earlier than previously reported, in the developing neural tube and underlying mesoderm. Loss of Map1b function using morpholinos (MOs) or δMap1b (encoding a truncated Map1b protein product) resulted in delayed NC and duplication of the neural tube, a defect associated with impaired NC. We observed a loss of stable MTs in these embryos that is likely to contribute to the NC defect. Lastly, we found that Map1b mediates cell elongation in a cell autonomous manner and polarized protrusive activity, two cell behaviors that underlie NC and are MT-dependent.ConclusionsTogether, these data highlight the importance of MTs in the early morphogenetic movements that shape the neural tube and reveal a novel role for the MT regulator Map1b in mediating cell elongation and polarized cell movement in neural progenitor cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0056-4) contains supplementary material, which is available to authorized users.

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly. Video LinkThe video component of this article can be found at https://www.jove.com/video/55792/ (Dclk) or Ensconsin (EMTB) 10,11 . Other lines (and constructs) have been generated that enable assessment of MT intrinsic polarity by specifically labeling MT plus ends or centrosome-anchored minus ends 11,12,13,14 . The power of these tools lies in the ability to study MT dynamics in live, developing organisms. Such studies have revealed, for example, the spatial and dynamic distribution of MTs in specific cell populations, the orientation of mitotic spindles in tissues undergoing morphogenesis (an indicator of the plane of cell division), the polarity of the MT polymer as it relates to processes such as cell elongation and migration, and MT growth rate determined by comet speed 9,13,15 . The limitation of these tools is that they do not readily discriminate between stable and dynamic MT populations.

The planar cell polarity (PCP) pathway plays a significant role in facilitating neural convergence (NC) – the narrowing of the neural plate before the formation of the neural tube. Evidence from the literature and our laboratory suggests that NC in zebrafish requires elongation and midline‐directed polarized migration of neural plate cells. Failure of NC or delayed stages of neural tube morphogenesis can result in severe neural tube defects (NTDs) which have been observed in all vertebrates studied. Although perturbation of the PCP pathway is associated with NTDs in model organisms and humans, the underlying neural cell behaviors remain elusive.In order to investigate the cellular effects of the PCP pathway, we used Knypek (Knyfr6), Van gogh‐like 2 (Vangl2vu67), and Frizzled 7a−/7b− (Fzd7ae3−; Fzd7bhu3495), three zebrafish lines carrying null mutations. We confirmed the published result that mutations in the PCP pathway delayed NC. We analyzed the width of the neural plate in 4–5 somite stage embryos to confirm NC by performing in situ hybridization using probes against the hindbrain and neural crest mRNAs Krox20 and Dlx3, respectively. Next, our comparative cellular analysis revealed how cell elongation, membrane dynamics, and trajectory are affected in homozygotes. We show that wild type (WT) neural plate cells elongate and medially restrict membrane protrusions, thus narrowing the neural tube. Our preliminary data show that cells in all mutants failed to elongate initially, specifically Vangl2 and Fzd7a/b mutant cells extend randomized protrusions while Kny mutant cells show temporally restricted protrusive activity. Furthermore, cell behavioral analysis revealed that neural plate cells in PCP mutant embryos are unable to polarize or migrate toward the midline effectively. Live cell behaviors were assessed by confocal fluorescence microscopy of embryos neural convergence extension movements.Evidence from the literature revealed that Kny is a Fzd co‐receptor thought to present Fzd with various Wnt ligands in the PCP pathway. Differences between Kny and Fzd7a/b cellular phenotypes could suggest a ligand‐independent aspect of PCP signaling during NC. While current literature understands that PCP genes regulate cell polarity and migration, the mechanisms of these genes in neural tissue and how they contribute to NTDs is poorly understood. By studying cell behaviors in the neural plate, our laboratory aims to further reveal how the PCP pathway promotes NC and identify additional genes affecting NTDs.Support or Funding InformationU.S. DOD W81XWH‐15‐RTR‐IDA NIH R01‐GM085290 NIH R03‐3HD076615 NSF DBI‐0722569 Meyerhoff Graduate Fellowship U.S. Department of Education GAANN Fellowship NIH/NIGMS MARC U*STAR T3408663 National Research Service Award to UMBC