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DEVELOPMENTAL DYNAMICS 245:197208, 2016

DOI: 10.1002/DVDY.24304

REVIEW

Coordinating Cell and Tissue Behavior During Zebrash


Neural Tube Morphogenesis
a

Claudio Araya,1,2,3* Laura C. Ward,4 Gemma C. Girdler,5 and Miguel Miranda1


1

Laboratory of Developmental Biology, Instituto de Ciencias Marinas y Limnol


ogicas, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja s/n, Valdivia,
Chile
UACh Program in Cellular Dynamics and Microscopy
3
Centro Interdisciplinario de Estudios del Sistema Nervioso (CISNe), UACh
4
University of Bristol, School of Physiology and Pharmacology, Medical Sciences, University Walk, Bristol, United Kingdom
5
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, United Kingdom

DEVELOPMENTAL DYNAMICS

The development of a vertebrate neural epithelium with well-organized apico-basal polarity and a central lumen is essential
for its proper function. However, how this polarity is established during embryonic development and the potential inuence
of surrounding signals and tissues on such organization has remained less understood. In recent years the combined superior
transparency and genetics of the zebrash embryo has allowed for in vivo visualization and quantication of the cellular and
molecular dynamics that govern neural tube structure. Here, we discuss recent studies revealing how co-ordinated cellcell
interactions coupled with adjacent tissue dynamics are critical to regulate nal neural tissue architecture. Furthermore, new
ndings show how the spatial regulation and timing of orientated cell division is key in dening precise lumen formation at
the tissue midline. In addition, we compare zebrash neurulation with that of amniotes and amphibians in an attempt to
understand the conserved cellular mechanisms driving neurulation and resolve the apparent differences among animals.
Zebrash neurulation not only offers fundamental insights into early vertebrate brain development but also the opportunity
to explore in vivo cell and tissue dynamics during complex three-dimensional animal morphogenesis. Developmental Dynamics
C 2015 Wiley Periodicals, Inc.
245:197208, 2016. V
Key words: zebrash; neurulation; morphogenesis
Submitted 22 April 2015; First Decision 15 June 2015; Accepted 3 July 2015; Published online 14 July 2015

Introduction
Vertebrate Neural Tube Formation
The embryonic stages of central nervous system (CNS) development that result in the formation of a tubular structure are called
neurulation. The neural tube is an epithelium with well-defined
apico-basal polarity, which is a fundamental feature for further
brain development and function. Despite many years of research,
the molecular details of neurulation are not fully understood,
conceivably because of significant variation in this morphogenetic process among animal models. The cellular basis of vertebrate neurulation has been extensively investigated in avian,
amphibian and mouse embryos due to its propensity for dysregulation, resulting in neural tube defects (such as anencephaly and
spina bifida), one of the most common forms of human birth
defects (Colas and Schoenwolf, 2001; Copp et al., 2003). These
studies show that neural tube formation begins from an initial
flat sheet of cells called the neural plate that rolls up and fuses
Grant sponsor: Fondecyt; Grant number: 11110106; Grant sponsor:
Conicyt/ECOS; Grant number: C13B03.
*Correspondence to: Claudio Araya, Laboratory of Developmental Biology,
Instituto de Ciencias Marinas y Limnol
ogicas, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja s/n, 5090000, Valdivia, Chile.
E-mail: claudio.araya@uach.cl

dorsally to directly generate the central canal. This canal will


subsequently form the ventricular system of the brain and spinal
cord. Although this mechanism to generate a neural tube, known
as primary neurulation, gives rise to the brain and trunk regions
in the CNS, in the future lumbosacral region (sinus romboidales)
the nerve cord is generated by a process known as secondary
neurulation, whereby a group of mesenchymal cells condenses to
form a transitory solid rod that eventually cavitates into an epithelial tube (Criley, 1969; Griffith et al., 1992; Yang et al., 2014).
Primary neurulation has been largely documented by present and
past embryologists from fixed material, and the lack of suitable
imaging approaches to visualize cell and tissue dynamics in an
intact living organism has limited our understanding of vertebrate neuroepithelial morphogenesis. The zebrafish embryo has
recently emerged as an excellent in vivo vertebrate model system
in which to study the cell and tissue dynamics during neural tube
development as it overcomes these limitations (Lowery and Sive,
2004; Clarke, 2009). The optical advantages of the zebrafish
embryo combined with its natural curvature allows live imaging
of neural tube morphogenesis through an entire transverse plane
Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.
24304/abstract
C 2015 Wiley Periodicals, Inc.
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198 ARAYA ET AL.

Fig. 1. Zebrafish neurulation. AD0 : Transverse brightfield time-lapse images to show tissue movements of neurulation in the zebrafish (Danio
rerio) at the levels of the hindbrain and spinal cord. In all panels, the neural plate has been pseudocolored in yellow. In E and E0 the enveloping
layer has been pseudocolored in pale blue while the mesoderm in highlighted in red. AA0 : At 1011 hpf the neural plate is sitting over an underlying mesoderm. BB0 : By 1213 hpf, the neural plate has already converged toward the midline to generate a solid neural keel. CC0 : At 1517 hpf,
the solid neural keel changes shape to form another solid structure called the neural rod. DD0 : By 1820 hpf, the solid neural rod transforms into
the neural tube. EE0 : Tissue organization in the zebrafish neural plate. For simplicity only half of the neural plate is shown. While in anterior
regions such as the hindbrain, the neural plate is a multi-layered tissue, in more posterior regions (spinal cord) the neural plate is single-layered.
Note that in brain regions the lateral head mesoderm is often a single-layer, whereas in more posterior regions the underlying mesoderm is
arranged into compact tissue giving rise to somites. hpf, hours post fertilization; pm, paraxial mesoderm; nt, notochord; evl enveloping layer; np,
neural plate; mes, mesoderm. Arrows in B indicate tissue movements. Scale bar 50 mm. F,G: Representative stages of zebrafish neurulation
showing major cell and tissue rearrangements. EVL cells are showing in blue, np cells are showing in yellow, and mes cells are showing in pale
red. Extracellular matrix (ECM) is showing in red and is deposited between the neural tissue and the underlying mesoderm throughout neurulation.
F: At neural plate stages, neural cells are irregular in shape and thought to be organized within the superficial-deep axis of the neural anlage and
few cells undergo division. G: By neural keel stages, neural cells undergo elongation and intercalation within its superficial-deep axis. H: At neural
rod stages, neural progenitors changes its mitotic spindle orientation in 90deg and undergo polarized midline crossing cell division (Tawk et al.,
2007). Apical information is enriched at cleavage plane of dividing cells (green) 90degrees. Midline apical deposition is also found in neural cells
prior the midline-crossing division (green dots, Buckley et al., 2013). I: Finally, by neural tube stages, neural cells show well-established apical
(green) and basal (purple) polarity and a central lumen is formed. In all pictures, dividing cells appear in gray.

along the rostrocaudal axis, which significantly increases the


level of detail at which cell and tissue dynamics can be visualized
(Fig. 1). Gene function is relatively easy to assess in these
embryos either by antisense morpholino knockdown or other
more precise genetic ablations (Lawson and Wolfe, 2011). In
addition cell transplantation is straightforward, enabling mosaic
analysis of experimental and wild-type cells in several contexts
(Ciruna et al., 2006; Zigman et al., 2011; Girdler et al., 2013).

Zebrash Neurulation
Live imaging shows that zebrafish neural tube formation occurs
through a series of steps that differs slightly from most amniote
and amphibian embryos. A simple brightfield microscopy
approach has revealed that zebrafish neural tube formation is initiated at around 1011 hr postfertilization (hpf), when both the left

and right sides of the neural plate begin to converge toward the
dorsal embryonic midline (Fig. 1AA0 ,F). Later on, at 12 hpf, neuroectodermal cells begin to internalize at the midline and have
generated a solid neural keel by around 13 hpf (Fig. 1BB0 ,G).
Then, by 1517 hpf, the solid keel progresses to another solid
structure, the neural rod, which presents with a more cylindricallike morphology (Fig. 1CC0 ,H). Gradually the neural rod develops
well-defined apico-basal polarity with opposing apical surfaces
along a midline seam and finally, by 18 hpf, the neural rod has
transformed into a hollow neural tube by generating a lumen (Fig.
1DD0 ,I) (Schimtz et al., 1993; Papan and Campos-Ortega, 1997;
Kunz, 2004; Lowery and Sive, 2005; Ciruna et al., 2006; Hong and
Brewster, 2006). Therefore, while teleost zebrafish embryo neurulation proceeds by tissue internalization and subsequent cell rearrangement at the midline to form a lumen instead of the dorsal
folding that occurs in amniotes, the end result is the generation of

DEVELOPMENTAL DYNAMICS

ZEBRAFISH NEURAL TUBE CELL AND TISSUE MORPHOGENESIS 199

Fig. 2. Cell and tissue organization during vertebrate neurulation. For simplicity only neural ectoderm (neural plate) and nonneural ectoderm (nnect) tissues have been considered. In all diagrams, grey cells with white nuclei represent a polarized epithelial organization while white cells with
gray nuclei represent nonpolarized tissue. Arrows indicate tissue movements A: Classical primary neurulation in the chick embryo (Gallus gallus).
This involves the invagination of an existing epithelium at the midline and lumen formation by rolling or folding up to form the neural tube. B: Neurulation in the frog Xenopus laevis involves the invagination of a bi-layered neural plate and the formation of a central lumen by tissue invagination.
C: Teleost neurulation in the zebrafish embryo (Danio rerio). This process shows similarities to both primary and secondary neurulation in other vertebrates. The neural plate converges and internalizes to form solid keel and subsequently rod primordia. Cell divisions occur at the midline of the
rod, the apical surface is established and cavitation generates a central lumen. The superficial grey epithelium here represents the enveloping layer
(EVL), a simple flattened epithelium of nonneural ectoderm, which covers the embryo. D: Classical secondary neurulation in the chick embryo (Gallus gallus). This is characterized by condensation of mesenchyme cells to form a solid primordium, which then undergoes an epithelial transition
to generate multiple lumens, which finally coalesce to form a continuous apical surface at the neural tube stage. In figures A and B, ng indicates
neural groove. Figure A adapted from Schoenwolf (1991), Figure B adapted from Schroeder (1970), Figure C adapted from a time-lapse movie (C.
Araya), and Figure D adapted from Colas and Schoenwolf (2001).

a conventional neural tube composed of a polarized epithelium,


forming a brain and spinal cord ventricular system comparable to
other vertebrate organisms (Lowery and Sive, 2004; Harrington
et al., 2009; Clarke, 2009).

Cell and Tissue Cytoarchitecture During


Early Neurulation
While cell and tissue organization of the early neural plate has
been characterized in great detail in amniotes and amphibian
embryos (Schroeder, 1970; Freeman, 1972; Wakely and Badley,
1982; Chan and Tam, 1986; Schoenwolf and Alvarez, 1989;
Schoenwolf, 1991; Colas and Schoenwolf, 2001), the structure of
the early zebrafish neural ectoderm is far less understood and
conflicting views exist concerning its organization (Harrington
et al., 2009; Clarke, 2009). In fact, some authors prefer to classify
the teleost neural plate as an amniote-like single cell layered
columnar epithelium structure (Miyayama and Fujimoto, 1977;
Reichenbach et al., 1990; Schmitz et al., 1993), while others have
strongly suggested that there are significant similarities between
the teleost and amphibian neural anlage (Hong and Brewster,
2006; Harrington et al., 2009). In the following section of this
review, we characterize the cell and tissue organization of the
early zebrafish neural plate and compare its cytoarchitecture
alongside amphibian and amniote models to illuminate conserved
and divergent developmental mechanisms of vertebrate neuroepithelial morphogenesis. A key point to consider in this discussion
is whether zebrafish neural tube formation begins from an existing polarized epithelial neural plate, or whether the zebrafish tis-

sue is more plastic and uses mechanisms other than epithelial


folding to internalize and build a lumen.

Neural Plate Architecture in Amniotes


The amniote neural plate of avian and mice embryos has typically been described as a pseudostratified columnar epithelial
monolayer (termed pseudostratified because nuclei are positioned
at different superficial-deep positions within the epithelium due
to interkinetic cell migration), with obvious apico-basal polarity
(Fig. 2A) (Freeman, 1972; Wakely and Badley, 1982; Chan and
Tam, 1986; Schoenwolf, 1991). Although at posterior regions of
the chick neural plate (likely prospective lumbosacral regions),
apico-basal polarity maturation is thought to occur throughout
the process of neural tube morphogenesis (during secondary neurulation for example) (Duband et al., 2009; Fournier-Thibault
et al., 2009), the polarized structure of the amniote neural anlage
is largely inherited from the epiblast during gastrulation (Stern,
2004). Of interest, however, far from been a rigid polarized structure several studies have indicated that the murine and avian
neural plate is a dynamic tissue that undergoes complex epithelial cellcell rearrangements that are critical for subsequent steps
of midline tissue folding (Duband et al., 2009; Fournier-Thibault
et al., 2009; Nishimura et al., 2012; Williams et al., 2014).

Amphibian Neural Plate Organization


In amphibian models, neural plate organization seems to be less
uniform among species. In urodelian newts, such as the mexican

200 ARAYA ET AL.

salamander Ambystoma mexicanun, the neural tube develops


from a neural plate that is already organized as a single-layered
columnar epithelium (Brun and Garson, 1983). On the other
hand, histological and ultra-structural studies in the anuran
amphibian Xenopus laevis indicate that the neural plate at spinal
cord levels is actually a bi-layered tissue composed of a polarized
superficial cell layer and an underlying nonpolarized cell layer
(Fig. 2B) (Schroeder, 1970; Davidson and Keller, 1999).

DEVELOPMENTAL DYNAMICS

Zebrash Neural Plate Structure


Unlike other vertebrates, which begin neurulation with an already
polarized epithelium, the early teleost neural plate does not
appear to be organized into a conventional epithelium (Figs. 1F,
2C). Early ultra-structural studies conducted in the 1990s state
that the initial teleost neural anlage is arranged as a columnar
epithelium, and, therefore, proposed that several common features of primary neurulation such as the presence of neural folds
and a midline groove might also be present during zebrafish neural tube formation (Reichenbach et al., 1990; Schmitz et al.,
1993). However, the lack of expression of apical markers of
mature epithelia at this particular stage has brought the existence
of such a polarized structure into question (Geldmacher-Voss
et al., 2003; Yang et al., 2009). In addition to its distinctive tissue
architecture, recent work has shown that regional differences in
tissue thickness exist along the anteriorposterior axis of the
developing zebrafish neural tube (Fig. 1EE0 ). In brain regions
(prospective mesencephalon and rhombencephalon), the neural
plate is a multi-layered stratified tissue of three to six cell layers
in depth (Figs. 1E, 2C) (Hong and Brewster, 2006; Tawk et al.,
2007). In contrast, the neural plate is reduced in thickness to a
single cell layer in posterior spinal cord levels (Fig. 1E0 ) (Araya
et al., 2014). In both cases, zebrafish neural plate cells are largely
cuboidal in shape (Fig. 1F), but the possibility that they retain
thin membranous connections across the superficial-deep axis of
the neural anlage rather than strictly forming a multi-layered
structure still awaits elucidation by high-resolution electron
microscopy. Therefore, because the zebrafish neural plate appears
to be a less rigid structure than an epithelium, a common view is
that teleost neural plate cells have both epithelial and mesenchymal characteristics (Lowery and Sive, 2004).

Mechanisms of Neural Plate Bending in Primary


Neurulation
Despite the differences in cell and tissue organization of the neural plate among species, the initial stages of vertebrate neurulation are characterized by the convergent movements of the
neural plate toward the midline of the developing embryonic
axis. In addition, zebrafish exhibit some cellular behaviors in
common with frogs and chick, such as cell intercalation, with
similar molecular components involved. We also examine in
detail the role of the extrinsic factors such as adjacent tissues to
correct tube morphogenesis. Finally, we focus on the specialized
cell behaviors such as oriented cell division that occur during
zebrafish neural tube formation, and consider the importance of
these processes for correct apical midline establishment.

Amniote Neural Plate Morphogenesis


In amniote embryos, ultrastructural analyses indicate that neural
plate shaping occurs in two phases; initially, neural progenitor
cells increase their height and consequently reduce their width
(Fig. 2A) (Wakely and Badley, 1982; Chan and Tam, 1986;
Schoenwolf and Alvarez, 1989; Schoenwolf, 1991; Copp et al.,
2003). Secondly, a series of coordinated cell shape changes take
place within particular medio-lateral regions of the neural plate
that are critical for the formation of morphological hinge points
along most of the anteriorposterior neural axis (Fig. 2A) (usually
between mesecenphalic and posterior spinal cord levels). The formation of a single median hinge point (prospective floor plate)
above the axial mesoderm, and paired dorsolateral hinge points
(future sulcus limitans) result from active apical constriction and
concomitant basal cell expansion within these regions (Fig. 2A)
(Schoenwolf, 1991; reviewed in Colas and Schoenwolf, 2001;
Suzuki et al., 2012). Although the above studies have largely
implicated that the amniote neural plate is a static polarized
structure, recent in vivo evidence suggests that this epithelial tissue undergoes extensive cellular rearrangements both within the
plane of epithelia (i.e., cell intercalation and apical neighbor
exchange) (Nishimura et al., 2012; Williams et al., 2014) and
across its apical-basal depth (i.e., basal medio-lateral elongation
and intercalation) (Williams et al., 2014).
At the molecular level, the genetic analysis of bio-mechanical
pathways controlling epithelial dynamics (i.e., apical constriction,
polarized cell intercalation, and apical neighbor exchange) during
vertebrate neurulation has led to the identification of several
actin-binding proteins such as Shroom (Haigo et al., 2003), the
Ena/VASP (Menzies et al., 2004), and MARCKS (Zolessi and
Arruti, 2001) families, as well as the Rho/ROCK, MRCK and
MLCK signaling proteins required for nonmuscle myosin-II activity (Shimizu et al., 2005; Rolo et al., 2009; Nishimura et al., 2012;
Williams et al., 2014). While neural plate bending mediated by
apical cell constriction has been faithfully described in many
amniote embryos, to what extent similar mechanisms may operate in other species with less rigid cell polarity and adhesion is
currently unknown.

Amphibian Neural Plate Bending


In the bi-layered frog neural plate most of the deep nonpolarized
neural cells exhibit a regular elongated morphology throughout
neurulation, but the overlying superficial cells dramatically
change their shape from cuboidal to midline-elongated suggesting that this layer may sustain most of the tissue constriction
required for medial groove formation (Fig. 2B) (Schroeder, 1970;
Davidson and Keller, 1999). Initial tissue infolding is followed by
the appearance of a set of bilateral shallow neural folds at the
edge of the neural plate that come into close apposition and fuse
over the nascent ventral lumen (Fig. 2B). Furthermore, tissuelabeling experiments suggest that this initially bi-layered frog
neural plate becomes resolved into a single layer by a series of
dorsal cellcell rearrangements including lengthening and narrowing at the tissue level and radially intercalating cell behaviors
between the polarized superficial layer and the underlying nonpolarized neural deep cells, promoting the characteristic pseudostratified polarized cytoarchitecture of the developing neural tube
(Fig. 2B) (Davidson and Keller, 1999).

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ZEBRAFISH NEURAL TUBE CELL AND TISSUE MORPHOGENESIS 201

Fig. 3. Intrinsic and extrinsic mechanisms driving zebrafish neural tube morphogenesis. AC: Intrinsic mechanisms of teleost neurulation. Transverse confocal sections taken from time-lapse movies of zebrafish embryos labeled with membrane-tagged GFP. In all pictures, arrows indicate
the presence of the apical midline seam. A: By 20 hpf, wild-type (wt) embryos develop a neural tube with a single apical midline seam. B: Noncanonical Wnt/PCP mutant trilobite (tri) generates an aberrant neural tube with duplicated apical midlines. C: N-cadherin/cdh2 mutants fail to
undergo tissue invagination and embryos develop a T-shaped neural tube with abnormal apical midline seam configuration. DG: Extrinsic mechanisms of zebrafish neurulation. D: Series of confocal images taken from a timelapse movie using Histone2B-GFP (H2B-GFP) to label nuclei and
pseudocoloured to highlight the relationship of the neural plate (yellow), mesoderm (red) and the enveloping layer (EVL in white) during zebrafish
neurulation. EE0 : Cell movements between the neural plate (yellow) and underlying mesoderm (red) are tightly coupled during the initial stages of
neurulation. Arrowhead indicates the position of the dorsal midline. F: Tracks contrasting cell movements between wild-type embryos (purple neural plate and blue mesodermal cells) and mesoderm-less embryos (right, purple neural plate cells). Note the lack of coordination in cell movements
in MZoep mutant embryos. G: By 20 hpf, mesoderm-less embryos (Nodal/MZoep) develop an aberrant neural tube morphology and disorganized
apico-basal polarity as judged by the localization of the apical marker ZO-1. In all pictures, hpf indicates hr postfertilization and yellow dots indicate neural tissue. Figure B, adapted from Tawk et al., (2007), Figure D-G adapted from Araya et al., (2014).

Frog neurulation may thus share more common elements with


teleost neural tube morphogenesis than with amniotes, including
the presence of a multi-layered neural tissue, radial cell intercalation behavior across the superficial-deep axis and midline cell
migration of deep neural cells. Importantly, however, the existence of a prepolarized superficial layer within the bi-layered
neural plate and the process of lumen formation by means of a
central groove in frogs strongly challenge any assumption that
early teleost neural tissue internalization and subsequent lumen
formation occurs by similar mechanisms.

Zebrash Neural Plate Morphogeneis


Intrinsic mechanisms of zebrafish neural tissue
internalization
How the behavior of zebrafish neuroectodermal cells is regulated
during the initial stages of dorsal convergence is uncertain. However, regulated convergence movements of the zebrafish neural
plate cells are equally critical for tissue internalization and the
development of a symmetric solid neural keel and neural rod (Fig.
2C) (Tawk et al., 2007; Araya et al., 2014). In addition, the bilateral apposition of left and right sides of the neural primordia is
crucial to generate a single midline of apical specializations

(Ciruna et al., 2006; Tawk et al., 2007; Yang et al., 2009). Early
observations from the Campos-Ortega laboratory elegantly demonstrated that although the early zebrafish neural anlage is not a
conventional polarized tissue, neural plate morphogenesis occurs
as a coherent epithelial-like tissue as cells maintain their relative
position among neighbors while undergoing midline internalization (Papan and Campos-Ortega, 1994). At the cellular level, these
convergence movements of neural plate cells are thought to be
partially mediated by the activity of the noncanonical Wnt-PCP
pathway (Tada and Kai, 2009, 2012). During neural tube formation in both mice and amphibians, for instance, Wnt-PCP pathway genes are required for the coordinated convergence of
neuroprogenitor cells toward the dorsal midline (Murdoch et al.,
2001; Darken et al., 2002; Goto and Keller, 2002). The noncanonical Wnt/PCP pathway has also been proposed to act during
zebrafish neuroepithelial morphogenesis. Confocal time-lapse
analysis shows that Wnt/PCP pathway trilobite/strabismus
mutant embryos generate a wider and thicker neural primordium
from the early stages of neurulation (Fig. 3B) (Ciruna et al., 2006;
Tawk et al., 2007). Ciruna and colleagues (2006) interpret the
abnormal neural tube of MZtri as a consequence of the impaired
intercalation of neural cells during the neural keel-rod transition
(1517 hpf), thus attributing a cell autonomous role for the PCP
pathway during neurulation. Furthermore, they saw an

DEVELOPMENTAL DYNAMICS

202 ARAYA ET AL.

asymmetric localization of the PCP effector molecule Prickle


(GFP-Pk) to the anterior of neural keel cells, suggesting that a
certain polarity may be present along the anteriorposterior neural primordium during neural tube formation. Thus the PCP pathway, in combination with other mechanisms, could maintain
cells in position within the developing tube, preventing cell mixing across the anteriorposterior neural axis, similar to the role
proposed for Ephrin-Ephrin signaling in rhombomere boundary
formation during hindbrain development (Kemp et al., 2009).
However, how spatially distinct anteriorposterior PCP activity
might be incorporated into the major tissue rearrangements of
zebrafish neurulation occurring mostly along the mediallateral
axis (i.e., cell intercalation and orientated cell division across the
midline) and for early establishment of apico-basal cell polarity
(i.e., polarized midline crossing division) waits to be fully understood. It has been suggested that the PCP component Vangl2 and
the apical Par complex member aPKC share common binding
partners, potentially linking these two polarity pathways (Cui
et al., 2007; Li et al., 2011).
The cellcell adhesion molecule N-cadherin (cdh2) has also
been proposed to play an important role during the polarized
migration of zebrafish neural plate cells (Lele et al., 2002; Hong
and Brewster, 2006). In the prospective hindbrain, where the tissue is multi-layered in structure, loss of cdh2 results in defective
internalization of the neural primordium and embryos undergo
aberrant morphogenesis, leading to a T-shaped neural tube (Fig.
3C) (Hong and Brewster, 2006). This phenotype is proposed to
result from abnormal interdigitation between superficial and
deep cell layers during late stages of dorsal convergence. At the
sub-cellular level, loss of N-cadherin is thought to be required for
cell protrusive activity of intercalating zebrafish neural cells
(Hong and Brewster, 2006). Loss of another N-cadherin family
member, Protochaderin-19 (pcdh19) also results in arrested neural tissue convergence and aberrant anterior neural tube morphology (Biswas et al., 2010). Of interest, however, analysis of
convergence and extension movements of cdh2 and pcdh19
mutants show that embryos undergo initially normal convergence and the aberrant phenotype appears only during late keel
stages (Hong and Brewster, 2006; Biswas et al., 2010), indicating
specific spatio-temporal mechanistic requirements for neural tissue internalization.

Extrinsic contributors to zebrafish neural plate


morphogenesis
While the identification of molecular pathways and adhesion
molecules have shown that neural tube morphogenesis results
from the coordinated movements of cells within the neural tissue
itself, the vast extent of tissue remodeling observed during the
early stages of this process is likely to be synchronized with adjacent tissues and the local environment. In fact, classical embryological experiments based on in vitro tissue isolation approaches
(Holtfreter, 1933; Takaya, 1956a,b; Jacobson and L
ofberg, 1969;
Schroeder, 1970; Jacobson and Moury, 1995; Hackett et al.,
1997; Elul and Keller, 2000) and recent studies using quantitative
live imaging in frog (Morita et al., 2012) and fish embryos (Araya
et al., 2014) have demonstrated that surrounding tissues might be
critical for key steps of neural tube morphogenesis.
In all amniotes and amphibian embryos, the early neural plate
sits over an underlying mesodermal layer and is laterally flanked
by nonneural ectoderm (Schroeder, 1970; Hartenstein, 1989). This

nonneural ectoderm is required to complete neural plate convergence and to subsequently close the neural tube dorsally
(Schoenwolf, 1988; Moury and Schoenwolf, 1995). Recent functional studies along with live tissue quantification in the bilayered amphibian neural plate suggest that extrinsic pulling forces
produced by deep cells on the overlying nonneural ectoderm
depend on the cellcell adhesion molecule E-Cadherin and the
extracellular matrix (ECM) receptor, Integrin-1 (Morita et al.,
2012). In teleost fishes, the nonneural ectoderm layer is organized
differently to other vertebrates (Kunz, 2004). At early stages of
neurulation, the zebrafish neural anlage is covered by an overlying squamous epithelium termed the enveloping layer (EVL),
which remains almost motionless during the whole process of
neuruation and is, therefore, highly unlikely to exert any forces
on the neural tissue (Figs. 1F, 2C). Recent findings, however,
reveal that adjacent mesoderm is critical to coordinate cell movements within the neural plate toward the dorsal midline to form a
normal neural keel structure and generate a coherent and correctly polarized midline seam (Fig. 3D) (Araya et al., 2014). Moreover, quantification of cell and tissue behaviors in wild-type
embryos demonstrates that neural ectoderm and the underlying
mesoderm exhibit tightly coupled dorsal movements toward the
embryonic midline (Fig. 3E,F).
At present, the molecular mechanisms by which adjacent tissues, such as mesoderm, influence vertebrate neurulation are far
from understood. One possibility is to consider the underlying
mesodermal tissue as a source of ventraldorsal molecular cues
that can directly influence neural tissue organization and dynamics. This is perhaps most relevant to the most anterior brain
regions where the neural plate is multi-layered and cells must
thus maintain their relative superficial/deep positions during dorsal convergence (Hong and Brewster, 2006). Such vertical cues
could help to control both cell displacement and tissue adhesiveness within the neural plate. So far, however, there is no direct
evidence for such cues and so future studies carefully examining
spatio-temporal changes in gene expression are needed.
Alternatively, the mesoderm could be envisaged as a physical
substrate for converging neural plate cells. The close proximity
between the mesoderm and the neural plate might be enhanced
by the early deposition of basal ECM components such as Laminin and Fibronectin, which become firmly assembled between
these two tissues just before neurulation in zebrafish (10 hpf)
(Fig. 1F) (Latimer and Jessen, 2010). Basal lamina proteins are
also progressively assembled at the deep surface of the neural
plate in amniote embryos, at the junction between neural and
nonneural ectoderm layers (Martins-Green and Erickson, 1986;
Tuckett and Morriss-Kay, 1986; Morita et al., 2012). Evidence
from mouse and chicken suggests that upon interaction between
these two adjacent tissues, a series of cell shape changes are triggered that are required to complete neurulation (Martins-Green,
1988; Colas and Schoenwolf, 2001).
A third mechanism by which surrounding tissues influence neural tube formation is that adjacent tissues significantly constrain
the space by which neural plate cells can undergo collective displacements. In teleost fish, the neural plate is sandwiched between
the overlying EVL layer and subjacent mesoderm, thus forming a
narrow corridor through which neural cells can move (Fig. 1F). In
this situation, migrating neural plate cells display parallel and
smooth trajectories across the developing superficial-deep axis
(Araya et al., 2014), resembling laminar flows of viscous liquids
(Constantinescu, 1995). By analogy, loss of mesoderm generates

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ZEBRAFISH NEURAL TUBE CELL AND TISSUE MORPHOGENESIS 203

Fig. 4. Apical cell polarity during zebrafish neurulation. A: Time series of a neural rod cell before, during and following C-division (12ss14ss).
Cells are labelled with Par3-GFP, a nuclear label (H2B-RFP) and a membrane label (Cherry-CAAX). Dotted lines show the tissue midline, dashed
lines show the basal edges of the tissue. ML, indicates midline. Before division, Par3-GFP accumulates in multiple relatively large puncta in the
cell cortex, approximately at the point where the cell intersects the tissue midline (20 mins). Upon entry into telophase, Par3-GFP is distributed to
the cleavage plane of dividing cells. As cells complete cytokinesis, Par3-GFP localises more distinctly to a narrow region at the midline (1 hr, 15
min). B: Model for establishment of cell polarity during zebrafish neurulation. During neural rod stages, contralateral cells integrate basal cues from
the basal lamina (laminin) and interdigitate across the tissue midline where nascent adhesions (magenta) may be formed. These adhesions may
then recruit puncta of apical polarity proteins (such as Par3 and ZO-1, green) to an approximate region around the tissue midline. Subsequently,
these puncta of Par3 could then recruit the centrosome (orange), which may then organize the microtubule cytoskeleton (blue arrows) from this
point. This microtubule cytoskeleton could then reinforce the localization of apical proteins to the midline, adding precision and allowing junctions
to mature. Blue arrows indicate direction of apical traffic. Figure A, adapted from Buckley et al., (2013).

abnormal or turbulent flows (directionality) of these cells as they


move toward the dorsal midline (Araya et al., 2014). Furthermore,
inter-tissue contact mediated by cellcell adhesion proteins and
transmitted by the ECM might drive major tissue deformation during morphogenesis (Dzamba et al., 2009). Tensile forces exerted by
the mesoderm could in principle, locally deform the physical
assembly of the ECM, regulating cell surface receptors involved in
cell mechanical signal transduction. Increasing cell tension within
neural plate cells could, in turn, lead to cell shape changes through
re-organization of microtubule and actin networks, contributing to
tissue internalization. Although recent studies have indicated that
cortical tension might be a major driving force in tissue morphogenesis (Krieg et al., 2008; Dzamba et al., 2009; Heisenberg and
Bellache, 2013), quantifying these forces in intact living organisms still remains a major challenge in the field.

Influence of orientated cell division during zebrafish


neurulation
The neural keel-rod period of zebrafish neurulation is uniquely
characterized by an event of midline crossing (C-division), during
which almost all neural progenitor cells divide and deposit one
daughter cell into each side of the developing neural tube (Figs.

1H, 4B) (Kimmel et al., 1994; Papan and Campos-Ortega, 1997).


As a result, both sides of the neural tube receive equal contributions from both left and right sides of the neural plate. In fact,
experimental work has suggested that the midline-crossing
behavior depends on cell division, because pharmacologically
blocking cell division strongly reduces the number of cells able to
cross the midline (Ciruna et al., 2006; Tawk et al., 2007). Highresolution microscopy indicates that the dividing mother cell
remains connected on its basal ipsilateral side by a narrow cytoplasmic process, while the midline crossing daughter must form
a new stalk to connect to the contralateral basal side of the neural
rod (Figs. 1H, 4C) (Ciruna et al., 2006; Buckley et al., 2013). Once
this contralateral connection occurs the C-division is completed
by cytokinesis, which, in zebrafish neurulation, can take several
hours as sister cells remain temporally connected by an intercellular bridge (Fig. 4C) (Buckley et al., 2013).
It is essential that cells undergo mirror-symmetric C-division
at the correct time and in the correct place to ensure that the apical ends of cells are positioned at the developing tissue midline,
as this subsequently demarcates the lumen of the neural tube. In
embryos with delayed convergence of the neural tissue, such as
the zebrafish PCP pathway mutant trilobite (Sepich et al., 2000),
mirror-symmetric cell divisions occur in ectopic lateral locations

DEVELOPMENTAL DYNAMICS

204 ARAYA ET AL.

either side of the midline leading to the generation of bilateral


ectopic apical planes and consequently, duplicated neural tubes
(Fig. 3B) (Tawk et al., 2007). Moreover, when C-divisions are
misoriented, and subsequently no longer coordinated across the
midline, a single continuous lumen does not form and neural
tube morphology is branched and disorganized (Quesada-Hernandez et al., 2010; Zigman et al., 2011). These examples clearly
indicate that the C-division has a powerful morphogenetic influence during zebrafish neural tube development.
To date, it is not entirely clear how this midline-crossing division is regulated during zebrafish neural tube morphogenesis.
Several studies have hypothesized that cell geometry itself is the
default mechanism determining division orientation in both single cells (Palmer et al., 1992; Tsou et al., 2003) and also in multicellular tissues (OConnell and Wang, 2000; Aigouy et al., 2010).
In many of these studies, Hertwigs rule (also known as the
long-axis rule) is invoked as a universal feature for dividing
cells, and cell elongation appears to be the critical factor in predicting division orientation (Hertwig, 1893; Minc and Piel, 2012).
Cell elongation heavily depends on microtubules and so it is
likely that cells can determine the axis of division by autonomously measuring the length of microtubules (Kozlowski et al.,
2007; Minc et al., 2009). Zebrafish neural progenitors undergo
medio-lateral elongation during the neural keel-rod transition,
which temporally coincides with the appearance of highly
midline-orientated microtubules and onset of C-division (Buckley
et al., 2013).
A growing body of work has also indicated that the mitotic
spindle apparatus is critical for orientated cell division and the
correct distribution of cortical determinants, including apicobasal polarity complexes in several morphogenetic contexts
(Siller and Doe, 2009; Gillies and Cabernard, 2011; Morin and
Bellache, 2011). In the zebrafish, several authors have already
addressed the role of the mitotic spindle orientation during apical
midline formation. Initial observations by Campos-Ortega and
colleagues (2003) showed that the neural cells rotate their mitotic
spindle by 90degrees at the prospective midline of the neural rod
(Geldmacher-Voss et al., 2003). This suggests that mitotic spindle
orientation could be important for the C-division and the appearance of apical information at the prospective midline. Genetic
analysis, however, reveals that upon abrogation of individual
components of the apical Pard3/aPKC/Pard-6 complex there is
little effect on spindle orientation during the C-division (Geldmacher-Voss et al., 2003; Munson et al., 2008). More recently,
the subcellular basal determinant Scribble has been proposed to
control the mitotic spindle machinery during zebrafish neurulation by regulating cadherin-based cellcell adhesion (Zigman
et al., 2011). Moreover, evidence suggests that loss of function of
N-cadherin results in the down-regulation of alpha-catenin foci
in dividing neural cells (Zigman et al., 2011). Noncanonical Wnt/
PCP signaling is also thought to influence the mirror-symmetric
cell division during fish neurulation (Ciruna et al., 2006; Tawk
et al., 2007). Although mutation of key components of this pathway show defects in dorsal convergence and thus abnormal cell
orientation (Tawk et al., 2007), whether permissive PCP signaling
per se is required for cell division orientation was, until few years
ago, unclear. Evidence has implicated the Wnt/PCP receptor
Frizzled 7 (Fz7) in controlling mitotic spindle orientation within
the developing rod primordium and, therefore, neural tube morphogenesis (Quesada-Hernandez et al., 2010). In contrast to the
characteristic bilateral apical midline seams phenotype reported

in PCP deficient zebrafish due to delayed convergence, elimination of Fz7 results in the complete absence of an apical midline.
While correct division orientation is clearly needed for normal
apical midline specialization, how this is coordinated in time and
space is totally unknown. It has been proposed that an intrinsic
timing mechanism operates to control cell polarization and
lumen formation in zebrafish. Using heterochronic cell transplantation experiments, Girdler and colleagues (2013) found that
C-division occurs within a specified time-window and that this
timing can even resist abnormal morphogenetic tissue environments and in vitro culture conditions. The mechanism by which
cells gauge and respond to developmental time is not yet known,
but cells do not seem to monitor time by counting the number of
cell cycles and in heterochronic environments, cell polarization
does not even require mirror-symmetric cell division (Girdler
et al., 2013). microRNAs have been suggested as possible molecular mediators of this developmental clock due to their roles in
several intracellular timers in C. elegans (Lee et al., 1993; Pasquinelli et al., 2000; Reinhart et al., 2000) and the requirement for
zebrafish brain morphogenesis (Giraldez et al., 2005). The above
example illustrates the importance of mechanisms that coordinate cell intrinsic programmes with morphogenetic movements
and environmental signals, both in time and space for correct
embryogenesis.

Apico-basal polarity development and lumen formation


during zebrafish neurulation
One important consequence of the midline-crossing division is
that as daughter cells cross the midline and integrate into the
contra-lateral neuroepithelium they must develop a mirror-image
apico-basal polarity with respect to their original side (Fig. 1H,I).
Coincident with this period of neural tube development, several
studies have reported the first visible accumulation of apical
markers such as the adherens junction protein b-catenin and the
tight junction component, ZO-1 in neural progenitor cells (Geldmacher-Voss et al., 2003; Tawk et al., 2007; Yang et al., 2009).
These observations suggest that the midline-crossing division is
closely related to the events controlling the initial deposition of
apico-basal information within the neural tissue (Tawk et al.,
2007). Moreover, it also suggests that through this C-division
mechanism, neural cells could ensure an equal distribution of
polarity information at the tissue midline, which is a key step for
the subsequent opening of a lumen.
During neurulation, zebrafish embryos form a central lumen de
novo through a series of complex cell and tissue rearrangements
occurring at the developing midline of the solid neural rod (Buckley and Clarke, 2014). Initial studies by Schmitz et al. (1993)
showed that luminal opening first appears at approximately 17
hpf above the floor plate, which subsequently extends dorsally as
small lumens localized along the midline begin to coalesce
(Schmitz et al., 1993). Time-lapse analysis has confirmed these
observations (Lowery and Sive, 2005), indicating that zebrafish
lumen formation is apparently connected along the developing
neuraxis. Lumen opening spatially coincides with the deposition
of markers of mature apical adherens and junctional proteins,
including aPKC, Pard3, ZO-1, and b-catenin, which also show a
progressive ventral to dorsal refinement to the neural rod midline
(Geldmacher-Voss et al., 2003). In fact a well-polarized neuroepithelial structure is critical for zebrafish lumen formation (Lowery
and Sive, 2005, 2009) as embryos lacking apical cell polarity

DEVELOPMENTAL DYNAMICS

ZEBRAFISH NEURAL TUBE CELL AND TISSUE MORPHOGENESIS 205

proteins such as aPKC (Horne-Badovinac et al., 2001), Pard6


(Munson et al., 2008), and the MAGUK protein nagie oko (BitAvragim et al., 2008) show impaired lumen development. A common feature of these mutant neural tubes is the interruption of
the continuous apical midline seam by tissue-bridges (BitAvragim et al., 2008; Munson et al., 2008; Buckley et al., 2013).
Cell division has also been indicated to play an important role in
zebrafish lumen development (Lowery and Sive, 2005; Tawk
et al., 2007; Buckley et al., 2013). Although zebrafish embryos
treated with cell division inhibitors from early stages of neurulation do develop neural lumens, the morphology of the lumen is
abnormal and they develop a similar tissue-bridge lumen phenotype to those mutants described above that lack epithelial
polarity (Lowery and Sive, 2005; Buckley et al., 2013). Cell division (and particularly the midline C-division) thus acts synergistically with cell polarization mechanisms to enhance the efficiency
of lumen formation.
Although cellcell interactions are critical to localize the apical
midline seam in the developing rod, the important question of how
neural cells actually recognize the tissue midline has only recently
been investigated. Buckley and colleagues (2013) show that cellular
spatial awareness is achieved through a combination of cell interactions between left and right sides of the maturing rod and the integration of anti-basal signals such as Laminin, an ECM component
required for polarized lumen formation in several morphogenetic
contexts (OBrien et al., 2001; Yu et al., 2005; Tseng et al., 2012). A
key observation from this work is that apical information (for
example ZO1 and Pard3) becomes enriched in a broad midline zone
before C-division actually occurs, a localization that is, therefore,
independent of cell division itself but dependent on microtubule
organization (Fig. 4D) (Buckley et al., 2013). The asymmetric subcellular localization of apical proteins toward the tissue midline
thus generates a bilaterally symmetric plane of reflection between
left and right sides of the maturing tube. Moreover, the midline cell
intercalation is accompanied by enrichment of alpha-catenin at the
interface of interdigitating cells, indicating that nascent contralateral cellcell adhesion could have an instructive role during both
initial apico-basal organization and midline crossing-division (Fig.
4D) (Zigman et al., 2011; Buckley et al., 2013). Remodeling of cell
cell junctions within the interdigitation zone of neural rod cells
across the midline is thought to add spatial precision to the future
position of apical complexes within cells at the midline. The authors
suggest that this could be partially achieved by positioning centrosomes toward the centre of the tissue where both apical information
(Pard3) and microtubule-dependent endosomal (Rab11) traffic are
required to initiate lumen opening (Fig. 4C) (Buckley et al., 2013).
Despite the fact that zebrafish neural tube formation and polarization can occur in the absence of C-division (albeit generating a less
precise lumen), this stereotypic cell behavior appears to confer morphogenetic advantages at the tissue level by efficiently eliminating
cellular processes that would otherwise bridge the developing
lumen. Whether cell division in these embryos enables a more
sophisticated mechanism to remodel nascent apical junctions whilst
retaining epithelial integrity among neighbors awaits further exploration. Alternatively, this midline crossing cell behavior and interdigitation across the midline might be especially important because
the high curvature of the teleost zebrafish embryo imposes an
inherent mechanical stress that might otherwise inhibit neurulation.
Indeed, in the mouse mutant curly-tail, increased curvature of the
body axis has been directly attributed to the failure of neural tube
closure (Brook et al., 1991). Moreover, when chick embryos are cul-

tured on curved substrata this leads to a delay in closure of the posterior neuropore, the extent of which correlates with the angle of
curvature (van Straaten et al., 1993). The degree of axial curvature
also negatively correlates with the rate of neural tube closure
between different vertebrate species (Peeters et al., 1998). Thus, it is
possible that the zebrafish uses alternative mechanisms to overcome
high mechanical stress and minimize energy requirements. Division
and cellular remodeling may require less force generation than the
formation of neural folds and a hinge point, as in primary neurulation. Additionally, it is possible that the neural tissue of the zebrafish may contain fewer cells relative to other vertebrates. If this
were the case, the relative contribution of each constituent cell in
tissue-wide force generation would be much greater. The prevalence
of neurulation defects in other vertebrates is high (Copp et al.,
2013) but appears to be low in zebrafish and so the existence of
redundant mechanisms in zebrafish may also ensure that if one
mechanism goes wrong the crucial process of brain and spinal cord
development can still proceed.

Future Perspectives
There are significant differences in the process of neurulation
between fish and other vertebrates and it is, therefore, important to
analyze how far they possibly rely on the same basic mechanisms.
We have stressed in this review that the early cell and tissue organization (i.e., polarized vs. nonpolarized structure) play a significant role in the mechanisms of tissue internalization. In zebrafish
embryos, apico-basal identity is gradually established during the
course of neurulation and recent evidence suggests that the mechanism organizing such polarity at the midline is based on the integration of contralateral cellcell interaction and reinforced by
signals from the basal lamina. Moreover, the apparent discrepancy
between the alternative mechanisms used by teleost fish to make a
neural lumen, either with or without cell division, could be linked
to the existence of parallel redundant cellular and molecular mechanisms that underlie the maturation of apical junctional complexes
and drive cellular rearrangements. The high plasticity of neural
cells and the time delay before onset of polarity allows cells to
undergo complex tissue morphogenesis before they assemble stable apical junctions. Further in vivo analyses of cell dynamics
between teleosts and other vertebrate should establish true similarities and differences between teleost and other vertebrate neurulation. Particularly intriguing questions concern the mechanisms
that drive internalization of the fish neural plate to form the keel,
and the establishment of cellcell junctions across the midline of
the neural rod, which are essential to build a functional lumen.

Acknowledgments
We thank Jonathan Clarke and the Araya lab for helpful discussion
and comments on the manuscript and apologize to colleagues and
earlier researchers whose work could not be cited due to space constraints. We thank also Carlos Carmona-Fontaine for schematic
diagram in Figure 1.

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