The Neurulation: neural tube and neural crest formation

Neurulation describes the developmental event which starts with the formation of the neural plate and ends with its neurulation. Neurulation includes different steps: the formation of the neural plate (1), and its folding (2); the convergence (3) and the fusion (4) of the dorsal surfaces to form the neural tube. Scanning electron microscopy pictures of the correspondent phases taken during chicken embryo-development are shown on the left. Figure modified from biology-forum.com

neurulation process begins when approximately half of the ectodermal cells modify their morphology, elongating into columnar neural plate cells, by response to the underlying dorsal mesoderm-induced signaling. The different shape distinguishes the neural plate from the surrounding pre-epidermal cells (Gilbert 2010).

At the border of the newly formed neural plate, hinge regions starts to form. Those cells move the lateral edges of the neural plate toward its midline, which is known as medial hinge point (MHP). Next, the MHP cells anchor to the beneath notochord, and undergo further morphological changes. At the border between neural plate and the ectoderm, the dorsolateral hinge points (DLHP) form, which cells undergo similar changes as the MHP cells, reducing their height and adopting wedge-shape. The neural plate begins to bend around those hinge regions, which direct the rotation of the cells around them. Additionally, the surface ectoderm pushes toward the neural plate midline, contributing to the neural plate folding.

When the peaks of the neural folds are brought closer, they adhere to each other and merge, forming the neural tube. In vertebrates, the cells at this junction give rise to the neural crest cell population. Finally, the newly formed neural tube will become the embryonic precursor of the central nervous system, which will develop into brain and spinal cord (Gilbert 2010).

1.2 Definition of neural crest

The neural crest (NC) was originally identified by His in 1868 and described as "the cord in between" because of its origin between the neural plate and non-neural ectoderm. More recently, the NC has been renamed the 4^th germ layer, representing a vertebrate-specific addition to the classic three germ layers, ectoderm, mesoderm and endoderm. The neural crest population is one of the most significant factors contributing to vertebrate diversity and evolution, adding features such as hinged jaw, special sense of organs and neural circuitry (Munoz and Trainor 2015).

The defining features of the neural crest cells (NCC) are their origin at the neural plate border, their multipotency and their ability to migrate and give rise to a plethora of cell types and tissues in vertebrates.

NCC execute their main developmental steps in separate regions of the embryo, and during different stages of the embryogenesis, characteristic almost exclusive of this specific cell type. NCC development can be divided in four distinct steps and events (Figure 2):

(1) pre-migratory phase, which includes induction and specification at the neural plate, beginning during gastrulation and early neurulation;

(2) delamination phase, via epithelial to mesenchymal transition (EMT) at the end of neurulation;

(3) migratory phase, from the neural plate through the embryo;

(4) differentiation phase, through the course of organogenesis and late embryogenesis.

Figure 2 Representation of maturation steps of the neural crest. NCC maturation included different steps: pre-migratory, delamination, migratory and differentiation phases. Finally, NCC will differentiate, based on their position (cranial or trunk), into different celly types, such as sensory neurons, pigment cells, connective tissue, cartilage and bones. Figure modified from Green et al. 2015 and Knecht and Bronner-Fraser 2002.

In most of the species, NCC placodal regions. They positively regulate each other and induce the following developmental steps (Simoes-Costa and Bronner 2015). At the end of the neural crest specification process, the orchestrated expression of those genes initiates drastic structural changes, resulting in the delamination of the neural crest from the neural tube. This process is named epithelial-to-mesenchymal transition (EMT) and it is a complicated mechanism which brings to extreme structural remodeling of the premigratory neural crest, including regulation of the adhesive characteristics of the cells, cytoskeletal rearrangement, degradation of basement membrane by metalloproteases and inducement of a mesenchymal phenotype which allows the NCC to separate from the neural tube and disperse through the embryo (Figure 3) (Sauka-Spengler and Bronner-Fraser 2008).

After delamination, migratory neural crest cells starts to colonize different areas of the embryo, responding to different kind of permissive and inhibitory stimuli, which vary along the axial position. During this process, NCC maintain a stem cell-like, multipotent state, including the capacity of self-renewal (Baggiolini et al. 2015). The process of neural crest

Figure 3 Epithelial to mesenchymal transition (EMT) process. Before the delamination phase, NCC undergo to the process of EMT. The characteristics of epithelial (left) and mesenchymal (right) phenotypes are listed below each graphical representation.

diversification starts with activation of different regulatory circuits in the diverse migratory subpopulations. The arising of the different derivatives depends on the combination between the regulatory state of the neural crest and the environmental signals which surround them.

Once NCC arrive at their final location, they often self-aggregate during initiation of terminal differentiation (Simoes-Costa and Bronner 2015).

NCC can be divided in cranial, cardiac, vagal, trunk and sacral according to their axial position of origin. Cranial NCC give rise to most of the bone and cartilage of the facial skeleton, neurons and glia of the cranial ganglia, smooth muscle and pigment cells. Cardiac NCC form the valves, septa and outflow tract of the heart. The vagal and sacral NCC form the enteric nervous system; while the trunk NCC differentiate in melanocytes, neurons and glia of the peripheral nervous system (Motohashi and Kunisada 2015).