Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures.

Chick cranial neural crest cells release extracellular vesicles that are critical for their migration.

The content and activity of extracellular vesicles purified from cell culture media or bodily fluids have been studied extensively; however, the physiological relevance of exosomes within normal biological systems is poorly characterized, particularly during development. Although exosomes released by invasive metastatic cells alter migration of neighboring cells in culture, it is unclear whether cancer cells misappropriate exosomes released by healthy differentiated cells or reactivate dormant developmental programs that include exosome cell-cell communication. Using chick cranial neural fold cultures, we show that migratory neural crest cells, a developmentally critical cell type and model for metastasis, release and deposit CD63-positive 30-100 nm particles into the extracellular environment.

The lysine methyltransferase SETD2 is a dynamically expressed regulator of early neural crest development.

SETD2 is a histone H3 lysine 36 (H3K36) tri-methylase that is upregulated in response to neural crest induction. Because the H3K36 di-methylase NSD3 and cytoplasmic non-histone protein methylation are necessary for neural crest development, we investigated the expression and requirement for SETD2 in the neural crest. SetD2 is expressed throughout the chick blastoderm beginning at gastrulation.

Profiling NSD3-dependent neural crest gene expression reveals known and novel candidate regulatory factors.

The lysine methyltransferase NSD3 is required for the expression of key neural crest transcription factors and the migration of neural crest cells. Nevertheless, a complete view of the genes dependent upon NSD3 for expression and the developmental processes impacted by NSD3 in the neural crest was lacking. We used RNA sequencing (RNA-seq) to profile transcripts differentially expressed after NSD3 knockdown in chick premigratory neural crest cells, identifying 674 genes.

Embryological and Genetic Manipulation of Chick Development.

The ability to combine embryological manipulations with gene function analysis in an amniote embryo makes the chick a valuable system for the vertebrate developmental biologist. This chapter describes methods for those unfamiliar with the chick system wishing to initiate experiments in their lab. After outlining methods to prepare chick embryos, protocols are provided for introducing beads or cells expressing secreted factors, and for culturing tissue explants as a means of assessing development in vitro.

DNA methyltransferase 3b is dispensable for mouse neural crest development.

The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined.

Paladin is an antiphosphatase that regulates neural crest cell formation and migration.

Although a network of transcription factors that specifies neural crest identity in the ectoderm has been defined, expression of neural crest transcription factors does not guarantee eventual migration as a neural crest cell. While much work has gone into determining regulatory relationships within the transcription factor network, the ability of protein modifications like phosphorylation to modulate the function of neural crest regulatory factors and determine when and where they are active also has crucial implications. Paladin, which was previously classified as a phosphatase based on sequence similarity, is expressed in chick neural crest precursors and is maintained throughout their epithelial to mesenchymal transition and migration.

Division of labor during trunk neural crest development.

Neural crest cells, the migratory precursors of numerous cell types including the vertebrate peripheral nervous system, arise in the dorsal neural tube and follow prescribed routes into the embryonic periphery. While the timing and location of neural crest migratory pathways has been well documented in the trunk, a comprehensive collection of signals that guides neural crest migration along these paths has only recently been established. In this review, we outline the molecular cascade of events during trunk neural crest development.

Neuropilin receptors guide distinct phases of sensory and motor neuronal segmentation.

The segmented trunk peripheral nervous system is generated by ventrally migrating neural crest cells that exclusively invade the anterior sclerotome and differentiate into metameric dorsal root and sympathetic ganglia. Meanwhile, ventral spinal motor axons also project through the somites in a segmental fashion. How peripheral nervous system segmentation is generated is unknown.

Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus.

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood.

Regulation of actin cytoskeleton architecture by Eps8 and Abi1.

Background: The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive.