2023 Project 1

Student Projects 2023: 1 Patterning neural border and NC | 2 NPB NEUcrest | 3 EMT and NC | 4 miRNA and NC | 5 Adrenal Gland and NC | 6 Melanocyte & Melanoma | 7 Neurocristopathies | Neural Crest These projects are the sole work of undergraduate science students and may contain errors in fact or descriptions.

The patterning of the Neural Crest

Progenitor Neural Border Domain and the Pre-migratory Neural Crest Cells

Neural crest (NC) development starts in the ectodermal germ layer during early embryogenesis, more precisely during gastrulation, simultaneously with the events of neural induction. It continues throughout neurulation and organogenesis (Eames et al., 2020). Here we focus on the first steps of NC formation. The induction of the NC fate takes place in a specific region of the ectoderm known as the neural plate border or better, the neural border, as part of this area is devoid of neural plate markers (NB/NPB). In the plane of the ectoderm, the NB is located between the non-neural ectoderm (prospective epidermis, positioned more laterally), and the neural plate (prospective CNS, positioned more medially). The NB ectoderm also lies above the paraxial and intermediate mesoderm. In addition, the NB ectoderm gives rise not only to NC cells but also to the cranial placodes (future sense organs), parts of the dorsal neural tube, and of the non-neural ectoderm.

Tissue interactions play an essential role during NB and NC induction. Thus, NB induction is regulated by coordinated activation of different signals secreted by the surrounding tissues: the non-neural ectoderm, the neural plate, and the underlying mesoderm. The three main signaling pathways mediating these tissue interactions and ensuring the proper development of NC cells are the canonical Wnt/β-catenin signaling (WNT), the Bone Morphogenetic Protein (BMP), and Fibroblast Growth Factor (FGF) pathways. Multiple studies have shown that the activation of each of them is necessary but not sufficient alone and that they synergize to ensure appropriate levels of signaling targeted to the NB (Garnett et al., 2012; Hong and Saint-Jeannet, 2007; Monsoro-Burq et al., 2005; Saint-Jeannet et al., 1997; Tribulo et al., 2003); reviewed in (Prasad et al., 2019; Sutton et al., 2021).

Globally, during vertebrate gastrulation, graded BMP signaling specifies the mediolateral body axis while graded WNT and FGF signaling specify the anteroposterior axis of the neural plate, with no/low levels acting at the anteriormost part and higher levels for posterior trunk regions (reviewed in (Sutton et al., 2021). BMP signals are secreted by the nonneural ectoderm and the underlying lateral mesoderm to maintain the nonneural ectodermal fate and together with WNT signals repress the neural fate (Faure et al., 2002; García-Castro et al., 2002). In turn, the axial mesoderm and the medial part of the neural plate secrete diffusible BMP antagonists allowing the expression of neural genes thus creating a BMP signaling gradient (Plouhinec et al., 2013). Thereby, the current textbook model is that high BMP activity induces an epidermal fate, moderate BMP levels induce NB genes, whereas low BMP activity determines a neural fate and represses both NB and ectodermal fates (Schumacher et al., 2011; Suzuki et al., 1997). However, recent findings highlight the action of BMP signaling potentiators which act in the paraxial and intermediate mesoderm and in the NB ectoderm, ensuring a high level of BMP signaling rather than an intermediate one. At the same time, inductive WNT and FGF signals are involved in the formation of the NB. FGF ligands are secreted by the mesoderm, the sources of WNT ligands could vary according to the species, and include ectoderm and mesoderm (García-Castro et al., 2002; Monsoro-Burq et al., 2003). In addition, there are also several auxiliary signaling participating in NB induction, e.g. retinoic acid and hedgehog signaling (Tribulo et al., 2003), but these have not yet been fully integrated into the gene regulatory network governing NB induction.

The combination of WNT, BMP, and FGF pathways activates a complex network of transcription factors (TFs) within the neural border ectoderm, essential for NB induction and called the "NB specifiers". The most studied NB specifiers are Tfap2a, Gbx2, Msx1/2, Zic1, Pax3/7, and Hes4 (de Crozé et al., 2011; Monsoro-Burq et al., 2005; Sato et al., 2005; Simões-Costa and Bronner, 2015a). Once activated, the NB specifiers, along with extracellular signaling inputs (sustained or reactivated WNT signals for example), trigger the expression of downstream "NC specifiers": the transcription factors that are essential for NC induction and its further development: TFAP2B, SNAI1/2, FOXD3, TWIST1, SoxE genes (SOX8/9/10). In addition, some NB specifiers such as TFAP2A also act reiteratively as NC specifiers, regardless of their previous role (de Crozé et al., 2011; Simões-Costa and Bronner, 2015b). NC specifiers regulate NC induction of neural crest and further delamination of neural crest cells from neural tube via EMT but also activate downstream lineage-specific gene regulatory networks for the differentiation into distinct cell types. EMT is a process that is essential for the proper development of the neural crest and for the formation of many different tissue types. During EMT, cells undergo a series of changes that allow them to become more motile and migrate to different locations in the developing embryo. These changes include the loss of cell-cell adhesion, the acquisition of a more spindle-like shape, and the upregulation of various mesenchymal markers.

There are several transcription factors that are known to play a critical role in the preparation of the neural crest for EMT. These include Snai1 and Snai2, transcriptional repressors, that have been extensively studied in the context of neural crest specification and EMT. Overexpression of Snail1/2 has been shown to increase the size of the neural crest population, while their inhibition blocks neural crest specification and migration. The regulatory regions of Snail2 contain binding sites for LEF/TCF and Smad1, which are directly regulated by WNT and BMP signaling (Nieto, 2018; Vallin et al., 2001). In addition, Snail1 and Snail2 are direct targets of Zic1 and Pax3 during frog neurulation.o defects in the development of the craniofacial skeleton and pigmentation (Plouhinec et al., 2014).

Other transcription factors that have been implicated in the preparation of the neural crest for EMT include Twist1, which plays a role in the acquisition of a more spindle-like shape of the cells (Sepporta et al., 2022, p. 1). Twist1 directly interacts with the Snail1 and Snail2 proteins through its WR domain, and phosphorylation of Twist1 leads to the inhibition of the activity of Snail1 and Snail2 (Lander et al., 2013). Also, recent single-cell RNA sequencing data in mice and experimental manipulation of Twist1 expression in chick have demonstrated that Twist1 plays a role in regulating and promoting the mesenchymal fate choice (Soldatov et al., 2019).

Another essential for the specification and maintenance of the NC genes is the SOXE family of transcription factors, including Sox8/9/10. However, each factor has distinct functions within the NC gene regulatory network. In frog development, Sox8 and sox9 are among the first NC specifier genes to be expressed in the neural plate region, prior to the expression of Snai2 and Foxd3 (Hong and Saint-Jeannet, 2007; Spokony et al., 2002). Sox10, on the other hand, is expressed later in pre-migratory NC cells. Both So9 and Sox10 are activated by the canonical WNT signaling pathway, and the expression of Sox10 is controlled by Sox9 and Snai2 (Spokony et al., 2002).

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