2023 Project 1

From Embryology
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[1][2]. 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.[3]

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[4][5] (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[2]. 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).


Alkobtawi M, Pla P, Monsoro-Burq AH. 2021. BMP signaling is enhanced intracellularly by FHL3 controlling WNT-dependent spatiotemporal emergence of the neural crest. Cell Reports 35:109289. doi:10.1016/j.celrep.2021.109289 PMID: 34161771

Brugger SM, Merrill AE, Torres-Vazquez J, Wu N, Ting M-C, Cho JY-M, Dobias SL, Yi SE, Lyons K, Bell JR, Arora K, Warrior R, Maxson R. 2004. A phylogenetically conserved cis-regulatory module in the Msx2promoter is sufficient for BMP-dependent transcription in murine and Drosophila embryos. Development 131:5153–5165. doi:10.1242/dev.01390 PMID15459107

de Crozé N, Maczkowiak F, Monsoro-Burq AH. 2011. Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network. Proc Natl Acad Sci U S A 108:155–160. doi:10.1073/pnas.1010740107 PMID: 21169220

Eames BF, Medeiros DM, Adameyko I. 2020. Evolving Neural Crest Cells. CRC Press.

Faure S, de Santa Barbara P, Roberts DJ, Whitman M. 2002. Endogenous patterns of BMP signaling during early chick development. Dev Biol 244:44–65. doi:10.1006/dbio.2002.0579 PMID: 11900458

García-Castro MI, Marcelle C, Bronner-Fraser M. 2002. Ectodermal Wnt function as a neural crest inducer. Science 297:848–851. doi:10.1126/science.1070824 PMID: 12161657

Garnett AT, Square TA, Medeiros DM. 2012. BMP, Wnt and FGF signals are integrated through evolutionarily conserved enhancers to achieve robust expression of Pax3 and Zic genes at the zebrafish neural plate border. Development 139:4220–4231. doi:10.1242/dev.081497 PMID: 23034628

Hong C-S, Saint-Jeannet J-P. 2007. The Activity of Pax3 and Zic1 Regulates Three Distinct Cell Fates at the Neural Plate Border. MBoC 18:2192–2202. doi:10.1091/mbc.e06-11-1047 PMID: 17409353

Lander R, Nasr T, Ochoa SD, Nordin K, Prasad MS, LaBonne C. 2013. Interactions between Twist and other core epithelial–mesenchymal transition factors are controlled by GSK3-mediated phosphorylation. Nat Commun 4:1542. doi:10.1038/ncomms2543 PMID: 23443570

Marchant L, Linker C, Ruiz P, Guerrero N, Mayor R. 1998. The inductive properties of mesoderm suggest that the neural crest cells are specified by a BMP gradient. Dev Biol 198:319–329. PMID: 9659936

Milet C, Monsoro-Burq AH. 2012. Neural crest induction at the neural plate border in vertebrates. Developmental Biology, Neural Crest 366:22–33. doi:10.1016/j.ydbio.2012.01.013

Monsoro-Burq A-H, Fletcher RB, Harland RM. 2003. Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals. Development 130:3111–3124. doi:10.1242/dev.00531 PMID: 12783784

Monsoro-Burq A-H, Wang E, Harland R. 2005. Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction. Dev Cell 8:167–178. doi:10.1016/j.devcel.2004.12.017 PMID: 15691759

Nieto MA. 2018. A snail tale and the chicken embryo. Int J Dev Biol 62:121–126. doi:10.1387/ijdb.170301mn PMID: 29616719

Plouhinec J-L, Roche DD, Pegoraro C, Figueiredo A-L, Maczkowiak F, Brunet LJ, Milet C, Vert J-P, Pollet N, Harland RM, Monsoro-Burq AH. 2014. Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers. Dev Biol 386:461–472. doi:10.1016/j.ydbio.2013.12.010 PMID: 24360906

Plouhinec J-L, Zakin L, Moriyama Y, De Robertis EM. 2013. Chordin forms a self-organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo. Proc Natl Acad Sci U S A 110:20372–20379. doi:10.1073/pnas.1319745110 PMID: 24284174

Prasad MS, Charney RM, García-Castro MI. 2019. Specification and formation of the neural crest: Perspectives on lineage segregation. Genesis 57:e23276. doi:10.1002/dvg.23276 PMID: 30576078

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Sato T, Sasai N, Sasai Y. 2005. Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm. Development 132:2355–2363. doi:10.1242/dev.01823 PMID: 15843410

Schlosser G. 2008. Do vertebrate neural crest and cranial placodes have a common evolutionary origin? BioEssays 30:659–672. doi:10.1002/bies.20775 PMID: 18536035

Schumacher JA, Hashiguchi M, Nguyen VH, Mullins MC. 2011. An intermediate level of BMP signaling directly specifies cranial neural crest progenitor cells in zebrafish. PLoS One 6:e27403. doi:10.1371/journal.pone.0027403 PMID: 22102893

Sepporta M-V, Praz V, Balmas Bourloud K, Joseph J-M, Jauquier N, Riggi N, Nardou-Auderset K, Petit A, Scoazec J-Y, Sartelet H, Renella R, Mühlethaler-Mottet A. 2022. TWIST1 expression is associated with high-risk neuroblastoma and promotes primary and metastatic tumor growth. Commun Biol 5:1–17. doi:10.1038/s42003-021-02958-6 PMID: 35022561

Simões-Costa M, Bronner ME. 2015a. Establishing neural crest identity: a gene regulatory recipe. Development 142:242–257. doi:10.1242/dev.105445 PMID: 25564621

Soldatov R, Kaucka M, Kastriti ME, Petersen J, Chontorotzea T, Englmaier L, Akkuratova N, Yang Y, Häring M, Dyachuk V, Bock C, Farlik M, Piacentino ML, Boismoreau F, Hilscher MM, Yokota C, Qian X, Nilsson M, Bronner ME, Croci L, Hsiao W-Y, Guertin DA, Brunet J-F, Consalez GG, Ernfors P, Fried K, Kharchenko PV, Adameyko I. 2019. Spatiotemporal structure of cell fate decisions in murine neural crest. Science 364:eaas9536. doi:10.1126/science.aas9536 PMID: 31171666

Spokony RF, Aoki Y, Saint-Germain N, Magner-Fink E, Saint-Jeannet J-P. 2002. The transcription factor Sox9 is required for cranial neural crest development in Xenopus. Development 129:421–432.

Sutton G, Kelsh RN, Scholpp S. 2021. Review: The Role of Wnt/β-Catenin Signalling in Neural Crest Development in Zebrafish. Frontiers in Cell and Developmental Biology 9.

Suzuki A, Ueno N, Hemmati-Brivanlou A. 1997. Xenopus msx1 mediates epidermal induction and neural inhibition by BMP4. Development 124:3037–3044. doi:10.1242/dev.124.16.3037

Tribulo C, Aybar MJ, Nguyen VH, Mullins MC, Mayor R. 2003. Regulation of Msx genes by a Bmp gradient is essential for neural crest specification. Development 130:6441–6452. doi:10.1242/dev.00878

Vallin J, Thuret R, Giacomello E, Faraldo MM, Thiery JP, Broders F. 2001. Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling. J Biol Chem 276:30350–30358. doi:10.1074/jbc.M103167200

Linked References

  1. Alkobtawi M, Pla P & Monsoro-Burq AH. (2021). BMP signaling is enhanced intracellularly by FHL3 controlling WNT-dependent spatiotemporal emergence of the neural crest. Cell Rep , 35, 109289. PMID: 34161771 DOI.
  2. 2.0 2.1 Milet C & Monsoro-Burq AH. (2012). Neural crest induction at the neural plate border in vertebrates. Dev Biol , 366, 22-33. PMID: 22305800 DOI.
  3. Schlosser G. (2008). Do vertebrate neural crest and cranial placodes have a common evolutionary origin?. Bioessays , 30, 659-72. PMID: 18536035 DOI.
  4. Brugger SM, Merrill AE, Torres-Vazquez J, Wu N, Ting MC, Cho JY, Dobias SL, Yi SE, Lyons K, Bell JR, Arora K, Warrior R & Maxson R. (2004). A phylogenetically conserved cis-regulatory module in the Msx2 promoter is sufficient for BMP-dependent transcription in murine and Drosophila embryos. Development , 131, 5153-65. PMID: 15459107 DOI.
  5. Marchant L, Linker C, Ruiz P, Guerrero N & Mayor R. (1998). The inductive properties of mesoderm suggest that the neural crest cells are specified by a BMP gradient. Dev Biol , 198, 319-29. PMID: 9659936