2023 Project 2
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
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Neural Plate/Neural Border
Decades ago, Carl Gans and Glen Northcutt presented an intriguing ‘New head’ theory, which proposed that one of the significant inventions of vertebrates, in the evolutionary context is the development of a complex head [1]. This complexity conferred vertebrates with a plethora of novel cranial sensory organs and skeletal structures, contributing to an active predatory lifestyle and evolutionary success [1–3]. Various studies revealed that many of these novel cranial structures originate from two unique embryonic tissues: the cranial placodes and the neural crest (NC) [1,4,5]. While being ectodermal in origin, the neural crest and the cranial placodes give rise to multiple different tissues, with the neural crest also being able to form tissues of a mesodermal origin [Fig 1]. The cranial placodes develop into many of the paired sensory organs. In contrast, NC gives rise to various derivatives such as craniofacial bones and cartilage, melanocytes, and the peripheral nervous system. The neural crest and the cranial placodes originate from the neural border, a strip of tissue positioned between the neural and non-neural ectoderm [6,8; Fig 2]. This tissue does not broadly express any specific transcription factors, but is demarcated by the overlapping expression of genes like Pax3, Zic1, Tfap2a, Dlx3, Msx1 and others. The relative expression levels of these major genes and differential activity levels of different signaling pathways lead to the differential fate specifications: high Wnt/low to intermediate BMP forms the neural crest while low Wnt/Low BMP forms the cranial placode progenitors. Although the timing of induction differs across species, the neural border gives rise to the neural crest and preplacodal ectoderm (progenitor to cranial placodes) between the end of gastrulation and the start of neurulation.
Due to the vast number of derivatives, defects in the neural crest or placodes during early embryonic stages can lead to multiple developmental disorders. The importance of NC development in humans can be exemplified by the fact that defects in the specification, migration and differentiation of NC cells result in an array of congenital disorders collectively known as neurocristopathies [12]. Neurocristopathies affect a considerable population of newborns resulting in various conditions such as cleft palate, Waardenburg syndrome, and Hirschsprung’s disease [12]. On the other hand, defective placodes lead to diseases such as Branchio-Oto-Renal (BOR) Syndrome. Understanding the signalling pathways and GRN that governs the specification of NC and placodes at the NB is critical for understanding the molecular mechanisms underlying neurocristopathies. Ultimately, investigating their specification at the molecular level will give more insight into the aetiology of such diseases and their better therapeutic management.
Various research studies and reviews have shed light on the development and derivatives of cranial placodes and the neural crest [3,6–8].
Neural Crest Specification/PRE-EMT
The neural crest is a fascinating population of transient multipotent and migratory cells exclusively acquired by vertebrates during evolution [8,9]. It was first observed more than 150 years ago (His, 1868). Over the years, intricate experiments by lots of scientists proved that the neural crest is indeed multipotent and although being ectodermal in nature, can give rise to mesenchymal tissues as well. Later, the identification of Slug (Snail2) gave the neural crest a distinct genetic identity (Nieto et al., 1994). The induction of neural crest from the anterio-lateral neural border occurs due to high Wnt signaling during the end of gastrulation (frog) or during early neurulation (chick). This is marked by the expression of certain neural crest-specific markers, such as Snail1/2, Sox8/9 and FoxD3. The neural crest can be classified into three different types depending upon its position relative to the body axis (and expression of hox genes) – the rostralmost cranial neural crest, the caudal trunk neural crest and the vagal neural crest in the middle. In most cases, the induction of the neural crest follows a rostral to caudal direction. The differentiation potential of the cranial and vagal neural crest is higher than the trunk neural crest, with derivatives of the former including both ectodermal and mesodermal tissues while derivatives of the latter are only ectodermal in nature. As neurulation progresses, we observe the expression of late neural crest genes like Sox10 and Twist1, which are required for maturation of the neural crest and EMT. As the neural folds meet and the neural tube closes, the neural crest cells start to migrate (rostral first) to their final destinations, where they will give rise to various derivatives such as the peripheral nervous system, craniofacial cartilage and bone, and melanocytes [9,10].
The formation of NC at the NB is a hierarchical process governed by various signalling pathways and a complex gene regulatory network (GRN) involving multiple genes and transcription factors (TFs) [6]. The first process in the NC formation is the establishment of NB by TFs, notably pax3, zic1, dlx3 and msx1, also termed as NB specifiers (or NB-TFs) [6]. The interaction of various inductive signalling molecules (BMP, Wnt and FGF) from the neighbouring tissues: the neural plate, the non-neural ectoderm and the paraxial mesoderm activate the NB specifiers [11]. Subsequently, the synergism between various NB specifiers in the presence of above mentioned inductive signals leads to the activation of another set of transcription factors known as NC specifiers [9]. Based on numerous studies in vertebrate animal models such as chick, Xenopus and lamprey, NC specifiers can be broadly classified into early and late groups [6,8,9].
During late gastrulation/early neurulation, the early NC specifiers such as snai2, foxd3, sox8 and sox9 induce and specify the premigratory NC cells in the dorsal neural tube [8,9]. In the second half of the neuration, the earlier NC specifiers lead to the induction of late NC specifiers (e.g., sox10, ets1, and twist1), which initiate the epithelial-mesenchymal transition (EMT) program in the premigratory NC cells. Following EMT, the NC cells migrate towards their destined locations in the embryo and differentiate into multiple derivatives [9,11,12].
Neural crest and Sox E proteins
Sox E proteins are neural crest specifiers downstream of and induced by neural plate border specifiers in vertebrates. In Xenopus laevis, Sox9 is expressed shortly after gastrulation at the lateral edges of the neural plate, in the NC-forming region (from stage 11 onwards). As development proceeds, Sox9 expression persists in migrating cranial crest cells as they populate the pharyngeal arches. Depletion of Sox9 protein in developing embryos, using morpholino antisense oligos, causes a dramatic loss of neural crest progenitors and an expansion of the neural plate [15]. Later during embryogenesis, morpholino-treated embryos have a specific loss or reduction of neural crest-derived skeletal elements, mimicking one aspect of the craniofacial defects observed in Campomelic Dysplasia patients. Sox9 is an essential component of the regulatory pathway that leads to cranial neural crest and cranial placode formation but, very little is known about its gene regulation mechanism. In Xenopus laevis, Sox10 expression accumulates from stage 13/14 onwards (early neurulation). It is expressed in prospective neural crest and otic placode regions from the earliest stages of NC specification and in migrating cranial and trunk neural crest cells. Loss-of-function experiments in Xenopus using morpholino antisense oligos against SOX10 produce a loss of neural crest precursors and an enlargement of the surrounding neural plate and epidermis [16]. This effect of Sox10 depletion is produced during some of the earliest steps of NC specification, as is shown by the inhibition in the expression of Slug and FoxD3, which are early markers of neural crest specification. In addition, it has been shown that SOX10 depletion leads to an increase in apoptosis and a decrease in cell proliferation in the neural folds, suggesting that SOX10 could work as survival as well as a specification factor in NC precursors during pre-migratory stages. Mutations in one Sox10 allele have been found in patients that suffer from congenital aganglionic megacolon (Hirschsprung disease), sometimes associated with a combination of pigmentation defects and deafness (Waardenburg–Shah syndrome). The phenotype associated with this pathology indicates an important role for Sox10 in neural crest and otic placode development [17, 18].
Cranial Placode Specification
The ectodermal placodes were first described around the same time, depicted as ectodermal thickenings (Van Kuppfer, 1891). Thereafter, lineage tracing studies revealed that the placodes can give rise to multiple tissues, including both neurogenic and non-neurogenic. The cranial placodes are preceded by the formation of a preplacodal ectoderm, which is denoted by the expression of Six (1,2 4 and 5) and Eya (1 and 2), two important transcription factors. The preplacodal ectoderm forms in the anterior zone of the neural border which has low Wnt and low BMP signaling. At the end of neurulation, preplacodal ectoderm cells also undertake migration to different areas of the head to form the different cranial placodes, which have slightly distinct genetic identities. However, unlike the neural crest, they do not undergo EMT and instead delaminate and move away. Moreover, these migrating cells are essential for neural crest migration as they define the prospective migration routes. Later, they form the different cranial placodes –non-neurogenic adenohypophysis and lens, and neurogenic epibranchial, otic, paratympanic, trigeminal and olfactory. Aquatic anamniote vertebrates also possess the lateral line placodes, which forms mechanosensory organs in the head and the trunk. The placode cells also interact with the neural crest cells to form the cranial sensory ganglia.
References
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2. Manzanares M, Nieto MÁ: A celebration of the new head and an evaluation of the new mouth. Neuron 2003, 37:895–898. PMID: 12670419
3. Schlosser G: Early embryonic specification of vertebrate cranial placodes. Wiley Interdiscip Rev Dev Biol 2014, 3:349–363. PMID: 25124756
4. Couly GF, Le Douarin NM: Mapping of the early neural primordium in quail-chick chimeras. I. Developmental relationships between placodes, facial ectoderm, and prosencephalon. Dev Biol 1985, 110:422–439. PMID: 4018406
5. Gammill LS, Bronner-Fraser M: Neural crest specification: Migrating into genomics. Nat Rev Neurosci 2003, 4:795–805. PMID: 14523379
6. Pla P, Monsoro-Burq AH: The neural border: Induction, specification and maturation of the territory that generates neural crest cells. Dev Biol 2018, 444:S36–S46. PMID: 29852131
7. Maharana SK, Schlosser G: A gene regulatory network underlying the formation of pre-placodal ectoderm in Xenopus laevis. BMC Biol 2018, 16:79. PMID: 30012125
8. Seal S, Monsoro-Burq AH: Insights Into the Early Gene Regulatory Network Controlling Neural Crest and Placode Fate Choices at the Neural Border. Front Physiol 2020, 11:1528. PMID: 33324244
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11. Gammill LS, Bronner-Fraser M: Neural crest specification: Migrating into genomics. Nat Rev Neurosci 2003, 4:795–805.
12. Vega-Lopez GA, Cerrizuela S, Tribulo C, Aybar MJ: Neurocristopathies: New insights 150 years after the neural crest discovery. Dev Biol 2018, 444:S110–S143.
13. Riddiford N, Schlosser G: Dissecting the pre-placodal transcriptome to reveal presumptive direct targets of Six1 and Eya1 in cranial placodes. Elife 2016, 5.
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15. Spokony RF, Aoki Y, Saint-Germain N, Magner-Fink E, Saint-Jeannet JP: The transcription factor Sox9 is required for cranial neural crest development in Xenopus. Development 2002, 129(2):421-32. PMID: 11807034
16. Honoré SM, Aybar MJ, Mayor R: Sox10 is required for the early development of the prospective neural crest in Xenopus embryos. Developmental biology 2003, 260(1):79-96. PMID: 12885557
17. Saint-Germain N, Lee YH, Zhang Y, Sargent TD, Saint-Jeannet JP: Specification of the otic placode depends on Sox9 function in Xenopus. Development 2004, 131(8):1755-63. PMID: 15084460
18. Aoki Y, Saint-Germain N, Gyda M, Magner-Fink E, Lee YH, Credidio C, Saint-Jeannet JP: Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. Developmental biology 2003, 259(1):19-33. PMID: 12812785
C. Gans, R.G. Northcutt. Neural crest and the origin of vertebrates: a new head. Science, 220 (1983), pp. 268-274
C. Von Kupffer. The development of the cranial nerves of vertebrates. J. Comp. Neurol., 1 (1891), pp. 246-332
Nieto MA, Sargent MG, Wilkinson DG, Cooke J. Control of cell behavior during vertebrate development by Slug, a zinc finger gene. Science, 264,5160 (1994), pp. 835-839 PMID: 7513443
Suggested reading
Neural crest history - Etchevers HC, Dupin E, Le Douarin NM. The diverse neural crest: from embryology to human pathology. Development, 146(5):dev169821 (2019). PMID: 30858200 Review.
Cranial placodes – Schlosser G. Early embryonic specification of vertebrate cranial placodes. Wiley Interdiscip Rev Dev Biol. 2014;3(5):349-363. doi:10.1002/wdev.142 PMID: 25124756 Review.
