PAX transcription factors in neural crest development

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Seminars in Cell & Developmental Biology xxx (2015) xxx–xxx

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Review

PAX transcription factors in neural crest development Anne H. Monsoro-Burq ∗ Univ. Paris Sud, Université Paris Saclay, Centre Universitaire, 15, rue Georges Clémenceau, F-91405 Orsay, FranceInstitut Curie Research Division, Centre Universitaire, 15, rue Georges Clémenceau, F-91405 Orsay, FranceUMR 3347 CNRS, U1021 Inserm, Université Paris Saclay, Centre Universitaire, 15, rue Georges Clémenceau, F-91405 Orsay, France

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Article history: Received 3 June 2015 Received in revised form 14 September 2015 Accepted 21 September 2015 Available online xxx Keywords: Pax1 Pax3 Pax7 Pax9 Splotch mutant Waardenburg syndrome Cardiac neural crest Craniofacial skeleton Melanocyte Schwann cell Sensory neuron

a b s t r a c t The nine vertebrate PAX transcription factors (PAX1–PAX9) play essential roles during early development and organogenesis. Pax genes were identified in vertebrates using their homology with the Drosophila melanogaster paired gene DNA-binding domain. PAX1-9 functions are largely conserved throughout vertebrate evolution, in particular during central nervous system and neural crest development. The neural crest is a vertebrate invention, which gives rise to numerous derivatives during organogenesis, including neurons and glia of the peripheral nervous system, craniofacial skeleton and mesenchyme, the heart outflow tract, endocrine and pigment cells. Human and mouse spontaneous mutations as well as experimental analyses have evidenced the critical and diverse functions of PAX factors during neural crest development. Recent studies have highlighted the role of PAX3 and PAX7 in neural crest induction. Additionally, several PAX proteins – PAX1, 3, 7, 9 – regulate cell proliferation, migration and determination in multiple neural crest-derived lineages, such as cardiac, sensory, and enteric neural crest, pigment cells, glia, craniofacial skeleton and teeth, or in organs developing in close relationship with the neural crest such as the thymus and parathyroids. The diverse PAX molecular functions during neural crest formation rely on fine-tuned modulations of their transcriptional transactivation properties. These modulations are generated by multiple means, such as different roles for the various isoforms (formed by alternative splicing), or posttranslational modifications which alter protein–DNA binding, or carefully orchestrated protein–protein interactions with various co-factors which control PAX proteins activity. Understanding these regulations is the key to decipher the versatile roles of PAX transcription factors in neural crest development, differentiation and disease. © 2015 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Pax3 mutation causes severe defects in neural crest development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 PAX3 and PAX7 transcription factors are essential for early neural crest development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Pax3 and Pax7 expression are activated in the neural crest-forming edges of the developing neural plate during gastrulation . . . . . . . . . . . . . 00 3.2. Pax3 and Pax7 are required for neural crest induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. PAX3 is essential for neural crest survival during emigration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Pax genes are essential for the differentiation of multiple neural crest derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. PAX3 and PAX7 control proliferation of craniofacial neural crest and response to environmental stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. PAX9 is involved in tooth and craniofacial skeleton development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.3. PAX1 and PAX9 are involved in thymus, thyroid and parathyroid development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abbreviations: BMP, bone morphogenetic protein; CDH syndrome, Craniofacial-Deafness-Hand syndrome; Cdx, gene of the caudal homeobox gene family; E n, mouse embryonic day n (e.g. E16); FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; GRN, gene regulatory network; HD, homeodomain; HDAC, histone deacetylases; LIF, leukemia inhibitory factor; MBP, myelin basic protein; MITF, microphthalmia-associated transcription factor; NC, neural crest; nmSCs, non-myelinating Schwann cells; Pax/PAX, paired-box containing gene/protein; PD, paired domain; splotch (Sp), splotch mouse mutant; Wnt, wingless family secreted factor; WS, Waardenburg syndrome; WSI, Waardenburg syndrome type I; WSIII, Waardenburg syndrome type III. ∗ Correspondence to: Institut Curie Research Division, Batiment 110, Centre Universitaire, F-91405 Orsay Cedex, France. E-mail address: [email protected] http://dx.doi.org/10.1016/j.semcdb.2015.09.015 1084-9521/© 2015 Elsevier Ltd. All rights reserved.

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4.4. PAX3 controls the balance between proliferation and differentiation of sensory neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.5. PAX3 controls the balance between proliferation and differentiation in Schwann cells precursors and during nerve regeneration . . . . . . . . 00 4.6. Pax3 controls the differentiation of pigment cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions: PAX transcription factors are multifacet regulators of neural crest development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction The neural crest, a transient and highly multipotent embryonic cell population, forms a variety of cells and tissues in vertebrates [1]. Neural crest cells arise at the edge between neural and non-neural ectoderm [2,3]. Recent studies have highlighted that neural crest induction starts early during gastrulation, as the axial and paraxial mesoderm migrate underneath the ectoderm and signal toward the prospective neural and neural crest tissues [4,5]. As the neural folds elevate, during neurulation, the neural crest cells emigrate from the dorsal aspect of the embryo and travel extensively through several routes to reach target tissues. Neural crest derivatives are patterned during organogenesis. The neural crest cells give rise to both neurons and glia of the peripheral nervous system, including sensory, autonomous, enteric ganglia and Schwann cells around axons. They also generate the pigment cells, such as melanocytes, xanthophores, iridophores and other pigmented cells types. The skeleton and the mesenchyme of the head, face and neck, including skull and teeth, come from cranial neural crest cells, which are issued from mid-diencephalon level to somite 5 level along the anteroposterior body axis. These skeletal and mesenchymal derivatives, although derived from the ectoderm, are thus similar to tissues and cells formed elsewhere by trunk mesoderm: they are called “mesectoderm” or “ectomesenchyme” [1]. Cranial neural crest cells issued from the neural tube located between the otic vesicle and somite 3 level, form the cardiac neural crest. This latter cell population is essential to form the connective tissues of the thymus, thyroid and parathyroid glands [6,7]. Furthermore, the cardiac neural crest populates the outflow tract of the heart, forms smooth muscles of the great arteries, and displays a critical role in the formation of the aorticopulmonary septum [8,9]. Finally, trunk neural crest cells also form chromaffin cells in the adrenal gland [10]. During the development of each neural crest-derived lineage, various developmental regulators are involved, including PAX proteins. PAX factors are essential transcriptional regulators, highly conserved in vertebrates, which play major roles during organogenesis. PAX transcription factors are involved in neural crest induction. They are also expressed in multiple developing neural crest-derived structures and participate in key steps of their formation: migration, proliferation and differentiation. Fig. 1 summarizes the list of neural crest derivatives, and indicates the main function displayed by PAX proteins during the development of each lineage. In this review, we also underline the impact of PAX factors malfunction in neural crest-linked congenital diseases. Nine Pax genes have been identified in mouse, based on the homology of their paired-type DNA binding domain (PD) with the paired domain of Drosophila melanogaster segmentation genes [11,12]. In addition to the PD, vertebrate Pax genes are grouped into four subfamilies according to the presence of additional conserved domains, such as the octapeptide and presence of a partial or complete paired-type homeodomain [13]. Subfamily I PAX proteins contain only the paired domain and the octapeptide, while subfamily III PAX members have a paired domain, a homeodomain and the octapeptide. The homeodomain forms a second DNA-binding motif, whereas the octapeptide mediates repressive protein–protein interactions [14,15]. A C-terminal transactivation

domain is found in all Pax genes (e.g. Pax3, Pax6 [16,17]). All these PAX-specific domains are highly conserved throughout chordate and vertebrate evolution [18]. Alternative splicing, alternative promoter use, posttranslational regulation and mutations in the conserved domains can alter and modulate DNA recognition by PAX proteins and transactivation of PAX target genes (e.g. for PAX3 [19]). Pax genes developmental expression is highly conserved in vertebrates. During early organogenesis, subfamily III Pax genes, Pax3 and Pax7, are strongly expressed in the dorsal neural tube and in the myotomes while Pax6 (Pax subfamily IV, containing paired- and homeo-domains) marks intermediate and ventral parts of the spinal cord [20–22]. The functional importance of PAX3 for neural and neural crest formation is highlighted by the neural tube and neural crest defects observed in mouse or human Pax3 mutants [23]. During neural crest cell migration, PAX3 is further critical for cell survival in cardiac neural crest [24]. At subsequent developmental steps, Pax3 and Pax7 are expressed in the proliferating progenitors of several neural crest lineages, including sensory neurons, melanoblasts and Schwann cell precursors [25–29]. Additionally, on top of their prominent expression in mesoderm-derived sclerotome derivatives, subfamily I Pax genes are found in cranial neural crest-derived mesectoderm: Pax9 is expressed in the prospective craniofacial skeleton, and teeth. Additionally, Pax1 and Pax9 are found in the epithelium lining the developing thymus and parathyroids, organs developing in close interaction with the mesenchymal neural crest of the pharyngeal pouches [30–32]. The critical function of PAX1/9 proteins in the development of these structures is evidenced by the defects caused by Pax1 and Pax9 mutations in mouse and human [31–33]. Here, we focus on Pax genes of subfamilies I and III, which encode the main PAX factors participating to various steps of neural crest development, and to neural crest-linked pathologies. 2. Pax3 mutation causes severe defects in neural crest development The link between subfamily III Pax genes and neural crest development was evidenced by the analysis of mouse mutants, first described by Russell [34] and Auerbach [35], and by the identification of the gene mutations causing the human Waardenburg (WS) syndrome [23]. Several dozens of mutations in mouse Pax3 gene are reported, being either spontaneous mutations (splotch mutant, Sp), chemically and radiation-induced mutations, or targeted mutations. Homozygous null Pax3 mutations are embryonic lethal around embryonic day 15 (E14-E16 according to the genetic background). The splotch mouse phenotype comprises spina bifida in the central nervous system (accompanied by exencephaly in half of the embryos), and abnormal neural crest derivative development, including defects in various components of the peripheral nervous system, in the heart and pigmentation alterations [36,37]. These mice also present severe muscle development defects. The splotch mutations were mapped to mouse Pax3 gene on chromosome 1. The different splotch alleles bear diverse alterations of Pax3 gene such as deletions or point mutations [38,39]. In heterozygous Pax3 mutant mice, a melanocyte development phenotype is observed, with white spotting on belly, feet and tail, giving its name to the

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Fig. 1. Neural crest derivatives and Pax genes involved in neural crest development. (A) Neural crest derivatives are listed: Cranial-specific neural crest derivatives (mesectoderm forming head cartilage, bone, teeth and mesenchyme), cardiac neural crest derivatives (forming cardiac outflow tract, wall of great arteries, mesenchyme of the parathyroid and thymus), neural crest lineages found at all levels of the body axis (pigment cells, sensory, autonomous and enteric neurons, peripheral glia) and chromaffin cells of the adrenal gland. (B) The Pax genes involved in each aspect of neural crest formation are indicated. According to the observation in mouse embryos, the role of Pax3 and Pax7 in neural crest induction and migration is indicated as more important posteriorly (gray).

mutation [35]. In the rest of this review, “splotch mutant” will refer to the homozygous mouse mutant unless otherwise stated. Importantly, restoring PAX3 function in the neural crest lineage but not in the muscle lineage, by neural crest-specific expression of Pax3 in a Pax3-null background rescues the neural tube and NC defects as well as the embryonic lethality [40]. These mice die later, at birth, due to muscle and respiratory defects. This demonstrates that the cause of the embryonic lethality in the splotch phenotype is the lack of Pax3 expression in the neural crest lineage. Detailed neural crest lineage tracing in splotch mice, using for example lacZ expression driven by the neural crest specific Wnt1cre promoter, or a Pax3-lacZ knock-in, shows that the severity of neural crest defects increases posteriorly along the body axis [41–43]. Cranial neural crest cells appear almost normal in the head including the first branchial arches. In contrast, the formation and migration of cardiac, trunk and caudal neural crest are severely impaired. Cranial ganglia develop, while dorsal root ganglia and sympathetic ganglia are reduced at anterior trunk levels, and fail to form at lumbar and caudal levels. In human, Pax3 mutations are found in the Waardenburg syndrome (WS) and occasionally in the Craniofacial-Deafness-Hand (CDH) syndrome, which shares symptoms with WS. WS is a clinically heterogeneous syndrome, generally transmitted in an autosomal dominant manner. The phenotype includes pigmentation defects in hair, skin and irises, congenital sensorineural

deafness, mild craniofacial dysmorphogenesis (dystopia canthorum) and occasional association with cardiac defects [44,45]. Most aspects of WS syndrome phenotype, e.g. pigmentation defects, dystopia canthorum and sensorineural deafness, are linked to defects in neural crest development. These include defective formation of the melanocyte involved in skin and hair pigmentation, as well as those participating to inner ear development [46]. Heterozygous Pax3 mutations cause WS I, the most common WS type, affecting about 1/42,000 births [45,47]. In contrast, homozygous or compound heterozygous Pax3 mutations are related to the rare WS III (or Klein–Waardenburg syndrome). WS III patients display additional severe musculoskeletal defects in the forelimb. In WS I-III, both missense and truncation mutations are found spanning the entire Pax3 gene. They include mutations in the conserved DNA-binding domains (paired domain, homeodomain)[45]. Although about 70 point mutations in Pax3 are recorded, no specific recurrence is found. Most mutations are thought to affect DNA binding and transactivation ability [48,49]. Interestingly, the genetic background and environmental factors may play important roles in the phenotype penetrance, as there is a high heterogeneity in phenotype, even within families bearing the same mutation [50]. During vertebrate evolution, an ancestral Pax3/7 gene has been duplicated in two paralogs, Pax3 and Pax7. In contrast to the critical requirement for PAX3 function in neural crest development, PAX7 does not seem to play essential roles in neural crest

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formation. Although 22 different mutations are reported in mouse Pax7 gene, no obvious neural crest phenotype seems to develop (apart from a modest reduction of maxillary and nasal skeleton in some genetic backgrounds) [51]. So far, no Pax7 mutation is associated with a human syndrome or disease. In addition, at trunk levels in mouse, Pax7 is expressed in the dorsal half of the neural tube, but is excluded from the dorsalmost part (the roof plate) and from neural crest derivatives [42]. At cranial levels however, a modest subpopulation of neural crest cells expresses Pax7 prior and during neural crest migration [51,52]. Recent re-examination of Pax7-positive cranial neural crest derivatives reveals that, during organogenesis, their contribution to craniofacial structures is more important than previously anticipated [52]. Interestingly, as described above, PAX3 loss does not affect cephalic neural crest development in the mouse, suggesting redundancy between PAX3 and PAX7. Indeed, when experimentally expressed in the trunk neural crest, PAX7 can compensate the loss of PAX3, showing functional redundancy between the two paralogs [42]. Moreover, at cranial levels, a clear compensation occurs between the two genes in single knockout mice: the recent analysis of cephalic neural crest formation in the double Pax3/Pax7 knockout found severe craniofacial malformations with lack of development and fusion of the facial prominences, resulting in a cleft face phenotype [53]. Pax3 and, to a lesser extent, Pax7 have thus been the focus of most studies related to Pax gene function in neural crest development. In contrast, PAX7 plays critical roles in muscle stem cell development and postnatal maintenance (see Buckingham and Relaix, this volume).

3. PAX3 and PAX7 transcription factors are essential for early neural crest development Neural crest developmental mechanisms are largely conserved in vertebrates, from lamprey to mammals [54,55]. The molecular basis of the requirement for PAX3 and PAX7 factors was recently explored using complementary approaches in diverse vertebrate developmental models. Frog and chick were used to explore the earliest steps of neural crest formation at the edge of the neural plate; while mouse, chick, zebrafish and frog allowed studying neural crest migration. Neural crest differentiation was primarily studied in chick and mouse embryos. Despite a few minor speciesspecific variations, these studies have collectively shed light on the molecular mechanisms involving PAX3/7 genes in the neural crest gene regulatory network (GRN).

3.1. Pax3 and Pax7 expression are activated in the neural crest-forming edges of the developing neural plate during gastrulation According to species, either Pax3 or Pax7 appears first at the prospective lateral and posterior edges of the neural plate, delineating the future neural plate from the non neural ectoderm, as neural induction is initiated in the early gastrula (e.g. ancestral Pax3/7 in lamprey, Pax3 in fish and frog, Pax7 in chick) [4,5,54,56]. It is important to note that only a few amino acids differ between PAX3 and PAX7, in the protein C-terminus and not in the conserved DNA binding domains, meaning that the encoded proteins likely share most of their functions. Thus gene swapping has occurred during evolution, without major change in the architecture of the neural crest GRN [4,5,58]. As neural induction proceeds, the lateral edges of the neural plate express increasing Pax3/Pax7 levels, which label the dorsal half of the closed spinal cord at the end of neurulation [20,57]. The Pax3-positive region extends anteriorly at brain levels, roughly corresponding to the anterior boundary of neural crest-forming areas, i.e. the posterior half of diencephalon

[4]. Posteriorly, either Pax3 or Pax7, or both are expressed all along the dorsal neuraxis in different vertebrates [5,26,42,54,58]. A complex and dynamic combination of secreted signals and transcriptional regulators controls Pax3/Pax7 gene induction at the border between neural and non-neural ectoderm. These regulations act on several enhancer elements in Pax3 gene, driving specifically Pax3 expression in the neural crest, and not in myotome [40,56]. These enhancers are evolutionary conserved, as mouse enhancers drive neural crest expression in transgenic zebrafish lines [40]. Paraxial mesoderm-derived signals are critical to activate Pax3 at the neural plate border [59–61]. As it is shown for neural crest-specific genes, both Wnt and FGF signaling activate Pax3/Pax7 in chick, frog and fish [4,56,60,61]. Pax3 is an immediateearly Wnt signaling target in frog [62]. Detailed analysis of two Pax3 neural-crest-specific enhancers in fish reveals that each enhancer is activated by a precise integration of Wnt, FGF and BMP signals [56]. Both in fish and mouse, these multiple enhancers act in a redundant fashion, suggested that robust regulation of Pax3 expression is encoded in the genome [40,56]. In mouse embryos, Caudal homeobox transcriptional regulators (Cdx1-2-4) mediate Wnt signaling on Pax3 neural crest-specific enhancer, with interaction with zinc-finger transcription factor Zic2, indicating additional indirect regulation on Pax3 regulatory elements [63,64]. In frog, Tfap2a and Gbx2 also mediate indirect Wnt activation on Pax3, via evolutionary conserved TFAP2a binding sites [62,65]. In addition, TEAD2 and its co-activator YAP regulate Pax3 expression at the neural plate border, via TEAD binding sites in Pax3 regulatory sites [66,67]. Furthermore, a positive feed-forward loop involves several transcription factors at the neural-non neural border, to maintain and strengthen Pax3 expression [62]. Such fine control of Pax3 activation is essential to pattern the different ectoderm domains in the embryo, between neural, placode, neural crest and non-neural ectoderm areas, during neurulation [68]. 3.2. Pax3 and Pax7 are required for neural crest induction Experimental PAX3/PAX7 depletion in lamprey, frog, and chick, using carefully controlled antisense morpholino RNAs, has revealed their essential function in neural crest induction during the early steps of gastrulation [4,5,54]. PAX3/PAX7 depletion results in failure of early neural crest specific genes activation (e.g. Snail2 or Foxd3), followed by lack of neural crest emigration and further development. The phenotype of morphant embryos closely resembles that of the Pax3 mutants in mammals, with open neural tube, craniofacial and pigmentation defects [4]. However, while PAX3 gain of function in vivo expands the pre-migratory neural crest domain, it does not activate the early neural crest fate outside the border between neural and non-neural ectoderm. Similarly, activation of PAX3 in pluripotent ectoderm of the frog blastula (animal cap), with or without neuralization by BMP antagonists, does not consistently induce neural crest markers. This suggests that PAX3 alone is not sufficient to initiate formation of a functional neural crest territory, and that PAX3 cooperates with additional co-factors or signals in vivo [4]. In the current view of the neural crest gene regulatory network [3,69,70], several transcription factors are essential for neural/nonneural border formation, and are collectively called the neural border specifiers (Fig. 2). Neural border specifiers include TFAP2a, MSX1, HES4, ZIC1, PAX3/7, and GBX2 [4,65,71–75]. They respond and mediate signals from surrounding tissues, including BMP, WNT, FGF and other secreted molecules, and activate the genes which will characterize the neural crest and activate its epithelial to mesenchymal transition: the neural crest specifiers Snail2, SoxE genes, and Foxd3. Several of neural border specifiers show a functional synergy with PAX3 to activate Snail2 or Foxd3 and cell emigration. In particular, in animal cap pluripotent ectoderm, MSX1 and

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Fig. 2. Overview of Pax functions in the neural crest gene regulatory network.

ZIC1 are the most efficient PAX3 partners to activate Snail2 expression and cell scattering, while TFAP2a or HES4 fail to do so [76]. When combined to ZIC1, PAX3 gain of function now activates Snail2 and Foxd3 expression in ectopically, e.g. in ventral ectoderm and in endoderm in frog, and in extra-embryonic ectoderm in chick [4,76]. This synergistic effect depends on additional secreted factors, since the effect is lost when the animal cap is dissociated in vitro [4]. The secreted signals include Wnt signaling, since blocking Wnt activity using a dominant-negative Frizzled receptor in animal caps induced by PAX3 and ZIC1 blocks neural crest specifier activation [4]. Blocking retinoic acid or Notch signaling does not prevent neural crest induction in this experiment [4]. Moreover, adding back soluble WNT3a after cell dissociation in vitro, restores Snail2 induction [77]. Finally, the synergy between PAX3 and ZIC1 not only initiates neural crest development by inducing a few neural crest specifiers, but also activates the complete neural crest developmental cascade, including early specification, epithelium-to-mesenchyme transition, migration and differentiation into the various neural crest derivatives [76]. This key action of PAX3 and ZIC1 is achieved by the simultaneous activation of multiple neural crest specifiers as immediate-early targets: Snail1, Snail2, Ets1, Twist, Foxd3 [78]. Finally, additional regulations on PAX3/PAX7 proteins have emerged recently since, in chick, posttranslational sumoylation of PAX7 participates to its function in neural crest specifiers induction [79]. 3.3. PAX3 is essential for neural crest survival during emigration In addition to its essential role in early neural border and neural crest specification, PAX3 also displays important functions in neural crest cell survival. To migrate throughout the embryo using

various routes and diverse microenvironments, neural crest cells acquire increased survival potential and resistance to apoptosis. The participation of PAX3 to the acquisition of these properties, has been illustrated by heretospecific grafting between mouse and chick embryos: the neural tube of splotch mutants can generate some emigrating cells when grafted instead of the neural tube of a normal chick embryo, i.e. in the wild type dorsal environment, but not if implanted in an ectopic location, while wild type neural tube forms neural crest cells ectopically [41]. In frog, while very rare, some dorsal neural tube cells initiate epithelial-mesenchymal transition in Pax3 morphant embryos. Live imaging shows a few Pax3 morphant cells dying shortly after delamination from the dorsal neural tube [4]. Splotch embryos display progressive loss of cardiac and trunk posterior neural crest [42,57]. A few cardiac neural crest cells delaminate from the postotic neural tube. These splotch cardiac neural crest cells undergo apoptosis after a short migration [80]. The resulting phenotype closely resembles the phenotype obtained by the experimental ablation of the cardiac neural crest, and heart malformations seen in congenital neurocristopathies such as DiGeorge syndrome [8,24,35,81]. PAX3 thus plays both a direct and an indirect role in cardiac neural crest development. First, in addition to controlling EMT, the neural crest specifiers activated by PAX3 (Snail2, Foxd3, Sox9, Twist) display complementary functions in cell survival and proliferation. In the absence of SOX9, chick trunk neural crest cells undergo massive apoptosis upon delamination [82]. SNAIL2 exerts anti-apoptotic roles in frog cranial neural crest [83]. Defective neural crest induction in absence of PAX3 implicates that, indirectly, the few cells emigrating outside the neural tube will have a poor survival [82].

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Secondly, PAX3 is expressed during the first steps of neural crest migration in mouse, chick and frog embryos ([42,80] and AHMB personal communication). In the heart, the neural crest provides parasympathetic innervation and mesectodermal derivatives contributing to the outflow tract mesenchyme, the conotruncal cushions, and the smooth muscle wall of the aortic arch arteries [6,8,81,85,86]. Lack of cardiac neural crest results in defective formation of the aorticopulmonary septum (persistent truncus arteriosus) and abnormal patterning of the aortic and pulmonary arteries. These cardiac defects cause embryonic death in mouse around E14.5 [35,87,88]. Elegant experiments used specific Pax3 enhancers to drive gene expression in the neural crest. When Pax3 is re-expressed in specifically in the neural crest of Splotch mice, both neural tube and heart defects are rescued, and embryonic lethality is abolished [40,89]. PAX3 directly blocks cell apoptosis during cardiac neural crest migration by preventing p53 activity. Blocking p53-dependent apoptosis by either crossing Sp mice with p53−/− mutants or treating Sp mice with p53 inhibitor pifithrin-a rescues the cardiac neural crest migration and apoptosis phenotype, and the outflow tract septation is restored [80]. In neuroepithelium-like cell culture, PAX3 directly engages protein–protein interactions with p53, and the ubiquitin ligase MDM2. This promotes MDM2-dependent p53 ubiquitination and degradation, without detectable effect on p53 gene expression. In contrast, mutant PAX3 protein fails to bind p53 or MDM2 [90]. It remains unclear if the vagal enteric neural crest, which does not form in Splotch mutants or PAX3/7 fish morphants, undergoes similar p53-dependent apoptosis [26,80].

4. Pax genes are essential for the differentiation of multiple neural crest derivatives After extensive migration during which they continue to proliferate, neural crest cells reach target tissues and organs and differentiate. Pax genes are involved in the differentiation of craniofacial mesectoderm, pigment cells, peripheral neurons and glia, as well as in formation of organs involving endoderm and neural crest interactions in the neck, such as thyroid, thymus, and parathyroids.

4.1. PAX3 and PAX7 control proliferation of craniofacial neural crest and response to environmental stress As mentioned above, cranial neural crest does form in Splotch mice, whereas in other species, more severe and earlier defects deplete neural crest cells by the time of induction or migration. This species-specific difference allows studying the later roles of PAX3 and PAX7 on craniofacial neural crest development in mouse embryos. In the double Pax3−/− ;Pax7−/− knockout mutant, initial cranial neural crest formation seems normal until E9.5. Migrating cells populate the facial prominences [53]. However, a severe cleft face phenotype arises from E11.5. In the double mutant facial buds, Zalc and colleagues found an upregulation of the dioxin-responsive AHR signaling, and of p21 expression, a regulator of cell cycle exit. AHR signaling alters fgf8 expression, a major mediator of craniofacial mesenchyme proliferation and patterning [91]. Loss of both Pax3 and Pax7 function decreased Fgf8 expression and cell proliferation, promoted cell cycle exit, resulting in atrophied frontonasal prominences [53]. Importantly, when Pax3−/− single mutants are exposed to dioxin, they present severe frontonasal malformations, indicating that these embryos, although developing normal craniofacial structures in control situations, are sensitized to pollutants such as dioxin [53].

4.2. PAX9 is involved in tooth and craniofacial skeleton development Neural crest-derived mesectoderm includes the craniofacial skeleton and teeth [1]. Teeth develop by a series of inductions between the epithelium and the underlying mesenchyme, involving secreted signals such as BMPs or FGFs. Pax9 is expressed in the early presumptive tooth mesenchyme [92]. In Pax9 mutant mice, tooth development is arrested at the early bud stage [31]. Activation of essential tooth patterning regulators, such as secreted factor BMP4 and transcription factors MSX1 and LEF1, fails or is strongly decreased in Pax9 mutant mice [31]. In human, Pax9 mutations are associated with tooth agenesis and hypodontia [33]. Moreover, mouse Pax9 mutants exhibit other craniofacial developmental defects such as a secondary cleft palate [31]. These defects are accompanied by malformations in the mesoderm skeletal derivatives, at the base of the skull and at trunk levels, which also express Pax9 and Pax1 [31,93]. 4.3. PAX1 and PAX9 are involved in thymus, thyroid and parathyroid development The pharyngeal organs of the neck include the thyroid gland, the thymus, the parathyroids and the ultimobranchial bodies (forming the calcitonin-producing cells of the thyroid). They are formed by complex inductive interactions between the endoderm-derived epithelium and the neural crest-derived mesenchyme that populate the pharyngeal pouches. The thymus, for example, is the primary site of T-lymphocyte differentiation. It is composed by an epithelium, derived from the endoderm, surrounded by a mesenchymal capsule derived from the neural crest [6,94]. The neural crest also provides pericytes of the thymus [6]. Thymocytes, of hematopoietic origin, populate the thymus. During thymus formation, interactions between the neural crest-derived mesenchyme and the epithelium are critical, although mesenchyme from other origins can also support thymus development [7,95]. The neural crest-derived mesenchyme expresses secreted signals such as BMP4 and FGFs which position the thymus-parathyroid boundary, and support thymus epithelial cells proliferation [96,97]. At early stage, the endoderm-derived epithelium of the third and fourth pharyngeal pouches expresses Pax1 and Pax9 [31,32]. Mice deficient for Pax1 have a reduced thymus and abnormal maturation of the thymocytes, while Pax9 mutant mice lack thymus, parathyroid glands and the ultimobranchial organs due to an early development arrest of the third and fourth pharyngeal pouches [31,32,98]. However, a role for the neural crest in regulating Pax1/9 epithelial expression remains unclear. In Hoxa3 mouse mutants, which are athymic, Pax1 expression is lost in the third pharyngeal pouch epithelium [99]. However, since Hoxa3 is expressed both in neural crest cells and in the endoderm-derived epithelium, the role of neural crest on Pax1 expression is still not demonstrated. At later stages, neither Pax1 nor Pax9 expression seems regulated by the neural crest derived FGF signals [96]. In conclusion, pharyngeal organ development depends on PAX1 and PAX9 activity, but, while the formation of these organs also include critical functions of the neural crest, it remains unclear if there is a direct link between neural crest cells and Pax genes regulation in these organs. 4.4. PAX3 controls the balance between proliferation and differentiation of sensory neurons During peripheral nervous system differentiation, Pax3 is specifically expressed in the forming dorsal root ganglia (DRG), but is not maintained in the differentiated sensory neurons [20]. In trunk neural tube culture, the neural crest cells delaminate, migrate on

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the substrate and eventually differentiate into multiple derivatives. Early emigrating neural crest cells express Pax3, while later on, Pax3 expression is retained only in a minor subset of cells, with neuronal morphology [100]. When cultivated under conditions favoring sensory neuron differentiation, using FGF2 and LIF, Pax3 expression is upregulated at the stage of neural crest migration. Finally, PAX3 depletion using antisense RNA-mediated in embryonic mouse DRGs organotypic culture, results in decreased generation of new sensory neurons in culture [100]. This suggests that Pax3 expression is specifically maintained in the DRG by environmental factors, and that PAX3 plays important roles in trunk sensory neuron differentiation as shown for cranial placodal sensory neurons [101]. In Splotch mutants, the rare neural crest cells formed seem to undergo a premature sensory neurogenesis. Moreover, PAX3 binds both to cis-regulatory elements of the Hes1 gene, involved in neuronal stem cell maintenance, and to the Neurogenin-2 gene (Ngn2), which is important for general neurogenesis and for sensory neuron differentiation [29,102]. The balance between Hes1 activation and Ngn2 activation by PAX3 is regulated by the acetylation of PAX3 on specific lysine residues of its C-terminal domain [103]. When acetylated, PAX3 would lower Hes1 expression, thereby decreasing proliferation of undifferentiated neurons, and activate Ngn2 expression, resulting in increased sensory neurons differentiation [103].

analysis reveals distinct requirements for PAX3 and PAX7 in different pigment lineages in fish [26]. Complex molecular mechanisms and multiple transcriptional regulators, such as PAX3, SOX2, SOX10, FOXD3, control mitf expression. PAX3 synergizes with SOX10 to activate mitf expression in the melanoblasts, by binding to melanoblast-specific regulatory elements in mitf gene [112,113]. Additionally, the coordinated binding of PAX3, YAP and TAZ, cofactors in the HIPPO pathway, to mitf promoter, achieves robust mitf activation [114]. By contrast, PAX3 binding to mitf promoter is inhibited by protein–protein interactions with FOXD3 [115]. In parallel, PAX3 inhibits the expression of the terminal differentiation marker Dct (Tyrp2). In presence of Wnt signaling, MITF displaces PAX3 from Dct promoter and activates its transcription [116]. Finally, the role of the seven human Pax3 isoforms has been addressed using stably transformed murine melanocyte cell lines, and measuring several cell biological parameters. The different isoforms modulate cell proliferation, survival and migration [117]. They also activate different sets of target genes [118]. Further analysis is needed to analyze the tissue-specific expression of each isoform and link these observations to their biological outcome in vivo.

4.5. PAX3 controls the balance between proliferation and differentiation in Schwann cells precursors and during nerve regeneration

In the past few years, intense research has revealed novel aspects of PAX transcription factors functions during neural crest development. First, the reiterated action of PAX3/PAX7 in the neural crest GRN has become clear, from the earliest steps of neural crest induction, during emigration, then in controlling the balance between proliferation and differentiation of multiple lineages (Fig. 2). These functions are largely conserved throughout vertebrate evolution, with minor variations between species. Secondly, how this diversity of activities is generated and controlled begins to emerge. PAX3/PAX7 factors act in coordinated action with cofactors, which modulate the transactivation of PAX target genes, either in the dorsal neural tube (TEAD2, YAP) or in specific lineages (YAP, FOXD3 in melanocytes). In addition, a few examples of posttranslational modifications, such as acetylation or SUMOylation, as well as usage of different isoforms, suggest an additional level of complexity. Finally, Pax genes appear to respond to environmental perturbations known to affect neural crest development and thereby responsible for congenital malformations, such as folate deficiency, fetal alcohol syndrome and diabetes-induced congenital defects. Migrating neural crest cells, treated with ethanol or high glucose in vitro, or deficient for folate transport, deregulate Pax3 expression [119,120]. In turn, Pax3-deficient neural crest is sensitized to environmental stress (e.g. caused by pollutants) [53] and more prone to p53-dependent apoptosis [90]. Collectively, these findings reveal the molecular links between Pax genes, key regulators of the neural crest GRN, and human congenital pathologies.

The neural crest cells form the myelin sheet along peripheral nerves. The transcription factor SOX10 is essential for development of the glial lineage from neural crest, and the zinc-finger protein KROX20 is the physiological inducer of myelination [104]. Homozygous splotch mice fail to form glial precursors [37]. In control mice embryos, Pax3 is expressed in the proliferating Schwann cell precursors at E14.5, in GFAP-positive non-myelinating Schwann cells (nmSCs), whereas MBP-positive nonproliferating myelinating Schwann cells downregulate Pax3 expression, both in late embryos and postnatally [27]. During nerve regeneration and remyelination, Pax3 expression is rapidly reactivated then disappears as glial differentiation takes place [27]. During glial specification, Pax3 expression is activated by the histone deacetylases 1 and 2 (HDAC1/2), which both bind to Pax3 promoter [105]. In turn, PAX3 promotes glial progenitor proliferation in culture, reverses KROX20-induced cell cycle arrest and mediates the HDAC2-dependent activation of Sox10 [28,105]. In contrast, PAX3 blocks the activation of the myelin differentiation markers, such as MBP, by KROX20, and activates nmSCs markers [27,28]. 4.6. Pax3 controls the differentiation of pigment cells Neural crest cells give rise to the pigment cell lineages: melanoblasts, xanthophores, iridophores. In chick and mouse embryos, the pigment cells progenitors migrate using the dorsolateral pathway between ectoderm and paraxial mesoderm, and, in mouse, some melanocytes also come from Schwann cells surrounding peripheral nerves [106,107]. In fish, melanoblasts are found both in the ventral and the dorsolateral pathways [106,108]. A major regulator of pigmentation in mammals is microphthalmiaassociated transcription factor (MITF), which controls key enzymes in the melanin differentiation cascade [109]. Heterozygous Pax3 mutations, as well as PAX3 or PAX7 depletion cause pigmentation defects, demonstrating acute sensitivity of this lineage for PAX3 dosage [26,35]. As Mitf mutations, Pax3 mutations are involved in large white patches of coat color in mammals [110,111]. Detailed

5. Conclusions: PAX transcription factors are multifacet regulators of neural crest development

Acknowledgements I am grateful to C. Borday, A. Figueiredo, and P. Pla for their comments on the manuscript. Research in the Monsoro-Burq team is supported by grants from Fondation pour la Recherche Médicale (FRM, DEQ20150331733), Agence Nationale pour la Recherche (ANR, Programmes blancs 2011 et 2015), and Association pour la Recherche contre le Cancer (ARC, PJA20131200185). References [1] Le Douarin N, Kalcheim C. The Neural Crest, 2nd ed; 1999.

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