the neural crest and neural crest cells in vertebrate development and evolution || neuronal cells...

Chapter 6Neuronal Cells and Nervous Systems

The following major points concerning the embryological origins of neurons havebeen made so far:

• Neurons of spinal ganglia arise from the neural crest (NC).• Neurons arise from the tail buds of embryos and not from a delaminated germ

layer.• All neuronal cells express the cell-adhesion molecule, N-CAM.• Neural crest cells (NCCs) and cells of the central nervous system are so closely

related that they share a common lineage; central neurons and NC derivatives canarise from the same cloned cells.

• Single cells from mouse TNC are multipotential and capable of forming neu-rons of the neural tube, dorsal root ganglia, and sympathoadrenergic system, inaddition to Schwann and pigment cells.

• Early in NCC migration, lineages of sensory and autonomic neurons segre-gate from pluripotential cells that can form sensory and autonomic neurons ormelanocytes.

• Single quail CNC are multipotential precursors capable of producing neurons,glia, and chondrocytes and a small number of precursors from which neurons,glia, chondrocytes, and pigment cells arise.

The following points concerning the evolutionary origins of neurons has beenmade:

• Ascidians possess pigment cells and are now regarded as the sister group to ver-tebrates.

• The new-head hypothesis proposed by Gans and Northcutt was introduced.• Lampreys do not possess sympathetic ganglia and lack a ventrally migrating pop-

ulation of NCCs.

Neuronal differentiation from uni-, bi-, or multipotential cells was discussed in somedetail in Chapter 3 in the context of the segregation of subpopulations of NCCs. Theemphasis in the current chapter is on evidence for the NC origin of these neuronsand of the nervous systems to which they belong.

B.K. Hall, The Neural Crest and Neural Crest Cells in VertebrateDevelopment and Evolution, DOI 10.1007/978-0-387-09846-3 6,C© Springer Science+Business Media, LLC 2009

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180 6 Neuronal Cells and Nervous Systems

The Neural Crest, Neurons, and Nervous Systems

Pioneering studies using amphibian embryos providing evidence for the NC ori-gin of a variety of types of neurons, parts of the nervous system—the peripheral andautonomic nervous systems—and neuron-supporting cells—Schwann cells and glia.Indeed, many of the basic elements of modern-day neurobiology were establishedin studies, in which amphibian NCCs were labeled, excised, or transplanted. Subse-quent studies on the neuronal derivatives of the NC concentrated on avian embryos,especially once the quail-chicken cell-marker system was discovered.1

Neuronal- and nervous-system derivatives discovered to be NC in origin include:

• spinal and cranial ganglia of the peripheral nervous system,• adrenergic and cholinergic sensory neurons of the autonomic (sympathetic

and parasympathetic) nervous system,• Schwann cells,• Glial cells, and• Rohon–Beard neurons (see Table 1.1).

The peripheral nervous system consists of sensory neurons organized into sen-sory ganglia that transmit information from peripheral tissues and organs to thebrain or spinal cord of the central nervous system. Sensory neurons extend one axonperipherally to the target organ and one centrally to the CNS. Two major classes ofneurons comprise the peripheral nervous system: (i) spinal ganglia, also known asdorsal root ganglia, organized in pairs along the spinal cord and that transmit infor-mation to the spinal cord and (ii) cranial ganglia organized along the cranial nervesand that transmit information to the brain.

The autonomic nervous system (sometimes known as the visceral nervous sys-tem), which controls involuntary functions such as breathing, digestion, and heartrate, consists of sensory and motoneurons in three subsystems: the sympathetic,parasympathetic, and enteric nervous systems. Studies initiated in the 1970s greatlyexpanded our knowledge of the NC origin of the autonomic nervous system, primar-ily using chicken embryos, with confirmatory studies with Japanese quail. Trans-planting quail NC into chicken embryos identified the regions of the NC from whichparticular portions of the autonomic nervous system and hormone-synthesizing cellsarise:

• Parasympathetic (cholinergic) enteric ganglia of the gut are derivatives of thevagal neural crest (VNC), corresponding to NC at the levels of somites 1–7.

• NC adjacent to somites 8–27 does not produce enteric ganglia but gives rise tothe sympathetic (adrenergic) ganglia of the adrenal gland.2

• Some regions of the NC—those adjacent to somites 6 and 7 and those caudal tosomite 18—produce cholinergic and adrenergic neurons.

• Adrenomedullary cells develop from NC that originates at the level of somites18–24.

The Peripheral Nervous System—Spinal and Cranial Ganglia 181

• NCCs caudal to somite 28 (the sacral neural crest) produce enteric ganglia forthe caudal (postumbilical) portion of the gut, including the ganglion of Remakof the ileum.⊕

Such precise cellular localizations, a necessary prelude to investigating the mecha-nisms of differentiation, neoplasia, and dysmorphogenesis (see Chapters 9 and 10),could not have been accomplished without the benefit of a ‘label’, such as the quailnuclear marker. Each of these cell types, NC regions, and nervous systems is nowdescribed in the context of the evidence for their NC origin and information on howtheir differentiation is controlled.

The Peripheral Nervous System—Spinal and Cranial Ganglia

The identification of spinal ganglia as NC derivatives goes back to Wilhelm His’discovery of the NC (see Chapter 1). Indeed, so readily was the claim of a crestorigin for spinal ganglia accepted that this embryonic region was known for a timeas the ganglionic crest.

The initial observations on the existence of the NC in elasmobranchs made inthe 1870s and 1880 included descriptions of neuronal NCC derivatives.3 Balfour(1876) identified a neural ridge (see Box 3.1) within elasmobranch neural tubesfrom which spinal ganglia arose. He thought that ganglia of the sympathetic ner-vous system arose as branches of the spinal nerves and therefore were products ofthe neural ectoderm. Balfour was adamant that His was wrong in his interpretationof the existence of an ‘intermediate cord’ (NC). By 1881, however, Balfour hadchanged his view and was illustrating and discussing ganglia as NC in origin (seeFig. 3.2 in Box 3.1).

Also in the 1880s, the dorsal roots of the spinal ganglia were described as arisingfrom the spinal cord in sharks, although no distinction was made between cells ofthe neural tube or NCCs as the source of the neurogenic cells. In what was perhapsthe next detailed study, Conel (1942) described the NC origin of the dorsal rootganglia of cranial and spinal nerves in the spiny dogfish, Squalus acanthias, and inthe electric ray (Fig. 6.1).

Wilhelm His made his observations using fixed embryos. Techniques to visualizecells in living embryos were not developed the 1930s. Detwiler stained local regionsof NC with pieces of agar impregnated with vital dyes to show that spinal gangliaarise from TNCCs that migrate between the somites and the spinal cord/notochord.Experimental proof was provided by Harrison and by DuShane, both of whomobserved that spinal ganglia and sensory nerves fail to form in tadpoles if the

⊕The ganglion of Remak or Remak ganglion, which is only found in birds, originates in thelumbosacral NC and is the major parasympathetic nervous system element in the hindgut. It isnamed after Robert Remak (1815–1865) who first described histological characters for each germlayer and provided the first detailed description of mesoderm, Pander’s ‘middle vessel’ layer (seeChapter 2).


Fig. 6.1 The neural crest origin of spinal ganglia as seen in sections of neural folds of a chickenembryo (A) and of the electric ray (B and C). ect, ectoderm; g, ganglion; sc, spinal cord. Modifiedfrom Volume II of Text-Book of Embryology, by Kerr (1919)

NC was removed at the time of neural fold closure. Extirpating the ventral por-tion of the neural tube results in larvae lacking motor nerves but has no effect onspinal ganglia or sensory nerves; that is, the NC was shown to be a derivative ofthe dorsal neural tube. Further confirmation was provided by Raven, who trans-planted NC between embryos of different species of amphibians, using such species-specific characters as differential cell or nuclear size to distinguish host from donorcells.4

A major source of the early confusion over whether ganglia arose from the neuralcrest, the dorsal neural tube, or adjacent ectoderm, was the observation that althoughthe NC always arises at the lateral borders of neural ectoderm, the NC arises atdifferent stages of neurulation in different vertebrates:

• NCCs do not migrate away from the trunk neural tube in sharks until late in neu-rulation, after the neural tube has invaginated and separated from the epidermalectoderm (see Fig. 3.1 in Box 3.1).

Placodal Ectoderm 183

• Neural crest appears at the open neural plate stage in amphibians and rodents,but at the closed-neural-fold stage in birds.

• In many teleost fish, neurulation is by cavitation of a neural keel, not by invagi-nation of a flat neural plate (see Fig. 3.1 in Box 3.1).

Further complexity exists, not the least of which, is that the most caudal embry-onic region arises by secondary neurulation without segregation into germ layers,often appearing as a tail bud (see Chapter 2). Furthermore, TNCC migration inteleost embryos is between the somites rather than through the rostral half of eachsomite as occurs in tetrapods. Using a combination of HNK-1 staining, SEM andDiI injection in the most complete analysis of the development of dorsal root gan-glia in any teleost, the Mozambique tilapia, Laudel and Lim (1993) demonstratedthat dorsal root ganglia contain sensory cells and motor fibers, and arise from NCCsthat have migrated between the neural tube and the somites.

Spinal ganglia do not self-differentiate but require influences from other cellsand tissues, primarily the somites. This conclusion goes back at least to the studiesby Detwiler in the 1930s, in which removing somites led to localized loss of spinalganglia and spinal nerves, while adding somites led to supernumerary ganglia andnerves.

An NC origin for the cranial ganglia in chicken embryos was one of the baseson which Wilhelm His identified the Zwischenstrang in 1868. Acceptance of theNC origin of cranial ganglia did not come quite as readily as acceptance of the NCorigin of spinal ganglia. Some maintained a NC, others a placodal origin. As itturned out, both were and are correct. As most extensively shown in the research ofLeon Stone and C. L. Yntema, many cranial ganglia receive contributions from NCand placodal cells (Fig. 6.2; and see Fig. 2.16), although cranial sensory ganglia inthe North American sea lamprey may be entirely placodal in origin (McCauley andBronner-Fraser, 2003). If confirmed with studies from other lamprey species, a NCcontribution to placodes may be a gnathostome innovation.

This section now discusses the nature and origin of and contributions that arisefrom placodal ectoderm before moving on to discuss the autonomic nervous system.

Placodal Ectoderm

The NC appears at the neural–epidermal border, whether that border is at the normalsite in vivo or created after ectopic induction of a neural tube within epidermalectoderm (Figs. 6.3 and 6.4).

Epidermal sensory placodes (hereinafter, placodes), which also arise at the bor-der of the neural plate (Fig. 6.3) give rise to cranial sense organs such as the noseand ear and represent an important source of neural tissue, especially for the centralganglia of the cranial nerves (Figs. 6.3 and 6.4) and in teleosts and amphibiansfor the lateral line, neuromasts, and mechanosensory systems (Box 6.1). Placodesdevelop as thickenings of the head ectoderm, either adjacent to the NC or from the


Fig. 6.2 The neural crest (A)and successive stages in thedevelopment of the roots andganglia of the spinal nerves(B and C) in a shark embryo.(A) Origin of the neural crest(pr) by proliferation from thedorsal surface of the neuraltube. (B) Initial formation ofthe root of the spinal nerve(pr). (C) An older embryo toshow the position of theposterior root (pr) and spinalganglion (sp.g) of the spinalnerve (n). Other abbreviationsare: ch, notochord; ao, aorta;nc, neural canal; sc, somaticmesoderm; sp, splanchnicmesoderm; x, subnotochordalrod. Modified from Balfour(1881)

Fig. 6.3 Fate maps of cranial placodes in a typical frog (Rana, A) and in Ambystoma (B and C)to show their relationships to the neural crest, neural plate (np), and epidermal ectoderm (epid).A and B as seen in dorsal view with anterior to the top, C in rostral view. Adapted from Baker andBronner-Fraser (2001)


Fig. 6.4 Fate maps of cranial placodes in chicken embryos at three H.H. stages (A, B, and C),all shown in dorsal view with anterior to the top. Both the rostral–caudal positioning of individualplacodes and their relationship to the neural crest, neural plate (np), and epidermal ectoderm (epid)can be seen. Adapted from Baker and Bronner-Fraser (2001)

neural folds themselves (Figs. 6.3, 6.4 and 6.5). In axolotls and in mice, perhapsin other groups, placodes arise from lateral neural fold ectoderm, while NC arisesfrom median neural folds.

Box 6.1 The lateral line system

In amphibians and in fish, neuronal cells form a specialized system of nerves, thelateral line nerves and specialized electro- and mechanoreceptive organs, neu-romasts, associated with the lateral line nerves. Both elements of the lateral linesystem, which arise from placodal ectoderm, have been lost from all amniotes.In fish, neuromasts are found either superficially in the epidermis or deep andassociated with bony canals of the lateral line system. All amphibian neuromastsare superficial.a Here I discuss the lateral line system of teleost fish.

Bony and cartilaginous fish have an extensive lateral line system. Eightbasic patterns, derived by heterochrony from an ancestral pattern, have beenproposed in groups such as cichlids, whose evolutionary history is wellresolved.

Neuromasts and lateral line nerves arise from ectodermal placodes.b NCCspattern neuromasts but have not definitively been shown to give rise to neu-romasts. On the basis of labeling zebrafish NCCs with DiI, however, it hasbeen claimed that neuromasts have a dual origin, arising from NC and fromnon-NC ectoderm. In the absence of histological evidence and because of thedifficulty of labeling NC in the neural folds without labeling placodal ectoderm


(Box 2.1), this claim remains unsubstantiated. As discussed in the text of thischapter, placodal ectoderm in some species arises from the lateral neural folds,NC arising from medial neural folds. In such species, placodal ectoderm maybe induced by NC rather than forming from NC. Axolotl lateral lines and neu-romasts express Msx2 and Dlx3, molecular markers that could be used to trackthe effects of NCCs on neuromast origins, differentiation and morphogenesis.c

In a recent study of the origin of neurogenic placodes and cranial sensoryganglia in the lesser-spotted dogfish, Tbx3, was identified as a specific markerfor lateral line ganglia in this species. Ngn1 is required for development oflateral line and cranial sensory ganglionic precursors in zebrafish.d

As described in salmon and other genera, preosteogenic cells derived fromthe NC accumulate under the sensory organs (neuromasts) of the lateral linesystem. Circumstantial evidence suggests that developing neuromasts inducescales or dermal bone in teleosts and cartilage in sharks. Once formed inteleosts, lateral line bone contains a canal for the lateral line nerve and poresfor the neuromasts. Canal neuromasts are distinctive; in the goldfish, forinstance, they are 50–100 �m in diameter. Superficial neuromasts are <50 �min diameter. Surprisingly, in two species of hexagrammid fish of the genusHexagrammis, some trunk lateral lines lack neuromasts. Although amphib-ians possess neuromasts, they lack the canal neuromasts associated with bonein fish.e

a For the induction and development of placodes in anurans and urodeles, see Schlosser andNorthcutt (2000, 2001) and Schlosser (2002a,b, 2006∗).b The story is more complicated than this; secondary neuromasts that develop postembry-onically in the posterior lateral line in fish, arise from new waves of precursors and not bybudding from the embryonic lateral line (Ledent, 2002).c See Collazo et al. (1994) for the DiI-labeling study, S. C. Smith et al. (1990∗) andNorthcutt (1996) for associations between placodal and NC ectoderm, and Metscher etal. (1997) for Msx2 and Dlx3. The role of the lateral line system, especially neuromasts,in specifying pigment patterns in anuran and urodele amphibian tadpoles is discussed inChapter 5.d See O’Neill et al. (2007) for Tbx3 and Andermann et al. (2002) for Ngn1. A neurogenicgene also has been cloned from amphioxus and the earliest known marker for amphioxusneuroectoderm, appearing as two segmental bands in early neurulae and in the dorsal neuraltube of mid-stage neurulae. Subsequent AmphiNg is expressed in epidermal chemosensorycells, and in the midgut along with an insulin-like peptide in a region that L. Z. Holland et al.(2000) surmise could be a homolog of the pancreas.e See Holmgren (1940) and Wonsettler and Webb (1997) for lateral line patterns in sharks,rays, and bony fish, and Webb and Northcutt (1997) for neuromasts in nonteleost bony fish,in which multiple canal neuromasts between pore positions is the evolutionarily primitivecondition. See Tardy and Webb (2003) and Webb and Shirey (2003) for lateral line canaland neuromast development in zebrafish, and Wilson et al. (2007) for Cad4, lateral line, andneuromast development in zebrafish. For the possible induction of bone or cartilage by organsof the lateral line, see Hall and Hanken (1985) and Webb and Noden (1993∗).


Fig. 6.5 A diagrammatic representation of the location of neurogenic placodal ectoderm (black)adjacent to the neural tube and of neural crest (gray) that contributes to placodes in a chickenembryo as seen from the dorsal side. Only the placodes on the right-hand side are shown. Themost rostral placodes are the nasal and lens. The cranial sensory ganglia are placodal (genicu-late, vestibulo-cochlear, petrosal, nodose ganglia), neural crest (root, superior jugular ganglia), orof mixed placodal and neural crest origin (Trigeminal ganglion). Note that ganglia can have aproximal component that is neural crest (superior jugular) and distal components that are placodal(petrosal as distal cranial ganglion IX; nodose as distal cranial ganglion X). Adapted from Webband Noden (1993)

Placodes have been proposed to form in association with weak competence atthe neural plate boundary, although the precise relationship between placodes andNC remains an active area of research. Lateral-line placodes in the Mexican axolotlare set aside at the late neural fold stage but can be induced as late as the earlytail bud stage, the temporal window of induction paralleling loss of competence forinduction by the ectoderm. The same group of experimenters examined develop-ment of direct-developing Puerto Rican frog, Eleutherodactylus coqui, which lacksneuromasts or ganglia associated with the lateral line. The loss is evident earlyin development; no lateral line placodes arise. Transplantation between coqui andaxolotl embryos demonstrated that coqui ectoderm lacks the competence to respondto inductive signals. Competence for induction emerges as an important mechanismregulating placode formation.5

All placodes except the adenohypophyseal and lens placode give rise to neurons;some give rise to other cell types as well (Table 6.1). The earliest jawed vertebratesare thought to have had seven pairs of cephalic dorsolateral placodes. Fish and manyamphibians have six dorsolateral lateral-line placodes, from which the lateral linesand lateral-line nerves arise (Box 6.1); the lateral-line system has been secondarily


Table 6.1 Overview of the major placodes, the sense organs to which they contribute, and the celltypes that arise from them

Placode Derivatives Cell types

Olfactory Nasal epithelia Olfactory neurons, supporting cellsolfactory epithelium

Brain Gonadotropin-releasing hormoneneuroendocrine (GnRH) cells, glia

Hypophyseal Adenohypophysis (anterior pituitary) Endocrine cellsLens Lens of eye Lens cellsTrigeminal Trigeminal ganglia Primary sensory neurons of the

ganglion of the Vth nerveOtica Inner ear Sensory hair cells, supporting cells,

sensory neurons of the ganglion ofthe VIII nerve

Lateral linea Neuromasts Mechanoreceptor and electroreceptorGanglia of lateral line nerves neurons, hair cells, supporting cells,

Epibranchialb Ganglia of cranial nerves VII Sensory neurons(the facial), IX (the glossopharyngeal)and X (the vagal)Taste buds, Afferent neurons (innervation)Heart Afferent innervation for receptorsc

Lungs, Intestine Afferent neuronsd

a.Together these make up the dorsolateral series of placodes.bThe three epibranchial placodes are the geniculate, petrosal, and nodosal.cThese neurons are involved in the transmission of information on heart rate and blood pressure.dThese neurons are involved in the transmission of information on distension of the gut epitheliumand stimuli in bronchii of the lungs.

lost from all amniotes. The seven classically recognized classes of dorsolateral pla-codes (otic and lateral-line placodes), along with the six ventrolateral placodes, areset out in Table 6.2, as are the sense organs to which each placode contributes andthe cell type(s) derived from placodal ectoderm in each sense organ.6

The adenohypophyseal placode (from which the anterior pituitary forms) is themost rostral, succeeded as one moves caudally by the hypophyseal, lens, trigeminal,

Table 6.2 Gene markers for individual placodes expressed early in placode specificationa

Placode Gene

Epibranchial Pax2, Sox2, Sox3, Phox2aLens Pax6, Sox2, Sox3Olfactory Sox2b, Sox3b, Pax6, Dlx3, Dlx5Otic Pax8b, Pax2, Dlx3, Dlx5, Dlx7, Sox2, Sox3, Spalt4Hypophyseal Six3, Pitx1Trigeminal Pax3b, Sox2, Sox3, Pax3Lateral line Eyal, Sox2, Sox3

aThese genes are the first markers to be expressed in the development of the particular placode(Source: Baker and Bronner-Fraser (2001∗), Schlosser (2005∗), Barenbaum and Bronner-Fraser(2007∗)).


and otic placodes (Figs. 6.3, 6.4, and 6.5 and Table 6.2). A century-old debate overwhether the first cranial nerve (Nerve 0) arises from the olfactory placode is dis-cussed in Box 6.2.

Box 6.2 The origin of cranial nerve 0

For more than a century, the most rostral cranial nerve, the nervus terminalis,traditionally named nerve 0, has been regarded as originating from the olfac-tory placode. A population of neurons within nerve 0 expresses gonadotropin-releasing hormone (GnRH). Known as GnRH neurons or luteinizing hormonereleasing (LhRH) neurons, defective migration of these neurons is suspected tobe the basis of Kallman syndrome in humans, in which the olfactory nerves failto develop (Carstens, 2002).

Earlier studies on mouse, chick, amphibian, and lungfish are consistent withan olfactory placodal origin for these GnRH-positive neurons, which have beeninterpreted as arising from the medial aspect of the olfactory placodes, migratingto the CNS along the vomeronasal and terminal nerves. A challenge to acceptedwisdom comes from a study tracing the origin of GnRH neurons in zebrafish,in which a caudal subset of GnRH neurons that contribute to the terminal nervewere traced back to the NC, and evidence provided for the origin within theadenohypophyseal placode of the rostral subset of neurons that migrate into thehypothalamus.a

a See the study by Whitlock et al. (2003) and a commentary on and discussion of it by vonBartheld and Baker (2004). See Box 4.2 and L. Z. Holland and N. D. Holland (2001∗) forhomologies of the adenohypophysis.

The lens placode (Figs. 6.3, 6.4, and 6.5), which segregates from head ectoderm in30-h-oldchickenembryos, sharesanintimateassociationwith thesubjacentNCCsandassociated extracellular matrix (ECM). Meier’s data (1978∗) convinced him that theplacode/NC association was so intimate that the two must interact developmentally.One difference between the two is that delamination of cells from placodal ectodermin chicken embryos does not involve the epithelial-to-mesenchymal transformationrequired for NCCs to delaminate (Graham et al., 2007).

Placodal Markers and Specification of Placodal Ectoderm

Until recently, one difficulty with assessing placodal development was the lackof specific markers. Thanks to more recent work we have genetic markers for allplacodes.7 All placodes with the exception of the maxillomandibular trigeminal pla-code express one or more members of the Pax gene family:

• Pax8 in the otic placode,• Pax3 in the ophthalmic trigeminal placode,


Fig. 6.6 Chicken embryos of 2.5 and 3.5 days of gestation to show otic and epibranchial placodes.(A) At 2.5 days Pax2 is expressed in the otic vesicle (ov) and ventral epibranchial placodal ectoderm.(B) At 3.5 days, Pax2 expression continues in the otic vesicle and labels the three epibranchialplacodes, the geniculate (g), petrosal (p), and nodose (n). (C) At 2.5 days, Phox2a is expressed ina small number of neurons in the three epibranchial placodes but not in the otic vesicle. (D) At 3.5days, Phox2a is expressed in the three epibranchial placodes. Figure kindly supplied by Clare Baker

• Pax6 in lens and olfactory placodes, and• Pax2 in the otic and epibranchial placodes in all gnathostomes analyzed (Table

6.2; Fig. 6.6).

As summarized in Table 6.2, a combination of Pax and other markers (partic-ularly members of the Sox gene families) enables early placode precursors to bedifferentiated from one another, even though individual markers may be expressedin more than one placode and function in other cell lineages. (Figure 6.6 shows Pax2in otic and epibranchial placodes and Phox2a in epibranchial but not in the otic pla-code in chicken embryos.) Nevertheless, judicious application of these markers hasexpanded our understanding of placodal origins, diversification, and relationshipsenormously in the past decade. One study on the formation of the otic placodes inchicken embryos and two studies on induction and specification of the otic placodeare used as examples: one in chicken embryos and another in mice.

The first detailed fate map of otic placodal ectoderm in chicken embryos wasproduced by Streit in 2002. Pax2 was used as the marker, although not all Pax2-positive cells contributed to the placode, a caution to be keep in mind when using asingle marker that is not uniquely expressed in one subset of cells. Initially, futureotic placodal cells are not adjacent but scattered in the ectoderm with future neural,NC, epithelial, and other placodal cells. Extensive cell movement brings otic placo-dal cells to the midline of the embryo adjacent to r5–6, where some cells from theneural folds contribute to the placode.


Using Pax2, Sox3, Bmp7, and Notch as markers, Groves and Bronner-Fraser(2000) localized the ectoderm from which the otic placode will arise in chickenembryos at the 4–5-somite stages, showed that the ectoderm was specified as oticby the 4–6-somite stages, that induction of the placode continued progressively tothe 10-somite stage, when the otic placode was committed (committed meaningthe earliest stage at which the placode could develop independently), and that theplacode formed between the 12- and 14-somite stages. In a subsequent study, Mar-tin and Groves (2006) showed that preplacodal ectoderm expressed some but not allotic placode markers in response to Fgf.

Pax2 is strongly expressed in the otic placode ectoderm in mouse embryos andweakly or not at all in the adjacent epidermal ectoderm. Wnt gene expression isenhanced in a subset of cells within the Pax2-positive placodal ectoderm. Wntsfunction through �-catenin. Inactivating �-catenin enhances the field of epidermalectodermal cells at the expense of the extent of the otic placodal domain. Activat-ing �-catenin produces the opposite result, the expanded otic placodal ectodermalexpressing exclusively dorsal otocyst markers (Ohyama et al., 2006).

Many more genes than included in Table 6.2 are expressed at later stages ofplacode formation; Figure 5 (pp. 319–321) in Schlosser (2006∗) contains a detailedlist of genes expressed at neural plate and tail bud stages in Xenopus embryos.

Following early studies showing that placodal ectoderm lies at the border betweencranial neural plate/neural crest and epidermal ectoderm, we now know that multipleplacodes can and do arise from individual regions within placodal ectoderm. As anexample, a continuous region of thickened placodal ectoderm in Atlantic cod embryosgives rise to the lens, otic, and lateral-line placodes, and to the ectodermal lining ofthe opercular cavity (Miyake et al., 1997). Such spatial patterning (regionalization)of placodal ectoderm may begin as early as gastrulation in some taxa.

The Panplacodal Domain

Perhaps the most important findings from recent studies is the recognition of a pre-or panplacodal domain at the border of the cranial neural tube within which indi-vidual placodes are induced at different times during development in response todifferent combinations of inductive molecules, competence to form individual pla-codes changing in space and over time.

The panplacodal domain is specified by members of two major families of tran-scription factors, the Six family (orthologous to sine oculis in Drosophila) and the Eyafamily (orthologous to the Drosophila gene eyes absent) are important, especially Six1and Eya1. Single copies of both genes are found through the metazoans, indicative ofthe ancient evolutionary origin of these transcription factors (see Figure 2 in Schlosser,2007). As evaluated by Andrea Streit and Gerhard Schlosser who have spearheadedanalysis of specification of the panplacodal domain, activation of Eya genes and mem-bers of the Six1/2 and Six 4/5 families,—the ‘panplacodal genes’—is under the controlof Bmps, Fgfs, and Wnts; Fgfs promoting and Bmps and Wnts suppressing Six /Eya.8

Target genes of Six1 and Eya1 are less well understood.


Table 6.3 Embryonic primordia providing inductive signals for individual placodesa

Placode Primordium—gene

Hypophyseal Diencephalon—Shh from midline tissues for induction; Bmp4(possibly aided by foregut endoderm) for patterning

Epibranchial Pharyngeal arch endoderm—Bmp7; FgfsLens Optic cup—Bmp4, Fgfs to induce; Bmp7 to maintainOlfactory Anterior, mesendoderm, aided by forebrainOtic Mesendoderm—Fgf3 (depending on species), other Fgfs, WntsTrigeminal Neural tubeLateral line Mesoderm, neural plate

a Source: Baker and Bronner-Fraser (2001∗).

Induction of Individual Placodes

The nature and source of the signals responsible for the specification of individ-ual placodes within the panplacodal domain is also being revealed. Signals from avariety of developing embryonic regions/tissues induce placode formation, the mostwell known of which are outlined in Table 6.3.

Details of these inductions and of how individual placodes arise from commonectodermal fields continue to emerge. For example, zebrafish epibranchial and oticplacodes are induced by a combination of signaling molecules, Fgf3 and Fgf8 (prob-ably from the hindbrain) and Foxi1, the latter acting via Pax8; placodal initiation isprevented in the hearsay mutant, a mutation of Foxi1, which is the earliest markerfor otic placodes in zebrafish. However, other signals may operate in vivo and,once formed, placodes evoked by the same signals come under independent con-trol; chicken Sox3 must be downregulated for epibranchial placodes to form fromSox3-positive preplacodal domains. If Dlx3 and Dlx4b are knocked out in zebrafish,epibranchial placode formation is unaffected, although otic placodes fail to form.Dlx3 and Dlx7 also have been shown to act in concert to initiate otic and epibranchialplacodal development in zebra fish.9

Such results are consistent with (i) shared signals for placode induction, (ii)what may be considerable redundancy of cascades of signals not yet sufficientlyunderstood, and (iii) divergent signals for later development. Supposedly, we areentering a decade of intensive research on placodal ectoderm.

Induction of individual placodes in the context of progressive restriction of a pan-placodal has just been elegantly demonstrated by Xu and colleagues (2008) usingDiI and DiO labeling to fate map the ophthalmic and maxillomandibular trigeminalplacodes in chicken embryos of 0–16 pairs of somites (24–48 h of incubation). Thethree major conclusions are that:

• precursors for the two placodes arise from a partially overlapping territory that isinitially distributed as a large region of ectoderm that includes precursors of thegeniculate and otic placodes and of epidermal ectoderm;

The Autonomic Nervous System 193

• no clear demarcation between the two placodes could be discerned, even at thelatest stage examined when neurogenesis is underway in both placodes;

• some cells at the border expressed both the ophthalmic placodal marker Pax3and the maxillomandibular placodal marker, Neurogenin1, consistent with somecells responding to inductive signals that initiate both placodes.

The Autonomic Nervous System

As with the origin of cranial ganglia (but for different reasons), authoritative figuresdiffered as to whether the NC contributed to the autonomic nervous system; as dis-cussed below, enteric and visceral ganglia of the sympathetic nervous system weremore convincingly shown to arise from NC.10

In part, the differences were technical; extirpation and transplantation studiesgave less reproducible results than did vital staining. The clearest interpretation ofthe early studies is that NC and ventral neural tube both contribute cells to the auto-nomic nervous system (see Horstadius, 1950∗). Resolution of the origin of the auto-nomic nervous system from dorsal neural tube (NC) had to await studies on othervertebrates, notably chicken embryos. That said, the ventral neural tube has reap-peared in a series of studies claiming that cells emigrating from the ventral neuraltube form derivatives regarded as the exclusive property of NCCs (see Box 6.3).

Box 6.3 The ventral neural tube: A ventral neural crest?

NCCs are derivatives of the dorsal neural tube. The ventral neural tube givesrise to neurons. However, as indicated in the text, cells of the ventral neuraltube can be switched to NCCs, if they are grafted into the migration pathwaytaken by NCCs.

Evidence from a series of studies in chicken embryos leads us to concludethat the ventral neural tube normally contributes chondrocytes to Meckel’s car-tilage and to the quadrate (Sohal et al., 1999), two skeletal elements that havebeen regarded as exclusively of NC origin.

The evidence comes from studies in which ventral neural tube cells ofchicken embryos were tagged with a viral LacZ marker and then injected intothe lumen of the rostral hindbrain. The injected cells moved out of the lumenof the hindbrain. Unlike NCCs, these cells were HNK-1 negative and so couldnot be confused with migrating NCCs. Furthermore, because they had beenmarked and injected 2 days after the completion of CNCC migration from thedorsal neural tube, dangers of confusing the migrating marked ventral neuraltube cells with NCCs were minimized. Subsequently, marked ventral neuraltube cells were found as perichondrial cells and as chondrocytes in Meckel’scartilage and in the quadrate. Consequently, they were readily distinguishable


from the HNK-1-positive NC-derived cells that comprise the majority of thechondrocytes in the cartilage.

This may not be the only situation in which non-NCCs contribute to skeletalelements thought to be entirely of NC origin. In their study, fate mapping theCNC of mouse embryos, Chai et al. (2000) observed a considerable numberof nonlabeled (i.e., non-NC) cells in the dental mesenchyme forming dentalpapillae and teeth and in Meckel’s cartilage. They considered the ventral neuraltube as a possible source of these cells, but could not rule out a subpopulationof NCCs not labeled with their Wnt1-Cre/R26R marker.

According to other studies by Sohal and colleagues, ventral neural tube cellsare even more versatile, forming smooth muscle cells of craniofacial arteriesand veins, hepatocytes in the liver, and contributing to the trigeminal ganglion,vestibulocochlear nerve, and otic vesicle. En route to contributing to the liver,cells derived from the ventral hindbrain of 2-day-old embryos were found incraniofacial muscles at 7 days and to migrate from the site of attachment ofthe vagus nerve, through the gut, into the smooth muscle of the stomach andintestine to form hepatocytes in the liver. DiI labeling and the expression of thehomeobox gene Islet-1 were used to show that cells delaminated from the neu-ral tube at site of attachment of the trigeminal nerve, migrated into the trigemi-nal ganglion, and then into the mesenchyme of the first pharyngeal arch.a

a See Ali et al. (2003) for the study on the otic vesicle and for references to the earlier studies,and Erickson and Weston (1999) for a critique of the possibility of accidentally labeling othercells in such studies.

We know from the evidence outlined in Chapter 3 that migrating NCCs requiresignals to differentiation into particular cell types. Bmp4 and Bmp7 within the dor-sal aorta of chick embryos trigger autonomic neuronal differentiation from migrat-ing NCCs (Reissmann et al., 1996); ectopic expression of these growth factorsinovo, or maintenance of NCCs with Bmp4 or Bmp7 in vitro, triggers autonomicdifferentiation.

The suspected dual NC and placodal origin of the autonomic nervous system alsoapplies to the origin of chromaffin cells of the adrenal g land and to ganglia asso-ciated with the aorta. Interestingly, lampreys lack chromaffin cells and peripheralsympathetic neurons, perhaps an indication that these two cell types have had a longevolutionary linkage within a single NC population that arose in jawed vertebrates.

During the 1920s and 1930s, a series of experimental studies performed onchicken embryos established the NC origin of the sympathetic ganglia. Althoughnot all early studies reported comparable results, subsequent experimental studiesaffirmed that these ganglia are indeed NC in origin. Enteric and visceral ganglia,chromaffin cells of the adrenal gland, and pigment cells were also shown to be NCin origin, and trunk, vagal, and sacral NCCs were identified (see section below, andsee Chapter 10 for defects affecting adrenal and ganglionic NC derivatives).11

Glial Cells 195

As with avian embryos (above), lysinated rhodamine dextran and DiI have beenused with effect to show that single TNCCs are multipotential and can form neuronsof the neural tube and contribute to the dorsal root ganglia, the sympathoadrener-gic system, Schwann, and pigment cells (Serbedzija et al., 1991; Serbedzija andMcMahon, 1977∗).

Schwann Cells

As neuronal support cells, Schwann cells—named after the co-founder of the celltheory, Theodor Schwann—provide an insulating sheath around neurons of theperipheral nervous system; each Schwann cell sheathing a single neuron and aid-ing in the conductance of electrical signals through that neuron.

Peripheral motor nerves arise from neural ectoderm. Evidence for the NC originof the Schwann cells that sheath peripheral motor nerves goes back to the extirpa-tion and tissue culture studies of Harrison and Muller and Ingvar in the early 20thcentury; Harrison’s were the first tissue culture experiments performed with any tis-sue. Subsequent vital dye staining of Ambystoma embryos confirmed the NC originof Schwann cells, which we now know arise from vagal and sacral NC, although inbirds the CarNC also provides Schwann cells for cranial nerve XII.12

Clonal cell culture has shown that Schwann cells share lineages with otherNC derivatives—sensory and sympathetic neurons, pigment cells, and neurons ofthe central nervous system (Fig. 3.17). Schwann cells are often found in embry-omas; malignant Schwannomas are composed of abnormal Schwann cells (seeChapter 10).

Glial Cells

Glial cells (neuroglia) also are supporting cells for neurons, in their case for neuronsin nerves and in the brain. Two types of glial cells are known.

(1) Oligodendrocytes secrete myelin that forms a sheath (the myelin sheath)around the axons of neurons of the CNS, providing an insulating functionand aiding the conduction of electrical signals along the nerve. Schwann cells(above) provide the same function to neurons of the peripheral nervous system,one Schwann cell per neuron. A single oligodendrocyte, however, can providethe sheath for dozens of neurons.

(2) Astrocytes (astroglia) are found in the brain where they provide support (espe-cially to the vascular cells that form the blood–brain barrier), metabolic prod-ucts, and aid in the transmission of electrical impulses.

Recent studies demonstrate that glial cells arise from multipotential NCC pre-cursors: The NCCs that delaminate early in zebrafish embryos differentiate as glialcells, pigment cells, and as neurons of the dorsal root ganglia. The CNC of early


quail embryos contains two classes of multipotential precursors, one that gives riseto glia, neurons, and chondrocytes, and a smaller population of cells that givesrise to these three cell types plus chromatophores (Baroffio et al., 1991, and seeFig. 3.17). Stabilization of �-catenin by Wnt (the canonical Wnt pathway; seeFig. 2.5) inhibits glial and neuronal differentiation as a side effect of enhancingchromatophore differentiation.13

As discussed in Chapter 2, Sox10 is essential for glial cell differentiation.Although Sox10 is expressed in premigratory NCCs along the entire neural axis, itis rapidly downregulated in the earliest stages of the differentiation of many NCCs,but not in glial precursors. Sox10 continues to be expressed at high levels throughoutglial differentiation in embryos and in adults, where, at least in rats, it functions tomaintain glial and neuronal potential in a subset of bipotential NCCs.

The differentiation of NCCs into glia and the maintenance of the glial pheno-type require continuous expression of Sox10 in glial cells. Sox10 maintains theexpression of Er3, a component of the Neuregulin receptor that regulates the expres-sion of neuregulin1. Sox10 is also involved in upregulating Notch1, which plays arole in specifying glial cell fate and rendering the cells unable to enter the neu-ronal lineage—or vice versa—it is unclear which occurs first. Sox10–/– mice lackall peripheral glia, autonomic and enteric neurons, and melanocytes because ofincreased cell death in premigratory TNCCs; Neuregulin serves as a postmitoticsurvival signal in Sox10+/+ and Sox10+/– mice.14

The requirement of Sox10 for initiation and maintenance of glial cell fate is aconsequence of the inhibition of autonomic neuronal cell fates by Sox10. Specifica-tion of NCCs as sensory neurons—which occurs in part via neurogenin transcriptionfactors such as Ngn1— is reduced in Sox10 mutants, although whether by direct orindirect action has to be determined. Two key transcription factors involved in thespecification of sympathetic neurons, Mouse achaete-scute homologue 1 (Mash1)and Phox2B, the expression of which inhibits sympathoadrenal differentiation, areactivated by Sox10. Therefore, Sox10 serves as a master switch; ‘global’ inhibitionof a switch into the autonomic neuronal lineage establishes a default in which theglial lineage is promoted.15

Multipotency, lineage specification, and phenotype specification are intimatelyrelated and under considerable shared control. Shared potency is defined byreceptors shared by NCCs early in their development (and so intrinsic?). Spec-ification reflects later activation of intrinsic switch genes, such as Sox10 andSox10-mediated transcription factors and receptors, although there is considerableredundancy among members of subfamilies such as SoxE. To take a recent example,deleting Sox9 from mouse glial (oligodendrocyte) cell precursors does not preventtheir terminal differentiation. Deleting Sox10 at the same time results in reducednumbers of glial cells because of induced apoptosis, although migration is also per-turbed. Sox9 and Sox10 both elicit their actions by regulating the expression of Pdgf-�, a receptor that regulates survival and migration of oligodendrocyte precursors.Identification of these upstream transcriptional and downstream signaling targets isbut the first step along a road to identifying target genes and gene cascades, only afew of which are known.16

Rohon–Beard Neurons 197

Vagal and Sacral Neural Crest

The VNC contributes enteric ganglia to the entire length of the gut, the sacral neuralcrest (SNC) predominantly to the hindgut; lysinated rhodamine dextran and DiI havebeen used with effect to label individual murine NCCs, follow their migration, anddemonstrate that enteric ganglia are SNC in origin. Simpson et al. (2007) developedand verified (with organ culture of embryonic chicken gut containing quail NCCs)a mathematical model demonstrating the key role of cell proliferation at the front ofthe wave of migrating NCCs in the invasion of the gut by NCCs destined to formenteric ganglia.17 Human syndromes associated with failure of development of allor parts of the enteric nervous system are discussed in Chapters 9 and 10, with someemphasis on Hirschsprung disease.

Because they come from different levels of the neural tube lack (somite lev-els 1–7 and caudal to somite 18, respectively), and because VNCCs migrate ros-trocaudally and SNCCs caudorostrally, avian sacral and vagal NCCs normallydo not interact. SNCCs can invade other regions of the gut, as demonstratedby transplanting chicken SNCCs more anteriorly. In chicken embryos, VNCCsmigrate ventrally and so they reach the developing gut several hours before SNCCs,because: the developing anterior intestinal portal is a barrier to SNCC migra-tion; the sacral environment permits cells from the vagal level to enter the gut;because enteric neurons require environmental cues from the hindgut but VNCCsdo not.18

In a recent screen of mutagenized zebrafish, Kuhlman and Eisen (2007) iden-tified four mutations with specific affects on enteric neurons, and nine mutationswith pleiotropic effects on enteric neurons and other NCC derivatives such asmelanophores or craniofacial mesenchyme, and/or mesodermal derivatives such assomites and fins.

Vagal and sacral NCCs express Sox10, EdnRB (the gene for endothelin receptorB) and the proto-oncogene Ret, which is expressed at a fourfold higher level invagal than sacral crest. Overexpressing Ret in SNCCs extends the regions of thedeveloping gut to which SNCCs contribute. As itappears to shift these NCCs towarda vagal fate, Ret is a potential regulator of vagal–sacral lineages in vivo. As discussedin Chapter 9, Hirschsprung disease in humans reflects mutations in RET and EDNRBgenes. In an interesting study using chick embryos, the same ECM signals wereshown to evoke different cell types in cranial (mesencephalic) and trunk (sacral)NCCs, in part because of differential upregulation of Hox genes.19

Rohon–Beard Neurons

Giant, transient, mechanosensory ganglionic cells known as Rohon–Beard (R–B)neurons form a network associated with the dorsal neural tube in some amphibianembryos and tadpoles, and in the embryos and larvae of all cartilaginous and bonyfish, described, for example, in the common skate, Mozambique tilapia, and stur-geon. Although called Rohon–Beard (R–B) neurons after Rohon (1884) and Beard


(1896), who described them most fully, they were reported first by Balfour (1878)in elasmobranchs.

Functionally, R–B neurons remain with the dorsal nervous system. Their cell pro-cesses (neurites) project to the hindbrain and to the skin as they function in medi-ating external sensations to the CNS. Ontogenetically, R–B neurons are transitoryand confined to larvae; they are replaced by later developing neurons of dorsal rootganglia in amphibian and fish embryos but persist into adulthood in lampreys. Asnoted in Chapter 4, the dorsal cells of Rhode may serve a similar function in adultamphioxus but their relationship to R–B neurons is untested.20

Apoptosis Removes R–B Neurons

Why and how R–B neurons disappear from larvae has not been thoroughly investi-gated. Cell death certainly is involved; Beard reported the death of R–B neurons inskate embryos in his original 1896 description.

All zebrafish R–B neurons are eliminated by programmed cell death (apoptosis)by 5 days after fertilization. The nerve growth factor, neurotrophin 3 (Nt3), andelectrical activity associated with Na+ currents, both regulate apoptosis. BlockingNt3 increases rates of apoptosis, while adding Nt3 enhances the rate at which R–Bneurons undergo cell death, two results consistent with neural activity playing a rolein the death of these neurons. Three markers of apoptosis in zebrafish R–B neuronsare reduced to similar levels in the macho (mao) phenotypic mutation (which isunresponsive to tactile stimulation) or if Na+ currents are blocked with the drugtricaine.21

Neural Crest Origin and Relationships to Other Neurons

Beard hypothesized that amphibian Rohon–Beard neurons arise from the NC; theydo not arise if TNC is removed. However, because R–B neurons and NC originatefrom separate lineages that can be identified as early as the 512-cell stage in Xeno-pus, their origin from the NC has been questioned. Separate lineages, combined withorigination at the gastrula stage before NC induction, are certainly inconsistent withhow we think of NC lineages arising. Lateral neural ectoderm has been proposed asthe site of origin of R–B neurons (see Box 3.3).

R–B neurons arise at the border between neural and epidermal ectoderm (theneural plate border), they can arise independently of NCCs, but they can arisefrom a common precursor of NCCs. A very recent study by Rossi and colleagues(2008) has identified Bmp4 as required for the induction of R–B neurons in Xeno-pus. Their approach was to transplant medial neural plate into lateral ectodermbetween pigmented and albino embryos to establish an ectopic neural plate border.The R–B neurons arose at the ectopic border, originating from both donor (neu-ral) and host (epidermal ectodermal) cells. Levels of Bmp4 lower than required to

Rohon–Beard Neurons 199

induce neural ectoderm were sufficient to induce R–B neurons in neural ectodermin vitro, consistent with the model discussed in Chapter 2 of intermediate levels ofBmp4 operating at the neural plate border. The Bmp-inhibitor Noggin inhibited R–Bformation.

HNK-1 was used to identify neurons within the dorsal neural tube of lampreysthat were taken to be homologs of R–B neurons. Although HNK-1-positive cells arefound in lamprey hearts, and HNK-1-positive mesenchymal cells are found in thedorsal fins of ammocoete larvae, and although independent evidence demonstratesthat NCCs contribute to the dorsal fin in lampreys, HNK-1 alone is not a sufficientmarker to allow us to conclude that cells are NC in origin.22

Amphioxus contains a population of primary intramedullary sensory neuronsknown as Retzius bipolar cells that either synapse with epidermal secondary neu-rons or terminate within the epidermis. As R–B neurons are primary intramedullarysensory neurons, amphioxus Retzius bipolar cells have been proposed as homologsof vertebrate R–B neurons, the only major difference being that R–B neurons do notsynapse with secondary neurons in the epidermis, although their neurites do ramifyextensively within the epidermis (Fritzsch and Northcutt, 1993∗). If this homologyis correct, then Retzius bipolar cells are precursors of NCCs or represent a parallelevolution.

The presence of R–B neurons in amphibians and fish (and lampreys?) and whatmay be their homologs in amphioxus have been used to propose that R–B neu-rons might be evolutionary precursors of the large proprioceptive neurons of themesencephalic trigeminal nucleus (MTN) that innervates the lower jaw (see Box3.3). Given that the two lineages lie adjacent to one another in the blastula, thatplacodal and NC ectoderm lie in close proximity, and that some placodal ecto-derm arises from the neural folds (see Figs. 2.10 and 2.12), a placodal origincannot be ruled out; the extirpation studies could have removed ectoderm otherthan NC from the neural folds. Further study of these interesting cells is clearlymerited.23

Genetic Control of R–B Neurons

Delta/Notch: Precursors of R–B neurons express Delta genes, which are ligands forNotch signaling. Misexpressing DeltaA leads to the loss of all R–B neurons, whilemutants with decreased Delta signaling have excess R–B neurons and decreasedTNCCs, results consistent with two cell populations or early segregation of a singlepopulation. Notch signaling inhibits the specification of R–B neurons in premigra-tory TNCCs, a result consistent with the inhibitory role of Notch over neuronalgene expression, although Notch signaling also leads to apoptosis in preneurogeniccells.24

Ap2:Under regulation by Wnt genes, Ap2 transcription factors regulate theexpression of Snail2 in Xenopus embryosand so play maintaining-induced NCCs.Once NCCs are induced, Ap2 is required for their segregation and survival (seeFig. 2.4).


Eliminating the genesap2a and ap2c from zebrafish, which encode the transcrip-tion factors Ap2� and Ap2�, eliminates all NCCs but leaves R–B neurons in almostnormal numbers. Two interpretations of these results are that R–B neurons do notarise from the NC (and so are not affected) and/or that there is a separate tran-scriptional control over R–B neurons and NCCs, in which case the results are notinformative about origins.

Midkine-b: A recent study in which expression of the heparin-binding growthfactor Midkine-b (Mdkb) was altered in zebrafish embryos is consistent with Mdkbestablishing the boundary between R–B neurons and NCCs. (Mdkb acts down-stream of retinoic acid.) Overexpressing Mdkb increases the numbers of NCCsand R–B neurons. Morpholino knockdown of Mdkb dramatically reduces the NCCpopulation and results in loss of all R–B neurons. Both findings are consistentwith the origin of both cell types from a boundary region, where specificationcan be altered to match local needs in individual embryos.25 Consequently, it maybe more appropriate to regard this region as a NC–RB neuron field than to askwhether it is a NC field that can form R–B neurons or an R–B neuronal fieldthan can form NCCs. Establishing more precise relationships between the twocell types will require more comparative analyses conducted within a phylogeneticcontext.

Notes

1. Readers interested in further details of the origin of neuronal cells from the amphibian NC willfind that Chapters 2 and 4 of Horstadius (1950∗), comprising some 40% of his monograph,provide an excellent evaluation and synthesis of the earlier work. See Le Douarin (1986) forearly studies on the neuronal NC derivatives in avian embryos. Reviews include Bronner-Fraser (1995) and Bronner-Fraser and Fraser (1997).

2. Because of cell surface differences, HNK-1-positive adrenergic subpopulations can be iso-lated using fluorescence-activated cell sorting (Maxwell and Forbes, 1991∗).

3. For early studies on NC contribution to dorsal root ganglia, see the literature discussed in Halland Horstadius (1988∗).

4. See the studies by Detwiler, Harrison, Raven, and DuShane cited in Hall (1999a∗) for vitaldye staining and experimental confirmation of the NC origin of spinal ganglia.

5. See Schlosser and Northcutt (2001) for induction of the lateral-line placodes in axolotls,Schlosser et al. (1999) for the studies on coqui, and Schlosser (2002a,b, 2006∗, 2007∗) forreviews.

6. See Le Douarin et al. (1986), Northcutt (1992, 1996), Webb and Noden (1993∗), Northcuttet al. (1994, 1995), Osumi-Yamashita et al. (1994), and Northcutt and Barlow (1998) foroverviews of placode development. See M. M. Smith and Hall (1990), S. C. Smith et al.(1994), Parichy (1996), Graham and Begbie (2000), Shimeld and Holland (2000), Baker andBronner-Fraser (2001), and O’Neill et al. (2007) for discussions of the developmental andevolutionary links between placodal and NC ectoderm. It appears that the NC does not con-tribute cells to the derivatives of the more caudal epibranchial placodes in the North Americansea lamprey (McCauley and Bronner-Fraser, 2003).

7. For recent overviews of our understanding of placodes, see Graham and Begbie (2000), Bakerand Bronner Fraser (2001∗), Streit (2004∗), Litsiou et al. (2005), Schlosser (2006∗, 2007∗),and Lassiter et al. (2007). For the first fine-grained mapping of ophthalmic and maxillo-mandibular trigeminal placodes, see Xu et al. (2008).

Notes 201

8. See Streit (2004∗) and Schlosser (2007∗) for summaries and analyses of co-option of genesinto placodal ectoderm and of their regulation of panplacodal ectoderm.

9. See Liu et al. (2003) and Nechiporuk et al. (2007) for Fgf3 and Fgf8, Sun et al. (2007) forDlx3 and Dlx4b, Solomon and Fritz (2002) for Dlx3 and Dlx7, Solomon et al. (2003) for thehearsay mutant, and Abu-Elmagd et al. (2001) for Sox3. A recent issue of the Int J Devel Biol(2007, 51[6/7]: 427–687) is devoted to ear development, including studies on otic placodes.

10. Yntema and Hammond (1947) provide a comprehensive review of the early studies on theautonomic and sympathetic nervous systems.

11. For the NC origin of autonomic ganglia, see Yntema and Hammond (1945) and the litera-ture summarized in Hall and Horstadius (1988∗), Le Douarin (1982), Anderson (1997), Hall(1999a∗), and Le Douarin and Kalcheim (1999∗). For enteric and visceral ganglia, see Yntemaand Hammond (1945) and Peters-van der Sanden et al. (1993).

12. See Harrison (1910) and Muller and Ingvar (1921, 1923) for studies on the NC origin ofSchwann cells, and Horstadius (1950∗) for a discussion of early studies that gave contraryresults.

13. See Raible and Eisen (1996) for migration and lineage restriction in zebrafish TNC, andRaible and Ragland (2005) for the role played by Wnt signaling.

14. See Paratore et al. (2001) for Sox10 and Neuregulin in mice.15. See Carney et al. (2006) for Sox10, neurogenin1 and specification of neuronal cell fate, and

Kelsh (2006∗) for an overview of the functions of Sox10. All sensory neurons of dorsal rootganglia require Ngn2 or Ngn1 to mediate the first and second waves of neurogenesis, respec-tively, the neurogenins being regulated by the canonical Wnt pathway (see Fig. 2.5).

16. See Hong and Saint-Jeannet (2005) and Kelsh (2006∗) for literature on the various roles ofSox10, and Finzsch et al. (2008) for the study on Pdgf�.

17. An even more recent study not only using an entirely different approach, but also usingchicken embryos, shows that leading edge CNCCs migrating from r4 into the pharyngealarches out-proliferate trailing NCCs by 3–1 (Kulesa et al., 2008).

18. See Pomeranz et al. (1993) and Serbedzija et al. (1991∗) for the SNC, Burns et al. (2000,2002) for lack of interaction between VNCCs and SNCCs of chicken embryos during migra-tion, Hearn and Newgreen (2000) for lumbo-sacral and VNCC interactions, and Erickson andGoins (2000) for the ability of SNCCs to invade anterior gut. These cells are multipotential;they can form neuronal or glial cells when cultured on laminin but are normally inhibitedfrom forming catecholaminergic neurons (Pomeranz et al. 1993).

19. See Delalande et al. (2008) for ret, and Abzhanov et al. (2004) for the CNC–SNC study.20. See Balfour (1878), Rohon (1884), and Beard (1892, 1896) for the discovery and descriptions

of Rohon–Beard neurons, Chibon (1974∗) for their NC origin, Laudel and Lim (1993) fortheir development in the Mozambique tilapia, Oreochromis mossambicus, and Kuratani et al.(2000) for their development in sturgeon embryos and regression as DRG develop.

21. See Williams et al. (2000∗), Cole and Ross (2001), and Svoboda et al. (2001∗) for removal ofRohon–Beard neurons from zebrafish, and Hunter et al. (2001) for R–B neuron origins andthe mesencephalic trigeminal nucleus. Both the trigeminal placode and R–B neurons providethe extensive network of epidermal neurites seen in sturgeon larvae (Kuratani et al., 2000).

22. See Langille and Hall (1988b) and McCauley and Bronner-Fraser (2003) for NCC contribu-tions to the dorsal fin in lampreys.

23. See M. Jacobson and Moody (1984) for lineage analysis of Rohon–Beard neurons, Lam-borghini (1980) for their origin during gastrulation, and Rao and Jacobson (2005∗) for ageneral discussion.

24. See Cornell and Eisen (2000, 2005∗) for Delta/Notch functioning in zebrafish, and Yeo andGautier (2004) for Notch and apoptosis of neurogenic cells.

25. See Li and Cornell (2007) for R–B neuron survival in zebrafish, Luo et al. (2003) and Saint-Jeannet (2006) for Xenopus, and Liedtke and Winkler (2008) for Midkine-b.

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