patterning and axon guidance of cranial motor...

© 2007 Nature Publishing Group

In humans, the cell bodies of cranial motor neurons lie in the brainstem, and their axons extend through the cranial nerves to control muscles in the head and neck. Other vertebrates (including fish, chicks and mice) show a high degree of conservation in both the arrangement of brainstem motor neurons and the muscles they inner-vate. Developing motor axons perform a spectacular feat, navigating over long distances from the CNS to their targets in the periphery.Early in development, the neural tube acquires a series of swellings at its rostral end, presaging the development of the forebrain, the midbrain and the hindbrain. Caudally, the neural tube remains narrow and elongates to form the spinal cord. Motor neurons differentiate ventrally, on either side of a midline structure, the floor plate. Cranial motor neurons reside in the midbrain and the hindbrain (which together constitute the brainstem), where they are partitioned into a series of nuclei. By contrast, spinal motor neurons form a number of dis-continuous columns along the length of the cord (for reviews, see refs 1,2). Cranial motor axons follow dorsal or ventral pathways from the brainstem; the axial posi-tion of this site of exit in turn dictates their peripheral paths to muscles of the eye, tongue, branchial arches or to parasympathetic ganglia3 (fIG. 1; TABLe 1).

Surprisingly, despite the functional significance of cranial motor nerves, an understanding of the molecular mechanisms that underlie their development is only just starting to emerge4,5. Exciting progress has been made in understanding cranial motor neuron development, par-ticularly from gene gain-of-function and loss-of-function

experiments. The specificity of cranial motor neuron projections is governed by rostrocaudal and dorsoven-tral patterning mechanisms that produce a diversity of motor neuron subpopulations with distinct differentia-tion programmes. Some of the guidance molecules that are involved in elaborating axon projections have also been characterized. However, many important ques-tions remain. The unique features of the differentiation programmes of each of the cranial nerves are only partly characterized. In particular, we know little about how patterning genes dictate the repertoires of receptors on axons, or how these receptors determine axon pathfind-ing behaviour to particular muscle targets. Deciphering these molecular mechanisms is a major challenge in developmental neurobiology.

In this context, it is fascinating that cranial dysinner-vation disorders, which reflect abnormalities of one or more cranial nerves, are starting to be genetically charac-terized in humans6. Clinical studies, together with studies in animal models, are now providing fresh impetus to understand how normal and abnormal cranial nerve wir-ing develops. In this Review, I describe the latest findings in cranial motor neuron patterning and axon guidance, focusing mainly on mouse and chick studies (as zebrafish studies have been reviewed elsewhere: see ref. 5).

Motor nuclei form at distinct axial levelsCranial motor neurons comprise three subsets: bran-chiomotor (BM), visceral motor (VM) and somatic motor (SM) neurons (fIG. 2; TABLe 1). Early in develop-ment, these neurons arise in longitudinal progenitor

MRC Centre for Developmental Neurobiology, King’s College, Guy’s Campus, London, SE1 1UL, UK.e‑mail: [email protected]:10.1038/nrn2254

Neural tubeThe primordium of the nervous system.

Floor plateThe ventral midline structure of the CNs. It has a role in patterning and axon guidance.

Branchial archesrepeated bars of mesenchymal tissue that contribute to the lower jaw and neck; each contains a cartilaginous component, a muscular component, a nerve and an artery.

Patterning and axon guidance of cranial motor neuronsSarah Guthrie

Abstract | The cranial motor nerves control muscles involved in eye, head and neck movements, feeding, speech and facial expression. The generic and specific properties of cranial motor neurons depend on a matrix of rostrocaudal and dorsoventral patterning information. Repertoires of transcription factors, including Hox genes, confer generic and specific properties on motor neurons, and endow subpopulations at various axial levels with the ability to navigate to their targets. Cranial motor axon projections are guided by diffusible cues and aided by guideposts, such as nerve exit points, glial cells and muscle primordia. The recent identification of genes that are mutated in human cranial dysinnervation disorders is now shedding light on the functional consequences of perturbations of cranial motor neuron development.

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FB

MB

HB

IV

V

VI

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X/XI

VII/VII

XII

III

BAsOV

Basal plateThe ventral half of the neuroepithelium.

Alar plateThe dorsal half of the neuroepithelium.

NeuroepitheliumA part of the early nervous system that consists of dividing progenitors arranged in a columnar epithelium.

HomeoboxA conserved 180 base pair sequence that encodes homeodomain regions of proteins that are involved in binding to DNA and regulating transcription.

domains in the hindbrain basal plate: BM and VM neuronal somata migrate dorsally into the alar plate, whereas SM somata remain ventral (in the basal plate). BM and VM axons extend dorsally through the neuroepithelium to large common exit points, whereas SM axons leave the neuroepithelium ventrally in small groups (fIG. 2c), with the exception of trochlear SM axons, which grow dorsally and cross the dorsal mid-line at the midbrain–hindbrain boundary to project contralaterally.

Individual motor nuclei can contain one or more of BM, VM and SM neuron subsets. The oculomotor nucleus (nucleus III), which contains SM and VM neurons, lies most rostrally in the midbrain. along the rostrocaudal axis, the hindbrain is divided into rhom-bomeres, segmental entities that contain repeating sets of neurons with distinct differentiation programmes at different axial levels7–9. Motor nuclei differentiate in individual rhombomeres or pairs of rhombomeres. Rostral rhombomere one (r1) contains the trochlear nucleus (nucleus IV), which contains SM neurons. The trigeminal nucleus (nucleus V; BM neurons) occupies

r1, r2 and r3 (in mice) or r2 and r3 (in chicks), the facial nucleus (nucleus VII; BM and VM neurons) lies in r4 and r5, the glossopharyngeal nucleus (nucleus IX; BM and VM neurons) lies in r6 (in mice) or r6 and r7 (in chicks), and the vagus nucleus (nucleus X; BM and VM neurons) and cranial accessory nucleus (XI; BM neurons) occupy r7 and r8 (ref.10) (fIG. 2). In the cau-dal hindbrain, the abducens nucleus (nucleus VI, SM neurons) occupies r5 in mice and r5 and r6 in chicks, with the extended hypoglossal nucleus (nucleus XII, SM neurons) found in r8. In both mice and chicks, the facial motor neurons of the BM subtype are segregated in r4, and those of the VM subtype are segregated in r5 (refs 11,12). In all except avian species, the facial bran-chiomotor (FBM) neurons are born in r4 and then undertake a striking caudal migration to r6 (refs 10,13), unlike most BM and VM neuron somata, which migrate dorsally14. Rhombomere 4 also contains a population of vestibuloacoustic neurons, which are efferent to the hair cells of the inner ear; a subset of these neurons (contral-ateral vestibuloacoustic neurons) translocate their cell bodies across the midline15.

Following their exit into the periphery, cranial motor axons converge to form components of the cranial nerves (fIG. 1). BM axons travel, through the trigeminal, facial, glossopharyngeal, vagus and cranial accessory nerves, towards branchial arches 1, 2, 3, 4 and 6, respectively, where they innervate muscles of the jaw and muscles that control facial expression, as well as the pharynx and the larynx. VM axons project towards parasympathetic ganglia, the neurons of which supply salivary and lacrimal glands, smooth muscle and visceral organs. Oculomotor, trochlear and abducens SM neurons innervate the six eye mus-cles, with an additional oculomotor VM component synapsing at the ciliary ganglion. Hypoglossal neurons project rostrally through the floor of the pharynx to the tongue muscles. Cranial motor nuclei conform to a theme, sharing common features, such as morphology and initial axon trajectory, but nevertheless possess-ing distinct positional identity, synaptic targets and functions.

Rostrocaudal patterning of the brainstemThe midbrain is divided into a series of ‘arcs’, which have been proposed to underlie the differentiation of nuclei and are distinguished by the expression of vari-ous homeobox genes and other molecular markers16–18. The most medial arc contains oculomotor neurons, and fibroblast growth factor 8 (FGF8), produced by the midbrain–hindbrain boundary, has been proposed to dictate the rostrocaudal position of the oculomo-tor nucleus, because misexpression of FGF8 shifts the nucleus rostrally17. Differentiating oculomotor neurons express the homeobox gene paired-like homeobox 2a (Phox2a)16, which is an important determinant of ocu-lomotor identity, as oculomotor neurons (as well as trochlear motor neurons) are absent in Phox2a-mutant mice19. Further details of the transcriptional hierarchy that underlies oculomotor neuron determination remain to be discovered.

Figure 1 |cranialnervesinthechickembryo.Alateral view of cranial nerves in the chick embryo at embryonic day four, showing the pathways from the hindbrain (HB), on the right, into the branchial arches (BAs) and other head structures (the midbrain (MB) and the forebrain (FB)), on the left. Roman numerals denote the nerves: III, oculomotor; IV, trochlear; V, trigeminal; VI, abducens; VII/VIII, facial/vestibuloacoustic; IX, glossopharyngeal; X, vagus; XI, cranial accessory; XII, hypoglossal. OV, otic vesicle. Figure modified, with permission, from ref. 3 (1990) Wiley-Liss.

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2253&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum



In the hindbrain, a large number of transcription factors and other genes pattern rhombomere territories through their segmental expression9,13,20. Many of these genes regulate motor neuron development, either directly or indirectly. For example, the zinc finger transcription factor early growth response 2 (EGR2; also known as KROX20) is expressed early in r3 and r5 in the mouse21 and determines many of the features of odd-numbered rhombomeres. In Egr2-mutant mice, r3 and r5 are largely missing, depleting the motor neurons at these levels22. The transcription factor MaFB (also known as Kreisler) is similarly expressed early and regulates r5 and r6 development23,24; in Mafb-mutant mice, r5 and r6 are lost, causing the deletion of the r5 VM facial and SM abducens neurons25.

Both Egr2 and Mafb lie upstream of, and activate the transcription of, Hox genes, which contain an antennapedia-class homeobox sequence and have a pre-eminent role in hindbrain patterning. In vertebrates there are four Hox-gene clusters (named a–d) on four separate chromosomes26. There are 13 paralogue groups, (although no individual cluster contains all 13 genes). Paralogous genes (for example, Hoxa3 and Hoxb3) often exhibit over-lapping functions; indeed, dosage-dependent effects have been shown in the case of Hoxa3 and Hoxd3 (ref. 27). Hox genes show nested domains of expression in the hindbrain (fIG. 3), with each paralogue group expressed from the spi-nal cord rostrally to particular rhombomere boundaries. Thus, group 2, 3 and 4 gene expression domains end at the r2–r3, r4–r5 and r6–r7 boundaries, respectively (although Hoxa2 expression terminates at the r1–r2 boundary). Group 1 genes show an anomalous rostral restriction at the r3–r4 boundary, and Hoxb1 is expressed at high levels in r4. The combination of Hox genes that are expressed

in a particular rhombomere, as well as the timing of the onset of the expression and the expression level, dictates segmentation and segment identity at that axial level.

The patterns of Hox gene expression in the hindbrain are established, at least in part, by the diffusible action of FGF8 and retinoic acid (Ra) at the rostral and caudal ends of the hindbrain, respectively28,29. Rostrally, FGF8 that is produced by the midbrain–hindbrain boundary sets the rostral boundary of Hoxa2 expression30. Ra that is derived from the mesodermal somites, which flank the caudal hindbrain, is thought to specify the caudal hind-brain (r5 to r8) through the induction of Hox genes, in a dose-dependent manner28. Experimental depletion of Ra in mice and chicks supports this idea: it causes caudal rhombomeres to assume more rostral identities (either r3 or r4)31,32. In some cases, the expression of caudal Hox genes is induced through upstream Retinoic acid Response Elements (RaREs)33,34. Ra is implicated in the production of SM neurons in the spinal cord35,36, raising the possibility that it might also generate SM neurons in r5 to r8 (the abducens and hypoglossal nuclei). Somatic motor neurons differentiate throughout the rostrocaudal extent of chick hindbrain explants following application of Ra37, whereas a reduction in Ra signalling in the zebrafish produces a loss of hindbrain cranial motor neurons38. The absence of SM neurons in the rostral hindbrain might be maintained by the action of the Ra-degrading enzyme CYP26, as inhibition of CYP26 caused the differentiation of SM neurons throughout hindbrain explants37. Thus, FGF8 and Ra, through their role in patterning Hox genes, and possibly through independent inductive mechanisms, pattern motor neurons such that BM and VM neurons differentiate throughout the hindbrain, whereas SM neurons are restricted to caudal rhombomeres.

Table 1 | Motor components of the cranial nerves and their targets in humans

nerve subtype nucleus Targetmusclesorganglia

III Somatic motor Oculomotor Superior, inferior and medial recti muscles; inferior oblique, levator palpebrae superioris

Visceral motor Edinger-Westphal Ciliary ganglion

IV Somatic motor Trochlear Superior oblique

V Branchiomotor Trigeminal motor Muscles of mastication, tensor tympani, anterior belly of digastric, others

VI Somatic motor Abducens Lateral rectus muscle

VII Branchiomotor Facial motor Muscles of facial expression, stapedius, posterior belly of digastric

Visceral motor Superior salivatory Pterygopalatine/sphenopalatine ganglion, submandibular ganglion

IX Branchiomotor Nucleus ambiguus Stylopharyngeus muscle

Visceral motor Inferior salivatory Otic ganglion

X Branchiomotor Nucleus ambiguus Laryngeal and pharyngeal muscles

Visceral motor Dorsal motor Non-striated muscle of thoracic and abdominal viscera

Cranial XI Branchiomotor Nucleus ambiguus Laryngeal and pharyngeal muscles

Spinal XI Branchiomotor Accessory nucleus, cervical spinal cord

Sternocleidomastoid and trapezius muscles

XII Somatic motor Hypoglossal Tongue muscles

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r8

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CVA

a Chick at embryonic day 4 b Mouse at embryonic day 11.5

c Transverse section of chick branchial region

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Hox genes control motor neuron identityas mentioned above, Hox genes are key controllers of rostrocaudal patterning in the head, including hind-brain segmentation and rhombomere identity4,39. as well as being expressed in neuroepithelial domains (fIG. 3), Hox genes are expressed in neural crest cells, which emigrate predominantly from even-numbered rhombomeres into the branchial arches (fIG. 4), generat-ing skeletal tissues and cranial ganglia40–42. as a result, several Hox-mutant mice show defects in the pattern-ing of branchial arch-derived structures39. There is positional registration, that is, motor neurons project into peripheral territory that expresses the same Hox repertoire as their rhombomere of origin. One expecta-tion of this pattern is that Hox genes dictate the expres-sion of matching axon guidance receptors and ligands, in neurons and their targets, respectively. But, hitherto, there is little data on this issue. Nevertheless, a number of studies with Hox-mutant mice have shown specific defects in the generation and patterning of cranial motor neurons. loss of rostrally expressed Hox genes in paralogue groups 1 and 2 leads to patterning defects of the trigeminal and facial BM and VM nuclei, which occupy rostral rhombomeres, whereas SM neurons in caudal rhombomeres are affected by the loss of group 3 paralogues.

Hoxa2 regulates trigeminal motor neuron differentiation. Hoxa2 is expressed up to the r1–r2 boundary and is the only Hox gene to be expressed in r2, whereas in r3 it is co-expressed with Hoxb2 (refs 39,43). In both mice and chicks, only r4 second branchial arch, not r2, (first branchial arch) neural crest cells express Hoxa2, and loss of Hoxa2 in mice produces a striking transfor-mation of the second branchial arch into a first-arch phenotype44–46. In Hoxa2-mutant mice, presumptive trigeminal motor neurons in r3, and a subset in r2, project through the r4 exit point46. This suggests that trigeminal motor neurons misroute to the transformed second arch, either because of a change in motor neuron identity and/or because of a change in axon guidance cues. The r4-derived facial nerve is also reduced in size later in development, possibly owing to a loss of second branchial arch character and arch-derived fac-tors46. when Hoxa2 is ectopically expressed in chick r1, which normally lacks motor neurons, trigeminal neurons are generated47, supporting the idea that Hoxa2 specifies trigeminal motor neurons. In Hoxb2-mutant mice there is also a mis-specification of some trigeminal motor neurons in r3, which project through the r4 exit point, but the effect on trigeminal axon projections is less striking than with Hoxa2 muta-tion (ref. 48). It is likely, therefore, that r2 trigeminal

Figure 2 |Motorneuronorganizationinthevertebratebrainstem.a | The organization of motor neurons in a flat-mounted chick brainstem at embryonic day four (E4). b | The organization of motor neurons in a flat-mounted mouse brainstem at E11.5. In parts a and b, rhombomere (r) levels are indicated; branchiomotor and visceral motor neurons are shown in red; somatic motor neurons are shown in blue. Roman numbers alone denote the nerves: III, oculomotor; IV, trochlear; V, trigeminal; VI, accessory abducens; VII/VIII, facial/vestibuloacoustic; IX, glossopharyngeal; X, vagus; XI, cranial accessory; XII, hypoglossal. Roman numerals with the letter g denote the cranial ganglia: gV, trigeminal ganglion; gVII, geniculate ganglion; gVIII, vestibuloacoustic ganglion; gIX, petrosal ganglion; gX, nodose ganglion. c | Cranial motor axon pathways in a transverse section of the branchial region in the chick embryo at E4 (for details, see ref. 10). aVI, accessory abducens nucleus;BA, branchial arch; CVA, contralateral vestibuloacoustic neurons; FP, floor plate; G, cranial sensory ganglion; OV, otic vesicle; PX, pharynx; VE, ventricle.

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neurons are patterned by Hoxa2, whereas those in r3 are patterned by a combination of Hoxa2 and Hoxb2.

Hoxb1 is a key regulator of FBM neurons in r4. Extensive insight has been gained into the genetic hierarchy that underlies FBM neuron development in r4. Hoxa1 and Hoxb1 are expressed up to the r3–r4 boundary in divid-ing progenitors, but the onset of Hoxa1 expression comes earlier (at embryonic day eight), and after neuronal dif-ferentiation, only Hoxb1 is maintained at high levels in r4 and in the postmitotic FBM neurons40. In Hoxa1-mutant mice, rhombomeric segmentation of r3 to r8 is disrupted, the r3–r4 boundary does not form correctly, r4 is reduced in size and r5 is absent49–51. By embryonic day 18.5, this early role of Hoxa1 in rhombomere segmentation and identity leads to loss of the abducens nerve and reduction or loss of the facial nerve. By contrast, Hoxb1 has a later role in FBM neuron specification, reflected in the obser-vation that in Hoxb1-mutant mice, segmentation occurs normally but there is a striking absence of FBM neurons (which fail to migrate caudally to r6 and eventually die); CVa neurons are also missing52,53. Motor neurons that do differentiate in r4 project axons to the first branchial arch, suggesting that they default to a trigeminal-like identity. The instructive role of Hoxb1 was demonstrated in chicks by overexpressing Hoxb1 in r2. This converts trigeminal motor neurons to an FBM fate, resulting in anomalous facial-like axon projections to the second branchial arch54. However, when Hoxb1 is misexpressed in both r2 and the first branchial arch, ectopic FBM axons navigate from r2

into the first arch54. Global Hoxb1 overexpression appears to cause the coordinated transformation of motor neurons and the first arch, such that they develop an FBM-like second arch identity. The molecular significance of this positional registration in Hox gene expression between BM neurons and their innervation territory was unclear for some time. However, recent studies show that Hoxb1-positive, r4-derived neural crest cells preferentially give rise to Schwann cells, which might provide the guid-ance cues and/or survival factors that are needed for the projection and maintenance of the facial motor nerve55. Conditional deletion of Hoxb1 only in the neural crest results in a significant proportion of animals showing facial paralysis, indicative of a loss of facial motor neurons (as in Hoxb1-null mice)56. The facial nerve fails to branch correctly and eventually the facial motor neurons die56. FBM axons might require an interaction with Schwann cells for their guidance and survival, perhaps through the production by the Schwann cells of neurotrophic factors. an intriguing piece of evidence in favour of this idea is the prolonged survival of mis-specified FBM neurons in Hoxb1-mutant mice that are also mutant for Bax (a gene that is required in cell death pathways that are associated with neurotrophic factor deprivation)57.

as with the pairing of Hoxa2 and Hoxb2 in trigeminal neuron specification, Hoxa1 and Hoxb1 appear to act synergistically to pattern facial motor neurons. Hoxa1;Hoxb1 double-mutants reflect this fact, and show more extensive FBM neuron defects than either of the single mutants58,59. Hoxb2 also functions in FBM specification, possibly because it is a downstream target of Hoxb1 (refs 48,60). The network of regulatory inter-actions that has been revealed so far for r4 provides a glimpse of how daunting it will be to achieve a thorough understanding of hindbrain motor neuron patterning. Following the activation of Hoxa1 and Hoxb1 by Ra, Hoxa1 transactivates Hoxb1, Hoxb1 auto-regulates its own expression and activates Hoxb2 and Hoxa2 (ref. 61). Mutation studies have also shown that the genes Gata2, Gata3 and T-box 20 (Tbx20) are among the downstream targets of Hoxb1 in FBM specification62,63.

One outstanding question is how the specificity of Hox proteins in binding to particular DNa target regions is conferred. an answer might lie in the fact that Hox gene-encoded homeoproteins form complexes with specific cofactors, possibly restricting the proteins’ binding spe-cificity. These cofactors are the Pbx and Meis homeopro-teins, which form a tripartite complex with Hox proteins to regulate downstream transcription64. In pbx4-mutant zebrafish, FBM neuron development is defective, and the resultant phenotype is identical to that which results from a deficiency in hoxb1a, the zebrafish Hoxb1 homo-logue65,66. Complete elimination of maternal and zygotic Pbx function produces a hindbrain r1 ‘ground state’ in r2 to r7, reflected by a lack of characteristic molecular markers and neuronal types, especially BM and VM neu-rons67. The specificity of the interactions between Pbx proteins and Hox proteins remains to be characterized, but HOXB1–Pbx-protein complexes have been shown to bind to specific regulatory regions of the Hoxb1 and Hoxb2 genes to regulate their expression in r4 (refs 60,68).

Figure 3 |expressionpatternsofHoxgenesinthevertebratehindbrain.The domains of mRNA expression of the Hox genes Egr2 and Mafb in the hindbrain of the mouse and chick embryo, at embryonic day 11.5 (E11.5) and E4, respectively. The bars labelled with different Hox genes show the genes’ expression domains, which extend from the caudal hindbrain up to particular rhombomere (r) boundaries4,168. Darker shading indicates higher levels of expression. HB, hindbrain; MB, midbrain.

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Hox3 genes regulate SM neuron differentiation. Hox3 paralogues are good candidates for genes that regulate the production of SM neurons in r5 to r8. Hoxb3, for example, is expressed at high levels in r5 and r6 in chicks, but only in r5 in mice69,70, corresponding with the location of abducens neurons in these two species. Knockout studies have shown that abducens neurons are lost in Hoxa3;Hoxb3 mouse double-mutants, but can be ectopically induced by rostral expression of Hoxa3 in chicks71,72. Future studies might reveal whether Hox4 paralogues, which are expressed in a caudal domain that extends up to the r6–r7 boundary, specify hypoglossal motor neurons in this region.

Dorsoventral patterning of HoxCombinations of other homeobox-containing tran-scription factors specify progenitor domains along the dorsoventral axis of the brainstem1,73. Sonic hedgehog protein (SHH) has been proposed to form a ventral-to-dorsal gradient that induces dose-dependent neuronal differentiation. although this model is based largely on experiments on the spinal cord, it is thought to apply to the hindbrain as well, and both cranial and spinal motor neurons are missing in Shh–/– mouse mutants74. SHH also controls the differentiation of the midbrain arc, which contains oculomotor neurons16. The model proposes that graded SHH signalling produces the graded activ-ity of Gli transcription factors75, which in turn activate or repress the expression of homeodomain proteins in

specific dorsoventral progenitor domains76. The expres-sion domains of these transcription factors show cross-repressive interactions at their boundaries, consolidating their identity and generating groups of postmitotic neu-rons that express repertoires of transcription factors that are involved in their further specification77. This model has given insight into the mechanism of differentiation of cranial motor neurons, the progenitors of which occupy distinct dorsoventral domains.

In the hindbrain, the progenitor domain that flanks the floor plate (known as the p3 domain) gives rise to BM and VM neurons78,79, whereas the dorsally adjoin-ing progenitor domain (known as the pMN domain) generates SM neurons (fIG. 5a). P3 and pMN progenitors express distinct ‘codes’ of transcription factors (fIG. 5), most of which act as transcriptional repressors to control neuronal identity80–83. The key regulators of BM and VM neuron fate are suspected to be Nkx2.2 and Nkx2.9, but mutation of either gene on its own leaves these neurons intact, probably because of redundancy78,84. Nkx6.1 and Nkx6.2 are not required to specify BM and VM neurons per se, but they act to repress alternative interneuron fates, and in double mutants, BM and VM neurons express some interneuron markers79. loss-of-function of Nkx6.1 alone or with Nkx6.2 also results in aberrant BM and VM neuron migration and axon pathfinding79,85. In the pMN domain, loss-of-function of Nkx6.1 and/or Nkx6.2, or of paired box gene 6 (Pax6), deletes abducens and hypoglossal SM neurons80,82,83,86. Combined Nkx gene activity in turn induces the expression of the basic helix-loop-helix transcription-factor-encoding gene oligodendrocyte transcription factor 2 (Olig2), which coordinates generic neuronal differentiation with motor neuron subtype87,88 and induces the homeobox-containing gene MNR2.

as a result of these early specification events, postmi-totic SM neurons express a combination of the homeo-box-containing genes MNR2 (which is chick-specific), motor neuron and pancreas homeobox 1 (MNX1; also known as HB9) and the lIM-homeobox genes Islet 1 (Isl1), Isl2, Lhx3 and Lhx4 refs 73,87,89,90 (fIG. 5b). Interestingly, hindbrain SM neurons show variations on this pattern, for example, subsets of abducens SM neurons express different combinations of these genes91. MNR2 and MNX1 are involved in specifying SM neuron fate and repressing interneuron fates, respectively90,92,93. Lhx3 and Lhx4 are determinants of ventral pathway choice; SM neurons, including abducens and hypoglos-sal subpopulations, are absent in Lhx3;Lhx4 double knockouts, and BM and VM neurons that misexpress these transcription factors extend axons ventrally rather than dorsally94. like SM neurons, postmitotic BM and VM neurons express Isl1 but, unlike SMs, they also express Tbx20 (ref. 95), Phox2a and Phox2b, with Phox2b expressed first19 (fIG. 5b). Phox2b is a key gene in BM and VM neuron generation96, and in Phox2b-mutant mice, all BM and VM neurons are absent97. Conversely, in mice that lack Phox2a, which is expressed before Phox2b in the oculomotor and trochlear nuclei, both of these nuclei are missing19. The generation of knock-in mouse lines, in which Phox2b was replaced by the Phox2a locus, or

Figure 4 |PatternsofneuralcrestmigrationandbranchialarchHoxgeneexpressioninchickandmouseembryos.Aschematic diagram of a chick head at embryonic day two, showing pathways of neural crest migration in the chick and mouse embryo and patterns of Hox gene expression in the branchial arches (BAs)42,102,169,170. FB, forebrain; HB, hindbrain; MB, midbrain; Md, mandibular part of BA1; Mx, maxillary part of BA1; OV, otic vesicle; r, rhombomere.

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N NSHH

a b

BM and VM neuronsSM neurons

BM/VM neuronsIslet-1, Phox2b,Phox2a, Tbx20.

p3Nkx2.2, Nkx2.9, Nkx6.1, Nkx6.2.

pMNPax6, Olig2, Nkx6.1, NKx6.2.

SM neuronsMNX1, MNR2, Islet-1, Islet-2, Lhx3, Lhx4.

Cranial paraxial mesodermThe population of mesoderm cells that originates adjacent to the brainstem and gives rise to many head muscles.

Sphenopalatine ganglionA parasympathetic ganglion that is innervated by VM neurons of the facial nerve.

vice versa, has revealed that the two genes are not func-tionally equivalent98. Oculomotor and trochlear motor neurons are only partially rescued by the substitution of Phox2b for Phox2a, and in the Phox2a into Phox2b knock-in line, FBM neurons differentiate but fail to migrate correctly 98.

Phox2b is regulated by converging streams of rostro-caudal and dorsoventral patterning information, which are dependent on Hox gene function and Nkx2.2 and Nkx6 gene function, respectively. loss-of-function stud-ies reveal that Nkx genes cooperate with Hoxb1 to main-tain Phox2b expression in r4 and favour an FBM over a serotonergic neuronal fate99. Hoxb1, Hoxb2 or Hoxa2 misexpression can all activate Phox2b ectopically in ventral regions of the hindbrain, but co-electroporation of Hoxb1 or Hoxa2 with Nkx2.2 is required to generate ectopic motor neurons in dorsal regions100. an upstream enhancer region that can be transactivated by Hoxb1, Hoxb2 or Hoxa2 has recently been identified in the Phox2b gene; this region contains conserved Pbx and Meis protein binding sites, and its transcriptional activity can be enhanced by Pbx and Meis cofactors99.

Diffusible cues guide cranial motor axonsThese early genetic programmes specify cranial motor neurons and dictate their responses to local guidance cues in the developing head (TABLe 2). The first pathway choice made by cranial motor axons is whether to project ventrally in small groups into the perinotochordal mes-enchyme (in the case of SM neurons) or dorsally (in the case of BM and VM neurons) through large common exit points. SM axons then innervate eye muscles derived from the prechordal mesoderm and cranial paraxial mesoderm (CPM), or tongue muscles derived from somites 1–4 (ref. 10). VM axons supply parasympathetic ganglia, such as the ciliary, sphenopalatine and otic ganglia, which are neural crest-derived structures101,102. BM axons project

into the branchial arch muscles, along paths that were previously followed by CPM cells; these CPM cells migrate dorsoventrally to form the branchial muscle plates103, which later split up into a characteristic set of muscles104.

Motor axons are repelled from the midline. The initial phase of cranial motor axon extension involves repul-sion of all subtypes of cranial motor axons by the floor plate105,106. Candidates for the mediation of this effect are the axon guidance molecules netrin 1, the Slit pro-teins and SEMa3a, all of which can repel BM and VM axons in vitro107–109. Cranial motor neurons express the uNC5a receptor (which mediates the repellent effect of netrin 1), the Slit receptors ROBO1 and ROBO2, and the SEMa3a receptor neuropilin 1 (refs 109–112). However, only netrin 1 and the Slit proteins are highly expressed in the brainstem floor plate at the time of axon extension109,110,113–115, suggesting that these are the relevant repellents in vivo. In particular, attenuation of Slit–ROBO signalling in chicks or mice in vivo leads to BM and VM axon pathfinding defects, suggesting an in vivo role for the Slit proteins109. a third Robo receptor, ROBO3 (also known as Rig1), has been shown to have a role in midline crossing of some neuronal types116, but might not be expressed in motor neurons.

There is as yet no clear evidence for a role for netrin 1 motor neuron repulsion in vivo, although trochlear motor neuron cell bodies enter the floor plate in netrin 1 mutants, suggesting a loss of repulsion from the mid-line117. Motor axon pathways have not been investigated in Unc5a mutants, but there are peripheral motor axon guidance defects in mutants for a related unc5 family member, Unc5c118,119. Unc5c is also prematurely expressed in FBM neurons that lack Nkx6.1 and fail to migrate85, suggesting that regulation of unc5 family members might be important in aspects of FBM guidance and

Figure 5 |Dorsoventralpatterningofcranialmotorneurons.a | A schematic diagram of a transverse section through the hindbrain, showing pMN and p3 progenitor domains, which give rise to branchiomotor (BM) and visceral motor (VM) neurons, and somatic motor (SM) neurons, respectively. The arrows extending from the floor plate (FP) and the notochord (N) show the presumed diffusion of Sonic hedghog protein (SHH) during motor neuron differentiation. For the pMN and p3 domains, the repertoires of transcription factors that are expressed by the progenitors are listed. b | A schematic diagram of a transverse section through the hindbrain, showing the location of postmitotic cranial motor neurons following dorsal migration by BM and VM neurons. The repertoires of transcription factors that are expressed by the neurons are listed. Lhx, LIM homeobox protein; MNX1, motor neuron and pancreas homeobox 1; Olig2, oligodendrocyte transcription factor 2; Pax6, paired box gene 6; Phox2b, paired-like homeobox 2b; Tbx20, T-box 20.

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migration. In some contexts, unc5a can also bind to the DCC (deleted in colorectal carcinoma) receptor, which normally mediates netrin 1 attraction, to mediate repul-sion120. In the fly embryo, uNC5a and DCC–uNC5a mediate short-range and long-range repulsion of dorsally projecting motor neurons, respectively121,122. a similar possible role of netrin 1 in the rat embryo is implied by the finding that BM and VM neurons express Unc5a during early axon projection but co-express Dcc during later axon extension110; however, this requires functional confirmation.

It is currently unknown what molecules mediate the floor plate repulsion of SM axons110, because only BM and VM axons are repelled by netrin 1 and Slit pro-teins108,109. SM neurons do respond to SEMa3a, which is expressed by the notochord and might have a function in spacing apart the exiting abducens and hypoglossal SM axons108,123 (fIG. 2c). Cranial SM axon exit from the hind-brain depends on the chemokine SDF-1 (also known as

CXCl12), which is expressed in the mesenchyme that underlies the neuroepithelium, at least in the trunk124. The receptor for SDF-1, CXCR4, is expressed by spinal and cranial SM neurons, but not BM or VM neurons, and in CXCR4 mutants, spinal, abducens and hypoglossal SM axons fail to project ventrally and instead extend across the floor plate or project dorsally124. In other neuronal types, SDF-1 has been shown to attenuate responses to repellents, such as Semaphorins or Slits, and so in motor axons, this molecule might attenuate the effects of Semaphorins or of unidentified SM floor plate-derived chemorepellents124,125.

The exit point is a key guidepost. The next step in path-finding is projection to the exit point. For BM and VM axons, exit from the hindbrain appears to require the presence of cranial sensory ganglia, which are apposed to the large dorsal exit points. ablation of these structures leads to a reduction in peripheral axon projections126.

Table 2 | Molecules involved in cranial motor axon guidance

Molecule effectoncranialmotoraxonguidance references

HOXB1 In Hoxb1 mutants, facial branchiomotor neurons project aberrantly to the first branchial arch. When Hoxb1 is ablated in the neural crest only, the facial nerve fails to branch correctly. Ectopic expression of Hoxb1 in r2 converts trigeminal motor neurons into facial motor neurons

52–54,56

HOXA2 In Hoxa2 mutants, trigeminal motor axons misroute into the second branchial arch 46

HOXB2 In Hoxb2 mutants, a subset of trigeminal motor axons misroute into the second branchial arch

48

NKX6.1, NKX6.2 In Nkx6.1 mutants, branchiomotor axons mistarget exit points and pathfind aberrantly

79,85

LHX3, LHX4 Direct cranial motor axons along ventral trajectories 94

PHOX2A In Phox2a mutants, visceral motor axons fail to project due to an absence of ganglionic targets

138

Netrin 1 Repels branchiomotor axons in vitro 107,108

UNC5C In Unc5c mutants, trochlear motor axons project aberrantly 118

Slits Repel branchiomotor axons in vitro. In Slit1;Slit2 double mutants, branchiomotor axons aberrantly enter the midline

109

Robos In Robo1 and Robo2 mutants, or in chick embryos that express dominant-negative Robo receptors, branchiomotor axons enter the midline and fail to reach exit points

109

SEMA3A Repels branchiomotor, visceral motor and somatic motor axons in vitro. Sema3A-mutant mice show defasciculation of cranial motor nerves

108,134

Neuropilin 1 Neuropilin 1 mutants show defasciculation of cranial motor nerves 135

SEMA3F SEMA3F repels trochlear motor axons in vitro or in the chick embryo in vivo. In Sema3F mutants, the trochlear nerve fails to exit and the oculomotor nerve is defasciculated

149,151,152

Neuropilin 2 In Neuropilin 2 mutants, the trochlear nerve fails to exit and the oculomotor nerve is defasciculated

149,150

FGF8 FGF8 attracts trochlear axons, causing them to exit the brain 148

Ephrin As Overexpression of ephrin As in branchial muscle causes trigeminal motor axon branching defects

143

EPHAs Trigeminal motor neurons that express dominant-negative EPHAs show axon branching defects

143

SDF-1/CXCL12 In SDF‑1 mutants, abducens and hypoglossal axons project dorsally rather than ventrally

124

CXCR4 The phenotype of CXCR4 mutants is identical to that of SDF‑1 mutants 124

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Rhombic lipThe structure at the dorsal extreme of the hindbrain.

The most proximal cells of the ganglion are specialized boundary cap cells which, in chicks, arise from a late-emigrating population of cadherin-7-expressing neural crest cells127,128. Transplantation experiments in chick embryos demonstrated that, when an odd-numbered rhombomere is rotated 180o, most BM axons reorien-tate to navigate rostrally to their correct exit points129. However, evidence for an exit point-derived chemoat-tractant has not been forthcoming, and the alar plate itself is chemorepellent130, possibly due to a narrow stripe of Slit2 expression at the rhombic lip109. Thus, BM and VM axon tracts might be hemmed in by floor plate repellents medially and alar plate repellents dorsally. The sensory ganglia exert only a weak chemoattract-ant influence on BM axons in vitro130, and the role of boundary cap cells has yet to be explored, but this role is likely to be pivotal, judging by the intriguing finding that these cells limit spinal motor neuron emigration from the neural tube131.

A balance of attraction and repulsion in the periphery. Cranial motor axon behaviour in the periphery depends on a balance of positive and negative influences. The perinotochordal mesenchyme, which is derived from the neural crest and differentiates into cartilage, repels spinal motor axons132. although this role has not been directly demonstrated for cranial SM axons, motor axons avoid the forming cartilage of the branchial arches, and the parachordal cartilages, by channelling into the develop-ing muscle plate103. Branchial arch neural crest cells also express Semaphorins, including SEMa3a, and in mouse mutants that lack Sema3A or neuropilin 1, cranial motor axons become defasciculated and invade inappropriate regions of the periphery133–135. The oculomotor nerve is normal in Sema3A mutants, consistent with the observa-tion that oculomotor axons are not repelled by SEMa3a in vitro108,134. Skeletogenic cells that express Semaphorins therefore influence peripheral axon pathways, and might interact with growing nerves to influence the position-ing of skull foramina, which are the mature pathways of the cranial nerves. Secreted Semaphorins, especially SEMa3a and SEMa3C, are expressed in particular subsets of cranial motor neurons, where their function remains to be explored111,112.

Chemoattraction by the branchial arches can also strongly orient the trajectories of BM and VM axons130. Hepatocyte growth factor (HGF) is expressed by the branchial muscle plate at the time of axon extension and accounts for most of this activity, as HGF presented on beads is chemoattractant, and HGF-blocking antibodies can eliminate a large portion of the arch-mediated che-moattraction. However, in HGF-mutant mice, BM and VM axon trajectories are normal, and only SM hypoglos-sal pathways are affected, suggesting that there are other branchial arch-derived chemoattractants130. Indeed, at least three other neurotrophic factors are capable of promoting cranial motor neuron outgrowth before they become crucial factors for survival136. Brain-derived neurotrophic factor (BDNF) has a strong effect on motor axon outgrowth136, is expressed in the branchial arches during early axon pathfinding, and has been shown to

chemoattract trigeminal sensory axons137. Therefore, other neurotrophic factors are likely candidates in BM and VM axon guidance.

The divergence of VM axons from their BM neigh-bours, for example, in the facial nerve, points to the existence of distinct VM guidance cues. Indeed, r5-derived VM axons navigate to their parasympathetic ganglion targets by following chemoattractant signals; genetic removal of the sphenopalatine ganglion in Phox2a mutants results in the specific loss of the relevant branch of the facial nerve138. The relevant chemoattract-ant molecules have not been identified, but the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors, which is implicated in the guid-ance and arborization of autonomic neurons, might be involved139.

Motor pools and their target muscleslittle is known about how cranial motor axons recog-nize their target structures at appropriate axial levels. For the spinal motor axon–limb system it has been pro-posed that major guidance information comes from the lateral plate mesenchyme140. In the head, embryological evidence strongly suggests that the neural crest from a particular axial level patterns muscles, the myogenic progenitors of which migrate from the same level102. Fate mapping has shown that neural crest cells form connective tissue sheaths and skeletal attachment points around muscles derived from the same axial level42. The neural crest might thus provide specific guidance infor-mation for BM axons and/or induce branchial muscles to produce such guidance signals. However, the nature of these guidance signals is currently unknown.

Following the rostrocaudal inversion of an odd-numbered rhombomere, trigeminal BM axons that project to incorrect target muscles are eliminated, sug-gesting a specific recognition between BM neurons and their targets141. Studies in fish and chicks suggest that r2- and r3-derived regions of the trigeminal nucleus contribute to separate subnuclei with different synaptic targets142,143. Rhombomere 2- and r3-derived trigeminal motor neurons express high and low levels of ephrin a receptors, respectively, whereas r3 target muscle showed patterned ephrin a expression143,144. Expression of dominant-negative ephrin a receptors in r3 trigemi-nal motor neurons, or overexpression of ephrin as in their target, led to aberrant axon branching patterns, suggesting a role for ephrins in this topographic axon targeting143.

Extraocular muscle innervation The six eye muscles that rotate the eyeball and pro-vide fine control of visual tracking movements are innervated by three nerves. This is a promising sys-tem in which to study the mechanism that controls nerve–muscle targeting, as well as a model that is of clinical relevance to humans. The abducens nerve innervates the lateral rectus muscle, the trochlear nerve innervates the superior oblique (the dorsal oblique in chicks) and the oculomotor nerve inner-vates the remaining four muscles, the medial, dorsal

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Congenital fibromatosis of the extraocular muscles(CfeOM). A group of congenital syndromes that involve cranial nerve miswiring and paralysis or paresis of the extraocular muscles, often associated with drooping of the upper eyelid.

Duane syndromeA congenital eye movement disorder characterized by impeded horizontal eye movements that resut from miswiring of the eye muscles.

Horizontal gaze palsy with progressive scoliosis(HGPPs). A rare congenital syndrome that is characterized by the absence of conjugate horizontal eye movements and by deformities in the spine.

Möbius syndromeA rare congenital disorder caused by abnormal development of the cranial nerves, which results in paresis or paralysis of the facial muscles and, in some cases, other abnormalities.

Marcus Gunn syndromeAlso known as jaw-winking syndrome. It consists of an elevation or depression of the eyelid on chewing and/or suckling, and is thought to be caused by aberrant innervation of branches of the trigeminal and oculomotor nerves.

and ventral recti, and the inferior oblique. In chicks, the lateral rectus primordium differentiates preco-ciously relative to the other extraocular muscles, in a position ventral to r2 and r3 (ref. 145). From embry-onic day three onwards, the abducens nerve extends rostrally from its origin in r5 and r6, contacting the lateral rectus primordium by embryonic day four146. a double immunohistochemical study contrasted the early abducens outgrowth with the delayed outgrowth of the oculomotor nerve, which contacts its first and most distant muscle target, the ventral oblique (VO), by embryonic day five147. after the oculomotor nerve reaches the VO, branches appear along the oculomotor nerve to its other targets (the medial, dorsal and ventral recti). It is not known whether this occurs by interstitial branching of pre-existing axons or by de novo growth of neurons. However, FGF8 and the Semaphorins have been shown to function in trochlear and oculomotor initial projections. The dorsal exit of trochlear axons from the neuroepithelium depends on attraction by the midbrain–hindbrain boundary and FGF8 (ref. 148). SEMa3F is expressed rostral and caudal to the midbrain–hindbrain boundary and acts through its receptor neuropilin 2 to guide the exit and initial trajectory of trochlear and oculomotor nerves in both mice and chicks149–152. SEMa3F repels trochlear axons in vitro, and in neuropilin-2- or Sema3F-mutant mice, trochlear axons fail to exit the brain correctly and the oculomotor nerve is defasciculated. The role of SEMa3F in extraocular innervation thus provides an interesting counterpart to the role of SEMa3a in guiding the branchiomotor nerves. Guidance cues for later growth and branching and for targeting of the nerves III, IV and VI to specific extraocular muscles remain to be characterized.

Dysinnervation disorders in humansIt will be vital to unravel in more detail the principles that underlie extraocular muscle wiring, in order to understand a group of human syndromes termed cranial dysinnervation disorders (CDDs6,153). These disorders are characterized most notably by deficits of horizontal eye movements (complex strabismus), and include defects such as congenital fibromatosis of the extraocular muscles (CfeOM), Duane syndrome, horizontal gaze palsy with progressive scoliosis (HGPPs), Möbius syndrome and Marcus Gunn syndrome. In some cases these disorders are congenital, and it has been proposed that they are caused primarily by defects in cranial motor neuron development and axon naviga-tion. Strabismus (squinting) often results from an imbalance in the function of the lateral (innervated by the abducens) and medial (innervated by oculomotor) recti muscles, which respectively abduct and adduct the eyeball. Familial studies, including linkage screens, and mapping to candidate genetic loci have implicated five different genes in five separate syndromes. These are HOXA1, PHOX2A, SALL4, KIF21A and ROBO3 (also known as RIG1)154–158. Three of these genes – HOXA1, PHOX2A and SALL4 – are transcription factors, one is involved in axonal transport (KIF21A)

and one is an axon guidance molecule (ROBO3), supporting the notion of a developmental basis for congenital CDDs.

Mutations in HOXA1 have been shown to result in two overlapping syndromes, Bosley-Salih-alorainy syn-drome (BSaS) and athabascan brainstem dysgenesis syndrome (aBDS)154. These disorders are character-ized by impaired horizontal eye movements, ear and vascular defects and, in some cases, autism or mental retardation. In Hoxa1-knockout mice, the abducens nerve is lacking50, and so it is likely that abducens devel-opment and consequent innervation of the lateral rectus muscle is aberrant in BSaS and aBDS patients. Clinical characterization of a loss of ocular motility, especially abduction (which is mediated by the abducens nerve), broadly agrees with this interpretation159. another congenital CDD, Duane syndrome type 2 (DuRS2), is also characterized by an absence or reduction of the abducens nerve, whereas in some cases the oculomo-tor nerve branches aberrantly into the lateral rectus muscle160,161. This innervation pattern bears a striking resemblance to the pattern that is seen in Hoxa3- and Hoxb3-mutant mice, which lack the abducens nerve and manifest aberrant innervation of the lateral rectus through another nerve of uncertain origin71. However, there is no published genetic evidence to link Hox3 or other genes with DuRS2.

CFEOM2, a CFEOM variant which involves fibro-sis of the extraocular muscles and severe loss of ocular motility, has been linked to mutations in PHOX2A155. Neuroimaging data show that patients lack the ocu-lomotor and trochlear nerves162, a phenotype that is identical to that of Phox2a-mouse mutants19. This suggests that the primary defect is neural, and that the extraocular muscle atrophy that occurs in CFEOM might occur secondarily to the lack of innervation. Similarly, patients with CFEOM1 were found to have hypoplasia of the oculomotor nerve, and in some cases of the abducens nerve163. CFEOM1 has been shown to result from mutations in the kinesin motor protein KIF21a156, which has a role in anterograde axonal transport164.

The rare syndrome HGPPS involves the absence of the conjugate horizontal eye movements that are medi-ated by the abducens and oculomotor nerves, as well as scoliosis, and is caused by mutations in ROBO3 (ref. 158). ROBO3 is required for axons to cross the midline and form commissures116, which are absent in the brainstems of HGPPS patients. It is likely that horizontal eye move-ment disturbances result from the failure of supranuclear tracts, such as the paramedian pontine reticular forma-tion, to cross the midline and innervate the abducens and oculomotor nuclei158.

a combination of clinical genetics and developmen-tal neurobiology might in future further illuminate the causes of CDDs in humans. In some CDDs, such as Möbius syndrome, an association with autism has been proposed165, suggesting that disorders of cranial motor neuron development might have far-reaching significance for understanding human disorders of brain wiring.

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Future prospects and challengesGreat progress been made in understanding how rostro-caudal and dorsoventral patterning processes regulate cranial motor neuron specification. However, there is a sizeable gap in our knowledge that concerns how combi-nations of transcription factors govern the expression of axon guidance receptors and motor axon pathway deci-sions. There are few characterized head-specific guid-ance cues, which stems from the lack of basic knowledge about tissue patterning in the head. Glial cells are likely to have a key role in cranial motor axon guidance, but the idea that they might be molecularly heterogeneous has not been addressed. Similarly, the specific charac-ter of the extraocular versus the tongue and branchial

arch muscles must be elucidated; recent studies point to differences in the molecular programme of differen-tiation in subpopulations of head mesoderm cells166,167. It is also currently unknown which combinations of neurotrophic factors maintain the survival of different cranial subpopulations; such factors might also have a role in axon guidance and branching. understanding the molecular hierarchy of development in motor neu-rons is an exciting and compelling problem, with many ramifications for human health. we need to elucidate the specific genetic hierarchies, signalling cascades and pathfinding strategies of the cranial nerves in order to address clinical problems, such as cranial nerve palsies, and pan-motor neuron diseases.

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49. Carpenter, E. M., Goddard, J. M., Chisaka, O., Manley, N. R. & Capecchi, M. R. Loss of Hox‑A1 (Hox‑1.6) function results in the reorganization of the murine hindbrain. Development 118, 1063–1075 (1993).

50. Mark, M. et al. Two rhombomeres are altered in Hoxa‑1 mutant mice. Development 119, 319–338 (1993).

51. Barrow, J. R., Stadler, H. S. & Capecchi, M. R. Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse. Development 127, 933–944 (2000).

52. Goddard, J. M., Rossel, M., Manley, N. R. & Capecchi, M. R. Mice with targeted disruption of Hoxb‑1 fail to form the motor nucleus of the VIIth nerve. Development 122, 3217–3228 (1996).

53. Studer, M., Lumsden, A., Ariza-McNaughton, L., Bradley, A. & Krumlauf, R. Altered segmental identity and abnormal migration of motor neurons in mice lacking Hoxb‑1. Nature 384, 630–634 (1996).This paper, together with reference 52, shows that facial branchiomotor (FBM) neurons in Hoxb1-mutant mice fail to differente and migrate and eventually die.

54. Bell, E., Wingate, R. J. & Lumsden, A. Homeotic transformation of rhombomere identity after localized Hoxb1 misexpression. Science 284, 2168–2171 (1999).This paper elegantly demonstrates that misexpression of Hoxb1 in presumptive trigeminal motor neurons converts them to an FBM fate.

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55. Arenkiel, B. R., Gaufo, G. O. & Capecchi, M. R. Hoxb1 neural crest preferentially form glia of the PNS. Dev. Dyn. 227, 379–386 (2003).

56. Arenkiel, B. R., Tvrdik, P., Gaufo, G. O. & Capecchi, M. R. Hoxb1 functions in both motoneurons and in tissues of the periphery to establish and maintain the proper neuronal circuitry. Genes Dev. 18, 1539–1552 (2004).This intriguing paper uses conditional deletion of Hoxb1 in the neural crest to show that r4 neural-crest-derived Schwann cells have a role in the maintenance and branching of FBM neurons.

57. Gavalas, A., Ruhrberg, C., Livet, J., Henderson, C. E. & Krumlauf, R. Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes. Development 130, 5663–5679 (2003).

58. Gavalas, A. et al. Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch. Development 125, 1123–1136 (1998).

59. Rossel, M. & Capecchi, M. R. Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development. Development 126, 5027–5040 (1999).

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61. Tumpel, S. et al. Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1. Dev. Biol. 302, 646–660 (2007).

62. Pata, I. et al. The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4. Development 126, 5523–5531 (1999).

63. Song, M. R. et al. T-box transcription factor Tbx20 regulates a genetic program for cranial motor neuron cell body migration. Development 133, 4945–4955 (2006).

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66. McClintock, J. M., Kheirbek, M. A. & Prince, V. E. Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention. Development 129, 2339–2354 (2002).

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76. Briscoe, J., Pierani, A., Jessell, T. M. & Ericson, J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435–445 (2000).This important study shows that progenitor domains along the dorsoventral axis of the spinal cord that express different combinations of homeodomain proteins are of key importance in

generating different neuronal types in response to SHH signalling. This model provides a conceptual framework for understanding motor neuron differentiation in the hindbrain.

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80. Ericson, J. et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90, 169–180 (1997).

81. Briscoe, J. et al. Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398, 622–627 (1999).

82. Sander, M. et al. Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates. Genes Dev. 14, 2134–2139 (2000).

83. Vallstedt, A. et al. Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31, 743–755 (2001).

84. Pabst, O., Rummelies, J., Winter, B. & Arnold, H. H. Targeted disruption of the homeobox gene Nkx2.9 reveals a role in development of the spinal accessory nerve. Development 130, 1193–1202 (2003).

85. Muller, M., Jabs, N., Lorke, D. E., Fritzsch, B. & Sander, M. Nkx6.1 controls migration and axon pathfinding of cranial branchio-motoneurons. Development 130, 5815–5826 (2003).

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97. Pattyn, A., Hirsch, M., Goridis, C. & Brunet, J. F. Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b. Development 127, 1349–1358 (2000).This paper uses knockout mice to show that PHOX2B has an important role in controlling the differentiation of BM and VM neuronal types in the hindbrain. In these mice, the neurons fail to differentiate and subsequently die.

98. Coppola, E., Pattyn, A., Guthrie, S. C., Goridis, C. & Studer, M. Reciprocal gene replacements reveal unique functions for Phox2 genes during neural differentiation. EMBO J. 24, 4392–4403 (2005).

99. Pattyn, A. et al. Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev. 17, 729–737 (2003).

This important paper shows the sequential generation of BM, VM and serotonergic neurons from the V3 domain in the hindbrain, and the genetic control mechanisms that control this process at different axial levels.

100. Samad, O. A. et al. Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins. Development 131, 4071–4083 (2004).This is an important paper in which the genes that regulate the patterning of the dorsoventral and rostrocaudal axes are found to converge in regulating PHOX2B expression and BM and VM neuron differentiation in r4.

101. Noden, D. M. Interactions and fates of avian craniofacial mesenchyme. Development 103 (Suppl.) 121–140 (1988).

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107. Colamarino, S. A. & Tessier-Lavigne, M. The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Cell 81, 621–629 (1995).

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109. Hammond, R. et al. Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain. Development 132, 4483–4495 (2005).This study uses a combination of electroporation in chicks, in vitro assays and, importantly, in vivo evidence, to demonstrate a role for Slit–ROBO signalling in the repulsion of BM and VM axons away from the midline and towards their exit points.

110. Barrett, C. & Guthrie, S. Expression patterns of the netrin receptor UNC5H1 among developing motor neurons in the embryonic rat hindbrain. Mech. Dev. 106, 163–166 (2001).

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114. Kennedy, T. E., Wang, H., Marshall, W. & Tessier-Lavigne, M. Axon guidance by diffusible chemoattractants: a gradient of netrin protein in the developing spinal cord. J. Neurosci. 26, 8866–8874 (2006).

115. Brose, K. et al. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795–806 (1999).

116. Sabatier, C. et al. The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117, 157–169 (2004).

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118. Burgess, R. W., Jucius, T. J. & Ackerman, S. L. Motor axon guidance of the mammalian trochlear and phrenic nerves: dependence on the netrin receptor Unc5c and modifier loci. J. Neurosci. 26, 5756–5766 (2006).

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AcknowledgementsThanks to A. Lumsden and R. Knight for critical comments on the manuscript.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBDNF | cadherin-7 | CXCR4 | CYP26 | DCC | EGR2 | FGF8 | Gata2 | Gata3 | HGF | Hoxa1 | HOXA1 | Hoxa2 | Hoxa3 | Hoxb1 | hoxb1a | Hoxb2 | Hoxb3 | Hoxd3 | Isl1 | Isl2 | KIF21A | Lhx3 | Lhx4 | MAFB | MNR2 | MNX1 | netrin 1 | neuropilin 1 | neuropilin 2 | Nkx2.2 | Nkx2.9 | Nkx6.1 | Nkx6.2 | Olig2 | Pax6 | pbx4 | Phox2a | PHOX2A | ROBO1 | ROBO2 | ROBO3 | ROBO3 | SALL4 | SDF-1 | SEMA3A | SEMA3C | SEMA3F | Slit2 | SHH | Tbx20 | UNC5A | Unc5c

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