Development, functional organization, and evolution of vertebrate axial motor circuits

Development, functional organization, and evolution of vertebrate axial motor circuits Neuronal control of muscles associated with the central body axis is an ancient and essential function of the nervous systems of most animal species. Throughout the course of vertebrate evolution, motor circuits dedicated to control of axial muscle have undergone significant changes in their roles within the motor system. In most fish species, axial circuits are critical for coordinating muscle activation sequences essential for locomotion and play important roles in postural correction. In tetrapods, axial circuits have evolved unique functions essential to terrestrial life, including maintaining spinal alignment and breathing. Despite the diverse roles of axial neural circuits in motor behaviors, the genetic programs underlying their assembly are poorly understood. In this review, we describe recent studies that have shed light on the development of axial motor circuits and compare and contrast the strategies used to wire these neural networks in aquatic and terrestrial vertebrate species. Keywords: Motor neuron, Spinal circuit, Neural circuit, Development, Evolution, Axial muscle Background invasion of land by vertebrates, axial muscles that were The neuromuscular system of axial skeleton plays crucial initially used in swimming were also adapted by the re- roles in basic motor functions essential to vertebrates, spiratory system to enable breathing in air. Since many of including locomotion, breathing, posture and balance. these diverse axial muscle-driven motor behaviors are While significant progress has been made in deciphering encoded by neural circuits assembled during develop- the wiring and function of neural circuits governing limb ment, insights into the evolution of axial circuits might control [1, 2], the neural circuits associated with axial emerge through comparisons of the genetic programs that muscles have been relatively under studied, particularly control neural circuit assembly in different animal species. in mammals. Despite comprising more than half of all In this review, we discuss studies which have investi- skeletal muscles in mammals, how axial neural circuits gated the development, evolution, and wiring of neuronal are assembled during development is poorly understood. circuits essential for control of axial muscle. Recent ad- Although all vertebrates share similar types of axial vances in genetically tractable systems, such as zebrafish muscle [3, 4], the nervous systems of aquatic and terres- and mouse, have provided novel insights into the mecha- trial species control these muscle groups in distinct ways. nisms through which axial circuits are assembled during In most aquatic vertebrates, rhythmic contraction of axial development, and have shed light on the wiring of the cir- muscle is essential for generating propulsive force during cuits essential for balance, breathing, and locomotion. We swimming, the predominant form of locomotion used by compare the strategies through which animals generate fish. In land vertebrates, axial circuits have been largely distinct classes of spinal neurons that coordinate axial dissociated from locomotor functions, and have been muscles, with particular focus on the spinal motor neuron modified throughout evolution to enable new types of subtypes that facilitate axial-driven motor behaviors. motor capabilities. In animals with upright postures, neur- onal control of axial muscles is essential to maintain bal- Functional organization and peripheral ance and proper alignment of the spine. During the connectivity of axial motor neurons Although used for fundamentally distinct motor functions, * Correspondence: jeremy.dasen@nyumc.org the axial neuromuscular systems of fish and tetrapods Neuroscience Institute, Department of Neuroscience and Physiology, NYU School of Medicine, New York, NY 10016, USA share many anatomic features and early developmental © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. D’Elia and Dasen Neural Development (2018) 13:10 Page 2 of 12 programs [3, 4]. In both fish and tetrapods, axial muscles lost. Both types of axial muscles receive innervation from can be broadly divided into two groups, epaxial and spinal motor neurons (MNs) and sensory neurons that hypaxial, which are initially separated by a horizontal project either along the dorsal (epaxial) or ventral (hypax- myoseptum (Fig. 1a). Epaxial muscles reside dorsal to the ial) branches of the spinal nerves. myoseptum and include muscle groups associated with In tetrapods, MNs targeting specific muscle groups are the vertebral column and base of the skull. Hypaxial mus- organized in discrete clusters, termed motor columns and cles are predominantly located ventral to the mysoseptum motor pools [5–8]. Spinal MNs projecting to functionally and give rise to diverse muscle groups including abdom- related muscle groups, such as epaxial, hypaxial, or limb inal and intercostal muscles, as well as the diaphragm in muscle, are contained within motor columns that occupy mammals. In tetrapods, migratory populations of hypaxial specific rostrocaudal positions within the spinal cord. muscle also generate all of the muscle in the limb. In fish Within these columnar groups, MNs further segregate and amphibians, the separation between dorsal and ven- into motor pools, each pool targeting a single muscle. tral axial muscles is maintained in adulthood, while in tet- Each pool occupies a specific position within the spinal rapods many of these positional differences have been cord, and its relative position along the dorsoventral, ab cd Fig. 1 Organization of axial MNs in tetrapods and fish. a In jawed vertebrates, axial muscles are separated into dorsal epaxial and ventral hypaxial groups, separated by the horizontal myoseptum (HM). Each muscle group is innervated by separate spinal nerves. Dorsal root ganglia (drg) and sympathetic chain ganglia (scg) are shown. b MN columnar subtypes at trunk levels. In tetrapods, as well as some cartilaginous fish, MNs innervating dorsal epaxial muscles are organized in the medial motor column (MMC). MNs projecting to ventral hypaxial muscles are contained within the hypaxial motor column (HMC). Autonomic preganglionic column (PGC) neurons, which project to scg, are shown in gray. c Organization of MN pools at thoracic levels. MNs innervating specific types of axial muscle are organized in pool-like clusters. Some MNs within the HMC project to dorsally located axial muscles, such as serratus, but are nevertheless supplied by axons originating from the ventral ramus. Abbreviations: tv, transversospinalis; long, longissimus; ilio, iliocostalis; lc, levator costae; sr, caudal serratus; ii, internal intercostal; sc, subcostalis; ei, external intercostal; eo, external oblique. Not all trunk muscles are shown. Diagram based on data from rat in [13]. d Organization of MNs in adult zebrafish. MNs innervating fast, intermediate, and slow muscle are organized along the dorsoventral axis. Fast MNs include primary MNs and some secondary MNs, intermediate and slow are all secondary MNs. These MN types project to specific types of trunk-level axial muscles. Diagram based on data in [14] D’Elia and Dasen Neural Development (2018) 13:10 Page 3 of 12 mediolateral, and rostrocaudal axes is linked to how MNs differences that emerged between certain fish species and project within a target region. The stereotypic organization other vertebrate classes. of MN position within the spinal cord therefore establishes a central topographic map which relates neuronal settling Genetic programs specifying early axial motor position to target specificity. neuron fates Studies on the developmental mechanisms controlling How are the distinct identities of MMC and HMC neu- MN columnar and pool organization have largely focused rons established during tetrapod development? As with on the diverse subtypes innervating limb muscles [9, 10]. other subtypes of spinal MNs, the progenitors that give Axial MNs also display a topographic organization that re- rise to axial MNs are specified through secreted signal- lates neuronal position to target specificity. The cell bodies ing molecules acting along the dorsoventral axis of the of MNs targeting epaxial and hypaxial muscles are orga- neural tube shortly after its closure [18]. These morpho- nized in specific columnar groups within the ventral spinal gens establish specific molecular identities through the cord (Fig. 1b). Dorsal epaxial muscles are innervated by induction of transcription factors in neuronal progenitors, MNs in the median motor column (MMC), while hypaxial whichsubsequentlyspecifytheidentityofeach ofthemajor musclesare innervated by MNsinthe hypaxial motorcol- classes of spinal neuron. In the ventral spinal cord, graded umn (HMC). MMC neurons occupy the most medial pos- Shh signaling induces expression of transcription factors ition of all spinal MNs, whereas HMC neurons, and all which specify MN and ventral interneuron progenitor iden- other MN subtypes, typically reside more laterally [11]. Like tities [19]. As progenitors differentiate, additional transcrip- limb MNs, both MMC and HMC neurons further differen- tion factors are expressed within postmitotic cells and act tiate into specific pool groups, and axial MN pool position to define specific neuronal class fates [20]. Spinal MN pro- is linked to the location of its muscle target (Fig. 1c). For genitors are derived from a domain characterized by ex- example, MMC neurons targeting more dorsal epaxial pression of Olig2, Nkx6.1, and Pax6. As postmitotic MNs muscles reside more medially than those targeting more emerge, they initially express the Lim homeodomain pro- ventral muscle [12]. A similar somatotopic organization has teins Islet1, Islet2 (Isl1/2), Lhx3, Lhx4 (Lhx3/4), as well as been observed for HMC pools targeting different intercos- the Mnx-class protein Hb9 (Fig. 2a). tal and abdominal muscles [13]. As MNs differentiate and migrate to their final settling In contrast to tetrapods, the organization of axial MNs positions, subtypes of axial MNs can defined by differen- into well-defined columnar groups has not been de- tial expression of Lim HD and Mnx factors [11, 21]. In scribed in zebrafish. Despite the absence of an obvious tetrapods, MMC neurons maintain expression of Hb9, columnar organization, zebrafish axial MNs are func- Isl1/2, and Lhx3/4, whereas the majority of other MN tionally organized along the dorsoventral axis of the subtypes, including HMC neurons, downregulate Lhx3 spinal cord (Fig. 1d). This organization is associated as they become postmitotic (Fig. 2b). The specific func- with how MNs are recruited at distinct swimming tions of Lhx3 and Lhx4 in MMC neurons are not com- speeds and correlated with the type of muscle a MN pletely understood, as both genes are required for the innervates, as opposed to the location of the muscle. differentiation of all spinal MN subtypes [22]. Neverthe- Axial MNs projecting to muscles activated at slow less, misexpression of Lhx3 can convert limb MNs to an swimming speeds reside ventrally, MNs recruited at MMC fate and redirect motor axons towards axial fast swimming speeds are located dorsally, and MNs muscle, indicating that Lhx3 plays an instructive role in involved in intermediate speeds sit between fast and determining the trajectories of MMC motor axons to- slow MNs [14–16]. wards epaxial muscle [23]. While trunk-level HMC neu- Although a clustered organization of axial MN has not rons can be also defined by expression of specific been described in zebrafish, in certain cartilaginous fish transcription factor combinations, whether these factors species, including the little skate and catshark, the cell bod- are required for columnar-specific differentiation pro- ies of MMC neurons are clustered and settle in a ventral grams is currently unknown. position [17]. These observations suggest that the A key step in the specification of axially-projecting MNs organization of axial MNs into columns was present in the is the segregation of newly born neurons into MMC and common ancestor to cartilaginous fish and tetrapods, and HMC subtypes. MMC neurons are thought to represent therefore to all jawed vertebrates with paired appendages. the ancestral “groundstate” of MNs from which all other Notably, unlike most fish species, skates do not use the subtypes subsequently evolved [24]. This idea is supported axial muscles to generate propulsive force during locomo- by the observation that MMC identity is the default differ- tion, which is instead provided by contraction of the pec- entiation state of MNs derived from embryonic stem cells toral and pelvic fins. The organization of MNs into (ESCs) generated through induction with retinoic-acid columnar and pool groups therefore does not appear to and Shh [25, 26]. In addition, MMC-like neurons drive have evolved with terrestrial locomotion, but rather reflects locomotor behaviors in limbless vertebrates such as the D’Elia and Dasen Neural Development (2018) 13:10 Page 4 of 12 bc Fig. 2 Specification of axial MNs in tetrapods and fish. a Specification of early axial MN identities. Graded sonic hedgehog (Shh) acts along the dorso (d)-ventral (v) axis to specify MN progenitors (pMN) and ventral interneuron fates. Graded Wnt signaling promotes sustained expression of Lhx3 in MMC neurons, while Hox signaling specifies segmentally-restricted MN columnar fates, including limb-innervating lateral motor column (LMC) neurons. b Axial MNs in tetrapods can be defined by expression of specific transcription factors. MMC neurons express Fgr1 and are attracted to mesodermally-derived FGF signaling. c Primary MNs in zebrafish. Four distinct axial MN types can be defined by their rostrocaudal position and muscle target specificity. dRoP, dorsal rostral primary; vRoP, ventral rostral primary; CaP, caudal primary; MiP, middle primary MN lamprey and insect larvae, suggesting that an MMC-like the cell cycle, the axons of MMC and HMC neurons MN population represents the ancestral condition of MNs begin to project outside the spinal cord, both initially in bilaterians. pursuing ventrolateral trajectories. The axons of MMC In tetrapods, an obligate step in MMC differentiation neurons separate from the main nerve and extend dor- is the sustained expression of Lhx3/4 in post-mitotic sally, while all other MN subtypes, including HMC neu- MNs; while in HMC neurons and all other MN subtypes rons, continue to extend ventrolaterally. The dorsal Lhx3/4 must be downregulated for proper differentiation trajectory of MMC neurons appears to rely on target- [21, 23]. The maintenance of Lhx3/4 in MMC neurons derived chemoattractant signaling emanating from a appears to be partially governed by Wnt signaling origin- somite-derived structure, the dermomyotome [29, 30]. ating from near the floorplate of the spinal cord (Fig. 2a) This region expresses fibroblast growth factors (FGFs) [27]. Overexpression of Wnt4 or Wnt5a promotes the which act on the axons of MMC neurons that selectively specification of MMC neurons at the expense of other express FGF receptor 1 (Fgfr1) (Fig. 2b)[31]. Mutation of MN subtypes in chick embryos, while combined genetic Ffgr1 in mice causes defects in the peripheral trajectory of removal of Wnt4, Wnt5a,and Wnt5b in mice leads to de- MMC axons. In addition, misexpression of Lhx3 leads to pletion in MMC number. Recent studies in ES cell- ectopic expression of Fgfr1 in non-MMC MNs and causes derived MNs suggest that additional signaling pathways limb motor axons to gain sensitivity to FGFs [31]. act in conjunction with Wnt signaling to promote MMC specification [28]. Inhibition of Notch signaling in ES-cell Specification of axial MNs in zebrafish derived MNs promotes the specification of HMC neurons In zebrafish, spinal MNs innervating axial muscle are spe- at the expense of MMC neurons, suggesting that Wnt4/5 cified by the same core groups of transcription factors that and Notch cooperate to specify MMC identity. act in tetrapods. Unlike amniotes, where all MNs are gen- While the extrinsic and intrinsic factors governing the erated during a single wave of neurogenesis, zebrafish specification of MMC and HMC neurons have been have two waves of MN birth, primary and secondary. Pri- characterized, the downstream effectors of their fate de- mary and secondary neurons are each important for dif- terminants are less well-understood. Soon after leaving ferent types of axial muscle-based behaviors, but not D’Elia and Dasen Neural Development (2018) 13:10 Page 5 of 12 distinguished by any known transcription factor [32, 33]. reflect the operation of MN-intrinsic genetic programs Primary MNs, which number three to four per hemi- acting during development. segment, are born between 10 and 14 hours post- fertilization (hpf), develop subtype-specific electrical Diversification of tetrapod axial motor columns membrane properties as early as 17 hpf, and begin axon While axial MNs of fish and mammals share several initiation at 17 hpf [34, 35]. Although one or two common common early developmental programs, in tetrapods MN markers such as Isl1, Isl2, and Mnx proteins can help these subtypes have undergone a significant degree of differentiate two or three primary MN subtypes at differ- modification throughout the course of vertebrate evolu- ent ages, these factors cannot distinguish them through- tion. All of the segmentally restricted subtypes of spinal out development and have dynamic expression patterns MNs, including the diverse MN populations innervating that make the subtypes challenging to track over time limb muscle, appear to have evolved from the ventrally- [36–38]. All early-born MNs require the Olig2 transcrip- projecting HMC-like population. This hypothesis is sup- tion factor [39], while Nkx6 proteins appear to be required ported by the observation that in genetic mutants with only in a subset of primary MNs [40]. Postmitotic primary disrupted specification of non-axial MN subtypes, af- MNs can be defined by differential expression of Mnx/ fected populations revert to a predominantly HMC-like Hb9, Isl1/2, and Lhx3 factors [37, 38, 41–43]. molecular profile. Genetic deletion of the limb MN fate Most genetic studies of axial MN specification in zeb- determinant Foxp1 in mice causes a loss of limb-specific rafish have largely focused on the specification of the MN programs and an expansion in the number of MNs four major types of primary MNs: the dorsal rostral pri- with an HMC-like molecular identity [21, 46]. Expres- mary (dRoP), ventral rostral primary (vRoP), caudal pri- sion of Foxp1 in limb-innervating lateral motor column mary (CaP), and middle primary (MiP) subtypes (Fig. (LMC) neurons is governed by Hox transcription factors 2c). dRoP and MiP MNs are similar to MMC neurons, expressed at specific rostrocaudal levels of the spinal in that they project to muscles located dorsal to the cord, and Hox genes are essential for generating the di- horizontal myoseptum, while CaP and vRoP project ven- verse motor pool populations targeting specific limb trally. However, unlike MMC and HMC neurons in tet- muscles [47–49]. MMC neurons appear to be insensitive rapods, these primary MN types cannot be distinguished to the activities of Hox proteins, likely due to the func- by differential expression of Lhx3. Nevertheless, disrup- tionally dominant actions of Lhx3 [21, 23]. The diversifi- tion of the core MN determinants Lhx3/4, Isl1/2, and cation of tetrapod spinal MNs appears to stem from Mnx leads to defects in primary MN specification and HMC-like precursors which co-opted Hox genes to gen- connectivity. For example, loss of Lhx3/4 leads to MNs erate more specialized populations. with hybrid MN/interneuron fates [41], while loss of Hox-dependent regulatory programs also contributed Mnx proteins affects the specification of MiP MNs [38]. to the diversification of MNs targeting specific hypaxial While much is known about primary axial MNs, the muscle types. An important step in the evolution of later-born secondary MNs have been particularly under- mammals was the appearance of a novel MN subtype studied. Although they make up the majority of spinal dedicated to control of respiratory muscles. MNs innerv- MNs in zebrafish, and are thought to be more similar to ating the diaphragm are contained within the phrenic mammalian MNs, very little is known about their differ- motor column (PMC) and require the actions of two entiation programs [44]. Secondary MNs are born start- Hox genes (Hoxa5 and Hoxc5) for their specification ing at 16 hpf, begin axon initiation at 26 hpf, and are [50]. Similar to the role of Foxp1 in limb MNs, loss of produced to an undetermined time after 25 hpf [35]. Hox5 genes disrupts PMC specification programs and Multiple studies have described up to ten different axial- diaphragm innervation, with the remaining MNs revert- muscle innervating subtypes, six of those are secondary ing to a thoracic HMC-like identity (Fig. 3a, b). As a MNs [45]. All MN subtypes can be differentiated based consequence, mice lacking Hox5 genes show severe de- on birthdate, muscle target, soma size and position, fects in respiratory function and perish at birth [50, 51]. presence or absence of intraspinal or intermyotomal col- Hox5 proteins act in conjunction with more MN- laterals, and firing properties. There are three distinct restricted fate determinants, including the POU-class types of firing patterns expressed by zebrafish axial MNs homeodomain protein Scip (Pou3f1), which is also es- at 4 dpf: tonic, chattering, and burst firing. Tonic firing sential for respiratory function [52]. Downstream targets patterns are specific to primary MNs, while chattering of Hox5 and Scip activities include genes encoding the and burst firing patterns are specific to secondary MNs. cell adhesion proteins Cdh10 and Pcdh10, which appear Each secondary MN subtype has a different distribution to be important for PMC neurons to cluster into colum- of these two firing patterns. While the distinct physio- nar groups [53]. logic and anatomic features of secondary MNs have been Whether MMC neurons targeting specific epaxial well-characterized, it is yet unknown whether they muscles show the same degree of molecular diversity as D’Elia and Dasen Neural Development (2018) 13:10 Page 6 of 12 cd Fig. 3 Diversification of axial MN subtypes in tetrapods. a At rostral cervical levels, HMC-like precursors give rise to phrenic motor column (PMC) neurons through the actions of Hoxa5 and Hoxc5 proteins. The activities of Hox5 proteins are inhibited by Lhx3 in MMC neurons, and Foxp1 in LMC neurons. Hox5 proteins work in conjunction with the Pou domain protein Scip to promote PMC-restricted gene expression. b In the absence of Hox5 genes, PMC neurons are disorganized and revert to an HMC-like state. c Pbx genes are required for the columnar organization of axial MNs. In the absence of Pbx genes, Hox-dependent MN subtypes (LMC and PGC neurons) are lost, and acquire an HMC fate. The remaining HMC and MMC subtypes are disorganized at all spinal levels. d Pbx proteins act in conjunction with other MMC-restricted factors such as Lhx3 to promote MMC specific gene expression HMC-derived MNs is less clear. While all MMC neu- include the transcription factor Mecom (MDS1/Evi1), rons can be defined by the maintenance of Lhx3/4 ex- which marks postmitotic axial MNs and can be induced pression, the specific determinants of MMC subtype- by forced misexpression of Lhx3 in non-MMC popula- specific properties are poorly defined. A recent study in- tions. The disorganization of axial MNs in Pbx mutants vestigating the function of Pbx transcription factors in therefore appears to be a consequence of the disruption of spinal MN differentiation identified a novel repertoire of regulatory programs acting in MMC neurons. genes selectively expressed in mature MMC neurons [54]. Pbx proteins are known to be important cofactors Development of locomotor axial motor circuits in for Hox proteins, and are essential for the specification fish of segmentally-restricted neuronal subtypes [55]. Muta- While the connections established between axial MNs tion of Pbx genes in spinal MNs disrupts the specifica- and muscle play important roles in shaping motor func- tion of all Hox-dependent subtypes, with the majority of tions, how the activities of different classes of MNs are the remaining MNs consisting of MMC and HMC neu- controlled during specific motor behaviors are less well- rons. Surprisingly, removal of Pbx genes also leads a loss understood. Activation of specific MN subtypes is or- of the somatotopic organization of the remaining Hox- chestrated through the inputs they receive from higher independent MMC and HMC populations. In Pbx mu- order “premotor” microcircuits within the spinal cord tants, MNs with MMC and HMC molecular identities and brain. In many cases, these premotor networks as- are generated at all rostrocaudal spinal levels, but MNs semble into rhythmically active central pattern generator of each type are randomly distributed within the ventral (CPG) networks to control basic behaviors such as walk- cord (Fig. 3c). ing, swimming, and breathing [1, 56, 57]. Much of our Loss of Pbx genes does not affect the ability of MMC understanding of the functional and electrophysiological and HMC neurons to select appropriate muscle targets properties of CPG networks stem from studies of axial [54], suggesting a specific function of Pbx targets in gov- muscle-driven motor circuits in the lamprey, which defined erning MN columnar organization. Gene targets acting the core neuronal constituents of CPGs [58]. Recent studies downstream of Pbx proteins are therefore essential for the in genetically tractable systems, such as zebrafish, have ability of axial MNs to coalesce into specific columnar drawn new attention to the role of axial MNs in shaping groups. Identification of genes differentially expressed be- functional properties of locomotor CPG networks. tween normal and Pbx mutant MNs uncovered a novel The first movements of the embryonic zebrafish begin repertoire of targets that are selectively expressed in at 17 hpf with altering coil contractions of the trunk that MMC neurons (Fig. 3d). These downstream targets increase in frequency until 19 hpf and decrease until 27 D’Elia and Dasen Neural Development (2018) 13:10 Page 7 of 12 hpf [32]. These early spontaneous coiling contractions in Zebrafish are able to modulate their swimming speed the embryo are not dependent on synaptic transmission, through the recruitment of distinct MN subtypes. While but involve electrically coupled networks of a subset of the MNs that drive different swimming speeds vary in premotor interneurons that are rhythmically active and anatomical size and excitability, studies suggest differen- dependent on gap junctions [33]. Ipsilateral neurons are tial recruitment of neurons along the dorso-ventral axis electrically coupled and active simultaneously, while is not dependent solely on intrinsic properties but also contralateral neurons are alternatively active [33]. At 21 on preferential excitatory drive [67]. Analogous to zebra- hpf, zebrafish will partially coil in response to touch and, fish spinal MNs, interneurons are organized on the at 27 hpf, zebrafish will swim in response to touch. These dorsal-ventral axis based on recruitment during swim- touch responses, and swimming thereafter, depend on glu- ming and birth order [62]. Dorsally positioned, early tamaterigic and glycinergic chemical synaptic drive and born V2a neurons are active during higher frequency descending inputs from the hindbrain [32, 33]. Propulsion swimming when ventral, late born V2a neurons are during swimming is generated by alternating, neural- inhibited. At least for V2a neurons, the relation between mediated waves of muscle contractions along the trunk of position and recruitment order does not persist into adult the fish. stages [14, 61, 68, 69]. However, experiments in adult zeb- The organization of MNs in the zebrafish spinal cord rafish have revealed preferential connections and reliable correlates with their functional role. This relationship is monosynaptic input from V2a neurons to proximal MNs because the MNs are grouped according to which type recruited at the same frequency of swimming, consistent of muscle fiber they innervate (Fig. 1d)[14]. For ex- with the idea that different V2a neurons govern different ample, the dorsal most MNs innervate fast muscle and speeds of locomotion [15, 61, 65, 69]. are involved in large, fast swimming. During swimming, While premotor inputs have a significant influence on MNs are recruited from slow to intermediate to fast, locomotor behavior, MNs are the ultimate gate to undu- and, therefore, from ventral MNs to dorsal MNs. Target lation in the zebrafish. Increasing evidence suggest MNs muscle is not the only defining factor between these serve in an instructive manner to control the output of groups of neurons, as firing pattern, input resistance, locomotor circuits. A recent study demonstrated that, in reliability, and oscillatory drive, are just a few of the in- addition to having chemical synapses, some V2a inter- trinsic properties suspected to contribute to their differ- neurons in zebrafish are also electrically coupled to MNs ential recruitment [14, 59, 60]. via gap junctions. This coupling permits the backward Primary MNs, which innervate fast muscle, are known propagation of electrical signals from MNs influencing to be responsible for the initial spontaneous coiling con- the synaptic transmission and firing threshold of V2a in- tractions and later escape behavior in zebrafish, while terneurons, and therefore their recruitment during loco- various subsets of secondary MNs are necessary for all motion [70]. These gap junctions allow the MNs to swim speeds. In a ned1 mutant where secondary MNs control locomotor circuit function in a retrograde man- degenerate, but primary MNs are preserved, normal ner, causing the V2a interneurons and the MNs to act as spontaneous coiling contractions are present, but the a unit, which may contribute to the maintenance of fish cannot swim [33]. Although the purpose of these locomotor rhythm generation. separate waves of neuronal birth remains elusive, some hypothesize primary MNs are necessary to form a base Functional diversity of axial motor circuits in for the development of locomotor CPG in the early em- tetrapods bryonic spinal cord [19]. While a primary function of axial MNs is to drive loco- Excitatory inputs onto axial MNs in zebrafish are pro- motion in zebrafish, in tetrapods MMC and HMC neu- vided by V2a interneurons defined by expression of the rons play essential roles in multiple non-locomotor Chx10 transcription factor [61–63]. It has been shown functions including breathing and maintaining spinal that distinct V2a populations drive dorsal and ventral alignment. Some features of the locomotor CPG in fish trunk musculature in zebrafish [60, 64, 65]. Studies in appear to have been preserved in tetrapods to assist in both zebrafish and lamprey disprove the previous notion limb-based locomotion. For example, in amphibian and that only left-right alternation CPGs existed in primitive reptile species undulation of spinal segments can be axial muscle control [64, 66]. This differential input con- used to facilitate movements of limbs [71]. In mammals, tributes to the non-synchronous activation of these particularly in bipedal species, axial MNs appear to have muscle groups important for behaviors such as postural been largely dissociated from locomotor CPG networks, control. Independent control of dorsal and ventral ipsi- which likely played an important role in enabling new lateral muscles is suggested to have been a template for types of axial muscle-driven motor behaviors. separate control of muscles on the same side of the An important step in the evolution of axial motor cir- body, such as those in limbs [67]. cuits in tetrapods was the utilization of hypaxial muscle D’Elia and Dasen Neural Development (2018) 13:10 Page 8 of 12 and its derivatives to support breathing on land. Expan- development of motor circuits controlling postural sion and contraction of the lungs during respiration is stabilization and spinal alignment have been more diffi- mediated by PMC and HMC neurons, which control the cult to study in mammals. In upright-walking bipedal diaphragm and body wall muscle, respectively. In mam- vertebrates, the spine is kept in a relatively rigid config- mals, PMC and HMC firing is governed by CPG circuits uration. Studies in humans indicate that coactivation of located in the brainstem. Neurons in the preBötzinger extensor and flexor axial muscles are essential for the (preBötz) complex and parafacial group provide the pre- load-bearing capacity and stability of the spine [74, 75]. dominant rhythmic drive to PMC and HMC neurons The circuits that stabilize spinal alignment are not well during inspiratory and expiratory breathing [57]. Brain- characterized, but presumably require axial neural control stem CPG networks target neurons in the ventral re- systems that are fundamentally distinct from those con- spiratory group (VRG) that in turn project to hypaxial trolling respiration in tetrapods and locomotion in fish. and phrenic MNs within the spinal cord (Fig. 4a). While A recent study in mice has provided evidence that sen- the developmental logic that determines connectivity be- sory neurons play important roles in maintaining align- tween preBötz, VRG, and spinal MNs is not fully under- ment of the spine. Mutation in the Runx3 transcription stood, a recent study has shown that connectivity factor, which is required for the development of muscle between preBötz and VRG neurons rely on a common proprioceptive sensory neurons (pSNs) [76], leads to a transcription factor, Dbx1 [72]. Expression of Dbx1 is progressive scoliosis of the spine (Fig. 4b-d)[77]. This absent from MNs, suggesting other intrinsic factors are phenotype does not appear to be a consequence of a re- involved in establishing connectivity between VRG and quirement for Runx3 function in other tissues, since axial MNs. Connections between brainstem respiratory similar results were observed after Runx3 deletion spe- centers and spinal MNs could rely on actions of cifically from pSNs. Although how this mutation affects segmentally-restricted fate determinants, such Hox the circuits involved in spinal stabilization is unclear, it genes, which differentiate PMC and HMC from other is likely due to altered connections between pSNs and spinal MN subtypes (Fig. 4a)[73]. the axial motor circuits essential for maintaining pos- While motor circuits controlling breathing and loco- ture. Loss and gain of function studies have shown that motion rely on rhythmically active neural circuits, the Runx3 is required for the ability of pSNs to establish bd c Fig. 4 Diverse function of axial motor circuits in tetrapods. a Simplified diagram of respiratory networks for inspirational breathing. Rhythm generation in the preBötzinger (preBötz) complex is relayed to rostral ventral respiratory group (rVRG) neurons. rVRG neurons target PMC neurons and HMC neurons in the spinal cord. Connections between preBötz and rVRG neuron relies on Dbx1 gene function. b-d Role of axial motor circuits in spinal alignment. b Axial muscles and nerves associated with vertebrae. Box indicates region magnified in panel c. c Consequences of Runx3 mutation on the projection of proprioceptive sensory neurons in the spinal cord. Loss of Runx3 leads to a loss of projections to MNs, and likely other classes of spinal interneurons. d Effect of Runx3 mutation on vertebral alignment in adult mice D’Elia and Dasen Neural Development (2018) 13:10 Page 9 of 12 connections with MNs and other neural classes [77–79], suggesting that the Runx3 mutant phenotype is due to a the disruption of local sensory-motor spinal reflex cir- cuits. In addition, mutations that affect the function of the MMC-restricted transcription factor Mecom also causes abnormal bending of the spine [80], raising the possibility that this phenotype is also consequence of al- tered connectivity between axial MNs and premotor neural populations. Developmental mechanisms of axial motor circuit assembly in tetrapods The distinct use of MMC neurons in locomotion and posture, while HMC and HMC-like MNs are essential for breathing, raise the question of how premotor cir- cuits dedicated to specific motor functions target the ap- propriate axial MN subtype. While the answer to this question is largely unknown, studies characterizing the distribution of spinal interneurons connected to specific MN columnar subtypes have provided a partial answer. Rabies-based monosynaptic tracing of interneurons con- nected to MMC and HMC neurons revealed that axial MNs receive local spinal premotor inputs that are evenly distributed across both sides of the spinal cord (Fig. 5a). In contrast, limb MNs receive inputs predominantly from premotor interneurons on the ipsilateral side of the Fig. 5 Developmental mechanisms of axial motor circuit assembly. a spinal cord [81]. Axial MN dendritic arborization pat- Dendritic morphology and premotor input pattern for MN columnar terns are also distinct from those of limb MNs, which subtypes. MMC neurons have dendrites that extend across the midline may help determine their specific connectivity with pre- and their monosynaptic premotor inputs are distributed across both sides of the spinal cord. Like MMC neurons, HMC neuron dendrites motor interneuron populations (Fig. 5a). MMC neurons extend medio-laterally and have a similar premotor input distribution have dendrites that extend across the midline, which ap- pattern. LMC neurons have radially organized dendrites and receive pears to enable them to capture a greater proportion of premotor inputs predominantly from ipsilateral spinal interneurons. inputs from contralateral interneuron populations, and Darker shading indicates higher density of interneurons connected to establish connectivity with interneurons distinct from MNs. b Effect of Hoxc9 mutation on premotor input pattern. In Hoxc9 mutants, thoracic HMC neurons are converted to LMC fate, while MMC those of HMC neurons. In contrast, limb-innervating neurons are grossly unaffected. In Hoxc9 mutants, ectopic LMC neurons LMC neurons are found in more lateral and dorsal re- still project to intercostal muscle. The dendritic pattern of thoracic MNs gions of the spinal cord and have radially-projecting in Hoxc9 mutants becomes more limb-like, and MNs projecting to dendrites, which may afford them greater input from ip- intercostal muscle receive a higher distribution of inputs from ipsilateral silateral interneuron populations. premotor interneurons. Diagram based on data in [84] Do the molecular identities and/or positional differ- ences between MN subtypes determine their premotor input pattern and function? The ability to genetically patterns and types of synaptic inputs that MNs receive alter the composition of MN subtypes within the mouse [84]. Transformation of thoracic HMC neurons to a spinal cord provides evidence that MN subtype identity limb-level LMC fate, through mutation of the Hoxc9 plays an important role in determining the functional gene [85], shifts spinal premotor inputs to predomin- properties of spinal circuits. Conversion of limb MNs to antly ipsilateral populations (Fig. 5b). In Hoxc9 mutants, an axial HMC fate, through deletion of limb MN deter- the transformed HMC populations also settle in a more minant Foxp1, leads to the loss of limb-specific motor dorsolateral position, and their dendrites project radially, output patterns [82, 83]. In the absence of Foxp1, the similar to those of limb-innervating MNs (Fig. 5b)[84]. normal alternation of limb-flexor and -extensor firing While these studies do not resolve the basic question of patterns is lost, and the remaining HMC-like popula- how differences between HMC and MMC inputs are tions fire in a predominantly flexor-like pattern. achieved, they suggest that intrinsic differences between Recent studies also indicate that determinants of MN MN molecular identity, dendritic morphology, and pos- columnar identity play crucial roles in defining the ition contribute to shaping the pattern of connection D’Elia and Dasen Neural Development (2018) 13:10 Page 10 of 12 within the motor circuits. How these genetic manipula- Abbreviations CaP: Caudal primary motor neuron; CPG: Central pattern generator; dpf: Days tions affect the function of axial motor circuits remains post fertilization; dRoP: Dorsal rostral primary motor neuron; ei: External to be determined. Nevertheless, analyses of Foxp1 and intercostal muscle; eo: External oblique muscle; ESC: Embryonic stem cell; Hoxc9 mutants indicate that the columnar identity of FGF: Fibroblast growth factor; FGFR1: Fibroblast growth factor receptor 1; HMC: Hypaxial motor column; hpf: Hours post fertilization; ii: Internal spinal MNs plays a significant role in determining the intercostal muscle; ilio: Iliocostalis muscle; lc: Levator costae muscle; architecture and output patterns of spinal circuits. LMC: Lateral motor column; long: Longissimus muscle; MiP: Middle primary motor neuron; MMC: Medial motor column; MN: Motor neuron; PGC: Preganglionic motor column; PMC: Phrenic motor column; pMN: Motor Conclusions neuron progenitor; pSN: Proprioceptive sensory neuron; sc: Subcostalis Studies on the development of neural circuits controlling muscle; Shh: Sonic hedgehog; sr: Caudal serratus muscle; axial muscles have provided valuable insights into how tv: Transversospinalis muscle; VRG: Ventral respiratory group; vRoP: Ventral rostral primary motor neuron specific motor functions develop and have evolved in the vertebrate lineage. While we have a fairly in depth un- Acknowledgements derstanding of the genetic programs controlling the spe- We thank David McLean and anonymous reviewers for feedback on the text. cification of tetrapod axial MN subtypes, how these Funding functionally diverse populations are connected to appro- Work in the lab is supported by grants R01NS062822 and R01NS097550 from priate higher order circuits remains to be determined. the NIH. Recent studies showing that MN-intrinsic programs contribute to differences in the patterns of premotor Authors’ contributions JD and KD wrote the manuscript and prepared the figures. Both authors connectivity between limb and axial MNs suggests a read and approved the final manuscript. general mechanism through which motor circuits are as- sembled, as a function of molecular differences in their Ethics approval and consent to participate target MN populations. Further functional studies on Not applicable. the consequences of disrupting MN differentiation could Competing interests provide a means to test the role of MN subtype identity The authors declare that they have no competing interests. in the development of axial circuits essential for breath- ing and spinal alignment. Publisher’sNote Comparisons between species that use axial MNs for dis- Springer Nature remains neutral with regard to jurisdictional claims in tinct functions have provided insights into how different published maps and institutional affiliations. motor behaviors are specified during development. Al- Received: 14 March 2018 Accepted: 26 April 2018 though this review has focused on vertebrate development, many of the intrinsic molecular features of axial MNs ap- pear to be conserved in invertebrates. Similar to verte- References brates, in Drosophila and C. elegans subtypes of MNs can 1. Kiehn O. Decoding the organization of spinal circuits that control locomotion. 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Development, functional organization, and evolution of vertebrate axial motor circuits

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Abstract

Neuronal control of muscles associated with the central body axis is an ancient and essential function of the nervous systems of most animal species. Throughout the course of vertebrate evolution, motor circuits dedicated to control of axial muscle have undergone significant changes in their roles within the motor system. In most fish species, axial circuits are critical for coordinating muscle activation sequences essential for locomotion and play important roles in postural correction. In tetrapods, axial circuits have evolved unique functions essential to terrestrial life, including maintaining spinal alignment and breathing. Despite the diverse roles of axial neural circuits in motor behaviors, the genetic programs underlying their assembly are poorly understood. In this review, we describe recent studies that have shed light on the development of axial motor circuits and compare and contrast the strategies used to wire these neural networks in aquatic and terrestrial vertebrate species. Keywords: Motor neuron, Spinal circuit, Neural circuit, Development, Evolution, Axial muscle Background invasion of land by vertebrates, axial muscles that were The neuromuscular system of axial skeleton plays crucial initially used in swimming were also adapted by the re- roles in basic motor functions essential to vertebrates, spiratory system to enable breathing in air. Since many of including locomotion, breathing, posture and balance. these diverse axial muscle-driven motor behaviors are While significant progress has been made in deciphering encoded by neural circuits assembled during develop- the wiring and function of neural circuits governing limb ment, insights into the evolution of axial circuits might control [1, 2], the neural circuits associated with axial emerge through comparisons of the genetic programs that muscles have been relatively under studied, particularly control neural circuit assembly in different animal species. in mammals. Despite comprising more than half of all In this review, we discuss studies which have investi- skeletal muscles in mammals, how axial neural circuits gated the development, evolution, and wiring of neuronal are assembled during development is poorly understood. circuits essential for control of axial muscle. Recent ad- Although all vertebrates share similar types of axial vances in genetically tractable systems, such as zebrafish muscle [3, 4], the nervous systems of aquatic and terres- and mouse, have provided novel insights into the mecha- trial species control these muscle groups in distinct ways. nisms through which axial circuits are assembled during In most aquatic vertebrates, rhythmic contraction of axial development, and have shed light on the wiring of the cir- muscle is essential for generating propulsive force during cuits essential for balance, breathing, and locomotion. We swimming, the predominant form of locomotion used by compare the strategies through which animals generate fish. In land vertebrates, axial circuits have been largely distinct classes of spinal neurons that coordinate axial dissociated from locomotor functions, and have been muscles, with particular focus on the spinal motor neuron modified throughout evolution to enable new types of subtypes that facilitate axial-driven motor behaviors. motor capabilities. In animals with upright postures, neur- onal control of axial muscles is essential to maintain bal- Functional organization and peripheral ance and proper alignment of the spine. During the connectivity of axial motor neurons Although used for fundamentally distinct motor functions, * Correspondence: jeremy.dasen@nyumc.org the axial neuromuscular systems of fish and tetrapods Neuroscience Institute, Department of Neuroscience and Physiology, NYU School of Medicine, New York, NY 10016, USA share many anatomic features and early developmental © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. D’Elia and Dasen Neural Development (2018) 13:10 Page 2 of 12 programs [3, 4]. In both fish and tetrapods, axial muscles lost. Both types of axial muscles receive innervation from can be broadly divided into two groups, epaxial and spinal motor neurons (MNs) and sensory neurons that hypaxial, which are initially separated by a horizontal project either along the dorsal (epaxial) or ventral (hypax- myoseptum (Fig. 1a). Epaxial muscles reside dorsal to the ial) branches of the spinal nerves. myoseptum and include muscle groups associated with In tetrapods, MNs targeting specific muscle groups are the vertebral column and base of the skull. Hypaxial mus- organized in discrete clusters, termed motor columns and cles are predominantly located ventral to the mysoseptum motor pools [5–8]. Spinal MNs projecting to functionally and give rise to diverse muscle groups including abdom- related muscle groups, such as epaxial, hypaxial, or limb inal and intercostal muscles, as well as the diaphragm in muscle, are contained within motor columns that occupy mammals. In tetrapods, migratory populations of hypaxial specific rostrocaudal positions within the spinal cord. muscle also generate all of the muscle in the limb. In fish Within these columnar groups, MNs further segregate and amphibians, the separation between dorsal and ven- into motor pools, each pool targeting a single muscle. tral axial muscles is maintained in adulthood, while in tet- Each pool occupies a specific position within the spinal rapods many of these positional differences have been cord, and its relative position along the dorsoventral, ab cd Fig. 1 Organization of axial MNs in tetrapods and fish. a In jawed vertebrates, axial muscles are separated into dorsal epaxial and ventral hypaxial groups, separated by the horizontal myoseptum (HM). Each muscle group is innervated by separate spinal nerves. Dorsal root ganglia (drg) and sympathetic chain ganglia (scg) are shown. b MN columnar subtypes at trunk levels. In tetrapods, as well as some cartilaginous fish, MNs innervating dorsal epaxial muscles are organized in the medial motor column (MMC). MNs projecting to ventral hypaxial muscles are contained within the hypaxial motor column (HMC). Autonomic preganglionic column (PGC) neurons, which project to scg, are shown in gray. c Organization of MN pools at thoracic levels. MNs innervating specific types of axial muscle are organized in pool-like clusters. Some MNs within the HMC project to dorsally located axial muscles, such as serratus, but are nevertheless supplied by axons originating from the ventral ramus. Abbreviations: tv, transversospinalis; long, longissimus; ilio, iliocostalis; lc, levator costae; sr, caudal serratus; ii, internal intercostal; sc, subcostalis; ei, external intercostal; eo, external oblique. Not all trunk muscles are shown. Diagram based on data from rat in [13]. d Organization of MNs in adult zebrafish. MNs innervating fast, intermediate, and slow muscle are organized along the dorsoventral axis. Fast MNs include primary MNs and some secondary MNs, intermediate and slow are all secondary MNs. These MN types project to specific types of trunk-level axial muscles. Diagram based on data in [14] D’Elia and Dasen Neural Development (2018) 13:10 Page 3 of 12 mediolateral, and rostrocaudal axes is linked to how MNs differences that emerged between certain fish species and project within a target region. The stereotypic organization other vertebrate classes. of MN position within the spinal cord therefore establishes a central topographic map which relates neuronal settling Genetic programs specifying early axial motor position to target specificity. neuron fates Studies on the developmental mechanisms controlling How are the distinct identities of MMC and HMC neu- MN columnar and pool organization have largely focused rons established during tetrapod development? As with on the diverse subtypes innervating limb muscles [9, 10]. other subtypes of spinal MNs, the progenitors that give Axial MNs also display a topographic organization that re- rise to axial MNs are specified through secreted signal- lates neuronal position to target specificity. The cell bodies ing molecules acting along the dorsoventral axis of the of MNs targeting epaxial and hypaxial muscles are orga- neural tube shortly after its closure [18]. These morpho- nized in specific columnar groups within the ventral spinal gens establish specific molecular identities through the cord (Fig. 1b). Dorsal epaxial muscles are innervated by induction of transcription factors in neuronal progenitors, MNs in the median motor column (MMC), while hypaxial whichsubsequentlyspecifytheidentityofeach ofthemajor musclesare innervated by MNsinthe hypaxial motorcol- classes of spinal neuron. In the ventral spinal cord, graded umn (HMC). MMC neurons occupy the most medial pos- Shh signaling induces expression of transcription factors ition of all spinal MNs, whereas HMC neurons, and all which specify MN and ventral interneuron progenitor iden- other MN subtypes, typically reside more laterally [11]. Like tities [19]. As progenitors differentiate, additional transcrip- limb MNs, both MMC and HMC neurons further differen- tion factors are expressed within postmitotic cells and act tiate into specific pool groups, and axial MN pool position to define specific neuronal class fates [20]. Spinal MN pro- is linked to the location of its muscle target (Fig. 1c). For genitors are derived from a domain characterized by ex- example, MMC neurons targeting more dorsal epaxial pression of Olig2, Nkx6.1, and Pax6. As postmitotic MNs muscles reside more medially than those targeting more emerge, they initially express the Lim homeodomain pro- ventral muscle [12]. A similar somatotopic organization has teins Islet1, Islet2 (Isl1/2), Lhx3, Lhx4 (Lhx3/4), as well as been observed for HMC pools targeting different intercos- the Mnx-class protein Hb9 (Fig. 2a). tal and abdominal muscles [13]. As MNs differentiate and migrate to their final settling In contrast to tetrapods, the organization of axial MNs positions, subtypes of axial MNs can defined by differen- into well-defined columnar groups has not been de- tial expression of Lim HD and Mnx factors [11, 21]. In scribed in zebrafish. Despite the absence of an obvious tetrapods, MMC neurons maintain expression of Hb9, columnar organization, zebrafish axial MNs are func- Isl1/2, and Lhx3/4, whereas the majority of other MN tionally organized along the dorsoventral axis of the subtypes, including HMC neurons, downregulate Lhx3 spinal cord (Fig. 1d). This organization is associated as they become postmitotic (Fig. 2b). The specific func- with how MNs are recruited at distinct swimming tions of Lhx3 and Lhx4 in MMC neurons are not com- speeds and correlated with the type of muscle a MN pletely understood, as both genes are required for the innervates, as opposed to the location of the muscle. differentiation of all spinal MN subtypes [22]. Neverthe- Axial MNs projecting to muscles activated at slow less, misexpression of Lhx3 can convert limb MNs to an swimming speeds reside ventrally, MNs recruited at MMC fate and redirect motor axons towards axial fast swimming speeds are located dorsally, and MNs muscle, indicating that Lhx3 plays an instructive role in involved in intermediate speeds sit between fast and determining the trajectories of MMC motor axons to- slow MNs [14–16]. wards epaxial muscle [23]. While trunk-level HMC neu- Although a clustered organization of axial MN has not rons can be also defined by expression of specific been described in zebrafish, in certain cartilaginous fish transcription factor combinations, whether these factors species, including the little skate and catshark, the cell bod- are required for columnar-specific differentiation pro- ies of MMC neurons are clustered and settle in a ventral grams is currently unknown. position [17]. These observations suggest that the A key step in the specification of axially-projecting MNs organization of axial MNs into columns was present in the is the segregation of newly born neurons into MMC and common ancestor to cartilaginous fish and tetrapods, and HMC subtypes. MMC neurons are thought to represent therefore to all jawed vertebrates with paired appendages. the ancestral “groundstate” of MNs from which all other Notably, unlike most fish species, skates do not use the subtypes subsequently evolved [24]. This idea is supported axial muscles to generate propulsive force during locomo- by the observation that MMC identity is the default differ- tion, which is instead provided by contraction of the pec- entiation state of MNs derived from embryonic stem cells toral and pelvic fins. The organization of MNs into (ESCs) generated through induction with retinoic-acid columnar and pool groups therefore does not appear to and Shh [25, 26]. In addition, MMC-like neurons drive have evolved with terrestrial locomotion, but rather reflects locomotor behaviors in limbless vertebrates such as the D’Elia and Dasen Neural Development (2018) 13:10 Page 4 of 12 bc Fig. 2 Specification of axial MNs in tetrapods and fish. a Specification of early axial MN identities. Graded sonic hedgehog (Shh) acts along the dorso (d)-ventral (v) axis to specify MN progenitors (pMN) and ventral interneuron fates. Graded Wnt signaling promotes sustained expression of Lhx3 in MMC neurons, while Hox signaling specifies segmentally-restricted MN columnar fates, including limb-innervating lateral motor column (LMC) neurons. b Axial MNs in tetrapods can be defined by expression of specific transcription factors. MMC neurons express Fgr1 and are attracted to mesodermally-derived FGF signaling. c Primary MNs in zebrafish. Four distinct axial MN types can be defined by their rostrocaudal position and muscle target specificity. dRoP, dorsal rostral primary; vRoP, ventral rostral primary; CaP, caudal primary; MiP, middle primary MN lamprey and insect larvae, suggesting that an MMC-like the cell cycle, the axons of MMC and HMC neurons MN population represents the ancestral condition of MNs begin to project outside the spinal cord, both initially in bilaterians. pursuing ventrolateral trajectories. The axons of MMC In tetrapods, an obligate step in MMC differentiation neurons separate from the main nerve and extend dor- is the sustained expression of Lhx3/4 in post-mitotic sally, while all other MN subtypes, including HMC neu- MNs; while in HMC neurons and all other MN subtypes rons, continue to extend ventrolaterally. The dorsal Lhx3/4 must be downregulated for proper differentiation trajectory of MMC neurons appears to rely on target- [21, 23]. The maintenance of Lhx3/4 in MMC neurons derived chemoattractant signaling emanating from a appears to be partially governed by Wnt signaling origin- somite-derived structure, the dermomyotome [29, 30]. ating from near the floorplate of the spinal cord (Fig. 2a) This region expresses fibroblast growth factors (FGFs) [27]. Overexpression of Wnt4 or Wnt5a promotes the which act on the axons of MMC neurons that selectively specification of MMC neurons at the expense of other express FGF receptor 1 (Fgfr1) (Fig. 2b)[31]. Mutation of MN subtypes in chick embryos, while combined genetic Ffgr1 in mice causes defects in the peripheral trajectory of removal of Wnt4, Wnt5a,and Wnt5b in mice leads to de- MMC axons. In addition, misexpression of Lhx3 leads to pletion in MMC number. Recent studies in ES cell- ectopic expression of Fgfr1 in non-MMC MNs and causes derived MNs suggest that additional signaling pathways limb motor axons to gain sensitivity to FGFs [31]. act in conjunction with Wnt signaling to promote MMC specification [28]. Inhibition of Notch signaling in ES-cell Specification of axial MNs in zebrafish derived MNs promotes the specification of HMC neurons In zebrafish, spinal MNs innervating axial muscle are spe- at the expense of MMC neurons, suggesting that Wnt4/5 cified by the same core groups of transcription factors that and Notch cooperate to specify MMC identity. act in tetrapods. Unlike amniotes, where all MNs are gen- While the extrinsic and intrinsic factors governing the erated during a single wave of neurogenesis, zebrafish specification of MMC and HMC neurons have been have two waves of MN birth, primary and secondary. Pri- characterized, the downstream effectors of their fate de- mary and secondary neurons are each important for dif- terminants are less well-understood. Soon after leaving ferent types of axial muscle-based behaviors, but not D’Elia and Dasen Neural Development (2018) 13:10 Page 5 of 12 distinguished by any known transcription factor [32, 33]. reflect the operation of MN-intrinsic genetic programs Primary MNs, which number three to four per hemi- acting during development. segment, are born between 10 and 14 hours post- fertilization (hpf), develop subtype-specific electrical Diversification of tetrapod axial motor columns membrane properties as early as 17 hpf, and begin axon While axial MNs of fish and mammals share several initiation at 17 hpf [34, 35]. Although one or two common common early developmental programs, in tetrapods MN markers such as Isl1, Isl2, and Mnx proteins can help these subtypes have undergone a significant degree of differentiate two or three primary MN subtypes at differ- modification throughout the course of vertebrate evolu- ent ages, these factors cannot distinguish them through- tion. All of the segmentally restricted subtypes of spinal out development and have dynamic expression patterns MNs, including the diverse MN populations innervating that make the subtypes challenging to track over time limb muscle, appear to have evolved from the ventrally- [36–38]. All early-born MNs require the Olig2 transcrip- projecting HMC-like population. This hypothesis is sup- tion factor [39], while Nkx6 proteins appear to be required ported by the observation that in genetic mutants with only in a subset of primary MNs [40]. Postmitotic primary disrupted specification of non-axial MN subtypes, af- MNs can be defined by differential expression of Mnx/ fected populations revert to a predominantly HMC-like Hb9, Isl1/2, and Lhx3 factors [37, 38, 41–43]. molecular profile. Genetic deletion of the limb MN fate Most genetic studies of axial MN specification in zeb- determinant Foxp1 in mice causes a loss of limb-specific rafish have largely focused on the specification of the MN programs and an expansion in the number of MNs four major types of primary MNs: the dorsal rostral pri- with an HMC-like molecular identity [21, 46]. Expres- mary (dRoP), ventral rostral primary (vRoP), caudal pri- sion of Foxp1 in limb-innervating lateral motor column mary (CaP), and middle primary (MiP) subtypes (Fig. (LMC) neurons is governed by Hox transcription factors 2c). dRoP and MiP MNs are similar to MMC neurons, expressed at specific rostrocaudal levels of the spinal in that they project to muscles located dorsal to the cord, and Hox genes are essential for generating the di- horizontal myoseptum, while CaP and vRoP project ven- verse motor pool populations targeting specific limb trally. However, unlike MMC and HMC neurons in tet- muscles [47–49]. MMC neurons appear to be insensitive rapods, these primary MN types cannot be distinguished to the activities of Hox proteins, likely due to the func- by differential expression of Lhx3. Nevertheless, disrup- tionally dominant actions of Lhx3 [21, 23]. The diversifi- tion of the core MN determinants Lhx3/4, Isl1/2, and cation of tetrapod spinal MNs appears to stem from Mnx leads to defects in primary MN specification and HMC-like precursors which co-opted Hox genes to gen- connectivity. For example, loss of Lhx3/4 leads to MNs erate more specialized populations. with hybrid MN/interneuron fates [41], while loss of Hox-dependent regulatory programs also contributed Mnx proteins affects the specification of MiP MNs [38]. to the diversification of MNs targeting specific hypaxial While much is known about primary axial MNs, the muscle types. An important step in the evolution of later-born secondary MNs have been particularly under- mammals was the appearance of a novel MN subtype studied. Although they make up the majority of spinal dedicated to control of respiratory muscles. MNs innerv- MNs in zebrafish, and are thought to be more similar to ating the diaphragm are contained within the phrenic mammalian MNs, very little is known about their differ- motor column (PMC) and require the actions of two entiation programs [44]. Secondary MNs are born start- Hox genes (Hoxa5 and Hoxc5) for their specification ing at 16 hpf, begin axon initiation at 26 hpf, and are [50]. Similar to the role of Foxp1 in limb MNs, loss of produced to an undetermined time after 25 hpf [35]. Hox5 genes disrupts PMC specification programs and Multiple studies have described up to ten different axial- diaphragm innervation, with the remaining MNs revert- muscle innervating subtypes, six of those are secondary ing to a thoracic HMC-like identity (Fig. 3a, b). As a MNs [45]. All MN subtypes can be differentiated based consequence, mice lacking Hox5 genes show severe de- on birthdate, muscle target, soma size and position, fects in respiratory function and perish at birth [50, 51]. presence or absence of intraspinal or intermyotomal col- Hox5 proteins act in conjunction with more MN- laterals, and firing properties. There are three distinct restricted fate determinants, including the POU-class types of firing patterns expressed by zebrafish axial MNs homeodomain protein Scip (Pou3f1), which is also es- at 4 dpf: tonic, chattering, and burst firing. Tonic firing sential for respiratory function [52]. Downstream targets patterns are specific to primary MNs, while chattering of Hox5 and Scip activities include genes encoding the and burst firing patterns are specific to secondary MNs. cell adhesion proteins Cdh10 and Pcdh10, which appear Each secondary MN subtype has a different distribution to be important for PMC neurons to cluster into colum- of these two firing patterns. While the distinct physio- nar groups [53]. logic and anatomic features of secondary MNs have been Whether MMC neurons targeting specific epaxial well-characterized, it is yet unknown whether they muscles show the same degree of molecular diversity as D’Elia and Dasen Neural Development (2018) 13:10 Page 6 of 12 cd Fig. 3 Diversification of axial MN subtypes in tetrapods. a At rostral cervical levels, HMC-like precursors give rise to phrenic motor column (PMC) neurons through the actions of Hoxa5 and Hoxc5 proteins. The activities of Hox5 proteins are inhibited by Lhx3 in MMC neurons, and Foxp1 in LMC neurons. Hox5 proteins work in conjunction with the Pou domain protein Scip to promote PMC-restricted gene expression. b In the absence of Hox5 genes, PMC neurons are disorganized and revert to an HMC-like state. c Pbx genes are required for the columnar organization of axial MNs. In the absence of Pbx genes, Hox-dependent MN subtypes (LMC and PGC neurons) are lost, and acquire an HMC fate. The remaining HMC and MMC subtypes are disorganized at all spinal levels. d Pbx proteins act in conjunction with other MMC-restricted factors such as Lhx3 to promote MMC specific gene expression HMC-derived MNs is less clear. While all MMC neu- include the transcription factor Mecom (MDS1/Evi1), rons can be defined by the maintenance of Lhx3/4 ex- which marks postmitotic axial MNs and can be induced pression, the specific determinants of MMC subtype- by forced misexpression of Lhx3 in non-MMC popula- specific properties are poorly defined. A recent study in- tions. The disorganization of axial MNs in Pbx mutants vestigating the function of Pbx transcription factors in therefore appears to be a consequence of the disruption of spinal MN differentiation identified a novel repertoire of regulatory programs acting in MMC neurons. genes selectively expressed in mature MMC neurons [54]. Pbx proteins are known to be important cofactors Development of locomotor axial motor circuits in for Hox proteins, and are essential for the specification fish of segmentally-restricted neuronal subtypes [55]. Muta- While the connections established between axial MNs tion of Pbx genes in spinal MNs disrupts the specifica- and muscle play important roles in shaping motor func- tion of all Hox-dependent subtypes, with the majority of tions, how the activities of different classes of MNs are the remaining MNs consisting of MMC and HMC neu- controlled during specific motor behaviors are less well- rons. Surprisingly, removal of Pbx genes also leads a loss understood. Activation of specific MN subtypes is or- of the somatotopic organization of the remaining Hox- chestrated through the inputs they receive from higher independent MMC and HMC populations. In Pbx mu- order “premotor” microcircuits within the spinal cord tants, MNs with MMC and HMC molecular identities and brain. In many cases, these premotor networks as- are generated at all rostrocaudal spinal levels, but MNs semble into rhythmically active central pattern generator of each type are randomly distributed within the ventral (CPG) networks to control basic behaviors such as walk- cord (Fig. 3c). ing, swimming, and breathing [1, 56, 57]. Much of our Loss of Pbx genes does not affect the ability of MMC understanding of the functional and electrophysiological and HMC neurons to select appropriate muscle targets properties of CPG networks stem from studies of axial [54], suggesting a specific function of Pbx targets in gov- muscle-driven motor circuits in the lamprey, which defined erning MN columnar organization. Gene targets acting the core neuronal constituents of CPGs [58]. Recent studies downstream of Pbx proteins are therefore essential for the in genetically tractable systems, such as zebrafish, have ability of axial MNs to coalesce into specific columnar drawn new attention to the role of axial MNs in shaping groups. Identification of genes differentially expressed be- functional properties of locomotor CPG networks. tween normal and Pbx mutant MNs uncovered a novel The first movements of the embryonic zebrafish begin repertoire of targets that are selectively expressed in at 17 hpf with altering coil contractions of the trunk that MMC neurons (Fig. 3d). These downstream targets increase in frequency until 19 hpf and decrease until 27 D’Elia and Dasen Neural Development (2018) 13:10 Page 7 of 12 hpf [32]. These early spontaneous coiling contractions in Zebrafish are able to modulate their swimming speed the embryo are not dependent on synaptic transmission, through the recruitment of distinct MN subtypes. While but involve electrically coupled networks of a subset of the MNs that drive different swimming speeds vary in premotor interneurons that are rhythmically active and anatomical size and excitability, studies suggest differen- dependent on gap junctions [33]. Ipsilateral neurons are tial recruitment of neurons along the dorso-ventral axis electrically coupled and active simultaneously, while is not dependent solely on intrinsic properties but also contralateral neurons are alternatively active [33]. At 21 on preferential excitatory drive [67]. Analogous to zebra- hpf, zebrafish will partially coil in response to touch and, fish spinal MNs, interneurons are organized on the at 27 hpf, zebrafish will swim in response to touch. These dorsal-ventral axis based on recruitment during swim- touch responses, and swimming thereafter, depend on glu- ming and birth order [62]. Dorsally positioned, early tamaterigic and glycinergic chemical synaptic drive and born V2a neurons are active during higher frequency descending inputs from the hindbrain [32, 33]. Propulsion swimming when ventral, late born V2a neurons are during swimming is generated by alternating, neural- inhibited. At least for V2a neurons, the relation between mediated waves of muscle contractions along the trunk of position and recruitment order does not persist into adult the fish. stages [14, 61, 68, 69]. However, experiments in adult zeb- The organization of MNs in the zebrafish spinal cord rafish have revealed preferential connections and reliable correlates with their functional role. This relationship is monosynaptic input from V2a neurons to proximal MNs because the MNs are grouped according to which type recruited at the same frequency of swimming, consistent of muscle fiber they innervate (Fig. 1d)[14]. For ex- with the idea that different V2a neurons govern different ample, the dorsal most MNs innervate fast muscle and speeds of locomotion [15, 61, 65, 69]. are involved in large, fast swimming. During swimming, While premotor inputs have a significant influence on MNs are recruited from slow to intermediate to fast, locomotor behavior, MNs are the ultimate gate to undu- and, therefore, from ventral MNs to dorsal MNs. Target lation in the zebrafish. Increasing evidence suggest MNs muscle is not the only defining factor between these serve in an instructive manner to control the output of groups of neurons, as firing pattern, input resistance, locomotor circuits. A recent study demonstrated that, in reliability, and oscillatory drive, are just a few of the in- addition to having chemical synapses, some V2a inter- trinsic properties suspected to contribute to their differ- neurons in zebrafish are also electrically coupled to MNs ential recruitment [14, 59, 60]. via gap junctions. This coupling permits the backward Primary MNs, which innervate fast muscle, are known propagation of electrical signals from MNs influencing to be responsible for the initial spontaneous coiling con- the synaptic transmission and firing threshold of V2a in- tractions and later escape behavior in zebrafish, while terneurons, and therefore their recruitment during loco- various subsets of secondary MNs are necessary for all motion [70]. These gap junctions allow the MNs to swim speeds. In a ned1 mutant where secondary MNs control locomotor circuit function in a retrograde man- degenerate, but primary MNs are preserved, normal ner, causing the V2a interneurons and the MNs to act as spontaneous coiling contractions are present, but the a unit, which may contribute to the maintenance of fish cannot swim [33]. Although the purpose of these locomotor rhythm generation. separate waves of neuronal birth remains elusive, some hypothesize primary MNs are necessary to form a base Functional diversity of axial motor circuits in for the development of locomotor CPG in the early em- tetrapods bryonic spinal cord [19]. While a primary function of axial MNs is to drive loco- Excitatory inputs onto axial MNs in zebrafish are pro- motion in zebrafish, in tetrapods MMC and HMC neu- vided by V2a interneurons defined by expression of the rons play essential roles in multiple non-locomotor Chx10 transcription factor [61–63]. It has been shown functions including breathing and maintaining spinal that distinct V2a populations drive dorsal and ventral alignment. Some features of the locomotor CPG in fish trunk musculature in zebrafish [60, 64, 65]. Studies in appear to have been preserved in tetrapods to assist in both zebrafish and lamprey disprove the previous notion limb-based locomotion. For example, in amphibian and that only left-right alternation CPGs existed in primitive reptile species undulation of spinal segments can be axial muscle control [64, 66]. This differential input con- used to facilitate movements of limbs [71]. In mammals, tributes to the non-synchronous activation of these particularly in bipedal species, axial MNs appear to have muscle groups important for behaviors such as postural been largely dissociated from locomotor CPG networks, control. Independent control of dorsal and ventral ipsi- which likely played an important role in enabling new lateral muscles is suggested to have been a template for types of axial muscle-driven motor behaviors. separate control of muscles on the same side of the An important step in the evolution of axial motor cir- body, such as those in limbs [67]. cuits in tetrapods was the utilization of hypaxial muscle D’Elia and Dasen Neural Development (2018) 13:10 Page 8 of 12 and its derivatives to support breathing on land. Expan- development of motor circuits controlling postural sion and contraction of the lungs during respiration is stabilization and spinal alignment have been more diffi- mediated by PMC and HMC neurons, which control the cult to study in mammals. In upright-walking bipedal diaphragm and body wall muscle, respectively. In mam- vertebrates, the spine is kept in a relatively rigid config- mals, PMC and HMC firing is governed by CPG circuits uration. Studies in humans indicate that coactivation of located in the brainstem. Neurons in the preBötzinger extensor and flexor axial muscles are essential for the (preBötz) complex and parafacial group provide the pre- load-bearing capacity and stability of the spine [74, 75]. dominant rhythmic drive to PMC and HMC neurons The circuits that stabilize spinal alignment are not well during inspiratory and expiratory breathing [57]. Brain- characterized, but presumably require axial neural control stem CPG networks target neurons in the ventral re- systems that are fundamentally distinct from those con- spiratory group (VRG) that in turn project to hypaxial trolling respiration in tetrapods and locomotion in fish. and phrenic MNs within the spinal cord (Fig. 4a). While A recent study in mice has provided evidence that sen- the developmental logic that determines connectivity be- sory neurons play important roles in maintaining align- tween preBötz, VRG, and spinal MNs is not fully under- ment of the spine. Mutation in the Runx3 transcription stood, a recent study has shown that connectivity factor, which is required for the development of muscle between preBötz and VRG neurons rely on a common proprioceptive sensory neurons (pSNs) [76], leads to a transcription factor, Dbx1 [72]. Expression of Dbx1 is progressive scoliosis of the spine (Fig. 4b-d)[77]. This absent from MNs, suggesting other intrinsic factors are phenotype does not appear to be a consequence of a re- involved in establishing connectivity between VRG and quirement for Runx3 function in other tissues, since axial MNs. Connections between brainstem respiratory similar results were observed after Runx3 deletion spe- centers and spinal MNs could rely on actions of cifically from pSNs. Although how this mutation affects segmentally-restricted fate determinants, such Hox the circuits involved in spinal stabilization is unclear, it genes, which differentiate PMC and HMC from other is likely due to altered connections between pSNs and spinal MN subtypes (Fig. 4a)[73]. the axial motor circuits essential for maintaining pos- While motor circuits controlling breathing and loco- ture. Loss and gain of function studies have shown that motion rely on rhythmically active neural circuits, the Runx3 is required for the ability of pSNs to establish bd c Fig. 4 Diverse function of axial motor circuits in tetrapods. a Simplified diagram of respiratory networks for inspirational breathing. Rhythm generation in the preBötzinger (preBötz) complex is relayed to rostral ventral respiratory group (rVRG) neurons. rVRG neurons target PMC neurons and HMC neurons in the spinal cord. Connections between preBötz and rVRG neuron relies on Dbx1 gene function. b-d Role of axial motor circuits in spinal alignment. b Axial muscles and nerves associated with vertebrae. Box indicates region magnified in panel c. c Consequences of Runx3 mutation on the projection of proprioceptive sensory neurons in the spinal cord. Loss of Runx3 leads to a loss of projections to MNs, and likely other classes of spinal interneurons. d Effect of Runx3 mutation on vertebral alignment in adult mice D’Elia and Dasen Neural Development (2018) 13:10 Page 9 of 12 connections with MNs and other neural classes [77–79], suggesting that the Runx3 mutant phenotype is due to a the disruption of local sensory-motor spinal reflex cir- cuits. In addition, mutations that affect the function of the MMC-restricted transcription factor Mecom also causes abnormal bending of the spine [80], raising the possibility that this phenotype is also consequence of al- tered connectivity between axial MNs and premotor neural populations. Developmental mechanisms of axial motor circuit assembly in tetrapods The distinct use of MMC neurons in locomotion and posture, while HMC and HMC-like MNs are essential for breathing, raise the question of how premotor cir- cuits dedicated to specific motor functions target the ap- propriate axial MN subtype. While the answer to this question is largely unknown, studies characterizing the distribution of spinal interneurons connected to specific MN columnar subtypes have provided a partial answer. Rabies-based monosynaptic tracing of interneurons con- nected to MMC and HMC neurons revealed that axial MNs receive local spinal premotor inputs that are evenly distributed across both sides of the spinal cord (Fig. 5a). In contrast, limb MNs receive inputs predominantly from premotor interneurons on the ipsilateral side of the Fig. 5 Developmental mechanisms of axial motor circuit assembly. a spinal cord [81]. Axial MN dendritic arborization pat- Dendritic morphology and premotor input pattern for MN columnar terns are also distinct from those of limb MNs, which subtypes. MMC neurons have dendrites that extend across the midline may help determine their specific connectivity with pre- and their monosynaptic premotor inputs are distributed across both sides of the spinal cord. Like MMC neurons, HMC neuron dendrites motor interneuron populations (Fig. 5a). MMC neurons extend medio-laterally and have a similar premotor input distribution have dendrites that extend across the midline, which ap- pattern. LMC neurons have radially organized dendrites and receive pears to enable them to capture a greater proportion of premotor inputs predominantly from ipsilateral spinal interneurons. inputs from contralateral interneuron populations, and Darker shading indicates higher density of interneurons connected to establish connectivity with interneurons distinct from MNs. b Effect of Hoxc9 mutation on premotor input pattern. In Hoxc9 mutants, thoracic HMC neurons are converted to LMC fate, while MMC those of HMC neurons. In contrast, limb-innervating neurons are grossly unaffected. In Hoxc9 mutants, ectopic LMC neurons LMC neurons are found in more lateral and dorsal re- still project to intercostal muscle. The dendritic pattern of thoracic MNs gions of the spinal cord and have radially-projecting in Hoxc9 mutants becomes more limb-like, and MNs projecting to dendrites, which may afford them greater input from ip- intercostal muscle receive a higher distribution of inputs from ipsilateral silateral interneuron populations. premotor interneurons. Diagram based on data in [84] Do the molecular identities and/or positional differ- ences between MN subtypes determine their premotor input pattern and function? The ability to genetically patterns and types of synaptic inputs that MNs receive alter the composition of MN subtypes within the mouse [84]. Transformation of thoracic HMC neurons to a spinal cord provides evidence that MN subtype identity limb-level LMC fate, through mutation of the Hoxc9 plays an important role in determining the functional gene [85], shifts spinal premotor inputs to predomin- properties of spinal circuits. Conversion of limb MNs to antly ipsilateral populations (Fig. 5b). In Hoxc9 mutants, an axial HMC fate, through deletion of limb MN deter- the transformed HMC populations also settle in a more minant Foxp1, leads to the loss of limb-specific motor dorsolateral position, and their dendrites project radially, output patterns [82, 83]. In the absence of Foxp1, the similar to those of limb-innervating MNs (Fig. 5b)[84]. normal alternation of limb-flexor and -extensor firing While these studies do not resolve the basic question of patterns is lost, and the remaining HMC-like popula- how differences between HMC and MMC inputs are tions fire in a predominantly flexor-like pattern. achieved, they suggest that intrinsic differences between Recent studies also indicate that determinants of MN MN molecular identity, dendritic morphology, and pos- columnar identity play crucial roles in defining the ition contribute to shaping the pattern of connection D’Elia and Dasen Neural Development (2018) 13:10 Page 10 of 12 within the motor circuits. How these genetic manipula- Abbreviations CaP: Caudal primary motor neuron; CPG: Central pattern generator; dpf: Days tions affect the function of axial motor circuits remains post fertilization; dRoP: Dorsal rostral primary motor neuron; ei: External to be determined. Nevertheless, analyses of Foxp1 and intercostal muscle; eo: External oblique muscle; ESC: Embryonic stem cell; Hoxc9 mutants indicate that the columnar identity of FGF: Fibroblast growth factor; FGFR1: Fibroblast growth factor receptor 1; HMC: Hypaxial motor column; hpf: Hours post fertilization; ii: Internal spinal MNs plays a significant role in determining the intercostal muscle; ilio: Iliocostalis muscle; lc: Levator costae muscle; architecture and output patterns of spinal circuits. LMC: Lateral motor column; long: Longissimus muscle; MiP: Middle primary motor neuron; MMC: Medial motor column; MN: Motor neuron; PGC: Preganglionic motor column; PMC: Phrenic motor column; pMN: Motor Conclusions neuron progenitor; pSN: Proprioceptive sensory neuron; sc: Subcostalis Studies on the development of neural circuits controlling muscle; Shh: Sonic hedgehog; sr: Caudal serratus muscle; axial muscles have provided valuable insights into how tv: Transversospinalis muscle; VRG: Ventral respiratory group; vRoP: Ventral rostral primary motor neuron specific motor functions develop and have evolved in the vertebrate lineage. While we have a fairly in depth un- Acknowledgements derstanding of the genetic programs controlling the spe- We thank David McLean and anonymous reviewers for feedback on the text. cification of tetrapod axial MN subtypes, how these Funding functionally diverse populations are connected to appro- Work in the lab is supported by grants R01NS062822 and R01NS097550 from priate higher order circuits remains to be determined. the NIH. Recent studies showing that MN-intrinsic programs contribute to differences in the patterns of premotor Authors’ contributions JD and KD wrote the manuscript and prepared the figures. Both authors connectivity between limb and axial MNs suggests a read and approved the final manuscript. general mechanism through which motor circuits are as- sembled, as a function of molecular differences in their Ethics approval and consent to participate target MN populations. Further functional studies on Not applicable. the consequences of disrupting MN differentiation could Competing interests provide a means to test the role of MN subtype identity The authors declare that they have no competing interests. in the development of axial circuits essential for breath- ing and spinal alignment. Publisher’sNote Comparisons between species that use axial MNs for dis- Springer Nature remains neutral with regard to jurisdictional claims in tinct functions have provided insights into how different published maps and institutional affiliations. motor behaviors are specified during development. Al- Received: 14 March 2018 Accepted: 26 April 2018 though this review has focused on vertebrate development, many of the intrinsic molecular features of axial MNs ap- pear to be conserved in invertebrates. Similar to verte- References brates, in Drosophila and C. elegans subtypes of MNs can 1. Kiehn O. Decoding the organization of spinal circuits that control locomotion. 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