Molecular and cellular development of spinal cord locomotor circuitry

1More than 20 distinct embryonic classes of neurons have been described in the spinal cord, and the developmental sources of their diversity have been elucidated over the past decade (Figure 1). This cellular diversity has been organized into a schema that defines major groups of neurons based on their expression of embryonic transcription factors. The major characteristics of these classes their generation, transcription factors, subsets, positions, neurotransmitters, connections, and functions are summarized here.

2Spinal cord development is subject to phylogenetically ancient organizing principles such as those that guide segmentation from the invertebrates, such as arthropods, to the vertebrates, such as mammals. Cellular identities in vertebrate spinal cord are specified during development along the three basic spatial axes of the embryonic body plan – rostral–caudal, dorsal–ventral, and medial–lateral. In addition, there is a temporal influence of development on these spatial coordinates such that distinct cell fates emerge at different times during development. This yields a four dimensional system for establishing spinal neuron cell fate that has been reviewed extensively.

3To summarize briefly, the rostral–caudal positional identities are coordinated by opposing gradients of fibroblast growth factor (Fgf, caudalizing) and retinoic acid (RA, rostralizing; Figure 2;;; ). The dorsal–ventral axis is governed by ventralizing Sonic hedgehog (Shh) produced by the floorplate, and dorsalizing signals from the roof plate such as bone morphogenetic proteins (BMPs) and Wnts (which are members of the Wingless + MMTV integrants, Int family). These diffusible morphogens form gradients that activate specific transcriptional responses at defined points in the gradient. These transcriptional programs first specify and reinforce the identities of progenitor cells, and second, act to oppose adjacent transcriptional programs and sharpen boundaries between progenitor zones. In the ventral cord, these transcription factors are grouped into two classes, those that are inhibited by Shh (Class I) and those that are activated by Shh (Class II; ). Spinal cord development is also organized along a medial–lateral axis dividing progenitor cells that are located adjacent to the lumen of the neural tube, medially, whereas differentiating progeny migrate laterally. Over time, a given progenitor domain defined by these spatial coordinates may sequentially produce distinct cellular classes.

4Within an idealized spinal cord segment, this system establishes thirteen progenitor pools along the dorsal–ventral axis (Figure 2). There are eight dorsal interneuron progenitor divisions, pd1–6 and the late-born pdILA and pdILB, four ventral interneuron progenitor divisions, p0–3, and one motor neuron progenitor domain, pMN. The identities of these domains are predominantly defined by basic-helix-loop-helix (bHLH) domain transcription factors, such as Ngn, Olig2, and Math (;;;; and homeodomain proteins, such as Pax3, Dbx1, and Nkx6.1 (;; Subsequently, additional transcription factors, predominantly of the LIM-homeodomain family, such as Lhx1 and Isl1, are expressed in sub-groups of these domains, further refining cell fate into at least 23 distinct classes.

5The pMN cell domain gives rise to: (1) 100s of genetically distinct groups of cholinergic alpha motor neurons clustered into motor pools that innervate specific skeletal muscles; (2) gamma motor neurons that innervate intrafusal fibers of specific skeletal muscles for proprioception; (3) the predominantly thoracic (T1–12, and to L1 and L2 in some species) cholinergic preganglionic sympathetic neurons; (4) the cholinergic parasympathetic motor neurons in the sacral (S2–4) cord; and (5) oligodendrocytes found throughout the spinal cord (Figure 3). The motor neurons that effect muscle movement are primarily alpha, with fewer beta, motor neurons.

6The recognition that a supernumerary notochord could induce the generation of additional motor neurons led to the identification of the diffusible morphogen, Shh, that induces neural precursors to a MN fate. An early marker of motor neuron development, the LIM-HD transcription factor, Isl1, indicated that motor neuron precursors are born between HH Stages 15 and 17 in chick and beginning at E9.5 (~25 somites) in mouse (Figure 4A;;;; ). These progenitors will give rise to somatic alpha motor neurons that innervate skeletal muscle in the medial and lateral motor columns (LMCs), gamma motor neurons that innervate intrafusal fibers of the muscle spindles, and preganglionic motor neurons of the autonomic nervous system. Generation of each of these classes and their organization into motor columns and motor pools requires the subsequent expression of additional transcription factors. These factors then drive the unique characteristics of that motor pool, such as guidance to the target and establishment of proper connectivity with sensory neurons and interneurons. Interestingly, this transcriptionally defined program is complemented by activity-dependent processes that control cellular connectivity and function. In the case of gamma motor neurons, the nuclear receptor Errγ is expressed in these motor neurons and their survival is dependent on GDNF signaling.

7The motor neuron progenitor domain is ventral to the Irx3 expressing p2 domain that delimits Olig2 expression and is dorsal to the p3 domain that expresses Nkx2.2 and Nkx2.9 to delimit Pax6 expression. The expression of Nkx6.1 and Nkx6.2 acts to limit transcription factor expression to Olig2, that in turn drives the expression of MN transcription factors Hb9 (Mnr2 in chick), and Ngn2. Hb9, expressed during the final cell division of pMNs, is sufficient to drive the expression of Isl1, Isl2, Lhx3, and ChAT—as well as its own expression—establishing pMN independence from Shh. Like Mnr2, the HD transcription factor, Hb9, can induce the formation of motor neurons when ectopically expressed. Loss of Hb9 in mouse, however, results in ectopic upregulation of a V2 IN marker gene, Chx10, but does not result in complete loss of motor neurons or of fictive locomotion (;; Alaynick, Pfaff unpublished observations).

8The medial sub-group of motor neurons innervates axial musculature and is found the length of the spinal cord. There are two divisions of this group, the medial motor column (MMC) and the hypaxial motor column (HMC or MMCl). Both express Isl1 and Isl2, although the ratio of expression varies, with greater Isl1 expression in the HMC than MMC at E11.5 and greater Isl2 in the MMC than HMC by E13.5 in mouse. The MMC innervates dorsal or epaxial musculature, while HMC innervates ventral or hypaxial musculature. Initially all motor neuron progenitors express the LIM homeodomain transcription factor, Lhx3. Lhx3 expression is maintained in the MMC while Lhx3 expression is downregulated in the HMC and LMC. Motor neuron (Hb9 promoter) dependent expression of Lhx3 results in conversion of LMC motor neurons to a MMC identity.

9At limb levels, the 50 or so muscles of the limb are innervated by motor neurons occupying a lateral motor column. Neurons of the lateral portion of the LMC (LMCl) are later born than the MMC motor neurons, and like the cortex, migrate in an inside-out arrangement such that LMC neurons are born in the proliferative ventricular zone of the pMN domain and then migrate through the MMC to form the LMC. While initially expressing Lhx3, a hallmark of MMC identity, these motor neurons down-regulate Lhx3 by an unknown mechanism and begin to express transcription factors not found in MMC that are definitive for LMC identity. The factors include Foxp1, Lim1, and the enzyme Raldh2. The lateral motor column has lateral (LMCl) and medial (LMCm) divisions that innervate the dorsal and ventral portions of the limb, respectively, and these cell fates are partially regulated by RA signaling. In the LMCl, Lim1 and Hb9 are expressed while Isl1 is downregulated. In the LMCm, there is low Hb9 and maintained Isl1 expression. The LMCm and LMCl both express Isl2, which is downregulated in the MMC and HMC. The LMCm and LMCl are further subdivided into motor pools, each innervating a specific muscle of the limb. These individual motor pools are defined by their expression of Ets and Nkx transcription factors that constitute a more refined transcriptional code.

10The rostro-caudal regions of the LMC appear to be determined in part by homeobox (Hox) genes. Hox6 is characteristic of brachial level, Hox9 of thoracic and Hox10 of lumbar. Disruption of the Hox genes in mouse or chick has shown that these boundaries can be profoundly altered to create an expansion of lateral motor columns into thoracic regions. More strikingly, loss of the Hox co-factor Foxp1 disrupts the ability of motor neurons to incorporate the homeobox code for spatial information, and results in a loss of defined motor pools in the LMC.

11Preganglionic motor neurons of the sympathetic nervous system are the most dorsal motor neurons and can be identified by their expression of ChAT, NADPH Diaphorase, and some members of the one-cut transcription factor class. Preganglionic motor neurons are also dependent on the downregulation of Lhx3 and are lost with continued, Hb9-dependent expression of Lhx3 in all motor neurons, or loss of Foxp1 or Isl2.

12A great deal has been learned about the development of discrete classes of interneurons by describing them by electrophysiology, behavioral output, and by expression of proteins involved in transcription, neurotransmitter signaling, and intracellular signaling. Currently, this schema has defined over 20 interneuron types in the spinal cord. While one can argue that every neuron has a unique molecular/genetic expression profile, dendritic arborization and axonal projection pattern, this grouping schema has been useful in organizing interneurons into functionally related groups.

13Historically, two broad groups have been defined: the “V” interneurons with progenitors that are found in the ventral cord and are grossly associated with motor function, and a dorsal Interneuron, dI class, associated predominantly with sensory processing. Most studies have examined development within a single or a few segments. A recent study examined rostro-caudal differences at one time point, e12.5. This showed that subclasses of ventral interneurons (V0, V1, V2, and V3) exhibit distinct organizational patterns at brachial, thoracic and lumbar levels of the developing spinal cord. Furthermore, each cardinal “V”’ class of ventral interneurons can be subdivided into several subsets according to further combinatorial expression of transcription factors. Given these caveats that likely apply to other interneuron classes, the V and dI interneuron classifications are a simplification with exceptions, some of which are listed below. Despite these limitations, the V and dI schema is a useful approach to the subject.

14Local projecting V0 neurons are a population of primarily contralateral, with some ipsilateral projecting neurons with inhibitory or excitatory identity that send axons 2–4 spinal segments rostrally. They receive inputs from ipsilaterally projecting Chx10+ glutamatergic V2a interneurons (; Figure 5). They are the dorsal-most ventral progenitor pool and are characterized by their expression of the Dbx (developing brain homeobox) homeodomain transcription factor, Evx1/2 (even-skipped homeobox 1; Figure 4B). Dbx1 and Dbx2 are expressed in dividing cells, although Dbx1 may be briefly expressed in post-mitotic cells (see V1 discussion ). Four V0 interneuron subclasses have been described to date: V0V, V0D, V0C, and V0G. Early studies addressed the V0 class by eliminating Dbx1 and showing that the Evx1+ V0V subclass was lost because these neurons become fated to an En1+ V1-like subclass and astrocytes. Because Dbx1 is transiently expressed, a Dbx1LacZ knock-in allele was used to show that with loss of Dbx1, E18.5 embryos retained 40% of the βgal+ cells and resulted in a 25% expansion in the number of Lbx1+ Pax2+ dl6-like commissural neurons. By perinatal time points, genetic strategies to track Dbx1+ cells using βgal find that most of these cells are neural by expression of NeuN and are found in lamina VIII where commissural interneurons reside. Lineage labeling of Dbx1-derived cell reveals a large abundance of glia. Moreover, the Dbx1 lineage includes many dorsal horn neurons as this transcription factor is also expressed in dorsal domains. Loss of Dbx1 results in loss of V0D and V0V subclasses, whereas loss of Evx1 results in a loss of only the V0V subclass. V0 and V1 classes both express Lhx1 and Lhx5, markers of inhibitory spinal interneurons.

15In mouse, the majority of Dbx1+ progenitors appear between E10 and E13 and give rise to V0D and V0V commissural interneurons. Dbx1/2 expression is found in the rostral CNS at stage 13 in chick and more caudally by stage 15. Evx1/2 positive V0 cells are generated at stages 17 and 18 and appear in the ventral domain of Dbx1 and Dbx2 expression. Ventral Evx1/2 expressing V0 neurons appear at stages 17–18 within the ventral expression domain of Dbx1 and Dbx2, and then migrate ventrally. The V0 class appears from a Pax6+, Dbx1/2+, Pax3/7- domain that is the dorsal-most ventral progenitor domain.

16The primarily inhibitory V0V class is distinguished by transient expression of the homeodomain transcription factor, Evx1. These cells arise from the ventral portion of the Dbx1+ progenitor domain, and like all post-mitotic cells arising from Dbx1+ progenitors, they share a similar post-mitotic migration and commissural axon pattern. The V0V interneurons are implicated in locomotion as indicated by increased c-fos immunoreactivity following fictive locomotion. However, Evx1 knockout mice have grossly normal locomotion patterns despite a ~70% reduction in the V0V interneurons and loss of appropriate contralateral intersegmental axonal projections in the remaining ~30% of interneurons. A subset of the V0V class has been reported to be excitatory in an unpublished observation.

17Unlike the V0V subclass, the more dorsal Dbx1+ progenitors of the glycinergic/GABAergic V0D class do not express Evx1. And while both V0D and V0V classes have similar axon guidance and cell body position, the loss of the V0D class, in conjunction with V0V class, does appear to alter locomotor behavior. When Dbx1 is knocked out, eliminating all V0 progenitors, a disruption of left–right coordination is observed at lumbar levels L2 and L5. These periods of left–right synchrony are intermittent and periods of normal left–right alternation are observed amidst episodes of synchrony. No disruption of flexor-extensor behavior, as indicated by alternating phasic activity of the L2 and L5 segments, was observed in a drug-induced isolated cord fictive locomotion assay. Recently, studies have showed that a cluster of V0D cells lateral to the central canal receive substantial input from primary afferents and preferentially project axons toward contralateral motoneurons via an oligosynaptic pathway, and are active during fictive locomotion. This suggests that this subset of V0 interneurons may be primarily responsible for coordination of left–right alternation during locomotion.

18The V0C and V0G subclass represent ~5% of V0 progenitors and are identified by expression of Pitx2 and occupy a medial position dorsal to the central canal. These cells were first observed in lumbar levels at E11.5–12.0 by Pitx2 immunoreactivity which, unlike many embryonic markers, could be detected until postnatal day 30. Neurotransmitter markers can subdivide the Pitx2+ cells into cholinergic (vAChT+ and ChAT+) and glutamatergic (vGluT2+) types that are distinct. While these are found at cervical and lumbar levels, within the lumbar cord, these two types are distributed in a gradient such that a greater number of cholinergic interneurons are found at more rostral levels and a greater number of glutamatergic interneurons at more caudal levels. The cholinergic cells are distinct from Pitx2- cholinergic C3 propriospinal interneurons. By genetic tracing, ~80% of these neurons were determined to be from a Dbx1+ progenitor domain at E12.5 and loss of Dbx1 eliminated the Pitx2 immunoreactivity in the intermediate cord. Because V0C and V0G Pitx2+ cells transiently express Evx1, they appear to be subsets of the V0V class. This relatively small ipsilaterally and bilaterally projecting class, however, is responsible for perhaps all c-boutons on motor neurons found in P8 to P25 mice. These interneurons provide relatively weak innervation to Sox14::eGFP+ V2a and calbindin+ V1 Renshaw cells. These cells appear to be involved in local circuitry as corticospinal and sensory vGluT1+ glutamatergic boutons were not found, whereas serotonergic and GAD67+ GABAergic boutons were observed. While previous experiments did not find a gross locomotor behavioral defect with loss of the V0V subclass in Evx1 mutant animals, Pitx2 mutant animals were found to have defects in locomotion revealed by EMG recordings during swimming. This deficit was argued to represent an abnormal integration of sensory inputs. It may, alternatively or in addition, represent a deficit in C-terminal modulation of motor neuron excitability. A survey in E12.5 mice showed that several V0 subclasses can be defined by expression of Pax2, Pax6, Evx1, Ptx2, Nurr1, HNF-6, BhlhB5, and Prdm8.

19As a population, this group appears to control burst durations and is comprised of cells physiologists defined as Ia inhibitory and Renshaw cells. Mice models without V1 and V2b showed significant difficulty with limb articulation in flexion and extension. The pV1 progenitor domain gives rise to important inhibitory subclasses of neurons that were previously described electrophysiologically: the Ia inhibitory interneurons that mediate reciprocal inhibition and the Renshaw cells that mediate inhibitory feedback to integrate limb and muscle length information into spinal circuitry. Renshaw cells and Ia inhibitory interneurons are V1 derived, but differ in morphology, location, calcium-binding protein expression, synaptic connectivity, and function. These differences are already present in neonates and their differentiation starts in the early embryo. In addition, 75% of V1 interneurons are non-Ia, non-Renshaw subclasses that await characterization. Short-range ipsilaterally and rostrally projecting glycinergic/GABAergic V1 neurons are characterized by transient expression of homeodomain transcription factor En1 (Figure 4C;;;,; ). Studies in embryonic chick indicate that these neurons project for only 1–2 segments and have been shown to make inhibitory contacts onto motor neurons and other interneurons, although this may not be the case in the mature mouse. Loss of Pax6 or En1-dependent DTA ablation eliminates the recurrent inhibition by Renshaw cells on motor neurons. Elimination of V1 interneurons results in a marked slowing on the drug-induced fictive locomotion period that is seen in conventional knockouts, targeted ablation, and acute inhibition with allatostatin. The mechanism by which elimination of an inhibitory class would prolong the locomotor cycle remains unknown and may result from the loss of inhibitory neurons to terminate MN firing. The V1 class, like V0, expresses the inhibitory spinal interneuron markers Lhx1 and Lhx5.

20Unlike cells in the dorsal-most p0 domain that expresses Dbx1 and Dbx2, the adjacent p1 domain only expresses Dbx2. The V1 class appears from a Pax6+, Dbx2+, Nkx6.2+, Dbx1- domain that is ventral to the Dbx1/2+ V0 domain. In chick, En1+, Lim1/2+ V1 neurons appear at stage 17, and most appear ventral to the domain of Dbx1 expression, within the ventral domain of these Dbx2+, Dbx1- progenitors. Dbx expression does not overlap with En1, perhaps due to the relatively late expression of En1. Genetic tracing studies using Dbx1nlsLacZ mice between ages E10 and E16.5 found that ~5–10% of En1+ cells did express a low level of βgal, perhaps a reflection of transient Dbx1 expression and more enduring βgal protein. The V1 class is marked by expression of Foxd3, found in the dI2 domain, as well. The transcription factor, Bhlhb5, which marks the V1, V2 and dI6 domains, is required at least partially for V1 identity assessed by En1 expression. Expression of Bhlhb5 in conjunction with Ngn2 facilitates V1 identity ectopically.

21Renshaw cells use both glycine and GABA as neurotransmitters, transiently express Gad65 early in embryonic development and have both motor neurons and Ia interneurons as targets. They also express calbindin D28K embryonically and continue to express this marker into adulthood. They receive input from motor neuron collaterals that release acetylcholine, glutamate, and aspartate. Renshaw cells modulate proprioceptive sensory input and motor neuron output. Genetic tracing studies showed that Renshaw cells are derived from an En1+ progenitor pool and, although they are not lost in the absence of En1, they do have fewer motor neuron recurrent inputs. They are, however, lost in the absence of Pax6. Recent study showed that selective activation of the Onecut transcription factors Oc1 and Oc2 during the first wave of V1 interneuron neurogenesis is a key step in the Renshaw cell differentiation; furthermore Renshaw cell development is dependent on the forkhead transcription factor Foxd3, which is more broadly expressed in post-mitotic V1 interneurons.

22Although Ia interneurons have been rediscovered as a V1 subclass, like Renshaw cells, the Ia INs were functionally described before the advent of molecular genetic dissection of interneuron development These inhibitory glycinergic cells receive input from muscle spindle Ia proprioceptive afferents carrying muscle length information and provide inhibitory input onto motor neurons innervating antagonist muscles. Like motor neurons, Ia receive inhibitory inputs from Renshaw cells. In neonatal mice, disynaptic glycinergic reciprocal inhibition is mediated by Ia interneurons, although this activity is preserved in the absence of Pax6, indicating that cells of more than one origin contributes to this functional class. Only when V1 and V2b are both ablated is reciprocal inhibition profoundly altered. Renshaw cells constitute 8–19% of V1 interneurons and the Foxp2+ (by immunohistochemistry) population accounts for around 33% of these neurons at P0 and 50% at E13. Because there are no universal markers of Ia interneurons, all Ia interneurons cannot be accounted for, leaving the physiologic properties and connectivity patterns of V1 interneurons unaccounted for. Of note, some interneurons with synaptic organization like Ia interneurons have been found that arise from the V1 population and are Foxp2 positive. A survey in E12.5 mice showed that several V1 subclasses can be defined by expression of Calbindin, OC1, OC2, OC3, Foxd3, En1, MafB, FoxP2, Foxd3, Foxp4, Pax2, Arx, Evx1, Nurr1, BhlhB5, Pou4F1, Pou3F1, and Prdm8.

23V2 interneurons become divided into V2a and V2b classes of ipsilaterally projecting interneurons that extend axons caudally across several segments. The excitatory V2a class is glutamatergic and expresses Chx10, while the Gata2/3+ V2b class is inhibitory and uses both glycine and GABA (Figure 4D;; ). The transcription factor, Bhlhb5, marks the V2, as well as V1 and dI6 domains.

24V2 interneurons arise from a progenitor pool just dorsal to the pMN domain and share expression of Lhx3 with the pMN domain. In addition, both domains share expression of NLI that forms homodimers. This NLI homodimer nucleates the formation of a higher-order tetramer with Lhx3 in the V2 progenitor domain, and in the case of pMNs this V2-defining tetramer (Lhx3-NLI-NLI-Lhx3) is disrupted by the insertion of Isl1 to form a hexamer (Lhx3-Isl1-NLI-NLI-Isl1-Lhx3). Transcriptional response elements that are active in V2 cells can bind both the motor neuron hexamers and the V2 associated tetramers, while response elements active in motor neurons are only responsive to the hexamers. Later the V2 domain expresses Chx10 that acts as a repressor of motor neuron associated hexamers in V2 progenitors, leaving only the LIM tetramers active. The progenitor pool of V2 neurons becomes post-mitotically segregated into V2a and V2b neurons.

25Time-lapse imaging in zebrafish showed that the majority of V2 progenitors give rise to a pair of V2a and V2b cells, indicating that V2a and V2b arise from the same progenitor. This segregation into V2a and V2b is mediated by Notch/delta signaling in zebrafish and mouse models. In mouse, Delta4, but not Delta 1, activates this signaling cascade and is downstream of Foxn4, which also induces expression of Mash1/Ascl1. Mind bomb-1 (Mib1) is an E3 ubiquitin ligase that ubiquitinates and promotes the endocytosis of Notch ligands. In mice model, Mib1 plays an important role in Notch activity and specific differentiation, neurogenesis and gliogenesis of V2 interneurons. Mice models with abnormal Mib1 resulted in unclear spinal progenitors, premature or unbalanced differentiation or loss of astrocytes and oligodendrocytes. In zebrafish embryos two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance; on the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. showed Foxn4 and proneural factors may serve as the trigger to initiate asymmetric Dll4-Notch and subsequent BMP/TGFβ signaling events required for neuronal diversity in the V2 domain. V2b fate is specified by active Notch1, Foxn4, Mash1, and Scl Notch-binding protein MAML is also required for this specification. Lack of active Notch1 results in V2a fate, shown in an increase of V2a interneurons at the expense of V2b in Psn1 KO mice or Notch1 KO mice. Transcription factor Gata2 is necessary in the normal development of V2a and V2b neurons and Gata2 promotes the selective activation of V2b at the expense of V2a fate. Progenitors that express the notch ligand, Delta-like 4 generate almost all V2a and V2c neurons while producing only a small fraction of neurons of other subtypes along the dorsoventral axis.

26The V2a class of ipsilaterally projecting interneurons expresses the transcription factors Chx10 and Sox14 and is glutamatergic. These interneurons are composed of cells with diverse firing properties and morphologies with local as well as long-range ipsilateral projection patterns. This class of interneurons has been shown to contact motor neurons and contralaterally projecting V0 interneurons. Loss of these cells has been shown to disturb locomotor function in a state-dependent manner. In a series of in vitro experiments it was found that Chx10-DTA V2a-ablated mice displayed more variable amplitude and period than wild-type controls during drug-induced fictive locomotion. Further, these mutant animals had incoherent left–right alternation during drug-induced fictive locomotion. Surprisingly, these animals failed to display coordinated brainstem stimulated or dorsal root stimulated fictive locomotion, suggesting that Chx10+ cells mediate descending and sensory activation of locomotor activity.

27A subsequent study, using a different strain of mice that avoided the neonatal lethality seen in previous work, showed that during treadmill running, Chx10-DTA mice can transition from alternating locomotion to synchronous hindlimb locomotion at higher speeds. High-speed synchronous left–right activity, or galloping, is not normally seen in mice, although it has been described in studies of Eph and ephrin signaling molecule mutant mice. The Eph/ephrin mutant mice, however, have synchronous activity at both slow and fast speeds. Some V2a interneurons express EphA4, but a compelling correlation has yet to be discovered. In zebrafish, alx, a zebrafish homolog of Chx10, is expressed in an ipsilateral descending excitatory interneuron population named CiD (circumferential descending) neurons that monosynaptically contact motor neurons. This population has been shown to be active during high-frequency swimming in larval zebrafish. Within this interneuron class, dorsally located cells are recruited at a high swimming frequency. As the frequency decreases, more ventral cells are recruited, accompanied by silencing of previously active dorsal cells. A survey in E12.5 mice showed that V2a subclasses can be defined by expression of BhlhB5, Pou3F1, OC1, OC2, OC3, Prdm8, MafA, and cMaf.

28Ipsilaterally projecting V2b interneurons express Gata2/3, are inhibitory GABAergic neurons, and appear to make direct connections onto motor neurons. Observations by the Goulding lab indicate they project caudally. These cells may underlie the retained reciprocal inhibitory pathways seen in V1 knockout mice. A survey in E12.5 mice showed that V2b subclasses can be defined by expression of BhlhB5, Pou3F1, OC1, OC2, OC3, Prdm8, MafA, and MafB. As pointed out earlier, V1- and V2b-derived neurons function as the core interneuronal components of the limb central pattern generator (CPG) that coordinate flexor-extensor motor activity.

29The V2 interneuron class has recently been shown to further diverge to a Sox1-expressing Gata3-negative population named V2c interneurons, function of which is still yet to be elucidated. A survey in E12.5 mice showed that V2c subclasses can be defined by expression of Sox1, OC1, OC2, and OC3.

30The Sim1+ VGluT2+ glutamatergic V3 interneurons send projections predominantly contralaterally and caudally. Genetic tracing, using a Sim1-eGFP or Sim1Cre and reporter lines, and viral tracing, using pseudorabies, shows that 80–85% of these cells project contralaterally and a minor proportion remain ipsilateral or project both contra- and ipsilaterally. As a population, Sim1+ V3 interneurons form 24% of glutamatergic connections on V1 Ia, 27% on Renshaw subclasses, 22% of glutamatergic synapses on lateral motor column motor neurons, as well as connections on Lhx3+ V2 interneurons, and lamina VIII commissural interneurons. Behaviorally, loss of V3 neuronal activity by genetic attenuation with tetanus toxin or allatostatin signaling resulted in a loss of CPG robustness. In isolated cord fictive locomotion, both dorsal root stimulation and drug-induced methods produced weak CPG activity in only some of the cords examined. The outputs were less consistent and had greater coefficients of variance. Although both right and left sides of the cord produced irregular outputs, the fidelity of left–right coordination was preserved suggesting that V3 interneurons do not regulate the coordination of left–right activity. In adult Sim1Cre AlstR192 animals, application of allatostatin to the cord produced locomotor disturbances in gait, as well. In Sim1 mutant mice, V3 interneurons are produced normally and maintain in the similar position and organizations as wild-type; however, there is significant reduction of interneurons in dorsal subgroup and there is significant reduction in the contralateral axonal projection. Therefore, Sim1 appears to be critical in migration and axonal projection of V3 interneuronal development. Mice that are mutant for Nkx2.2 and Nkx2.9 lose V3 interneurons and Nkx2.2+/- Nkx-/- mice display intermittent or permanent hopping gait. indicate that this mutation affects floor plate, and therefore likely affects commissural interneuron projections that mediate left–right coordination. A survey in E12.5 mice showed that V2c subclasses can be defined by expression of Olig3, Prox1, BhlhB5, and Nurr1.

31These V3 interneurons arise from the ventral-most p3 progenitor domain defined by homeobox transcription factors Nkx2.2 and Nkx2.9 and the PAS-bHLH transcription factor Sim1 (simple-minded homolog 1; Figure 4E;; ). Genetic tracing techniques using a Sim1TauLacZ knock-in reporter mouse or Sim1Cre and reporter lines (R26floxstop-GAP43-GFP and R26floxstop-lacZ) have shown similar expression at E11.5 to in situ hybridization data for Sim1 expression that appeared just lateral to the Nkx2.2 progenitors. Nkx2.2 also regulates the expression of Olig3 in V3 neurons. While Olig3 plays a key role in respecification of dl2 and dl3 neurons into dl4 interneurons in dorsal spinal cord (see below), it does not appear to affect the generation and migration of the ventral neurons.

32Each class of interneurons can likely be further subdivided. The existence of V3 subtype heterogeneity defined by cell body positions was first reported in a review of locomotor circuitry by the Goulding group. This group recently examined both electrophysiological and morphological properties of mature V3 interneurons in adult mouse and were able to identify two V3 subpopulations with distinct intrinsic properties and distributions (ventral and dorsal), as well as an important intermediate subgroup. They reported V3V, primarily located in lamina VIII, possessed a few branching processes and were capable of generating rapid tonic firing spikes and V3D had a more complex morphology with relatively slow average spike frequency with strong adaptation. A survey in E12.5 mice showed that V3V express Olig3, Prox1, BhlhB5, and Nurr1, and V3D can be defined by expression of OC1, OC2, and OC3.

33A group of glutamatergic, rhythmically active interneurons with possible connections to motor neurons can be found along either side of the ventral midline in thoracic and upper lumbar segments. These Hb9+ and VGluT2+ interneurons are found in lamina VIII, although the developmental origin of these cells is unknown (Figure 4F). These cells have oscillatory behavior, make potential contacts with motor neurons, and are associated with motor rhythms. These interneurons were the first to show oscillatory properties and efforts have been made to discover a relationship to rhythm generation or a pacemaker property for the CPG. No cell class, however, has been found to act as a pacemaker for CPG activity. Remaining questions for the VX include: what is the progenitor domain that gives rise to the VX domain; and why are they not found below the L2 segment at E18.5.

34There are eight canonical classes of dorsal progenitors, dI1–6 and dILA and dILB. Of these, the dorsal-most dI1–3 progenitors are dependent on signals from the roof plate and termed Class A. The remaining dI4–6 and dILA and dILB are independent of roof plate signals and termed Class B. The dorsal-most progenitors, pd1–pd3, are born between days E9.5 and 10.5 and become post-mitotic and begin to migrate ventrally between E10.5 and E11.5. These cells will eventually form the deeper layers of the dorsal horn. The more ventral Class B dI4–6 cells are born between E10 and 12.5 and then post-mitotically express Lbx1 and migrate either dorsally to form the more superficial layers of the dorsal horn or migrate ventrally to the deep dorsal horn and the ventral spinal cord. The later born dILA and dILB classes are born between E11 and E13 and are inter-mixed with each other. They then migrate dorsally and constitute a significant portion of the cells in the superficial dorsal horn, including the substantia gelatinosa. As with the ventral interneuron classes, each of these classes, or their subgroups, has characteristic features. For instance, each interneuron subclass appears to have a unique axonal projection that produces a tight fascicle within white matter tracts.

35The dorsal-most progenitor domain pd1 expresses the bHLH transcription factor Math1+ (Mouse atonal homolog 1, also known as Atoh1) and gives rise to at least two VGluT2+ glutamatergic subclasses: dI1A and dI1B, characterized by Lim-HD expression and their spinocerebellar tract (SCT) contributions (Figure 4G). Recent study shows that Msx1 and Msx2, two homeodomain transcription factors that are induced earlier than bHLH transcription factors, likely play a role as transcriptional activators of Math1/Atoh1 in spinal cord development. The dI1A (also known as dI1comm) neurons express the Lim-HD transcription factors Lhx2high and Lhx9low, while dI1B (also known as dI1ipsi) express the Lim-HD TF Lhx9. The dI1 interneurons migrate to the deep dorsal horn and intermediate gray where they receive proprioceptive input from the periphery and form commissural projections of dorsal and ventral SCTs. Using an Atoh1LacZ allele to trace the fate of pd1 progenitors in developing mouse, at least two subsets of the dI1 class have been identified: (1) a medial cluster of vertically oriented neurons that are Cbln2+ and Smarca2+ and projects to the SCT in the contralateral (Tag1+) lateral funiculus; (2) a more lateral Sox6+, cluster of horizontally oriented neurons that contributes to the SCT in the ipsilateral lateral funiculus. In chick, data with an enhancer that labels these cells suggests that both fascicles coalesce in the lateral funiculus ventral to the fascicle formed by the dI2 projections.

36The roof-plate-dependent Class A dp1 progenitors of the dI1 class express the bHLH transcription factors Olig3 and Math1. The dI1 neurons in mouse are born between E10 and E12.5 and express Lhx2/9, Barhl1 (bar homeobox like 1) and Brn3a (Pou4f1, a class IV POU domain-containing transcription factor; ). Loss of function experiments with BMP7 in chick and Bmp7 mutant mice results in loss of dI1, dI3, and dI5.

37dI2 interneurons are ascending, contralaterally projecting, relay interneurons that migrate to the intermediate spinal cord and ventral horn. These interneurons have been suggested to convey sensory information via the spinothalamic tract to the thalamus, based on their location (Figure 4E;;; ). The projections likely occupy the lateral funiculus and are dorsal to the dI1 fascicle, as analyzed by enhancer expression in chick. Arising from bHLH transcription factor Ngn1 (neurogenin 1) and Ngn2 expressing progenitors, these neurons express LIM-HD transcription factors Lhx1, Lhx5 and winged-helix domain Foxd3 (forkhead homeobox D3) post-mitotically. These interneurons were previously known as D3A interneurons.

38The roof-plate-dependent Class A dI2 progenitor domain, pd2, is characterized by the expression of the bHLH transcription factors, Olig3, Ngn1, and Ngn2 and are born between E10- and E12.5 (Figure 4H;; ). Two SoxD transcription factors, Sox5 and Sox6, are expressed in restricted domains of dorsal progenitors. Sox5 controls cell fate specification of dp2 and dp3 progenitors and, as a result, controls the correct number of the corresponding dorsal interneurons (dI2 and dI3; ).

39The dI3 neurons are excitatory interneurons in the deep dorsal horn and intermediate spinal cord. These cells target motor neurons monosynaptically, as revealed by recent rabies tracing experiments. They have axons that project rostrally, ipsilaterally, and longitudinally in two fascicles. A ventral fascicle enters the ventral lateral funiculus (VLF) and the dorsal fascicle enters the dorsal funiculus (DF; ). The dorsal projecting axons re-enter the cord when they encounter axons sensory axons at the dorsal root entry zone (DREZ; ). Similarly, the ventrally projecting neurons re-enter the cord at ventral root exit points. In mice model, dl3 appears to convey input from low threshold cutaneous afferents to the motor neurons that is critical in hand/forelimb grip. The dI3 pool also expresses Tlx3 (T-cell leukemia homeobox 3) and LIM-HD transcription factor Isl1-expressing cells (Figure 4I; ). The turning behavior of dI3 neurons is dependent on Isl1, and expression of Isl1 in dI1 neurons conferred dI3-like axon choice points to dI1 neurons. Tlx1 (also known as Hox11) and Tlx3 (also known as Rnx and Hox11L2) are markers of glutamatergic signaling. Tlx3 functions cell-autonomously to specify a glutamatergic neurotransmitter phenotype.

40The roof-plate dependent Class A dP3 progenitors express the basic helix-loop-helix (bHLH) transcription factor Mash1 (Ascl1, Mouse Achaete-scute complex-like 1), as do adjacent pd4 and pd5 domains. They also express Olig3, Pax7 and Ngn2 and Gsh2. In chick spinal cord electroporation experiments it has been shown that over-expression of Olig3 increases dI3 interneurons at the expense of other Classes A and B neuron classes and this effect is enhanced by Mash1. Over-expression of Mash1 results in more dI3 and dI5 neurons at the expense of dI2 and dI4, while loss of Mash1 causes a decrease in dI3 and dI5 populations while dI4 is maintained. As mentioned above, loss of function experiments with BMP7 in chick and Bmp7 mutant mice results in loss of dI1, dI3, and dI5.

41The early born (E10.5–E11) dI4 interneurons become Pax2+, Lhx1+, and Lhx5+ GABAergic ipsilaterally projecting somatosensory associative neurons that migrate laterally to the deep dorsal horn (Figure 4J;;; ). In addition, both dI4 and dI5 interneurons also express Gsh1 (Gsx1) and Gsh2 post-mitotically, while dI3 only express Gsh2. They are GABAergic, calbindin+ and express the nociceptive marker PLCγ. The dI4 fate is dependent on Ptf1a and loss of this gene results in loss of all GABAergic dorsal neurons and respecification to dI5 fate. Loss of Lhx1 and Lhx5 results in a loss of Pax2, Viaat, and Gad1. In addition, Pax2 is required for the maintained expression of Lhx1, Lhx5, Pax5, and Pax8.

42The roof-plate independent Class B dP4 domain expresses Lbx1, Mash1 and higher levels of Pax7 than the dorsally adjacent pD3 domain. These progenitors are born between E10.5 and E11 and this domain is distinct from the dIL progenitor domain that produces dILA and dILB progenitors, although both have very similar transcription factor expression patterns (below). Olig3 over-expression can inhibit formation of dI4, and loss can result in expansion of this domain. While these cells express Mash1, loss of Mash1 does not block dI4 formation, yet it does disrupt dI3 and dI5.

43The roof-plate independent Class B dI5 neurons become contralaterally projecting glutamatergic somatosensory interneurons of the deep dorsal horn (nucleus proprius) and ventral horn that express the homeodomain transcription factors Lbx1, Brn3a, Tlx1, Tlx3, and Lmx1b (Figure 4K;;;;; ). In addition, a subset expresses PhoxA2. These interneurons were previously known as D4.

44These cells are born between E10.5–E11 and arise from a Mash1+ and Pax7+ dP5 progenitor domain that express Lbx1 post-mitotically to both reinforce Class B fate and oppose Class A fates (Figure 4K;; ). The dI5 domain expresses Gsh1 and Gsh2, as does the adjacent dI4 domain. As noted previously, loss of function experiments with BMP7 in chick and Bmp7 mutant mice results in loss of dI1, dI3, and dI5.

45The roof-plate independent Class B dI6 commissural inhibitory interneurons express Lbx1, Lhx1, Lhx5, and are Pax2 positive, indicating a GABAergic fate (Figure 4L;;;;; ). These cells may also use glycine for neurotransmission. Although arising from a dorsal progenitor pool, and not being part of the “V” interneurons, the dI6 group of interneurons gives rise to more than one subtype and appears to contribute to motor function. These inhibitory neurons are reported in unpublished observations to be commissural and may be involved in right–left alternation, as well. Dmrt3, a novel marker in dl6 interneuron was traced to play a key role in locomotor circuitry and in development of commissural interneurons, and mutation in dmrt3 result in divergent in gait pattern in mice models. Double knockout of Lhx1 and Lhx5 results in a loss of Pax2, Viaat, and Gad1 expression. Furthermore, Pax2 is required for the maintained expression of Lhx1, Lhx5, Pax5, and Pax8. These cells also express WT1 (Wilms’ tumor 1; ). The transcription factor, Bhlhb5, marks the dI6, V1 and V2 domains. Electrophysiologic characteristics of the dl6 interneurons around a central canal reveal two possible subtypes: one firing trains of action potentials that are loosely coupled to the ventral root output and expressing intrinsic rhythmic activity which suggests a role in locomotor rhythm generation. The other subtype fires action potentials that are tightly coupled to the ventral root output.

46The dI6 neurons are born around E10.5–E11 and originate from a Pax7+, Dbx2+, Ngn1+ and Ngn2+ pD6 progenitor domain. Post-mitotically they express Bhlhb5, Wt1, Lbx1, Lhx1, Lhx5, and Pax2 (references above).

47The dIL neurons represent a second wave of neurogenesis from the dIL progenitor domain that constitutes most of the interneurons in the superficial dorsal horn in Rexed laminae II–IV. These cells are formed from common progenitors, and their cell fates are controlled by Ascl1/Mash1 (Figure 4M; ).

48The roof-plate independent Class B ILA interneurons are ipsilaterally projecting association neurons that occupy the superficial dorsal horn in Rexed laminae I–III. These inhibitory neurons are GABAergic and are calbindin+, and a subset express Gbx1.

49The dILA interneurons are late born (E.13.5) and arise from the dIL progenitor domain that is Lbx1+, Lhx1/5+, and Pax2+. The sensory relay interneuron maker Foxd3 is downregulated in these cells. Pax2 has been shown to be necessary for GABAergic differentiation and 98% of these cells also express Gad1 (GAD67; ). The dILA subclass is dependent on Ptf1a and loss of this gene results in trans-fate to the dILB identity.

50The roof-plate independent Class B are DRG11+ ipsilaterally projecting association interneurons that integrate input from cutaneous sensory neurons that detect noxious stimuli. This last-born subclass gives rise to glutamatergic neurons expressing Tlx1 and Tlx3 and Lmx1b. The dILB neurons migrate dorsolaterally to settle in the superficial dorsal horn in Rexed laminae I–III. Over 96% of the dorsolateral population of these cells expresses both Tlx3 and VGluT2.

51These neurons are born later than the dI1–6 class and express Lbx1 post-mitotically. Mash1 controls the upregulation of Notch signaling to direct formation of dILB from common dIL progenitors.

52Combinatorial transcriptional control of cell fate is a mature perspective for understanding spinal cord development. This focus on transcription factors has been powerful in two major respects. First, it has allowed the identification of downstream factors that direct cell-specific characteristics, such as neurotransmitter status. Second, it has permitted a powerful genetic analysis of spinal neurons, using transcription factors as class-specific tools to drive changes in cell fate and function. Novel techniques are emerging for tracing neuronal circuitry and for the sophisticated manipulation of neuronal activity, including selective cellular ablation, optogenetic activation and silencing, and chemically induced activation and silencing. These techniques may reach their most exciting potential when coupled with the increasingly specific genetic control available in the spinal cord.

53The further refinement of developmental neuronal classes is showing that subclasses may reflect functionally coherent groups of cells that can be mapped onto physiologically identified populations (Figure 5). Therefore, as the spinal cord development field grows to incorporate circuitry and behavior, it is merging with the rich field of adult spinal cord electrophysiology that has uncovered major mechanisms of spinal cord function. The combination of these disciplines will advance spinal cord biology to a state that fully encompasses both form and function.

54The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.