From Motor Neuron Specification to Function: Filling in the Gaps

*Mudassar Nazar Khan and Till Marquardt*

### **Abstract**

Motor neurons operate at the interface between nervous system and movement apparatus and play several roles in movement generation. During development, motor neurons emerge from progenitor cells in the ventral neural tube and eventually settle into stereotypic position that predict the identity of their target muscles. The specification of these 'positional' identities has been studied in detail and involves a coordinate grid of intersecting extrinsic signals that result in the activation of unique combinations of transcription factors acting as cell-autonomous determinants. Eventually, motor neurons diversify into 'functional' (e.g., fast/intermediate/ slow alpha, beta, and gamma) subtypes essential for proper movement execution, a process linked to the acquisition of unique sets of functional properties. Recent progress has provided insights into the molecular composition and specification of motor neuron functional identities, but little is known about their relationship to the mechanisms underlying the specification of positional identities. In this chapter, we attempt to provide a framework for consolidating both aspects of motor neuron diversification, in addition to outlining the gaps in our knowledge to guide future research directions aiming at understanding the events on a motor neuron's journey from specification to function.

**Keywords:** spinal motor neurons, motor neuron functional specification, motor neuron positional identities, motor neuron development, neurogenesis, movement control, neuronal development, gamma, beta, alpha motor neurons, fast, slow motor neurons

## **1. Introduction**

To paraphrase Sherrington, motor neurons represent the final common pathway in the generation of behaviors by linking the nervous system with the movement apparatus [1]. This role is reflected by two levels of organization, one spatial and one functional, that together allow motor neurons to serve as the interface through which the brain can engage with and act upon the external world. On the one hand, motor neurons are somatotopically organized, with the position of motor neuron somas in the spinal cord predicting the specific muscle it controls (**Figure 1**) [2]. These 'positional' identities of motor neurons are specified early in development, prior to the establishment of neuromuscular connections [2]. On the other hand, motor neuron diversity is defined by different roles in movement generation and by the different

#### **Figure 1.**

*Schematic summarizing the different levels of motor neuron specification. All spinal motor neurons originate from neuron progenitor cells in the ventral neural tube. Motor neuron positional identities (columns, divisions innervating certain muscle groups and motor pools innervating individual muscles) are acquired after cell-cycle exit. A typical motor pool of tetrapod vertebrates contains a mixture of different 'functional' motor neuron types: Alpha motor neurons (fast/f-MNs, intermediate/i-MNs and slow/s-MNs) as well as beta and gamma motor neurons, which all possess different biophysical properties such as firing rates (such as high firing rates for f-MNs and gamma motor neurons) and roles in movement execution and control. It remains unclear, when exactly motor neuron functional diversity is generated and to what extent, if at all, this is coordinated with the specification of motor pool identities.*

muscle fiber types that are innervated (**Figure 1**) [3]. In tetrapod vertebrates, a skeletal muscle typically contains a mixture of different muscle fiber types and is supplied by a range of motor neuron types involved in different aspects of movement generation or control [2, 4–6]. To avoid confusion, we will refer to the first instance of motor neuron diversity as 'positional' diversity and the second as 'functional' diversity. While the mechanisms promoting positional or functional diversity may operate independently form each other, an individual motor neuron can eventually be assigned both by a positional and a functional identity (**Figure 1**). Several excellent reviews have appeared in recent years covering either positional or functional motor neuron diversification [2, 3, 7]. To avoid redundancy, we will in this chapter just briefly touch upon the fundamentals of both spatial and functional motor neuron diversity and mostly focus on what is known—or rather what is not—about the link between the mechanisms that underlie the development of both types of motor neuron diversity necessary for proper movement execution and control.

### **2. Motor neuron positional identities**

#### **2.1 Initial specification of motor neuron identity**

Positionally distinct classes of motor neurons emerge in the developing spinal cord whose fates are specified under the influence of intersecting gradients of signaling molecules called morphogens along the dorsoventral (d-v) and rostrocaudal (r-c) axes of the neural tube [8, 9]. Morphogens are secreted from several embryonic structures to the neural tube: Sonic hedgehog (Shh) from the notochord and floor plate and bone morphogenetic protein (BMP) and wingless (Wnt) from the dorsal roof plate and surface ectoderm, retinoic acid (RA) from the paraxial mesoderm

#### *From Motor Neuron Specification to Function: Filling in the Gaps DOI: http://dx.doi.org/10.5772/intechopen.114298*

and fibroblast growth factors (FGFs) from the caudal mesoderm and tail bud, that together contribute to the specification of molecular identities of neural progenitor cells or domains along the d-v and r-c axes of the neural tube [8, 9]. Shh drives the specification of motor neuron progenitor (pMN) and interneuron populations (V0, V1, V2, V3) in the ventral spinal cord, while RA and FGFs also contribute to motor neuron progenitor identities [10–12]. The graded expression of Shh in the d-v axis regulates Gli (Glioma-associated oncogene) family transcription factors activity, which establishes the neural progenitor domains via the expression of Class I and II homeodomain transcription factors (TFs) [9].

Progenitor domain boundaries develop and are sustained through the crossrepressive actions of class I (Pax7, Pax6, Irx3, Dbx1, Dbx2) and Class II TFs (Nkx6.1 and Nkx2.2) [13]. The co-expression of Pax6 and Nkx6.1 activates the expression of basic helix-basic-helix (bHLH) protein Olig2, which together mark the motor neuron progenitor domain [14–16]. The expression of Olig2 and its repressive activities (specifically, the repression of a repressor that normally represses Ngn2 expression) leads to the indirect upregulation of Ngn2. This results in the expression of post-mitotic motor neuron genes of Lhx3/Isl1 and motor-neuron specific gene Mnx1 (Hb9) [13, 17, 18]. Moreover, the expression levels of Olig2/Ngn2 must be balanced to ensure timely motor neuron specific gene expression, since high expression of Olig2 sustains the pMN state, while high levels of Ngn2 activates the conversion of pMN to post-mitotic motor neurons [19]. Post-mitotic neuron classes, like the progenitor classes, are specified by the combinatorial expression of transcription factors, and in addition, develop specific patterns of connectivity, a neurotransmitter system and electrophysiological properties [8, 9]. Newly born post-mitotic motor neurons express a set of TFs like Lhx3, Isl1 and Mnx1 and send axons peripherally to muscles, use acetylcholine and glutamate as neurotransmitters [20, 21]. This set of early post-mitotic TFs bind to specific enhancers to specify and maintain motor neuron identity [22, 23].

### **2.2 Specification of motor neuron positional identities: columns, divisions and pools**

A second level of motor neuron organization within the spinal cord is the clustering of somas into columns within the r-c axis (**Figure 1**). These positional identities are patterned by reciprocally graded concentrations of fibroblast growth factors (FGFs) and retinoic acid (RA) that regulate the temporal and spatial expression of homeobox (*Hox*) family transcription factors [24–26]. *Hox* family transcription factors are sequentially arranged on chromosomes in four clusters (*HoxA, HoxB, HoxC, HoxD*) that encode 39 genes that are expressed in the spinal cord and hindbrain during both progenitor and post-mitotic phases of motor neuron differentiation [27, 28]. RA regulates the expression of *Hox4-Hox6* genes at the cervical/brachial levels of the spinal cord [7, 26]. While FGF induces *Hox4-Hox10* in cervical/brachial, thoracic and lumbar levels of the spinal cord and Gdf11/FGF8 induce *Hox10* in the lumbar spinal cord [7, 26].

*Hox* gene expression pattern in post-mitotic motor neurons determines their columnar subtype identity. For example, in the phrenic motor column (PMC), motor neurons at the cervical level of the spinal cord express *Hox5* [29]. Loss of *Hox5* in motor neurons results in failure of dendritic arborization in the diaphragm muscle and neuronal death leading to respiratory failure and perinatal death in mice [29]. The lumbar motor column (LMC) motor neurons of the brachial and lumbar levels express *Hox6* and *Hox10*, respectively [25, 30–34]. The specification of LMC depends

on the expression of *Hox* genes and their regulation of Foxp1 expression pattern [30, 32]. The preganglionic motor column (PGC) motor neuron identities depend on *Hoxc9* expression [35]. While, motor neurons that innervate axial muscles do not depend on *Hox* gene programs. For example, the medial motor column (MMC) columnar subtype motor neurons identity depends on the *Wnt* genes and are marked by the expression of Prdm family transcription factor Mecom [7, 36, 37]. Still, the programs involved in the specification of other motor columns, like hypaxial motor column (HMC) which innervates axial muscles are unknown.

A third level of motor neuron organization is known as divisional identity which is defined by the motor axons and the muscles they target. The genetic programs that are involved in the specification of muscle targets have been studied extensively in the lateral motor column (LMC) motor neurons. While the generation of LMC motor neurons depends on *Hox* genes and the expression pattern of transcription factors they regulate, the maturation of LMC neurons is dependent on both the limb-derived cues and molecular programs intrinsic to motor neurons. *Hox* genes promote high *Foxp1* expression levels, which is required for the expression of Raldh2, an enzyme that catalyzes retinoic acid (RA) synthesis. Localized synthesis of RA in motor neurons at the brachial and lumbar spinal cord levels establish LMC divisional identities [38–40]. The LMC motor neurons express specific Lim homeodomain (HD) proteins and their axons target specific muscles groups of the limbs, which confers their divisional identity: the lateral division (LMCl) motor neurons express Lhx1 and send axons to the muscles within the dorsal compartment, while the medial division (LMCm) motor neurons express Isl1 and send axons to the muscles within the dorsal compartment [41]. To ensure LMCl identity, Lhx1 expression is maintained by the repressive interaction between Lhx1 and Isl1, the protein signaling controlled by the sources of RA from Raldh2+ motor neuron and the paraxial mesoderm [7, 38–40]. Moreover, the routes that LMCl motor neuron axons select within the limbs is dependent upon Lhx1 expression. Lhx1 expression within LMCl regulates the expression of axonal guidance receptor Eph4 (which repels ventral axons that express ephrin), leading to the dorsal projection of axons [7, 42, 43]. Moreover, ventrally projecting LMCm axons use similar signaling strategies: Lim HD proteins regulate the expression of axonal guidance molecules like ephrin and Eph receptors that ultimately determine axonal trajectories [7, 44].

A fourth level of motor neuron organization observed in the spinal cord are motor pools (**Figure 1**). Motor neurons within a specific motor pool cluster in a specific position in the spinal cord, innervate a single muscle, have specific type (alpha/gamma) ratios and show motor pool-specific molecular marker expression, morphology, central and peripheral connectivity, and electrophysiological patterns. These features are determined by *Hox* genes, in part, and by molecular cues from peripheral targets. Early on in development, motor pool diversity is regulated by *Hox* genes and their regulation of transcription factor expression levels. Combinatorial expression of *Hoxc8* and *Hoxc6* determines the expression of several proteins, including ETS domain transcription factor Pea3, the Pou domain protein Scip (also known as Pou3f1), and Nkx6.1, which ultimately characterize LMC motor pool identities [7]. Thus, the loss of *Hoxc8* and *Hoxc6* in mice leads to the decrease in Pea3-expressing motor neurons and decrease in axonal arborization of targeted muscles [31, 45, 46]. Moreover, specific muscle innervation in mice is disrupted in motor pools that no longer express Nkx6.1, a downstream target of *Hox* signaling [47], while *Foxp1* mutant mice show reduced expression of motor pool markers *Pea3*, *Scip* and *Nkx6.1* [32]. Motor pool identities are also regulated by the molecular

cues from peripheral muscles. For example, neurotrophic factors like glial-derived neurotrophic factor (GDNF), regulate the expression of Pea3 and thus, determine motor pool soma position and motor pool targeted muscle innervation [48].
