REVIEW ARTICLE

published: 09 October 2014

CELLULAR NEUROSCIENCE doi: 10.3389/fncel.2014.00293

Motor neurons and the generation of spinal motor neuron

diversity
Nicolas Stifani *
Medical Neuroscience, Dalhousie University, Halifax, NS, Canada

Edited by: Motor neurons (MNs) are neuronal cells located in the central nervous system (CNS)
Lachlan Thompson, Florey controlling a variety of downstream targets. This function infers the existence of MN
Neuroscience Institute, Australia
subtypes matching the identity of the targets they innervate. To illustrate the mechanism
Reviewed by:
involved in the generation of cellular diversity and the acquisition of specific identity, this
Asuka Morizane, Kyoto University,
Japan review will focus on spinal MNs (SpMNs) that have been the core of significant work
Frederic Clotman, Université and discoveries during the last decades. SpMNs are responsible for the contraction of
Catholique de Louvain, Belgium effector muscles in the periphery. Humans possess more than 500 different skeletal
*Correspondence: muscles capable to work in a precise time and space coordination to generate complex
Nicolas Stifani, Atlantic Mobility
movements such as walking or grasping. To ensure such refined coordination, SpMNs
Action Project, Life Science
Research Institute, Dalhousie must retain the identity of the muscle they innervate. Within the last two decades,
University, 1344 Summer Street, scientists around the world have produced considerable efforts to elucidate several
Halifax, NS B3H 4R2, Canada critical steps of SpMNs differentiation. During development, SpMNs emerge from dividing
e-mail: [email protected]
progenitor cells located in the medial portion of the ventral neural tube. MN identities are
established by patterning cues working in cooperation with intrinsic sets of transcription
factors. As the embryo develop, MNs further differentiate in a stepwise manner to form
compact anatomical groups termed pools connecting to a unique muscle target. MN
pools are not homogeneous and comprise subtypes according to the muscle fibers they
innervate. This article aims to provide a global view of MN classification as well as an
up-to-date review of the molecular mechanisms involved in the generation of SpMN
diversity. Remaining conundrums will be discussed since a complete understanding of
those mechanisms constitutes the foundation required for the elaboration of prospective
MN regeneration therapies.
Keywords: motor neurons, development, central nervous system, spinal cord, transcription factors, spinal motor
neuron, lower motor neuron

INTRODUCTION tuning of gestures. To ensure such refined coordination, SpMNs

Motor neurons (MNs) are neuronal cells located in the cen- must acquire and retain the identity of muscles they innervate
tral nervous system (CNS) controlling a variety of downstream as well as be integrated in a coherent and functional neuronal
targets. There are two main types of MNs, (i) upper MNs circuitry. Hollyday et al. (1977) and Landmesser (1978) initially
that originate from the cerebral cortex and (ii) lower MNs described the anatomical organization of SpMNs with respect
that are located in the brainstem and spinal cord. Among to their muscle targets. Authors acknowledged an association
the latest, spinal MNs (SpMNs) have been intensively stud- between SpMNs’ positions and their respective muscle target in
ied during the last decades and therefore provide an interest- the periphery. Ultimately these findings led to the concept of MN
ing framework for further molecular characterization. SpMNs pool, which is defined as a compact anatomical group of MNs
are located in the ventral horn of the spinal cord and con- sharing similar intrinsic characteristics and connecting to a single
trol effector muscles in the periphery. They form the ulti- target in the periphery. Because of their unique and irreplaceable
mate and irreplaceable component of the neuronal circuitry function, diseases that involve loss of MNs such as progressive
since there is no alternative route to convey the commands muscular atrophy, spinal muscular atrophy, primary lateral scle-
from the processing centers located in the CNS to the effector rosis, and amyotrophic lateral sclerosis, are rapidly debilitating,
muscles in the periphery. Their axon extending through sev- as only symptomatic treatments are available. Understanding the
eral meters in mammals constitute an exceptional and unique molecular mechanisms underlying SpMN diversity is among the
anatomical feature. SpMNs are therefore the longest known fundamental steps required to elaborate successful regenerative
cell type. therapies in the future. Here, we provide a complete description
Complex movements such as walking or grasping require of MN classification to then review in depth the organization as
the cooperation of several dozens of muscles. Additionally, well as the molecular mechanisms involved in the generation of
sensory-motor feedback loops are essential for the real-time SpMNs.

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Stifani Spinal motor neuron diversity

MOTOR NEURON CLASSIFICATION embryological origin since they do not derive from the somites,
MNs are exceptional cell types that can be divided into two main but instead from the branchial arches. Such developmental differ-
categories according to the location of their cell body: (i) upper ence is mirrored by specific characteristics reviewed in depth by
and (ii) lower MNs. Upper and lower MNs must be considered Chandrasekhar (2004).
as distinct entities despite of their shared nomenclature. Table 1
summarizes the differences between the two in terms of cell body Visceral motor neurons
location, neurotransmitter, targeting, and symptoms upon lesion Visceral MNs belong to the autonomic nervous system (ANS)
and emphasizes the inappropriateness of a similar appellation to responsible for the control of smooth muscles (i.e., heart and
name both entities. arteries) and glands. The ANS can be described as the association
of two components: (i) preganglionic MNs located in the CNS
UPPER MOTOR NEURONS connected to ganglionic neurons belonging to the peripheral ner-
Upper MN cell bodies are located in the pre-motor and primary vous system (PNS). In turn, peripheral ganglionic neurons target
motor region of the cerebral cortex also known as the “motor to the final effector organ. Additionally, the ANS is anatomically
strip.” Since upper MNs make glutamatergic connections with and functionally divided into two structures: (i) the sympathetic
lower MNs located in the CNS, they are exclusively confined to system and (ii) the parasympathetic system.
the latter. Typical clinical symptoms of upper MN lesion include
uncontrolled movement, decreased sensitivity to superficial reflex Motor neurons of the sympathetic system. The sympathetic
stimulation and spasticity (Ivanhoe and Reistetter, 2004). The nervous system is involved in the traditional “fight or flight”
organization of upper MNs is complex and can’t be completely responses, recruiting energy storage, increasing awareness, and
and accurately described in this review that primarily focuses on leading to a global activation of the body metabolism. Central
molecular mechanisms that generate SpMN diversity. Readers are MNs of the sympathetic system are located in the spinal cord
invited to refer to the chapter 16 entitled “Upper Motor Neuron from the thoracic segment 1 (T1) to the lumbar segment 2 (L2).
Control of the Brainstem and Spinal Cord” from Purves and These MNs have an intermedio-lateral position and constitute the
Williams (2004) for more information. preganglionic column (PGC) that will be described below. They
connect to 3 different targets: two chains of ganglia adjacent to
LOWER MOTOR NEURONS the spinal cord named (i) paravertebral and (ii) prevertebral as
Lower MN cell bodies are located in specific nuclei in the brain- well as directly to (iii) the chromaffin cells of the adrenal medulla
stem as well as in the ventral horn of the spinal cord and therefore, responsible for the release of the catecholamines (i.e., adrenaline
alike upper MNs, are settling within the CNS. The remarkable and noradrenaline) in the circulation, in response to stress stim-
characteristic of lower MNs is their axonal extension and con- uli. On the other hand, paravertebral and prevertebral ganglia
nection outside of the CNS. Lower MNs are cholinergic and connect to a wide variety of targets including the heart, lungs,
receive inputs from upper MNs, sensory neurons (SNs) as well as kidneys, intestines and the colon.
from interneurons (INs). Paralysis is a typical clinical symptom of
Motor neurons of the parasympathetic system. The parasympa-
lower MN lesions since once damaged there is no alternative route
thetic system controls glands secretion and activates the gastroin-
to convey the information to the muscle targets in the periph-
testinal tract as well as sexual behavior, which are summarized as
ery. Lower MNs are classified into three groups according to the
“rest and digest” functions. Central MNs of the parasympathetic
type of target they innervate: (i) branchial, (ii) visceral, and (iii)
system are located in the brainstem and contribute to the forma-
somatic MNs.
tion of the cranial nerves (III, VII, IX, and X). Parasympathetic
Branchial motor neurons MNs are also found in sacral segments 2 to 4 (S2–S4) of the
Branchial MNs are located in the brainstem and form, together spinal cord. They innervate ganglia located in the proximity of
with SNs, the cranial nuclei. They innervate branchial arch the peripheral targets such as the heart, bladder, lungs, kidneys,
derived muscles of the face and neck through 5 cranial nuclei: the and pancreas.
trigeminal (V), facial (VII), glossopharyngeal (IX), vagus (X) and In summary, visceral central MNs from the sympathetic and
accessory (XI) nerves. Despite their similar function, muscles of parasympathetic systems relay information from the CNS to gan-
the neck and the face differ from other skeletal muscles in their glionic neurons of the PNS. In turn those ganglia antagonistically
control a large number of various visceral targets. In contrast
to branchial mentioned previously and somatic MNs described
Table 1 | Comparison between upper and lower MNs. below, visceral MNs do not directly connect to the final effec-
tor. As a result, they constitute an anatomical and functional
Upper MNs Lower MNs
exception among lower MNs.
Location Cortex Brainstem and SC
Somatic motor neurons
Neurotransmitter Glutamate Acetylcholine
Somatic MNs are located in the Rexed lamina IX in the brainstem
Targeting Within the CNS Outside the CNS
and the spinal cord and innervate skeletal muscles responsible
Symptoms upon lesion Spasticity Paralysis
for movements (Rexed, 1954). MNs form coherent groups con-
Upper and lower MNs diverge in their cell body location, neurotransmitter, necting to a unique muscle target defined as MN pools. Somatic
targeting and symptoms upon lesion. MNs can be divided into 3 groups: (i) alpha, (ii) beta, and (iii)

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Stifani Spinal motor neuron diversity

gamma according to the muscle fiber type they innervate to Analogously, extrafusal fibers are divided into 3 types according
within a specific muscle target (Figure 1). A motor unit defines to their physiological and molecular properties: (i) slow-twitch
a single MN together with all the muscle fibers it innervates. fatigue-resistant (SFR), (ii) fast-twitch fatigue-resistant (FFR)
Interestingly, motor units are homogeneous: a MN innervates and (iii) fast-twitch fatigable (FF). Table 2 summarizes the prin-
muscle fibers of a single type. This observation suggests selec- cipal characteristics of the three extrafusal muscle fibers.
tivity in the establishment of neuromuscular connectivity and/or Mirroring the diversity of both intra- and extrafusal fiber types
a coordinated maturation between a MN and its targeted fibers. in a muscle, somatic MNs are further sub-divided into 3 types:
Intuitively, the diversity of MNs mirrors the diversity of targets (i) alpha, (ii) beta and (iii) gamma that will be further described
they innervate. Therefore, to better describe somatic MN diver- below.
sity, a brief description of skeletal muscle physiology will be
provided. Alpha motor neurons. Alpha MNs exclusively innervate extrafusal
Three classes of muscles can be anatomically and functionally muscle fibers and are the key of muscle contraction (Figure 1).
distinguished: (i) cardiac muscles, (ii) smooth muscles and (iii) Anatomically, alpha MNs are characterized by a large cell body
skeletal muscles. Cardiac muscles are responsible for the rhyth- and a well-characterized neuromuscular ending. They have an
mic contraction of the heart while smooth muscles control the important role in the spinal reflex circuitry by receiving monosy-
diameter of blood vessels and the internal digestive and secre- naptic innervation directly from SNs thus minimizing the delay
tion organs. Both smooth and cardiac muscles are innervated by of the response (Eccles et al., 1960). Alpha MNs can be further
the ANS (described above). In contrast, somatic MNs exclusively divided into 3 different subtypes depending on the extrafusal
innervate skeletal muscles that are the most abundant muscle fiber type they innervate: (i) SFR, (ii) FFR, and (iii) FF (Burke
class, with around 639 different muscles in the human body et al., 1973) (Figure 2). There is no universal criteria distinguish-
(Stone and Stone, 2009). Skeletal muscles are firmly attached to ing alpha MNs subtypes; however, some trends are observed in
the skeleton by the tendons and are responsible for both pos- term of size, excitability, and firing pattern. SFR MNs tend to
ture and movement. Developmentally, skeletal muscles derive have a smaller cell body diameter and thus a higher input resis-
from the paraxial mesoderm that produces the somites, which tance making them responsive to a lower stimulation threshold.
in turn generate muscle precursor cells called myoblasts. Those As a result, SFR MNs are recruited first during muscle contrac-
cells migrate toward the periphery and fuse to form the body of tion. They also have the capacity of maintaining a persistent
the muscle. Physiologically, skeletal muscles are composed of 2 activity even after the stimulation ceased (Lee and Heckman,
structures: (i) extrafusal fibers, generating the force and (ii) mus- 1998). On the other hand, FF MNs have often a larger cell
cle spindles providing proprioceptive information on the position body and are firing after the initial recruitment of SFR neurons
and extension status of the muscle. Muscle spindles are composed giving extra strength to the activated muscle. In terms of conduc-
of several intrafusal fibers enveloped by a collagen sheath named tion velocity, MNs innervating fast fibers are substantially faster
the outer capsule. There are three kinds of intrafusal fibers with (100 m/s) than SFR MNs (85 m/s) (Burke et al., 1973). Lastly, lit-
specific characteristics: (i) dynamic nuclear bag fibers (B1), (ii) tle is known about FFR MNs physiology; yet, they are considered
static nuclear bag fibers (B2 fibers) and (iii) nuclear chain fibers. to have intermediate characteristics between FF and SFR MNs
(Figure 2).

Alpha MN Beta motor neurons. Beta MNs are smaller and less abundant
than other somatic MN subtypes. As a result beta MNs are poorly
Gamma MN characterized. They innervate both intrafusal and extrafusal mus-
cle fibers (Bessou et al., 1965) (Figure 3). Therefore, beta MNs
Sensory Neurons constitute an exception to the homogeneity observed in motor-
units and control both muscle contraction and responsiveness of
the sensory feedback from muscle spindles. They are further sub-
divided into two subtypes depending on the type of intrafusal
MS fibers they innervate: (i) static, innervating nuclear chain fibers
IF and (ii) dynamic, innervating the nuclear bag fibers of muscle
OC spindles. Static beta MNs increase the firing rate of type Ia and
type II sensory fibers at a given muscle length whereas dynamic
EF beta MNs increase the stretch-sensitivity of the type Ia sensory
fibers by stiffening the nuclear bag fibers. Beta MNs are mainly
characterized anatomically and functionally, further molecular
FIGURE 1 | Muscle innervation. Schematic of muscle fibers on the
longitudinal section (adapted from Purves and Williams, 2004). Alpha MN and electrical properties remain to be identified.
(red) innervates (incoming arrow) extrafusal muscle fibers (EF, brown)
whereas gamma MN (purple) connects to intrafusal fibers (IF, blue) within Gamma motor neurons. Gamma MNs control exclusively the
the muscle spindle (MS, light gray) surrounded by the outer capsule (OC,
sensitivity of muscle spindles. Their firing increases the tension
dark gray). Sensory neurons (green) carry information from the intrafusal
fibers to the central nervous system (outgoing arrow).
of intrafusal muscle fibers and therefore mimics the stretch of the
muscle. Like beta MNs, gamma MNs are functionally divided into

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Stifani Spinal motor neuron diversity

Table 2 | Characteristics of extrafusal muscle fiber types.

SFR FFR FF

Anatomical properties Fiber Diameter Small Intermediate Large

Color Red Light red White
Capillaries Many Many Few
Myoglobin level High High Low
Mitochondria Many Many Few

Physical properties Duration use Hours Minutes < Minute

Power produce Low High Very high
Recruitment order First Second Third
Fatigue sensitivity Slow Intermediate Fast
Contraction velocity Slow Fast Fast
Function activity Posture Normal movements Intense movements

Metabolic properties Myosin ATPase activity Slow Fast Fast

ATP synthesis Aerobic Intermediate Anaerobic
Glycogen stores Low Intermediate High
Oxidative capacity High Intermediate Low
Glycolytic capacity Low Intermediate High
Energy storage Triglycerides Creatine Phosphate Creatine Phosphate
Glycogen Glycogen

Slow-twitch fatigue-resistant (SFR), Fast-twitch fatigue-resistant (FFR), and Fast-twitch fatigable (FF) fibers differ in term of anatomical, physical, and metabolic
properties.

Alpha MNs
αFF Extraf
usal fi
ber Muscle fibers
αFFR
IIb Fast-twitch fatigable
αSFR
IIa Fast-twitch fatigue-resistant
Gamma MN I Slow-twitch fatigue-resistant
Intraf
usal fi
ber

Ventral SC Muscle spindle

FIGURE 2 | Characteristics of alpha and gamma MNs. Schematic three types of extrafusal fibers: fast-twitch fatigable muscle fibers (light
showing the principal characteristics of alpha and gamma MNs (adapted brown, IIb) are innervated by αFF MNs, fast-twitch fatigue-resistant muscle
from Kanning et al., 2010). Within the ventral spinal cord (SC light gray), fibers (dark brown, IIa) are innervated by alpha αFFR MNs and slow-twitch
MN pools (dashed lines) are composed of gamma MNs (blue) as well as fatigue-resistant muscle fibers (green, I) are innervated by αSFR MNs.
three type of alpha MNs: αFF (light brown), αFFR (dark brown), αSFR Intrafusal muscle fibers (blue) reside within a muscle spindle (gray) and are
(green). Alpha MNs have a larger diameter than gamma MNs. Beta MNs exclusively innervated by gamma MNs. A single MN innervate multiple
are not represented for simplicity. The proportion of alpha MN subtypes fibers all of the same type; however, for the schematic simplicity only one
varies between MN pools. In the periphery, a muscle is composed of fiber is represented.

two subtypes: (i) static, innervating nuclear chain fibers and static SUMMARY OF MOTOR NEURON CLASSIFICATION
nuclear bag fibers and (ii) dynamic, innervating the dynamic As seen above, the term “motor neuron” groups a significant
nuclear bag fibers (Figure 3). Gamma MNs receive only indirect diversity of cell types and does not ideally reflect biological real-
sensory inputs and do not possess any motor function. Therefore, ity. Upper and lower MNs are fundamentally different and their
gamma MNs do not directly participate to spinal reflexes (Eccles shared nomenclature can easily be misleading. For instance, if
et al., 1960) but instead contribute to the modulation of muscle we define a MN by being a “neuronal cells settling within but
contraction. projecting outside of the CNS,” upper MNs would be excluded.

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Stifani Spinal motor neuron diversity

In fact, upper MNs would be more accurately defined by the (Figure 4). Over the last three decades, many studies have shaded
terminology “imbuo-neurons” derived from Latin imbuo that light on important mechanisms governing MN differentiation in
signifies “give initial instruction” or by the terminology “didactic- the spinal cord. A comprehensive and up-to-date review of those
neurons” derived from the Greek didaktikós for instructive. In studies will be presented below.
contrast, lower MNs, with the exception of visceral MNs, con-
nect directly to their muscle targets and constitute the last step DEVELOPMENTAL ORIGIN
of the neuronal circuitry. SpMNs are divided into functional During the early phase of embryogenesis, the egg cell undergoes
groups, termed pools, mirroring the diversity of muscle targets in a series of divisions until forming a sphere made of a single layer
the periphery. In addition, a single muscle is composed of sev- of cells called the blastula. Subsequently, during a process called
eral fiber types that are innervated by specific classes of MNs. gastrulation, a group of cells will enter the blastula cavity lead-
Therefore MN pools should not be considered as a set of identical ing in triploblastic animals to the formation of the three primary
cells but instead as a mosaic of MN cell types covering a broad germ layers: (i) the endoderm, (ii) the mesoderm, and (iii) the
range of functions. The generation of this complex architecture ectoderm. Individual layers generate progenies restricted to a lim-
must rely on precise mechanisms ensuring the establishment of ited number of distinct fates. The ectoderm undergoes a process
the correct connections between matching MN - target pairs. We called neurulation in which it folds inward and leads to the for-
will review the functional organization of SpMNs as well as the mation of three ectodermic masses: (i) the neural tube, (ii) the
molecular mechanisms leading to their generation. neural crest cells, and (iii) the external ectoderm. The external
ectoderm generates the epidermis whereas the neural crest cells
GENERATION OF SPINAL MOTOR NEURONS form the peripheral ganglion, the pigments of the skin as well
The spinal cord offers a relatively simple, yet, powerful experi- as the dorsal root ganglia. Finally, the neural tube gives rise to
mental model to study neuronal development. It can be schema- the CNS, composed of the brain and the spinal cord (Purves and
tized as a circuitry formed by three different neuron types. Williams, 2004) (Figure 5A).
Sensory neurons located in the dorsal root ganglia (DRG) receive
GENERATION OF DEDICATED SPINAL CORD PROGENITOR DOMAINS
input information from the periphery and transmit it either
Soon after neurulation, the neural tube is surrounded by sev-
directly to alpha MNs located in the ventral horn (monosynap-
eral inductive signals stimulating the subsequent differentiation
tic connections) or to association neurons (commissural and
process. Members of the wingless-type MMTV integration site
interneurons) that, in turn, process and convey the information
family (WNT) (Alvarez-Medina et al., 2008) and of the bone
toward the MNs. MNs then stimulate their respective effector that
morphogenetic protein family (BMPs) (Mehler et al., 1997) and
will generate the appropriate output response (Eccles et al., 1957)
their regulators Noggin (NOG), Chordin (CHRD), and Follistatin
(FST) (Zimmerman et al., 1996; Streit et al., 1998) are expressed
in a decreasing dorsal to ventral gradient. Additionally, the
Gamma MN Sensory Fibers Alpha MN
static dynamic Ia II
Beta MN

DRG SC SN
IF EF
IN
MS B2 MNs Ex
OC B1 ten
CH so
rm
u
sc
Fle MS le
FIGURE 3 | Detailed innervation of a muscle spindle. Schematic of an xo
rm
adult muscle spindle (MS, light gray) on the longitudinal section (adapted us Limb
from Maier, 1997). Alpha MN (red) exclusively innervates (incoming arrow) cle
extrafusal fibers (EF, brown). Beta MNs (green-brown) innervate both EF
and intrafusal fibers (IF, blue). Gamma MNs are divided into two subtypes:
static (blue) connecting to nuclear chain (CH, light blue) and nuclear bag 2 FIGURE 4 | The spinal cord reflex circuitry. Schematic of a myotatic reflex
(B2, dark blue) fibers and dynamic (purple) connecting to nuclear bag 1 illustrating the spinal cord (SC) circuitry (adapted from Purves and Williams,
fibers (B1, intermediate blue). Sensory afferent axons Ia (light green) and II 2004). Sensory neuron (SN, blue) located in the dorsal root ganglia (DRG)
(pink) convey information (outgoing arrows) to sensory neurons located in transmits a stretch stimulus sensed by the muscle spindle (MS, gray) to an
the dorsal root ganglia. The outer capsule (OC) is a dedicated membrane interneuron (IN, purple) as well as directly to motor neurons (MNs, dark and
isolating the muscle spindle from the extrafusal fibers. A single MN light green). In turn, MNs stimulate the contraction of extensor muscle (red)
innervate multiple fibers all of the same type; however, for the schematic and ensure the concomitant relaxation of the antagonist flexor muscle
simplicity only one fiber is represented. located in the limb.

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Stifani Spinal motor neuron diversity

A B
Wnt
Neural crest BMP
Neural tube
Ectoderm
Somite

RA
Shh
Notochord Floor plate
FIGURE 5 | Early anatomy and inductive signals in the neural tube. (B) Schematic summarizing signals involved in the dorso-ventral
(A) Schematic of the anatomy of the neural tube after neurulation pattering of the mouse neural tube shown in transverse section
(adapted from Purves and Williams, 2004). The ectoderm (light blue) is (adapted from Dessaud et al., 2008). Wnt and BMP secreted by the
positioned on the external side whereas neural crest (orange) resides roof plate (blue) as well as retinoic acid (RA) produced by the somites
underneath. The notochord (gray) induces the differentiation of the floor (green) cooperate with Shh expressed by the floor plate and the
plate (red). The somites (green) give rise to muscles and bones. notochord (red) to pattern the neural tube.

surrounding paraxial mesoderm expresses the aldehyde dehy- most dorsal domain (Briscoe et al., 2000). Thus, cross-repressive
drogenase 1 A2 (ALDH1A2 or RALDH2) (Niederreither et al., interactions between pairs of class-I and class-II proteins guaran-
1997), which converts retinaldehyde into retinoic acid (RA) a tee the formation of sharp boundaries between adjacent domains
well-characterized regulator of neuronal differentiation (Pierani and ensure that they remain mutually exclusive. Ultimately, this
et al., 1999; Novitch et al., 2003; Lee et al., 2009). Together those process leads to the emergence of five ventral progenitor domains
signals collaborate with an increasing ventral to dorsal gradient (p0, p1, p2, pMN, and p3) defined by the expression of a unique
of sonic hedgehog morphogen (SHH) secreted by the underlying combination of transcription factors (Ericson et al., 1997; Briscoe
notochord as well as the floor plate (Yamada et al., 1991, 1993; et al., 2000; Vallstedt et al., 2001). This simple mechanism is in
Roelink et al., 1994; Marti et al., 1995a,b; Ericson et al., 1996) fact more complex as additional molecules ensure the integrity of
(Figure 5B). each individual progenitor domains. For example both WNT sig-
Molecularly, SHH binds to the patched homolog 1 recep- naling pathway (Lei et al., 2006; Alvarez-Medina et al., 2008; Yu
tor (PTCH1) (Stone et al., 1996) and releases its constitutive et al., 2008) and the transducin-like enhancer of split 1 (TLE1)
inhibition of the smoothened homolog (SMO) (Quirk et al., (Todd et al., 2012) contribute to reinforce the ventral boundary
1997) thereby, preventing the degradation of the GLI-Kruppel of the pMN domain. Another level of complexity arises from the
family (GLI) proteins (Chen et al., 2011b; Niewiadomski et al., interpretation of SHH gradient that is modulated by the down-
2014). Hence, SHH signaling correlates directly with GLI activity stream network of transcription factors. Hence, tis mechanism
(GliA) (Figure 6). Conversely, signals from the roof plate induce creates a feedback loop during the developmental period allowing
the expression of GLI repressors (GliR). Together, ventral and the modulation of progenitor domain formation (Balaskas et al.,
dorsal signals lead to a net decreasing gradient of GLI activity 2012).
from the ventral to the dorsal. In turn, GLI proteins promote Ultimately, the five ventral progenitor domains will generate
or repress in a concentration dependent manner homeodomain neuronal cells restricted to a specific lineage (V0, V1, V2, V3,
transcription factors that can be sorted into two classes: (i) Class- INs, and MNs) (Alaynick et al., 2011). Conceptually, the strat-
I; paired box 3/6/7 (PAX3/6/7), developing brain homeobox 1 egy used for the establishment of the progenitor domains involves
and 2 (DBX1/2), and Iroquois related homeobox 3 (IRX3) are inductive gradients interpreted into the expression of specific
repressed by GliA and thus expressed dorsally whereas (ii) Class- combinations of transcription factors. Cross-repressive interac-
II NK2 homeobox 2 and 9 (NKX2.2/2.9), NK6 homeobox 1 tions between pairs of transcription factors ensure the creation
and 2 (NKX6.1/6.2), and oligodendrocyte transcription factor 2 of mutually exclusive domains. Each progenitor domain then
(OLIG2) are induced by GliA and therefore, located ventrally generates progenies restricted to a specific lineage.
(Shirasaki and Pfaff, 2002). This initial patterning is subsequently
refined by cross-repression between pairs of class-I and class- ACQUISITION OF MOTOR NEURON FATE
II proteins. Studies using systematic gain or loss of function All SpMNs arise from the unique pMN progenitor domain that
approaches have identified the specific pairs of class-I and class- expresses the unique combination of the homeodomain proteins
II proteins. For example, inactivation of OLIG2 leads to a ventral NKX6.1, PAX6, and the basic helix-loop-helix (bHLH) protein
expansion of IRX3 (Zhou and Anderson, 2002) whereas ectopic OLIG2 (Tanabe et al., 1998; Novitch et al., 2001; Vallstedt et al.,
expression of NKX6.1 restrains the expression of DBX2 to the 2001). To become a mature MN, progenitors need to exit the

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Stifani Spinal motor neuron diversity

GliR Class-I Dbx1 Dbx2 Irx3 Pax6

Wnt
BMP p0 V0
p1 V1
V2
p2
pMN MN
p3 V3
Nkx6.2 Olig2
Shh GliA Class-II Nkx6.1 Nkx2.2
FIGURE 6 | Generation of ventral spinal progenitor domains. Schematic induce Class-I proteins (light blue) in the dorsal portion of the ventral spinal
summarizing the mechanisms of progenitor domain formation in the ventral cord. This initial expression pattern is subsequently refined by
spinal cord (adapted from Ulloa and Marti, 2010). Opposing gradients of Shh cross-repressive interactions between pairs of Class-I and Class-II proteins to
(red) and Wnt/BMP proteins (blue) are transduced into Gli protein activity. Gli generate five exclusive progenitor domains (p0, p1, p2, p3, and pMN). V0, V1,
activators (GliA, brown) in the most ventral region induce the expression of V2, V3, interneurons arise from the p0, p1, p2, and p3 respectively whereas
Class-II proteins (light brown) whereas Gli repressors (GliR, dark gray-blue) all MNs derive from the pMN progenitors.

cell cycle and enter the differentiation process. These events from the pMN domain (Richardson et al., 2000; Lu et al., 2002;
must be tightly regulated in order to generate an appropriate Zhou and Anderson, 2002). Therefore, the dynamic regulation
number of differentiated cells at a particular time during neuro- of OLIG2 and NEUROG2 during neurodevelopment allows the
genesis. Several mechanisms involved these transitions have been sequential generation of MNs and oligodendrocytes at different
characterized and will be described here. time from a common progenitor domain (Lee et al., 2005). An
First, RA described previously as a regulator of progenitor important downstream target of OLIG2 and NEUROG2 signaling
domain formation, is also involved in the acquisition of the MN is the motor neuron and pancreas homeobox 1 (MNX1 or HB9)
fate (Novitch et al., 2003). This process illustrates a principle com- (Tanabe et al., 1998; Lee et al., 2005, 2009). Remarkably, MNX1
monly seen in developmental biology and in biology in general, stimulates its own expression (Tanabe et al., 1998) providing to
namely, the use of a single cue at multiple steps during devel- developing MNs their independence from SHH and RA signaling.
opment as a mean to reduce the biological cost in energy. RA Therefore, MNX1 has been used as a reliable and specific marker
induces in MN progenitors the expression of glycerophosphodi- of post-mitotic SpMNs.
ester phosphodiesterase domain containing 5 (GDPD5 or GDE2) Although cell fates seem to be established early in devel-
(Jacobson and Rao, 2005; Rao and Sockanathan, 2005). In turn, opment, some evidences suggest that additional mechanisms
GDPD5 complexes with the peroxiredoxin 1 (PRDX1) (Yan et al., that ensure their maintenance are required. For example, MNs
2009) and with the GDP form of the G protein alpha subunit and V2 INs are generated by two adjacent progenitor domains
i2 (GNAI2) (Hammerle and Tejedor, 2007; Periz et al., 2010; (Figure 6). Inactivation of MNX1 in developing MNs induces a
Sabharwal et al., 2011; Park et al., 2013) to promote the MN dif- switch toward V2 IN fate (Arber et al., 1999; Thaler et al., 1999).
ferentiation program. Similarly, the cut-like homeobox 2 (CUX2) Comparably, the runt-related transcription factor 1 (RUNX1),
is involved in progenitors’ cell cycle progression and cell cycle exit whose expression is restricted to selected post-mitotic cervical
(Iulianella et al., 2008). MNs (Theriault et al., 2004; Stifani et al., 2008; Guizard et al.,
In parallel, OLIG1 and 2 contribute to the expression 2010) is important for the consolidation of MN phenotype by
of another bHLH protein named neurogenin 2 (NEUROG2) ensuring the persistent suppression of the IN program (Stifani
(Sommer et al., 1996; Novitch et al., 2001, 2003; Scardigli et al., et al., 2008). The molecular mechanism underlying the diver-
2001; Lu et al., 2002; Zhou and Anderson, 2002; Lee and Pfaff, gence between V2 INs and MNs have been remarkably revealed
2003; Lee et al., 2004). NEUROG2 interacts with the RA receptor by Pfaff and colleagues and involves the transient expression
(RAR) and recruits the histone acetyl transferases CREB bind- of the LIM homeobox 3 (LHX3) in developing MNs and V2
ing protein (CREBBP) and E1A binding protein p300 (EP300) INs (Thaler et al., 2002). In prospective V2 INs, LHX3 forms a
(Lee et al., 2009) to promote the transcription of downstream complex with the LIM domain binding 1 (LDB1 or NLI) and pro-
MN genes (Mizuguchi et al., 2001; Novitch et al., 2001; Scardigli motes the IN fate via the LIM domain only 4 (LMO4) (Thaler
et al., 2001; Lu et al., 2002; Zhou and Anderson, 2002; Lee et al., 2002; Lee et al., 2008). In prospective MNs, the ISL1 tran-
and Pfaff, 2003). Interestingly, during the early stage of MN scription LIM homeodomain (ISL1) is induced by SHH secreted
generation OLIG2 and NEUROG2 collaborate to promote MN by the notochord and floor plate (Yamada et al., 1991; Ericson
fate (Mizuguchi et al., 2001). At later stages, the persistence of et al., 1992) and inserts into the LHX3-LDB1 complex to induce
OLIG1/2 expression and the concomitant down-regulation of a switch toward MN specification (Thaler et al., 2002; Song et al.,
NEUROG2 allow the emergence of oligodendrocyte progenitors 2009). Most importantly ISL1-LHX3 complex directly binds and

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Stifani Spinal motor neuron diversity

induce the expression genes involved in cholinergic neurotrans- of the polycomb repressive complex (Golden and Dasen, 2012).
mission, a fundamental characteristic of SpMNs (Cho et al., 2014; At the caudal edge, cross-repressive interactions between pairs of
Kania, 2014a). Despite these important findings the acquisition HOX proteins (Dasen et al., 2003, 2005) lead to non-overlapping
and the maintenance of MN fate remain to be fully understood. domains. For instance, HOX6, 9, and 10 expression correlates
A recent study has shed light on the mechanisms by which with the brachial, thoracic, and lumbar segments, respectively.
MN precursors detach from neuroepithelium and migrate lat- Subsequently, HOX patterning induces the formation of
erally as they exit the cell cycle (Rousso et al., 2012). What are anatomical columns termed motor columns along the rostro-
the molecular mechanisms controlling such migration? Rousso caudal axis (Shah et al., 2004; Dasen et al., 2005; Jung et al.,
et al. (2012) remarkably identify the role of the forkhead box P2 2010). The underlying mechanisms have been partially defined
and 4 (FOXP2/4) in promoting the detachment of newly born since HOX patterning converges toward the expression of FOXP1
MNs from the ventricular zone. Additionally, the authors ele- (Arber, 2008; Dasen et al., 2008; Pfaff, 2008; Rousso et al., 2008;
gantly linked nuclear gene regulation to effector protein at the Palmesino et al., 2010). Mechanistically, HOX6 and 10 at brachial
membrane. Namely, FOXP2/4 repress the expression cadherin 2 and lumbar segments respectively direct the expression of FOXP1,
(CDH2) responsible for the attachment of developing progenitors which in turn cooperate with HOX proteins to induce the for-
to the neuroepithelium. mation of limb specific MNs at the expense of thoracic MNs
In conclusion, the emergence of newborn MNs from the pMN (Dasen et al., 2008; Rousso et al., 2008). Additionally, HOXC9 has
progenitor domain relies on the precise control of the balance a critical role in restricting appendage specific MNs to the limb
between proliferation and differentiation. Although OLIG2 and innervating segments by selectively excluding them from thoracic
NEUROG2 have prominent roles into MN fate commitment, segments (Jung et al., 2010, 2014). This effect is at least partially
additional mechanisms are required to ensure the consolidation mediated by direct and indirect repression of FOXP1 in thoracic
of this phenotype. Following the acquisition of their general iden- segments.
tity, MNs need to differentiate and acquire features required for In summary, after the formation of dedicated progenitor
their respective function. This process, termed patterning, will be domains, intrinsic and extrinsic molecules cooperate to pro-
described hereafter. mote a general MN fate. Inductive signals along the rostro-caudal
axis profile developing MNs to adjust to specific local needs.
PATTERNING IN SPINAL MOTOR NEURON DEVELOPMENT Together these mechanisms lead to the formation of anatomically
Following the initial acquisition of their general fate, newborn defined motor columns. We will describe hereafter each column
SpMNs are required to further differentiate to adopt an iden- by providing information on their molecular specificity as well as
tity reminiscent of their respective muscle targets. The general mechanisms of their formation.
strategy is, at least in part, comparable to the mechanisms lead-
ing to the emergence of spinal progenitor domains. Globally, COLUMNAR ORGANIZATION OF SPINAL MOTOR NEURONS
SpMN specification follows a temporal gradient along the ventro- SpMNs are organized into distinct anatomical columns extend-
dorsal and rostro-caudal axes (Nornes and Carry, 1978). MNs ing along the rostro-caudal axis and called motor columns
located more ventrally and more rostrally are generated earlier. (Figure 7). Previous studies have described four main columns:
This temporal regulation reflects two mechanisms: (i) the pro- the median motor column (MMC), the lateral motor column
gressive expansion of the total volume of the neural tissue and (LMC), the hypaxial motor column (HMC), and the pregan-
(ii) the generation of specific cell types along the rostro-caudal glionic column (PGC) (Prasad and Hollyday, 1991; Tsuchida
axis. et al., 1994; Jessell, 2000; Alaynick et al., 2011; Francius and
Several proteins including the fibroblast growth factor (FGF), Clotman, 2014) Each column possesses a coherent gene expres-
the growth differentiation factor 11 (GDF11 or BMP-11), mem- sion profile as well as a uniform axonal projection pattern
bers of transforming growth factor beta (TGFB) family as well (Figure 8). We will hereafter describe their molecular identity as
as RA (Durston et al., 1989; Muhr et al., 1999; Liu et al., 2001; well as the developmental mechanisms required for their forma-
Bel-Vialar et al., 2002; Sockanathan et al., 2003; Liu, 2006) tion. Moreover, we will complete this picture by describing the
form a gradient along the rostro-caudal axis and induce, in a less well-characterized spinal accessory column (SAC) and the
concentration-dependent manner, the expression of protein of phrenic motor column (PMC).
the homeobox (HOX) family (Ensini et al., 1998; Lance-Jones
et al., 2001). Hox genes are arranged into genomic clusters and The median motor column
their response to FGF and RA concentration is correlated to their MMC MNs are located in the medial region of the ventral spinal
position within a cluster (Liu et al., 2001; Bel-Vialar et al., 2002). cord and target to the dermomyotome (Gutman et al., 1993;
Genes located at the 5 end are induced by high concentration of Tsuchida et al., 1994), which gives rise to the axial musculature
FGF and thus expressed in more caudal regions. Conversely, genes later in development (Fetcho, 1987; Gutman et al., 1993). Axial
at the 3 end are induced by low concentrations of FGF and there- muscles are mainly involved in the maintenance of the body pos-
fore expressed in more rostral regions (Liu et al., 2001; Bel-Vialar ture and are found all along the body axis. Therefore, MMC
et al., 2002; Dasen et al., 2003; Mazzoni et al., 2013b). After the MNs are not segmentally restricted and are found all along the
initial activation of HOX protein expression, further refinement spinal cord (Figure 7). MMC MNs are characterized by the co-
is achieved at the rostral boundary by histone modifications per- expression of MNX1, ISL1/2, and LHX3 (Tsuchida et al., 1994)
formed by the Bmi1 polycomb ring finger oncogene (BMI1) part (Figure 8). In mature MNs, LHX3 is unique to MMC MNs and its

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Stifani Spinal motor neuron diversity

Cervical Thoracic Lumbar Sacral

1 2 3 4 5 6 7 8 1 12 1 2 3 4 5 1 2 3 4 5

MMC
SAC
PMC
PGC
HMC
LMC
FIGURE 7 | Segmental organization of spinal motor columns. Schematic preganglionic column (PGC, orange) extends through the thoracic segments
summarizing the segmental distribution of spinal motor columns (adapted until the second lumbar segments (L2) as well as well as between sacral
from Dasen and Jessell, 2009). While the medial motor column (MMC, segments 2 and 4 (S2–S4). The hypaxial motor column (HMC, light blue) is
brown) is present all along the rostro-caudal axis, the spinal accessory exclusive of the thoracic segment where as the lateral motor column (LMC,
column (SAC, purple) is restricted to the five first cervical segments (C1–C5). dark and light green) is located at limb levels: brachial (C5-T1) and lumbar
The phrenic motor column (PMC, red) is confined between C3 and C5. The segments (L1–L5).

forced expression is sufficient to impose MMC identity (Sharma MNs (vMNs) exiting classically via the ventral root. Molecularly,
et al., 1998, 2000). LHX3 is therefore commonly used as a reliable SAC MNs have been successfully distinguished from other MNs
marker of MMC MNs; however, as mentioned earlier, LHX3 is by the use of different markers such as activated leukocyte cell
also transiently expressed developing MN in which it contributes adhesion molecule (ALCAM or BEN) (Schubert and Kaprielian,
to the establishment of their identity at the expense of the V2 2001; Dillon et al., 2005) as well as ISL1, RUNX1 and the paired-
INs. Interestingly, MMC MNs present an exception to the rostro- like homeobox 2b (PHOX2B) (Pattyn et al., 2000; Amiel et al.,
caudal patterning of HOX proteins. How do MMC MNs escape 2009; Dubreuil et al., 2009; Stifani and Ma, 2009; Kobayashi et al.,
HOX rostro-caudal patterning? Molecularly, proteins from the 2013; Mazzoni et al., 2013a; Laumonnerie et al., 2014).
WNT family (WNT4/5A/5B) are expressed in a ventral to dorsal Developmentally, the NK2 homeobox 9 (NKX2.9) has been
decreasing gradient (Agalliu et al., 2009) and permit the persis- shown to be required for proper SAC MN generation (Pabst et al.,
tence of LHX3/4 expression (Shirasaki and Pfaff, 2002; Agalliu 2003) as well as for SAC axonal projection (Dillon et al., 2005).
et al., 2009) in the most ventral region. In turn, LHX3/4 make Conversely, LHX3/4 inactivation leads to an increase number of
MMC MNs unresponsive to HOX patterning (Dasen et al., 2005, SAC MNs (Sharma et al., 1998) whereas LHX3 is sufficient to pro-
2008). As suggested by Dasen and colleagues, this unique feature mote vMNs at the expense of dMNs (Lieberam et al., 2005; Hirsch
likely reflects the ancestral properties of the MMC from which et al., 2007; Kobayashi et al., 2013). Together these results sug-
other motor column have derived during evolution (Dasen et al., gest that the expression of LHX3/4 allows vMNs generation at the
2008; Dasen, 2009; Dasen and Jessell, 2009; Jung et al., 2010; expense SAC MNs.
Philippidou and Dasen, 2013). SAC MNs are a very peculiar population of SpMNs as they
are the exclusive cells of branchial type in the spinal cord.
The spinal accessory column Several characteristics described above are reminiscent of hind-
SAC MNs are located in the intermedio-lateral region of the brain branchiomotor and visceromotor MN populations and
spinal cord and expand from the end of the medulla until largely differ from other SpMNs. SAC MNs may appear there-
the 5th cervical segment (C1–C5) (Jacobson and Marcus, 2007; fore as a transitional population between the hindbrain and
Ullah et al., 2007) (Figure 7). SAC MNs innervate mastoid cervical MNs.
muscles as well as four muscles of the neck (Sternomastoid,
Cleidomastoid, Cleidotrapezius, and Acromiotrapezius) (Brichta The phrenic motor column
et al., 1987; Watanabe and Ohmori, 1988). While SAC MNs inner- Phrenic MNs are located in the cervical segments C2–C6 at
vating the mastoid muscles are located in the rostral portion, MNs embryonical stages and become progressively confined between
innervating the trapezius muscles are located in the most cau- cervical levels C3–C5 by birth (Webber and Pleschka, 1976;
dal segments of the C1–C5 segment of the spinal cord (Ullah Allan and Greer, 1997a,b; Song et al., 2000) (Figure 7). They
et al., 2007; Stifani et al., 2008). SAC MNs are different from connect to a particular muscle: the diaphragm. This muscle is
other SpMNs because they innervate muscles that derive from essential for respiration and therefore is under constant rhyth-
branchial arches (Pabst et al., 1998; Aldskogius et al., 2009) and mic activity. This characteristic also applicable to the cardiac
because their axons penetrate the periphery by exiting through muscle differs diametrically from skeletal muscles required to
the lateral exit point (LEP) located midway along the dorso- generate unsystematic contraction. The diaphragm is involved
ventral axis of the spinal cord (Sharma et al., 1998; Schubert and in both inspiration and expiration, both conscious and uncon-
Kaprielian, 2001; Pabst et al., 2003; Dillon et al., 2005; Bravo- scious. Because of its unique function, phrenic MNs are required
Ambrosio and Kaprielian, 2011) (Figure 8). Therefore, SAC MNs to produce a perpetual rhythmic firing as early as the first
are also referred as dorsal MNs (dMNs) as opposed to ventral instant after birth and throughout life. Although phrenic MNs

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Stifani Spinal motor neuron diversity

MMC PMC SAC

Cervical
SAC Epaxial
Mastoid
PMC LEP Neck

MMC

Mnx1 Mnx1 Isl1

Isl1/2 Isl1/2 Alcam Diaphragm
Lhx3 Alcam Phox2b
Scip

B
Brachial / Lumbar

MMC LMC
LMC Epaxial
m
l
Limb
MMC d
v
Mnx1 Foxp1 Foxp1
Isl1/2 Isl1/2 Isl2
Lhx3 Lhx1
Mnx1
C
Thoracic

MMC HMC PGC

PGC Epaxial

HMC
MMC
Hypaxial SCG
Mnx1 Mnx1 Isl1 AdrG
Isl1/2 Isl1 nNos
Lhx3 Isl2low Foxp1low
Etv1 pSmad
Zeb2

FIGURE 8 | Organization of SpMNs at cervical, brachial/lumbar and divisions medial (m, dark green) and lateral (l, light green). LMCm MNs
thoracic levels. Schematic summarizing the characteristics of spinal connect to the ventral (v) part of the limb whereas LMCl MNs innervate
motor columns at cervical (A), brachial/lumbar (B) and thoracic (C) levels the dorsal (d) region. HMC MNs (light blue) are located in the
(adapted from Dasen and Jessell, 2009). MMC MNs (brown) are located medio-lateral region and connect to the body wall and intercostal
medially and connect to the axial musculature (Epaxial). PMC MNs (red) muscles (Hypaxial). PGC MNs (orange) are positioned dorso-laterally and
have an inter-medio-lateral position and connect to the diaphragm. SAC innervate to the sympathetic chain ganglia (SCG) and chromaffin cells of
MNs (purple) exit the CNS via the lateral exit point (LEP) and connect to the adrenal gland (AdrG). Proteins expressed by each column are
mastoid and neck muscles. LMC MNs (green) are divided into two depicted with their respective color code.

have been well characterized in terms of cell body position and of phrenic MNs (Philippidou et al., 2012). Namely, phrenic MNs
anatomical properties, until recently little was known about their are under the control of HOX5 patterning and are expressing high
molecular characteristics. In a well-detailed study, Philippidou levels of the POU domain class 3 transcription factor 1 (POU3F1
and colleagues identified for the first time the molecular profile or SCIP) as well as ISL1/2, and MNX1 (Thaler et al., 1999; Rousso

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Stifani Spinal motor neuron diversity

et al., 2008; Castellani and Kania, 2012; Philippidou et al., 2012; located in the proximity of the spine, sacral PGC MNs connect
Philippidou and Dasen, 2013) (Figure 8). Interestingly, soon after to ganglia in the vicinity of the effector targets (kidney, blad-
birth, phrenic MNs as well as the diaphragm muscle itself undergo der, gonads). Therefore, the molecular properties of sacral PGC
significant anatomical and functional transformations including MNs that remain largely unknown are presumably substantially
dendritic arborization and electrical activity properties (Cameron different from thoracic PGC MNs.
et al., 1991; Prakash et al., 2000). These changes reflect the
passage from the aquatic intrauterine gestation to the aerobic The hypaxial motor column
life. Therefore, although phrenic MNs are established early in Initially the MMC had been separated in two divisions: (i) a
development to ensure their functionality for the time of birth, medial MMC (MMCm), described above as MMC, targeting to
additional mechanisms, yet to be characterized, are likely occur- axial musculature and present all along the rostro-caudal axis
ring after birth to guarantee further post-natal maturation. The and (ii) a lateral MMC (MMCl) targeting to the body wall and
vital role of the PMC coupled to the recent molecular findings present only in the thoracic segments (Gutman et al., 1993; Jessell,
cited above encouraged Machado and colleagues to induce the 2000). However, recent molecular findings have associated MMCl
differentiation of embryonic stem cells into phrenic MNs in vitro MNs with PGC and LMC MNs rather than with MMC MNs
(Machado et al., 2014). This prodigious achievement carries great (Dasen et al., 2008; Rousso et al., 2008). Therefore, the MMCl has
hope for stem cell based regenerative therapies as patients suffer- been referred to as the hypaxial motor column (HMC) (Dasen
ing from SpMN diseases ultimately face respiratory impairments. et al., 2008; Agalliu et al., 2009). This new nomenclature better
Nevertheless, additional efforts are still required to establish a reflects HMC MN molecular nature and avoids confusion with
viable therapy from this initial breakthrough. MMC MNs. HMC MNs are located in the ventro-lateral spinal
cord and innervate muscles derived from the ventral mesenchyme
The preganglionic motor column (Smith and Hollyday, 1983). The ventral mesenchyme gives rise
PGC MNs also known as spinal visceral MNs constitute the CNS to the body wall musculature composed of the intercostal and
component of the ANS. They are located in the thoracic and abdominal muscles present only at thoracic level (Prasad and
upper lumbar spinal segments (T1–L2) (Figure 7) where they Hollyday, 1991). Therefore, HMC MNs are only present at tho-
occupy an intermedio-lateral location (Figure 8). They do not racic level (Tsuchida et al., 1994; Sharma et al., 2000) (Figure 7).
innervate skeletal muscles as other somatic SpMNs do but instead Molecularly, HMC MNs are characterized by the expression of
connect to the sympathetic ganglia. Thus, PGC MNs are involved MNX1, ISL1, ETS variant 1 (ETV1 or ER81) and low levels of ISL2
in stimulation of smooth muscles as well as in control of glands (Dasen et al., 2008; Rousso et al., 2008) (Figure 8). Interestingly,
secretions. They can be molecularly identified by the expression of FOXP1 inactivation converts both PGC and LMC MNs to a HMC
the SMAD family member 1 (SMAD1 or pSMAD1) (Dasen et al., phenotype (Dasen et al., 2008). As suggested by Dasen and Jessell
2008), nitric oxide synthase 1 neuronal (NOS1 or nNOS) (Saito (2009), HMC and MMC MNs likely reflect the vestige of an ances-
et al., 1994; Wetts and Vaughn, 1994; Dasen et al., 2003), zinc fin- tral spinal motor column organization from which other motor
ger E-box binding homeobox 2 (ZEB2 or SIP1) (Roy et al., 2012), columns derived (Jung et al., 2014). Finally, because intercostal
as well as low level of FOXP1 (Dasen et al., 2008; Rousso et al., and abdominal muscles are involved in respiration, HMC MNs
2008; Morikawa et al., 2009). could presumably be somehow related to PMC MNs described
Despite extreme functional differences, somatic and visceral previously. To our knowledge no experiment has been reported
SpMNs arise from common precursors expressing ISL1/2 (Prasad to address this suggestion that remains to be tested.
and Hollyday, 1991; Thaler et al., 2004). The maintenance of
ISL2 in maturing MNs leads to the generation of somatic MNs The lateral motor column
whereas its down-regulation together with the persistence of ISL1 LMC MNs are located in the most lateral portion of the ven-
guides maturing MNs toward the visceral fate (Thaler et al., 2004). tral spinal cord (Bueker, 1944). They connect to muscles of
Recently, the one cut domain family members (ONECUT1/2/3), the appendages and therefore are present only at limb levels
expressed in newly born MNs (Francius and Clotman, 2010, 2014; also defined as brachial (C5 to T1) and lumbar levels (L1–L5)
Audouard et al., 2012), have been found to bind directly to a (Hollyday and Hamburger, 1977; Hollyday and Jacobson, 1990)
specific enhancer region of Isl1 gene to maintain its expression (Figure 7). This segmentation reflects the rostro-caudal pattern-
(Roy et al., 2012) resulting in a limitation of PGC MN formation. ing of HOX proteins (Kessel and Gruss, 1991; Liu et al., 2001;
This consequence is challenged by the effect of ZEB2 promoting Dasen et al., 2003) controlled by local inductive signals (Ensini
PGC formation. Therefore, opposing and cooperating mecha- et al., 1998).
nisms ensure the proper divergence between somatic and visceral LMC MNs are further separated into two divisions: medial
SpMNs. and lateral (Tosney et al., 1995). These divisions retain a topo-
As mentioned earlier, spinal visceral MNs are also found in graphic correspondence with the localization of their target in
the sacral segments (S2–S4). However, these cells belong to the the periphery. Medial LMC (LMCm) MNs target to the ventral
parasympathetic system (rest and digest) while thoracic PGC part of the limb whereas lateral LMC (LMCl) MNs innervate
MNs belong to the sympathetic autonomic system (fight or the dorsal limb musculature (Landmesser, 1978; Tosney and
flight). In addition to these functional differences, thoracic and Landmesser, 1985a,b; Kania et al., 2000) (Figure 8). Molecularly,
sacral PGC MNs also differ in terms of axonal projections. While LMC MNs are characterized by the expression of ISL2, FOXP1,
thoracic PGC MNs connect to the sympathetic chain ganglia and ALDH1A2 and do not sustain LHX3 expression (Tsuchida

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Stifani Spinal motor neuron diversity

et al., 1994; Sockanathan and Jessell, 1998; Dasen et al., 2008; interactions between HOX members produce a unique combi-
Rousso et al., 2008). Sockanathan and Jessell (1998) have remark- natorial code that directs MN pool identity (Dasen et al., 2005;
ably revealed the molecular mechanism leading to the emergence Lacombe et al., 2013). This identity is revealed by the activation
of LMC divisions. At limb levels, the paraxial mesoderm secretes of pool specific proteins such as the ETV1 and ETV4 (or PEA3)
RA that induces the generation of LMC MNs (Ensini et al., 1998; (Lin et al., 1998; Ladle and Frank, 2002; Livet et al., 2002), RUNX1
Sockanathan et al., 2003; Vermot et al., 2005). Early born LMC (Theriault et al., 2004; Dasen et al., 2005; Stifani et al., 2008;
MNs co-express ISL1/2 as well as ALDH1A2 and in turn secrete Zagami et al., 2009; Lamballe et al., 2011) and POU3F1 (Dasen
RA. This additional signal induces the down-regulation of ISL1 et al., 2005; Rousso et al., 2008). By doing so, Dasen et al. (2005)
to the profit of the Lim homeobox 1 (LHX1) in later born LMC. have remarkably linked the intrinsic HOX combinatorial network
Furthermore, cross-repressive interactions allow both divisions to to extrinsically induced factors whose expressions are dependent
remain mutual exclusive (Kania and Jessell, 2003). ISL1 and LHX1 on a signal from the periphery (Lin et al., 1998; Haase et al.,
also control the differential segregation of the cell body posi- 2002) described in more detail below. However, to date the entire
tion of LMC divisions (Sockanathan and Jessell, 1998; Kania and mapping of HOX proteins in SpMN pools remains unpublished.
Jessell, 2003; Rousso et al., 2008). Interestingly, matured LMCm Furthermore, molecular effectors of pool specificity downstream
MNs down-regulate MNX1 expression (Kania and Jessell, 2003; of the HOX combinatorial network remain elusive.
Rousso et al., 2008); a unique characteristic among SpMNs. Yet, In parallel to intra-segmental HOX combinatorial network,
the functional relevance of this distinct observation remains to be NKX6.1 contributes to the intrinsic mechanisms of MN pool
elucidated. Further information about LMC will be provided in specification (De Marco Garcia and Jessell, 2008). First, NKX6.1
the section dedicated to axonal targeting. is expressed in subsets of LMC MNs independently of the pres-
To date, 6 different motor columns have been identified in ence of its muscle target. Second, NKX6.1 inactivation leads to
mouse the spinal cord. The SAC located in the rostral cervi- persistent muscle targeting errors. These results strongly suggest
cal segments is the only representative of the branchial category that NKX6.1 participates in controlling MN pool specificity and
whereas the PGC in the thoracic and sacral segments is the only uncover two sequential roles of NKX6.1 in MN development.
visceral motor column. In contrast, MMC, HMC, PMC, and LMC In the early phase, it takes part in the specification of progen-
are somatic and innervate skeletal muscles belonging to different itor domains in response to SHH gradient whereas in the late
groups. However, to date SpMNs haven’t been mapped at the sin- phase, it contributes to the specification of discrete MN pools.
gle cell resolution levels (Wichterle et al., 2013). Therefore, the Strategically, intrinsic cues allow the development and the mat-
possibility of having uncharacterized discrete SpMN populations uration of MNs independently of their location. This approach
can’t be excluded. Furthermore, SpMN diversity expands beyond provides plasticity and tolerance to adapt to changes in the
the columnar organization described above. In fact, SpMNs form peripheral environment.
muscle specific groups termed pools. We will review hereafter the Unlike NKX6.1 and the HOX combinatorial intra-segmental
mechanisms driving motor pool formation. network described above, extrinsically induced players are
expressed in developing MNs upon reception of a specific sig-
SPECIFICATION OF MOTOR NEURON POOLS nal. This mechanism can be considered as a checkpoint ensuring
A remarkable event in SpMN development is the acquisition of further developmental refinements only after the completion of
MN pool identity, assigning to a given group a specific muscle prerequisite steps. What are the extrinsic signals allowing further
target. The coordination between more than 50 different mus- MN differentiation? So far, only one factor has been unam-
cles in the typical amniotes’ limb required to perform complex biguously identified. Namely, the glial cell derived neurotrophic
movements implies a precise mechanism to assign to each mus- factor (GDNF) is secreted by both Cutaneous maximus (CM)
cle a corresponding MN pool (Romanes, 1941; Sullivan, 1962). and Latissimus dorsi (LD) muscles and induces the expression of
Previous studies have described the localization of individual MN ETV4 in the corresponding MN pools (Lin et al., 1998; Haase
pools according to specific targets (Landmesser, 1978; Hollyday et al., 2002; Livet et al., 2002). The analysis of ETV4 mutant
and Jacobson, 1990; Choi and Hoover, 1996; Ryan et al., 1998) animals revealed that even though some aspects of MN devel-
and suggest that MN pools respect a topographic organization opment are pre-established by intrinsic cues, later signals are
(reviewed by Kania, 2014b). The more rostral a MN pool is posi- further required for the maintenance of MN pool characteristics
tioned, the more anterior and proximal the target is located. such as cell body position, axonal arborization and dendritic pat-
Interestingly, MNs possess predetermined intrinsic features inde- terning ensuring the establishment of correct input connections
pendent of the presence of peripheral targets that control at (Ladle and Frank, 2002; Livet et al., 2002; Vrieseling and Arber,
least partially pool specification (Phelan and Hollyday, 1990). 2006). Additionally, after the initial expression of ETV4 induced
Therefore, MN pool determination can be divided in two phases by GDNF, CM MNs recruits adjacent MNs and induce in a non-
(i) purely intrinsic and (ii) extrinsically induced (Dasen, 2009). cell autonomous manner the expression of ETV4 (Helmbacher
The intrinsic molecular mechanisms of MN pool specifica- et al., 2003). Therefore, one of the strategy initial differentiation
tion are not yet fully understood, however it appears to rely followed by the recruitment in situ of neighboring MNs.
on the combinatorial expression of HOX proteins. Dasen et al. Together these results illustrate the coordination between
(2005) have performed an extensive screen of the expression of 39 intrinsic and extrinsically-induced cues. While the first group
Hox genes as well as HOX cofactors. Their results demonstrate allows MN development independently of the environment, the
that within a specific rostro-caudal segment, cross-repressive second ensures the completion of essential steps. Together these

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Stifani Spinal motor neuron diversity

mechanisms create a flexible process allowing MNs to adapt to novel molecules involved in SAC MNs axonal targeting could
environment variability. By definition, MN pool specification is presumably be restricted to the first cervical segments. Unbiased
intimately linked to axonal targeting. Intensive works have iden- differential screenings of genes downstream of transcription fac-
tified various molecules involves in SpMN axonal targeting. We tors exclusive of dMNs (PHOX2B) or vMNs (LHX3/4) may
will dedicate the next section to the review the known molecular identify new effector molecules involved in their divergence.
mechanisms controlling SpMN axonal targeting. The second step in MN axonal growth consists in selecting
the orientation toward their forthcoming muscle target (termed
MOTOR NEURON AXONAL TARGETING “Columns” in Figure 9). Schematically, growing vMN axons can
Axonal targeting is a critical process of MN development. MN adopt three directions: (i) dorsal, toward the axial musculature
axons emerge within the CNS and transit through different tissues (MMC), (ii) lateral, invading the limb (LMC) or (iii) ventral,
to reach and connect to their specific muscle target in the periph- toward the sympathetic chain or to the body wall muscula-
ery. Axonal targeting not only provides to MNs their unique ture (PGC and HMC, respectively). This schematic intentionally
anatomical characteristic and therefore their irreplaceable func- omits PMC targeting for simplicity. These decisions are com-
tion but also ensures their persistence through the action of prised within the identity of a particular motor column and
trophic signals. In order to complete such critical process, MNs therefore considered as intrinsic. Presumably, the unique combi-
combine several mechanisms in a stepwise manner (Figure 9). natorial expression of transcription factors controls downstream
Several “checkpoints” are established along the axonal route, each effectors and modulators of axonal growth. Although the molec-
one requiring a choice to orientate toward a particular direc- ular mechanisms remain largely unknown, MMC MNs express
tion. While the initial steps rely on intrinsic mechanisms, the the fibroblast growth factor receptor 1 (FGFR1) and are attracted
late aspects of MN axonal targeting rely on signals received by the dermomyotome secreting FGF (Shirasaki et al., 2006;
at the growth cone, and inducing molecular and anatomical Soundararajan et al., 2010). Additionally, MMC axons express-
modifications. ing the Eph receptor A3 and 4 (EPHA3 and 4) are constrained by
The very first choice SpMN axons make occurs within the repellent contact with sensory DRG neurons expressing ephrin-
spinal cord (termed “CNS exit” in Figure 9). vMNs leave the As (EFNA1) (Gallarda et al., 2008). Together these mechanisms
CNS via the ventral root whereas dMNs exit more dorsally via lead MMC axons to bypass the DRG and target to the axial
the LEP. This decision is at least partially controlled by LHX3 musculature (Figure 10B). The molecules leading LMC axons
and 4 (Sharma et al., 1998; Bravo-Ambrosio and Kaprielian, to initially target the limb are unknown, however Huber et al.
2011). Yet, LHX3/4 are transcription factors and therefore are (2005) revealed the role of Semaphorin-Neuropillin in control-
unlikely the effectors of this axonal targeting decision made at ling the timing and the fasciculation of LMC axons. Neuropilin
the growth cone. Instead, the chemokine (C-X-C motif) recep- 1 (NRP1) expressed by LMC axons mediates the repulsion from
tor 4 (CXCR4) is expressed by vMN axons and its ligand CXCL12 the limb mesenchyme expressing semaphoring 3A (SEMA3A).
localizes in the ventral mesenchyme surrounding the spinal cord. Inactivation of SEMA3A-NRP1 signaling results in a premature
This molecular signal attracts vMN axons toward the ventral invasion of the limb bud. Interestingly, NRP1 is expressed by
root (Lieberam et al., 2005). Conversely, dMNs express the netrin both MN and SN axons and contributes to MN axon fascicula-
receptor deleted in colorectal carcinoma (DCC) and are repelled tion along the sensory axons (Huettl et al., 2011). This example
away from the midline expressing netrin 1 (NTN1) (Dillon et al., illustrates the use of a single molecule to synchronize sensory and
2005) (Figure 10A). The complete molecular mechanisms allow- motor development (Wang et al., 2011; Fukuhara et al., 2013).
ing dMNs to escape the classical ventral root exit are yet to be Such strategy ensures the formation of a coherent and functional
characterized. As dMNs are absent outside of the cervical regions, circuitry. Lastly, PGC and HMC axons specifically turn ventrally

CNS exit Columns Divisions Pool intrinsic Pool extrinsic

Epaxial LMCl d
Lhx1 Etv4
LEP
LMCm Nkx6.1
Isl1 v Gdnf
dMN MMC LMC
Lhx3 Foxp1 Limb
vMN Dorsal vs ventral Muscle connection Terminal arborization
Lhx3/4

FIGURE 9 | Steps of MN axonal targeting. Schematic summarizing the choice made by the medial and lateral divisions of the LMC. LMCl MNs (light
steps of MN axonal targeting (adapted from Dasen and Jessell, 2009). The green) invade the dorsal part of the limb (d) whereas LMCm (dark green) MNs
first step termed “CNS exit” reflect the choice of developing MNs to exit the target to the ventral region (v). The fourth step termed “Pool intrinsic” refers
CNS via the ventral root (vMNs, blue) or through the lateral exit point (LEP) to the selection of a specific muscle target (red) and is controlled by intrinsic
(dMNs, red). The second choice labeled “Columns” return to the motor cues. The last step named “Pool extrinsic” illustrates the induction of specific
column: MMC MNs (brown) target to epaxial musculature whereas LMC MNs protein expression upon a signal from the muscle target, which coordinates the
(green) project to the limb. The third step named “Divisions” refers to the terminal arborization of MN axons. Proteins involved in each step are indicated.

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Stifani Spinal motor neuron diversity

A B C
Ephrin-As
dMN +
DCC + Fgf LMCm LMCl
+
LMC FgfR1 EphA4
Ntn1 MMC EphA3/4 Ephrin-Bs Ephrin-As
+
vMN + Sema3F EphAs
CxcR4 + CxcL12 Sema3A +
Npn1 EphB1 +
Npn2 +
Ephrin-As

FIGURE 10 | Guiding cues of SpMN axonal targeting. Schematic further growth. This pause is mediated by Npn1-Sema3A repellent
summarizing guiding cues important for MN axonal targeting. (A) signaling expressed by LMC MNs and the limb respectively. (C) LMCm
Ventral exiting MNs (vMN, blue) express CxcR4 and are attracted (plus MN (dark green) axons express EphB1 and Npn2 and are constrained
signs) by CxcL12 expressed by the mesenchyme (dark green). Dorsal into the ventral limb by Sema3F and Ephrin-Bs expressed by the dorsal
MNs (dMN, purple) express DCC and are repelled (minus sign) away limb mesenchyme (dark brown). Conversely, LMCl MN (light green)
from the midline expressing Ntn1 (light green). (B) MMC MNs (brown) axons express Ephrin-As and EphA4 and are restricted to the dorsal
expressing both FgfR1 and EphA3/4 are attracted by Fgf secreted by part of the limb by a combination of Ephrin-As repulsive signal from
the dermomyotome but repelled by Ephrin-As expressed by the dorsal the ventral limb mesenchyme (light brown) and EphAs (red) attractive
root ganglion. LMC MNs (green) target to the limb and pause before signal from the dorsal part of the limb.

toward the sympathetic chain and the body wall musculature, in cis (Kao and Kania, 2011) regulate LMC MN axonal targeting.
respectively. To date the mechanisms of such decision remain Furthermore, the tyrosine kinase receptor Ret proto-oncogene
unidentified. (RET) acts co-receptor for both GDNF and ephrin-As modulat-
The lateral and medial divisions of the LMC have provided a ing their response and thus adding another layer of complexity
powerful framework to study MN axonal decisions. After enter- in LMC MN axonal targeting (Bonanomi et al., 2012). Together
ing the base of the limb LMC axons pause before targeting these results demonstrate that LMC targeting is complex and
toward the dorsal or the ventral parts of the limb (Tosney and tightly regulated. Further experiments will permit a better under-
Landmesser, 1985a; Wang and Scott, 2000). LMCm MNs express standing of this multifaceted process.
ISL1 and target to the ventral part of the limb whereas LMCl MNs After making their initial decisions MN axons need to select
express LHX1 and connect to the dorsal part of the limb (termed their specific muscle target. This step is closely related to the
“Division” in Figure 9). Interestingly, LHX1 inactivation does not formation of MN pools discussed above. MNs are programmed
perturb LMCl formation but instead impairs the dorsal/ventral to recognize their muscle target (Lance-Jones and Landmesser,
axonal projection specificity (Kania et al., 2000). Reciprocally, the 1980). Intrinsic cues are expressed in a pool specific manner
LIM homeobox transcription factor 1 beta (LMX1B) expressed in to direct MN axons toward their specific muscle target (termed
a decreasing dorsal to ventral gradient in the limb mesenchyme “Pool Intrinsic” in Figure 9). NKX6 (De Marco Garcia and Jessell,
is also important for LMC divisions axonal targeting (Kania 2008) as well as the HOX combinatorial network (Dasen et al.,
et al., 2000). The molecular mechanisms of LMC axonal target- 2005) have been proposed as intrinsic regulators of muscle tar-
ing rely prominently on Ephrin-Eph signaling and have been the get selection. Presumably, other molecules, yet to characterize,
source of recent exciting discoveries summarized by Bonanomi play a role in the establishment of specific connections between
and Pfaff (2010) and reviewed in depth by Kao et al. (2012). a MN pool and its respective muscle target. Among them, the
In brief, LMCl MNs express LHX1 that induces the expression downstream molecular effectors that regulate axonal path finding
of EPHA4. LMCl axons are repelled away from the ventral limb remain to be identified.
mesenchyme expressing EFNAs (Helmbacher et al., 2000; Kania Finally, after reaching their appropriate muscle, MN
and Jessell, 2003). Similarly, LMCm MNs express EPHB1 are axons need to form functional connections with their tar-
repulsed from the dorsal limb mesenchyme expressing EFNBs get. Interestingly, studies focusing on the CM and LD muscles
(Luria et al., 2008). Therefore, cross-repulsive Ephrin-Eph sig- have revealed that this process is initiated upon receiving a signal
naling mediates the correct segregation of LMCl and LMCm from the peripheral target and therefore is considered as an
(Figure 10C). However, additional mechanisms contribute as well extrinsic event (termed “Pool extrinsic” in Figure 9). MN pools
to LMC MNs axonal targeting. For example, GDNF and GDNF innervating these two muscles are characterized by the expression
family receptor alpha 1 (GFRA1) cooperate with EFNAs-EPHAs of ETV4 (Ladle and Frank, 2002). It has been remarkably shown
signaling to control LMC MN dorso-ventral choice (Kramer et al., that the initial expression of ETV4 is induced by GDNF expressed
2006). More recently, new discoveries have enriched Ephrin-Eph by the CM and LD muscles (Haase et al., 2002; Helmbacher et al.,
signaling with additional levels of complexity. Trans forward and 2003). In turn, ETV4 is responsible for inducing the terminal
reverse signaling (Dudanova et al., 2012) as well as interaction axonal arborization (Livet et al., 2002) as well as the dendritic

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Stifani Spinal motor neuron diversity

refinement of these specific MN pools (Vrieseling and Arber, the mechanisms controlling axon guidance and MN survival into
2006). The molecular effectors of terminal arborization are still a unified and coherent process.
unknown; however, downstream targets of GDFN and/or ETV4 Since this article does not primarily focus on MN cell death
signaling could be good candidates for further investigation. and selective survival, the following reviews are recommended to
Recently, Audouard et al. (2012) have identified for the first provide a detailed description of this complex and indispensable
time a transcriptional regulator of neuromuscular junction process (Oppenheim, 1991; Hamburger, 1992; Henderson, 1996;
formation. The analysis of ONECUT1 inactivated animals Pettmann and Henderson, 1998; Gould and Enomoto, 2009).
demonstrates a peculiar hind limb locomotion pattern result-
ing from impairments in neuromuscular junction formation. SPECIFICATION OF MOTOR NEURON SUBTYPES WITHIN A POOL
These findings open new opportunities to further characterize The diversity of SpMNs is not limited to the specification of MN
downstream molecular effectors important for the formation of pools but also impinges on muscle fiber structural and functional
functional connections between MNs and their respective muscle diversity. Despite the detailed columnar classification of SpMNs,
targets. little is known about the mechanisms causing a specific MN to
recognize and connect to a unique fiber type within its individual
PERIOD OF NATURAL CELL DEATH muscle target. We will describe recent studies that shed light on
MNs are generated in excess and then progressively decrease in the mechanisms controlling alpha and gamma MN differentiation
number during a natural cell death period (Oppenheim, 1991). as well as between fast and slow alpha MNs.
This process ensures the generation of the appropriate number
of MNs and guarantees the elimination of aberrant cells. This Alpha vs. Gamma MNs
strategy can also result from the requirement of a temporary The divergence between alpha and gamma MNs is poorly char-
function; for example, certain MNs may initially be generated to acterized (Eccles et al., 1960; Bryan et al., 1972; Westbury, 1982).
ensure a particular developmental function and are subsequently Evidence from several studies suggests that alpha and gamma MN
eliminated. Regardless of the reasons, natural MN death leads identities are fated early during embryonic stages. For example,
to the removal of around 40% of the initially generated MNs inactivation of the programmed cell death in MNs leads to an
(Hamburger, 1975). This loss can be comprehensively divided increased number of MNs with gamma characteristics (Buss et al.,
into two phases (Yaginuma et al., 1996). The early phase is inde- 2006). This result implies that alpha and gamma MN are differen-
pendent of any peripheral signal and likely reflects a negative tiated prior to axon outgrowth and trophic support requirement.
selection of unsuitable MNs. The subsequent phase has been During the first weeks after birth, alpha and gamma MNs can
described more intensively and is dependent on survival signals be molecularly identified by the differential expression of the
from the periphery and thus reflects the refinement of mature RNA binding protein, fox-1 homolog 3 (RBFOX3 or NeuN),
MN innervations. Temporally, natural MN cell death in mice the estrogen-related receptor gamma (ESRRG) (Friese et al.,
starts progressively from embryological day (E) 11.5 in most ros- 2009), the GDNF family receptor alpha 1 (GFRA1) (Shneider
tral segments and spreads gradually to the caudal levels with a et al., 2009), the serotonin receptor 1D (HTR1D) (Enjin et al.,
peak occurring at E14 (Yamamoto and Henderson, 1999). The 2010) as well as the ATPase, Na+/K+ transporting, alpha 1
absence of MN cell death postnatally suggests a necessity to reach (ATP1A1) (Edwards et al., 2013). Alpha MNs maintain high
completion of MN development before birth (Oppenheim, 1986). levels of RBFOX3 expression after birth whereas gamma MNs up-
Numerous molecules have been involved in MN survival signal- regulate ESRRG and GFRA1 and simultaneously down-regulate
ing. The initial discoveries of the nerve growth factor (NGF), RBFOX3. These markers are segregated only at post-natal stages
neurotrophins (NTFs) and brain derived neurotrophic factor and are therefore unlikely participate in the early phase of alpha
(BDNF) (Snider, 1994) led to the characterization of additional and gamma MN divergence. A recent study identified the first
molecules involved in neuronal survival, including cytokines (cil- embryological marker of gamma MNs (Ashrafi et al., 2012).
iary neurotrophic factor CNTF, leukemia inhibitory factor LIF) Namely, WNT7A is selectively expressed in gamma MNs at late
(Dechiara et al., 1995; Li et al., 1995), the TGFB family (GDNF, embryological stages. The authors also revealed that its expres-
neurturin NRTN, persephin PSPN) (Henderson et al., 1994; sion is dependent on a muscle spindle-derived signal that is not
Poulsen et al., 1994; Oppenheim et al., 1995, 2000), the hepato- GDNF, previously characterized as required for their survival
cyte growth factor (HGF) as well as FGF1, 2 and 5 (Henderson, (Gould et al., 2008; Shneider et al., 2009). These results open new
1996; Oppenheim, 1996). perspectives to further characterize the molecular mechanisms
Interestingly, in parallel of the general survival mechanisms controlling alpha vs. gamma MN divergence.
introduced above, results suggest the existence of pool specific
survival signals (Gould and Oppenheim, 2004). Gu and Kania Fast vs. Slow MNs
(2010) undertook the profiling of survival receptors expression in Alpha MNs can be classified according to the type of extrafusal
lumbar LMC MN pools as well as survival molecules in the cor- fiber they innervate (FF, FFR, SFR). MNs are intrinsically compe-
responding limb muscles. Although their results did not reveal a tent to recognize and connect to either fast or slow muscle fibers
general mechanism linking MN pool specific survival and com- (Rafuse et al., 1996; Landmesser, 2001). Studies have proposed
bination of trophic factors expressed in the muscles, they empha- that the synaptic vesicle glycoprotein 2a (SV2A) (Chakkalakal
sized the complexity of MN survival. Indeed, the authors discuss et al., 2010) as well as the estrogen-related receptor beta (ESRRB)
several indications supporting a plausible convergence between (Enjin et al., 2010) are restricted to slow MNs soon after birth.

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Stifani Spinal motor neuron diversity

Conversely, the calcitonin-related polypeptide alpha (CALCA) the integrity of SpMN development, naturally programmed cell
and the chondrolectin (CHODL) are restricted to fast MNs (Enjin death induces the elimination of inadequate MNs and ensure the
et al., 2010). More recently, Muller et al. (2014) elegantly identi- formation of a coherent circuitry.
fied the non-canonical Notch ligand delta-like homolog 1 (DLK1) Although, the overall strategy as well as the intrinsic tran-
as a regulator necessary and sufficient to promote fast MN pheno- scription factors governing the generation of SpMN diversity
type. These results identify for the first time a molecular regulator have been, at least partially characterized and summarized here,
of the fast vs. slow MN divergence. This initial breakthrough will our review emphasizes the poor knowledge about the down-
indubitably facilitate further identification of the mechanisms of stream molecular effectors of MN development. In fact, the more
fiber-type-specific alpha MN differentiation. differentiated SpMNs become the more fragmentary our under-
In summary, SpMN diversity expends beyond the formation standing is. This is particularly important in regard to prospective
of MN pools. In fact, SpMN identity impregnates into muscle MN regeneration therapies for which understanding MN general
fiber types characteristics. Recent findings lead to the identifi- identity will not be sufficient. Instead, tweaking subtype-specific
cation of key players as well as molecular markers of MN sub- effector molecules may be a powerful strategy to regenerate
type populations. These discoveries open new avenue for further functional MNs in fully developed adults. The identification of
characterization. additional effectors can be achieved in two ways: (i) oriented
investigation of downstream targets of known intrinsic regula-
CONCLUSION AND PERSPECTIVES ON THE GENERATION OF SPINAL tors such as LIM and/or HOX proteins for example and (ii)
MOTOR NEURONS unbiased screenings combining, viral retrograde tracing (Stepien
SpMNs are unique and irreplaceable neuronal cells connecting et al., 2010; Tripodi et al., 2011), laser capture micro-dissection
the CNS to targets in the periphery. While visceral SpMNs of the (Bandyopadhyay et al., 2014) and RNA sequencing (Enjin et al.,
thoracic and sacral regions control autonomic functions, somatic 2010). Such approaches would indubitably unveil new regulators
SpMNs regulate movements by controlling the contraction of and effectors of SpMN subtype specification. Furthermore, sev-
individual muscles. These crucial roles lead to inexorable impair- eral studies have already shed light on the role of non-coding
ments when affected by diseases. Thus, intensive research has micro RNAs (miRNAs) in MN development (Cao et al., 2007;
focused on understanding MN biology and diseases. Visvanathan et al., 2007; Otaegi et al., 2011a,b). For example,
Over the years, studies have accumulated data and revealed Chen and Wichterle (2012) demonstrate that the inactivation
mechanisms driving MN properties and behaviors. Remarkably, of the Endoribonuclease Dicer (DICER1), an important player
the diversity of SpMNs mirrors the variety of targets they inner- of double strand RNA post-transcription gene silencing, per-
vate but also impinges within individual muscle fiber types. This turbs the formation of PGC and LMC MNs. Similarly, OLIG2
exceptional diversity is acquired progressively during develop- repression initiated at the p2-pMN border relies on mir-17–3 p
ment and has been reviewed here. The ventralization of the miRNA-mediated silencing of Olig2 mRNA (Chen et al., 2011a).
neural tube has been described as a consequence of surround- The implication of non-coding miRNAs is likely more complex
ing molecules expressed in a gradient fashion and inducing in and numerous findings will likely arise from this recent and
a concentration dependent manner the expression of sets of mostly unexplored field of research. The unbiased screenings
homeodomain proteins leading the emergence of exclusive pro- mentioned above could identify novel regulatory mechanisms of
genitor domains. All SpMNs arise from the pMN domain from SpMN diversity involving non-coding RNAs.
which SpMN precursors exit the cell cycle and migrate away from SpMNs are anatomically well organized. This morphological
the neuroepithelium while acquiring post-mitotic MN features. arrangement correlates with the position of their respective tar-
Concomitantly, patterning molecules along the rostro-caudal axis get in the periphery as reviewed by Kania (2014b). Thus, SpMN
induce in a concentration-dependant manner the expression of settling position and axonal targeting must be somehow molec-
several transcription factors notably members of the HOX fam- ularly connected. An ingenious strategy to further understand
ily. In turn, these proteins define exclusive rostro-caudal seg- the mechanisms driving SpMN specification consists in uncou-
ments (brachial, thoracic, lumbar). Subsequently, while SpMNs pling MN differentiation processes such as column formation, cell
strengthen their motor identity, they segregate into anatomical body positioning, and axonal targeting. One naturally occurring
columns termed motor columns. Combinations of LIM home- opportunity to study MN differentiation processes independently
odomain proteins provide a unique molecular profile for each from one another could lie on the analysis of rhomboideus MN
motor column. In parallel, the LIM code induces the initial pool. These neurons constitute, in fact, the only known exception
steps of a crucial process: MN axonal targeting. SpMN axonal to the MN columnar organization described earlier. Although
targeting and further differentiation occurs in a step-wise man- innervating an axial muscle, this MN pool is located in the lat-
ner. Checkpoints are established along the route to ensure the eral component of the ventral horn at caudal brachial segments; a
completion of critical steps. Furthermore, these checkpoints are position typical of LMC MNs (Straznicky and Tay, 1983; Hollyday
informative and instruct developing SpMNs of the environment and Jacobson, 1990; Tsuchida et al., 1994; Rousso et al., 2008).
at the growth cone. The SpMN target can be seen as the last check- Therefore, molecular profiling of this particular MN pool may
point of the chain. Upon reaching their final destination, SpMNs be interesting to identify new effectors and regulators of SpMN
are required to complete their differentiation process and form organization.
functional connection with their target. SpMN identity echoes Finally, this review deliberately focused on SpMN develop-
muscle fiber type properties. Finally, as a mechanism controlling ment from a motor perspective. However, SpMNs are “only”

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Stifani Spinal motor neuron diversity

one constituent of a larger coherent circuitry. Complex move- in the vertebrate neural tube. Cell 148, 273–284. doi: 10.1016/j.cell.2011.
ments require the control of individual muscles in a collaborating 10.047
Bandyopadhyay, U., Fenton, W. A., Horwich, A. L., and Nagy, M. (2014).
manner. This coordination relies on a highly organized circuitry
Production of RNA for transcriptomic analysis from mouse spinal cord motor
between SNs, association neurons, and SpMNs as reviewed by neuron cell bodies by laser capture microdissection. J. Vis. Exp. 13:e51168. doi:
Ladle et al. (2007). In a perspective of regeneration therapies, 10.3791/51168
SpMNs with the correct identity should insert in a pre-existing Bel-Vialar, S., Itasaki, N., and Krumlauf, R. (2002). Initiating Hox gene expres-
neuronal circuitry. Such possibility infers that (i) regenerated sion: in the early chick neural tube differential sensitivity to FGF and RA
signaling subdivides the HoxB genes in two distinct groups. Development 129,
SpMNs settle at their appropriate location, (ii) that SpMNs’
5103–5115.
inputs are plastic to form new functional connections and (iii) Bessou, P., Emonet-Denand, F., and Laporte, Y. (1965). Motor fibres innervating
that regenerated SpMNs project to their appropriate target across extrafusal and intrafusal muscle fibres in the cat. J. Physiol. 180, 649–672.
a fully developed living organism. These are the challenges the Bonanomi, D., Chivatakarn, O., Bai, G., Abdesselem, H., Lettieri, K., Marquardt,
scientific MN community will have to resolve in the coming T., et al. (2012). Ret is a multifunctional coreceptor that integrates
diffusible- and contact-axon guidance signals. Cell 148, 568–582. doi:
future. 10.1016/j.cell.2012.01.024
Bonanomi, D., and Pfaff, S. L. (2010). Motor axon pathfinding. Cold Spring Harb.
AUTHOR CONTRIBUTIONS Perspect. Biol. 2:a001735. doi: 10.1101/cshperspect.a001735
Nicolas Stifani reviewed the relevant literature, wrote the Bravo-Ambrosio, A., and Kaprielian, Z. (2011). Crossing the border: molec-
ular control of motor axon exit. Int. J. Mol. Sci. 12, 8539–8561. doi:
manuscript and generated the figures. 10.3390/ijms12128539
Brichta, A. M., Callister, R. J., and Peterson, E. H. (1987). Quantitative analy-
ACKNOWLEDGMENTS sis of cervical musculature in rats: histochemical composition and motor pool
organization. I. Muscles of the spinal accessory complex. J. Comp. Neurol. 255,
I would like to acknowledge Dr. Stefano Stifani, Dr. Artur Kania,
351–368. doi: 10.1002/cne.902550304
Dr. Ed Ruthazer, Dr. Tim Kennedy, Dr. Donald Von Meyel and Dr. Briscoe, J., Pierani, A., Jessell, T. M., and Ericson, J. (2000). A homeodomain pro-
Christine Vande Velde for comments and corrections. Gene and tein code specifies progenitor cell identity and neuronal fate in the ventral neural
protein names and symbols follow HUGO Gene Nomenclature tube. Cell 101, 435–445. doi: 10.1016/S0092-8674(00)80853-3
Committee at the European Bioinformatics Institute freely avail- Bryan, R. N., Trevino, D. L., and Willis, W. D. (1972). Evidence for a com-
mon location of alpha and gamma motoneurons. Brain Res. 38, 193–196. doi:
able at http://www.genenames.org. 10.1016/0006-8993(72)90602-6
Bueker, E. D. (1944). Differentiation of the lateral motor column in the avian spinal
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