Molecular and Extracellular Cues in Motor Neuron Specification and Differentiation

Introduction

Motor neurons (MN) are a diverse group of cells without which complex life would not be possible. MNs are responsible for integrating signals from the brain and the sensory systems to control voluntary and involuntary movements. Though MNs can be split into cranial and spinal subsets, this chapter will focus on spinal MNs, as they are a key target of disease and injury. As such, MNs are the focus of regenerative efforts to alleviate these public health burdens. During late gastrulation and neurulation, the developing spinal cord, termed the neural tube, is patterned into distinct progenitor domains. MNs are specified from progenitors in the ventral neural tube. Once specified, newly born MNs are further specified into columns, pools, and subtypes, forming a unique topography. From these columns and pools, axons reach out to their targets under varying guidance cues. All MNs are cholinergic cells which integrate with the motor control circuit, the sensory system, and their outlying targets to control movement. MNs are unique in that their targets lie outside the central nervous system (CNS), meaning that they require novel methods for seeking out and synapsing on them. Here, we present an overview of MN differentiation and development. We will focus mainly on signaling events, transcription factor markers, and the extracellular matrix (ECM) as they pertain to MN development. These cells are targets of permanent and often deadly diseases including amyotrophic lateral sclerosis, spinal muscular atrophy, multiple sclerosis, and injuries such as spinal cord injury. Only by understanding how these cells progress through development can we understand how to treat these maladies which currently have little hope of a cure. Further, by decoding the major events and players in development, we can better recapitulate them in vitro for cell replacement therapy, or harness the underlying principles for regeneration in the adult. Given the growing importance of the MN–glia interaction in a number of neurodegenerative diseases, we will also discuss the initial specification of oligodendrocyte precursor cells (OPCs) in detail, as they share a common progenitor with MNs.

Specification of Neuroectoderm

Vertebrate embryos specify the ectoderm in late gastrulation. This germ layer will become the epidermis and the nervous system. The anterior neural ectoderm is distinguished from the epidermis by its inability to bind bone morphogenic proteins (BMPs) due to the inhibitors secreted from the Spemann–Mangold organizer region of the gastrula. These inhibitors—noggin, chordin, and follistatin—bind and neutralize the effects of BMPs, creating a permissive transcriptional environment for neural progression. Posteriorly, the neural plate is specified by fibroblast growth factors (FGFs) and Wingless-related integration site (Wnt) proteins that also suppress BMP activity. Additionally, retinoid signaling from the paraxial mesoderm specifies the cells of the future spinal cord. This newly specified neural plate then thickens as cells proliferate and invaginates through the convergent extension, forming the neural groove. The neural groove forms hinge points which will ultimately close to form the neural tube – the precursor for the entire CNS. For an in-depth review, see Massarwa et al.

Spinal Cord Patterning

The spinal cord is a two-way information conduit that connects the brain with the sensory and motor systems. To do this, it must generate a highly diverse set of neurons during development. The neural tube provides a three-dimensional template which is patterned by gradients of morphogens to generate this diversity. The early neural tube is composed of multipotent neural stem cells expressing Sex determining region Y box 1 (Sox1). The dorsal neural tube will generate cells linking the CNS to the sensory peripheral nervous system (PNS). The ventral neural tube will ultimately give rise to the motor control circuit responsible for controlling MNs. Bone morphogenetic proteins specify the dorsal portion of the neural tube, including neuronal subtypes involved in integration of the peripheral sensory nervous system. Ventrally, an initial wave of Sonic hedgehog (Shh) from the notochord patterns the cells into distinct progenitor domains. These domains arise due to cross-repressive actions of two types of transcription factors downstream of Shh signaling: Type I transcription factors are repressed at threshold Shh concentrations, while Type II are expressed below threshold Shh concentrations ( Fig. 1.1A ). The type I transcription factor paired box protein 6 (Pax6) represses the activity of type II homeobox protein Nkx2.1. Similarly, type II homeobox Nkx6.1 cross-represses developing brain homeobox 2 (Dbx2). The most ventral progenitor domain is the floor plate, which is induced to secrete Shh in a second wave of patterning, followed by the progenitor domains p3, pMN, p2, p1, and p0 ( Fig. 1.1B ). The combinatory actions of these two classes of proteins yield the five spatially distinct ventral progenitor domains.

The Motor Neuron Progenitor Domain and Initial Neurogenesis

The MN progenitor (pMN) domain is responsible for generating MNs. In mice and chick models, this domain is identified by the expression of the homeobox transcription factors Nkx6.1 and Pax6 and the basic helix–loop–helix (bHLH) oligodendrocyte transcription factor 2 (Olig2). Olig2 expression is obligate for MN specification, as Olig2 ^null mice fail to generate MNs. Initially, Olig2 plays a key role in progenitor proliferation; however, it also drives the expression of neurogenin 2 (Ngn2), a key neural determinant. The first murine MNs are born around E9.5. The homeobox transcription factor Nkx2.2, important for the glial switch and a marker for p0 cells, shows variable expression in humans compared to mouse and chick models: the human pMN domain appears to include both Olig2+/Nkx2.2− as well as Olig2+/Nkx2.2+ cells. This could potentially add to the diversity of human MNs.

Molecular Programs in Newborn Motor Neurons

As mentioned above, Olig2 drives Ngn2 expression. However, Ngn2 is ultimately responsible for cell cycle exit and neurogenesis, in direct contrast to the role of Olig2. Once Ngn2 protein levels surpass those of Olig2, cell cycle exit occurs and cells commit to the neuronal lineage. Olig2 binds and sequesters the MN transcription factor homeobox gene 9 (Hb9, also called MNX1), which is necessary for MN development. LIM homeobox gene Isl1 and LIM homeobox 3 (Lhx3) form a complex with the nuclear LIM interactor which suppresses interneuron fate and specifies MN. Along with Ngn2, this complex stimulates Hb9, which self-stimulates its own expression, while forming a positive feedback loop with Isl1. Isl1 and 2 work in concert to further specify MN cell fate. Lhx3 and Isl1 expressions are necessary for MN generation and the expression of cholinergic genes common to all MNs. However, little is known about potential negative feedback mechanisms in this differentiation process that would limit MN number and organ size. We will discuss this further in the Glial Switch section.

In summary, Shh secreted from notochord drives the expression of Pax6 and Nxk6.1, which in turn drive Olig2 expression. Olig2 expression delineates a mitotic pMN progenitor. Olig2 induces the expression of Ngn2, which is responsible for cell cycle exit, of Lhx3/Isl1 transcription factors, as well as MN-specific Hb9 in newborn, postmitotic MNs ( Fig. 1.2 ).

Migration

The topography of MNs is largely correlated with their function. MNs cluster in columns with similar transcription factor expression and like targets. Within muscle-innervating columns, there are MN pools which innervate specific muscle groups. In order to specify this topography, MNs must migrate away from the ventricular progenitor cells to their final destination in the ventral horn of the spinal cord. Newly born neurons detach from the epithelium and migrate radially to the medial and lateral areas of the neural tube. Critical to this migration is cadherin expression driven by beta and gamma catenin signaling in a Wnt-independent manner. In knockout models of either cadherin, MNs fail to properly align to their proper column, although their ultimate muscular targets are not disrupted. This implies that the stereotypic and highly organized topology of MNs is not a modulator of identity or function. The role of this highly specific organization has yet to be elucidated. Transcriptionally, the forkhead box P (Foxp) genes regulate cadherin expression for migration to occur. Specifically, the Foxp2/4 genes allow for the detachment of MN from the neuroepithelium by downregulating cadherin 2 and allow them to migrate toward their final location by further modulation. Although cell bodies migrate within the spinal cord, all MN soma are exclusively contained within the spinal cord. Recently, Isl1/2 has been shown to play an integral part in preventing the cell body from exiting the spinal cord. In knockout animals, MN soma successfully exited the spinal cord into the periphery. One potential mechanism is through the semaphorin–neuropilin repulsive signaling pathway common to axon guidance, as neuropilin was found to be regulated by Isl1/2. For an in-depth review of migration and topography, see Kania (2005).

Motor Neuron Subtypes and Targets

MNs that innervate similar regions of the body group together in columns with identical molecular properties. The rostrocaudal axis is specified externally by retinoic acid (RA) at the cervical and brachial regions and by growth differentiation factor (GDF11) and FGF8 at the thoracic and lumbar regions. Like much of the developing embryo, this rostrocaudal positional identity is specified internally by Hox gene activation in response to these external cues to delineate the types of MNs in a given region. Hox expression is summarized in Fig. 1.3 . Hoxc9 plays a critical role in the organization of the spinal cord by repressing limb-specific Hox genes, thus specifying the thoracic column. Within each column, MNs organize into pools which innervate distinct muscles. Hox genes work in concert with the Hox accessory factor Foxp1 to specify many of the columns and establish motor pools within columns. Notably, in the absence of Foxp1 in knockout animals, there is a lack of defined rostrocaudal motor columns, further strengthening the case for its role in the positional identity of motor columns.