The vertebrate spinal cord is an incredibly complex structure and layers of neurons along its dorso-ventral (D-V) axis control the physiological functions essential for an animal to be able to feel and respond to various external stimuli such as pain, touch and temperature. Understanding how the spinal cord is formed during embryonic development will provide a fascinating insight into these functions and, therefore, many research laboratories are currently focused on elucidating this mechanism.
In order to explain the process by which the spinal cord is patterned it is first necessary to describe its anatomical organization. Broadly speaking, the motor neurons and interneurons lie in the ventral grey matter while the somata of the sensory neurons are concentrated in the dorsal root ganglia (DRGs) (Caspary & Anderson, 2003). The gray matter of the spinal cord is organised into nine discrete parallel layers plus the region surrounding the central canal (Patestas & Gartner, 2006). These are termed Rexed laminae I-X, after the Swedish neuroanatomist who mapped out their distribution. The axons of the sensory neurons project both to the periphery and to specific laminae in the spinal cord. Establishing correct connections between the neurons of the grey matter and the sensory neurons requires prior specification of the laminae as a target for incoming axons (Figure 1).
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Figure 1. Organization of the spinal cord. Sensory neurons project from the DRG to specific laminae. Nociceptive neurons (red) sense pain and temperature. Mechanoreceptors (green) in the periphery mediate touch while sensory neurons that project to ventral motor neurons mediate proprioception. Motor neurons connect back to the muscles and control movement.
The spinal cord is a model of a patterned structure and its D-V organization is structured early in embryonic development. Vast research in numerous organisms, particularly in the chick and mouse, has led to a plausible mechanism of spinal neuron formation (Jessell, 2000). In broad terms, spinal cord formation consists of four major stages (figure 2).
At the very beginning, two signalling centres are formed at opposite poles of the D-V axis of the neural tube. Axial mesodermal structures generate a ventral floorplate, while signals from the overlying ectoderm induce a dorsal roofplate. Although these structures are morphologically distinct they both generate molecules that non cell-autonomously control neuronal development (Zhuang & Sockanathan, 2005).
Figure 2. Four stages of spinal cord development a. Neural cells are laterally lined by the epidermal ectoderm (ECT). Underlying the midline and the lateral region of the neural plate are notochord cells (N) and segmental plate mesoderm (S), respectively. b. The floorplate (F) is induced ventrally and the somitic mesoderm starts to form. c. The roofplate (R) develops dorsally and neural crest cells (NC) delaminate from the dorsal neural tube. d. Commissural (C) and association (A) neurons develop in the dorsal spinal cord, while motor neurons (M) and ventral interneurons (V) form in the ventral. Dorsal root ganglion (DRG) neurons form from neural crest progenitors. Â
In ventral patterning, the floorplate secretes a gradient of sonic hedgehog (Shh) that results in the formation of five separate progenitor domains (Figure 3). Shh is a molecule of the Hh family which is first expressed in the notochord. The floorplate then activates the expression of Shh, and becomes itself a new source of Shh. Shh is defined as a morphogen, meaning it is an extracellular signalling molecule that acts at a distance from its origin and differentiates distinct cell types in a concentration- dependent mode (Wilson & Maden, 2005).
The process by which Shh patterns the ventral spinal cord can be summarised in a three-stage mechanism. First, a gradient of Shh controls, according to its concentration, the secretion of homeodomain (HD) and basic Helix- Loop-Helix (bHLH) transcription factors. These are either class I or class II proteins if they are inhibited or activated by Shh, respectively. Second, cross-regulatory interactions between pairs of class I and II proteins sharpen their D-V borders of expression. Last, the combined expression of HDs and bHLH transcription factors ultimately defines the identity of the neural progenitor cells, which will in turn form postmitotic neuronal subtypes (Figure 3).
Data from a recent study has allowed the proposal of a mechanism by which the ventralizing activity of Shh is antagonized by the activity of Wnt (Ulloa & Marti, 2010). Wnt are a family of secreted palmitoylated glycoproteins which, like Shh, have key functions in embryonic development. Wnt is secreted from the roofplate and is believed to induce the expression of Gli3, a potent inhibitor of Shh. Consequently, antagonistic interactions between Wnt, which induces dorsal identities, and Shh, which promotes ventral ones, partly define D-V patterning.
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Figure 3. Ventral patterning of the spinal cord. Five progenitor domains (p0-3 and pMN) in the ventricular zone differentiate into five neuronal subtypes (V0-3 and MN). A ventral to dorsal gradient of Shh is established which is transduced into an intracellular gradient of Gli. The net Gli activity results from the combination of activator and repressor forms of Gli, promoted or inhibited by Shh, respectively (MN, motor neuron; FP, floor plate).
How are neuronal subtypes induced dorsally? A class of secreted factors, the bone morphogenetic
proteins (BMPs), have been shown to actively promote dorsal cell formation. Inhibition of BMP by its specific inhibitors, noggin and follistatin, inhibits the ECT from inducing the roofplate. Furthermore, as indicated by electroporation of BMP4, BMP7, BMP inhibitors and an activated BMP receptor into early chick neural plate, BMP signalling is both essential and adequate for roofplate formation (Chizhikov & Millen, 2004).
It is known that BMPs activate the transmembrane serine-threonine kinase receptors and BMP signals are transduced from the membrane to the nucleus by the transcription factor SMAD. When BMP receptors are activated, the SMADs are phosphorylated and pass into the nucleus where they regulate the expression of transcription factors that determine the fate of specific dorsal cells (Kandel et. al, 2000).
Figure 4. Dorsal patterning of the spinal cord. Six progenitor domains (dp1-6) result in six early born (dI1-6) and two late born (dILA and dILB) dorsal interneurons. The eight subtypes are grouped into two classes: roofplate-dependent Class A and roofplate-independent Class B neurons. Cross repression between bHLH transcription factors has a key role in patterning the progenitor cells (RP, roofplate).
In summary, both dorsal and ventral cell formation is controlled by inductive signals. Ventral patterning is regulated by a single protein, Shh, which induces different cell types at distinct concentrations, while dorsal patterning involves several types of BMPs, each inducing a different set of cells. Both types of patterning have, however, one common feature. In both cases nonneural cells primarily express inductive signals which through homeogenetic induction pass to specialized glial cells at the midline of the neural tube. This process guarantees that future sources of inductive signals are properly positioned to regulate cell fate at the late stages of development.
This essay has emphasized both the progress and the gaps in our knowledge of D-V patterning. The numerous studies have not only helped to suggest a plausible mechanism for the differentiation of neuronal cells in the spinal cord, but have also raised various new questions. For instance: what other signals besides Shh induce ventral neuronal cell formation? In what way do cells assimilate the various signals they are exposed to? What particular function do Gli proteins have? Despite the gaps in our understanding, the success of elucidating how the neural tube cells associate with the laminae and functional neuronal pathways does not only depend on the extent of our imagination, but also on our capacity to observe and experiment.
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