The vertebrate nervous system initially develops from a thickening of the ectoderm referred to as the neural plate. The folding of this flat sheet of progenitor stem cell eventually ensues to form the neural tube. Through the process of lineage restriction, these progenitors follow distinct differentiation pathways to generate post-mitotic neurons with particular anatomy and functional properties. Commitment to a specific cell fate does not, however, occur at random. Spatial patterning in response to chemical signals gives rise to the laminar organisation of the different neuronal cell types. Neurons of different cell densities and cell body size sequentially emerge so that they are grouped according to their function. Cells in the dorsal half of the neural tube give rise to the different types of sensory neurons, whereas cells in the ventral half form motor neurons and ventral interneurons that are necessary to generate coordinated motor output. This arrangement allows a smooth flow of information within the functional entity of the central nervous system that is the spinal cord. One primary mechanism that dictates spatial order during development is the establishment of morphogenetic fields. Sonic Hedgehog (Shh) is an essential signalling molecule, known to pattern distinct neuronal subtypes within the neural tube. This protein is produced by a mesodermal structure underlying the neural tube, the notochord and subsequently by the floor plate, and diffuses ventrally towards the roof plate. A gradient of Shh is therefore established along the dorsoventral axis of the neural tube and acts to specify progenitor cell boundaries. In this essay, I will discuss how can Shh as a single factor act to confer precise positional information to specific clusters of cells within the ventral horn of the spinal cord.
Sonic Hedgehog as a gradient morphogen
The notochord has been identified as the source of the ventralising signal of the neural tube (Yamada et al., 1993). Through the localised release of a paracrine factor, the notochord induces the floor plate of the neural tube (Ericson et al., 1996). This latter was identified as a second signalling centre capable of secreting the ventralising agent. Roelink (1994) determined Shh to be the inductive cue by inducing ectopic floor plate differentiation in frog embryos through ectopic expression of a vertebrate homologue of Shh. Motor neurons and ventral interneuron differentiation is also dependent on Shh (Ericson et al.,1996). These latter, however, appear at a distance from the notochord. Although Shh seems to act as a pure inductive signal when specifying the floor plate, it can operate over a wide area along the dorsoventral axis. When part of the notochord is removed, only cells overlying the remaining mesodermal tissue form floor plate cells. (Placzek et al., 1990) This result could suggest that its signalling function is contact-mediated, and following neuronal induction would result from contact with the floor plate and so on. Placing ectodermal tissue at a distance from the notochord, however, does result in the generation of motor neurons (Tanabe et al., 1995). Motor neuron induction is thus independent of floor plate induction and relies on secretions from the primary signalling centre to a greater extent. Additionally, substantial evidence from neural explant studies shows that high concentrations of Shh induce floor plate formation, while lower concentrations produce motor neurons (Dessaud et al., 2007). It can, therefore, be speculated that Shh diffuses from the inducer tissue forming a morphogen gradient. Morphogens are active at very low concentrations – to – which makes the detection of the Shh gradient difficult (Gurdon et al., 2001). The gradient can be visualised indirectly by following Patched(Ptc) gene expression, which encodes for Shh receptors (Stone et al., 1996). A clear morphogenetic field along the dorsoventral axis can be inferred from such studies. Chamberlain (2008) confirmed this finding using genetically modified mice that produce Shh tagged with green fluorescent protein. Visualising the fluorescence points to a dynamic gradient of Shh where transitions from defined values coincide with progenitor cell boundaries.
Transduction of the Shh signal
The threshold concentration of Shh required to induce floor plate has been identified to be five times that for motor neuron induction. (Roelink et al., 1995). Interestingly, high concentrations of Shh appear to specify floor plate cells at the expense of all other ventral neuronal cell types in neural explant studies. Shh acts in a graded manner (Dessaud et al., 2007). Cells are capable of interpreting the continuous gradient of Shh by spatially arranging into distinct progenitor domains as follows along the dorsoventral axis: VO, V1, V2 interneurons, motor neurons, V3 interneurons and neural floor plate cells (Jessel, 2000). Signal transduction involves the transcription factors of the Gli family. Ptc receptor is bound to the inhibitor protein Smoothened(Smo) which cleaves Gli molecules by protein kinase A activity rendering the transcription factor to act as a repressor of Shh target genes. Binding of Shh to Ptc causes a conformational change that releases Smo, Gli is then released and serves as an enhancer (Yao et al., 2015). It is believed that Shh signalling is directly interpreted by cells as they respond to a corresponding gradient of Gli activity (Persson et al., 2004). Furthermore, Shh concentrations that identify distinct progenitor domains are reflected by changes in Gli activity within a cell. It was demonstrated in in vitro study that generating a gradient of Gli through gain-of-function mutations and no involvement of Shh, was sufficient to mimic the patterns created in the neural tube (Lei et al., 2004). Three Gli proteins have been identified in vertebrates (Gli1, Gli2 and Gli3). Their individual roles as enhancers or repressors appear to be different for given concentrations of Shh. (Aza-Blanc et al., 2000). Hence, the response elicited in cells to Shh signalling is determined by the combination of differential activity of the Gli proteins. A continuous gradient of Shh permits the discernment of spatially organised cells by defining differential Gli activity. Each protein will act as an activator at high concentrations of Shh, suppressor at low concentrations of Shh. A combination of enhancing and repressing activity is generated at intermediate concentrations. Contextually, suppressing Gli3 activity induces V0-V2 interneurons, a combination of Gli2 and Gli1 activity generates motor neurons, and a combination of Gli2 and low Gli1 activity produces V3 interneurons and floor plate cells in a concentration-dependent manner (Persson et al., 2002).
Differential gene expression
Progenitor domains in the ventral neural tube are discerned through the interpretation of a particular combination of the following homeodomain transcription factors: Nkx2.2, Olig2, Pax6 and Pax7. These proteins show differential sensitivity to Shh: Pax 6 and Pax7 are repressed by Shh signalling while Nkx2.2 and Olig2 require exposure to Shh to be synthesised (Ericson et al., 1997). Explant studies where the individual transcription factors can be identified by using fluorescent labels allow us to map their spread of along the axis. Progenitors placed in high concentrations of Shh, which is analogous to most ventral cell populations of the spinal cord produce Nkx2.2 and differentiate into V3 interneurons. Subjecting the explant to a lower concentration result in the synthesis of Pax 6 and Olig2, forming motor neurons. Finally, the lower concentrations of Shh, equivalent to the most dorsal set of progenitors within neural tube induces V2 and V1 interneurons through the expression of Pax6 alone (Lee et al., 2001). The gene expression pattern is thought to be specified by the level of Gli activators and repressors within the individual domains. Interaction of Gli proteins is such that low Gli activity results in the synthesis of Pax6 but appears not to be sufficient to produce Olig2. Higher Gli activity triggers Olig2 production while inhibiting synthesis of Pax6 and even higher levels of Gli activity induce Nkx2.2 synthesis while suppressing Olig2(Balaskas et al., 2012). Positional information can be conferred by the overlap of these different transcription factors, adding a higher level of complexity to the process. The inhibition effect of some transcription factors on others as identified by Balaskas(2012) further refines boundaries in between the progenitor cell domains. These interactions make it possible to generate a wide range of progenitor cell types from a continuous crude gradient. Not only that, but they could also be responsible for the maintenance of the domain boundaries as the neuroepithelium grows in size rendering the gradient less distinct. At a later stage of neurulation, the progenitors exit their cell cycle. Fate-determining genes are transcribed by the action of the various transcription factor combinations mentioned above, conferring neuronal identities to the distinct collections of post-mitotic cells.
Temporal aspects of Sonic Hedgehog signalling
Shh signalling relies in fact on a very complex regulatory network requiring the integration of both spatial and temporal distribution of Shh. Explant studies carried out by Dessaud(2007) demonstrate how the duration of Shh exposure on intermediate neural tube cells is a crucial factor in defining progenitor domains. Expression of Olig2 initially characterises the explant tissue which would give rise to motor neurons. However, when it is subjected to a high concentration of Shh, both Olig2 and Nkx2.2 are detected. Over longer exposure times, Nkx2.2 becomes the factor predominantly expressed. Effectively, the cell fate of the intermediate neural cells has been altered by changing the culture environment, and it now follows a different differentiation pathway corresponding to more ventral progenitors. This reflects a property of plasticity linked to the Shh signalling pathway. It also accounts for how the most ventrally located tissue respond to Shh as these are subjected to gradually higher concentrations while it is secreted and diffuses away to establish the continuous gradient. Consequently, transcription factors must be induced sequentially as Shh concentration increases at the ventral end of the neural tube. Pax6 is essentially expressed throughout the neural plate before being repressed in most ventral regions (Pieranni et al.,1999). As individual combinations of transcription factors can be generated with respect to the amount and time of exposure to Shh, it is critical to consider the duration of signalling to interpret a morphogen gradient correctly. This adds a temporal dimension to the traditional definition of a morphogen where only the concentration of the inductive signal determines the tissue response.
All in all, cell fate specification is determined by characteristic gene expression. Selective action of transcription factors that bind gene promoter to either enhance or repress fate determining gene expression results in the production of selective proteins. These confer the structural characteristics and functional traits that define progenitor domains and ultimately individualise the discrete populations of cells within the spinal cord. Emission of Shh from its source leads to the formation of correctly located motor neurons and interneurons. Positional information is inferred from Shh concentration gradient along the dorsoventral axis. Defined threshold values allocate sharp boundaries to the ventral progenitor domains of the developing neural tube. Dorsal progenitor domains are mainly derived from an opposing gradient of TGF-? signal secreted from the floor plate defining the various sensory neurons within the dorsal horn. Morphogenetic fields constitute highly efficient systems to pattern the complex cell arrangements found in the spinal cord and ensure the correct formation of neuronal networks. Similar inductive mechanisms are involved in patterning the various anatomical axis of the spinal cord, notably Fgf8 and Wnt signals along the rostrocaudal axis. Eventually, positional information from all axes is integrated to a high degree of specificity. In that way, converging all signals allows fine adjustments to the shape the spinal cord corresponding to its function at particular points, distinctly the enlargement of the ventral horns at limb attachment points.