Introduction

During the formation and maturation o f the nervous system there are a number o f characteristic changes in protein expression in different regions o f the developing SC. I chose to use immunohistochemical methods to study the in vivo expression levels o f a number o f proteins chosen either for tlieir particular association with regenerating systems, or because they are markers for

undifferentiated and differentiated neural cells. The aim o f this study was to examine changes in protein expression with increasing gestational age in human SC between six and sixteen weeks gestation.

I have focused on intermediate filaments as various members o f this family of cytoskeletal proteins, such as nestin, vimentin and GFAP are good markers o f neural development. The 200kDa neurofilament protein provided an indication o f the pattern o f axonal development in the SC, and NCAM, abundant in neuronal membranes in both the developing and mature nervous system, provided an additional marker for developing neurons (Terkelsen et a l, 1992). Although some scattered information is available no systematic analysis o f the expression o f these proteins in early human SC has been published.

4.2 Results

I carried out an immunohistochemical analysis to study changes in protein expression during SC development in the thoracic section o f developing human SC using SC from embryos o f 40, 70 and 112 days gestation. (For clarity, haematoxylin and eosin stained sections o f 37, 49 and 70 day embryonic SC with labelled structures and regions are shown in figure 4.1).

Figures 4.9, 4.10 and 4.11 show a schematic view o f the protein expression patterns described in my results.

4.2.1 Nestin

At all developmental stages analysed, nestin expression levels were below the threshold for satisfactory detection by fluorescence immunohistochemistry, although some expression could be detected using this technique. In order to more readily visualise this protein immunocytochemistry was carried out using an alkaline phosphatase conjugated secondary antibody on longitudinal

sections o f thoracic SC. SC from embryos o f 40, 56, 70 and 84 days gestation was used.

At 40 days gestation nestin expression is strong in the embryonic

neuroepithelium but there are no obvious radial structures extending from the neuroepithelial layer to the cord’s periphery (fig. 4.2A). By 56 days gestation expression in the developing ependymal zone is strong and nestin expressing radial glia can be seen to extend from the central canal to the pial surface (fig.

4.2B). Two weeks later, at 70 days gestation, the pattern o f expression is similar, though the fibres appear more numerous (fig. 4.2C). By 84 days gestation there are no nestin expressing fibres contacting the central canal (fig. 4.2D1), a more dorsal longitudinal section reveals nestin staining in the dorsal midline (fig. 4.2D2).

4.2.2 Vimentin

At 40 days gestation vimentin is expressed throughout the developing SC including the neuroepithelial cell layer (fig. 4.3.1A-E). At 70 days gestation expression is less widespread. The protein is strongly expressed in the ventral- most part o f the marginal layer, showing the same expression pattern as

observed for GFAP at this stage but also appearing in the floor plate cells and their processes that form the ventral median septum (fig. 4.3.2A1). As with GFAP, strong expression is also observed in roof plate cells and their processes that form the dorsal median septum (fig. 4.3.2A2). In the subpial region,

radially orientated, vimentin expressing filaments contact the glia limitans, but do not extend into the grey matter. By 112 days gestation the radially

orientated filaments are no longer visible and apart from some scattered expression at the lateral edges o f the cord the strongest vimentin expression is observed in the roofplate cells forming the dorsal median septum and the floorplate cells forming the ventral median septum (fig. 4.3.2 B 1-4). Very little expression is seen in the ependymal layer surrounding the central canal.

4.2.3 GFAP

GFAP expression is not observed in human SC at 40 days gestation. At 70 days gestation GFAP is strongly expressed in the ventral-most part o f the marginal layer in a similar pattern to that observed for vimentin, but is absent from the floor plate cells and the long processes that form the ventral median septum (fig. 4.4A2). As noted for vimentin, strong expression is observed in the roof plate cells and their basal processes that clump together to form the dorsal median septum (fig. 4.4A1). In the subpial region radially orientated, GFAP expressing filaments contact the pial surface, their GFAP expression levels decreasing as they reach the grey matter (fig. 4.4A3). By 112 days gestation the radially orientated filaments are no longer visible and GFAP expression is strongest in the rooQ)late cells forming the dorsal median septum, with a low level o f expression remaining in the white matter surrounding the ventral median septum, and around the ventral half o f the ependymal layer (fig. 4.4B1-4).

4.2.4 Cytokeratin 8

At 40 days gestation cytokeratin 8 is not expressed in the developing SC. As observed for vimentin and GFAP expression at 10 weeks gestation, in the subpial region, radially orientated, cytokeratin 8-expressing filaments contact the glia limitans, their expression levels decreasing to nothing before they reach the grey matter (fig. 4.5A1 & A3). There is also low-level expression throughout the grey matter and the ependymal layer, in a honeycomb pattern

(fig. 4.5A2). After 16 weeks gestation, cytokeratin 8 expression is limited to a small area around the outer edge o f the ventral-most part o f the ventral horns (fig. 4.5B).

4.2.5 Cytokeratin 18

At 40 days gestation cytokeratin 18 is not expressed in the developing SC, though expression was noted in the embryo’s skin (fig. 4.6A). At 10 weeks gestation, in the subpial region, radially orientated, cytokeratin 18-expressing filaments contact the glia limitans, their expression levels decreasing to

nothing before they reach the grey matter (fig. 4.6B1). There is also low-level expression throughout the grey matter and the ependymal layer, in a

honeycomb pattern (fig. 4.6B2). After 16 weeks gestation, cytokeratin 18 expression is much less widespread. Diffuse expression is observed in the ventral half o f the ependymal zone and in the ventral horns (fig. 4.6C1 & C2).

4.2.6 200kDa neurofilament protein (antibody RT97)

At 40 and 42 days gestation cross sections o f the SC show neurofilament protein expressed in axon bundles that occupy two large dorsolateral channels running along either side o f the developing cord (fig. 4.7. IG l, G2 & H). Longitudinal sections o f the SC show expression in the dorsal root ganglia and at a lower level in the intermediate zone of the cord. There is no expression in the neuroepithelial layer (fig. 4.7. lA-E). At 10 weeks gestation the SC is surrounded by a thick band o f RT97-positive axons with a small gap on both

sides o f the cord at the dorsolateral level (fig. 4.7.2A). Expression is similar at 16 weeks gestation, there is a thick band o f axons around the grey matter surrounding the ventral half o f the cord, and the dorsal area between the dorsal horns. Two small areas o f neurofilament expression are visible in the grey matter, dorsolateral to the dorsal most part o f the ependymal layer (fig. 4.7.2B).

4.2.7 NCAM

At 40 days gestation longitudinal sections o f SC show that NCAM (ERIC antibody) is strongly expressed in the marginal layer, and there is also expression throughout the mantle layer though the neuroepithelial cell layer and the dorsal root ganglia are negative (fig. 4.8.1A-D). At 77 days gestation, human NCAM is strongly expressed throughout the white matter in a thick band encircling the grey matter when viewed in cross section, and there is scattered expression throughout the grey matter (fig. 4.8.2A). By 16 weeks gestation human NCAM appears to be expressed throughout the SC, but expression remains strongest in the white matter between the dorsal horns and the white matter between the ventral horns (fig. 4.8.2B1 & B2).