A radial glial scaffold surrounds the developing dorsal funiculi
Firstly, we examined the organisation of radial glial processes in the vicinity of the emerging dorsal funiculi. From E16, vimentin- and nestin-immunoreactive radial glial processes course from the central canal to the pial surface of the dorsal and ventral spinal cord (Fig. 1A,B). These cells express the glial-specific marker GLAST (Fig. 1C). A band of vimentin- and nestin-immunoreactive processes, termed the DMS, extends from the dorsal part of the central canal to the dorsal pial surface (arrow in Fig. 1D). This septum does not express any of the glial markers used to differentiate radial glial cells from their neuroepithelial cell precursors (Barry & McDermott, 2005). As such, the cells composing the DMS can be considered distinct from typical radial glia. The DMS clearly divides the growing GAP43-immunoreactive axons of the emerging dorsal funiculi into right and left halves (Fig. 1D). Nestin- (data not shown) and vimentin-immunoreactive fibres emerge laterally from the DMS ventral to the pioneering GAP43-immunoreactive dorsal funiculi axons in the regions of the presumptive ascending cuneate and gracile fasciculi (arrows in Fig. 1E,F). As with the DMS, these fibres do not express the radial glial markers GLAST or BLBP and are, therefore, in this study classified as similar to DMS-fibres. Considering that the dorsal funiculi develop gradually along the rostrocaudal axis of the spinal cord, the structural arrangement of DMS and its laterally projecting fibres suggests that they may provide a framework along which axons will grow. From early E17, the axons of the dorsal funiculi, as identified by GAP43, begin to segregate into the cuneate fasciculus and the more medial gracile fasciculus (Fig. 1G). This process appears to be facilitated by the dispersed network of laterally projecting DMS-fibres condensing into distinct dorsolaterally projecting fascicles extending from the septum to the dorsal pial surface through the emerging cuneate and gracile fasciculi, thereby physically separating them from one another (arrows in Fig. 1H,I). These boundary-forming fibres are transient and dissipate around the perinatal period once the cuneate and gracile fasciculi have become established (Barry & McDermott, 2005). At late E17, the cuneate fasciculus extends ventrally and is divided clearly along the midline by the DMS (arrow in Fig. 1J). Another network of DMS-derived fibres arch bilaterally from the septum and extend dorsolaterally first and then medially to form an almost closed loop (arrows in Fig. 1K,L). These processes seem to form a tube-like scaffold that surrounds the dorsal funiculus throughout the longitudinal extent of the cord, thereby creating a physical sleeve through which the axons project, potentially helping to prevent misdirection of axons as they extend rostrally.
Figure 1. DMS-fibres facilitate the formation of the dorsal funiculi. (A,B) At E16 vimentin- and nestin-immunoreactive radial glial cells are present in dorsal and ventral regions of the spinal cord extending from the central canal (cc) to the pial surface (ps). GAP43 is evident in the growing ventral, lateral, dorsolateral and dorsal funiculi. (C) Nestin is coexpressed with the glial specific marker GLAST in radial glial cell processes. (D) At E16, the vimentin-immunoreactive DMS extends from the dorsal central canal to the dorsal pial surface and divides the emerging GAP43-immunoreactive dorsal funiculi bilaterally. (E) Vimentin-immunoreactive collaterals (DMS-fibres) emanating from the DMS appear to provide a scaffold around which the dorsal funiculi grow (arrows). (F) Panel (E) converted to grey-scale, which clearly highlights vimentin-immunoreactive processes emanating laterally from the DMS on both sides of the spinal cord and then extending dorsally (arrows) to the pial surface. (G) Early at E17, the GAP43-immunoreactive cuneate and gracile fasciculi begin to separate in the dorsal spinal cord (box). (H) A higher magnification image of a region similar to that boxed in (J) showing vimentin-immunoreactive DMS-fibres appearing to segregate the more medial gracile fasciculus (Fg) from the cuneate fasciculus (Fc) (arrows). (I) Panel (H) converted to grey-scale, highlighting how fascicles of vimentin-immunoreactive DMS-fibres extend from the DMS to the dorsal pial surface (arrows). (J) At late E17, the cuneate fasciculus enlarges ventrally (arrow). (K) A higher magnification image of a similar region indicated by the arrow in (J) showing a band of vimentin-immunoreactive processes radiating from the DMS bilaterally to surround the growing cuneate fasciculus, seemingly forming a framework through which axons grow (arrows). (L) Panel (K) converted to grey-scale, showing vimentin-immunoreactive DMS-fibres extending laterally from the DMS first then extending dorsally (arrows). (Images A–C, D, G and J are confocal projections of 15–20 images captured at 0.5-μm intervals. Images E, F, H–I and K and L are confocal projections of 65–70 images captured at 0.5-μm intervals. Scale bars are in microns as indicated. Dorsoventral orientations are consistent for all images as indicated in A).
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Radial glial cell processes display a highly conserved structural pattern in the developing spinal cord WM
To determine how radial glial cells or their precursors relate to and perhaps segregate fascicles of nascent axons throughout the spinal cord WM, we examined their distribution and organisational pattern in the ventral and lateral funiculi throughout the rostrocaudal axis of the spinal cord, during the periods of peak WM growth from E13 to E18. Firstly, we examined the scaffold formed by radial neuroepithelial cells (radial glia precursors) in the WM at E13, which is approximately the time of onset of axon ingrowth in the rat spinal cord (Oudega et al. 1995). At this stage, 2F7-immunoreactive mature and immature neuronal cell bodies and axons are present in the presumptive grey matter (GM) ventrally, and in axon fascicles emerging in the subpial regions of the ventral and lateral spinal cord (Fig. 2A). At this stage, the WM here consists largely of the pioneering axons of the rubrospinal, reticulospinal and vestibulospinal tracts. Neuroepithelial cells in the ventral ventricular zone have radially arranged nestin-immunoreactive processes, many of which course fully from the ventricular zone to the pial surface. These neuroepithelial processes frequently change direction, crossover and branch (Fig. 2B). In the ventrolateral WM, some of these neuroepithelial radial fibres appear to exit the spinal cord and pass through the presumptive ventral root (Fig. 2C).
Figure 2. The organisation of radial neuroepithelial and radial glial cells at the onset of white matter (WM) formation in the spinal cord. (A) At E13, nestin-immunoreactive neuroepithelial cells are present ventrally and dorsally. 2F7-immunoreactive mature and immature neurons and axons are present in the presumptive ventral horn and WM, respectively, in the presumptive lateral WM and crossing over the ventral midline. (B) Nestin-immunoreactive radial cells extend from the central canal to the pial surface through the emerging ventral grey matter (GM) and WM. (C) In the ventrolateral spinal cord, some nestin-immunoreactive processes extend past the pial surface and appear to enter the ventral root (vr; arrows), possibly guiding motor axons exiting the spinal cord. (D–F) At E14, the emerging ventral and lateral WM tracts are present in the rostral, middle and caudal spinal cord. These tracts correspond to the growing vestibulospinal tracts (vst) ventrally, the spinothalamic tracts (stt) and the reticulospinal tracts (rtt) laterally. A band of axons entering from the dorsal root are evident dorsally. Nestin-immunoreactive processes radiate from the central canal to the pial surface in the rostral, middle and caudal spinal cord. (G) At E14, brain lipid-binding protein (BLBP) is coexpressed with nestin in the ventral portion of the spinal cord in radial glial processes. (H) The proportion of BLBP and nestin expressing radial glial cells compared with nestin expressing radial neuroepithelial cells decreases along a rostral to caudal gradient. (I) Two-hundred-micrometer-thick slices show how nestin- and BLBP-immunoreactive radial glial cells ventrally adopt a uniform orientation as they pass through the GM/WM interface (dotted line) and radiate toward the pial surface. Occasionally, spaces are observed between radial glial cell processes that persist through the thickness of the slice (asterisks). (I–K) Radial glial cells often branch at the GM/WM interface (dotted lines) resulting in an increased number of processes entering the presumptive WM. DiI labelling of axons shows how discrete fascicles seem to be contained within channels of nestin-immunoreactive processes in the WM (arrowhead in J). (K) Near the ventral floor plate (fp), nestin- and BLBP-immunoreactive radial glial cell processes display a striking change of direction as the transition from the GM/WM interface (dotted line) to the pial surface. (L) The densities of radial glial cell processes in the dorsal and lateral WM are maintained throughout the rostrocaudal axis of the spinal cord. (Images A–F are confocal projections of 15–20 images captured at 0.5-μm intervals, images G, I–K are confocal projections of 400 images captured at 0.5-μm intervals. Scale bars are in microns as indicated. Dorsoventral orientations are consistent for all images as indicated in A).
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By E14, the GAP43-immunoreactive ventral and lateral funiculi of the spinal cord have increased in size in the rostral and caudal spinal cord (Fig. 2D–F). Dorsally, neurons are present in the GM, and a band of axons entering from the dorsal rootlets is present both rostrally and caudally. Nestin-immunoreactive processes extend from the central canal to the pial surface. Ventrally, these processes now also express BLBP, identifying them as classical radial glial cells of astroglial lineage (Fig. 2G; Feng et al. 1994; Shibata et al. 1997). The ratio of nestin- and nestin/BLBP-immunoreactive cells along the rostrocaudal axis was quantified. In rostral regions of the cord, nestin/BLBP-immunoreactive cells represent 35% of the entire radial cell population, while 65% remain nestin-immunoreactive-only radial cells (Fig. 2H) and do not express BLBP. At mid spinal cord levels, 30% of radial processes have transformed to radial glial cells at E14, while in caudal regions of the spinal cord, 27% are radial glial cells.
We next analysed 200-µm slices of immunostained spinal cords using confocal microscopy to visualize BLBP- and nestin-immunoreactive radial glial cells, and more accurately assess their three-dimensional structural organisation. Using this approach, BLBP- and nestin-immunoreactive radial glial processes appeared uniformly organised as they radiate from the ventricular zone to the pial surface (Fig. 2G). Ventrally, the processes often change direction once they encounter the GM/WM interface, and adopt an orientation more perpendicular to the pial surface (Fig. 2I,K). BLBP expression along these processes appears upregulated along the portion of the radial glial cell process that extends through the WM (Fig. 2G,I–K). We regularly observed ‘corridors’ in these 200-μm slices formed by a periodic spatial arrangement of radial glial processes (asterisks in Figs 2I and S1). This was particular strikingly in the ventral and lateral WM, and these corridors extended through the longitudinal axis of the spinal cord. Additionally, radial glial processes branched extensively at the GM/WM interface (dotted line in Figs 2I–K and S1), such that significantly more processes passed through the WM than through the GM. Furthermore, we used Dil to label axons in smaller numbers than conventional antibody-based immunohistochemistry, and to visualise the structural relationship between developing axonal fasciculi and glial processes more clearly. Using this approach the segregation of axons in the WM by the finely branched glial processes was clearly evident, and frequently axons appeared to be bundled between the corridors formed by radial glial processes (arrowhead in Fig. 2J). To determine the spatial distribution of radial glial processes in the WM, we quantified the radial glial fibre density per mm of the ventral and lateral WM through the rostrocaudal axis of the E14 spinal cord (Fig. 2L). At E14, radial glial fibre density is similar in the ventral WM of the rostral (75.3 ± 4.1 fibres mm−1) and caudal (85.1 ± 4.6 fibres mm−1) regions of the spinal cord. At this stage, the descending axons of the rubrospinal tract, reticulospinal tract and vestibulospinal tract are present in the ventral and lateral WM in rostral and caudal regions (Fig. 2D–F). The conservation of the pattern of radial glial processes through these growing axon tracts suggests that careful regulation of process density along the rostrocaudal axis of the spinal cord may be required for correct tract formation. Fibre densities are also conserved in the rostral (106.4 ± 5.0 fibres mm−1) and caudal (115.2 ± 5.7 fibres mm−1) lateral WM (Fig. 2L).
At E16, a longitudinal section through the dorsolateral spinal cord showed that the GAP43-immunoreactive WM, presumably containing ascending spinothalamic and dorsal spinocerebellar tracts, is present the entire length of the spinal cord (arrows in Fig. 3A). As at E14, nestin-immunoreactive radial glial processes course radially from the central canal to the pial surface both ventrally and dorsally in rostral, middle and caudal regions (Fig. 3B–D). At this age, in contrast to E14, these cells express GLAST and BLBP in both dorsal and ventral regions of the spinal cord (Barry & McDermott, 2005). As at E14, these processes appear uniformly arranged as they pass from the GM/WM junction through the developing WM to the pial surface (Fig. 3E,F). Moreover, the density of these radial glial processes was constant through the rostrocaudal axis of the cord both dorsally (rostral: 77.5 ± 3.2 fibres mm−1; caudal: 74.81 ± 3.3 fibres mm−1) and ventrally (rostral: 71.6 ± 2.4 fibres mm−1; caudal: 69.3 ± 2.61 fibres mm−1; Fig. 3G).
Figure 3. Radial glial cells coursing through the WM display a high degree of organisation in all regions of the E16 spinal cord. (A) Longitudinal section of an E16 embryo showing how GAP43-immunoreactive axon tracts are present the entire length of the spinal cord (arrows). Nestin-immunoreactive radial glial cells radiate from the central canal to the pial surface at all levels. (B–D) At E16, GAP43 expression is increasing in the ventral and lateral WM of the rostral, middle and caudal spinal cord. The presumptive WM of the dorsal horn and the dorsal funiculi is also emerging at all levels of the spinal cord. Nestin-immunoreactive radial glial cells radiate from the central canal to the pial surface both dorsally and ventrally. (E,F) Radial glial processes appear evenly distributed in the ventral (E) and dorsal (F) WM. (G) The densities of radial glial processes in the ventral and dorsal WM are maintained throughout the rostrocaudal axis of the spinal cord. (All images are confocal projections of 15–20 images captured at 0.5-μm intervals. Scale bars are in microns as indicated. Dorsoventral orientations are consistent for images B–F as indicated in B).
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At E18, GAP43-immunoreactive axons were extensive in ventral, lateral and dorsal WM funiculi and in the GM (Fig. 4A). The pattern resembles that of the mature spinal cord (data not shown), indicating that much of the structural organisation of the adult WM is already in place by this stage of development. The radial glial cell–astrocyte transformation is underway in most regions of the spinal cord by E18 (Barry & McDermott, 2005). As a consequence, many radial glial cells are beginning to lose their ventricular zone attachment and appear to branch at the GM/WM junction (arrows in Fig. 4B). However, the density of radial glial processes in the WM is still relatively constant within the E18 spinal cord. There is no statistical variation in fibre density evident in the ventral WM of the rostral (70.3 ± 2.2 fibres mm−1) and caudal (69.2 ± 2.1 fibres mm−1) spinal cord, or in the dorsal WM of the rostral (61.6 ± 3.3 fibres mm−1) and caudal spinal cord (61.8 ± 2.8 fibres mm−1; Fig. 4C).
Figure 4. (A) GAP43 is expressed in the ventral, lateral and dorsal funiculi and in the GM. Nestin-immunoreactive radial glial cells still radiate from the central canal to the pial surface. (B) Radial glial cells exhibit a high degree of branching at the GM/WM interface (arrows), but remain evenly distributed in the WM. (C) The densities of radial glial cell processes in the ventral and dorsal WM are maintained throughout the rostrocaudal axis of the spinal cord. (D) The mean densities of radial glial cells in both the rostral (*P < 0.05) and caudal (+P < 0.05) WM are significantly reduced from E14 to E16, and from E16 to E18, in line with axon tract formation. (E) The combined mean densities of radial glial cell processes in all regions of the WM decline significantly from E14 to E16, and from E16 to E18 (*,**P < 0.05). (F) Western blot showing vimentin and GAP43 expression from E14 to P6. Β-actin was used as a control. (G) Densitometrical analysis showing how GAP43-immunoreactive axons increase postnatally, while vimentin-immunoreactive radial glial cells decline. (Both images are confocal projections of 15–20 images captured at 0.5-μm intervals. Scale bars are in microns as indicated, the dorsoventral orientation is consistent for B as indicated in A).
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Overall, the density of radial glial processes in the WM decreases substantially throughout the spinal cord with increasing age (Fig. 4D,E; P < 0.05). To confirm the loss of radial glial cell processes from the spinal cord during development, Western blots were carried out on spinal cord lysates isolated from E14, when radial glial cells first differentiate from radial precursor neuroepithelial cells, to P6 when WM radial glia are no longer present in the cord (Fig. 4F). Levels of vimentin are greatest at E14 and decrease with increasing age. Vimentin becomes lost from the postnatal spinal cord, in accordance with the loss of the radial glial cell phenotype during development and the postnatal restriction of vimentin to some WM astrocytes (Fig. 4F,G). The concomitant level of maturation of axons and consequently of the spinal cord was revealed by Western blot analysis of GAP43 expression in lysates ranging in age from E14 to P6 (Fig. 4F,G). Levels of GAP43 increase with increasing age, reflecting the establishment of axon pathways in WM regions during development.