GAP43 expression in the developing rat lumbar spinal cord

Neuroscience Vol. 41, No. 1, pp. 187-199, 1991 Printed in Great Britain

0306-4522/91 $3.00 + 0.00 Pergamon Press plc

0 1991 IBRO

GAP-43 EXPRESSION IN THE DEVELOPING RAT LUMBAR SPINAL CORD

M. FITZGERALD,*? M. L. REYNOLDS* and L. I. BENOWITZ~

*Department of Anatomy and Developmental Biology, University College London, Gower Street, London WClE 6BT, U.K.

IMailman Research Center, McLean Hospital, Harvard Medical School, Belmont, MA 02178, U.S.A.

Abstract-The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period El 1 (embryonic day 1 1), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (El l-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned ipsilateral and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (El4-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During this period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At El7-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At El9-P2, GAP-43 is concentrated in axons of substantia gelatinosa.

Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.

GAP-43 is a growth-associated protein which is synthesized by developing neurons, transported to their growing axons4.5x37 and concentrated in the axonal growth cones where it is apparently incorporated into the membrane.‘6,2’,36

The expression of GAP-43 has been mapped in a number of developing CNS regions’3,20,24*26 and here we have studied its expression during the development of the rat lumbar spinal cord. The aim was to map the distribution of this protein during the growth of dorsal and ventral roots and axons from spinal cord interneurons and projection neurons as well as descending axons from brainstem neurons within the developing rat lumbar spinal cord. The changes in GAP-43 expression in these groups of axons were studied during their initial outgrowth, axon elongation and terminal arborization within their target.

EXPERIMENTAL PROCEDURES

Tissue for this study was obtained from Sprague-Dawley rats and processed for immunocytochemistry as described in the preceding paper. 29 Briefly, time-mated embryos from embryonic day (E) 10 to postnatal day (P) 29 were obtained; tissue from embryos between El0 and El7 were fixed by immersion and from all older animals by trans- cardial perfusion. Tissues from all ages were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer or in Bouins fixative. Embryos up to El4 were fixed and sectioned whole. Between El5 and E18, blocks of tissue containing the lumbar enlargement of the spinal cord were removed and

tTo whom correspondence should be addressed. Abbreviations: E, embryonic day; P, postnatal day.

the dorsal surface of the spinal canal opened to expose the cord during postfixation. At all other ages, the lumbar enlargement of the spinal cord was dissected out before postfixation.

Frozen sections were cut in transverse and sagittal planes at 50-1OOpm and immunostained free-floating for GAP-43 using antisera prepared by Benowitz et ~1.~ The Vectastain ABC kit was used and the stain visualized with 3,3’-diaminobenzidine plus H,O,

RESULTS

GAP-43 expression during early fetal spinal cord development: embryonic days 11-14

GAP-43 immunoreactivity was first detected in the spinal cord, dorsal root ganglia and brainstem at E 11. At El2 the density of GAP-43 increased considerably as explosive growth of axons began. This period is characterized by an absence of GAP-43 expression within the spinal cord gray matter other than the motoneuron pool and a concentration of GAP-43 staining in surrounding white matter and roots.

Dorsal root ganglia and dorsal roots. Dorsal root ganglia display weak GAP-43 immunoreactivity at El 1, more clearly at rostra1 levels than caudal levels (Fig. 1). The ganglion cells are dispersed and bipolar in shape but are just beginning to grow weakly GAP-43-positive processes which at caudal levels are sparse and growing in all directions (Fig. lC, D), but at rostra1 levels have grouped together into dorsal roots and reached the cord (Fig. lA, B). The majority grow towards the dorsal cord, but some weakly stained dorsal root fibres turn ventrally and run down

187

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central canal FC spmal cord dorsal roots sg substantia gelatinosa dorsal columns vc ventral column dorsal root ganglia vcm ventral commissure dorsal horn vh ventral horn bundle of His vm ventromedial columns k4t’Xdl columns vr ventral roots motoneuron pool VZ ventricular zone mesencephalon

Fig. 1. El 1 spinal cord. (A) Transverse section at rostra1 level Dorsal roots (arrows) grow oul from the _ _ _ ^ . . . . ._ . . . . dorsal root ganglia and down the lateral srdes ot the cord towards the motoaeuron pool. Lateratly placSa spinal cord neurons also appear to send axons via this route. x 320. (B) Sagittal section at rostra1 level showing the rostrocaudal growth of dorsal roots in the early bundle of His as well as the ventrally directed growth (arrowheads) of some dorsal root tibres towards motoneurons. x 320. (C). (D) As (A) and (B) but at caudal levels. Note the less well-developed dorsal roots and bundle of His but already the clear

ventrally directed growth of some fibres. The ventral roots can be seen in (C). Y 320

188

GAP-43 expression in the developing rat lumbar spinal cord 189

the lateral side of the cord towards the motoneuron pool (Fig. lA, C).

At El2 the intensity of GAP-43 expression of ganglia and roots increases markedly. The loosely bundled GAP-43-positive roots have reached the cord where they turn and travel rostrally or caudally along the cord in the early bundle of His (Fig. 2B). The roots do not penetrate the gray matter, which is totally free of GAP-43 except for the occasional small group of axons heavily labelled with GAP-43, which leaves the bundle of His and grows towards the ventricular zone (Fig. 2A, C). The presence of GAP-43 along the length of these axons right to the growth cone is evident (Fig. 2E). A few GAP-43-positive lumbar dorsal root fibres can also be detected turning in the ventral direction and growing into the motoneuron pool and these are not seen again after this age (Fig. 2A).

At El3-14 the dorsal root ganglia are still well stained with GAP-43 (Fig. 2F) but this is much reduced by El5 (Fig. 2H). Over this period more dorsal roots grow towards the cord to join the bundle of His, which expands medially (Fig. 2E) and by El5 forms the dorsal columns (Fig. 2G). The high levels of GAP-43 in these growing roots and the increasingly tighter bundling or fasciculation results in intense GAP-43 labelling of the bundle of His (Fig. 2D, E). Occasional axons still grow into the dorsal horn gray matter towards the ventricular zone, some reaching it and taking a U-turn back again (Fig. 2E), but the general pattern is still one of axons avoiding entering the cord.

Motoneurons and ventral roots. A few motoneurons express GAP-43 at El1 in the rostra1 cord. Their weakly stained ventral root axons have just grown out of the cord, fanning out dorsally into the surrounding tissue (Fig. 3A).

At E12, the lumbar ventral horn motoneuron pool is densely stained with GAP-43. The immunoreactivity, although sometimes detectable in cell bodies, is largely restricted to axons which form a densely stained tangle within the ventral horn (Fig. 3B). Some motoneuron axons even appear to grow into the ventral commissures (Fig. 3B, C).

By E14, medial and lateral motoneuron pools can be distinguished (Fig. 3E, F), although overall GAP-43 immunoreactivity in the ventral horn has begun to decrease and is absent by El5 (Fig. 3G).

Axons of interneurons and projection neurons. At E 11, GAP-U-positive fine axons arising from a group of neurons in the very lateral cord, just caudal to the bundle of His, can be observed running ventrally down the lateral edge of the spinal cord towards the motoneuron pool (Fig. 1). Their course is around the margin of the cord and they do not penetrate it. By El2 the GAP-43 expression in these axons has increased considerably and they form the ventral commissure. Horizontal sections show these commissural axons and their growth cones all intensely stained with GAP-43 as they reach the ventral mid-

line floor plate (Fig. 3C). At El2 a further, new group of axons begin to express GAP-43 arising again from laterally positioned cell bodies but projecting to the ipsilateral white matter. These ipsilateral projecting axons are very densely stained and form thick, twist- ing bundles of GAP-43 in the lateral white matter (Fig. 3B) as they wind and loop their way up the cord (Fig. 3D). GAP-43-positive collaterals can be seen branching from these bundles and growing down to the ventral cord, again taking a course around the margin of the cord rather than penetrating it (Fig. 3D).

White matter tracts. At Ell, when GAP-43 expression is first detectable in the rostra1 spinal cord, it is also clearly expressed in a group of axons growing down from the mesencephalon coursing round the primitive pons and medulla towards the spinal cord. No other GAP-43 immunoreactivity is detectable in the brain at this time (Fig. 6A). By El2 this descending tract, strongly stained with GAP-43, growing down the lateral funiculus as a widespread collection of axons headed with large GAP-43-positive growth cones, has reached the thoracic cord (Fig. 6B) and by El4 reaches the lumbar cord, contributing to dense GAP-43 staining in the lateral white matter at this age (Fig. 3E).

GAP-43 expression during later fetal spinal cord development: embryonic days 15-19

This period is characterized by a reduction in GAP-43 expression in dorsal root ganglion and motoneurons, dorsal and ventral roots. At the same time, GAP-43 expression within the spinal cord gray matter greatly increases as axons of intrinsic interneurons and projection neurons and collaterals from white matter tracts develop.

Dorsal root ganglia and dorsal roots. By El5 the dorsal roots are only weakly stained for GAP-43 and there is apparently very little in the dorsal root ganglion (Fig. 2H). GAP-43 expression in dorsal root ganglion remains weak at El6 and El7 and is gone at El 8 (Fig. 4B, C). The bundle of His is still strongly GAP-43-positive at El5 and extends medially to become the dorsal columns (Fig. 2G) which merge at the midline at El7 (Fig. 4A). At El8 the dorsal columns have enlarged and pushed down ventrally so that true dorsal horns are formed (Fig. 4C, D). They remain densely stained with GAP-43 at this stage.

Also at El5, the growth of very fine dorsal root collaterals, only weakly stained with GAP-43, begins into the dorsal horn gray matter (Fig. 2G). These are quite different from the occasional strongly GAP-43- stained bundle of axons seen leaving the bundle of His at earlier ages. They are far more numerous, of finer diameter and are clearly directed towards a target. The first collaterals grow from the most lateral part of the bundle of His in an arc, curving towards the very lateral part of the dorsal horn (Fig. 2G), and continue to grow into the dorsal horn gray matter in a lateral to medial progression (Fig. 4A). At El7-18 a further, more medially positioned bundle of weakly

190 M. FITZGERALD et ~1

Fig. 2.

GAP-43 expression in the developing rat lumbar spinal cord

Fig. 3. El l-15 ventral lumbar spinal cord. (A) El 1. Weak staining of motoneurons (closed arrow) and early spread out growth of ventral roots (open arrow). (BHD) E12. Showing, in (B), the densely stained motoneuron pool and ventral roots. On the lateral side of the cord are the densely stained ipsilateral projection axons (open arrow) and ventral to the central canal is the beginning of the ventral commissure (closed arrow). (C) Shows axons crossing in the ventral commissure in the horizontal plane (closed arrows). (D) Shows the meandering route taken in the rostrocaudal direction by the ipsilateral projection axons (open arrows). (A) Transverse. x 304. (B) Transverse. x 190. (C) Horizontal. x 190. (D) Sagittal. x 190. (E), (F) E14. Showing, in (E), the altered pattern of staining in the ventral horn picking out a medial and lateral motoneuron group (open arrows). The growth of axons medially from the lateral side of the cord is also evident (closed arrows). The ventral columns and ventral commissure are not well developed and contralateral projection axons cross ventromedially through the gray matter to join the ventral commissure. The lateral columns are small but clear. In (F) the individual commissural axons can be detected growing towards the ventral columns (open arrows). (E) Transverse. x 190. (F) Detail of (E). x 475. (G) El 5 showing that staining in the ventral horn has now disappeared and is weak in the proximal ventral roots. The lateral and ventral columns are well established but there are no axons in the dorsolateral column yet (closed

arrow). Transverse. x 190.

Fig. 2. El2-15 dorsal lumbar spinal cord. (AHC) El2 showing loosely bundled dorsal roots growing towards the cord and forming the rostrocaudally directed bundle of His. Note the single dorsal root axon straying into the gray matter (open arrows) and another growing ventrally towards the motoneuron pool (closed arrow). (A) Transverse. x 304. (B) Sagittal. x 190. (C) Transverse. x 190. (DHF) El4 showing, in (D), the increasing size and tighter bundling of the bundle of His. In (E) stray axons (open arrows) still leave the bundle and grow towards the ventricular zone or loop right round and grow back laterally (closed arrow). (F) shows that dorsal root ganglion cells are still well stained. (D) Transverse. x 304. (E) Transverse. x 475. (F) Transverse. x 304. (G), (H) El5 showing, in (G), the medial spread of the bundle of His to form the dorsal columns and the growth of dorsal root collaterals into the gray matter (closed arrows). The collaterals grow in a ventrolateral arc towards the lateral edge of the cord. (H) shows that staining in the dorsal root ganglia is reduced.

(G) Transverse. x 304. (H) Sagittal. x 76.

192 M. FITZGERALD~I al.

Fig. 4. El&P2 lumbar dorsal spinal cord. (A), (B) El6 showing, in (A). the extensive development of the dorsal columns and the continued growth of dorsal root collaterals, more medtally than at E15, into the dorsal gray matter (closed arrows). Note that there is now considerable axonal growth within the cord, including some (open arrow) that arises just dorsal to the central canal. In (B) tt can be seen that dorsal root and dorsal root ganglion staining is fading. (A), (B) Transverse. x 190. (C), (D) El 8 showing the expansion of the dorsal columns and formation of dorsal horns. Dorsal root collaterals contmue to grow (closed arrows), now from the most medial dorsal columns including a ventrally directed bundle of collaterals, presumably la afferents (open arrows). Clumped staming of individual dorsal horn cells is also apparent (ringed). Dorsal root ganglia and dorsal roots no longer stain. (C) Transverse. x 122. (D) Detail of (C). x 304. (E) E19. General axonal staining is evident in the gray matter with a higher concentration in the most dorsal laminae of the spinal cord (open arrow), Dorsal root collaterals can still be picked out (closed arrows). Individual dorsal horn cells in deeper laminae can also be seen (ringed). Transverse. x 304. (F) P2. Axonal staining in the dorsal horn is still high, concentrated in the dorsal laminae (open arrows). The cells of substantia gelatinosa are free of stain (closed arrows) but large cells in deeper dorsal

horn are still well stained (ringed). Transverse. x 304.


GAP-43-positive dorsal root collaterals, presumably Ia afferent collaterals (Fig. 4C, D), can be seen growing directly down to the motoneuron pool.

At El9, the most dorsal part of substantia gelatinosa, which until this age has been free of GAP-43 immunoreactivity other than that due to passing collaterals growing to deeper laminae, becomes stained with a fine network of fibres which form a thin cap over the dorsal horn (Fig. 4E, F).

Motoneurons and ventral roots. By El5, GAP-43 immunoreactivity is not longer detectable in lumbar motoneuron pools (Fig. 3G). The ventral roots are weakly stained but in the peripheral nerve motor axons continued to stain heavily with GAP-43 until postnatal life (see Ref. 29).

Axons of interneurons and projection neurons. At E1.5, GAP-43 staining appears in axons and growth cones in the gray matter core of the spinal cord rather than around its perimeter or restricted to the ventral horn (Fig. 3E). Strongly GAP-rlZpositive contralateral projection axons arc ventromedially through the cord and new axon growth fans out medially from neuronal pools in the lateral and ventrolateral cord (Fig. 3E, F), while staining in the ventral horn fades (Fig. 3G). Weakly staining axons from the medial intermediate gray matter grow laterally towards the lateral white matter. Ipsilateral projection axons merge with other axons to form the lateral white columns (Fig.

A

3G).

Detectable at El6 and prominent at El7 are a group of GAP-43~positive cell bodies distributed throughout the dorsolateral gray matter as dark clumps (Fig. 5). Axons can be traced a short distance and the majority grow ventrolaterally towards the ipsilateral lateral columns. Some more dorsally positioned cells send their axons into the dorsolateral funiculus. These clumps of GAP-43-positive cells become even more prominent at El%19 (Fig. 4C-E) but are more ventrally positioned in deeper laminae of the dorsal horn, although still occasionally seen in more superficial laminae (Fig. 5).

Other less well-defined areas of GAP-43 immunoreactivity appear at E16-17 just dorsal to the central canal (Fig. 4A) and bilaterally on the ventrolateral borders of the central canal, but have disappeared at El8 (Fig. 4C). At El8 and El9 GAP-43-positive axons arising just dorsal to the central canal can be seen growing towards the lateral columns and forming the dorsal commissure.

In general, however, El%19 is characterized by the appearance of a general network of GAP-43-stained axons within the spinal cord gray matter making tracing of individual axons and fibre tracts no longer possible.

White matter tracts. El5 marks a great increase in the relative size of the white matter at lumbar levels, much of it strongly GAP-43-positive. There are large numbers of densely stained fibres in the lateral

Fig. 5. Diagram of the location of individual GAP-43-stained neurons in the lumbar dorsal horn at E17, (A), El8 (B), E21 (C) and P2 (D). The results in two 50 pm sections were plotted on each diagram with

the aid of a camera lucida.

194 M. FITZGERALII et ul

columns (Figs 3G and 6C) but a conspicuous absence of GAP-43-stained axons in the dorsolateral funiculus (Fig. 3G). GAP-43-positive axons do not appear in the dorsolateral funiculus until E16, coinciding with the appearance of GAP-43-stained dorsal horn projection cells described in Fig. 4A. At E16, therefore, only the most dorsal and most ventral midline white matter has no GAP-43-positive axons at all.

Starting at El6 (Fig. 6D) and established on El7 (Fig. 6E) is the growth of weakly GAP-43-positive stained collaterals from the ventral and lateral white matter into the ventral and intermediate gray, some fibres having an irregular beaded appearance along their length. This increases at El 8-21 and contributes to the general overall axonal staining in the gray matter at this time.

GAP-43 expression during perinatal spinul cord

development: embryonic days 21-P29

GAP-43 is no longer expressed in the dorsal root ganglia, dorsal roots, motoneurons and ventral roots at this stage. Expression is concentrated in substantia gelatinosa of the dorsal horn in the early postnatal period, but then declines. White matter staining also fades postnatally except for the late appearance of GAP-43 in growing axons of the corticospinal tract.

Axons qf interneurons andprojection neurons. GAP- 43-positive cell bodies in the deeper laminae of the dorsal horn are still present but are restricted to the lateral two-thirds of the dorsal horn. At P2 some of the clumps are very large and may well represent two

Fig. 6. White matter tracts El 1-17. (A) El I, showing axons arising from the mesencephalon and growing down towards the cord (closed arrows). Sag&al. x 76. (B) E12. The descending axons travel@ down in a loose lateral bundle have reached the lumbar cord (closed arrows). Sagittal. x 122. (C) El 5 The lateral columns are now well developed and contain both ascending and descending axons. Sagittal. x 122 (D) E16. Axon collaterals with large growth cones (closed arrows) can be seen growing in from the lateral and ventral columns towards the ventral and intermediate gray matter. Transverse. x 304. (E) El 7. Axon collaterals from the lateral and ventral columns continue to grow into the gray matter Note that the

ventral commissure is no longer stained. Transverse. x 304.


or three cells together (Fig. 4F). individual axons are hard to pick out amongst the general network of fibres that are now stained throughout the dorsal horn. By PI0 they are no longer present.

Also at E19-21 the densely packed small cell bodies within substantia gelatinosa begin to stain with GAP-43, their short axons oriented in all directions (Fig. 4E). This is quite unlike the clumped staining of projection cells described above but is evenly distributed amongst all the intrinsic cells of substantia gelatinosa. By P2 these cell bodies are free of GAP-43 but the dense network of axon staining remains, surrounding “empty” cell bodies (Fig. 4F). At E21-P2 substantia gelatinosa is the most intensely stained area of the lumbar gray matter.

By Pi0 there is only a light background stain within the neuropil which is almost completely gone at P29. At neither age are cell bodies or well-stained axons detectable (Fig. 7B, D).

White matter tracts. Generally, GAP-43 stain decreases considerably postnatally in the white matter, such that at PI0 it is weak everywhere (Fig. 7A-C). At PlO, a distinctive column in the ventral part of the dorsal columns, the corticospinal tract, becomes GAP- 43-positive (Fig. 7B, C). There is also a very small, densely staining bundle in the ventromedial columns adjacent to the ventral median sulcus, the uncrossed part of the corticospinal tract. These two tracts are still detectable although faint at P29 (Fig. 7D, E).

DISCUSSION

A number of earlier studies on vertebrate spinal cord development using Golgi impregnation,28~“~4s silver stain,& horseradish peroxidase labelling,9J2J4J7+~ thymidine labelling,3s25 cytological techniques3 or immunocytochemical mapping with antibodies to neurofilament and neuropeptides’9*34 provide inform- ation which allows us to be confident that GAP-43 i~un~~~he~st~ stains all growing axons. There is no evidence here that it is staining neuroglia as has been reported in culture.43*47

Its expression in growing axons make it likely that GAP-43 participates directly in some aspect of axonal growth and synaptogenesis. Intra- and extracellular signals, such as neurotransmitters, membrane de- polarization and second messengers, all regulate the phospho~lation state of this protein’aJ1*22 and this in turn influences level of important signalling molecules of the nerve ending. ‘J’ Moreover, the protein may serve to link events between the membrane and cytoskeleton*,” and seems to be particularly important in areas of the brain where synaptic remodelling occurs6~’

As a general rule we found that GAP-43 was expressed in neuron cell bodies at the beginning of axon outgrowth and then as axons elongated and turnover increased, staining was restricted to the axons. GAP-43 staining within the axons peaked at a time when axons were actively growing and decreased as they formed terminal arbors, consistent

with the transport peaking at this time,” and then it disappeared altogether at the cessation of growth.

These conclusions are drawn partly from previous studies on developing rat spinal cord and partly from the present study, where it was possible to trace the pathways of the growing GAP-43-stained axons to their targets. They are discussed in relation to specific pathways below.

GAP-43 expression in early spinal cord development

GAP-43 expression in the very early phase of spinal cord development coincides with the onset of axon growth.3 The great increase in GAP-43 expression at El2 indicates that there is an explosion of neuronal production and axon outgrowth in dorsal root ganglion motone~on pools, ipsilateral and contra- laterally projecting neurons at this time which con- tinues actively until E14, decreasing at E15. Again this is consistent with the results of Altman and Bayer.’

El2 marks of the arrival of strongly GAP-43- positive dorsal root axons to the lumbar dorsal horn and the formation of the rostrocaudally running bundle of His. This is in agreement with studies in several species3”.28*45*46 and consistent in timing with the findings of Windle and Baxter& in the rat. As in the chick,9 sensory axons extend rostrally and caudally in the white matter for several days before invading the spinal gray matter and this period of axon elongation is marked by intense GAP-43 expression along the axons and a gradual fading within the dorsal root ganglion. GAP-43 staining in the ventral roots and motor nerves is intense at E14-15 as the axons grow, but is fading from the motoneuron pool.

Axons of ipsilateral and contralateral projecting neurons express GAP-43 consistent with the time of their axonal growth3 and continue to do so until the postnatal period, suggesting that they do not reach their targets until then. It is interesting to note that whereas in the chick, and to a lesser extent the mouse, many of the ipsi- and contralateral projection cells are dorsally positioned, some right up in the most dorsal part of the mantle layer,28s45 our results indicate that in the rat all these neurons are located in the lateral intermediate cord. No GAP-43-positive axons were ever seen growing down from the dorsal spinal cord at this age.

In view of current studies on chemotaxis and inhibitory factors during axonal development,27~4i some interesting points emerge from the study of GAP-43 expression in the developing spinal cord. Firstly, at El 1, when axonal growth is just beginning, it is clear that a number of different axonal groups are attracted to the floor plate region; i.e. dorsal roots, motoneuron axons, ipsilateral and contralateral projection cell axons. It therefore appears that the proposed specific chemotactic factor in the embryonic floor plate acting to induce contralateral projecting axons to cross the midline4i may have a role in deciding which axons

196 M. FITZGERALD et al

Fig. 7. P2-29 spinal cord. (A) P2, showing general axonal staining in the gray matter and white matter tracts. Transverse. x 128. (B), (C) PIO, showing that staining in the gray and white matter is considerably reduced. The corticospinal tract in the dorsal and ventromedial columns is now detectable (arrows). (B) Transverse, x 128. (C) Detail of (B). x 200. (D), (E) P29, showing the virtual absence of staining everywhere in the cord except for the corticospinal tract (arrows). (D) Transverse. x 80. (E) Transverse.

x 200.


actually remain in the commissure rather than which ones initially join it.

Secondly, the early spinal cord gray matter appears to provide an example of active inhibition to axonal growth. The dorsal roots grow into the cord but run up and down its length for days rather than penetrate the gray matter. Other than a few exceptional axons that appear to break through and head straight for the ventricular zone, the bundle of His appears to form a sharp boundary to growth until El5 Another example of this is the pathway taken by the contra- laterally projecting axons before El4 around the outside perimeter of the cord to reach the floor plate commissural region, although it would be a much shorter route to cross through the gray matter. It is possible that the early fetal gray matter actively inhibits ~netration by growing axons and that this is an important guidance factor. At each stage, however, there are a few axons that display aberrant growth. These were often strongly stained with GAP- 43 and it is possible that their large and extra active growth cones made them resistant to inhibitory factors or guidance cues.

At EIS, after 3 days of growth restricted to the bundle of His, dorsal root collaterals enter the dorsal horn and this coincides with a decline in the expression of GAP-43 in these axons. The timing is in good agreement with previous horseradish peroxidase studies in the rat’7.38 and compares to events at E6 in the chick cord.’ It is also consistent with the onset of reflex responses recorded from ventral roots following dorsal root stimulation of fetal rat cord.17,30 The growth of dorsal root collaterals and the reduction in GAP-43 expression exactly coincides with the innervation of their peripheral target, the skin,*’ and the pattern of growth is somatotopically appropriate. The first dorsal root collaterals grow into the lateral cord, the location of proximal limb afferents in the adult, and this is followed by collateral growth into the medial cord, the final position of distal limb afferents.35 This is exactly what would be predicted from the proximal to distal progression of innervation of limb skinz9 and supports the proposal that central collateral growth is triggered by peripheral innervation.p,3p

GAP-43 expression in later spinal cord d~e~opment

The expression of GAP-43 in dorsal horn cells and their axons from El7 to P2 corresponds well with Golgi studies demonstrating axonal growth of such neurons at this time.8 Many neurons in Bicknell and Beal’s study formed nests of four or more aggregated cells which may explain the clumped nature of the GAP-43 staining observed

here. These cells are likely to represent sensory projection neurons making up the spinocervical, spinovestibular, spinotectal and in some cases the spin~re~llar tracts.” Those in the marginal zone may contribute to the projection to the midbrain via the ipsilateral dorsolateral funiculus.” It is tempting to speculate that the maturation and axonal growth of these cells beginning at El6 is triggered by the arrival of sensory afferent input. The reduction in GAP-43 staining of these neurons and their axons by P2 suggests that they have now reached their targets and are forming terminal arborizations.

The build up of GAP-43 staining in the white matter from El5 onwards indicates not only the growth of ascending tracts but also of descending tracts. A prominent group of axons originating in the midbrain reaches the lumbar cord at El4 and other descending tracts soon follow. By birth, all projections from the first cervical segment, brainstem, deep cerebellar and diencephalic nuclei are present, if not complete,is which explains the heavy GAP-43 white matter staining in the late fetal period. Our findings that fine collaterals from the lateral and ventral white matter only begin to grow in to the cord at E17-18 (consistent with Windle and Baxtep) suggest another example of a delay between axon arrival and collateral growth. The apparent varicosities seen in some of the more lightly stained fibres may be due to the membrane-bound vesicular transport of GAP-43 towards the large growth cones. The clear and much later development of GAP-43 expression in the pyramidal tract agrees with the well-described anatomical development of this tract3”3 and with previous studies using GAP-43 immunocytochemistry,l’ This tract is known to reach lumbar cord by P9 and the bottom of the cord by P14 but growth cones may be found in the tract up to a week after first axonal arrival,32 and growth of collaterals into the gray matter is delayed for 3 days after axon arrivaL3i It is therefore not surprising to see GAP-43 staining so late in development.

CONCLUSION

The expression of GAP-43 in growing axons reflects their stage of growth from initial outgrowth to axon elongation through to formation of terminal collaterals and arbors. The intensity and specificity of GAP-43 expression has provided a clear picture of the temporal and organizational aspects of axonal growth in the spinal cord.

Acknowledgements-We thank Jaqueta Middleton and Penney Ainsworth for their valuable tecimical help and Clifford Woolf for his advice and en~~~rnent.

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(Accepted 14 September 1990)

GAP43 expression in the developing rat lumbar spinal cord

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