/. Embryol. exp. Morph. 82, 241-251 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
241
The pattern of alkaline phosphatase activity in the
developing mouse spinal cord
By W. H. KWONG AND P. P. L. TAM
Department of Anatomy, Faculty of Medicine, The Chinese University of Hong
Kong, Shatin, N.T., Hong Kong
SUMMARY
The localization of alkaline phosphatase activity in the lumbosacral region of the developing
spinal cord was studied in 9-5- to 17-5-day mouse embryos. The activity was uniformly
distributed in the pseudostratified neuroepithelium of the 9-5-day cord. In the 11-5-day cord
in which the lateral motor columns were being formed, the enzymatic activity was localized
in the ventrolateral sector of the cord. The enzyme-positive ventricular cells tended to be
located medially whereas radially oriented enzyme-positive processes extended into the
marginal layer. The 13-5-day cord displayed a similar distribution pattern, but there were
many more radial processes and the enzyme-positive cells had spread laterally. Close
apposition between the processes and the ventricular cells was observed. By 15-5 and 17-5
days, when the intermediate layer was fully developed and the ventricular layer had regressed
to a thin ependyma, the activity had become diffusely located in the ventral half of the cord.
The enzyme-positive cells and processes became less conspicuous. The silver-stained
processes in the cord were found to be organized in an entirely different pattern from that of
the enzyme-positive processes, suggesting that the enzyme-positive processes were not
neuronal processes. The enzymatic activity found in the developing spinal cord may be
associated with the migration of neuroblasts along the radially aligned processes.
INTRODUCTION
The early embryonic neural tube is made up of pseudostratified epithelial cells
(Hernan & Kaufman, 1966; Sturrock, 1981a) whose nuclei undergo intermitotic
migration (Smart, 1972). The formation of the basic layers of the spinal cord
begins at day 10-11 with the appearance of the ventricular, intermediate and
marginal layers (Sturrock, 19816; Boulder Committee, 1970). The ventricular
layer forms the germinal tissue from which neuroblasts and glioblasts of the
foetal cord are generated (Smart, 1972; Nemar, Sakla & Mahran, 1974; Wentworth & Hinds, 1978; Sturrock, 1981a). The earliest neurones that appear on day
9 are of medium size and reside in the lateral and the ventral horns (McConnell,
1981). Motor neurones emerging on day 10 and day 11 migrate from the basal
plate to the adjacent intermediate layer. Those neurones formed on day 12-14
come from the alar plate and settle in the dorsal portion of the intermediate layer
(Nornes & Carry, 1978). Some of the earliest neurones from the alar plate send
out circumferential axons which are the forerunners of commissural fibres
(Holley, 1982). The mechanism whereby this pattern of histogenesis is brought
EMB82
242
W. H. KWONG AND P. P. L. TAM
about in the developing cord is not fully known. However, it has been proposed
that the organization of layers of neural cells and the formation of neuronal connections are dependent on the information perceived by the neuroblasts via contact guidance with the pre-existing fascicles of cell processes (Sidman & Rakic,
1973; Henrikson & Vaughn, 1974; Rakic, 1978; Holley, Nornes & Morita, 1982).
The histogenesis of the spinal cord has been correlated with changes in the biochemical profiles of the various cell types involved in this developmental process.
For example, the formation of morphologically discernible neurones in the neural
tube of early embryos is related to the distribution of acetylcholinesterase
activities (Burt, 1975; Miki & Mizoguti, 1982). Acid phosphatase is mainly
localized in the sensory neurones in the dorsal horn of the rat spinal cord (Knyihar
& Csillik, 1977) and this group of enzyme is present in different forms in the substantia gelatinosa and in the motor neurones (Sanyal & Rustioni, 1974). The activity of alkaline phosphatase has been localized in the developing cord of the human
foetuses. At an early stage when the cord is entirely composed of the ventricular
layer, the enzymatic activity is present in every cell but cells in the ventricular
layer show a stronger activity than those in the intermediate and marginal layers.
With development the enzymatic activity progressively diminishes and by 4-5
months the enzymatic activity becomes confined to the blood vessels of choroid
plexuses and meninges of the brain and spinal cord (Rossi & Reale, 1957).
Alkaline phosphatase activity has been found in many tissues of the adult
spinal cord and brain of the mouse and the rat, primarily on the wall of arterial
vessels (Shimizu, 1950; Leduc & Wislocki, 1952). Adult neurones generally show
a moderate enzymatic activity (Sanyal & Rustioni, 1974; Sood & Mulchandani,
1977); however, no information is available for early embryonic mouse neural
tube. In other embryonic tissues, alkaline phosphatase is initially present in a
high concentration during the early stage of cell differentiation, decreases during
differentiation and stabilizes at a low adult level which usually shows no regional
pattern of distribution. A high enzymatic activity is present in embryonal carcinoma cells (Bluthman et al. 1983) and in embryonic ectoderm (Damjanov,
Solter & Skreb, 1971); in both cases, the differentiated derivatives exhibit a low
enzyme level (Bernstine, Hooper, Grandchamp & Ephrussi, 1973; Solter, Damjanov & Skreb, 1973). A strong enzymatic activity has been found at the early
organogenetic stage in the somitic mesoderm, intestinal epithelium and migrating primordial germ cells (Rossi & Reale, 1957; Tarn & Snow, 1981). The present
study is to investigate the developmental changes of alkaline phosphatase activity
in the foetal spinal cord. This enzyme pattern is interpreted in relation to the
migration of neuroblasts and the formation of the neuronal pattern.
MATERIALS AND METHODS
ICR strain mouse embryos at 9-5-17-5 days of gestation were used and the
morning when the vaginal plug was detected was designated as 0-5 days post
Alkaline phosphatase in foetal spinal cord
243
coitus. The pregnant mice were killed by cervical dislocation and the embryos
were dissected out of the uterus into cold phosphate-buffered saline. Whole
embryos at 9-5-13-5 days, and the lumbosacral region of the embryos at
15-5-17-5 days, which was identified as the region corresponding to the hindlimb, and of the adult spinal cord were processed according to the following
methods. For general histology of the spinal cord, the specimens were fixed in
Bouin's fluid, and serial transverse sections were obtained and stained by
Mallory's acid fuschin-aniline blue-orange G triple staining method. To stain
neuronal processes, the specimens were fixed with DeCastro's fixative (100 ml
distilled water, 100ml 9 5 % ethanol, 6gm chloral hydrate, 3 ml concentrated
nitric acid) and then processed with a modified silver method based on Cajal and
DeCastro (Levi-Montalcini, 1949). For the demonstration of alkaline
phosphatase, serial paraffin sections of ethanol-fixed tissues were processed with
the azo-coupling technique (Gabe, 1975), using sodium a-naphthyl phosphate as
the substrate and Fast TR Red salt for the coupling-staining reaction. Control for
the histochemical reaction was performed either by adding tetramisole hydrochloride (1 ITIM; Borgers, 1973) or EDTA (10 mM) to the incubation medium to
inhibit the enzyme, or by removing the substrate from the medium.
RESULTS
When tetramisole hydrochloride was added to the incubation medium, no
enzymatic reaction was detected in any part of the spinal cord at all stages.
Removal of the substrate from the incubation medium or addition of EDTA to
the medium also drastically reduced the intensity of the reaction. The control
thus indicated that the activity of alkaline phosphatase was responsible for the
staining reaction.
The neural tube of the 9-5-day embryo was made up of a tall pseudostratified
neuroepithelium, which had not differentiated into the definitive layers. A
strong alkaline phosphatase activity was found uniformly distributed throughout
the transverse section of the tube (Fig. 1). No stained processes were evident in
the silver preparations.
The differentiation of neuroblasts occurred primarily in the basal plate of the
neural tube so that by 11-5 days, the marginal layer and the intermediate layer
containing the lateral motor column had developed (Fig. 7). A band with a
strong alkaline phosphatase activity was observed in the ventricular layer of the
basal plate (Fig. 2). It extended from the luminal surface to the vicinity of the
dorsal half of the lateral motor column. The lateral motor column itself showed
little activity. The band consisted of enzyme-positive ventricular cells and
radially aligned processes (Figs 8, 9). In the marginal layer, a high enzymatic
activity was found in the craniocaudally oriented processes (Fig. 9), which
appeared to be continuous with the radial processes. In addition, a few strands
were seen along the border between the ventricular layer and the lateral motor
244
W. H. KWONG AND P. P. L. TAM
*v
< • * . '
-^r
dr
/^"t,."
Figs 1-6. The pattern of distribution of alkaline phosphatase activity in the spinal cord
at different stages: 9-5-day (Fig. 1), 11-5-day (Fig. 2), 13-5-day (Fig. 3), 15-5-day (Fig.
4) and 17-5-day (Fig. 5). In the adult spinal cord, the blood capillaries (arrow heads)
showed a strong activity (Fig. 6). The boundary between the ventricular (ependymal)
and the intermediate layers was demarcated by a broken line, ml = marginal layer.
dc = dorsal columns, sg = substantia gelatinosa. Bar equals 100 jum.
Alkaline phosphatase in foetal spinal cord
Figs 7-10 for legend see p. 246
245
246
W. H. KWONG AND P. P. L. TAM
column. The remaining part of the spinal cord did not show any enzymatic
activity
Silver staining revealed the presence of many processes which were organized
into five groups according to their location and orientation (Fig. 10). It can be
seen that the pattern of silver-positive processes differed notably in two aspects
from the pattern of alkaline phosphatase activity. Firstly, no silver-positive
processes were present in the strong enzyme-positive band of the ventricular
layer, and secondly, in the lateral motor column, the silver-positive processes
were dc rsoventrally oriented whereas the enzyme-positive processes were radially oriented.
In the 13-5-day embryo, both the intermediate and the marginal layers were
well developed on both the ventral and dorsal parts of the cord. The ventricular
Fig. 7. Triple-stained transverse section of the 11-5-day cord. The ventricular layer
with mediolaterally aligned cells was demarcated (broken line) from the intermed.ate layer. The marginal layer (ml) was well developed on the periphery of the
prominent lateral motor column (Imc). Bar equals 50/mi.
Figs 8, 9. Alkaline phosphatase activity in transverse (Fig. 8) and horizontal (Fig.
9) sections of the 11-5-day cord. A few processes (arrow) arose from the band of
enzyme-positive cells in the ventricular layer and extended across the lateral motor
column (Imc) to the marginal layer (ml). Bar equals 50//m.
Fig. 10. Silver-stained transverse section of the 11-5-day cord, showing five groups
of neuronal processes: (A) Dorsolateral group, consisting of craniocaudally oriented
processes at the dorsolateral margin of the spinal cord. (B) Ventrolateral group,
cons sting of craniocaudally oriented processes on the ventrolateral margin of the
motor column. (C) Lateral motor column processes, which were dorsoventrally
oner ted and were aggregated on the lateral part of the motor column. (D) Circumferer.tial group, which intervened between the ventricular layer and the lateral motor
column, and crossed to the opposite side ventral to the neural canal; some of these
processes diverged from the circumferential pathway to enter the lateral motor
column as Group C processes. (E) Ventral root axons. Bar equals 50 /mi.
Fig. 11. A transverse section of the 13-5-day cord, showing the enzyme-positive
transverse band and its radial processes (arrow). Imc = lateral motor column.
ml = marginal layer. Bar equals 50/mi.
Fig.: .2. A horizontal section of the 13 • 5-day cord. Numerous enzyme-positive radial
processes (arrow) of the transverse band joined the craniocaudally oriented
processes (arrow head) of the marginal layer (ml). Bar equals 50/mi.
Fig. 13. A horizontal section of the 13-5-day cord, showing enzyme-positive
processes traversing through stained and unstained cells from the luminal side (lu)
to ths marginal layer (ml). The cells with high enzymatic activity were especially
densi sly packed near the luminal side. The processes appeared in close apposition with
adja( ent cells where the cell surface often appeared flattened. Bar equals 25 /mi.
Fig. 14. A silver-stained half-section of the 13-5-day cord, and a camera-lucida
drawing to illustrate the stained fine processes. Groups of processes are labelled
similarly to those in the 11-5-day cord. Bar equals 50 /mi.
Fig. ] 5. Alkaline phosphatase activity in the ventral part of the 15-5-day cord, which
showsd radial processes (arrow head) and mediolaterally oriented processes
(arrow). Some of the processes extended to the marginal layer (ml) and formed
partiiions (pt) between groups of longitudinal processes. Bar equals 50/mi.
Alkaline phosphatase in foetal spinal cord
Figs 11-15 for legend see p. 246
247
248
W. H. KWONG AND P. P. L. TAM
layer remained thick dorsally, but had become much narrower on its ventral part.
The cord contained a transverse band of strong alkaline phosphatase activity
(Fig. 3) in a similar location as the 11-5-day cord. However, this band extended
from the luminal surface through the dorsal part of the lateral motor column and
reached the marginal layer (Figs 11-13). The same five groups of silver-stained
processes as those in the 11-5-day cord were observed in the 13-5-day cord (Fig.
14). In the marginal layer, the dorsolateral and ventrolateral groups of processes
had thickened. The originally dorsoventrally oriented processes of the lateral
motor column had become obliquely oriented and spread out to the whole motor
column.
Further differentiation resulted in an increase in thickness of the intermediate
layer in the 15-5-day cord, while the ventricular layer became a thin ependymal
layer. The cord at this stage differed markedly from that of the 13-5-day embryo
in both the distribution and intensity of the alkaline phosphatase activity. Instead
of being confined to a transverse band, the activity was distributed throughout
the ventral two-thirds of the cord (Fig. 4). The overall activity was substantially
lower than that of the transverse band in the previous stage and only a few cells,
mostly ependymal cells, were stained. From the ependyma, faintly to moderately
stained processes extended laterally and ventrally (Fig. 15). The remaining
dorsal part of the cord and the floor plate area did not have any detectable
activity.
In the silver preparation, processes of various orientations were organized in
the same basic pattern as those in the 13-5-day cord, except that a well-defined
circumferential group was no longer recognizable. Like the earlier stages, the
organization of the stained processes did not correspond at all to that of the
enzyme-positive processes.
By 17-5 days, the neural canal and the ependymal layer had become restricted
to the ventral part of the cord. The alkaline phosphatase activity at this stage was
located in a meshwork of neuropil in the ventral half of the cord and in the
longitudinal processes of the marginal layer (Fig. 5). A stronger activity was
found on either side of the ventral white commissure and in the marginal layer.
The organization of silver-stained processes followed the same pattern as that in
the 15-5-day cord.
The alkaline phosphatase activity in the adult spinal cord was low, except for
the substantia gelatinosa which was weakly stained (Fig. 6).
DISCUSSION
The distribution of the alkaline phosphatase activity changed during the
development of the embryonic neural tube. At the earliest stage, the enzymatic
activity was found in the neuroepithelial cells. With further development, the
activity became confined to a band of cells and processes in the ventricular layer
of 11-5- and 13-5-day spinal cord. The enzymatic activity remained evident in the
Alkaline phosphatase in foetal spinal cord
249
ventral grey matter and the adjacent marginal layer of the late foetal spinal cord
but the overall activity had diminished. The enzymatic activity is generally low
in the adult mouse spinal cord except for the cells of blood capillaries. The
association of the enzyme with the ependyma of the neural canal and the blood
vessels in the grey horns, the choroid plexus and the meninges of the adult central
nervous system (Shimizu, 1950; Leduc & Wislocki, 1952; Sood & Mulchandani,
1977) has led to the suggestion that the enzymatic activity is related to the trophic
function of the blood-neural tissue barrier and that the enzyme pattern in the
embryonic neural tube reflects the process of angiogenesis (Ciani, Contestabile,
Minelli & Quaglia, 1973). However, in the foetal spinal cord there were no
significant variations in the vascular density between the dorsal and ventral areas
of the spinal cord (Sturrock, 19816; Simon-Marin, Vilanova, Aguinagalde &
Barbera-Guillem, 1983) that could account for a regional localization of the
enzymatic activity described in the present study. Furthermore, the enzymatic
activity diminished during the late foetal stage when the vascular pattern is
maximally developed (Sturrock, 19816).
A comparison of the pattern of enzyme-positive processes with that of silverstained processes suggests that the enzyme-positive processes were not neuronal
processes. Observations of the present study suggested that the differentiating
neuroblasts were in intimate apposition with these enzyme-positive processes.
Similar radially oriented processes, which were termed radial glial processes or
ependymoglial processes, have been described in the spinal cord and
telencephalon of the mouse embryo (Henrikson & Vaughn, 1974; Hinds & Ruffett, 1971; Sturrock, 1981a, 1982;Holley,Nornes&Morita, 1982). Many of these
ependymoglial fibres later degenerate or become astrocytes, but some may persist
in the dorsal median septum and in the floor plate of the late foetal cord (Sturrock,
1981a). In the lateral marginal layer of the embryonic mouse spinal cord, the growing dendrites from the lateral motor column make a close association with the
radial glial processes (Henrikson & Vaughn, 1974). A similar association between
the radial processes and the migrating neuroblasts has been observed in the
developing cortex of the cerebrum (Sidman & Rakic, 1973). This structural
specialization which seems to favour an orderly radial migration of the neuroblasts
to a distant position is crucial to the organization of neurones in a cortical column
(Rakic, 1978). The exact mechanism for such a contact guidance of neuronal
patterning is not known. The ependymoglial processes may provide a mechanical
and/or chemical pathway for neurites to follow (Singer, Norlander & Egar, 1979).
These glycogen-rich glial processes may also serve as a trophic medium from which
the neuroblasts obtain the energy for migration and growth (Ciani, Contestabile,
Minelli & Quaglia, 1973; Sturrock, 1981a,6). The elevated level of alkaline
phosphatase activity may reflect the active metabolic state of these processes.
During the development of the lumbosacral segment of the spinal cord, a high
alkaline phosphatase activity occurred concomitantly with the formation of the
ventral grey matter from the basal plate. The ventricular cells of the basal plate
250
W. H. KWONG AND P. P. L. TAM
always show a lower mitotic activity than the dorsal counterpart (Smart, 1972) and
the newly formed ventricular cells spend a short time in the ventricular layer
before migrating laterally to form the lateral motor column (Nornes & Carry,
1978;McConnell, 1981;Sturrock, 1981a). In the dorsal portion of the neural tube,
the thickness of the ventricular layer increases as a result of the accumulation of
daughter cells. Transformation of these cells to those of the intermediate layer
does not occur progressively as in the ventral portion but takes place rapidly
(Smart, 1972; Sturrock, 1981a). The lower enzymatic activity in the dorsal portion
may therefore not be due to fewer ependymoglial processes but to a lower rate of
neuroblast migration. It may not be coincidental that the band of highest alkaline
phosphatase activity was found in the ventral region of the ventricular layer where
the mitotic activity is declining (Smart, 1972) and the intermediate layer
neuroblasts begin to emerge in recognizable numbers (Sturrock, 1981a). Many
radial processes are already existing in the ventricular layer prior to the birth of
motor neurones (Nornes & Das, 1974; Wentworth & Hinds, 1978) and they seem
to enhance the dispersion of the neuroblasts to the intermediate layer (Holley,
Nornes & Morita, 1982). A high metabolic activity of the radial process may be
heralding an accelerated exodus of neuroblasts out of the ventricular layer.
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LEDUC,
{Accepted 9 April 1984)