Developmental Brain Research, 39 (1988) 225-232 225 Elsevier

BRD50705

The development of acetylcholinesterase activity in the embryonic nervous system of the frog, Xenopus laevis

Sally A. Moody and David B. Stein Department of Anatomy and Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908 (U.S.A.)

(Accepted 6 October 1987)

Key words: Primary motoneuron; Rohon-Beard neuron; Neuronal differentiation; Primary interneuron; Trigeminal ganglion cell; Neuronal development; Acetylcholinesterase

Histochemical detection of acetylcholinesterase (ACHE) activity in Xenopus embryos was found first in primary motoneurons, Ro- hon-Beard neurons and somitic myotubes at early tail bud stages. At late tail bud stages all primary neurons, including primary inter- neurons, cranial ganglion cells and ventral brainstem cells expressed this enzyme. The onset of detectable AChE activity in some pri- mary neurons occurred near the time of initial axon outgrowth, whereas in others it occurred at much later stages. At early indepen- dent-feeding and continuous-swimming stages nearly all seemingly postmitotic neurons began to express AChE activity, and by the beginning of limb bud stages, when many secondary neuronal populations were going through their final rounds of mitosis, nearly all CNS cells outside the ventricular zone were intensely stained. Thus, the onset of detectable AChE activity in secondary neurons oc- curred near the time of their final mitoses. In trunk somites the enzyme activity initially was located diffusely throughout the myotube, and with progressing development it became localized to the myocommata. From the onset of AChE activity both head somites and head muscles had discrete patches of reaction product all over their surfaces. The onset of detectable AChE activity occurred in mus- cles near the time that they were contacted by motor axons. These data demonstrate that the primary neurons are the first to express AChE activity, and that as the secondary neurons begin to proliferate, AChE is expressed by nearly all embryonic neuronal popula- tions.

INTRODUCTION

Acetylcholinesterase (ACHE), the catabolic en- zyme for the neurotransmitter acetylcholine, is an in-

teresting molecule because it may have several enzy- matic activities and functions during embryonic de- velopment 3°.37. Al though it is found in great abun-

~dance in cholinergic and cholinoreceptive cells, it

also has been detected in cells with no known associa-

tion with this neurotransmitter system. For example,

A C h E activity has been detected transiently in an- eural embryonic systems and in the undifferentiated nervous system (see reviews3°'33'52). In fact, it has

been proposed that there are two acetylcholine sys- tems in embryost6: an early system that may be in-

volved in neuronal growth and maturation, and a late

system that may be involved in cholinergic synapto-

genesis. It follows that early embryonic A C h E activi-

ty may be linked to terminal cell division, cell growth processes and early morphological differentiation 3°,

rather than specifically to neurotransmitter catabo- lism.

We have studied the development of A C h E ex- pression in frog embryos because their nervous sys-

tem develops in what may be considered a two-step

process, possibly reflecting the early and late acetyl-

choline systems mentioned above. After neurula- tion, a primitive circuitry comprised of the so-called primary neurons matures to control escape-swim-

ming and other early embryonic functions that are necessary prior to metamorphosis 9'2t'44. A few days

later, coincident with the onset of feeding and contin-

Correspondence: S.A. Moody, Department of Anatomy and Cell Biology, Box 439, Medical Center, University of Virginia, Char- lottesville, VA 22908, U.S.A.

0165-3806/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

226

uous-swimming, secondary neuronal populations are born that will remain through adulthood. We have found that AChE activity becomes detectable in all primary neurons at the time of their morphological differentiation, often during the time of initial axon outgrowth. At the later developmental stages when the secondary neurons begin to differentiate, virtual- ly all cells in the CNS express AChE activity. Thus, these two periods of Xenopus development both dis- play the early pattern of AChE activity that is not specifically related to cholinergic neurotransmission.

The pattern of expression in the embryonic neurons is consistent with the hypothesis that AChE activity may be linked to terminal cell divisions and early dif- ferentiation 33.

MATERIALS AND METHODS

Embryos were obtained from matings of adult pairs of Xenopus laevis that were induced by injec- tion of human chorionic gonadotropin (Sigma). Jelly coats were removed at early cleavage stages as pre- viously described 4°. Embryos were raised in Stein- berg's solution at 20 °C until stages 19-46 (ref. 41), and fixed overnight at 4 °C in a solution of 4% para- formaldehyde, 1% CaCl2, and 0.25% dimethyl sul- foxide in phosphate buffer. They were washed in 5% sucrose in a 0.1 M sodium hydrogen maleate buffer for 12 h and in 15% sucrose in the same buffer for 1 h, quickly frozen in OCT medium (Tissue Tek) and se- rially sectioned at 16~m in a cryostat. The tissue sec- tions were incubated on slides according to the methods of Karnovsky and Roots 23 for the histo-

chemical visualization of AChE activity. For e a c h age group 3 control incubations were done simulta- neous to the AChE reaction 42. In order to distinguish between AChE activity and butyrylcholinesterase (BChE) activity, butyrylthiocholine iodide (0.5 mg/ml) instead of acetylthiocholine iodide (0.5 mg/mt) was used as the substrate. To distinguish be- tween cholinesterase activity and non-specific ester- ase activity, eserine (32.4 mg/ml; Sigma) was added to the reaction solution. Finally, a third reaction solu- tion without any substrate was used to check for non- specific staining by the chromagen. All sections from at least two embryos per age group were incubated in each of the 3 control solutions, and all of this tis- sue contained no reaction product (Fig. 1). Thus, the

cholinesterase activity in the developing Xenopu,s embryo reported below specifically was due to ACHE. Similar results for control incubations have been reported in other amphibia <~5~s.

RESULTS

No AChE activity was detected in stage 19 or 20 (early neural tube) embryos (Fig. 2). At stage 21 (early tail bud) faint reaction product was detected near the spinomeduUary junction in primary moto-

neurons, a few neural crest cells dorsal to the spinal cord and a few myotubes (Fig. 3). At stage 22 many more of the cells in these populations contained reac- tion product. For example, most myotubes in the

head somites and rostral trunk somites had diffuse reaction product over the entire cell. In addition, 60% of the embryos had reaction product in a few in- terneurons and Rohon-Beard neurons near the spi- nomedullary junction. A similar pattern of AChE staining occurred at stage 23 (Fig. 4). However, ac- tivity extended rostraily to ventral hindbrain neurons and caudally to the level of the cloaca. The most in- tensely stained cell bodies were those of primary mo- toneurons (Fig. 4). The spatial patterns of AChE ac- tivity at stages 25, 26, and 29/30 were the same as that at stage 23. However, more cells within these popula- tions were stained and the reaction product grew more intense with increasing age. Furthermore, at stage 29/30 the staining of the myotubes became more intense at the myocommata than over the rest of the cell.

At stage 33/34 new populations displayed AChE activity. The trigeminal ganglion (Fig. 5) and the fa- cio-acoustic ganglion (Fig. 6) contained stained cell somas. Reaction product was contained within the hindbrain ventrolateral tracts, some axons of which belong to the trigeminal ganglion cells 19. Ventral di- encephalon and mesencephalon contained a few labeled cells and ventral rhombencephaton con- tained many labeled cells. In the rostral spinal cord all of the primary motoneurons, many, but not all, of the Rohon-Beard neurons and many primary inter- neurons were stained (Fig. 7). The primary interneu- rons were identified by the position of their cell bodiesa4; however, since neither their dendritic nor axonal arbor was clearly defined, we could not dis- tinguish between the various classes of primary inter-

227

Fig. 1. a: stage 46 spinal cord in which butyryltbiocholine was used as the substrate, nt, neural tube; no, notochord; s, somite, x 188. b: stage 46 spinal cord in which eserine was added to the reaction medium, x 172. c: stage 46 spinal cord in which no substrate was added to the reaction medium, x188.

Figs. 2-14. Tissue sections were incubated for the histochemical detection of AChE activity. Fig. 2. Transverse section of a stage 19 trunk region that has no detectable level of AChE activity in any of the tissues. The dark granu- lar coloration is due to melanin granules in many of the cells, e, epidermis; g, gut; no, notochord; nt, neural tube; s, somite, x 175. Fig. 3. Arrowheads point out faintly stained myotubes in a transverse section of a stage 21 embryo. The small dark granules in sur- rounding ~l ls contain melanin. ×700. Fig. 4. Transverse section of a stage 23 rostral spinal cord demonstrating reaction product in the somite (s) and ventral neural tube (nt). Arrows point to faintly stained interneurons and the arrowhead points to an intensely stained primary motoneuron. Granular col- oration in dorsal neural tube, epidermis (e) and gut (g) is due to melanin granules, no, notochord, x205. Fig. 5. In a transverse section many cells in the stage 33/34 trigeminal ganglion (Vg) are stained. Arrows in the hindbrain (hb) point to reaction product in axons in the ventrolateral tracts. Epidermal ceils (e) are colored because they contain melanin granules. × 520. Fig. 6. In a transverse section 4 populations of cells in the stage 35/36 hindbrain (hb) region contain AChE reaction product: myotubes in the head somite (hs), cells in the facio-acoustic ganglion (VIIg), axons in the ventrolateral tracts of the hindbrain (black arrows) and a ventral cluster of neurons lateral to these tracts (open white arrows), e, epidermis; no, notochord; oto, otocyst, x300.

neurons that have b e e n desc r ibed 44. In the caudal

spinal cord all of the p r i m a r y m o t o n e u r o n s and Ro-

h o n - B e a r d neu rons w e r e s ta ined. Labe l l ed axons in

the la teral t racts e x t e n d e d into the pos tc loaca l spinal

cord. A few neura l crest cells w e r e s ta ined faint ly at

these caudal segments . Al l s e g m e n t e d muscu l a tu r e

was in tensely s ta ined; occas iona l ly l abe l led m o t o r

axons w e r e d i sce rned a m o n g the m y o t u b e s (Fig, 7).

The s taining pa t t e rn at s tage 35/36 was s imilar to that

for s tage 33/34 with the add i t ion of m a n y clusters of

228

Fig. 7. Transverse sections of stage 34/35 spinal cord. Note that no cells surrounding the central canal {c) contain reaction product, a: faint AChE staining in a primary interneuron (1), whose axon (arrows) joins the lateral tract (bracket). P, pigment cell. x3fllk b: AChE staining in a Rohon-Beard neuron (R), primary interneurons (arrows), and a primary motoneuron (M). P, pigment cell. ×30ft. c: arrowheads point to motor axons growing into the adjacent somite, the cell bodies of which are in a rostral section, n. notochord. x250. Fig. 8. Transverse section of a stage 41 spinal cord demonstrating heavy staining in an extramedullary neuron (E), Rohon-Beard neu- ron (R), several primary interneurons (arrows) and primary motoneuron (M). The heavy deposition dorsal and lateral to the spinal cord (P) is composed of melanocytes. Note that the cells surrounding the central canal (c) do not contain reaction producl, x 500. Fig. 9. Transverse section of the depressor mandibulae muscle in a stage 46 embryo. Note the punctate staining along the length of the myotubes and the intense band of staining at their ends. x200. Fig. 10, Transverse section of a stage 46 diencephalon. Staining occurs in nearly all cells in the intermediate zone (i), in many fibers in the marginal zone (m) and in the optic nerve (on). Note that cells surrounding the third ventricle (III), i.e. those in the ventricular zone (v), are not stained. P, melanocytes surrounding the brain, x210. Fig. 11. Transverse section of a stage 46 rhombencephalon. Staining occurs in nearly all cells in the intermediate zone (i) and in many fibers in the marginal zone (m). Note that the cells surrounding the fourth ventricle (IV), i.e. those in the ventricular zone (v) do not contain any reaction product, Arrows point to an intensely stained tract deep to the optic tectum (tee). P, melanocytes surrounding the brain, x260. Fig. 12. Transverse section of the retina of a stage 46 embryo. Note that most of the cells in the inner nuclear (in) and the ganglion cell (g) layers, and many fibers in the inner lip) and outer lop; arrows) plexiform layers are stained, r, photoreceptors; P, pigmented reti- na; 1, lens. x 190. Inset: note the stained optic nerve axons (on). x 140. Fig. 13. Transverse section of a stage 46 spinal cord demonstrating that all cells except those surrounding the central canal (c) contain reaction product. E, e×trameduUary neuron and its labeled axon (arrow); M, primary motoneurons; P, melanocytes surrounding the spinal cord; R, Rohon-Beard neuron, x380. Fig. 14. Transverse section of a stage 46 spinal cord demonstrating an intensely stained Rohon-Beard axon (arrow) coursing between the spinal cord and the somite (So). Note the characteristic loop in the Rohon-Beard axon at the tip of the arrow. P, melanocyte; c. central canal, x450.

229

stained cells in the ventral rhombencephalon (Fig. 6) and of stained extramedullary neurons next to the spinal cord.

At stage 41 the intensity of staining greatly in- creased in the above mentioned populations. It was especially heavy in the ventral roots and myocomma- ta of the myotomes. Many cells outside of the ventric- ular zone in the brain and spinal cord displayed AChE activity (Fig. 8). In half of the embryos there was faint staining in both plexiform layers of the reti- na. At stage 46 the intensity of staining further in- creased. All of the jaw and extraocular muscles were stained intensely in patches and in bands at their tips (Fig. 9). Many cells were labelled in the intermediate zone of the ventral and lateral diencephalon (Fig. 10), mesencephalon and rhombencephalon (Fig. 11). Many fibers in both plexiform layers of the retina and in the optic nerve, and many cell somas in the inner nuclear and ganglion cell layers were labelled (Figs. 10 and 12). Many, but not all cells were labeled inten- sely in the cranial ganglia. All spinal cord cells, ex- cept those directly surrounding the central canal, were heavily stained (Fig. 13), and their peripheral axons were prominent (Fig. 14). In one embryo labelled dorsal root ganglion cells were observed.

DISCUSSION

Comparison to other amphibia There are several previous reports of AChE activi-

ty in developing amphibia. Most investigators 3'5'26'34, including ourselves, first detect AChE activity around the time of initial spinal peripheral axon out- growth (stages 21-22 in Xenopus25"5°), although it has been biochemically detected at the end of neuru- lation 18'31. A few reports detected activity in early gastulae 1 or early neurulae 46, but these findings have not been substantiated. While one study 1 reported a sharp drop in AChE activity after stage 24, other studies 18"31'36'43'47 detected a steady increase in bio- chemical activity, continuing through larval stages when the embryo begins to feed and swim continu- ously. In agreement with these latter studies we de- tected a steady increase in the total number of cells and in the types of cells that expressed AChE activity through the tail bud and larval stages.

We did not detect any BChE activity in Xenopus embryos using histochemical methods. However ,

Atherton and Lee 1 reported BChE biochemical ac- tivity between stages 11 (gastrulation) and 35 (begin- ning of active swimming); it has been suggested that their data were derived from non-specific esterases rather than from BChE TM. Furthermore, in other em- bryonic 15.18 and adult 49 amphibia BChE appears to be very low in abundance or not detectable. In con- trast, BChE activity is commonly detected in the de- veloping brain of other vertebrates 52'53, and it seems

.. to be confined primarily to glia 3° and their precursors in the ventricular zone 24"32"33.

The time of AChE expression and the specific cell populations in which this activity is localized differ somewhat between our study and a few previous ones. For example, we first observed AChE activity in both plexiform layers of the retina at stage 41, whereas previous authors 36 detected faint histochem- ical staining in these layers at stages 37/38, about the time of initial axon outgrowth from the retina 2°. Ear- lier reports detected staining only in the plexiform layers 13,36, whereas we additionally observed stain- ing in both the inner nuclear and the ganglion cell lay- ers. In another case, we observed AChE activity in virtually all postmitotic cells in independently feed- ing Xenopus tadpoles (stages 40-46), whereas in in- dependently feeding bullfrog tadpoles, only primary neurons contained AChE activity 15. We found that AChE activity was restricted to primary neurons only at younger stages (tail bud) of Xenopus.

Significance of embryonic AChE expression It is well known that the activity of AChE changes

during the development of the nervous system 3°'52. There are many examples of neuronal populations that express AChE activity during development that are neither cholinergic, cholinoreceptive nor ACHE- reactive in the adult 3°. It has been proposed that an early acetylcholine system may be expressed by all neurons, be involved in growth processes and early differentiation, and disappear with synaptic matura- tion, while a later acetylcholine system may appear at the onset of synaptogenesis only in cholinergic and cholinoreceptive neurons 16. In support of this idea, AChE activity has been reported during non-neural organogenesis, in morphologically undifferentiated cells that will become neuroblasts, in early neuro- blasts shortly after terminal mitotic division, in mito- tically blocked neuroblastoma cells, and in proliferat-

230

ing and migrating cells in the developing nervous sys- tem14.24,3o,33.48.

The primary nervous system of the frog is a func- tionally complete, yet transient embryonic nervous system. During the embryonic period it is solely re- sponsible for the nervous activity of the embryo, i.e. escape swimming behavior. It consists of Rohon- Beard neurons, extramedullary neurons, primary motoneurons, trigeminal and facio-acoustic ganglion cells, ventral plate neurons in the hindbrain and pri- mary spinal interneurons 9'2~'4~. We were interested

in whether the early or later acetyicholine system ~c' would be exhibited by the primary neurons because they are functionally mature, yet are found within a very immature nervous system. These neurons all ex-

pressed AChE activity, but the onset of expression differed among cell types and did not correlate with any specific aspect of their growth or differentiation. Unlike mammalian 5~ and avian 3238'39 neurons,

AChE histochemical activity in Xenopus was not temporally coordinated with terminal mitosis in all of the primary neurons; many primary neurons become postmitotic during the late stages of gastrulation 2~, many hours before AChE activity was detected. In some primary neurons (motoneurons and Rohon- Beard neurons) the onset of AChE activity occurred near the onset of the outgrowth of their a x o n s 22"25'5°.

However, for other primary neurons its onset was much later than the time of their axons' outgrowth. For example, trigeminal ganglion cells and extrame- dullary cells sprout peripheral axons at stage 24 (refs. 12, 50), and yet were not AChE-positive until a day later (stages 34-35). Possibly all of the primary neu- rons express AChE activity during the same phase of their differentiation program, and either we were un- able to detect it with our histochemical procedures, or to monitor the appropriate developmental event. In general, all primary neurons showed AChE reac- tivity at the time when their characteristic morpholo- gy was distinct. Thus, the pattern of AChE expres- sion in the primary neurons was most similar to the pattern of the early acetylcholine system ~6, in that all of these cells were AChE-positive. But, the onset of AChE activity was not uniformly correlated with the earliest phases of primary neuron growth and matu- ration.

The pattern of AChE expression in primary neu- rons also did not correlate with eholinergic neuro-

transmission. The probable neurotransmitters of pri- mary neurons have been reported: some dorsal con> missural primary interneurons contain glycine ~, sev- eral primary interneurons contain GABA 45, many

Rohon-Beard neurons, some primary interneurons and some cranial ganglion cells contain substance pS.17 Rohon-Beard neurons respond to GABA and

~glycine 2 and Rohon-Beard neurons and some pri-

mary interneurons have high-affinity GABA uptake systems 29. In fact, the primary motoneurons are the only likely cholinergic candidates. We have at-

tempted to demonstrate this point using antibodies against choline acetyltransferase, but have not been successful in identifying an antibody that will cross-

react with embryonic frog tissue. It is possible that the functional role of AChE in primary neurons is not related to acetylcholine catabolism since both AChE and BChE have the ability to hydrolyse peptide neurotransmitters such as substance P and enkepha- lin 6'7'35. Furthermore, there are many molecular

forms of AChE 27'37'53 that are not expressed concom-

itantly 31, and therefore, the embryonic function of

AChE probably is not restricted to its AChE activ- ity.

Between stages 39 and 41 the Xenopus embryo be- gins to feed, to constantly swim (i.e. not just in re- sponse to tactile stimulation), to use its gill structures and to use its visual and lateral line systems. This de- velopment of the secondary nervous system is a pre- lude to metamorphosis and produces many of the neurons present in the adult brain. Although we did not follow the pattern of AChE activity to metamor- phosis and adulthood, the pattern of enzyme activity observed during the beginnings of this period, name- ly stages 41 and 46, is very similar to the pattern of the early acetylcholine system 16. Staining was pres- ent in nearly all postmitotic cells (judged so because they were not located in the ventricular zones of the brain nor surrounding the central canal of the spinal cord), only some of which were morphologically dif- ferentiated (see Figs. 13 and 14). Thus, these neu- rons probably begin to show AChE activity shortly after their terminal mitotic division, and maintain it during their growth and maturation phases. Further- more, at these later stages there was no correlation between AChE activity and the time of initial axon outgrowth, which may occur for many hours after ini- tial AChE staining. Thus, the secondary embryonic

231.

nervous system of Xenopus displays the typical pat-

tern of the early acetylcholine system 16.

In our study, myotubes and motoneurons of the

same segments expressed A C h E at the same stages,

suggesting that an interaction between the two, pos-

sibly dependent upon axon contact, may influence

A C h E expression. In fact, growth cone contact with

muscle membranes causes a persistent release of ace-

tylcholine 54. However, several studies have demon-

strated that A C h E activity can develop at the neuro-

muscular junct ion in the absence of activity 1°'31 and

without prior axonal contact 31. Thus, if there is a con-

tact-mediated expression of A C h E in myotubes, it is

not an obligatory one.

ACKNOWLEDGEMENTS

We would like to thank Mrs. Kathryn Kersey for

her photographic assistance. This work was sup-

ported by NINCDS Grants NS20604 and NS23158

and March of Dimes Basil O ' C o n n o r Grant 5-527.

S.A.M. is a Sloan Foundat ion Research Fellow.

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