Development of the nervous system
217
Immunohistochemical study on the differentiation of cranial sensory
and autonomic ganglia in the rat
C. Ayer-Le Lievre and A. Seiger, Department of Histology, Karolinska Institutet, S-10401 Stockholm,
Sweden
The differentiation of cranial peripheral ganglia was studied in the rat using anti-substance P (SP) and
anti-neurofilament (NF) antisera. NF immunoreactivity, as well as silver impregnation, appear first in
cranial sensory ganglia in 2-day-old rat fetuses. At this stage, only a perinuclear ring of reactive material is
visible in some neurons while numerous brightly fluorescent fibres are present in cranial nerves. By day 16 of
gestation the first SP-immunoreactive (SPLI) cell bodies appear in cranial sensory and parasympathetic
ganglia. At this stage the amount of NF-immunoreactive material clearly increases in some cell bodies of the
trigeminal, petrous, nodose and cervical dorsal root ganglia. At day 17 of gestation the NF immunoreaction
in sympathetic (superior cervical) and parasympathetic (sphenopalatine, otic, submaxillary, lingual and
enteric) ganglia is reduced to a thin perinuclear ring in some cell bodies.
In later prenatal and in postnatal stages there is no marked change in the distribution of NF
immunoreactivity in peripheral cranial ganglia. On the contrary there is a marked increase of SPLI in
sensory and parasympathetic ganglia between days 16 and 21 of gestation. The number of positive cells
becomes higher, the amount and morphology of the immunoreactive material also varies. After birth, fewer
cells are SPLI in sensory ganglia and SPLI cannot be demonstrated in parasympathetic ganglion cells, in
normal conditions. However the presence of SPLI in parasympathetic neurons after birth can be proved in
experimental conditions.
In conclusion, the appearance of SPLI in peripheral neurons (unlike central neurons) in the rat takes
place 5 days after that of the first histochemical sign of neural differentiation in the ganglia, i.e., the
acquisition of NF. 16 days of gestation appears as a critical stage in the differentiation of peripheral sensory
and autonomic neurons regarding both criteria used here, SPLI and NF immunoreactivity. At birth a second
significant change takes place in intraneuronal distribution of SPLI, related to functional maturation,
whereas NF immunoreactivity does not seem to vary.
The authors thank Dr D. Dahl and Dr I. Black, Dr. J. Kessler for kindly providing the NF antiserum and
the SP antiserum respectively.
This work was supported by the Swedish MRC and the French CNRS.
Pattern formation in the embryonic nervous systems of grasshoppers and
flies
Michael Bate*, Department of Zoology, Downing Street, Cambridge CB2 3EJ
We would like to understand the mechanisms which underlie the oriented growth of nerve fibres to form
patterns in embryonic nervous systems. Growth cones appear to respond to a hierarchy of adhesive cues: the
preferred substrate for neural growth is an existing nerve, so that the first pioneer axons are crucial
determinants of a pattern of nerves which aggregates about the paths they form. The first axons in the
pattern are guided by cues in the epithelium over which they grow. In the grasshopper, the precise shapes of
individual neurons unfold as the growth cones of differentiating neurons choose between, and grow along
alternative pioneer pathways in the developing central nervous system. Each cell executes a predictable
sequence of choices and turns which lays out a characteristic axon shape. We now find that the embryonic
nervous system of Drosophila is a miniature replica of the grasshopper: the same neurons can be identified
in both making the same choices and turns within an identical framework of pathways, with the implication
that the guidance mechanisms can now be studied with genetic and molecular methods which are unique to
the fly. Also in Drosophila we have studied the formation of adult nerve pathways in the imaginal discs. In
the leg disc, larval nerves act as guides for growing axons. In the antennal disc there are no such larval
pathways. Homoeotic transformations of antennal discs into leg discs allow us to assess the significance of
cues in the disc epithelium for the guidance of growing adult axons.
218
Development of the nervous system
Expression of neural antigens by Kupffer cells
Hilary P. Benton \ Michael R. Hartley 2, Amanda J. Suitters 1, Graham P. Wilkin 2.1Departmentof
Histopathology, R.P.M.S. Du CaneRd, London W12 0HS. 2 Department of Biochemistry, Imperial College,
London SW72AZ
Kuppfer cells, the resident macrophages of the liver, inducibly express the immune accessory marker la
antigen. Recently this characteristic has also been shown in central nervous system astroglia, prompting us
to investigate more closely the immunochemical similarities between these cell types. We have shown, in
three different experimental systems, that Kupffer cells express several markers previously thought to be
unique to astrocytes.
1) In primary cultures of adult rat liver cells, Kupffer cells can be identified by their characteristic process
bearing morphology and can be stained for the intermediate filament, glial fibnllary acidic protein (GFAB)
and the glial specific isoenzymes, arar-enolase and aldolase c. Moreover these cells co-ordinately express
vimentin and GFAB, a combination of intermediate filaments previously only described in astrocytes.
2} In fixed sections of normal rat liver the same markers are expressed.
3) In an experimental model of graft versus host disease, where la antigen expression is fully induced on
susceptible cells, these markers can be found on la positive process bearing cells of rat liver sections.
These observations point to the possibility that Kupffer cells and astrocytes may be tissue specific subtypes
of the same population of cells.
Morphogenetic aspects of the relation of the left vagus nerve and the
stomach in the rat
A. B. Boekelaar and J. Bloot, Department of Anatomy and Embryology of the University of Amsterdam,
Meibergdreefl5,1105 AZ Amsterdam, The Netherlands
The growth of the vagus nerve and the stomach - especially their topographical relation - has been
studied in the rat during development.
The stomachs of rats of 15 days of gestation until 1 day after birth have been used. At least 10 specimens
of either stage were examined. The stomachs were stained according to the AChE in toto technique
described by Baljet and Drukker (1975). This technique made it possible to examine the organ in toto under
the dissecting microscope. So we were able to ascertain the three dimensional relation of the nerve with its
target organ.
The results obtained can be summarized as follows. On day 15 the branches of the left vagus nerve
towards the stomach reach as far as the greater curvature of the antrum and the corpus. From day 15 until
day 17 this relation does not alter very much. From day 17 the stomach grows faster than the vagus nerve
and its branches. Consequently the gastric branches in the neonatal rat are then restricted to an area of the
antrum and the corpus half-way the lesser and greater curvature. This pattern does not differ essentially
from the situation in the adult rat as described earlier (Boekelaar, 1983).
BALJET, B. & DRUKKER, J. (1975). Stain Technol. 50, 31-36.
BOEKELAAR, A. B. (1983). Verh. Anat. Ges. 77, 347-348.
Development of the nervous system
219
Selection of pathways by regenerating and developing spinal cord axons
5. M. Bunt*, F. G. InglisandP. F. Moebs, Department of Anatomy, University of Dundee,
Dundee DD1 4HN
While there has been much investigation of the selection of pathways and termination sites by fibres
growing from the CNS to the periphery (e.g. growth of fibres from ventral spinal roots to the muscles) or
from the periphery to the CNS. (e.g. from the retina to the optic tectum), there has been little investigation
of the growth of fibres within the CNS. We have traced the pathways taken by fibres regenerating along the
goldfish spinal cord and fibres growing down and through a rotated section of the developing spinal cord in
Xenopus laevis tadpoles.
Six weeks to six months after a total transaction of the juvenile (7 cm) golkdfish spinal cord at the level of
the caudal edge of the dorsal fin, horse-radish peroxidase (HRP) was applied to a second partial cut made
either 1 cm above, or below, the site of the previous operation. Serial sectioning of operated animals
verified a total transection. Other control studies showed that the HRP could not cross a transection by
diffusion or intravenous transport. Histological processing of the sectioned brain with the chromogens DAB
or TMB revealed the pathways taken by the fibres filled with the HRP. At the site of the transection the
fibres were seen to take tortuous and abnormal routes through the scar tissue, however on each side of the
scar tissue the regenerating fibres re-entered the appropriate funiculi of the cord. Other identifiable groups
of fibres did not regenerate across the scar tissue.
In Xenopus laevis embryos (stage 22) we have rotated a section of the neural tube about its vertical axis so
that the originally rostral part of the graft was replaced caudally, thusfibresgrowing down the left side of the
tube were faced with the right side of the rotated graft. E. M. studies showed that no more than 10fibresare
present in the neural tube at this stage. One week to three months later the spinal cord was injected with
HRP. In contrast to the regenerating fibres in the fish we have not observed any disturbance of the fibre
pathways following a simple cut of the neural tube. However, in the animals with a rotated spinal cord, we
have observed a crossing over of fibres in the ventral funiculi of the cord at both the rostral and caudal ends
of the transplant indicating that these fibres can detect the rotation of the graft and select their pathways in
the distal spinal cord.
An x-ray microanalytical study of the postnatal development of otoconia
F. J. Canizares, P. V. Crespo, F. Revelles and A. Campos, Departamento de Histologia y Embryiologia
General, Facultad de Medicina de Granada, Spain
In the present work the morphostructural and microanalytical characteristics of otoconia in the maccula
during postnatal development are studied.
A total of 16 Wistar rats were divided into two groups of eight animals such representing two different
stages of postnatal development: 10 and 15 days.
In this period of development a process of association was observed between different types of otoconia,
possibly suggestive of existence of complementary interrelationships which may ultimately lead to the
formation of structural units of a higher order. In the smaller Type I otoconia, flattened surface areas were
observed not infrequently which corresponded to zones of adhesion between otoconia of the same or of a
different type. The larger Type II and Type III otoconia were generally associated with otoconia of the same
type.
An especially interesting observation was the presence of characteristically patterned associations which
most frequently distinguished the adhesions between Type III otoconia: their flattened distal faces were
often joined, forming groups generally consisting of three units. Each otoconia in these units conserves its
respective double edges in the zone of contact, which consequently showed a typical Y configuration. These
groups of three otoconia present a characteristic 'cats paw' configuration.
Postnatal otoconia were also studied microanalytically with X-ray dispersion in order to analyze units of
identical volume. Calcium content increased along with otoconial size. Microanalysis also confirmed that
centrally-located otoconia contained higher levels of calcium than in otoconia of the same type in peripheral
regions of the maccula.
In conclusion, these microanalytical findings as well as the process of association between otoconia could
be related to morphogenesis and metabolism of these structures.
220
Development of the nervous system
The role of the cerebrospinal fluid during embryonic development. A
biochemical study
Checiul. 1, Oltea Prelipceanu 1, O. Popescu 2.1Laboratory ofEmbryology, Center of Hygiene and Public
Health, 1900 Timisoara, Romania. 2Department ofCell Biology, Institute of Medicine and Pharmacy,
Cluj-Napoca, Romania
The total protein content and the protein fractions were determined in the blood serum and in the
cerebrospinal fluid (CSF) of chick embryos and foetuses between the 4th and 20th day of incubation and of
2-day-old hatched chickens (by Lowry's method and by polyacrylamide gel electrophoresis respectively).
The main results were as follows:
1. The total protein content of the CSF increases from 1-8 mg/ml on day 4 to 3-1 mg/ml on day 10. After
a slight decrease to 2-6 mg/ml on day 12, no further changes are detected. In the blood serum, the total
protein content was 4-3 mg/ml on day 4 and 4-7 mg/ml on day 9. Beginning with day 10 it increases
constantly to 23-2 mg/ml on day 2 after hatching (daily rate 1-1-2 mg/ml). The result is that the 'protein
barrier' between blood and CSF begins to function at 10-12 days of incubation.
2. From the protein fractions determined, the light fraction I in the CSF represents 50 % of the similar
blood serum fraction (on day 10). As to the heavy fractions XI-XIV, their concentration in the CSF
represents, beginning with day 10, 51 % of the similar blood serum fraction (until the end of the period
investigated).
The result is that the 'protein barrier' has an active, selective character. Some comparative aspects with
literature data are discussed.
The early development of the olfactory nerve in Discoglossus pictus
(Amphibia, Anura)
P. Clairambault and C. Pairault, Neuroembryologie, Universite" Paris VII, 2 Place Jussieu, Paris Cedex 5,
France
Previous work (Clairambault, 1976) has shown that the olfactory nerve in Discoglossus induces
telencephalon differentiation which occurs at stage 26 (according to Gallien and Houillon, 1951), but it is
noteworthy that this induction begins as early as stage 23.
To investigate the modalities of the olfactory induction, we have undertaken a preliminary study of the
growth of the olfactory fibres from stage 23 to stage 26, using electronic microscopy.
At stage 23, a few bundles of olfactory fibres leave the olfactory placode and reach the peripheral part of
the anlage of the bulk, where numerous filopods have been seen. These bundles are surrounded by glial
processes and a basal lamina.
As early as stage 24, synaptic contacts have been observed in the bulb, between axonal and dendritic
profiles.
Olfactory glomeruli (i.e. the typical bulbar differentiation) become obvious from stage 26; in addition, the
perikaryons of the mitral cells show some axo-dendritic synapses.
These results led us to conclude that, even though the actual differentiation of the bulb does not take
place before stage 26, some of the olfactory fibres may have synaptic contacts with bulbar cells prior to stage
23.
Development of the nervous system
221
Developmental synchrony of innervation, cutaneous field production of
NGF and neuronal responsiveness
Alun M. Davies and Andrew G. S. Lumsden, Departments of Anatomy, St. George's and Guy's Hospital
Medical Schools, London
Only a proportion of neurons survive beyond the stage of innervating their terminal fields. It has been
suggested that for certain peripheral neurons nerve growth factor (NGF), transported from the peripheral
terminal-field by axonal flow, mediates survival during this critical phase of development (Theonen et al.,
1981). To determine the relationship of peripheral terminal-field encounter to NGF production and
responsiveness we have studied the mouse trigeminal ganglion and its cutaneous field at closely staged
intervals throughout development. Peripheral fibres initially contacted the epithelium of the mandibular
process by E10-5 and the maxillary process by E l l . This coincided with the stage in vitro during which the
magnitude of neurite outgrowth from ganglion explants was significantly increased by NGF (Davies and
Lumsden, 1984). NGF responsiveness persisted throughout and just beyond the period of natural neuronal
death (E13 to E19).
Neurite outgrowth elicited from E10 and E l l ganglia by co-cultured maxillary process was not affected by
antiserum to isogeneric NGF. Neurite outrgrowth in E12 co-cultures, however, was virtually abolished by
anti-NGF (Lumsden & Davies, 1983).
The synchrony of peripheral innervation and NGF responsiveness together with the demonstration of
NGF production by presumptive cutaneous tissue at an appropriate stage of development substantiate the
hypothesis the NGF acts as a selective maintenance factor during sensory neurogenesis.
THOENEN, H., BARDE, Y. A. & EDGAR, D. (1981). The role of nerve growth factor (NGF) and related
factors for the survival of peripheral neurons. In Neurosecretion and Brain Peptides (ed. J. M. Martin, S.
Reichlin and K. L. Bick), pp. 263-273, New York: Raven Press.
DAVIES, A. M. & LUMSDEN, A. G. S. (1984). Relation of target encounter and neuronal death to nerve
growth factor responsiveness in the developing mouse trigeminal ganglion. /. Comp. Neurol. 223,
124-137.
LUMSDEN, A. G. S. & Davies, A.M. (1983). Earliest sensory nervefibresare guided to peripheral targets by
attractants other than nerve growth factor. Nature 306, 786-788.
Differing responses of neural crest and placode derived cranial sensory
neurons to nerve growth factor (NGF)
AlunM. Davies 1 and Ronald M. Lindsay 2.]Department of Anatomy, St. George's Hospital Medical
School, Cranmer Terrace, Tooting, London SW 17 ORE. 2Unit of Neurobiology, National Institute for
Medical Research, Mill Hill, London
We have undertaken a comprehensive in vitro study of all cranial sensory ganglia from E4 to E16 chick
embryos to ascertain whether NGF responsiveness is related to neuronal ontogeny
NGF elicited pronounced neurite outgrowth from neural crest derived explants; the dorso-medial part of
the trigeminal ganglion and the superior ganglion of cranial nerves DC and X. This response was first evident
at E6, reached a maximum between E8 and E l l and gradually declined through E16. Explants in which the
neurons were of placodal origin varied in their response to NGF. Neurite outgrowth from explants of the
ventro-lateral part of the trigeminal ganglion and vestibular ganglion was negligible and uninfluenced by
NGF. The geniculate, petrosal and nodose ganglia exhibited an early moderate response to NGF which was
first evident by E5, reached a maximum by E6 and declined rapidly through later ages. In dissociated
neuron-enriched cultures at E6 and E9, however, only neural crest derived neurons survived and grew in the
presence of NGF.
We conclude that of sensory neurons only those derived from neural crest require NGF during
development whereas those of placode origin do not. We suggest that the NGF-promoted neurite outgrowth
from early geniculate, petrosal and nodose explants emanates from neural crest derived cells which survive
or differentiate within explants under these particular culture conditions.
222
Development of the nervous system
Functionally distinct sensory neurons have different trophic
requirements during development
Alun M. Davies 1 and Ronald M. Lindsay 2. ^Department of Anatomy, St. George's Hospital Medical
School, Cranmer Terrace, Tooting, London SW 17 ORE. 2Unit of Neurobiology, National Institute for
Medical Research, Mill Hill, London
Almost all studies of the trophic requirements of developing sensory neurons have been confined to dorsal
root ganglia (DRG). The survival and growth of embryonic DRG neurons is promoted by nerve growth
factor (NGF) and a number of other neurotrophic agents (reviewed by Barde et al., 1983). These ganglia,
however, consist of a functionally heterogeneous collection of neurons which innervate a variety of
cutaneous and visceral receptors and proprioceptors. In the absence of specific markers for these
functionally different types of sensory neurons it is not possible to determine whether a relationship exists
between the innervation target of sensory neurons and specific neurotrophic factor requirement.
To investigate this possibility we have compared the neurotrophic requirements of embryonic chick DRG
with the dorso-medial part of the trigeminal ganglion (DM-TG). The DM-TG like DRG is neural crest
derived but unlike DRG has a mainly cutaneous distribution. Over the age range studied (E6 to Ell), NGF
promoted survival and pronounced neurite outgrowth from explants and dissociated neuron-enriched
cultures of both DRG and DM-TG. Whilst extracts of chick eye, liver and spinal cord also elicited a marked
response from E8 and older DRG neurons, DM-TG neurons were almost entirely unresponsive to the
neurotrohic activity of these extracts. We suggest that this difference is a consequence of the more restricted
sensory distribution of DM-TG neurons.
Y. A.,
601-612.
BARDE,
EDGAR,
D. &
THOENEN,
H. (1983). New neurotrophic factors. Ann. Rev. Physiol. 45,
Synthesis and cloning of complementary DNA to polyadenylated RNA
from chick embryo spinal cord: identification of tissue specific
sequences
J. G. Dickson, S. E. Coulson, J. Kenimer and F. S. Walsh, Institute of Neurology, Queen Square,
London WC1N3BG
In order to study expression and regulation of genes involved in the development of the vertebrate spinal
cord we have constructed a cDNA plasmid library representing expressed sequences of 7-day embryonic
chick spinal cord. Double-stranded cDNA synthesized from spinal cord poly(A)+ RNA was annealed into
the Pst-1 site of PBR322 using dG - dC homopolymeric tails and annealed vector-cDNA then used to
transform competent E. Coli HB101 cells (4xlO4 transformants per fig DNA). To identify sequences
exhibiting tissue-specific expression patterns the library
was screened by colony hybridization of 1)
32
P-labelled cDNA probes synthesized from poly(A)+ RNA of spinal
cord and liver tissues, and 2) spinal
cord cDNA depleted of sequences complementary to liver poly(A)+ RNA (cDNA difference probes). For
screening with spinal cord versus liver cDNA probes (5x10 dpm per fig), randomly selected colonies were
replicated in ordered arrays and duplicated nitrocellulose filters processed for colony hybridization. In the
case of difference probe analyses, P-labelled spinal cord cDNA was hybridized with excess liver poly(A)+
RNA to a Cot value of 2000, and cDNA-RNA hybrids removed by hydroxyapatite chromatography. The
single-stranded fraction representing sequences unique to spinal cord tissue was then used to probe the
spinal cord cDNA library. Some 3-5 % of clones in the library were found to represent sequences expressed
in spinal cord, but not detectable in liver tissue. Plasmid DNA isolated from 10 putative spinal-cord-specific
clones was restricted with Pst-1 endonuclease and subjected to agarose gel electrophoresis. All plasmids
were linearized by Pst-1 digestion and 5 had excisable cDNA inserts of >300 bp. Thus we+have identified
expressed sequences of 7-day chick embryo spinal cord which are not present in poly(A) RNA of liver
tissue. Further studies are required to determine the molecular nature and sequence of corresponding RNA
species. In addition to studies on tissue-specific gene expression, the approach described here may be
applied to analysis of cellular subpopulations from spinal cord e.g. motorneurones, for which suitable bulk
separation methods exist.
Development of the nervous system
223
Amphibian neuronal precursor cells express neurofilament proteins
D. Paulin 2, F. Foulquier 1 and P. Kan 1.1ERA~CNRS327, Laboratoire de Biologie, University P. Sabatier,
31062 Toulouse-Cedex, France, institute Pasteur, 25 R. Dr. Roux, Paris, France
Neurofilaments, the intermediate-sized filaments of neurons (NIF) are composed of three polypeptides of
different molecular weights (200 Kda, 160 Kda and 70 Kda). Described in mammals and birds, are these
neurofilament components present in amphibian neuronal cells and at what developmental stage do they
appear?
Our findings are based on immunofluorescent localization of NIF proteins using two previously
characterized antisera against 200 Kda and 70 Kda components (1). Embryonic undifferentiated neuronal
cells from P. waltl neural primordium: neural plate+ neural fold (early neurula stage) are cultured isolated
in vitro (2). They differentiate progressively and exhibit phenotypical and biochemical characteristics of
neurons (3).
At the beginning of the culture none of the undifferentiated neuronal precursors bind antibodies against
the 200 Kda or 70 Kda proteins. This binding is detected when morphological differentiation takes place (2/3
day cultures). Both the cell bodies and the processes were stained. After 2-3 weeks, immunostaining of the
neurons was very distinctive and bright. The non-neuronal cultured cells do not exhibit any specific labelling
with these two NIF antibodies.
These observations indicate the presence of NIF in amphibian neurons and the early acquisition of NIF
expression by neuronal precursor cells (late gastrula stage).
(1) PROCHIANTZ, A., DELACOURTE, A., DAGUET, M. C , PAULIN, D. (1982). Exptl. Cell Res, 139, 404-410.
(2) DUPRAT, A. M., ZALTA, J. P., BEETSCHEN, J. C. (1966). Exptl. Cell Res., 43, 358-366.
(3) DUPRAT, A. M., GUALANDRIS, L., KAN, P., FOULQUIER, F. (1984). in The role of cell interactions in early
neurogenesis, Plenum Publ. Co.
Soluble, muscle-specific trophic activity enhances choline
acetyltransferase levels in cultures of enriched spinal motoneurons
T. P. Flanigan*, J. G. Dickson andF. S. Walsh, Institute of Neurology, Queen Square, London WC1N3BG
Recent evidence from in vitro studies suggests that skeletal muscle may supply soluble retrogradely-acting
factors which influence the survival and development of spinal motorneurons. We have previously described
the survival and cholinergic development in vitro of a motorneuron-enriched cell population from E7 chick
spinal cord. When grown on hydrated collagen gels, these cells extend neurites and continue to express
choline acetyl-transferase activity in the absence of exogenous sources of trophic influence. In addition,
since these cultures are virtually devoid of non-neuronal components, this in vitro cholinergic expression
occurs in the apparent absence of endogenous feeder cell activity.
To examine the possible existence of retrograde, soluble factors exerting a trophic influence on
motorneurons, primary cultures enriched in motorneurons were established on collagen gels in the presence
and absence of soluble extracts from a variety of E12 chick tissues, including skeletal muscle. Cultures were
harvested after 5 days and assayed for CAT activity. In the presence of whole embryo extract (CEE, 2 %)
or a soluble fraction from hind limb muscle (50 fig/ml) cultures exhibited a 2-5-fold increase in CAT level
compared to controls. Soluble extracts from other tissues e.g. skin and liver, did not influence significantly
the level of CAT in the cultures. All tissue extracts, at levels above 200 /u,g/ml, did however exert a
generalised cytotoxicity in vitro.
This study shows that while significant levels of CAT activity are maintained in the motorneuron-enriched
cultures in the apparent absence of any exogenous trophic influence, a soluble factor(s) from skeletal muscle
can produce a further enhancement in the level of the enzyme. It remains as yet to be defined whether this
trophic activity increases the survival of motorneurons or exerts an inductive effect on CAT expression. The
tissue-source specificity of this soluble neurotrophic activity is compatible with a muscle-derived
retrogradely-acting growth factor, and in view of the more generalised cytotoxicity of crude tissue extracts,
it will be of importance to attempt a partial purification of this activity.
224
Development of the nervous system
Origins of the ipsilateral retinal projections in prenatally-enucleated
mice
P. Godementx andJ. Salatin 2.1Laboratoire de Neurobiologie du Diveloppement, University Paris XI,
Orsay, France. 2Laboratoire d'Embryologie du College de France et du CNRS, Nogent-sur-Marne, France
The temporal retina is the only retinal sector which sends fibers to the ipsilateral side of the brain in
mammals. It has been shown that destruction of one eye at birth in rodents leads to modifications in the
distribution and number of ipsilaterally-projecting ganglion cells (IGcs) in the remaining eye. The present
experiments were done to study the effects of enucleations done at earlier (prenatal) steps of development
in C57BL/6 pigmented mice.
One optic tract in normal adult mice, and the optic tract ipsilateral to the remaining eye in mice that had
been enucleated at E12, E13, E16, or birth (= E19) was cut and filled with HRP gel. 48 h later the retinas
were reacted for HRP in flat mounts and the location of all IGcs was charted. In some cases also the optic
nerves were cut and stained.
In normal mice a mean of 932 ganglion cells (784-1073) are filled in a narrow region along the inferior
temporal periphery of the retina ipsilateral to the HRP injection, and 10-25 in the rest of the retina. In all
monocular mice, most (76-93 %) IGcs lie within the appropriate temporal crescent of the retina. However
following enucleations at E16 or birth their number is increased: as many as 1700 cells within, and 300
outside of the temporal crescent can occur. In mice enucleated at E12 or at E13 the number of IGcs is in all
cases less than in later-enucleated mice, and is even in many cases less than in normal mice: a mean of 625
cells (349-875) lie in the temporal crescent, and 103 (8-275) outside. In some of these mice (with few IGcs)
the width and shape of the crescent are slightly distorted.
In normal and birth-enucleated mice the density of IGcs is relatively uniform within the temporal
crescent; in mice enucleated at E16, it increases towards the internal border of the crescent. Most ipsilateral
fibers in normal and monocular mice course along the lateral and ventral edges of the optic nerve between
the eye and the chiasm.
These results are consistent with the hypothesis that temporary excessive ipsilateral projections (from
temporal+nasal retina) retract through competition with crossed projections, and show that they are
partially retained in the absence of crossed fibers. The decrease in the uncrossed projections when one eye is
destroyed prior to arrival of optic fibers at the chiasm region (E12, E13) suggested however that the
presence of an early crossed projection is required for full development of the uncrossed projections in the
mouse. Finally our findings indicate that the property of temporal retina to project ipsilaterally is to a large
extent independent of interactions between fibers from each eye at the chiasm or in the visual centers. It
may be due to retinotopic order in the optic nerve.
Uptake and release of [3H] GABA by a growth cone-enriched fraction
from neonatal rat brain
Phillip R. Gordon-Weeks, R. Owen Lockerbie and Brian Pearce, Brain Research Group, The Open
University, Milton Keynes MK76AA
A growth cone enriched fraction was prepared from 5-day-old rat forebrain(l). This fraction has a
sodium-dependent, high affinity uptake system for [3H] GABA with a Km of 4-5 f*M and a Vmax of
59-4 pmol/min/mg. Electron microscopic
autoradiography unequivocally demonstrated the ability of
of isolated growth cones pre-labelled with
isolated
growth cones to take up [3H] GABA. Supervision studies
3
[ H] GABA demonstrated a K (25mM)-induced release ofJ 3 H] GABA which at its3 peak was 100 % above
the pre-stimulus release level. About 50 % of othe K -induced release of [ H] GABA was Ca + +
dependent.
The functional role of uptake and release systems for GABA in growth cones is unknown. They may be
merely precocially acquired before synaptogenesis or, more interestingly, actually play a part in synapse
formation.
(1) GORDON-WEEKS, P. R. & LOCKERBIE, R. O. (1984). Isolation and partial characterisation of neuronal
growth cones from neonatal rat forebrain. Neuroscience, in press.
Development of the nervous system
225
The formation of the axonal pattern in the avian embryonic retina
Willi Halfter*, Max-Planck-lnstitut fur Entwicklungsbiologie, D-7400 Tubingen, West Germany
The mechanisms underlying axonal navigation during embryogenesis are still not understood.
Environmental and intrinsic properties of the neurons as well as mutual interactions between axons are
thought to play a role in the formation of the neural network found in the adult animal. I studied the growth
behavior of axons and the formation of the optic fiber pattern in early retinal morphogenesis. Whole mounts
of embryonic chick, quail and pigeon retinae were examined with silver staining, Golgi impregnation,
scanning and transmission electron microscopy. Area measurements show that the retina as well as the optic
fiber layer increase in size exponentially, but with different rates. Consistently, in none of the species the
retinal fiber layer expands symmetrically. It enlarges with polarities along the dorso-ventral and the
naso-temporal axes. The very first axon-bearing ganglion cells appear at stage 16 in the dorsal and central
retinal portion and grow ventrally to merge at the optic disk. As development proceeds, the retina and the
optic fiber layer expand peripherally and ventrally so that the optic nerve head, starting from an initially
ventral position, comes to lie close to the geometrical center of the retina. From stage 23 on, the optic fiber
layer expands faster in the temporal than in the nasal side. Quantitative measurements on the initial
polarisation of young axonal processes show that the axonal growth is highly directed towards the optic
fissure and the optic nerve head. The growth polarisation is found at the very onset of growth cone
formation and in axons far distant from the next ganglion cells and axons. This excludes fiber-fiber
interaction to be essential for the initial fiber orientation.
ae3Proliferation of embryonic rat sympathetic ganglion cells following
destruction of motoneurons is not due to NCF
A. J. Harris*1 and C. D. McCaig 2 Department of Physiology, University ofOtago, Dunedin, New Zealand.
Present addresses: lLab. Zoologie, University Grenoble I, 38402 St. Martin d'Htres, France, department
Physiology, University Medical School, Edinburgh
Total destruction of embryonic rat motoneurons with beta-bungarotoxin (bBTX) on embryonic day 14
results in increased numbers of thoracic sympathetic ganglion cells and proliferation of axons in peripheral
sympathetic nerve trunks. Destroying half the embryonic motoneurons resulted in a lesser increase;
increasing the number of motoneurons reduced sympathetic neuron and axon numbers to below normal.
These effects are not due to NGF. Cell body and nuclear diameters of sympathetic neurons from treated
embryos were reduced; NGF causes hypertrophy. In controls, 3-5 % of ganglionic ChAT activity was
post-synaptic; in embryos treated chronically with NGF, 5-6 %; and in bBTX-treated 20 %. NGF increased
ganglion cell ChAT to 170 % control and TH to 200 % control; hence the ratio ChAT/TH was little
changed. Ganglion cell ChAT activity in bBTX treated embryos was 295 % control, and when given NGF in
addition, 328 % control. TH activity was not changed by bBTX treatment. The increased ChAT/TH ratio
indicates a partial transformation of transmitter phenotype in sympathetic ganglion cells. NGF and bBTX
both increased sympathetic axon numbers. These effects were additive and embryos given both treatments
had 4x the normal number.
We conclude that if the proliferation and the transformation of transmitter phenotype of sympathetic
neurons which follows destruction of motoneurons is due to a growth factor released from denervated
skeletal muscle this factor is not NGF. It may be a motoneuron growth factor destined to be taken up by
motor nerve terminals and hence normally inaccessible to the autonomic nervous system.
226
Development of the nervous system
A new method isolating actin filaments
Mechthild Harders-Opolka, M.P.I, fur Entwicklungsbiologie, 74 Tubingen, West Germany
The development of neural networks during embryogenesis requires the interaction of growing axons with
their environment. Growth of axons involves assembly and disassembly of actin filaments. This process
appears to be modulated by so called actin associated proteins controlled by external signals. Isolation of
the actin filament system is therefore a prerequisite for the study of this process. I developed a simple
procedure to isolate native actin filaments from embryonic neural tissue. This procedure includes gentle
homogenizing of the embryonic chick brain (E12 to E14) tissue followed by short centrifugation to remove
cell debris. The supernatant was centrifuged through a sucrose gradient (25 to 45 %). The resulting pellet
contains well preserved filaments of different thickness. This was demonstrated by electron microscopy.
Furthermore two dimensional gel electrophoresis resolved actin as the prominent component (85 % of total
protein) and some twenty minor proteins. Some of these proteins were isolated and further characterized.
Their function in filament assembly was tested. Antibodies against native actin filaments were obtained by
injection of the purified filaments into a rabbit. The serum reacted strongly with actin as shown by
immunoblotting.
Expression of proopiomelanocortin-related peptides in the motoneuron
in murine dystrophy
L. W. Haynes and Margaret E. Smith, Department of Physiology, The Medical School, Vincent Drive,
Birmingham B152TJ
. /3-endorphin immunoreactivity has been demonstrated in motoneurons of immature but not of adult rats,
suggesting that its expression undergoes developmental regulation in these cells. Both /J-endorphin and
tf-melanotropin were detected in motor nerve axons in 12- and 25-day-old normal C57BL/6J mice. These
peptides were absent in normal adults, but were present in a proportion of motoneurons in adult dystrophic
mice. A greater proportion of terminals were immunoreactive for both peptides in slow (soleus) than in fast
(EDL) muscles. The presence of pro-proopiomelanocortin mRNA in spinal cords of dystrophic mice was
investigated by dot-blot hybridisation using two cDNA probes encoding parts of its sequence.
The results suggest that, in motoneurons of dystrophic mice, the expression of proopiomelanocortin
peptides, normally a transient event in development, is prolonged into adulthood. The inappropriate
neurotrophic influences of these peptides in the adult dystrophic mouse may contribute to myopathic
symptoms.
Development of the nervous system
227
T. Holstein and C. N. David, Zoologisches Institut der Universitdt, Luisenstrasse 14, D-8000 Munchen 2,
West Germany
Nerve cell differentiation in hydra exhibits a spatial pattern and is due to nerve commitment localized in
head and foot tissues, which express high levels of activation for head and foot formation, respectively. In
body column tissue, nerve cell differentiation is induced at sites of head or foot regeneration due to a
significant increase in levels of activation at these sites. (Venugopal, G. & David, C. N. (1981). Dev.
Biol.83, 353-360). We have now obtained evidence that in addition to a high level of activation, nerve cell
differentiation requires a second morphogenetic signal, suggesting that it involves at least two steps.
Incubation of whole animals (H. attenuata) in crude extracts of hydra tissue (OD280nm 0*25) causes
commitment of stem cells to nerve cell differentiation. Actual differentiation of these cells, however,
requires a second step: either isolation of small segments of the body column or simply placement of cuts in
the body column. Differentiation of committed precursors does not occur in control hydra treated with
extract but not wounded or in control hydra which were wounded but not pretreated with extract. The
committed state is unstable, decaying within 24-36 h following treatment with extract. The wounding effect
is relatively short range: three well-spaced cuts per body column are required to induce differentiation of all
committed precursors in an animal containing 9000 epithelial cells.
Our data indicate that nerve differentiation in hydra requires two morphogenetic signals in sequence: (1)
a high activation level induced by regeneration or treatment with crude extract which causes commitment of
nerve precursors and (2) a signal released by wounding which leads to differentiation of these precursors.
Supported by the Deutsche Forschungsgemeinschaft.
Retinotectal projection: on position-specific properties of cell
membranes and directional cues for growing axons
B. Kern-Veits, S. Thanos and F. Bonhoeffer*, Max-Planck-Institutfur Entwicklungsbiologie,
Spemannstr. 35, D7400 Tubingen, West Germany
Axons mechanically or chemically deflected from their normal route can sense directional cues within the
target organ and can correct their routes correspondingly towards the specific target area.
During the search for guiding cues it has been shown in vitro that certain properties of cell membranes
depend on the position of the cells from which the membranes are derived. Using living growth cones as
tools to investigate cell surfaces it has been established that there are several differences between
membranes of anterior and of posterior tectal cells and that, furthermore, nasal and temporal axons
themselves carry different recognition markers.
Possible connections between position-dependent membrane properties, directional cues for growing
axons, and map formation will be discussed.
228
Development of the nervous system
Segmentation of the peripheral nervous system in the chick embryo
R. J. Keynes* and C. D. Stern, Department of Anatomy, Downing Street, Cambridge
Zinc iodide-osmium tetroxide staining of stage 17 chick embryos reveals that motor and sensory axons
grow from the neural tube region exclusively through the anterior (rostral) half of each successive somite.
180 ° antero-posterior rotation of the neural tube relative to the somites does not alter this relationship,
showing that segmented outgrowth is not due to an intrinsic property of the neural tube. Furthermore, if the
somitic mesoderm is rotated 180 ° about an antero-posterior axis, prior to somite segmentation, axons now
grow through the posterior (original anterior) half of each somite. These experiments show that there must
exist some difference between anterior and posterior cells of the somite, which is undisturbed by rotation
and which determines the position of axon growth.
Neural crest and placode derived sensory neurons have different
neurotrophic requirements: placode neurons are unresponsive to NGF,
lack NGF-receptors but do require trophic factors
R. M. Lindsay* andH. Rohrer, Neurobiology Laboratory, National Institute of Medical Research,
Mill Hill, London NW71AA
Peripheral sensory neurons are derived from either the neural crest or neural placodes. The neurotrophic
requirements of crest-derived dorsal root ganglion (DRG) sensory neurons have been studied extensively,
and it is now well established that DRG neurons have an absolute requirement for nerve growth factor
(NGF) during embryonic development. Whilst the mode of action of NGF remains to be elucidated, it is
clear that NGF activity is mediated through a neuronal cell surface receptor, the NGF-receptor. In marked
contrast to crest-derived sensory neurons, little is known about the trophic requirements, of placode-derived
neurons. To ascertain whether crest and placode-derived sensory neurons have similar or different
neurotrophic requirements, we have carried out a tissue culture study comparing the response of DRG
(crest) and nodose ganglion (NG: placode derived distal ganglion of the Xth cranial nerve) neurons to NGF.
In the chick over the embryonic age range E7-E16 nodose ganglion neurons in either explant or dissociated
neuron enriched culture showed virtually no response to NGF. Over most of the same age range the
majority of DRG neurons respond to NGF. At ages earlier than E7 (E4-E6) NGF elicited a modest fibre
outgrowth from NG explants but did not support the survival of dissociated NG neurons in the absence of
non-neuronal cells or their precursors. In contrast to the lack of effect of NGF over a broad concentration
range, extracts of liver, brain and spinal cord from one-week-post-hatched chicks supported the survival of
up to 50-60 % of NG neurons in dissociated neuron enriched cultures. Over the age range E6-E12 virtually
no NGF-receptors (autoradiographic analyses of cultures treated with radiolabelled NGF:I-I25 NGF) were
detected on any NG neurons surviving in culture with liver extract (in the presence or absence of NGF). At
similar ages 80-95 % of DRG neurons have abundant NGF-receptors whether supported in culture with
NGF or liver extract. These results suggest that placode derived sensory neurons do not respond to NGF,
but do require distinct neurotrophic molecules present in their peripheral and central target tissue.
Development of the nervous system
229
Chemotactic guidance of developing sensory neurites by appropriate
target epithelium
A. G. S. Lumsden* and A. M. Davies, Departments of Anatomy, Guy's and St George's Hospital Medical
Schools, London
When developing trigeminal ganglia, explanted from E10-E11 mouse embryos, are co-cultured with
homologous explants of the superficial rostral part of the maxillary process (an area which later develops
mystacial vibrissae), neurite outgrowth is elicited by and exclusively directed towards the target tissue(l).
Co-cultured explants of an inappropriate cutaneous field (the limb bud) neither elicit nor direct trigeminal
outgrowth. The attraction of developing neurites in this in vitro model appears to be due to a target-derived
chemotactic agent, unrelated to nerve growth factor, which is active during the normal period of initial
peripheral outgrowth from the trigeminal ganglion but which declines in activity or influence following
normal growth cone-target encounter [Ell onward(2)]. We refer to this agent as trigeminal neurotropic
factor (TNF).
We have recently used the same procedure to co-culture early trigeminal ganglia with isolated component
tissue layers of the presumptive vibrissa field after their separation by treatment in the cold with crude
trypsin. Whereas there is no outgrowth towards the mesenchymal moeity, outgrowth towards the epithelium
is as profuse and as oriented as that which grew towards the intact maxillary process in the earlier
experiments.
The emanation of TNF activity from target epithelium indicates that the target itself is capable of
attracting appropriate innervation at a distance ana that nerve fibre outgrowth need not require direction by
any property of the mesenchyme - through which cutaneous nerves normally grow to reach epithelial targets
and which, in other developing and regenerating systems, has been assumed to play a specific guiding
role(3).
(1) LUMSDEN, A. G. S. & DAVIES, A. M. (1983). Earliest sensory nerve fibres are guided to peripheral
targets by attractants other than nerve growth factor. Nature 306, 786-788.
(2) DAVIES, A. M. & LUMSDEN, A. G. S. (1984). Relation of target encounter and neuronal death to nerve
growth factor responsiveness in the developing mouse trigeminal ganglion. /. Comp. Neurol. 223,
124-137.
(3) HONIG, M. G. (1982). The development of sensory projection patterns in embryonic chick hind limb. /.
Physiol. 330, 175-202.
PURVES, D. (1982). Guidance of axons during development and after nerve injury. In Repair and
Regeneration of the Nervous System (ed. J. Nicholls), pp. 107-125, Berlin: Springer.
Histotypic reorganization of cultured nerve cells
/. Maher l and D. James 2 . l Unite de neuroimmunologie, Faculte de Midicine, Angers 49045, France.
2
University College London, London WC1
Cells dissociated from various regions of the brain from embryos of mouse or chick were reported to
reaggregate and reconstruct into patterns morphologically similar to the regions from which they were
derived (Delong, 1970; Moscona, 1979).
We dissociated spinal cord cells from 7-day-old chick embryos, according to the method of Bird and
James (1973). Cells were plated out on polylysine coated glass cover slips. Cultures were observed by time
lapse cinematography for periods up to three days during the first week in vitro. During the same period,
cultures were fixed and stained for light and electron microscopic study.
Cinematographic observations demonstrated that cells began at once to reaggregate. Reaggregates
moved and coalesced forming larger ones. Coalescing reaggregates were always interconnected with neurite
processes. Semithin sections showed that many reaggregates consisted of a cortical layer of loosely arranged
cells, and a medullary region that was difficult to elucidate. Electron microscopy demonstrated that the
cortical layer contained mainly neurons, while glial cells were scattered in the medullary and basal parts
among a complex network of interweaving processes. Segregation of neurons into an identifiable layer in the
reaggregates would indicate that neurons have a greater affinity for their own type than for other cells.
However, neither the coalescence of reaggregates nor their internal morphology could be interpreted on the
basis of histotypic resemblance to the developing spinal cord.
The hypothesis that embryonic nerve cell reaggregates in vitro reflect with fidelity normal histogenesis in
vivo could not be confirmed in our study.
BIRD, M. & JAMES, D. (1973). Development of synapses in vitro between previously dissociated chick spinal
cord neurons. Z. Zellforsch. 140, 203-216.
DELONG, R. G. (1970). Histogenesis of foetal mouse isocortex and hippocampus in reaggregating cell
cultures. Develop. Biol. 22, 563-583.
MOSCONA, A. A. (1979). Cell aggregation: Construction of multicellular systems from cell suspensions. In
Introductory concepts in Develop. Biol. by A. Monroy and A. A. Moscona, pp. 164-204. The Univ. of
Chicago Press, Chicago and London.
230
Development of the nervous system
Effect of organophosphorus compound on tryptophan level in brains of
suckling rabbits
Danuta Maslinska, Laboratory of Developmental Neuropathology, Medical Research Centre,
Polish Academy of Sciences, Warsaw, Poland
Organophosphates are known as the agents which have a fairly specific mechanism of action by inhibiting
acetylcholinesterase (AChE) at the central and peripheral cholinergic synapses. Recently the modulating
effect of these compounds on the concentration of serotonin (5-HT) in brain has been found. Since
serotonin plays a part in the neurogenesis and differentiation of the cells, the present work was performed
on suckling rabbits, in which the availability of tryptophan for the synthesis of serotonin in brains was
studied.
Animals were treated for 10 days with dichlorvos (DDVP, 0,0-dimethyl-0-(2,2 dichlorovinyl)-phosphate)
by oral gavage starting on the 6th day of life. In 16-day-old rabbits the concentration of (1) non-esterified
fatty acids (NEFA) (titration method); (2) whole and free tryptophan (TP) (fiuorimetric method (dialysis));
(3) longchain neutral amino acids (LNNA) by means of Beckman Amino Acid Analyser, in plasma and
brain were studied. In plasma of treated animals increases of NEFA concentration of about 48 %o and free
tryptophan of about 44 % were found. The concentration of whole tryptophan was not changed. The
decrease of some LNNA levels (valine, isoleucine, phenylalanine, methionine by up to 65, 50, 63, 65 %
respectively) led to the increase of the tryptophan/LNNA ratio. In brain of treated rabbits the tryptophan
level was higher than in controls by about 59 %.
The results obtained in the present study suggest the following pathomechanism of changes. In plasma of
animals treated with dichlorvos, the high NEFA level binds more albumin and thus competes with
tryptophan for these proteins. The concentration of free tryptophan increases. The high availability of free
tryptophan in plasma increases the binding of tryptophan with the transport carrier through the blood-brain
barrier (BBB). Moreover, the level of LNNA, which compete with tryptophan for this carrier, decreases
and tryptophan can more easily cross BBB. In consequence the concentration of brain tryptophan
significantly increases.
Embryonic loss of spinal motoneurons: competition or
predetermination?
C. D. McCaig 1 and A. J. Harris 2. Department of Physiology, University of Otago, Dunedin, New Zealand.
Present address:1Department of Physiology, University Medical School, Edinburgh. 2Lab. Zoologie,
University Grenoble 1,38402 St. Martin D'Heres, France
Axons in C5 ventral roots of rat embryos, aged E15-E21, were counted from E. M. photomontages to
assay motoneuron numbers. 83 % of motoneurons present on E15 (6940) were lost by E21 (1168). Of this
loss, 79 % occurred between E15-E16. Some motoneurons normally destined to die were rescued from cell
death by paralysing embryos (TTX filled capillary in the amnion) from E15 (26 %), E16 (45 %), or E17
(48 %) until E21. The number surviving until E21 was, in each case, that normally present in controls about
24 h after paralysis was initiated. This indicates that motoneurons are committed to die one day before their
loss and paralysis rescues all those not so committed. Thus most motoneurons are determined to die on E14
or earlier, when only a small proportion of primary myotubes have differentiated, end plates have not
formed and there can be little or no 'competition for synaptic sites'.
I.p. injection of 1 /ug of beta-bungarotoxin (bBTX) in embryos in E12 killed half the motoneurons
projecting axons through C5 before E15. Despite this considerable pruning of the motoneuron pool,
subsequent cell death occurred with the same time course and to the same proportionate extent as in
controls. 79 % of the E15 depleted number of motoneurons was lost by E21 and 78 % of death occurred
within 24 h. By E21 the ventral root contained 60 % of othe control number of axons. Cell death did not
adjust the numbers of embryonic motoneurons to the normal final goal by E21 and we suggest that the death
of individual motoneurons may be predetermined.
Development of the nervous system
Cell surface antigens and CGAP filaments in non-myelin forming
Schwann cells
231
R. Mirsky andK. R. Jessen, MRC Neuroimmunology Unit, Department of Zoology, University College,
London
Surprisingly little is known about the molecular properties of the non-myelin forming glia of the P.N.S.
Further, the relationship between these cells and CNS glia is poorly understood. In this communication we
show that a group of three antigens are co-expressed by the non-myelin forming Schwann cells of peripheral
nerve trunks. While these antigens are absent from myelin forming Schwann cells they are expressed by
astrocytes and enteric glia.
We compared the distribution of two surface proteins, Ran-2 and A5E3 antigen, both defined by
monoclonal antibodies, with the distribution of the intracellular intermediate filament protein GFAP in
peripheral nerve trunks of adult rats using double label immunofluorescence. It was clear that the expression
of all three proteins is confined to the same population of Schwann cells, namely, the Schwann cells
surrounding the unmyelinated axons. The myelin forming Schwann cells surrounding the larger axons did
not express immunohistochemically detectable amounts of any of the proteins. In teased nerve preparations
from the preganglionic sympathic trunk, sciatic nerve, brachial plexus, dorsal and ventral roots, a complete
correspondence was found between expression of Ran-2 and GFAP, and between A5E3 and GFAP in
non-myelin forming Schwann cells. Schwann cells positive for Ran-2 alone, A5E3 antigen alone, or GFAP
alone were not found.
In development A5E3 disappears from Schwann cells during the first few days of myelination (postnatal
days 3-5) being retained only on those cells not forming myelin. Conversely, if myelin forming Schwann
cells are taken into culture, they acquire A5E3, as they gradually lose the myelin components. In contrast,
RAN-2 disappears from Schwann cells when they are maintained in culture.
The same constellation of antigens is also expressed by astrocytes in the adult rat C.N.S. and enteric glia
in the P.N.S. and not by other cell types, suggesting that this may reflect some common function for these
three cell types.
Choice of motoneuron paths after removal of target muscle during
postembryonic development of an insect
D. R. Ndssel and P. Sivasubramanian, Department of Zoology, University of Lund, Sweden. Department of
Biology, University of New Brunswick, Fredericton, Canada
Leg motoneurons survive limb bud removal in embryonic locusts, differentiate and form axons that
choose new routes out of the CNS (Whitington & Seifert (1982), Devi Biol. 93, 206-215.). We are
interested in where the motoraxons grow after removal of their normal target muscle. Left mesothoracic leg
discs were extirpated from prepupae of thefleshflySarcophaga. The left mesothoracic leg nerve is missing in
the resulting adult pentaped flies, but histology shows that the motoneurons deprived of leg muscle survive.
Horseradish peroxidase or cobalt chloride was applied to the lesioned nerves of adjacent legs on the ipsiand contralateral side, or intramuscularily into different sets of indirect flight muscle. We found labelling of
normal-appearing motoneurons in the deprived neuromere after either of the following marker
applications: ipsilateral pro- or metathoracic leg nerves, or dorsal longitudinal- or dorso-ventral flight
muscles. The axons chose only one nerve root in each specimen. When the axons run to pro- or
metathoracic legs, almost the full compliment of leg motoneurons were labelled centrally. Injections into
indirect flight muscle, however, revealed fewer than normal number in the deprived neuromere, indicating
that some motoneurons might run to other muscle supplied by the same root. Extirpation of a leg disc
followed by implantation of another leg disc only rarely led to motorinnervation of the ectopic leg. This was
when the sensory neurons of the ectopic leg found the neuromere that was originally deprived of its leg
(normally ectopic legs send sensory axons to the metathoracic neuromere via an abdominal nerve). Our data
suggests that motoneurons deprived of targets follow adjacent axonal tracts of sensory or motoneurons and
that innervation of muscles of an ectopic leg occurs only if the sensory neurons of this leg find the correct
neuromere in the thoracic ganglia. We do not know yet whether the motoneurons form functional synapses
with muscle in their new paths.
232
Development of the nervous system
P Nerve growth factor (/JNGF) receptors in chick embryonic
development
G. Raivich, A. Zimmermann and A. Sutter, Institut fur Pharmakologie, Freie Universitat Berlin,
Thielallee 69-73, D1000 Berlin 33, West Germany
For a direct evaluation of jSNGF receptor expression in vivo during development specific binding of
I-j3NGF to thin sections of chick embryos was monitored autoradiographically between embryonic day 3
(E3) and hatching (E21). Expression of pNGF receptors was not seen before E4 when specific high affinity
binding was observed to dorsal root ganglia (drg), to dorsal and ventral roots, to sympathetic ganglia (sg), to
peripheral nerves, to myotome
and to spinal cord (sc). At E4 anterior roots and myotome were especially
heavily labelled with 1*Ij8NGF. With development labelling of the drg increased and was particularly
prominent in the mediodorsal region of the ganglion. The same labelling pattern was observed for cranial
sensory ganglia. Parasympathetic ganglia showed weak but distinct jSNGF binding at E8, but were negative
at E12 and onward. In contrast to the drg and sg (cell somata) peripheral nerves had
lost their NGF
receptors by E20. The gross labelling of skeletal muscle in early development gave way to 125I/8NGF binding
to a restricted zone at the muscle-connective tissue border by E12, from where it was lost by E20. In the sc
the lateral motor column was NGF receptor positive between E4 and E12. Labelling of the area of the
dorsolateral tract of the sc, reaching into the zona terminalis and substantia gelatinosa was seen at E6 and
was still present at the time of hatching. Labelling of the cranial nerve entry zones in the brain stem and the
dorsal root entry zone in the sc was observed between E4 and E18. The results of this mapping of the spatial
and temporal distribution of specific jSNGF binding sites raise a number of unexpected questions concerning
the role of /8NGF or /JNGF like factors in embryonic development.
125
Cell interactions in development of visual system
Paslco Rakic*, Section of Neuroanatomy, Yale University School of Medicine, New Haven, Conn. 06510,
USA
Genesis of normal and experimentally altered visual system in the rhesus monkey offers an excellent
model to study the role of cell interaction in formation of complex synaptic circuits. Soon after the last cell
divisions, which occur during the first half of gestation, retinal ganglion cells send axons to the optic stalk.
Reconstruction from EM serial sections shows that growth cones in the embryonic optic nerve have large,
sheath-like lamellipodial tips which do not adhere to a particular set of neighbouring axons, indicating that
orderly growth of retinal axons cannot explain topography of retinofugal fibers in the visual centres.
Furthermore, there are more than double (2*85x10 ) the retinal axons present during midgestation than
in the adult monkey. Over one million axons are rapidly eliminated within the first 3-4 weeks of the second
half of gestation - the remaining half million are depleted at a slow rate during infancy. Monocular
enucleation during midgestation reduces elimination of axons in the remaining eye, demonstrating that the
number of axons depends on the competition between terminals originating from the two eyes. Initially
intermixed axons subserving the two eyes sort out to form synapses in the appropriate laminae of the lateral
geniculate nucleus (LG) and appropriate patches of the superior colliculus. Coincidence of terminal
segregation and a rapid wave of axonal depletion indicates that this selective elimination plays a role in
establishment of appropriate synaptic connections. Similar development from the diffuse to segregated
phase is observed during development of geniculcortical connections. After completion of the segregation
phase, synaptic density rapidly increases in the retina, LG, and the visual cortex where it transiently exceeds
adult values. To our surprise, synaptic connectivity in central structures is initiated in advance of the
peripheral centres. Furthermore, synapses between amacrine and ganglion cells in the retina occur before
development of true line projections from the receptors via bipolar and ganglion cells. The connections are
modifiable: enucleation of an eye during the first half of gestation results in contacts between the axons from
the remaining eye and neurons in appropriate laminae of othe LG and in inappropriate ocular dominance
columns. However, the rate of synaptogenesis, ratio and size of various synaptic classes is similar in controls
and animals prematurely exposed to light. These studies collectively reveal the dynamic nature of
establishment of synaptic architecture that requires a balance between various inputs and co-operation
between genetic and epigenetic factors. Although the molecular mechanisms of these events are not
understood, the available data reveal the multifactorial nature of the system that cannot be explained by a
pairing of recognition molecules without taking into account competitive interactions among all the neurons
involved in the formation of these circuits in vivo.
Development of the nervous system
Use of WGA-HRP to assess the progress of map refinement after
regeneration of the goldfish optic nerve
233
E. C. C. Rankin andJ. E. Cook, Department of Anatomy & Embryology, University College London,
Gower Street, London WC1E6BT
Wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), injected iontophoretically
into the normal goldfish tectum, is taken up locally by optic terminals and labels a small cluster of ganglion
cells in the retina (Cook & Rankin, 1984). This method of demonstrating retinotopy can be used to follow
the progress of map refinement after regeneration of the optic nerve. The right optic nerves of 47 goldfish
(Carassius auratus; 55-65 mm long) were cut under MS-222 anaesthesia. Fish were kept at 20 ± 0-5 °C in
diurnal lighting. After 28-91 days, WGA-HRP was injected iontophoretically at a caudodorsal site on the
left tectum.
Early in regeneration (day 28, five fish), labelled cells were scattered across the appropriate (ventronasal)
quadrant of the retina and were occasionally found in other parts. No localized clusters were visible,
implying that retinotopic terminals were rare or absent. Cluster formation was detectable at day 35 (five
fish), though scattered cells were still common. At day 42 (seven fish) and day 49 (five fish), large clusters,
often radially extended, were clearly visible. At day 56 (six fish), clusters were still large: two fish now
showed a pair of irregular aggregates, loosely linked along a radial line. Later in regeneration (day 70, two
fish; day 91, three fish), clusters were mostly more compact (though still larger than in normal controls)
implying that the terminals were becoming more retinotopic: and there were fewer scattered cells.
It is not certain whether label found in scattered cells early in regeneration represents uptake by
non-retinotopic terminals, growth cones of misrouted axons, or both (Cook & Rankin, 1984). The first, at
least, is likely since synaptogenesis is known to begin as soon as regenerating axons reach the tectum
(Stuermer & Easter, 1984).
COOK, J. E. & RANKIN, E. C. C. (1984). Use of a lectin-peroxidase conjugate (WGA-HRP) to assess the
retinotopic precision of goldfish optic terminals. Neurosci. Lett, (in press).
STUERMER, C. A. O. & EASTER, S. S., JR. (1984). A comparison of the normal and regenerated retinotectal
pathways of goldfish. J. comp. Neurol., 223, 57-76.
Differential innervation of dermal and epidermal explants by sensory
neurons in tissue culture: a time-lapse analysis
R. SaxodandJ. M. Verna, Laboratoire de Biologie Animate, ERA CNRS621, University de Grenoble 1, BP
6838042 Saint-Martin-d'Heres Cedex, France
In establishing skin innervation, sensory nerve fibers interact with cellular and acellular cutaneous
components. In order to determine the part played by these interactions in neuritic growth and guidance,
the behaviour of nerve fibers was studied in serum-free co-cultures of lumbar spinal ganglia and either
dermis or epidermis from 7-day chick embryos.
After a few days of culture, two different patterns of neuritic growth were obtained: in co-cultures with
dermis, the initial orientation of neurite elongation is maintained even after encountering cells migrating at
the periphery of the dermal explants. On the contrary, neurites tend to avoid epidermis and so deviate
around the explant.
Morphometric time-lapse analysis of cultures show that, on a collagen substrate, neuritic growth cones
contact epidermal cells but then quickly withdraw, without establishing the close associations with the cell
surface observed with dermal cells. Nerve fibers then progress along the edge of the epidermal layer. On a
polylysine substrate, on the other hand, neurites do not contact the epidermis explant but deviate around it
at a distance.
These differences as a function of the culture substrate suggest the existence of diffusible factor(s)
released by epidermal cells which bind to a polylysine substrate. Moreover, these results indicate cell
membrane incompatibility between neurites and epidermal cells, the nature of which is not yet known.
234
Development of the nervous system
Cell surface analysis in multilineage systems
Fritz Sieber and Maya Sieber-Blum, Departments of Medicine and Cell Biology and Anatomy, The Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
Many experimental systems for the study of cell differentiation are not readily amenable to direct
biochemical analysis because the relevant cell populations are present in small numbers and difficult to
purify. However, when clonal assays for progenitor cells are used in conjunction with suitable molecular
probes, insights into some molecular aspects of cell differentiation are possible. In this communication, we
report on the use of merocyanine 540 (MC 540)-mediated photosensitization in the analysis of plasma
membrane changes during hematopqiesis and neural crest cell development. MC 540 is a negatively charged
fluorescent dye that binds preferentially to fluidlike or cholesterol-free domains in the outer leaflet of the
lipid bilayer. Photoexcitation of membrane-bound dye leads to an impairment of membrane functions and,
eventually, cell death. By illuminating stained cells for graded time intervals, we were able to establish
characteristic kinetics of photosensitization for different classes of quail neural crest cells and murine and
human hematopoietic stem cells. Among quail neural crest cells, heterogeneity with regard to their
sensitivity to MC 540-mediated photosensitization was observed already on the first day of secondary
culture, i.e. four days before the onset of melanin and catecholamine synthesis. Neural crest cells that gave
rise to pigmented cells only were markedly less sensitive than cells giving rise to unpigmented and mixed
progeny. Among murine hematopoietic stem cells, late erythroid progenitors were the most sensitive cells
followed in order of decreasing sensitivity by early erythroid progenitors, megakaryocyte progenitors, day
7-spleen colony-forming cells, granulocyte/macrophage progenitors and day 11-spleen colony-forming cells.
Human hematopoietic cells followed the same rank order of sensitivity. Significant differences in sensitivity
were found between developmental stages that are thought to be only one or two cell divisions apart. In
summary, these results indicate the fluorescent dye MC 540 is a useful probe for developmentally regulated
plasma membrane constituents. Since MC 540 expresses little, if any, cell cycle specificity and reacts with
the lipid portion of the plasma membrane, it complements cell cycle specific drugs and lectins and antibodies
which recognize carbohydrates and proteins. (Supported by NIH grants AM 27157 and HD 15311).
Proliferation and differentiation of quail neural crest cells in a defined
culture
Maya Sieber-Blum* and Hitesh R. Chokshi, Department of Cell Biology and Anatomy, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205, USA
Cultured avian neural crest cells give rise to several differentiated phenotypes and thus represent an
attractive experimental system for the study of cell differentiation. However, so far the analysis of the
underlying regulatory mechanisms has been hampered by two factors: 1) the contamination of cultures with
cells of noncrest origin and 2) the complex growth medium that contained two ill-defined components, horse
serum and chick embryo extract. The introduction of clonal cultures (Sieber-Blum, M. and Cohen, A. M.
(1980). Develop. Biol. 80,96-106) eliminated the first problem. We now report the formulation of a defined
culture medium that supports the proliferation and differentiation of quail neural crest cells. Neural tubes
were explanted into culture dishes that had been coated with collagen and fibronectin and were cultured in
medium MCDB 202 supplemented with insulin, transferrin, cortisone, gonadal hormones, vitamins, and
trophic factors. The explanted neural tubes adhered to the substratum and the neural crest cells emigrated in
the usual fashion. Cell proliferation was, however, slower than in complex medium. After 4-6 days all
cultures contained pigmented cells that were densely packed with melanin granules and had the typical
appearance of melanocytes. Twenty-five percent of the cultures contained adrenergic neurons as indicated
by intense formaldehyde/glyoxylic acid-induced catecholamine fluorescence. Neurons had characteristic
long, varicose processes with growth cones and rounded cell bodies that tended to aggregate with each
other. We believe that the development of this defined medium will prove useful in the study of neural crest
cell differentiation under controlled conditions. (Supported by USPHS Grant HD15311).
Development of the nervous system
235
Development of the peripheral nervous system from the neural crest:
establishment of phenotypic diversity
Julian Smith*, Mireille Fauquet, Jose" Garcia-Arraras, Catherine Ziller and Nicole Le Douarin, Institut
d'Embryologie du CNRS et du College de France, 49bis, Avenue de la Belle-Gabrielle, 94130
NOGENT-sur-MARNE, France
The major part of the peripheral nervous system in vertebrates develops from neural crest cells that
migrate to appropriate sites in the body. Much current interest centres on the mechanisms whereby a wide
variety of differentiated cell types arise from this apparently homogeneous embryonic structure. The
developmental potential of neural crest at all levels of the neural axis is significantly greater than that
actually expressed during normal ontogeny, indicating an important role for the embryonic
microenvironment during the early phases of nervous system differentiation. However, it is not clear a priori
whether external factors act on pluripotential precursor cells or on cells that are already largely
predetermined on leaving the neural primordium. A number of experimental approaches to this problem
have been made, including attempts to identify differentiation markers on premigratory crest cells,
examination of the relationship between differentiation and cell division, studies on the restriction of
developmental potential with time and analysis of neural crest differentiation in culture in vitro. As a result,
it is becoming increasingly apparent that at least certain crest cells are limited in their developmental
capabilities and are partly or totally committed to a particular phenotype before, or shortly after, starting to
migrate. Thus, data from transplantation experiments in vivo suggest an early, irreversible segregation of
sensory and autonomic lineages. Phenotypic diversity increases as definitive neurotransmitter-and
neuropeptide-related properties are acquired by developing autonomic neurons. A number of these events
can be reproduced in vitro, where evidence has been obtained for the existence of a least two types of
neuronal precursor in neural crest.
Quantitative and qualitative measurement of acetylcholinesterase
changes in the central nervous system and reproductive organs of mice
during low and high doses of a-chlorohydrin
P. P. SoodandV. K. Kakaria, Department of Biosciences, Saurashtra University, Rajkot-360005, India
The effect of a-chlorohydrin (3 chloro-l,2-propanediol; U-5897) has been studied on the
acetylcholinesterase (AChE) changes in the reproductive organs and central nervous system of mice with
special reference to the hypothalamic nuclei and related structures, in order to see the mechanism of this
antifertility drug action.
The drug was administered in chronic low dose (15 mg/kg body weight/day) for 20 and 30 days and a
single high dose (90 mg/kg body weight). The enzyme was studied using histochemical, cytophotometric and
biochemical parameters.
Histochemical studies reveal a sharp and progressive decrease of AChE in most of the thalamic and
hypothalamic nuclei of the brain, Leydig cells of testis and epiothelium of cauda and caput epididymis
during low dose treatment of 20 days. The enzyme further decreased when the doses and duration were
prolonged up to 30 days. On the contrary, single high dose effect was comparatively less. Biochemical
estimations of tissues also verified these findings.
On the basis of the above studies, two possible ways of the or-chlorohydrin action have been suggested: a)
the drug may affect the Leydig cells of the testis and control the androgen production, which simultaneously
affect the spermatogenesis and sperm maturation. In such case, the effect on the central nervous system may
be secondary; b) the drug may directly affect the central nervous system and thereby alter the cholinergic
mechanism, specifically of the hypothalamic nuclei controlling the anterior pituitary and exert a control over
the gonadotropins (FSH and LH) secretion leading to affect the Leydig cells function and thereby alter the
spermatogenesis. It may be added here that serum FSH and LH levels are reported to be increased in
ar-chlorohydrin treated rats(l).
(1) MORRIS, I. & JACKSON, C. M. (1975). /. Endocinol 67, 21-22.
236
Development of the nervous system
How retinal axons grow in the tectum: branching analysis of axonal
arbors of single physiologically identified frog retinal ganglion cells
R. Victoria Sterling*1 and E. G. Merrill2.1National Institute for Medical Research, Mill Hill,
London NW71AA, 2Laboratory of Physiology, University of Oxford
The interaction between growing axons and their environment is reflected in the three dimensional shape
of their arbors. We have made intracellular recordings and injected single axons of several different classes
of retinal cell.
The filled cells and their tectal arbors are stained in sections or whole mount preparations using a modified
ion intensified DAB reaction. The morphology of the tectal axonal tree in whole mounts is analysed using a
motor driven stage and interactive programme (J. Green et al. (1979). /. Physiol., 300,13) which records X,
Y and Z coordinates of selected points along the tree. A reconstruction of the tree can be viewed in stereo
from any angle, and the distribution of branch points and internodal distances easily computed.
Many axons give off both very fine as well as large caliber branches before the principal tree is reached.
Analysis of the positions of these branches indicates whether they represent the ghosts of earlier
terminations made in the younger, smaller tadpole brain (Constatine-Paton, etal. (1983) /. Comp. Neuroi,
218, 297-313). These data will illustrate the constraints acting on the growth of axons from particular types
of ganglion cell as they make successive terminations in the maturing frog tectum.
The behaviour of motor axons invading reversed chick wings
R. Victoria Stirling and Dennis Summerbell, National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7IAA
Early limb buds were uniaxially reversed in the dorsoventral (DV) or anteroposterior (AP) axis before
axons invade, by grafting to the contralateral side of host embryos. The resulting embryos survive well and
healing between graft and host is good, reliably producing clearly reversed wings. The motor pools and
trajectories of the motor axons were traced after retrograde filling from muscle injection of horseradish
peroxidase followed by whole mount staining and wax sectioning. The axon trajectories were analysed and
visualized using a bit pad and interactive three dimensional serial section reconstruction programme.
After DV axis reversal there is no evidence of course correction by the axons which innervate functionally
anappropriate targets in the graft. Thus motor axons which normally would innervate ventral limb flexors
now supply extensors, while those that normally innervate dorsal extensors supply flexors.
However, after AP axis reversal axons do change their position in the plexus to leave it in a position
appropriate to reach their normal targets in the reversed wing. Rostral segments therefore innervate biceps
despite its abnormal caudal location and caudal segments likewise innervate triceps.
If this difference is related to the distance of displacement of axons from their normal route one would
expect reversal proximal to the confluence of the three segmental roots into the plexus to give a greater
mismatch in the AP rather than DV axis. However we consistently find axon correction with AP reversals
but no correction after proximal DV reversals. We are presently examining the level of reversal using
chick/quail chimaeras.
It seems that axons are able to detect mismatch in the AP but not the DV axis. This may be related to the
displacement distance or may be due to an absence of positional cues for the DV axis. The AP axis is still
subject to positional signals from the zone of polarising activity during the critical period, but there is little
evidence for similar signalling across the DV axis.
Development of the nervous system
The pathways of fibres from translocated eyes in Xenopus
237
J. S. H. Taylor, D.J. Willshaw and R. M. Gaze, Department of Zoology, University of Edinburgh,
West Mains Road, Edinburgh EH93JT
In normal Xenopus the retinal ganglion cell fibres arising from each quadrant of the retina travel in a
characteristic fashion to the optic tectum, where they terminate in retinotopic order(l). We present a recent
investigation of the distribution of fibres in the optic tract of Xenopus in which the eyes have been
translocated in embryonic life from a right to a left orbit without rotation. This operation results in a
visuotectal projection which, when recorded electrophysiologically, is normal dorsoventrally but inverted
nasotemporally(2). Labelling of small groups of retinal axons with HRP showed that the fibre trajectories
from dorsal and ventral retina were normal, whereas fibres from nasally placed retina had diencephalic
pathways and tectal terminations typical of temporal fibres, and fibres from temporally placed retina had
diencephalic pathways and tectal terminations typical of nasal fibres. Thus from just beyond the chiasma the
fibres seem to have already achieved the major uniaxial rearrangement necessary to establish a normal tract
distribution despite the eye translocation. The fibre-sorting required to permit the formation of a
naso-temporally inverted visuotectal projection appears to occur not on the tectum or in the optic tract, but
either within the nerve or at the chiasma.
(I)FAWCETT, J. W. & GAZE, R. M. (1982). The retinotectal fibre pathways from normal and compound eyes
in Xenopus. J. Embryol. exp. Morph., 72, 19-37.
(2) GAZE, R. M., FELDMAN, J. D., COOKE, J. & CHANG, S.-H. (1979). The orientation of the visuotectal map
in Xenopus: developmental aspects. /. Embryol. exp. Morph., 53, 39-66.
Morphogenesis of neuromeres observed at the TEM level
Fiona Tuckett, Department of Human Anatomy, University of Oxford, South Parks Road, Oxford OXI3 QX
In coronal section of the cranial neural tube, the epithelium has a characteristic segmental patterning;
each of these segments represents a neuromere. A model has been proposed to explain the development of
this segmental patterning, based on TEM observation of transversely orientated microtubules and
longitudinally orientated microfilament bundles, together with known kinetic behaviour of the neural
epithelium. The feasibility of the model was investigated by TEM examination of embryos which had been
treated in culture with either cytochalasin D or colchicine.
The model proposes that the neural epithelium, which should expand longitudinally due to the
predominant orientation of the spindle axes in this plane, cannot expand due to the localised strengthened
regions created by the transversely orientated microtubules and hence the epithelium has to bulge. The
bulging occurs along the line of least resistance; the microfilament-rich luminal (apical) border provides a
greater resistance than the basal lamina and as a result the epithelium bulges downwards into the underlying
mesenchyme.
238
Development of the nervous system
Methylmercury induced enzymatic changes in the trigeminal ganglia and
hind-brain of rats
K. R. Unnikumar and P. P. Sood, Department of Biosciences, Saurashtra University, Rajkot - 360 005', India
Methylmercury is known to be the most potent neurotoxicant among the organomercurials(l). The
present attempt is focused to elucidate the enzymatic changes in trigeminal ganglia and hind brain of rats
due to methylmercury poisoning. The animals were given 10 mg/kg body weight methylmercuric chloride
(MMC) for 2, 7 and 15 days, and were sacrificed on 3, 8 and 16 days respectively along with the controls.
The trigeminal ganglia and hind brain were quickly removed, weighed and processed for biochemical
estimations of acid-phosphatase (ACP), alkaline phosphatase (ALK), adinosine triphosphatase (ATPase)
and succinic dehydrogenase (SDH).
All the enzymes showed decreased activities both in trigeminal ganglia and hind brain in 7 and 15-day
treated animals, though the decrease was more pronounced in trigeminal ganglia. 2-day treated animals
showed insignificant variations. In 15-day treated animals there was a sharp increase in ACP levels.
On account of these studies, it is concluded that the onset of biochemical lesion is dependent upon the
duration of exposure, and vulnerability of trigeminal ganglia to MMC is more conspicuous than in hind
brain. Since MMC binds with the SH groups of enzymes(2), it may bring about decreased activity of the
latter as well as psychosomatic disturbances. The possible disruption of lysosomes by MMC may account for
an increased ACP level. Though it has been reported that mitochondnal immunity to MMC(3) in the
present investigations, a marked decrease in SDH levels were observed in 15-days treated animals.
(1) TAKEUCHI, T. (ed. M. Kutsuma) (1968). Pathology of Minimata Disease. In Minimata Disease.
(2) WEBB, J. L. (1966). Enzyme and Metabolic inhibitors, 2, 729-1070. New York; Academic Press.
(3) YOSHINO, Y., MOZAI, T. & NAKAO, K. (1966). /. Neurochem, 13, 1223-1230.
Effect of a notochordal implant on the neural tube and neuroblasts
Histological, histometrical and enzymehistochemical results
H. W. M. van Straaten, F. Thors, E. L. Hoessels, J. W. M. HekkingandJ. Drukker, Department of
Anatomy and Embryology, National University Limburg, 6200 MD Maastricht, The Netherlands
The early development of motoneuroblasts is morphologically characterised by increase of cellular and
nuclear size, outgrowth of a lateral cell process, migration in lateral direction and arrangement in the ventral
horn; they originate by proliferation of the matrix. In the chick most basal plate neuroblasts originate
between day 2 and 4 of incubation. Regulation of this early development is hardly understood, but evidence
has been presented that it proceeds independently of peripheral interaction. Several data suggest that
chordomesodermal material exerts an effect on the early mitotic activity of the neural tube. In the present
study we investigated whether the notochord exerts a specific effect on the development of neuroblasts.
Material and Methods. In chick embryos of 2 days, an additional notochord fragment was implanted near
the right side of the thoracic neural tube. At 4 days the embryos were fixed in Bodian's fluid (for histology
and histometry) or in Holt's fluid (for enzymehistochemistry) and embedded in Technovit 7100 (Kulzer).
From the experimental area transverse sections were cut and serially sampled. The location of the implant
and the sectional area of the right and left neural tube halves were determined histometrically. By
performing a reaction on AChE, neuroblasts and efferent axons become clearly recognizable.
Results. If the implant is located directly lateral to the neural tube, the sectional area of the right half of
the neural tube is increased, concomitant with an enlargement of the neuroblast area. Efferent axons leave
the neural tube over a considerable dorsoventral trajectory. When the notochord is located at the
ventrolateral side of the neural tube, bulging of the ventral horn is not present, whereas a population of
neuroblasts is present more dorsally. In longitudinal direction the effects are completely absent within
200 /urn. The effects neither occur if the implant is situated at a distance of more than 100 /um from the
neural tube, nor in sham-operated embryos.
Conclusion. The implanted notochord affects developmental processes in the early neural tube within a
restricted level. Morphogenesis of the tube, total volume of neuroblasts and arrangement of neuroblasts in
the ventral horn as well as the sites where axons leave the neural tube are influenced. This conclusion
supports our view that the natural notochord plays a regulating role in the early development of neuroblasts.
Development of the nervous system
239
Proliferation and differentiation in histotypic aggregates of embryonic
chick retinal cells in the presence of pigmented epithelial (PE) cells
G. Vollmer* and P. G. Layer, Max-Planck-Institutfur Entwicklungsbiologie, Spemannstr. 35,
D-7400 Tubingen, West Germany
Dissociated E5-E6 chick retinal cells in the presence of PE-cells have the capacity to reaggregate and
reconstruct all main layers of an intact E10-E14 retina after 14-21 days in rotary culture(l). Now we have
tried to correlate patterns of proliferation with differentiation phenomena occurring during the formation of
these 'retinoids'.
Determination of cell numbers in aggregates after 9 days in culture reveals a 4-fold increase in
R-aggregates (composed of retinal cells alone) and a 5-fold increase in RPE-aggregates
(retinal cells and
PE-cells). Compared with retinae in vivo, the time course and the amount of 3H-thymidine uptake in
aggregates shows a decrease to 20 % during the first day and then is similar (R-Aggregates) or even
accelerated (RPE- aggregates) for the next three days. A double-label procedure showed striking
differences in the spatial order of thymidine- and AChE-positive cells within RPE- and R-aggregates. In
R-aggregates thymidine- incorporating cells are distributed over the whole aggregate. They express high
amounts of AChE at the periphery, but no layered arrangement in the center of the aggregates. In
RPE-aggregates proliferating cells are arranged in a concentric band in the outer part and differentiated
cells are localized in the inner part of the aggregate. Concomitantly an AChE-positive layer arises in the
middle of the RPE-aggregate, which possibly is composed of INL cells. It marks a clear border between
differentiated and proliferating cells after 2-4 days in culture.
We conclude that the high degree of organization in these aggregates is accompanied by pronounced rates
of proliferation. In RPE-aggregates proliferation and differentiation (AChE) occur in separate lamina
showing similarities to the sequence observed in an in vivo retina.
(1) VOLLMER, G., LAYER, P. G. & GIERAR, A. (1984). Reaggregation of embryonic chick retina cells:
pigment epithelial cells induce high order of stratification. Neuroscience Letters (in press).
Neuronal differentiation of a cloned human teratoma cell line
Michael Webb 1 , Frank S. Walsh * and Christopher F. Graham 2. 1Molecular Neurobiology Laboratory,
Institute of Neurology, Queen Square, London WC1N3BG. 2Department of Zoology, South Parks Road,
Oxford OX13PS
The differentiation of a cloned human teratoma cell line (Tera-2) after treatment with retinoic acid (RA)
has been analysed using well characterised immunological reagents. The cells were treated with RA in
suspension, conditions which favour the formation of large aggregates, and then plated in gelatin coated
tissue culture dishes 7-10 days after aggregation. Cells with a variety of morphologies were found in such
cultures, and approximately 1-3 % extended long branching processes from a retractile cell body. These
cells all expressed neurofilament proteins as detected with two different monoclonal antibodies (McAb's)
against neurofilament (BF10 and RT97). They also expressed cell surface receptors for tetanus toxin (tt) and
were labelled by the McAb A2B5 (Eisenbarth et al. 1979): these markers are expressed by neurons and a
subset of glial cells and their precursors in the mammalian brain. Expression of these markers was low or
absent in undifferentiated populations of Tera-2 stem cells. In time course studies, tt receptors were
consistently expressed earlier than A2B5 antigen after treating the cells with RA, although the proportion of
cells expressing these markers was variable between experiments, ranging from 17 %-98 % (tt) and
6 %-92 % (A2B5) by 10 days after RA treatment. The expression of A2B5 antigen and tt receptors is not
confined to neurons in the mammalian nervous system. Since there were many cells in the differentiated
populations which expressed these markers but lacked both a neuronal morphology and the neuron-specific
neurofilament proteins, we examined the expression of glial markers in these cultures. In the differentiated
populations, up to 6 % of the cells expressed the marker of immature oligodendrocytes recognised by McAb
04 (Sommer and Schachner, 1982). A cell surface antigen expressed only on astrocytes inprimary human
brain cultures (recognised by McAb Ml/Nl) was expressed on a variable proportion (9 %>-43 %) of the
cells, although no staining with anti-GFAB antibodies was found. These results suggest that the human
teratoma may provide a useful model system for the study of early events in the differentiation of a variety
of neural cell types.
EISENBARTH, G. S., WALSH, F. S. & NIRENBERG, M. (1979). Monoclonal antibody to a plasma membrane
antigen of neurons. Proc. Natl. Acad. Sci. USA 76, 4913-4917.
SOMMER, I. & SCHACHNER, M. (1981). Monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: an
immunological study in the central nervous system. Dev. Biol. 83, 311-327.