Development of the nervous system 217Immunohistochemical study on the differentiation of cranial sensory

and autonomic ganglia in the ratC. 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) andanti-neurofilament (NF) antisera. NF immunoreactivity, as well as silver impregnation, appear first incranial sensory ganglia in 2-day-old rat fetuses. At this stage, only a perinuclear ring of reactive material isvisible in some neurons while numerous brightly fluorescent fibres are present in cranial nerves. By day 16 ofgestation the first SP-immunoreactive (SPLI) cell bodies appear in cranial sensory and parasympatheticganglia. At this stage the amount of NF-immunoreactive material clearly increases in some cell bodies of thetrigeminal, petrous, nodose and cervical dorsal root ganglia. At day 17 of gestation the NF immunoreactionin sympathetic (superior cervical) and parasympathetic (sphenopalatine, otic, submaxillary, lingual andenteric) 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 NFimmunoreactivity in peripheral cranial ganglia. On the contrary there is a marked increase of SPLI insensory and parasympathetic ganglia between days 16 and 21 of gestation. The number of positive cellsbecomes higher, the amount and morphology of the immunoreactive material also varies. After birth, fewercells are SPLI in sensory ganglia and SPLI cannot be demonstrated in parasympathetic ganglion cells, innormal conditions. However the presence of SPLI in parasympathetic neurons after birth can be proved inexperimental conditions.

In conclusion, the appearance of SPLI in peripheral neurons (unlike central neurons) in the rat takesplace 5 days after that of the first histochemical sign of neural differentiation in the ganglia, i.e., theacquisition of NF. 16 days of gestation appears as a critical stage in the differentiation of peripheral sensoryand autonomic neurons regarding both criteria used here, SPLI and NF immunoreactivity. At birth a secondsignificant 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 andthe SP antiserum respectively.

This work was supported by the Swedish MRC and the French CNRS.

Pattern formation in the embryonic nervous systems of grasshoppers andflies

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 formpatterns in embryonic nervous systems. Growth cones appear to respond to a hierarchy of adhesive cues: thepreferred substrate for neural growth is an existing nerve, so that the first pioneer axons are crucialdeterminants of a pattern of nerves which aggregates about the paths they form. The first axons in thepattern are guided by cues in the epithelium over which they grow. In the grasshopper, the precise shapes ofindividual neurons unfold as the growth cones of differentiating neurons choose between, and grow alongalternative pioneer pathways in the developing central nervous system. Each cell executes a predictablesequence of choices and turns which lays out a characteristic axon shape. We now find that the embryonicnervous system of Drosophila is a miniature replica of the grasshopper: the same neurons can be identifiedin both making the same choices and turns within an identical framework of pathways, with the implicationthat the guidance mechanisms can now be studied with genetic and molecular methods which are unique tothe fly. Also in Drosophila we have studied the formation of adult nerve pathways in the imaginal discs. Inthe leg disc, larval nerves act as guides for growing axons. In the antennal disc there are no such larvalpathways. Homoeotic transformations of antennal discs into leg discs allow us to assess the significance ofcues in the disc epithelium for the guidance of growing adult axons.

218 Development of the nervous systemExpression of neural antigens by Kupffer cells

Hilary P. Benton \ Michael R. Hartley 2, Amanda J. Suitters 1, Graham P. Wilkin 2.1DepartmentofHistopathology, 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 laantigen. Recently this characteristic has also been shown in central nervous system astroglia, prompting usto investigate more closely the immunochemical similarities between these cell types. We have shown, inthree different experimental systems, that Kupffer cells express several markers previously thought to beunique to astrocytes.1) In primary cultures of adult rat liver cells, Kupffer cells can be identified by their characteristic processbearing 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 expressvimentin 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 onsusceptible 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 subtypesof the same population of cells.

Morphogenetic aspects of the relation of the left vagus nerve and thestomach 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 beenstudied 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 specimensof either stage were examined. The stomachs were stained according to the AChE in toto techniquedescribed by Baljet and Drukker (1975). This technique made it possible to examine the organ in toto underthe dissecting microscope. So we were able to ascertain the three dimensional relation of the nerve with itstarget organ.

The results obtained can be summarized as follows. On day 15 the branches of the left vagus nervetowards the stomach reach as far as the greater curvature of the antrum and the corpus. From day 15 untilday 17 this relation does not alter very much. From day 17 the stomach grows faster than the vagus nerveand its branches. Consequently the gastric branches in the neonatal rat are then restricted to an area of theantrum and the corpus half-way the lesser and greater curvature. This pattern does not differ essentiallyfrom 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 219Selection 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 fibresgrowing from the CNS to the periphery (e.g. growth of fibres from ventral spinal roots to the muscles) orfrom the periphery to the CNS. (e.g. from the retina to the optic tectum), there has been little investigationof the growth of fibres within the CNS. We have traced the pathways taken by fibres regenerating along thegoldfish spinal cord and fibres growing down and through a rotated section of the developing spinal cord inXenopus laevis tadpoles.

Six weeks to six months after a total transaction of the juvenile (7 cm) golkdfish spinal cord at the level ofthe caudal edge of the dorsal fin, horse-radish peroxidase (HRP) was applied to a second partial cut madeeither 1 cm above, or below, the site of the previous operation. Serial sectioning of operated animalsverified a total transection. Other control studies showed that the HRP could not cross a transection bydiffusion or intravenous transport. Histological processing of the sectioned brain with the chromogens DABor TMB revealed the pathways taken by the fibres filled with the HRP. At the site of the transection thefibres were seen to take tortuous and abnormal routes through the scar tissue, however on each side of thescar tissue the regenerating fibres re-entered the appropriate funiculi of the cord. Other identifiable groupsof 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 sothat the originally rostral part of the graft was replaced caudally, thus fibres growing down the left side of thetube were faced with the right side of the rotated graft. E. M. studies showed that no more than 10 fibres arepresent in the neural tube at this stage. One week to three months later the spinal cord was injected withHRP. In contrast to the regenerating fibres in the fish we have not observed any disturbance of the fibrepathways following a simple cut of the neural tube. However, in the animals with a rotated spinal cord, wehave observed a crossing over of fibres in the ventral funiculi of the cord at both the rostral and caudal endsof the transplant indicating that these fibres can detect the rotation of the graft and select their pathways inthe distal spinal cord.

An x-ray microanalytical study of the postnatal development of otoconiaF. 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 macculaduring postnatal development are studied.

A total of 16 Wistar rats were divided into two groups of eight animals such representing two differentstages 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 theformation of structural units of a higher order. In the smaller Type I otoconia, flattened surface areas wereobserved not infrequently which corresponded to zones of adhesion between otoconia of the same or of adifferent type. The larger Type II and Type III otoconia were generally associated with otoconia of the sametype.

An especially interesting observation was the presence of characteristically patterned associations whichmost frequently distinguished the adhesions between Type III otoconia: their flattened distal faces wereoften joined, forming groups generally consisting of three units. Each otoconia in these units conserves itsrespective double edges in the zone of contact, which consequently showed a typical Y configuration. Thesegroups of three otoconia present a characteristic 'cats paw' configuration.

Postnatal otoconia were also studied microanalytically with X-ray dispersion in order to analyze units ofidentical volume. Calcium content increased along with otoconial size. Microanalysis also confirmed thatcentrally-located otoconia contained higher levels of calcium than in otoconia of the same type in peripheralregions of the maccula.

In conclusion, these microanalytical findings as well as the process of association between otoconia couldbe related to morphogenesis and metabolism of these structures.

220 Development of the nervous systemThe role of the cerebrospinal fluid during embryonic development. A

biochemical studyCheciul. 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 thecerebrospinal fluid (CSF) of chick embryos and foetuses between the 4th and 20th day of incubation and of2-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. Aftera slight decrease to 2-6 mg/ml on day 12, no further changes are detected. In the blood serum, the totalprotein content was 4-3 mg/ml on day 4 and 4-7 mg/ml on day 9. Beginning with day 10 it increasesconstantly to 23-2 mg/ml on day 2 after hatching (daily rate 1-1-2 mg/ml). The result is that the 'proteinbarrier' 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 similarblood serum fraction (on day 10). As to the heavy fractions XI-XIV, their concentration in the CSFrepresents, beginning with day 10, 51 % of the similar blood serum fraction (until the end of the periodinvestigated).

The result is that the 'protein barrier' has an active, selective character. Some comparative aspects withliterature 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 inducestelencephalon differentiation which occurs at stage 26 (according to Gallien and Houillon, 1951), but it isnoteworthy that this induction begins as early as stage 23.

To investigate the modalities of the olfactory induction, we have undertaken a preliminary study of thegrowth 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 ofthe anlage of the bulk, where numerous filopods have been seen. These bundles are surrounded by glialprocesses and a basal lamina.

As early as stage 24, synaptic contacts have been observed in the bulb, between axonal and dendriticprofiles.

Olfactory glomeruli (i.e. the typical bulbar differentiation) become obvious from stage 26; in addition, theperikaryons 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 takeplace before stage 26, some of the olfactory fibres may have synaptic contacts with bulbar cells prior to stage23.

Development of the nervous system 221Developmental synchrony of innervation, cutaneous field production of

NGF and neuronal responsivenessAlun 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 beensuggested that for certain peripheral neurons nerve growth factor (NGF), transported from the peripheralterminal-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 andresponsiveness we have studied the mouse trigeminal ganglion and its cutaneous field at closely stagedintervals throughout development. Peripheral fibres initially contacted the epithelium of the mandibularprocess by E10-5 and the maxillary process by El l . This coincided with the stage in vitro during which themagnitude of neurite outgrowth from ganglion explants was significantly increased by NGF (Davies andLumsden, 1984). NGF responsiveness persisted throughout and just beyond the period of natural neuronaldeath (E13 to E19).

Neurite outgrowth elicited from E10 and El l ganglia by co-cultured maxillary process was not affected byantiserum to isogeneric NGF. Neurite outrgrowth in E12 co-cultures, however, was virtually abolished byanti-NGF (Lumsden & Davies, 1983).

The synchrony of peripheral innervation and NGF responsiveness together with the demonstration ofNGF production by presumptive cutaneous tissue at an appropriate stage of development substantiate thehypothesis 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 nervegrowth factor responsiveness in the developing mouse trigeminal ganglion. /. Comp. Neurol. 223,124-137.

LUMSDEN, A. G. S. & Davies, A.M. (1983). Earliest sensory nerve fibres are guided to peripheral targets byattractants other than nerve growth factor. Nature 306, 786-788.

Differing responses of neural crest and placode derived cranial sensoryneurons to nerve growth factor (NGF)

AlunM. Davies 1 and Ronald M. Lindsay 2.]Department of Anatomy, St. George's Hospital MedicalSchool, 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 chickembryos to ascertain whether NGF responsiveness is related to neuronal ontogeny

NGF elicited pronounced neurite outgrowth from neural crest derived explants; the dorso-medial part ofthe trigeminal ganglion and the superior ganglion of cranial nerves DC and X. This response was first evidentat E6, reached a maximum between E8 and E l l and gradually declined through E16. Explants in which theneurons were of placodal origin varied in their response to NGF. Neurite outgrowth from explants of theventro-lateral part of the trigeminal ganglion and vestibular ganglion was negligible and uninfluenced byNGF. The geniculate, petrosal and nodose ganglia exhibited an early moderate response to NGF which wasfirst evident by E5, reached a maximum by E6 and declined rapidly through later ages. In dissociatedneuron-enriched cultures at E6 and E9, however, only neural crest derived neurons survived and grew in thepresence of NGF.

We conclude that of sensory neurons only those derived from neural crest require NGF duringdevelopment whereas those of placode origin do not. We suggest that the NGF-promoted neurite outgrowthfrom early geniculate, petrosal and nodose explants emanates from neural crest derived cells which surviveor differentiate within explants under these particular culture conditions.

222 Development of the nervous systemFunctionally distinct sensory neurons have different trophic

requirements during developmentAlun M. Davies 1 and Ronald M. Lindsay 2. ^Department of Anatomy, St. George's Hospital MedicalSchool, 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 dorsalroot ganglia (DRG). The survival and growth of embryonic DRG neurons is promoted by nerve growthfactor (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 ofcutaneous and visceral receptors and proprioceptors. In the absence of specific markers for thesefunctionally different types of sensory neurons it is not possible to determine whether a relationship existsbetween the innervation target of sensory neurons and specific neurotrophic factor requirement.

To investigate this possibility we have compared the neurotrophic requirements of embryonic chick DRGwith the dorso-medial part of the trigeminal ganglion (DM-TG). The DM-TG like DRG is neural crestderived but unlike DRG has a mainly cutaneous distribution. Over the age range studied (E6 to El l ) , NGFpromoted survival and pronounced neurite outgrowth from explants and dissociated neuron-enrichedcultures of both DRG and DM-TG. Whilst extracts of chick eye, liver and spinal cord also elicited a markedresponse from E8 and older DRG neurons, DM-TG neurons were almost entirely unresponsive to theneurotrohic activity of these extracts. We suggest that this difference is a consequence of the more restrictedsensory distribution of DM-TG neurons.

BARDE, Y. A., EDGAR, D. & THOENEN, H. (1983). New neurotrophic factors. Ann. Rev. Physiol. 45,601-612.

Synthesis and cloning of complementary DNA to polyadenylated RNAfrom chick embryo spinal cord: identification of tissue specific

sequencesJ. 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 spinalcord we have constructed a cDNA plasmid library representing expressed sequences of 7-day embryonicchick spinal cord. Double-stranded cDNA synthesized from spinal cord poly(A)+ RNA was annealed intothe Pst-1 site of PBR322 using dG - dC homopolymeric tails and annealed vector-cDNA then used totransform competent E. Coli HB101 cells (4xlO4 transformants per fig DNA). To identify sequencesexhibiting tissue-specific expression patterns the library was screened by colony hybridization of 1)32P-labelled cDNA probes synthesized from poly(A)+ RNA of spinal cord and liver tissues, and 2) spinalcord cDNA depleted of sequences complementary to liver poly(A)+ RNA (cDNA difference probes). Forscreening with spinal cord versus liver cDNA probes (5x10 dpm per fig), randomly selected colonies werereplicated in ordered arrays and duplicated nitrocellulose filters processed for colony hybridization. In thecase 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. Thesingle-stranded fraction representing sequences unique to spinal cord tissue was then used to probe thespinal cord cDNA library. Some 3-5 % of clones in the library were found to represent sequences expressedin spinal cord, but not detectable in liver tissue. Plasmid DNA isolated from 10 putative spinal-cord-specificclones was restricted with Pst-1 endonuclease and subjected to agarose gel electrophoresis. All plasmidswere linearized by Pst-1 digestion and 5 had excisable cDNA inserts of >300 bp. Thus we have identifiedexpressed sequences of 7-day chick embryo spinal cord which are not present in poly(A)+ RNA of livertissue. Further studies are required to determine the molecular nature and sequence of corresponding RNAspecies. In addition to studies on tissue-specific gene expression, the approach described here may beapplied to analysis of cellular subpopulations from spinal cord e.g. motorneurones, for which suitable bulkseparation methods exist.

Development of the nervous system 223Amphibian 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 ofdifferent molecular weights (200 Kda, 160 Kda and 70 Kda). Described in mammals and birds, are theseneurofilament components present in amphibian neuronal cells and at what developmental stage do theyappear?

Our findings are based on immunofluorescent localization of NIF proteins using two previouslycharacterized antisera against 200 Kda and 70 Kda components (1). Embryonic undifferentiated neuronalcells from P. waltl neural primordium: neural plate+ neural fold (early neurula stage) are cultured isolatedin vitro (2). They differentiate progressively and exhibit phenotypical and biochemical characteristics ofneurons (3).

At the beginning of the culture none of the undifferentiated neuronal precursors bind antibodies againstthe 200 Kda or 70 Kda proteins. This binding is detected when morphological differentiation takes place (2/3day cultures). Both the cell bodies and the processes were stained. After 2-3 weeks, immunostaining of theneurons was very distinctive and bright. The non-neuronal cultured cells do not exhibit any specific labellingwith these two NIF antibodies.

These observations indicate the presence of NIF in amphibian neurons and the early acquisition of NIFexpression 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 cholineacetyltransferase 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-actingfactors which influence the survival and development of spinal motorneurons. We have previously describedthe survival and cholinergic development in vitro of a motorneuron-enriched cell population from E7 chickspinal cord. When grown on hydrated collagen gels, these cells extend neurites and continue to expresscholine 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 expressionoccurs in the apparent absence of endogenous feeder cell activity.

To examine the possible existence of retrograde, soluble factors exerting a trophic influence onmotorneurons, primary cultures enriched in motorneurons were established on collagen gels in the presenceand absence of soluble extracts from a variety of E12 chick tissues, including skeletal muscle. Cultures wereharvested 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 levelcompared to controls. Soluble extracts from other tissues e.g. skin and liver, did not influence significantlythe level of CAT in the cultures. All tissue extracts, at levels above 200 /u,g/ml, did however exert ageneralised cytotoxicity in vitro.

This study shows that while significant levels of CAT activity are maintained in the motorneuron-enrichedcultures in the apparent absence of any exogenous trophic influence, a soluble factor(s) from skeletal musclecan produce a further enhancement in the level of the enzyme. It remains as yet to be defined whether thistrophic activity increases the survival of motorneurons or exerts an inductive effect on CAT expression. Thetissue-source specificity of this soluble neurotrophic activity is compatible with a muscle-derivedretrogradely-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 systemOrigins of the ipsilateral retinal projections in prenatally-enucleated

miceP. 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, FranceThe 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 thedistribution and number of ipsilaterally-projecting ganglion cells (IGcs) in the remaining eye. The presentexperiments were done to study the effects of enucleations done at earlier (prenatal) steps of developmentin C57BL/6 pigmented mice.

One optic tract in normal adult mice, and the optic tract ipsilateral to the remaining eye in mice that hadbeen enucleated at E12, E13, E16, or birth (= E19) was cut and filled with HRP gel. 48 h later the retinaswere reacted for HRP in flat mounts and the location of all IGcs was charted. In some cases also the opticnerves were cut and stained.

In normal mice a mean of 932 ganglion cells (784-1073) are filled in a narrow region along the inferiortemporal periphery of the retina ipsilateral to the HRP injection, and 10-25 in the rest of the retina. In allmonocular mice, most (76-93 %) IGcs lie within the appropriate temporal crescent of the retina. Howeverfollowing enucleations at E16 or birth their number is increased: as many as 1700 cells within, and 300outside of the temporal crescent can occur. In mice enucleated at E12 or at E13 the number of IGcs is in allcases less than in later-enucleated mice, and is even in many cases less than in normal mice: a mean of 625cells (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 temporalcrescent; in mice enucleated at E16, it increases towards the internal border of the crescent. Most ipsilateralfibers in normal and monocular mice course along the lateral and ventral edges of the optic nerve betweenthe eye and the chiasm.

These results are consistent with the hypothesis that temporary excessive ipsilateral projections (fromtemporal+nasal retina) retract through competition with crossed projections, and show that they arepartially retained in the absence of crossed fibers. The decrease in the uncrossed projections when one eye isdestroyed prior to arrival of optic fibers at the chiasm region (E12, E13) suggested however that thepresence of an early crossed projection is required for full development of the uncrossed projections in themouse. Finally our findings indicate that the property of temporal retina to project ipsilaterally is to a largeextent independent of interactions between fibers from each eye at the chiasm or in the visual centers. Itmay be due to retinotopic order in the optic nerve.

Uptake and release of [3H] GABA by a growth cone-enriched fractionfrom neonatal rat brain

Phillip R. Gordon-Weeks, R. Owen Lockerbie and Brian Pearce, Brain Research Group, The OpenUniversity, Milton Keynes MK76AA

A growth cone enriched fraction was prepared from 5-day-old rat forebrain(l). This fraction has asodium-dependent, high affinity uptake system for [3H] GABA with a Km of 4-5 f*M and a Vmax of59-4 pmol/min/mg. Electron microscopic autoradiography unequivocally demonstrated the ability ofisolated growth cones to take up [3H] GABA. Supervision studies of isolated growth cones pre-labelled with[3H] GABA demonstrated a K (25mM)-induced release of J3H] GABA which at its peak was 100 % abovethe pre-stimulus release level. About 50 % of othe K -induced release of [3H] 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 synapseformation.

(1) GORDON-WEEKS, P. R. & LOCKERBIE, R. O. (1984). Isolation and partial characterisation of neuronalgrowth cones from neonatal rat forebrain. Neuroscience, in press.

Development of the nervous system 225The formation of the axonal pattern in the avian embryonic retinaWilli 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 arethought to play a role in the formation of the neural network found in the adult animal. I studied the growthbehavior of axons and the formation of the optic fiber pattern in early retinal morphogenesis. Whole mountsof 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 opticfiber layer increase in size exponentially, but with different rates. Consistently, in none of the species theretinal fiber layer expands symmetrically. It enlarges with polarities along the dorso-ventral and thenaso-temporal axes. The very first axon-bearing ganglion cells appear at stage 16 in the dorsal and centralretinal portion and grow ventrally to merge at the optic disk. As development proceeds, the retina and theoptic fiber layer expand peripherally and ventrally so that the optic nerve head, starting from an initiallyventral position, comes to lie close to the geometrical center of the retina. From stage 23 on, the optic fiberlayer expands faster in the temporal than in the nasal side. Quantitative measurements on the initialpolarisation of young axonal processes show that the axonal growth is highly directed towards the opticfissure and the optic nerve head. The growth polarisation is found at the very onset of growth coneformation and in axons far distant from the next ganglion cells and axons. This excludes fiber-fiberinteraction to be essential for the initial fiber orientation.

ae3Proliferation of embryonic rat sympathetic ganglion cells followingdestruction 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, EdinburghTotal 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 peripheralsympathetic 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 treatedembryos were reduced; NGF causes hypertrophy. In controls, 3-5 % of ganglionic ChAT activity waspost-synaptic; in embryos treated chronically with NGF, 5-6 %; and in bBTX-treated 20 %. NGF increasedganglion cell ChAT to 170 % control and TH to 200 % control; hence the ratio ChAT/TH was littlechanged. Ganglion cell ChAT activity in bBTX treated embryos was 295 % control, and when given NGF inaddition, 328 % control. TH activity was not changed by bBTX treatment. The increased ChAT/TH ratioindicates a partial transformation of transmitter phenotype in sympathetic ganglion cells. NGF and bBTXboth increased sympathetic axon numbers. These effects were additive and embryos given both treatmentshad 4x the normal number.

We conclude that if the proliferation and the transformation of transmitter phenotype of sympatheticneurons which follows destruction of motoneurons is due to a growth factor released from denervatedskeletal muscle this factor is not NGF. It may be a motoneuron growth factor destined to be taken up bymotor nerve terminals and hence normally inaccessible to the autonomic nervous system.

226 Development of the nervous systemA 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 withtheir environment. Growth of axons involves assembly and disassembly of actin filaments. This processappears to be modulated by so called actin associated proteins controlled by external signals. Isolation ofthe actin filament system is therefore a prerequisite for the study of this process. I developed a simpleprocedure to isolate native actin filaments from embryonic neural tissue. This procedure includes gentlehomogenizing of the embryonic chick brain (E12 to E14) tissue followed by short centrifugation to removecell debris. The supernatant was centrifuged through a sucrose gradient (25 to 45 %). The resulting pelletcontains 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 totalprotein) 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 byinjection of the purified filaments into a rabbit. The serum reacted strongly with actin as shown byimmunoblotting.

Expression of proopiomelanocortin-related peptides in the motoneuronin 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 andtf-melanotropin were detected in motor nerve axons in 12- and 25-day-old normal C57BL/6J mice. Thesepeptides were absent in normal adults, but were present in a proportion of motoneurons in adult dystrophicmice. 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 wasinvestigated 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 proopiomelanocortinpeptides, normally a transient event in development, is prolonged into adulthood. The inappropriateneurotrophic influences of these peptides in the adult dystrophic mouse may contribute to myopathicsymptoms.

Development of the nervous system 227T. 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 inhead and foot tissues, which express high levels of activation for head and foot formation, respectively. Inbody column tissue, nerve cell differentiation is induced at sites of head or foot regeneration due to asignificant 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 celldifferentiation 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) causescommitment 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 inthe body column. Differentiation of committed precursors does not occur in control hydra treated withextract but not wounded or in control hydra which were wounded but not pretreated with extract. Thecommitted state is unstable, decaying within 24-36 h following treatment with extract. The wounding effectis relatively short range: three well-spaced cuts per body column are required to induce differentiation of allcommitted 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 ofnerve 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 cellmembranes 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 thetarget 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 membranesdepend on the position of the cells from which the membranes are derived. Using living growth cones astools to investigate cell surfaces it has been established that there are several differences betweenmembranes of anterior and of posterior tectal cells and that, furthermore, nasal and temporal axonsthemselves carry different recognition markers.

Possible connections between position-dependent membrane properties, directional cues for growingaxons, and map formation will be discussed.

228 Development of the nervous systemSegmentation 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 axonsgrow 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 thesomitic mesoderm is rotated 180 ° about an antero-posterior axis, prior to somite segmentation, axons nowgrow through the posterior (original anterior) half of each somite. These experiments show that there mustexist some difference between anterior and posterior cells of the somite, which is undisturbed by rotationand which determines the position of axon growth.

Neural crest and placode derived sensory neurons have differentneurotrophic requirements: placode neurons are unresponsive to NGF,

lack NGF-receptors but do require trophic factorsR. M. Lindsay* andH. Rohrer, Neurobiology Laboratory, National Institute of Medical Research,

Mill Hill, London NW71AAPeripheral 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 isclear that NGF activity is mediated through a neuronal cell surface receptor, the NGF-receptor. In markedcontrast to crest-derived sensory neurons, little is known about the trophic requirements, of placode-derivedneurons. To ascertain whether crest and placode-derived sensory neurons have similar or differentneurotrophic 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 dissociatedneuron enriched culture showed virtually no response to NGF. Over most of the same age range themajority of DRG neurons respond to NGF. At ages earlier than E7 (E4-E6) NGF elicited a modest fibreoutgrowth from NG explants but did not support the survival of dissociated NG neurons in the absence ofnon-neuronal cells or their precursors. In contrast to the lack of effect of NGF over a broad concentrationrange, extracts of liver, brain and spinal cord from one-week-post-hatched chicks supported the survival ofup to 50-60 % of NG neurons in dissociated neuron enriched cultures. Over the age range E6-E12 virtuallyno NGF-receptors (autoradiographic analyses of cultures treated with radiolabelled NGF:I-I25 NGF) weredetected on any NG neurons surviving in culture with liver extract (in the presence or absence of NGF). Atsimilar ages 80-95 % of DRG neurons have abundant NGF-receptors whether supported in culture withNGF 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 229Chemotactic guidance of developing sensory neurites by appropriate

target epitheliumA. G. S. Lumsden* and A. M. Davies, Departments of Anatomy, Guy's and St George's Hospital Medical

Schools, LondonWhen 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 developsmystacial 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 trigeminaloutgrowth. The attraction of developing neurites in this in vitro model appears to be due to a target-derivedchemotactic agent, unrelated to nerve growth factor, which is active during the normal period of initialperipheral outgrowth from the trigeminal ganglion but which declines in activity or influence followingnormal growth cone-target encounter [Ell onward(2)]. We refer to this agent as trigeminal neurotropicfactor (TNF).

We have recently used the same procedure to co-culture early trigeminal ganglia with isolated componenttissue layers of the presumptive vibrissa field after their separation by treatment in the cold with crudetrypsin. Whereas there is no outgrowth towards the mesenchymal moeity, outgrowth towards the epitheliumis as profuse and as oriented as that which grew towards the intact maxillary process in the earlierexperiments.

The emanation of TNF activity from target epithelium indicates that the target itself is capable ofattracting appropriate innervation at a distance ana that nerve fibre outgrowth need not require direction byany property of the mesenchyme - through which cutaneous nerves normally grow to reach epithelial targetsand which, in other developing and regenerating systems, has been assumed to play a specific guidingrole(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 andRegeneration of the Nervous System (ed. J. Nicholls), pp. 107-125, Berlin: Springer.

Histotypic reorganization of cultured nerve cells/. Maher l and D. James 2. lUnite de neuroimmunologie, Faculte de Midicine, Angers 49045, France.

2University College London, London WC1

Cells dissociated from various regions of the brain from embryos of mouse or chick were reported toreaggregate and reconstruct into patterns morphologically similar to the regions from which they werederived (Delong, 1970; Moscona, 1979).

We dissociated spinal cord cells from 7-day-old chick embryos, according to the method of Bird andJames (1973). Cells were plated out on polylysine coated glass cover slips. Cultures were observed by timelapse 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. Reaggregatesmoved and coalesced forming larger ones. Coalescing reaggregates were always interconnected with neuriteprocesses. Semithin sections showed that many reaggregates consisted of a cortical layer of loosely arrangedcells, and a medullary region that was difficult to elucidate. Electron microscopy demonstrated that thecortical layer contained mainly neurons, while glial cells were scattered in the medullary and basal partsamong a complex network of interweaving processes. Segregation of neurons into an identifiable layer in thereaggregates 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 thebasis of histotypic resemblance to the developing spinal cord.

The hypothesis that embryonic nerve cell reaggregates in vitro reflect with fidelity normal histogenesis invivo could not be confirmed in our study.

BIRD, M. & JAMES, D. (1973). Development of synapses in vitro between previously dissociated chick spinalcord neurons. Z. Zellforsch. 140, 203-216.

DELONG, R. G. (1970). Histogenesis of foetal mouse isocortex and hippocampus in reaggregating cellcultures. Develop. Biol. 22, 563-583.

MOSCONA, A. A. (1979). Cell aggregation: Construction of multicellular systems from cell suspensions. InIntroductory concepts in Develop. Biol. by A. Monroy and A. A. Moscona, pp. 164-204. The Univ. ofChicago Press, Chicago and London.

230 Development of the nervous systemEffect of organophosphorus compound on tryptophan level in brains of

suckling rabbitsDanuta 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 inhibitingacetylcholinesterase (AChE) at the central and peripheral cholinergic synapses. Recently the modulatingeffect of these compounds on the concentration of serotonin (5-HT) in brain has been found. Sinceserotonin plays a part in the neurogenesis and differentiation of the cells, the present work was performedon suckling rabbits, in which the availability of tryptophan for the synthesis of serotonin in brains wasstudied.

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-esterifiedfatty 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 andbrain were studied. In plasma of treated animals increases of NEFA concentration of about 48 %o and freetryptophan of about 44 % were found. The concentration of whole tryptophan was not changed. Thedecrease 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 tryptophanlevel was higher than in controls by about 59 %.

The results obtained in the present study suggest the following pathomechanism of changes. In plasma ofanimals treated with dichlorvos, the high NEFA level binds more albumin and thus competes withtryptophan for these proteins. The concentration of free tryptophan increases. The high availability of freetryptophan in plasma increases the binding of tryptophan with the transport carrier through the blood-brainbarrier (BBB). Moreover, the level of LNNA, which compete with tryptophan for this carrier, decreasesand tryptophan can more easily cross BBB. In consequence the concentration of brain tryptophansignificantly increases.

Embryonic loss of spinal motoneurons: competition orpredetermination?

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 toassay motoneuron numbers. 83 % of motoneurons present on E15 (6940) were lost by E21 (1168). Of thisloss, 79 % occurred between E15-E16. Some motoneurons normally destined to die were rescued from celldeath 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 about24 h after paralysis was initiated. This indicates that motoneurons are committed to die one day before theirloss and paralysis rescues all those not so committed. Thus most motoneurons are determined to die on E14or earlier, when only a small proportion of primary myotubes have differentiated, end plates have notformed 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 motoneuronsprojecting 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 incontrols. 79 % of the E15 depleted number of motoneurons was lost by E21 and 78 % of death occurredwithin 24 h. By E21 the ventral root contained 60 % of othe control number of axons. Cell death did notadjust the numbers of embryonic motoneurons to the normal final goal by E21 and we suggest that the deathof individual motoneurons may be predetermined.

Development of the nervous system 231Cell surface antigens and CGAP filaments in non-myelin forming

Schwann cellsR. 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 weshow that a group of three antigens are co-expressed by the non-myelin forming Schwann cells of peripheralnerve trunks. While these antigens are absent from myelin forming Schwann cells they are expressed byastrocytes and enteric glia.

We compared the distribution of two surface proteins, Ran-2 and A5E3 antigen, both defined bymonoclonal antibodies, with the distribution of the intracellular intermediate filament protein GFAP inperipheral nerve trunks of adult rats using double label immunofluorescence. It was clear that the expressionof all three proteins is confined to the same population of Schwann cells, namely, the Schwann cellssurrounding the unmyelinated axons. The myelin forming Schwann cells surrounding the larger axons didnot express immunohistochemically detectable amounts of any of the proteins. In teased nerve preparationsfrom the preganglionic sympathic trunk, sciatic nerve, brachial plexus, dorsal and ventral roots, a completecorrespondence was found between expression of Ran-2 and GFAP, and between A5E3 and GFAP innon-myelin forming Schwann cells. Schwann cells positive for Ran-2 alone, A5E3 antigen alone, or GFAPalone were not found.

In development A5E3 disappears from Schwann cells during the first few days of myelination (postnataldays 3-5) being retained only on those cells not forming myelin. Conversely, if myelin forming Schwanncells 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 gliain the P.N.S. and not by other cell types, suggesting that this may reflect some common function for thesethree cell types.

Choice of motoneuron paths after removal of target muscle duringpostembryonic development of an insect

D. R. Ndssel and P. Sivasubramanian, Department of Zoology, University of Lund, Sweden. Department ofBiology, University of New Brunswick, Fredericton, Canada

Leg motoneurons survive limb bud removal in embryonic locusts, differentiate and form axons thatchoose new routes out of the CNS (Whitington & Seifert (1982), Devi Biol. 93, 206-215.). We areinterested in where the motoraxons grow after removal of their normal target muscle. Left mesothoracic legdiscs were extirpated from prepupae of the fleshfly Sarcophaga. The left mesothoracic leg nerve is missing inthe 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 ipsi-and contralateral side, or intramuscularily into different sets of indirect flight muscle. We found labelling ofnormal-appearing motoneurons in the deprived neuromere after either of the following markerapplications: ipsilateral pro- or metathoracic leg nerves, or dorsal longitudinal- or dorso-ventral flightmuscles. The axons chose only one nerve root in each specimen. When the axons run to pro- ormetathoracic legs, almost the full compliment of leg motoneurons were labelled centrally. Injections intoindirect flight muscle, however, revealed fewer than normal number in the deprived neuromere, indicatingthat some motoneurons might run to other muscle supplied by the same root. Extirpation of a leg discfollowed by implantation of another leg disc only rarely led to motorinnervation of the ectopic leg. This waswhen 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 datasuggests that motoneurons deprived of targets follow adjacent axonal tracts of sensory or motoneurons andthat innervation of muscles of an ectopic leg occurs only if the sensory neurons of this leg find the correctneuromere in the thoracic ganglia. We do not know yet whether the motoneurons form functional synapseswith muscle in their new paths.

232 Development of the nervous systemP Nerve growth factor (/JNGF) receptors in chick embryonic

developmentG. 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 of125I-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 affinitybinding was observed to dorsal root ganglia (drg), to dorsal and ventral roots, to sympathetic ganglia (sg), toperipheral nerves, to myotome and to spinal cord (sc). At E4 anterior roots and myotome were especiallyheavily labelled with 1*Ij8NGF. With development labelling of the drg increased and was particularlyprominent in the mediodorsal region of the ganglion. The same labelling pattern was observed for cranialsensory ganglia. Parasympathetic ganglia showed weak but distinct jSNGF binding at E8, but were negativeat E12 and onward. In contrast to the drg and sg (cell somata) peripheral nerves had lost their NGFreceptors by E20. The gross labelling of skeletal muscle in early development gave way to 125I/8NGF bindingto a restricted zone at the muscle-connective tissue border by E12, from where it was lost by E20. In the scthe lateral motor column was NGF receptor positive between E4 and E12. Labelling of the area of thedorsolateral tract of the sc, reaching into the zona terminalis and substantia gelatinosa was seen at E6 andwas still present at the time of hatching. Labelling of the cranial nerve entry zones in the brain stem and thedorsal root entry zone in the sc was observed between E4 and E18. The results of this mapping of the spatialand temporal distribution of specific jSNGF binding sites raise a number of unexpected questions concerningthe role of /8NGF or /JNGF like factors in embryonic development.

Cell interactions in development of visual systemPaslco Rakic*, Section of Neuroanatomy, Yale University School of Medicine, New Haven, Conn. 06510,

USAGenesis 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 celldivisions, 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 thatorderly 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 thanin the adult monkey. Over one million axons are rapidly eliminated within the first 3-4 weeks of the secondhalf of gestation - the remaining half million are depleted at a slow rate during infancy. Monocularenucleation during midgestation reduces elimination of axons in the remaining eye, demonstrating that thenumber of axons depends on the competition between terminals originating from the two eyes. Initiallyintermixed axons subserving the two eyes sort out to form synapses in the appropriate laminae of the lateralgeniculate nucleus (LG) and appropriate patches of the superior colliculus. Coincidence of terminalsegregation and a rapid wave of axonal depletion indicates that this selective elimination plays a role inestablishment of appropriate synaptic connections. Similar development from the diffuse to segregatedphase is observed during development of geniculcortical connections. After completion of the segregationphase, synaptic density rapidly increases in the retina, LG, and the visual cortex where it transiently exceedsadult values. To our surprise, synaptic connectivity in central structures is initiated in advance of theperipheral centres. Furthermore, synapses between amacrine and ganglion cells in the retina occur beforedevelopment of true line projections from the receptors via bipolar and ganglion cells. The connections aremodifiable: enucleation of an eye during the first half of gestation results in contacts between the axons fromthe remaining eye and neurons in appropriate laminae of othe LG and in inappropriate ocular dominancecolumns. However, the rate of synaptogenesis, ratio and size of various synaptic classes is similar in controlsand animals prematurely exposed to light. These studies collectively reveal the dynamic nature ofestablishment of synaptic architecture that requires a balance between various inputs and co-operationbetween genetic and epigenetic factors. Although the molecular mechanisms of these events are notunderstood, the available data reveal the multifactorial nature of the system that cannot be explained by apairing of recognition molecules without taking into account competitive interactions among all the neuronsinvolved in the formation of these circuits in vivo.

Development of the nervous system 233Use of WGA-HRP to assess the progress of map refinement after

regeneration of the goldfish optic nerveE. 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 iontophoreticallyinto the normal goldfish tectum, is taken up locally by optic terminals and labels a small cluster of ganglioncells in the retina (Cook & Rankin, 1984). This method of demonstrating retinotopy can be used to followthe 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 indiurnal lighting. After 28-91 days, WGA-HRP was injected iontophoretically at a caudodorsal site on theleft 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 (fivefish), 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 nowshowed a pair of irregular aggregates, loosely linked along a radial line. Later in regeneration (day 70, twofish; 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 bynon-retinotopic terminals, growth cones of misrouted axons, or both (Cook & Rankin, 1984). The first, atleast, 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 theretinotopic 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 retinotectalpathways of goldfish. J. comp. Neurol., 223, 57-76.

Differential innervation of dermal and epidermal explants by sensoryneurons in tissue culture: a time-lapse analysis

R. SaxodandJ. M. Verna, Laboratoire de Biologie Animate, ERA CNRS621, University de Grenoble 1, BP6838042 Saint-Martin-d'Heres Cedex, France

In establishing skin innervation, sensory nerve fibers interact with cellular and acellular cutaneouscomponents. 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 eitherdermis or epidermis from 7-day chick embryos.

After a few days of culture, two different patterns of neuritic growth were obtained: in co-cultures withdermis, the initial orientation of neurite elongation is maintained even after encountering cells migrating atthe periphery of the dermal explants. On the contrary, neurites tend to avoid epidermis and so deviatearound the explant.

Morphometric time-lapse analysis of cultures show that, on a collagen substrate, neuritic growth conescontact epidermal cells but then quickly withdraw, without establishing the close associations with the cellsurface observed with dermal cells. Nerve fibers then progress along the edge of the epidermal layer. On apolylysine substrate, on the other hand, neurites do not contact the epidermis explant but deviate around itat 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 cellmembrane incompatibility between neurites and epidermal cells, the nature of which is not yet known.

234 Development of the nervous systemCell surface analysis in multilineage systems

Fritz Sieber and Maya Sieber-Blum, Departments of Medicine and Cell Biology and Anatomy, The JohnsHopkins University School of Medicine, Baltimore, Maryland 21205, USA

Many experimental systems for the study of cell differentiation are not readily amenable to directbiochemical analysis because the relevant cell populations are present in small numbers and difficult topurify. However, when clonal assays for progenitor cells are used in conjunction with suitable molecularprobes, insights into some molecular aspects of cell differentiation are possible. In this communication, wereport on the use of merocyanine 540 (MC 540)-mediated photosensitization in the analysis of plasmamembrane changes during hematopqiesis and neural crest cell development. MC 540 is a negatively chargedfluorescent dye that binds preferentially to fluidlike or cholesterol-free domains in the outer leaflet of thelipid 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 establishcharacteristic kinetics of photosensitization for different classes of quail neural crest cells and murine andhuman hematopoietic stem cells. Among quail neural crest cells, heterogeneity with regard to theirsensitivity to MC 540-mediated photosensitization was observed already on the first day of secondaryculture, i.e. four days before the onset of melanin and catecholamine synthesis. Neural crest cells that gaverise to pigmented cells only were markedly less sensitive than cells giving rise to unpigmented and mixedprogeny. Among murine hematopoietic stem cells, late erythroid progenitors were the most sensitive cellsfollowed in order of decreasing sensitivity by early erythroid progenitors, megakaryocyte progenitors, day7-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 sensitivitywere found between developmental stages that are thought to be only one or two cell divisions apart. Insummary, these results indicate the fluorescent dye MC 540 is a useful probe for developmentally regulatedplasma membrane constituents. Since MC 540 expresses little, if any, cell cycle specificity and reacts withthe lipid portion of the plasma membrane, it complements cell cycle specific drugs and lectins and antibodieswhich recognize carbohydrates and proteins. (Supported by NIH grants AM 27157 and HD 15311).

Proliferation and differentiation of quail neural crest cells in a definedculture

Maya Sieber-Blum* and Hitesh R. Chokshi, Department of Cell Biology and Anatomy, The Johns HopkinsUniversity School of Medicine, Baltimore, Maryland 21205, USA

Cultured avian neural crest cells give rise to several differentiated phenotypes and thus represent anattractive experimental system for the study of cell differentiation. However, so far the analysis of theunderlying regulatory mechanisms has been hampered by two factors: 1) the contamination of cultures withcells of noncrest origin and 2) the complex growth medium that contained two ill-defined components, horseserum 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 definedculture medium that supports the proliferation and differentiation of quail neural crest cells. Neural tubeswere explanted into culture dishes that had been coated with collagen and fibronectin and were cultured inmedium MCDB 202 supplemented with insulin, transferrin, cortisone, gonadal hormones, vitamins, andtrophic factors. The explanted neural tubes adhered to the substratum and the neural crest cells emigrated inthe usual fashion. Cell proliferation was, however, slower than in complex medium. After 4-6 days allcultures contained pigmented cells that were densely packed with melanin granules and had the typicalappearance of melanocytes. Twenty-five percent of the cultures contained adrenergic neurons as indicatedby intense formaldehyde/glyoxylic acid-induced catecholamine fluorescence. Neurons had characteristiclong, varicose processes with growth cones and rounded cell bodies that tended to aggregate with eachother. We believe that the development of this defined medium will prove useful in the study of neural crestcell differentiation under controlled conditions. (Supported by USPHS Grant HD15311).

Development of the nervous system 235Development of the peripheral nervous system from the neural crest:

establishment of phenotypic diversityJulian 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, 94130NOGENT-sur-MARNE, France

The major part of the peripheral nervous system in vertebrates develops from neural crest cells thatmigrate to appropriate sites in the body. Much current interest centres on the mechanisms whereby a widevariety of differentiated cell types arise from this apparently homogeneous embryonic structure. Thedevelopmental potential of neural crest at all levels of the neural axis is significantly greater than thatactually expressed during normal ontogeny, indicating an important role for the embryonicmicroenvironment during the early phases of nervous system differentiation. However, it is not clear a prioriwhether external factors act on pluripotential precursor cells or on cells that are already largelypredetermined on leaving the neural primordium. A number of experimental approaches to this problemhave 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 ofdevelopmental 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 developmentalcapabilities and are partly or totally committed to a particular phenotype before, or shortly after, starting tomigrate. Thus, data from transplantation experiments in vivo suggest an early, irreversible segregation ofsensory and autonomic lineages. Phenotypic diversity increases as definitive neurotransmitter-andneuropeptide-related properties are acquired by developing autonomic neurons. A number of these eventscan be reproduced in vitro, where evidence has been obtained for the existence of a least two types ofneuronal precursor in neural crest.

Quantitative and qualitative measurement of acetylcholinesterasechanges in the central nervous system and reproductive organs of mice

during low and high doses of a-chlorohydrinP. 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 theacetylcholinesterase (AChE) changes in the reproductive organs and central nervous system of mice withspecial reference to the hypothalamic nuclei and related structures, in order to see the mechanism of thisantifertility drug action.

The drug was administered in chronic low dose (15 mg/kg body weight/day) for 20 and 30 days and asingle high dose (90 mg/kg body weight). The enzyme was studied using histochemical, cytophotometric andbiochemical parameters.

Histochemical studies reveal a sharp and progressive decrease of AChE in most of the thalamic andhypothalamic nuclei of the brain, Leydig cells of testis and epiothelium of cauda and caput epididymisduring low dose treatment of 20 days. The enzyme further decreased when the doses and duration wereprolonged up to 30 days. On the contrary, single high dose effect was comparatively less. Biochemicalestimations 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 simultaneouslyaffect the spermatogenesis and sperm maturation. In such case, the effect on the central nervous system maybe secondary; b) the drug may directly affect the central nervous system and thereby alter the cholinergicmechanism, specifically of the hypothalamic nuclei controlling the anterior pituitary and exert a control overthe gonadotropins (FSH and LH) secretion leading to affect the Leydig cells function and thereby alter thespermatogenesis. It may be added here that serum FSH and LH levels are reported to be increased inar-chlorohydrin treated rats(l).

(1) MORRIS, I. & JACKSON, C. M. (1975). /. Endocinol 67, 21-22.

236 Development of the nervous systemHow retinal axons grow in the tectum: branching analysis of axonalarbors 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 shapeof their arbors. We have made intracellular recordings and injected single axons of several different classesof retinal cell.

The filled cells and their tectal arbors are stained in sections or whole mount preparations using a modifiedion intensified DAB reaction. The morphology of the tectal axonal tree in whole mounts is analysed using amotor 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 stereofrom 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 earlierterminations 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 typesof 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 beforeaxons invade, by grafting to the contralateral side of host embryos. The resulting embryos survive well andhealing between graft and host is good, reliably producing clearly reversed wings. The motor pools andtrajectories of the motor axons were traced after retrograde filling from muscle injection of horseradishperoxidase followed by whole mount staining and wax sectioning. The axon trajectories were analysed andvisualized 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 functionallyanappropriate targets in the graft. Thus motor axons which normally would innervate ventral limb flexorsnow 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 positionappropriate to reach their normal targets in the reversed wing. Rostral segments therefore innervate bicepsdespite 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 wouldexpect reversal proximal to the confluence of the three segmental roots into the plexus to give a greatermismatch in the AP rather than DV axis. However we consistently find axon correction with AP reversalsbut no correction after proximal DV reversals. We are presently examining the level of reversal usingchick/quail chimaeras.

It seems that axons are able to detect mismatch in the AP but not the DV axis. This may be related to thedisplacement distance or may be due to an absence of positional cues for the DV axis. The AP axis is stillsubject to positional signals from the zone of polarising activity during the critical period, but there is littleevidence for similar signalling across the DV axis.

Development of the nervous system 237The pathways of fibres from translocated eyes in Xenopus

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 acharacteristic fashion to the optic tectum, where they terminate in retinotopic order(l). We present a recentinvestigation of the distribution of fibres in the optic tract of Xenopus in which the eyes have beentranslocated in embryonic life from a right to a left orbit without rotation. This operation results in avisuotectal projection which, when recorded electrophysiologically, is normal dorsoventrally but invertednasotemporally(2). Labelling of small groups of retinal axons with HRP showed that the fibre trajectoriesfrom dorsal and ventral retina were normal, whereas fibres from nasally placed retina had diencephalicpathways and tectal terminations typical of temporal fibres, and fibres from temporally placed retina haddiencephalic pathways and tectal terminations typical of nasal fibres. Thus from just beyond the chiasma thefibres seem to have already achieved the major uniaxial rearrangement necessary to establish a normal tractdistribution despite the eye translocation. The fibre-sorting required to permit the formation of anaso-temporally inverted visuotectal projection appears to occur not on the tectum or in the optic tract, buteither within the nerve or at the chiasma.

(I)FAWCETT, J. W. & GAZE, R. M. (1982). The retinotectal fibre pathways from normal and compound eyesin 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 mapin Xenopus: developmental aspects. / . Embryol. exp. Morph., 53, 39-66.

Morphogenesis of neuromeres observed at the TEM levelFiona 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 ofthis segmental patterning, based on TEM observation of transversely orientated microtubules andlongitudinally orientated microfilament bundles, together with known kinetic behaviour of the neuralepithelium. The feasibility of the model was investigated by TEM examination of embryos which had beentreated in culture with either cytochalasin D or colchicine.

The model proposes that the neural epithelium, which should expand longitudinally due to thepredominant orientation of the spindle axes in this plane, cannot expand due to the localised strengthenedregions created by the transversely orientated microtubules and hence the epithelium has to bulge. Thebulging occurs along the line of least resistance; the microfilament-rich luminal (apical) border provides agreater resistance than the basal lamina and as a result the epithelium bulges downwards into the underlyingmesenchyme.

238 Development of the nervous systemMethylmercury induced enzymatic changes in the trigeminal ganglia and

hind-brain of ratsK. 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). Thepresent attempt is focused to elucidate the enzymatic changes in trigeminal ganglia and hind brain of ratsdue 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 biochemicalestimations 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-daytreated animals, though the decrease was more pronounced in trigeminal ganglia. 2-day treated animalsshowed 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 theduration of exposure, and vulnerability of trigeminal ganglia to MMC is more conspicuous than in hindbrain. Since MMC binds with the SH groups of enzymes(2), it may bring about decreased activity of thelatter as well as psychosomatic disturbances. The possible disruption of lysosomes by MMC may account foran increased ACP level. Though it has been reported that mitochondnal immunity to MMC(3) in thepresent 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 neuroblastsHistological, histometrical and enzymehistochemical results

H. W. M. van Straaten, F. Thors, E. L. Hoessels, J. W. M. HekkingandJ. Drukker, Department ofAnatomy and Embryology, National University Limburg, 6200 MD Maastricht, The Netherlands

The early development of motoneuroblasts is morphologically characterised by increase of cellular andnuclear size, outgrowth of a lateral cell process, migration in lateral direction and arrangement in the ventralhorn; they originate by proliferation of the matrix. In the chick most basal plate neuroblasts originatebetween day 2 and 4 of incubation. Regulation of this early development is hardly understood, but evidencehas been presented that it proceeds independently of peripheral interaction. Several data suggest thatchordomesodermal material exerts an effect on the early mitotic activity of the neural tube. In the presentstudy 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 nearthe right side of the thoracic neural tube. At 4 days the embryos were fixed in Bodian's fluid (for histologyand 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 implantand the sectional area of the right and left neural tube halves were determined histometrically. Byperforming 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 ofthe neural tube is increased, concomitant with an enlargement of the neuroblast area. Efferent axons leavethe neural tube over a considerable dorsoventral trajectory. When the notochord is located at theventrolateral side of the neural tube, bulging of the ventral horn is not present, whereas a population ofneuroblasts is present more dorsally. In longitudinal direction the effects are completely absent within200 /urn. The effects neither occur if the implant is situated at a distance of more than 100 /um from theneural tube, nor in sham-operated embryos.

Conclusion. The implanted notochord affects developmental processes in the early neural tube within arestricted level. Morphogenesis of the tube, total volume of neuroblasts and arrangement of neuroblasts inthe ventral horn as well as the sites where axons leave the neural tube are influenced. This conclusionsupports our view that the natural notochord plays a regulating role in the early development of neuroblasts.

Development of the nervous system 239Proliferation and differentiation in histotypic aggregates of embryonic

chick retinal cells in the presence of pigmented epithelial (PE) cellsG. 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 andreconstruct all main layers of an intact E10-E14 retina after 14-21 days in rotary culture(l). Now we havetried to correlate patterns of proliferation with differentiation phenomena occurring during the formation ofthese 'retinoids'.

Determination of cell numbers in aggregates after 9 days in culture reveals a 4-fold increase inR-aggregates (composed of retinal cells alone) and a 5-fold increase in RPE-aggregates (retinal cells andPE-cells). Compared with retinae in vivo, the time course and the amount of 3H-thymidine uptake inaggregates shows a decrease to 20 % during the first day and then is similar (R-Aggregates) or evenaccelerated (RPE- aggregates) for the next three days. A double-label procedure showed strikingdifferences in the spatial order of thymidine- and AChE-positive cells within RPE- and R-aggregates. InR-aggregates thymidine- incorporating cells are distributed over the whole aggregate. They express highamounts of AChE at the periphery, but no layered arrangement in the center of the aggregates. InRPE-aggregates proliferating cells are arranged in a concentric band in the outer part and differentiatedcells are localized in the inner part of the aggregate. Concomitantly an AChE-positive layer arises in themiddle of the RPE-aggregate, which possibly is composed of INL cells. It marks a clear border betweendifferentiated and proliferating cells after 2-4 days in culture.

We conclude that the high degree of organization in these aggregates is accompanied by pronounced ratesof proliferation. In RPE-aggregates proliferation and differentiation (AChE) occur in separate laminashowing 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 lineMichael Webb 1, Frank S. Walsh * and Christopher F. Graham 2. 1Molecular Neurobiology Laboratory,

Institute of Neurology, Queen Square, London WC1N3BG. 2 Department 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 insuspension, conditions which favour the formation of large aggregates, and then plated in gelatin coatedtissue culture dishes 7-10 days after aggregation. Cells with a variety of morphologies were found in suchcultures, and approximately 1-3 % extended long branching processes from a retractile cell body. Thesecells 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) andwere labelled by the McAb A2B5 (Eisenbarth et al. 1979): these markers are expressed by neurons and asubset of glial cells and their precursors in the mammalian brain. Expression of these markers was low orabsent in undifferentiated populations of Tera-2 stem cells. In time course studies, tt receptors wereconsistently expressed earlier than A2B5 antigen after treating the cells with RA, although the proportion ofcells expressing these markers was variable between experiments, ranging from 17 %-98 % (tt) and6 %-92 % (A2B5) by 10 days after RA treatment. The expression of A2B5 antigen and tt receptors is notconfined to neurons in the mammalian nervous system. Since there were many cells in the differentiatedpopulations which expressed these markers but lacked both a neuronal morphology and the neuron-specificneurofilament proteins, we examined the expression of glial markers in these cultures. In the differentiatedpopulations, up to 6 % of the cells expressed the marker of immature oligodendrocytes recognised by McAb04 (Sommer and Schachner, 1982). A cell surface antigen expressed only on astrocytes inprimary humanbrain cultures (recognised by McAb Ml/Nl) was expressed on a variable proportion (9 %>-43 %) of thecells, although no staining with anti-GFAB antibodies was found. These results suggest that the humanteratoma may provide a useful model system for the study of early events in the differentiation of a varietyof neural cell types.

EISENBARTH, G. S., WALSH, F. S. & NIRENBERG, M. (1979). Monoclonal antibody to a plasma membraneantigen of neurons. Proc. Natl. Acad. Sci. USA 76, 4913-4917.

SOMMER, I. & SCHACHNER, M. (1981). Monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: animmunological study in the central nervous system. Dev. Biol. 83, 311-327.