Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina

Pergamon

ht. J. Devl Neuroscience,

Vol. 12, No. 6, pp. 567-578, 1994 Elsevier Science Ltd Copyright 0 1994 ISDN Printed in Great Britain. All rights reserved 0736-5748/94 $7.oO+O.W

CHOROID COAT EXTRACT AND CILIARY NEUROTROPHIC FACTOR STRONGLY PROMOTE NEURITE OUTGROWTH IN THE EMBRYONIC CHICK RETINA NESTOR G. CARRI,*tS PETER RICHARDSON~and TED EBENDAL~ *IMBICE,CC 403,190o La Plats,Argentina;SDepartment of NeurologyandNeurosurgery, Montreal

General

Hospital,

Montreal, Quebec, Canada; TDepartment of Development Uppsala University, S-751 23 Uppsala, Sweden

(Received 7 February 1994: revised

McGill University and Biology, Biomedical Center.

26 April 1994; accepted 3 May 1994)

Abstract-Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retina1 explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the El8 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways. Key words: CNTF, CNS, visual system,

retina,

neuritogenesis,

axon elongation,

chick embryo.

Trophic factors stimulate neurite outgrowth and neuronal survival. For the chick retina in vivo and in vitro it has been demonstrated that neurons require trophic support of molecules not only to survive, but also to initiate and support neurite extension. 1~10~57 Ciliary neurotrophic factor (CNTF) is a trophic molecule which was originally identified in conditioned media and embryonic tissue extracts in the late 1970s due to its survival, neurite stimulating and cholinergic differentiating properties.3,20,33,34 The main bioassay system to measure the presence of CNTF involves the use of ciliary ganglion neurons from the ES9 chicken embryo 3J8,23,33where CNTF acts as a cholinergic neuronotrophic factor.67 CNTF bioactivity was first found to be enriched in intraocular tissues, mainly the choroid layer including the pigment epithelium and the iris.3,1s,22,39In particular, extracts of the choroid coat of the chicken embryo at embryonic days 15-18 have been found to be prominent sources for neurotrophic activity ascribed to CNTF. In retrospect, some of the observed effects may also be attributable to fibroblast growth factors (FGFs). CNTF bioactivity was subsequently also found to be present in the rat sciatic nerve59,68 and is known also from a variety of other mammalian and avian tissues including heart, kidney, skeletal muscle and brain tissue.18 The CNTF protein has been purified from the intraocular tissues of the chick eye6 and later from the sciatic nerves of rabbit and rat, 5obased on partial amino acid sequences obtained from the CNTF protein purified from rabbit and rat sciatic nerves.3345@ CNTF encoding cDNA was cloned from these two species45@,65 and the sequence information was used to isolate DNA encoding also the human CNTF.52,54 A growth promoting activity (GPA) for ciliary neurons has also been isolated from the chicken sciatic nerve and corresponding DNA clones isolated and sequenced!3 The GPA shows considerable similarities to the mammalian CNTFs and it may represent an avian gene homologous to the mammalian ciliary neurotrophic factor gene. In spite of the fact that the role of CNTF still remains to be fully characterized, it has been shown that CNTF acts on central and peripheral neurons as well as glial cells.4,51,66At present, CNTF is

$To whom all correspondence 587, S-751 23 Uppsala, Sweden.

should be addressed

at: Department

of Developmental

Biology,

Uppsala

University,

Box

N. G. <‘arri rt d.

56X

also regarded as a factor responding to injury of the CNS.2 The biological effects of CNTF have been extended to include effects such as survival of sensory and sympathetic chick neurons6,24 proliferation and differentiation of E7 chick sympathetic neurons. 2x cholinergic differentiation in developing sympathetic neurons ‘“and enhanced survival of motoneurons.s.ht CNTFalso stimulates survival in other central populations of neurons put into culture such as rat embryo hippocampal neurons,37.47 cortical neurons,“‘( medial septal neurons3t and rat El6 cerebellar neurons.“(’ It has also been demonstrated that some populations of CNS glial cells are affected by CNTF.3’.J4~“” CNTF has been localized in sciatic nerve of adult rat after injury and at the time of regeneration,“’ been shown to prevent cell death in preganglionic sympathetic spinal cord neuron8 and to delay motoneuron death in the pmn mouse mutant showing progressive motor neuronopathy.h3 Considering the broad actions of CNTF. the present study was undertaken with the aim to study the developing retina as a potential target for CNTF. CNTF immunoreactivity has been described in astrocytes in the optic nerve of the postnatal rat. ‘7.6s CNTF has been demonstrated in myelin-related Schwann cells in vivo2” and the CNTF receptor cymRNA found to be abundant in the adult rat retina.3x Hofmanr?” reported CNTF stimulation of choline acetyltransferase activity in cultured retinal cholinergic, possibly amacrine, cells from the ES chicken embryo in culture. Lehwalder et ~1.~~showed survival of purified retinal ganglion cells from the El0 chick embryo in response to CNTF and to choroid extract. Now we have used a bioassay based on the cultivation of organotypic explants from the E6 chick retina on gels of collagen. The system reveals neurite stimulation of retinal ganglion cells and has been extensively studied to characterize tissue extracts from the chick visual pathways and also to examine substratum effects on neurite outgrowth. 10-‘5 We are now using this system also to characterize the growth-stimulating neurotrophic effects of the different neurotrophins on retinal development (Carri et al., to be submitted) and we considered it important to compare the responses to optic tectum extracts and neurotrophins also with the effects of CNTF in its original form as a choroid coat extract and as purified recombinant CNTF on the retinal ganglion cells.

EXPERIMENTAL

PROCEDURES

Bioassay and culture procedures

White Leghorn chicken embryos were staged according to Hamburger and Hamilton.32 A complete description of the bioassay and preparation methods is provided in our previous papersl(“2~14.1S.19.26 Briefly, ~6 embryos (stages 28-29) were dissected in Basal Medium according to Eagle (BME). The collagen type I used in the present study was obtained from rat tail tendons according to the procedure described by Elsdale and Bard.*’ Experiments were performed in 35 mm plastic dishes (Nunc). The collagen coated area was kept under a 100 ~1 drop of BME. Before plating, the explants medium was renewed (the collagen preparation is fully described in a review’“). Retinal explants were taken from the posterior pole of the eye, placed on collagen and incubated for four days at 37S’C in BME and tissue extract (crude extracts or recombinant protein). Embryos E9 were dissected and ciliary ganglia were also collected in BME. Then ganglia were treated for dissociation with 0.12% trypsin Ca2+ and Mg2+-free saline. Ciliary cells were preplated for 90 min on culture plastic to remove flat cells. The neuron-enriched suspension was mixed with collagen type I and placed as a 100 u.1drop in the culture dishes. As positive controls, cells and explants were cultured with the trophic support of the tissue extracts that were obtained from the choroid coat or tectum opticum of El8 chick embryo. Negative controls consisted of cultures with the cells in plain culture medium only. Extraction was done by homogenization in a chilled Potter-Elvehjem homogenizer (1 g of wet tissue in 5 ml of BME, 10 strokes) followed by centrifugation. Protein content of the extracts were determined according to Bradford.” To corroborate that the stimulative effect of a crude extract was due to the CNTF, we performed the same experiments with recombinant protein. The explants were studied with dark-field or phase contrast in an inverted microscope (Fluovert, Leitz). As detailed in our previous studies’s*‘* the length from the margin of the explant to the distal tip of the 10 longest fibre fascicles was determined using a micrometer scale and used as a measure of maximum neurite length. This mean length of maximum neurite fascicles is expressed relative (in %) of the highest values obtained here under

CNTF stimulates retinal fibre outgrowth

569

optimal conditions as a function of time or as a function of concentration in all the figures. For the ciliary neuron cultures, the number of seeded cells being of large, rounded, phase-bright appearance was counted as neurons. Nearly all of them survived in the presence of choroid extract as the neuronal counts obtained were set as 100% for the calculations. The definition of the trophic unit was the concentration of extract required to evoke 50% of the maximum response under each test condition. ~re~uration ~~recumbi~a~t rat CNTF

A PCR fragment encoding the entire rat CNTF was isolated as described ebewhere.30The CNTF was expressed in E. coli as a fusion protein with glutathione S-transferase linked to the CNTF via a sequence that is recognized and cleaved by thrombin. The recombinant CNTF was affinity purified on glutathione-agarose beads and released by thrombin digestion30 Chemicals and antibodies

Basal Medium Eagle (BME) with Earle’s Salts was from Inter-Med AB (Sweden), streptomycin sulfate from Sigma Chemicals (St. Louis, MO, U.S.A.) and Fungizone from E.R. Squibb and Sons (London, U.K.) A monoclonal anti-200 kD neurofilament antibody was obtained from Amersham (Buckinghamshire, U.K.). The secondary antibodies were affinity purified biotinylated goat anti-mouse IgG (heavy and light chain reactive) immunoglobu~n both obtained from Vector Laboratories ~Burlington, CA, U.S.A.). Streptavidin fluorescein isothiocyanate conjugate was from Bethesda Research Laboratories (Gaithersburg, MD, U.S.A.). Fluoromount G was from Southern Biotechnology Associates (Birmingham, AL, U.S.A.). Itnrnunohistochemistry

Immunohistochemistry was performed on the whole-mount organotypic explants or on collagen gels containing ciliary ganglion cells. The staining was carried out maintaining the collagen substrate and explants or cells in the dish. They were rinsed in PBS 10 min and then fixed in 2% paraformaldehyde for 1 h. Dishes were incubated overnight at 4°C with the primary antibody {diluted l:lO-1:200; the control IgG was tested from 12 to 75 pg/mI). Biotinylated secondary antibodies at 10 Fg/ml were applied on the dish, followed by streptavid~-fluorescein isothiocyanate diluted 1:260. Specimens were studied in aNikon microscope. Staining for neuro~lament expression in situ was performed on ciliary E9 dissociated cells and whole-mount explant essentially as described above for the explants. The nuclei were stained with Hoechst-33258 binding to DNA.

RESULTS Retinal fibre outgrowth in response to choroid tissue extract: quantitative comparkons tive extracts and other developing neuron populations

with alterna-

We have previously demonstrated that chick retina responds to tissue extract from the embryonic optic lobe by extension of Iong neurite fascicles. 1oX2S4~15 We now wanted to see if this effect was also elicited by extract of the choroid coat of the chick eye, a known source for neurotrophic activity for the parasympathetic chick ciliary ganglion neurons.3,22,23*25*67In order to compare the two tissues in their capacity to evoke neurite outgrowth in the E6 retina, we prepared extracts from the optic tectum and from the choroid of El8 chicken embryo, grew retinal explants on a collagen gel for four days in the presence of these extracts at different concentrations to examine the length of the resulting nerve fibre fascicles as a function of the protein concentration (Fig. 1). The outgrowth in response to optic lobe extract was as expected from our earlier studies: a pronounced stimulation of long retinal fibre fascicles occurred and a dose-dependent survival effect on the dissociated ciliary ganglion neurons was also evident. Unexpectedly, it was found that choroid coat extracts had an even more potent effect than optic tectum extracts on the formation of long and dense fibre fascicles emanating from the retinal explants (Fig. 1). An example of the mo~hology of the cultures responding to the choroid extract is shown in Fig. 2. The mo~hology of relevant control cultures have been presented previously.t0~r2

N. G. (‘arti

570 100

h

E 6 ncurd

et cd

retina

&F--

t-

I

_

E 18 choroid coat

e-4

E 18optictcctum

,

/

I

I I I

Fig. 1. Comparison of the growth-promoting effects on retinal explants by optic tectum and choroid extracts. The maximum neurite lengths of retinal explants are expressed as percentage relative to highest values obtained. The measurements on the explants were performed in living cultures using phase-contrast optics after four days of culture. In each case data from 10 experiments were included in the figure (mean va1uesrtS.E.M. are shown).

Fig. 2. View of embryonic retinal explant cultured on a collagen gel substratum in medium supplemented with choroid coat extract from chick El8 embryos. The explant faces down towards the collagen gel. Thick extending fascicles are seen. Phase-contrast micrograph. Calibration bar, 100 pm.

We also wanted to know whether a similar relationship in trophic activity existed also in the classical target cells for the neurotrophic activity in the choroid, i.e. the ciliary neurons of the E9 chick embryo. A similar percentage of stimulation in the two cases could indicate, but not establish, that the same trophic molecule is present in both optic tectum and choroid coat extracts and is acting on both neuronal populations tested. The results again showed the choroid extract to be more efficient than optic tectum in enhancing survival in the ciliary neurons (Fig. 3). The best neuritogenic response to choroid coat extracts in retina in vitro was found at a total protein concentration of

571

CNTF stimulates retinal fibre outgrowth

I

Oh1

I

I

I

10

100

1000

Protein cow. @g/ml)

Fig.3. The survival counts

of dissociated ciliary neurons in response to choroid coat and optic lobe extract. Cell were performed in living cultures using phase-contrast optics after two days of incubation. Data from six cultures in each case. The figure shows mean values2S.E.M.

93 pg/ml (Fig. 1). In this case neurite elongation occurred at a mean rate of 13.5 Fm/hr. Ciliary neurons were effectively stimulated by optic tectum extracts at a concentration of 311 kg/ml (Fig. 3). The controls were done with BME medium and survival was below 5%. Ciliary neurons were also tested with choroid coat and survival reached a level of 100% at 187 kg/ml total protein concentration. For both extracts the background values for retinal fibre outgrowth and ciliary neuron survival were approached at a total protein concentration of around 10 kg/ml and the neurotrophic responses thus only obvious at higher concentrations. The better stimulation of ciliary neurons obtained with choroid extract is shown in Fig. 4. Based on the found dose-response relationships, values for the trophic activity units (TU) per mg of protein in the extracts were calculated and are shown in Fig. 5. One trophic unit is the concentration of extract required to evoke 50% of the maximum response in the particular test. The data show that the choroid extract has more trophic units per mg of total protein as determined for both retinal and ciliary ganglion neuron populations. The proportions in neurotrophic activity between the extracts are similar but not identical when tested in the retinal outgrowth and the ciliary neuron survival assays, suggesting that if the active trophic factor is the same in the two extracts, the linearity in evoked response differs when determined as retinal fibre outgrowth length and ciliary neuron survival, respectively. The level of TU per mg determined using the retina thus surpasses the activity indicated by the ciliary ganglion neurons (Fig. 5). Total protein concentrations above 1000 p,g/ml using the optic tectum extract gave clear, negative effects on neurons. This effect may reflect the increased concentration of inhibitory substances. The chick retina responds to recombinant rat CNTF

The strong stimulation of retinal nerve fibre outgrowth by the choroid coat extract concomitant with the ciliary neuronal survival effect evoked by this extract suggested that the active neurotrophic factor may be CNTF (ciliary neurotrophic factor). CNTF has hitherto not been demonstrated to stimulate retinal nerve fibre outgrowth in developing retinal ganglion cells. We therefore tested recombinant rat CNTF purified after expression in E. coli30 on retinal explants for a comparison with its survival effects on dissociated ciliary ganglion neurons. The stimulative effect of recombinant CNTF is shown in Fig. 6, using the maximum values obtained with choroid coat as 100%. Addition of the recombinant protein resulted in a clear stimulation in a dose-dependent manner. Although it is obvious that the recombinant CNTF in our hands mimics only 30-50% of the response evoked by the choroid extract it is nevertheless clear that recombinant CNTF has a strong effect to promote nerve fascicle outgrowth from the explanted E6 chick retina (Fig. 7a). In fact, the proportion of stimulation relative to the choroid extract was higher in the retinal assay than the

572

N. G.

C’arric-‘tcd

Fig. 4. View of dissociated E9 ciliary neurons included in a collagen gel. Culture medium for control (a) or supplemented with El8 optic rectum(b) or El8 choroidcoat extract (c). Longer arrows show prominent elongated neurites. Shorter arrows show cells. Phase-contrast micrograph. Calibration bar. 100 km.

cihary neuron assay (Fig. 6). The SO% level of maximum fibre outgrowth length was thus reached in the retina at a concentration of 1-2 ng/ml of the recombinant CNTF. If CNTF of the same specific activity accounts for all of the trophic activity detected with by the retina in the choroid extract, it can be estimated that around 70 ng CNTF is present in the choroid per mg of protein and that the corresponding value for the optic tectum extract would be around 10 ng of CNTF per mg protein (cf. Fig. 5). Finally, it was considered important to demonstrate that the CNTF-evoked retinal fibre outgrowth emanated from neuronal cells within the retina. This is even more important as it is becoming evident that CNTF may directly influence development of glial cells in addition to neuronal lineages in the embryo. 36*44Thus we used an antibody directed to the 200 kd neurofilament protein for an immunohistochemical examination of fixed retinal cultures. The results show that all fibre bundles protruding from the retinal explants are distinctly positive for the neurofi~ament

573

CNTF stimulates retinal fibre outgrowth Cilianf

Retina

1LIL r *

n

homid

Tcctum

Tcctum

Chomi

Fig. 5. Trophic activity of tectum opticum and choroid coat (expressed per mg of total protein) shown as histograms. One trophic unit (TU) is defined as the activity at which half of the maximum survival or fibre outgrowth responses are evoked.

Reoombiwt

RATCNTF (e. cd)

Cont. of CNTF (q/ml) Fig. 6. Retinal outgrowth and ciliary neuron survival in response to recombinant CNTF. Both maximum neurite length of retinal explants and the survival of dissociated ciliary neurons were expressed in percentages of the maximum values obtained as a function of CNTF concentration. The measurements were performed as described in Figs 2 and 4. In each case data included were from 10 retinal and 10 ciliary experiments. The figure shows mean2S.E.M.

protein (Fig. 7a). The antibody was also checked for its specificity on the easily identified based on their characteristic neuronal morphology. Also under the used conditions (Fig. 7b, c). It is thus clear that the fibre fascicles all have neuronal origin, most likely emanating from the retinal ganglion

ciliary ganglion neurons these cells were positive from the retinal explants cells.

DISCUSSION This paper reports on a strong neurotrophic activity of CNTF on retinal and ciliary ganglion neurons (Figs 14). Data showing this activity on both neurons or comparisons between retinal and target ciliary cells have not been presented earlier. It was previously demonstrated that half of the chick retinal ganglion cells at El0 when cultured for one day were supported by CNTF.42 Moreover, it was recently shown that CNTF can support survival in adult rat retinal ganglion cells in Go56

N. Cr. Carri

574

Fig. 7 FITC neurc

a al

lost?linin ~g of whole-mount gel cultures stimulated by recombinant rat CNTF. Sta linil lg with proteins. Intense staining is seen over neuriter j grc )win g fl.om 1ciliary anti I-200 kD neurofilament and retir lal t:ells. The nuclei are blue due to staining with Hoechst 332158 Ibind ling to DNA. Calibration bars, 100 km.

CNTF stimulates retinal fibre outgrowth

57.5

and that CNTF can rescue rat photoreceptors from the effect of injury.41 A component of the CNTF receptor, CNTFRa, that is required for signaling is also present in the adult rat retina,38 and we have recently shown, by in situ hybridization, that mRNA encoding the CNTFRa is present also in the developing rat retina at El6 and E21 (Carri and Ebendal, in preparation). In the chicken embryo, intraocular tissues have been found to be particularly rich in cholinergic neuronotrophic activity by comparative screening of different eye-parts.3 This trophic activity has so far not been tested on neural retinal explants. As evident from Figs 14, all extracts used in the present study supported survival and outgrowth in both retinal and ciliary neurons assayed as previously described (for retinal explants, see Refs 10-14, 25; regarding ciliary neuron survival assay, see Refs 17, 18). The results thus show the neurotrophic activity of the choroid extract to have similar effects on both ciliary and retinal neurons. Parallel response patterns were also confirmed with recombinant CNTF. Retinal explants attached, survived well and generated neurites in a dose-dependent fashion under the stimulation of the choroid coat extract or the recombinant CNTF protein. The best neuritogenic response was found there at low concentrations of the extract (Fig. 2). It has earlier been found that CNTF is affecting central neurons in rat substantia nigra4” and moreover that CNTF present in the sciatic nerve will stimulate retinal ganglion cell survival in the adult rat. Recently it has been demonstrated that injury down-regulates the expression of CNTF.58 It has also been demonstrated that CNTF has an effect on survival motor neurons of the spinal cord,5 hippocampa137 and cerebellar4’ rat neurons. To extend these observations, we studied both the El8 optic tectum and choroid coat extracts for a comparison with the recombinant CNTF protein in two assays using the E6 retina and the E9 ciliary ganglion neurons. The ciliary cells showed excellent survival with strong fibre outgrowth seen in all neurons identified by immunostaining. This effect was present for both choroid and optic tectum extracts and for the recombinant CNTF protein. It is clear that optic tectum extract gave a trophic response similar to that of the choroid coat but at higher concentrations of the extract (see Figs 3 and 5). El8 optic tectum extract was previously shown to effectively promote extension of embryonic retinal neurites.10,12-15 We now show that this extract also stimulates survival and neurite elongation in dissociated ciliary neurons E9 (Figs 1 and 4). Information is limited regarding trophic support of visual cells and the distribution of neurotrophic molecules in the developing visual system.1,7,25$26We now show that molecules supporting neurite outgrowth from retinal ganglion cells are highly concentrated in both the choroid coat and the optic tectum of El8 chicken embryos. These findings are consistent with the suggestion that retinal ganglion cells derive trophic support from cells near the neuronal cells bodies as well as target tissues and glial cells along the course of their axons. In accordance with previous observations,” the choroid coat was also found to be a rich source of ciliary neurotrophic activity. Although ciliary neurotrophic activity increases in concentration during development of the chick embryo, it can be detected already in E6 embryos. 24 The chicken CNTF remains to be cloned and at present the information on the developmental appearance of CNTF is scanty. The time of appearance of retinotrophic activity in the optic tectum has not been determined. It is clear that the outgrowth in the bioassay could be related to the normal developmental time in which retinal axons reach the anterior part of the optic tectum. This is the time when retinal ganglion cells need the trophic support to survive and differentiate a proper neurite that will reach the target tectum opticum. We can relate this time of outgrowth of retinal ganglion cells axons to the retinotectal projection moment. It is clear enough that CNTF stimulates survival and promotes neurite outgrowth from E9 dissociated ciliary neurons in culture so we cannot disregard the fact that the E6 retinal ganglion cells respond to CNTF in the same way, but it cannot be concluded that this represents neurite outgrowth rather than survival. Previous studies42 showed that CNTFmaintained the viability of dissociated ElOretinal ganglion cells albeit at higher dosage than required for neurite outgrowth from E6 embryos. CNTF at a concentration of 40 rig/ml supported survival of 49% of the retinal ganglion cells 24 hr after initiation of the cultures with half maximum effects being reached at a CNTF concentration of 13 ng/ml. The choroid extract was able to support 36% of the retinal ganglion cells at a total protein concentration of 11 pg/ml with half maximum being reached at 3 tJ_g/m1.42Thus, because the profiles of dose-response curves to CNTF are similar to responses of ciliary and retinal neurons (Figs 1 and DN 12:6-C

576

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Carri et al.

3), it is reasonable to assume that high-affinity receptors for CNTF are found on E6 chick retinal ganglion cells. What is the nature of retinotrophic activity in the tectum and choroid coat of El 8 chick embryos? It seems probabie but unproven that at least some of this stimulus comes from the chick homologue of mammalian CNTF.43 However, the fact that the maximal responses of retinal explants to CNTF is not as good as the maximal response to embryo extracts suggest that factors in addition to CNTF are present in the chick choroid coat and optic tectum. These additional factors could include acidic or basic FGF which also act on ciliary neurons as well as BDNF which does not. The evidence that choroid coat has more retinotrophic activity than ciliary neurotrophic activity (see Fig. S) is consistent with the suggestion that BDNF or another neurotrophin may also be present. We have recently shown that El8 chicken tectum is rich in BDNF mRNA (Hallbook et nE.,to be submitted). Evidence to suggest the presence and function of CNTF in embryonic developmen t is scanty. Stockli ef a[.64 found that CNTF mRNA was not detectable in the rat peripheral nerve until after birth, suggesting that CNTF becomes expressed only after the period of target-regulated neuronal death in the spinal cord motoneurons. A similar developmental pattern was found also in the rat optic nerve.65 In contrast, the CNTF-related GPA was found in the embryonic chick choroid, increasing in expression from E9 to El 1 and remaining high at E19.43 Furthermore, CNTFlacks a typical signal peptide for secretion whereas GPA was demonstrated to be secreted from cells. Recently, mice with the CNTF gene disrupted by homologous recombination show slowly progressing loss of motoneurons postnatally but no interference with development.s3 However, the retinal development in these mice remains to be examined before conclusions can be drawn regarding a function for CNTF in establishing and maintaining the visual pathways. To sum-up, the present report shows that the choroid coat of the El8 chick embryo as well as recombinant CNTF supports a vigorous retinal neurite outgrowth. This pattern of neuritogenesis is well related in length and density to the stimulation achieved by the appropriate target.“’ Moreover, an extract of the optic lobe, comprising a retinal target, stimulates survival of the ciliary neurons in culture. Besides, the survival of ciliary neurons reach the values of the cells culture under the support of the choroid coat. The stimulative effects of molecules contained in the choroid extract gave a characteristic outgrowth pattern higher in values than the recombinant protein. Nothing contradicts the assumption that the choroid coat extract could contain more than the CNTF, as well as the possibility that neurons could express more than one class of receptors which act to bind neurotrophic molecules. Acknort,Ie~ge~zenrs-Nestor G. Carri held a Wenner-Gren Center Research Fellowship IYY3. The work was granted by the Swedish Natural Science Research Council B-BU 4024 and CONICET-PID-BID 1431-91-01 I and Third World Academy of Science Research Grant BC 90-oY9. We are grateful for technical assistance given by Annika Kylberg and Vibeke Nilsson.

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