The dynamics of neurite outgrowth elicited by embryonic chick heart explants in sympathetic, spinal, ciliary and Remak’s ganglia were investigated in collagen gel cocultures. Neurites emerged preferentially on the side facing the heart explants even after only 6 h and continued to increase in density and length for the next 2 days. Removal of the heart explants after only initial stimulation resulted in less-dense neurite outgrowth. Washing of such cultures led to retraction or degeneration of neurites, effects which could be countered by again adding heart explants. Addition of a second set of heart explants on the back of ganglia initiated a second wave of neuritic outgrowth locally. Ganglia extracted from gels separate from their fibre halos and transferred to a second gel did not regenerate neurites unless again stimulated by heart explants. Neurites from additional, distally positioned ganglia failed to advance into parts of the gel shadowed from the heart explants by proximal ganglia. The asymmetry of neurite outgrowths may be explained by local chemokinetic stimulation of extension, possibly in combination with chemotactic orientation of fibre tips up concentration gradients. The results show that the extension of several categories of ganglionic neurites was reversible, being controlled by the concentration of a soluble neuronotrophic factor released from a developing end organ.

The theory that developing tissues emit chemical signals which attract outgrowing nerve axons (neurotropism) was presented by Ramón y Cajal already in the last century (see Jacobson, 1978, for a review of nerve fibre guidance). Such mechanisms received little experimental support until it was demonstrated that implants of tumour tissues producing nerve growth factor (NGF) cause hyperinnervation of viscera in the chick embryo (Levi-Montalcini & Hamburger, 1953; for reviews of NGF see Levi-Montalcini & Angeletti, 1968; Bradshaw, 1978), and that NGF injected into the brain of newborn rats attracts massive ingrowth of sympathetic fibres along central pathways normally devoid of such axons (Menesini Chen, Chen & Levi-Montalcini, 1978). Observations of favoured outgrowth of neurites towards certain tissues (Chamley, Goller & Burnstock, 1973; Eränkö & Lahtinen, 1978) and towards localized sources of NGF in culture (Charlwood, Lamont & Banks, 1972; Ebendal & Jacobson, 1977,a; Campenot, 1977; Letourneau, 1978) were also taken to support the notion that NGF may act as a neurotropic factor (Chamley & Dowel, 1975; Levi-Montalcini, Menesini Chen & Chen, 1978). Little is known, however, of the actual distribution of NGF in developing tissues (see Harper & Thoenen, 1980).

Other growth factors besides NGF which affect survival and neurite extension in neurons (neuronotrophic factors, Varon & Bunge, 1978), were also implicated in embryonic tissues (Ebendal & Jacobson, 1977,a; Helfand, Riopelle & Wessells, 1978; McLennan & Hendry, 1978; Collins, 1978; Adler, Landa, Manthorpe & Varon, 1979). Several reports independently established, through the use of ganglionic or single-cell bioassays of nerve growth activity in embryonic tissues, a developmentally regulated appearance of such non-NGF trophic factors in the heart, liver and intraocular tissues of the chick (Ebendal, 1979; Lindsay & Tarbit, 1979: Landa, Adler, Manthorpe & Varon, 1980). Similar neurite-inducing activities were also present at various levels in other embryonic chick organs (Ebendal & Jacobson, 1977,b). Partial purification of the active material was carried out in the case of heart (Ebendal, Belew, Jacobson & Porath, 1979) and intraocular tissues (Manthorpe et al. 1980).

In order to confirm the developmental role, if any, of these factors, it is vital to discover not only their intraembryonic distribution and chemical properties but also the extent to which they can control density and direction of neurite outgrowth. In an earlier paper (Ebendal, 1979), I reported that chick heart explants co-cultured with different chick ganglia in collagen gels elicited outgrowth of neurites preferentially towards, rather than away from, the stimulatory explants. The present experiments were designed to study the interaction between the heart and ganglia by removing, inserting or repositioning explants at various intervals, in order to gain a better understanding of the possibilities and limitations for a peripheral tissue to control its innervation by way of neuronal growth factors.

Sympathetic ganglia, ciliary ganglia, spinal ganglia and Remak’s ganglia were collected from 8- to 10-day-old chick embryos (White Leghorn), Ganglia were exposed from one side to three explants of 16- to 18-day-old embryonic chick heart ventricle in collagen gels (Elsdale & Bard, 1972) as described earlier (Ebendal, 1979; see e.g. Figs 1 and 2). The chosen age combination results in maximum or near maximum outgrowth of neurites (Ebendal, 1979). The distance between ganglionic and heart explants was about 1 mm. Eagle’s basal medium plus 1 % fetal calf serum was used for the gels. The 35 mm culture dishes containing the gels were incubated for 2 days at 37 °C in a humidified atmosphere with 5 % CO2. Cultures were examined at intervals through an inverted microscope.

Fig. 1.

Sympathetic chick ganglion cultured for 2 days in a collagen gel. Three heart explants were present (left) for the first 24 h and then removed (arrows). An asymmetrical outgrowth of neurites is evoked by the heart tissue.

Fig. 1.

Sympathetic chick ganglion cultured for 2 days in a collagen gel. Three heart explants were present (left) for the first 24 h and then removed (arrows). An asymmetrical outgrowth of neurites is evoked by the heart tissue.

Fig. 2.

Effects on density of neuritic outgrowth of gradually increasing the period when heart tissue was present in the gel during 2 days of culture. The heart explants (HT) were present initially, then removed at the intervals indicated. Neuritic outgrowth from the ganglion (G) was measured as number of intersections between fibres and an eye-piece grid line on the side facing (line a) or opposite (line b) the heart explants. Each point represents the mean ± S.E.M. of eight ganglia of each type.

Fig. 2.

Effects on density of neuritic outgrowth of gradually increasing the period when heart tissue was present in the gel during 2 days of culture. The heart explants (HT) were present initially, then removed at the intervals indicated. Neuritic outgrowth from the ganglion (G) was measured as number of intersections between fibres and an eye-piece grid line on the side facing (line a) or opposite (line b) the heart explants. Each point represents the mean ± S.E.M. of eight ganglia of each type.

A technique of extricating explants from, or inserting new explants into, an already-precipitated gel with the aid of fine watchmaker’s forceps, was used in this study. After pieces were removed from the gel (thickness about 0·2–0·5 mm) a hole was left (Fig. 1). The gel readily accommodated new explants if these were inserted with care. The method also allowed transfer of explants from one gel to another (see Fig. 7). For some experiments the gels were gently washed by several changes of extra culture medium, superimposed on the collagen layer.

Neurite outgrowth was scored after 1 or 2 days by counting the number of intersections seen between nerve fibre bundles and a straight line of an eyepiece graticule positioned through the outgrowth at half the distance between explant boundary and leading fibre tips as shown, e.g. in Fig. 2. Using dark-field illumination, outgrowth was determined, independently for the side of the ganglion facing the original heart explants (side a in Figs 2, 6 and 7) and the opposite side (side b in corresponding Figs). It was previously shown that this method is a reliable measure of neurite densities evoked in ganglia by a series of NGF concentrations (Ebendal, 1979). Measurements were carried out in eight dishes for each combination and are given as means ± S.E.M. in the graphs. For statistical analysis the individual scores were compared using the Mann-Whitney U test (Siegel, 1956).

Neuritic outgrowth and the presence of heart explants

In order to examine how neurite outgrowth depends upon the initial exposure to heart tissue, the heart explants were removed from the gels after various periods (Fig. 2). The outgrowth observed after 2 days on the exposed side (a side) was increased when the heart explants were present for a longer period. This held good for all four types of ganglia tested (Fig. 2). A similar but less marked tendency was seen for the side facing away from the heart (b side). The 12 h scores for the b side are thus significantly higher than for background outgrowth (P < 0·01 for all ganglia) while significantly lower than those for the a side (P < 0·01 for all ganglia). Nearly maximum outgrowth was reached with the explants present during the first day of the two-day culture period (Figs 1 and 2). These results suggest that the density of neurite outgrowth is determined by the level of a soluble stimulant, most probably that partly defined biochemically by Ebendal et al. (1979), and that this stimulant probably accüihulates in the gel with time in consequence of release from the heart explants. Observations of rapid and vigorous neurite outgrowth in ganglia inserted into gels preincubated with heart explants for one or two days support this notion. In the case of preincubated gels the stimulus can indeed be maintained within a local conditioned volume for at least 24 h between removal of heart and insertion of ganglia.

Intermittent observations of cultures showed that normally the first fibres in coculture appeared after 6 h and even then projected towards the heart explants. After 12 h a fairly dense outgrowth of short neurites had normally emerged on the a side (Fig. 3). In order to test whether the outgrowth of neurites progresses autonomously after initial excitation such gels were carefully washed with several changes of culture medium after removal of heart explants. After the initial 12 h of stimulation a subsequent 12 h of washing led to a marked cessation of extension and even to a retraction of the neurites (Fig. 4). After a further 24 h of washing of the gel, outgrowth was totally abolished. Degenerative changes were, furthermore, manifest in the neurite outgrowth after 24 h of stimulation followed by 24 h of washing. However, the negative effects of washing could invariably be overcome by introducing new heart explants to the gel (Fig. 5). In this situation neurite extension was again stimulated and outgrowths formed readily within 24 h toward the new heart explants whether inserted in the original position or on the opposite side of the ganglion. These facts suggest that the stimulative factor can be eliminated from the gel by washing and is thus likely to exert its effects without forming insoluble precipitates on the substratum. Furthermore, neurite extension appears to be a reversible process which requires the continuous presence of the trophic factor to persist.

Fig. 3.

Outgrowth of neurites from a sympathetic ganglion after 12 h of co-culture with heart explants (left). A number of short neurites extends into the gel from the side of the ganglion (side a) facing the heart tissue whereas no neurites leave the opposite side of the ganglion (side b).

Fig. 3.

Outgrowth of neurites from a sympathetic ganglion after 12 h of co-culture with heart explants (left). A number of short neurites extends into the gel from the side of the ganglion (side a) facing the heart tissue whereas no neurites leave the opposite side of the ganglion (side b).

Fig. 4.

The same ganglion cultured for a further 12 h with the heart explants removed (arrows) and the gel carefully washed and left with culture medium on top. The neurites, initially extending toward the heart explants, are retracting.

Fig. 4.

The same ganglion cultured for a further 12 h with the heart explants removed (arrows) and the gel carefully washed and left with culture medium on top. The neurites, initially extending toward the heart explants, are retracting.

Fig. 5.

The ganglion after a further 24 h with new heart explants inserted (arrows) in the original position and the fluid culture medium poured off the gel. Neurite outgrowth is again supported towards the heart tissue (some single migrating cells, but not neurites, can be seen on the opposite side of the ganglion). Phase contrast of living cultures. All at same magnification.

Fig. 5.

The ganglion after a further 24 h with new heart explants inserted (arrows) in the original position and the fluid culture medium poured off the gel. Neurite outgrowth is again supported towards the heart tissue (some single migrating cells, but not neurites, can be seen on the opposite side of the ganglion). Phase contrast of living cultures. All at same magnification.

On the other hand, it was also evident that without initial stimulation by heart tissue the ability of ganglia to extend neurites rapidly diminished: the response to heart explants inserted after a 6 h delay was thus lowered in all ganglia and when 12 h had elapsed the ability to respond was seriously impaired (data not shown).

Effects of additional heart explants

To examine the response to a second competing source of stimulation, additional sets of heart explants were inserted on the other side of the ganglia at various intervals (Fig. 6). This evoked a marked additional outgrowth towards the new explants but did not interrupt outgrowth towards the original heart explants (the values for outgrowth on the a side at 0 and 48 h do not differ significantly for any of the ganglia, P > 0·1). The sooner the new heart explants were introduced (i.e. the longer they were present during the 2-day culture period) the longer and denser became the extra outgrowth of neurites (Fig. 6). The same effects were found even if the original set of heart explants was removed from the gel after the first 24 h.

These results demonstrate that an increased level of the trophic factor on the back of ganglia rapidly evokes a massive extra outgrowth of neurites which would not normally have emerged. Nevertheless it is not clear whether these neurites belong to a separate population of neurons or are collaterals to neurites which were already present in the original outgrowth.

Fig. 6.

Effects on neurite outgrowth of introduction of a second set of heart explants. Determination of fibre density and symbols as in Fig. 2.

Fig. 6.

Effects on neurite outgrowth of introduction of a second set of heart explants. Determination of fibre density and symbols as in Fig. 2.

Transfer of stimulated ganglia

In one series of cultures the ability of ganglia to reform fibre halos lost after initial stimulation was studied. Ganglia responding to heart explants were thus transferred to new gels after 24 h of co-culture (Fig. 7). Upon transfer the fibre halos remained attached to the original gel. Prior to transfer, the outgrowth of neurites was significantly higher towards the heart tissue (P < 0·01 in all ganglia). During an additional 24 h period in the new gel without heart explants, the ganglia failed to generate neurites (Fig. 7) despite prior stimulation (a similar lack of fibre outgrowth was evident also in sympathetic and spinal ganglia which had been incubated floating in NGF-solutions before insertion into control gels). On the other hand, ganglia transferred to newly prepared gels for a second exposure to heart explants regenerated fibre outgrowths of normal appearance, preferentially directed towards the heart tissue irrespective of whether this was positioned on the original a or b side (data not shown). Ganglia transferred to gels preincubated with heart explants for one day, also developed within 24 h dense outgrowths of neurites directed toward the source of stimulation, irrespective of its position relative to the original a and b sides (Fig. 7). However, the outgrowth away from the preincubated heart explants was always higher than from newly inserted heart tissue. Thus, in the situation illustrated in Fig. 7 the shift in fibre outgrowth mainly from the originally exposed a side to the b side exposed to the preincubated heart explants after transfer is significant for the sympathetic and Remak’s ganglia (P < 0·01 and 0·05, respectively) but not wholly so for the spinal and ciliary ganglia (P ∼ 0·1 and 0·2, respectively). These results suggest firstly that the ganglia retained no memory of the signal which initiated and directed neurite outgrowth in the first place and, secondly, that the outgrowth of neurites from the back of ganglia is higher if the stimulant has been spread in advance without being hindered by the bulk of ganglionic tissue.

Fig. 7.

Effects on neurite outgrowth of transfer of initially stimulated ganglia to control gels or gels preincubated with heart for 24 h. Outgrowths were measured after the initial 24 h (left). Ganglia were then transferred and neurite extension scored again after a further 24 h of control culture (centre) or of co-culture with heart tissue (right). Ganglia taken to the gels preincubated with heart tissue were given a reverse orientation with reference to the heart explants. Symbols as in Fig. 2.

Fig. 7.

Effects on neurite outgrowth of transfer of initially stimulated ganglia to control gels or gels preincubated with heart for 24 h. Outgrowths were measured after the initial 24 h (left). Ganglia were then transferred and neurite extension scored again after a further 24 h of control culture (centre) or of co-culture with heart tissue (right). Ganglia taken to the gels preincubated with heart tissue were given a reverse orientation with reference to the heart explants. Symbols as in Fig. 2.

Stimulation of ganglia arranged in tandem

To test further the notion that ganglionic explants may hinder spreading of the trophic factor, doublets of sympathetic ganglia were arranged in tandem facing a triplet of heart explants (Fig. 8). The proximal ganglia presented completely normal neurite outgrowths with fibres extending from the side facing the heart and from both ends of the ganglion. In contrast, the distal ganglia emitted only a few fibres, mainly from their protruding ends, but consistently failed to send neurites into the gel behind the proximal ganglia (Fig. 8). The distal ganglia were positioned less than 2 mm away from the heart explants. This is within a distance normally bridged by the spreading factor although the density of fibres of the asymmetric outgrowth is reduced with an increase in spacing between the co-cultured explants (Fig. 9; Ebendal, 1979).

Fig. 8.

Sympathetic ganglia co-cultured for 24 h in tandem with heart explants (left). The proximal ganglion shows the normal asymmetrical pattern of outgrowth. This pattern is not repeated in the distal ganglion which fails to extend fibres towards the adjacent rear but shows some scattered neurites at both ends. The left and right framed areas indicate positions similar to those shown in section in Figs 11 and 12, respectively.

Fig. 8.

Sympathetic ganglia co-cultured for 24 h in tandem with heart explants (left). The proximal ganglion shows the normal asymmetrical pattern of outgrowth. This pattern is not repeated in the distal ganglion which fails to extend fibres towards the adjacent rear but shows some scattered neurites at both ends. The left and right framed areas indicate positions similar to those shown in section in Figs 11 and 12, respectively.

Fig. 9.

A ganglion at a distance (about 1·8 mm) from the heart explants comparable to that for the distal ganglion of Fig. 8 but with no shielding, proximal ganglion. Although at this distance fibre outgrowth is less dense, it is not impaired as seen in tandem combinations. Darkfield.

Fig. 9.

A ganglion at a distance (about 1·8 mm) from the heart explants comparable to that for the distal ganglion of Fig. 8 but with no shielding, proximal ganglion. Although at this distance fibre outgrowth is less dense, it is not impaired as seen in tandem combinations. Darkfield.

These observations support the idea that ganglia themselves act as barriers locally shadowing the gel from the trophic factor spreading from the heart explants. A model experiment in which methylene blue was allowed to spread through the gel from a piece of filter paper confirmed that only cells at the front and the ends of ganglia were heavily stained (Fig. 10) and that, furthermore, the ganglia effectively prevented dense staining of the gel immediately behind them (not seen well in photographs as in Fig. 10). One possibility suggested by such patterns of labelling is that selective neuronal survival in favoured parts of the ganglia exposed to the heart tissue may account for the asymmetry of neurite outgrowth. To test this, horizontal sections were cut of four ganglia, drawn from a pool of eight ganglia showing preferential outgrowth towards heart explants, and read on a blind basis. The distribution of neurons did not match the asymmetry in outgrowth of fibres but, rather, neuronal survival was excellent throughout the stimulated ganglia (examples of areas are given in Figs 11 and 12). Fibre bundles run parallel to the surface at the back of ganglia whereas in parts more exposed to the heart explants they followed courses perpendicular to the ganglionic surface and extended out into the gel (Figs 11 and 12). In control ganglia cultured without the heart tissue almost no neurons survive. Hence it seems likely, as was concluded also in an earlier study on silver-impregnated whole mounts of ganglia showing preferential outgrowths (Ebendal & Jacobson, 1977b), that a direct influence on fibre guidance causes the asymmetrical outgrowth of fibres.

Fig. 10.

Model experiment showing the absorption of methylene blue by a ganglion in the gel. Methylene blue was allowed to spread from a square of filter paper (arrow) for about 4 h before photography. Staining of cells is heavy at the front and both ends of the proximal ganglion (dark areas). It is also evident in the microscope (but not seen well in the photograph) that the gel behind the ganglion is only lightly stained. Brightfield. Figs 8 to 10 at same magnification.

Fig. 10.

Model experiment showing the absorption of methylene blue by a ganglion in the gel. Methylene blue was allowed to spread from a square of filter paper (arrow) for about 4 h before photography. Staining of cells is heavy at the front and both ends of the proximal ganglion (dark areas). It is also evident in the microscope (but not seen well in the photograph) that the gel behind the ganglion is only lightly stained. Brightfield. Figs 8 to 10 at same magnification.

Fig. 11.

Horizontal section of a sympathetic ganglion co-cultured with heart explants for 2 days. The side facing the heart tissue is shown (a side; see Fig. 8, left frame, for a corresponding position). Numerous surviving neurons are visible together with fibre bundles projecting into the gel.

Fig. 11.

Horizontal section of a sympathetic ganglion co-cultured with heart explants for 2 days. The side facing the heart tissue is shown (a side; see Fig. 8, left frame, for a corresponding position). Numerous surviving neurons are visible together with fibre bundles projecting into the gel.

Fig. 12.

A corresponding section through the part of ganglion facing away from the heart explants (b side; cf. Fig. 8, right frame). Neuronal survival is as good as in Fig. 11 but no fibres extend into the gel. Phase contrast of toluidine-blue-stained 2 μm plastic sections. Same magnification in Figs 11 and 12.

Fig. 12.

A corresponding section through the part of ganglion facing away from the heart explants (b side; cf. Fig. 8, right frame). Neuronal survival is as good as in Fig. 11 but no fibres extend into the gel. Phase contrast of toluidine-blue-stained 2 μm plastic sections. Same magnification in Figs 11 and 12.

The present results demonstrate the strict dependence of ganglionic neurite extension on the presence of a factor spreading from the embryonic chick heart explants. A heat-labile molecular species with an apparent weight of about 40000 dalton extracted from the chick heart presumably mediates this stimulation (Ebendal et al. 1979). Accumulation of the stimulus in the gel is evidently time-dependent (Fig. 2), and once the gel is conditioned the heart explants are no longer needed to evoke the graded neurite outgrowth responses.

Moreover, neurite outgrowth in response to the heart factor would seem reversible since washing of the gels, which is likely to remove the factor, resulted in a neurite retraction (Figs 3 and 4) and this retraction could be reversed by again introducing heart explants (Fig. 5). The progressive development of neurites thus depended upon a continuous trophic support and did not persist after initial triggering. This is analogous to the need to renew the supply of NGF repeatedly in order to maintain ectopic sympathetic fibres which invade the brain of newborn rats upon intracerebral injection of NGF (Levi-Montalcini et al. 1978; Menesini Chen et al. 1978). The observation that extra heart explants will provoke additional outgrowth of neurites on the rear of the ganglia (Fig. 6) indicates that the stimulation of neurites is exerted locally. This accords with the concept of local control of neurite development by NGF presented by Campenot (1977). The fact that either the heart factor or NGF must be present in the gels to propagate neurites in transferred ganglia despite prior stimulation (Fig. 7) lends further support to their functional equivalence in local control of neurite extension.

The neurite outgrowth is easily observed to be stimulated in the main direction towards the heart explants (Figs 1 and 8). A series of definitions relating to migratory behaviour in response to chemical stimuli were proposed by Keller et al. (1977). These referred chiefly to polymorphonuclear leucocytes but they should apply to the locomotory behaviour of extending neurites not withstanding that complications arise in that the stimulants may directly affect survival of neurons. Positive chemokinesis thus denotes an increased rate of locomotion, in random directions, in the presence of a chemical stimulus while positive chemotaxis signifies directional movement up a concentration gradient of a chemical attractant. A combination of these two responses is also feasible and the best way to clarify a specific situation was considered to be direct microscopic observation of the behaviour of individual cells in relation to gradients of chemical stimulants (Zigmond, 1977, 1978). A series of circumstances favours biased chemokinesis as a major mechanism causing the asymmetrical outgrowth of neurites seen in the present co-cultures. Firstly, as noted above (see e.g. Fig. 2) the rate of extension is obviously related to the level of the heart factor present in the gel. Secondly, all fibres extend more or less radially, possibly due to contact inhibition (Dunn, 1971), which means that some fibres seek the source of stimulation whereas others, at the ends of the ganglion, extend perpendicular to this direction (Figs 1 and 8). This pattern hardly supports the theory that neuronal growth cones actively orient up a gradient which should have its highest concentration close to the heart explants. Thirdly, a number of observations (see e.g. Figs 8 and 9) show that the concentration of the heart factor is low behind the ganglia, whether acting as barriers or sinks, which would itself suffice to explain why neurites do not occupy this region. In line with this view, outgrowths in ganglia only partially inserted into the gel, and thus probably less effective as barriers, were markedly less asymmetrical (not shown).

Chemotactic orientation cannot, however, be excluded. It may be that the barrier effect (Fig. 10) by the ganglionic tissue itself locally sets up a gradient of the trophic factor that is steep enough for the neurites to detect. According to observations in leucocytes, chemotaxis is a rapid response, occurring within 15 to 30 min after stimulation and may, for example be at work during initial stages of extension before neurites are visible outside the ganglia. Especially after the recent demonstration of chemotactic orientation in sensory growth cones within 9 to 21 min after application of gradients of NGF (Gundersen & Barrett, 1979) chemotaxis combined with chemokinesis may perhaps be postulated in directed fibre outgrowth.

The present results suggest that developing peripheral tissues have the ability locally to control neurite extension by releasing neuronal trophic factors (Varon & Bunge, 1978). The mechanism in the observed behaviour of neurites may be envisaged as involving binding of the heart factor, at the tips of growing neurites, to cell surface receptors in some way exerting transmembrane control of the mesh work of actin microfilaments (Yamada, Spooner & Wessells, 1971 ; Kuczmarski & Rosenbaum, 1979) and microtubular system both of which may be expected to be directly involved in extension/retraction of neurites (Roisen & Murphy, 1973). Moreover, the time and distance scales (h and mm, respectively) found effective in the present experiments are those considered realistic by Crick (1970) for setting up gradients of morphogens in an embryo. Finally, the control of fibre extension seen here is in general agreement with the direct observations by Speidel (1933) of neuronal growth cones seemingly attracted towards specific regions in the tails of living frog tadpoles.

This work was supported by the Swedish Natural Science Research Council. The cultures were prepared by Ms Annika Jordell-Kylberg and Ms Stine Söderström. Thanks are due to Ms Kerstin Ahlfors for mounting the photographs and to Ms Vibeke Nilsson for secretarial help.

Adler
,
R.
,
Landa
,
K. B.
,
Manthorpe
,
M.
&
Varon
,
S.
(
1979
).
Cholinergic neuronotrophic factors: Intraocular distribution of trophic activity for ciliary neurons
.
Science
204
,
1434
1436
.
Bradshaw
,
R. A.
(
1978
).
Nerve growth factor
.
Ann. Rev. Biochem
.
47
,
191
216
.
Campenot
,
R. B.
(
1977
).
Local control of neurite development by nerve growth factor
.
Proc. natn. Acad. Sci. U.S.A
.
74
,
4516
4519
.
Chamley
,
J. H.
&
Dowel
,
J. J.
(
1975
).
Specificity of nerve fibre ‘attraction’ to autonomic effector organs in tissue culture
.
Expl Cell Res
.
90
,
1
7
.
Chamley
,
J. H.
,
Goller
,
I.
&
Burnstock
,
G.
(
1973
).
Selective growth of sympathetic nerve fibers to explants of normally densely innervated autonomic effector organs in tissue culture
.
Devl Biol
.
31
,
362
379
.
Charlwood
,
K. A.
,
Lamont
,
D. M.
&
Banks
,
B. E. C.
(
1972
).
Apparent orientating effects produced by nerve growth factor
.
In Nerve Growth Factor and its Antiserum
(ed.
E.
Zaimis
&
J.
Knight
), pp.
102
107
.
London
:
Athlone Press
.
Collins
,
F.
(
1978
).
Induction of neurite outgrowth by a conditioned-medium factor bound to the culture substratum
.
Proc. natn. Acad. Sci. U.S.A
.
75
,
5210
5213
.
Crick
,
F. H. C.
(
1970
).
Diffusion in embryogenesis
.
Nature, Lond
.
225
,
420
422
.
Dunn
,
G. A.
(
1971
).
Mutual contact inhibition of extension of chick sensory nerve fibres in vitro
.
J. comp. Neurol
.
143
,
491
508
.
Ebendal
,
T.
(
1979
).
Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia
.
Devl Biol
.
72
,
276
290
.
Ebendal
,
T.
&
Jacobson
,
C.-O.
(
1977
).
Tests of possible role of NGF in neurite outgrowth stimulation exerted by glial cells and heart explants in culture
.
Brain Res
.
131
,
373
378
.
Ebendal
,
T.
&
Jacobson
,
C.-O.
(
1977b
).
Tissue explants affecting extension and orientation of axons in cultured chick embryo ganglia
.
Expl Cell Res
.
105
,
379
387
.
Ebendal
,
T.
,
Belew
,
M.
,
Jacobson
,
C.-O.
&
Porath
,
J.
(
1979
).
Neurite outgrowth elicitep by embryonic chick heart: Partial purification of the active factor
.
Neurosci. Lett
.
14
,
91
95
.
Elsdale
,
T.
&
Bard
,
J.
(
1972
).
Collagen substrata for studies on cell behavior
.
J. Cell Biol
.
54
,
626
637
.
Eränkö
,
O.
&
Lahtinen
,
T.
(
1978
).
Attraction of nerve fiber outgrowth from sympathetic ganglia to heart auricles in tissue culture
.
Acta physiol. Scand
.
103
,
394
403
.
Gundersen
,
R. W.
&
Barrett
,
J. N.
(
1979
).
Neuronal chemotaxis: Chick dorsal-root axons turn toward high concentrations of nerve growth factor
.
Science
206
,
1079
1080
.
Harper
,
G. P.
&
Thoenen
,
H.
(
1980
).
Nerve growth factor: Biological significance, measurement, and distribution
.
J. Neurochem
.
34
,
5
16
.
Helfand
,
S. L.
,
Riopelle
,
R. J.
&
Wessells
,
N. K.
(
1978
).
Non-equivalence of conditioned medium and nerve growth factor for sympathetic, parasympathetic, and sensory neurons
.
Expl Cell Res
.
113
,
39
45
.
Jacobson
,
M.
(
1978
).
Developmental Neurobiology
. 2nd ed., pp.
562
.
New York, London
:
Plenum Press
.
Keller
,
H. U.
,
Wilkinson
,
P. C.
,
Abercrombie
,
M.
,
Becker
,
E. L.
,
Hirsch
,
J. G.
,
Miller
,
M. E.
,
Scott Ramsey
,
W.
&
Zigmond
,
S. H.
(
1977
).
A proposal for the definition of terms related to locomotion of leucocytes and other cells
.
Cell. Biol. Int. Rep
.
1
,
391
397
.
Kuczmarski
,
E. R.
&
Rosenbaum
,
J. L.
(
1979
).
Studies on the organization and localization of actin and myosin in neurons
.
J. Cell Biol
.
80
,
356
371
.
Landa
,
K. B.
,
Adler
,
R.
,
Manthorpe
,
H.
&
Varon
,
S.
(
1980
).
Cholinergic neuronotrophic factors. III. Developmental increase of trophic activity for chick embryo ciliary ganglion neurons in their intraocular target tissues
.
Devl Biol
.
74
,
401
408
.
Letourneau
,
P. C.
(
1978
).
Chemotactic response of nerve fiber elongation to nerve growth factor
.
Devl Biol
.
66
,
183
196
.
Levi-Montalcini
,
R.
&
Angeletti
,
P. U.
(
1968
).
Nerve growth factor
.
Physiol. Rev
.
48
,
534
569
.
Levi-Montalcini
,
R.
&
Hamburger
,
V.
(
1953
).
A diffusible agent of mouse sarcoma, producing hyperplasia of sympathetic ganglia and hyperneurotization of viscera in the chick embryo
.
J. exp. Zool
.
123
,
233
288
.
Levi-Montalcini
,
R.
,
Menesini Chen
,
M. G.
&
Chen
,
J. S.
(
1978
).
Neurotropic effects of the nerve growth factor in chick embryos and in neonatal rodents
.
In Formshaping Movements in Neurogenesis
(ed.
C.-O.
Jacobson
&
T.
Ebendal
), Zoon6,201-212.
Stockholm
:
Almqvist & Wiksell International
.
Lindsay
,
R. M.
&
Tarbit
,
J.
(
1979
).
Developmentally regulated induction of neurite from immature chick sensory neurons (DRG) by homogenates of avian or mammalian heart, liver and brain
.
Neurosci. Lett
.
12
,
195
200
.
Manthorpe
,
M.
,
Skaper
,
S.
,
Adler
,
R.
,
Landa
,
K.
&
Varon
,
S.
(
1980
).
Cholinergic neuronotrophic factors: Fractionation properties of an extract from selected chick embryonic eye tissues
.
J. Neurochem
.
34
,
69
75
.
McLennan
,
I. S.
&
Hendry
,
I. A.
(
1978
).
Parasympathetic neuronal survival induced by factors from muscle
.
Neurosci. Lett
.
10
,
269
273
.
Menesini Chen
,
M. G.
,
Chen
,
J. S.
&
Levi-Montalcini
,
R.
(
1978
).
Sympathetic nerve fibers ingrowth in the central nervous system of neonatal rodent upon intracerebral NGF injections
.
Archo Ital. Biol
.
116
,
53
84
.
Roisen
,
F. J.
&
Murphy
,
R. A.
(
1973
).
Neurite development in vitro. II. The role of microfilaments and microtubules in dibutyryl adenosine 3’,5’-cyclic monophosphate and nerve growth factor stimulated maturation
.
J. Neurobiol
.
4
,
397
412
.
Siegel
,
S.
(
1956
).
Nonparametric Statistics for the Behavioral Sciences
.
New York
:
McGrawHill
.
Speidel
,
C. C.
(
1933
).
Studies of living nerves. II. Activities of ameboid growth cones, sheath cells, and myelin segments, as revealed by prolonged observation of individual nerve fibres in frog tadpoles
.
Am. J. Anat
.
52
,
1
79
.
Varon
,
S. S.
&
Bunge
,
R. P.
(
1978
).
Trophic mechanisms in the peripheral nervous system
.
Ann. Rev. Neurosci
.
1
,
327
361
.
Yamada
,
K. M.
,
Spooner
,
B. S.
&
Wessels
,
N. K.
(
1971
).
Ultrastructure and function of growth cones and axons of cultured nerve cells
.
J. Cell Biol
.
49
,
614
635
.
Zigmond
,
S. H.
(
1977
).
Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors
.
J. Cell Biol
.
75
,
606
616
.
Zigmond
,
S.
(
1978
).
Chemotaxis by polymorphonuclear leukocytes
.
J. Cell Biol
.
77
,
269
287
.

Erratum

Volume 61, pp. 303-316.

Karlsson, Jane: The distribution of regenerative potential in the wing disc of Drosophila

The headings to the second parts of Tables 2 and 3 (pages 309 and 311 respectively) were inadvertently omitted. These headings are supplied below so they can be appended to the appropriate tables.

Table 2.
graphic
graphic
Table 3.
graphic
graphic