DEVELOPMENTAL

Target

BIOLOGY

Organ

43, l‘%158

(1975)

Stimulation

the Developing

of Parasympathetic Mouse

Submandibular

Nerve

Growth

in

Gland

MICHAEL D. C OUGHLIN' Department

of Biological

Sciences,

Stanford

Accepted

University,

November

Stanford,

Californiu

94305

15, 1974

Parasympathetic nerve growth from the mouse submandibular ganglion is stimulated and directed by the glandular epithelium. Cultured alone, the submandibular ganglion shows little axon extension, but in the presence of salivary epithelium, either cisfilter or transfilter, stimulation of axon outgrowth occurs. The capacity to stimulate such outgrowth from the ganglion is restricted, but not completely specific to salivary epithelium. Stimulation of directed outgrowth occurs even through a 0.1 pm pore size filter and over distances of up to 0.5 mm. Preliminary studies with the parasympathetic ganglia of the pelvic plexus show that axon outgrowth in this case is dependent on a target issue, with salivary epithelium being capable of directing nerve outgrowth from this source. INTRODUCTION

It has long been known that proliferation and differentiation of neurons can be affected by manipulating the size of the peripheral zone of innervation (reviewed recently by Jacobson, 1970, Ch. 5). For example, grafting of an extra limb onto an Amblystoma larva will cause hyperplasia of adjacent spinal sensory ganglia, while amputation of a limb bud will cause hypoplasia of the ganglia (Detwiler, 1920). Despite the long history and ample documentation of such studies in L&O, the phenomenon has proved to be very refractory to analysis in uitro. Selective outgrowth of axons between paired explants has been suggested by only a few studies (Chen and Levi-Montalcini, 1970; Seshan and Levi-Montalcini, 1973; Charnley et al., 1973). The latter studies raise the possibility of a “tropic” effect of certain glandular explants on the explanted ganglia, as judged by selective outgrowth of axons toward a given tissue. Such a “tropic” effect of target tissue on nerve growth was postulated by Ramon y Cajal in the early part of this century in regard to the innervation of epithelial elements (1919). ’ Present address: Department McMasters University, Medical Ontario, Canada L85 459.

of Neurosciences, Centre, Hamilton, 140

Copyright All rights

0 1975 by Academic Pregs. Inc. of reproduction in any form reserved.

The studies of Attardi and Sperry (1963) on optic nerve regeneration demonstrated a highly specific influence of target cells on the regrowth of nerve fibers and suggested a chemotactic explanation of nerve specificity. The discovery of the nerve growth factor (Levi-Montalcini and Hamburger, 1953; reviewed by Levi-Montalcini, 1972) provided an example of how a tissue (sarcoma 180) could produce a diffusible factor that would stimulate directional nerve growth. Nevertheless, with the possible exception of the studies of Charnley et al. (1973) and Petersen and Crain (1970), there have been no reports of selective outgrowth of nerve between paired explants of tissue in vertebrate systems (Levi-Montalcini and Seshan, 1973), although various such experiments to test this question have been made over the past 60 yr (Harrison, 1910; Levi-Montalcini and Angeletti, 1961; Masurovsky and Benitez, 1967; Crain, 1968; Silberstein et al., 1971). All the studies of vertebrates heretofore mentioned have made use of isolated sensory or sympathetic ganglia, or central nervous tissue. A previous report (Coughlin, 1975) has shown that there is a close correspondence between epithelial and parasympathetic neuronal development in the mouse sub-

MICHAEL

D. COUCHLIN

Target

mandibular gland, both in time and in space. Is there a causal relationship between these elements? It is known, for instance, that there is a mesenchymal requirement for epithelial development in the gland (Borghese, i950), and the mesenchyme contains the parasympathetic ganglion. There are systems in which nervous tissue is necessary for development (limb regeneration: Singer, 1952; taste bud formation: Guth, 1958; Herbst and Grandy corpuscles: Saxod, 1972; review of such “trophic” relations in Guth, 1968). The present study will examine possible “trophic” (dependence of epithelial development on nerve) and “neurotropic” (dependence of nerve development on epithelium) relationships in the developing gland. Certain factors controlling axon outgrowth from the submandibular ganglion will be reported. A possible “neurotropic” influence of epithelium on axon growth and distribution is examined by means of cisfilter and transfilter recombinations, using methods that have been employed in now classical studies of tissue interactions (Grobstein, 1953). Specificity of interactions will be questioned by means of recombinations of submandibular ganglia with various other tissue types, and by looking at the interaction of the submandibular epithelium with another parasympathetic ganglion (the pelvic plexus). MATERIALS

Culture

AND

METHODS

Methods

Mouse embryos were obtained from pregnant Balb/c females mated with C3H males with the day of discovery of the vaginal plug being considered day 0. The general culture methods are as described in a previous report, the usual culture medium being the modified F12 with 10% fetal calf serum (F12FCSlO; Coughlin, 1975). Several of the later experiments were run with Eagle’s Basal Medium (BME, Pacific Biological Co.) containing 10% horse serum and 10% embryo extract

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(prepared according to Wessells, 1967). (Although the epithelium of the submandibular gland developed better in the enriched medium than in FlBFCSlO, especially in transfilter cultures, and the ganglia showed more extensive nerve outgrowth, the medium did not alter the basic responses of the tissue.) Glands normally were cultured on 25 pm thick, 0.45 pm pore size, Millipore filters, but several experiments were run on plastic filters (Nucleopore Corp.) with a reported pore diameter of 0.1 pm. Wartiovaara et al. (1972) report seeing essentially no penetration of these filters by cell processes. It was very difficult to obtain good adhesion of tissues to the Nucleopore filters and so Millipore filters were routinely used. For recombination experiments, the epithelial rudiment was separated from the mesenchymal capsule (which, in the case of the submandibular gland, contains the ganglion) by treating the glands with 0.3% trypsin-pancreatin (Wessels, 1967) for 0.5-l hr in a CO, incubator at room temperature, and then gently easing the rudiment out of the capsule with iridectomy knives. Epithelia were gently flushed in a small bore pipet to dislodge any remaining mesenchymal cells. Tissues were then transferred to a mixture of equal parts Hank’s balanced salt solution (HBSS) and horse serum and after 15-30 min transferred to culture assemblies or held in medium. Epithelial rudiments of lung (ll-day embryos) were obtained by mechanical separation with iridectomy knives or by trimming off some of the mesenchyme and then using the same trypsin-pancreatin procedure as for the salivary glands (Shib Banerjee, personal communication). Tissues for transfilter recombinations were washed free of horse serum in HBSS before use. The mesenchymal capsule containing the ganglion was stranded in the well of the filter assembly (Wessells, 1967) and covered immediately with medium or HBSS. It is important to cover the tissue

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with fluid immediately, since the surface specific activity of 49.5 mCi/mmole. The tension caused by remaining fluid seeping acetylcholine was dissolved in triple disback in through the filter tends to loosen tilled water, the specific activity adjusted the stranded tissue or pull it free of the with nonradioactive acetylcholine iodide filter. The mesenchyme was then incu(Sigma) to 1.18 mCi/mmole to give approxbated, well side up, for 1-5 hr to allow it to imately 100,000 cpm per reaction at 10m3 M thoroughly attach. The assembly was then concentration. The substrate was then carefully turned over and a single epithelial buffered to pH 7.0 with 0.01 M phosphate, rudiment was pipetted into position. In the loaded onto an AG 1 X-2 column (Biorad, last few experiments it was found that chloride form) to remove free acetate, and there was no need to incubate mesenchyme eluted with the same buffer. High activity well side up for attachment, and that the fractions were pooled and the resultant assembly could be turned over and epithemolarity was determined from sample lium added immediately after the mesen- counts. Passage through the column rethyme had been stranded as described. duced background in subsequent reactions The medium was changed daily and from about 3% of the total counts to 0.3%. cultures were fixed and stained for observaHomogenates were prepared as prevition or removed from the filters for enzyme ously described (Coughlin, 1975) in buffer assays, ordinarily on day 3 of culture. containing 0.05 M phosphate, pH 6.8, 0.001 For experiments with nerve growth fac- M EDTA, pH 6.8, and Triton X-100, 0.1% tor (NGF), the factor was prepared as an v/v. Protein was determined by Rutter’s (1967) modification of the Lowry-Folin homogenate of adult male mouse (Balb/c) salivary glands in HBSS and serially di- procedure. The assay procedure was based on a luted to appropriate concentrations. The effectiveness of the preparations was as- modification (Russell, personal communisayed with control cultures of 8-day chick cation) of the microassay method reported dorsal root ganglia. Lyophilized horse by Potter (1967). Reactions were carried out in scintillation vials in a total volume serum containing antibodies to NGF (Antiof 100 ~1. Unless otherwise stated, the NGF) was obtained from Burroughs-Wellcome. The serum was reconstituted in reaction contained the following components (modified from Wilson et al., 1972): 1 distilled water at 100 mg/ml. Effectiveness of the anti-serum was determined on cul- mM SH-acethylcholine iodide (1.18 mCi/ mmole), 0.2 M NaCl, 0.05 M potassium tures of chick dorsal root ganglia containphosphate buffer, pH 6.8, 0.001 M EDTA, ing 1:500,000 of the crude NGF. pH 6.8, 0.1% v/v Triton X-100, and 5-30 Microscopy pg of the protein to be assayed. For each Tissues were selectively stained for reaction a control was run containing a nerves by supravital staining with methylfinal concentration of 10e4 M BW284C51. ene blue or by histochemical localization of Reactions were incubated at 37°C for 20 cholinesterase using a modification of the min, then but on ice and terminated with Karnovsky-Roots method (1964). Details the addition of 100 11 monochloroacetic of the staining procedures and also of the acid (1 M, pH 3.0). This stops enzyme preparation of the tissues for electron mi- activity, protonates the acetate, and stops croscopy are given in a previous report. hydrolysis of acetylcholine. To the scintillation vials containing the reaction mixture Radioassay for Acetylcholinesterase was added 10 ml of scintillation fluid conSH-acetylcholine iodide was obtained sisting of a mixture of nine parts toluenefrom New England Nuclear Corp. with a Omnifluor (4 g/liter) to one part isoamyl

MICHAEL

Target Organ Control of Nerve Growth

D. COUCHLIN

143

alcohol. Vials were tightly capped, shaken RESULTS thoroughly to insure extraction of the aceEpithelial Development Independent of tate into the organic phase, and counted in Nerve Elements a Nuclear Chicago Mark 1 at 36% efficiency. To test for dependence of epithelial morTotal counts present in the reactions phogenesis on nerve, salivary mesenchyme were determined by adding aliquots of the was divided into cap mesenchyme (resubstrate solution to a mixture of 10 ml of moved from the side of the lobules distal to toluene-Omnifluor and 0.5 ml NCS solubi- the stalk, which has been shown not to lizer (Nuclear Chicago). This mixture gave contain nervous elements) and stalk mesmore consistent and accurate readings enchyme (removed from around the stalk than either toluene-Triton X-100 or Aquaand containing the ganglia.) Epithelia were sol (New England Nuclear). Under these cleaned of mesenchyme and recombined conditions, using column-purified sub- directly with different types of mesenstrate, background was 0.2-0.4s of total thyme and cultured on agar to prevent counts added, and 80-857’ of the acetate excessive spreading of mesenchyme (Fig. was extracted into the counting fluid. The 1). activity is based on the extracted acetate The results are shown in Fig. 2. After 48 and is expressed as the difference in activhr, the extent of morphogenesis is equivaity between the sample and the control lent in the two different kinds of recombipreincubated for 5 min with inhibitor. The nations. After 3 days the epithelium in cap control very rarely showed any significant mesoderm has continued to develop while activity over background. Electric eel epithelium in stalk mesenchyme has AChE (Worthington) showed greater than started to degenerate. 99% inhibition when preincubated with Recombinations with stalk mesenchyme 10e4 M BW284C51 dibromide. stained positively for nerve with both

CAP

Q

MESENCHYME

CULTURED ON AUERBACH RAFTS FILLED WITH 1% AGAR

ion

STALK

YESENCHYME

FIG. 1. Recombinations of salivary epithelia and salivary mesenchyme with and without the ganglia. Whole glands were treated for 0.5-l hr in 0.3% trypsin-pancreatin and the tissues separated as indicated. Recombinations were made on agar-filled plastic rafts and cultured in Grobstein dishes.

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MICHAEL

D. COUGHLIN

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145

11 were grown in the embryo extract enmethylene blue and the cholinesterase stain (Figs. 3 and 6), while those with cap riched medium and likewise showed better mesenchyme showed no staining of nerve growth. by either method, though the epithelium of Nerve Growth on itself stains slightly for cholinesterase (Fig. Dependence Epithelium 4). (Elements believed to be vascular, as previously described, were still found in In recombinations involving stalk mesthese recombinations.) In cases where the enchyme it was obvious that, though the distal end of the epithelial lobule had original positions of the ganglia relative to the direction of epithelial growth was usudegenerated, there were abundant necrotic cells. Extensive areas of the lobule near ally altered from that found in intact glands this necrotic zone were almost devoid of in vivo, the nerve outgrowth from the ganglia was highly oriented. Thus, the basal lamina, so that axons and mesenchyma1 cells abutted on the naked parenchyaxonal bundles tended to converge on the ma1 cells at the periphery. Axons were also area of the epithelium from which lobular outgrowth occurred; furthermore, the found deep within the degenerating epithelium. axons extended outward from that region Of 25 recombinations with stalk meso- in a basically normal pattern (Figs. 3 and derm, nine were cultured with FlBFCSlO 6). This observation suggested that epitheenriched with 20% embryo extract. None of these latter cultures showed the degeneralium might be directing nerve outgrowth. tive changes and all showed greatly in- To test for this possibility, salivary mesencreased growth and bud formation. Of 35 thyme was cultured with and without epirecombinations made in cap mesenchyme, thelium both cis- and transfilter and subse-

FIG. 3. Salivary epithelium recombined with ganglion-containing mesenchyme, cultured on Millipore filter for 3 days and stained with methylene blue. Large axon bundles (Ax) extend over some distance from the elements of the ganglion (SG) to reach the area from which initial epithelial outgrowth occurs (C). This area usually becomes cystic. Axons then extend out from this point to practically encapsulate the epithelial lobules (SEp). x 56. FIG. 4. Salivary epithelium recombined with cap mesenchyme (no ganglion) and cultured for 3 days. Stained for cholinesterase. No nerve elements can be detected, and the epithelium (SEp) is well developed. x 56.

FIG. 2. Epithelia cultured in salivary mesenchyme, with and without the the time course of development of an epithelium cultured on agar with cap D-F show development with the ganglion present. At 48 hr morphogenesis some degeneration is noted in epithelium cultured with the nerve element

submandibular ganglion. A-C show mesenchyme devoid of the ganglion. in both cases is equivalent. At 72 hr (arrows in F). x 83.

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quently assayed for nerve outgrowth, both by histochemical staining and by radioactive assay for acetycholinesterase. Results

from Staining

In none of the 58 cases where ganglioncontaining mesenchyme was cultured without epithelium did the AChE stain show there to be other than very minimal outgrowth of axons. In medium F12FCSlO very few ganglion cells stained for AChE and no axons could be seen; even under optimal conditions of culture (medium containing horse serum and embryo extract) the ganglion showed no marked outgrowth beyond what might have been present at the time of culture (Fig. 5), certainly in no way approaching the outgrowth in the presence of epithelium (Fig. 6).

Transfilter cultures prepared with the mesenchyme and ganglion below the filter and epithelium above likewise showed marked outgrowth of axons, with growth being confined to (or at least oriented toward) the area beneath the epithelium (Figs. 7 and 8). In cross sections of the transfilter cultures, cellular extensions project from areas of concentrations of nerve

VOLUME

43, 1975

toward the epithelium (Figs. 9 and 10). Several transfilter recombinations were made on 0.1 pm Nucleopore filters. A 0.1 pm pore size has been shown to effectively block the passage of cellular processes (Grobstein and Dalton, 1957; Wartiovaara et al., 1972). Salivary tissues did not adhere to the Nucleopore filters as well as to Millipore, so that the mesenchymal mass moved or slid over the filter surface; further, the edges of mesenchyme tended to retract. In those cases where tissues remained attached to the filter, nerve outgrowth could be seen, even though epithelial growth was minimal (Fig. 11). Effect

of Distance

on the Interactions

In a number of cases where the salivary epithelium was recombined with cap mesenchyme and the ganglion-containing mesenchyme was added to the two tissues, the normal pattern of axonal outgrowth did not occur. Therefore, the distance from the near edge of the ganglion to the center of the epithelium was measured in a representative number of the recombinations. Results are given in Table 1. The maximum distance over which the epithelium can act on axonal outgrowth is approxi-

FIG. 5. Whole, ganglion-containing salivary mesenchyme cultured on Millipore filter for 3 days, fixed and stained for choline&erase. Even though grown on BME medium enriched with 10% horse serum and 10% embryo extract, there is essentially no axon outgrowth from the ganglion (SG); it does remain cholinesterase positive. x 75. FIG. 6. Mesenchyme recombined with epithelium and cultured for 3 days on Millipore filter in medium FlPFCSlO. There is abundant axon outgrowth from the ganglion over the epithelium. x 75. FIGS. 7-11. Transfilter cultures of salivary epithelia and mesenchyme, set up as described in the text: FIG. 7. Transfilter recombination on 0.45 rrn pore size Millipore filter. Cultured for 3 days. Centripetally directed arrows outline the epithelium. Some parts of the ganglion (SG) are readily identifiable. Large bundles of axons (Ax) extend out to the periphery of the epithelium and even axons that initially extend out randomly seem to be redirected toward the epithelium. The dense, reticulated area from which the axon bundles extend is similar to that seen in ganglia of some stained preparations of whole glands. x 80. FIG. 8. Same culture situation. Arrows indicate epithelium. A smaller part of the ganglion (SG) has been carried through the culture procedure, but axons extend from it only in the area beneath the epithelium. x 80. FIG. 9. Section through a transfilter culture. 0.45 pm pore size Millipore filter. Stained epon section shows elongate epithelial cells (Ep), and a loosely packed mesenchyme transfilter (Mes). The arrow indicates an area where cell processes extend through the filter. At the electron microscope level there is an accumulation of axons in this area. x 400. FIG. 10. A several-millimeter-thick section of the same culture as shown in Fig. 9 viewed with Nomarski optics. Cell processes extend all the way through the filter, from the mesenchyme side. x 400.

MICHAEL

D.

COUCHLIN

Target Organ Control

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Nerve Growth

147

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DEVELOPMENTAL

BIOLOGY

mately 0.5 mm. Figure 12 shows an example of such a recombination in which axons have extended through 0.45 mm of mesenthyme to reach the epithelium.

VOLUME

Radioassay

43, 1975

for AChE

In order to quantitate the differences in axon outgrowth with and without epithelial stimulation, the activity of acetylcholinesterase was determined in the various cultures. Acetylcholinesterase activity has been used as a marker for neuronal differentiation in several studies (Blume et al., 1970; Wilson et at., 1972; Peterson et al., 1973). As already shown, the cholinesterase stain used in these studies shows that cholinesterase activity is limited to ganglia, axonal processes, and to a much lesser degree the epithelium itself (reported TABLE DISTANCE

DEPENDENCE

1 OF EPITHELIAL-NERVE

INTERACTION

Distance from ganglion to epithelium FIG. 11. Transfilter culture using a 0.1 pm pore Nucleopore filter, cultured 3 days and stained cholinesterase. The epithelium rounded up but not develop. Though it was washed off the filter in -staining process, it occupied the area indicated by dark circle. Axons (arrow) from the ganglion in mesenchyme extend out and converge on the area neath the epithelium. A few exploratory fibrils tend out forming a halo around the area. x 80.

size for did the the the beex-

Number Abundant

up to 0.35 mm 0.4 - 0.5 mm more than 0.5 mm

17 3 0

giving degree” outgrowth

of

Little

None

1 2 0

0 3 8

n Based on the number and size of the axon extending from the ganglion to the epithelium days of culture.

bundles after 3

FIG. 12. Submandibular epithelium was first recombined with cap mesenchyme and then to this was added ganglion-containing mesenchyme. An axon bundle (Ax) extends from the ganglion (SG) over a distance of 0.45 mm before reaching the epithelium. There it branches and extends out over the epithelium. AChE stain. x 85.

MICHAEL

D. COUCHLIN

Target

also by Snell and Garrett, 1958). Since the enzyme activity occurs along the length of the axonal processes and throughout the ganglia, it was considered to be useful as a marker for axon elongation as long as such activity was correlated with the staining results. Characterization

of

AChE

in Embryonic

Salivary The rate of AChE activity is linear with homogenate protein concentrations between 1 and 35 pg per reaction and is linear with time between 10 and 40 min. The substrate concentration curve (Fig. 13 A) is similar to that reported by Wilson et al. (1972) for mouse brain AChE, with substrate inhibition occurring somewhat above 5 x lOma M. The K, for the salivary enzyme is approximately 2 x lo-’ (Fig. 13B), confirmed by several experiments. This agrees with the K, value of 2 x lo-’ reported by Ellman et al. (1961) for erythrocyte AChE. The activity of the enzyme at !G)

Organ

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4°C was 20-30% of that at 37°C (also Wilson et al., 1972). The change in activity in the salivary gland system with time can be seen in Fig. 14. The specific activity (Fig. 14B) of the AChE at 14 and 15 days may be considered a measure of the ratio of axon to epithelium since the epithelium contributes most to the total protein at these stages yet stains only slightly for AChE. The increase in total activity (Fig. 14A) is then an indication of the total axon outgrowth which must occur to maintain and increase the specific activity. The epithelium itself may account for some activity, but epithelium recombined with cap mesenchyme, cultured for 3 days and assayed, shows an activity/gland of 13 and an activitylpg protein of 2.2, activities comparable to those of the 13-day gland. AChE in

Transfilter

Cultures

The results of the radioassay for AChE activity agree with staining results as indi.E.o

T

B

FIG. 13. AChE activity in homogenates of submandibular glands from 15-day embryos. Picomoles 3H-acetate hydrolized from 3H-acetylcholine iodine assayed as in “Materials and Methods”. Each reaction mixture contains 8 rg protein and reaction time is 20 min. Rates of activity were linear between 1 and 35 pg protein per reaction and between 10 and 40 min of reaction time. A. Reaction velocity as a function of substrate concentration. Substrate inhibition occurs somewhat above 5 mM. B. Relation between reaction velocity and substrate concentration, plotted according to the Lineweaver and Burk method. Two separate experiments both give a K, of approximately 2 x lo-‘.

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DEVELOPMENTAL

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5oc 4oc H ,” c E ,

3oc

9 E :

zoc

c

100

1 J ?d c

1

FIG. 14. Increase in AChE activity as a function of developmental stage. Glands were taken from 13, 14, and 15day embryos, pooled, frozen, homogenized and assayed. Glands from 13-day embryos were cultured for 2 days on Millipore filters (13d and 2d cul), and then assayed. A and B measure the activity in relation to the number of glands and the amount of protein respectively. Since the epithelium contributes most to the increase in protein, the activity/gland is considered the more reliable measure of axon outgrowth. Error bars represent the range between two experiments.

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To determine the specificity of the epithelial effect on nerve outgrowth and on enzyme activity, ganglion-containing salivary mesenchyme was combined with various other tissues. Of those surveyed, the lung epithelium-salivary mesenchyme recombination was the most thoroughly studied. The 11-day lung rudiment is at approximately the same stage of morphogenetic development as the 13-day salivary and is known to branch and grow well in culture (Taderera, 1967). In combination with lung, the ganglion shows enhanced outgrowth of axons over controls and a more intense staining reaction within the ganglion itself (Fig. 17). Nevertheless, outgrowth of axons is still much less than that obtained with salivary epithelium, and the outgrowth is not directed toward the epi-

cated by the values in Fig. 15. Mesenthyme containing ganglia cultured transfilter from salivary epithelium shows twoto threefold increase in AChE activity over controls. Specificity

of the Stimulator-y

t

Effect

It is well known that among the inductive interactions described over the years some require the presence of a specific “inducing” tissue (e.g., in lung development), while others (e.g., metanephrogenic mesenchyme or pancreas) respond to a range of tissues, or even, as in the case of the pancreas, to factors isolated from given tissue types (reviewed by Wessells, 1968). Which type of “inductive” interaction are we dealing with in the case of the submandibular ganglion? Is the stimulatory effect specific to the target tissue, or is it a common response to a variety of tissues?

13d MES

13d MES +3d

13d MES TRANS TO EP +3d

FIG. 15. Increase in AChE activity in ganglia cultured transfilter to salivary epithelium. Ganglioncontaining mesenchyme of submandibular glands taken from 13-day embryos was separated from the epithelium by trypsin-pancreatin treatment and (1) assayed immediately (13d Mes), (2) cultured alone on Millipore filter discs for 3 days and assayed (13d Mes + 3d), mean * 1 SE from three experiments and 21 glands, or (3) cultured transfilter from salivary epithelium for 3 days and assayed (13d Mes trans to ep + 3d), mean * 1 SE from five experiments and 40 glands.

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Though there was no obviously directed outgrowth to the lung, neither was there any significant morphogenesis of lung epithelium in salivary mesenchyme in these experiments (also reported by Taderera, 1967, and by Spooner and Wessells, 1970). Nerve outgrowth from the submandibular ganglion might depend on processes occurring during morphogenesis. Therefore, to allow morphogenesis to occur, 11-day lung anlagen were partially cleaned mechanically only around the area of the tracheo.bronchial junction. Ganglion-containing salivary mesenchyme was placed in close contact with the bared epithelium. In the several such recombinations made, lung developed normally but axons showed no outgrowth along the branching lung epithelium. There was, however, fairly good axon growth between the clusters of ganglion 13d MES 13d MES 13d MES + 3d *LUNG EP +SALlV EP cells (Fig. 19). Several other tissues were 3d + 3d also surveyed for a stimulatory effect on FIG. 16. AChE activity in submandibular ganglia nerve outgrowth, two to five recombinain response to cisfilter recombination with lung and salivary epithelia and in response to different media. tions were examined for each case, the age Ganglion-containing salivary mesenchyme (13d Mes) of the embryo donor is indicated in parenwas cultured for three days alone, or with lung thesis: (1) Jaw mesenchyme (13-day); (2) epithelium or with salivary epithelium. Each of the heart atrium (13-day); (3) vas deferens three types of culture was done in medium FlZFCSlO (13-day); (4) mammary gland (13-day); (5) (m ) and Eagle’s Basal Medium with 10% horse serum and 10% embryo extract (I ). Eight to ten ureteric bud (ll-day); (6) urethra (l&day); cultures were pooled for each assay. (7) urogenital sinus (l&day); and (8) preputial gland (&day). (Whole explants of thelium. These results are confirmed by these tissues were untreated and combined directly with submandibular ganglia). The assay for AChE activity in such recombinations (Fig. 16). It is also apparent from effect of the urogenital sinus could not be determined, since it is itself associated these latter results that culture medium with horse serum and embryo extract is with ganglionic elements, presumably the pelvic plexus. Of all the much more conducive to growth in this parasympathetic system than FlSFCSlO. Despite the appar- other tissues, the only one to which axon ent stimulatory effect of lung epithelium on outgrowth from salivary gland ganglia was axon growth, if the nerves are given a choice directed was the preputial gland. Such of lung or salivary epithelium (21 recombioutgrowth occurred even in the presence of nations), almost all apparent outgrowth is competing salivary epithelium (Fig. 20) associated with the salivary epithelium and without cleaning the preputial epithe(Fig. 18). lium of its associated mesenchyme. The lung is known to receive parasympaEffect of Salivary Epithelium on Pelvic thetic innervation, and electron microsPlexus Ganglia copy shows that there are normally large Ganglionic material from the pelvic nerves along the developing trachea. 250

l

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plexus cultured alone behaves similarly to the submandibular ganglion. There is no outgrowth of axons, although the cells retain some staining capacity for AChE. In recombination with salivary epithelium in salivary cap mesenchyme, the ganglion cells send out axons that surround the central area of the salivary epithelium at 2 days (Fig. 21) and extend to the periphery of the epithelial buds by three days of culture (Fig. 22). It is apparent, then, that at least one other parasympathetic ganglion requires an “inductive” stimulus for axonal outgrowth and that salivary epithelium is capable of providing that stimulus in the case of the pelvic ganglion. Effects of NGF and Anti-NGF

As expected from previous reports (LeviMontalcini and Angeletti, 1968), the para-

153

sympathetic submandibular ganglion showed no outgrowth of axons (as judged by methylene blue stain) when the ganglion and salivary mesenchyme were cultured in vitro, despite the presence of NGF in the medium (1:500,000 dilution). This concentration of crude NGF produces optimal axon outgrowth in dissociated cell cultures of chick dorsal root ganglia (Luduefia, 1973). Ganglion-containing mesenchyme was next grown in NGF (1:50,000) and assayed for acetylcholinesterase. In control cultures the 1:50,000 dilution used elicited a dense outgrowth of axons from chick embryonic dorsal root ganglia at a level characteristic of lo-100 standard units of NGF (LeviMontalcini and Angeletti, 1968). The results of this level of NGF on salivary ganglia are given in Table 2. The increase

FIG. 17. Axon outgrowth from the submandibular ganglion (SG) is slightly stimulated in the presence of lung epithelium (LEp). Eleven-day lung epithelium (trachea and bronchus) separated from its mesenchyme by treatment with 0.3% trypsin-pancreatin and recombined with ganglion-containing salivary mesenchyme. The recombination was cultured in minimum essential medium supplemented with 10% horse serum and 10% embryo extract, fixed with formalin after 3 days and stained for cholinesterase. Lung epithelium shows very little morphogenesis. There is some axonal outgrowth (Ax) from the ganglion, but it is not directed toward the epithelium. x 80. FIG. 18. Lung epithelium (LEp) does not successfully compete with salivary epithelium (SEp) in attracting axons from the submandibular ganglion (SG). Eleven-day lung epithelium (trachea and bronchus) separated mechanically from its mesenchyme by teasing, and 13-day salivary epithelium separated from its mesenchyme with 0.3% trypsin-pancreatin. Epithelia were placed near each other on Millipore filter rafts, surrounded by salivary cap mesenchyme (see Fig. l), and then ganglion-containing mesenchyme was placed near the two epithelia. Recombination was cultured in Eagle’s Basal Medium (BME) supplemented with 10% horse serum and 10% embryo extract; fixed and stained for AChE after 3 days. Almost all axon outgrowth (Ax) is directed toward the branching salivary epithelium. The developing muscle layer (Mu) around the trachea (T) also stains for AChE. x 80. FIG. 19. Submandibular ganglion combined with morphogenetically active lung. Lung mesenchyme was cut away from the epithelium at the junction of the trachea and bronchial rudiment of the 11-day lung. Ganglion-containing mesenchyme from 13.day salivary was placed in this bared area and the recombination cultured for 3 days, fixed and stained. Axon bundles (Ax) join elements of the submandibular ganglion (SG), but axon outgrowth is not directed along the branching epithelium (Br.). Muscle cells (arrows) along the trachea (T) also stain for AChE. x 80. FIG. 20. Outgrowth from submandibular ganglion (SG) to salivary epithelium (SEp) and intact preputial gland (Pp). Preputial gland excised from 15-day embryo successfully competes with salivary epithelium for nerve fibers (arrows) from the salivary ganglion. x 70. FIG. 21. Outgrowth from pelvic plexus ganglion to salivary epithelium. 13-day salivary epithelium was trypsin isolated and recombined with salivary cap mesenchyme (see Fig. 1) and ganglionic material from the urogenital sinus region of the 15-day embryo. Fixed and stained after 2 days of culture. Axons (Ax) from the pelvic ganglion (PG) extend out and envelope the central area of the salivary epithelium (SEp). x 75. FIG. 22. Same type of recombination as in Fig. 21. After 3 days of culture. Axons (arrows) from the pelvic ganglion (PG) extend along the salivary epithelium (SEp) in a manner similar to that shown by axon bundles from the submandibular ganglion (see Fig. 18); however, groups of axons are much less compactly arranged into bundles. x 70.

154

DEVELOPMENTAL TABLE EFFECT OF NGF

BIOLOGY

ACTIWW

Ganglia with

NGFD

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protein accumulation in both these over that found with FlBFCSlO.

2

ON AChE

VOLUME

Ganglia cultured transfilter to epithelia

DISCUSSION

Epithelial

Stimulation

of Axon Outgrowth

In the presence of salivary epithelium, either cisfilter or transfilter, stimulation of 19 30 132 axon outgrowth occurs. Such stimulation is defined by (1) a marked increase in the number and length of nerve fibers found by histochemical staining of whole cultures, o Ganglion-containing mesenchyme was trypsinseparated from the epithelium of glands from 13-day and (2) by a three- to fourfold increase in embryos. Tissues were cultured for 3 days on the AChE activity of the experimental cultures bottom of Millipore filters in the Auerbach filter when compared with controls. That the assembly, alone in media with and without NGF, or elongation of axons is in some way dependwith epithelia placed on top of the filter. ent on the epithelium is also suggested by OMedium contained crude NGF as a 1:50,000 dilution. the fact that even in whole cultured glands L “Gland” in this instance refers to the whole outgrowth from the ganglion occurs preganglion-containing mesenchyme of the gland less the dominantly in the direction of epithelial epithelium. outgrowth and does not extend beyond the d Substrate concentration in assay was lo-’ M, sp. epithelium even when the rate of outact. 9.9 mCi/mmole. growth of the latter has slowed or come to a in AChE activity in medium with NGF is halt. Most previous studies on axonal guidvery small compared to that with the and ganglion transfilter to epithelium. How- ance in uiuo or on the stimulation ever, the NGF-treated tissue showed a guidance of axonal growth in culture have doubling in protein concentration, suggest- made use of systems in which axons are ing that the effect may be on general levels regenerating after having been cut during manipulation (Attardi and of protein synthesis, not on the enzyme per experimental Sperry, 1963; Levi-Montalcini et al., 1954; se. The salivary homogenate (“crude NGF”) has been reported to contain a TABLE 3 factor that stimulates in vitro the growth of EFFECT OF ANTI-NGF ON AChE ACTIVITY” mesenchymal cells (Attardi et al., 1965). Anti-NGF was effective in preventing Medium axon outgrowth from chick dorsal root FlZFCSlO Horse Anti-NGP Serum ganglia at concentrations of 10% and 1% in Control” F12FCSlO but not at 0.1%. Crude NGF in cultures of dorsal root ganglia was present Protein/gland 10.4 15.6 14.4 at a dilution of 1:300,000 (Yamada et al., (rd 233 306 287 1971). Whole salivary glands grown in AChE activity (Apmoleslminl medium containing 5% Anti-NGF (as gland) judged by AChE stain) over controls in F12 with 5% FCS and 5% horse serum. Results n Whole submandibular glands from Id-day emfilters in the of the radioassay for AChE in such cultures bryos cultured for 2 days on Millipore are given in Table 3. There is practically no different media. bMedium F12 with 5% fetal calf serum and 5% difference in AChE activity between Anti- horse serum. NGF containing medium and medium ~Medium F12 with 5% fetal calf serum and 5% with horse serum. There is an increase in antiserum to NGF. Protein/gland’

(rg) AChE activity” (Apmoles/min/ gland)’

3.3

6.7

3.6

MICHAEL

D.

COUCHLIN

Target

Charnley et al., 1973). The axons in the submandibular ganglion studied in this work are engaged in primary outgrowth, not in regeneration. Therefore, the guidance of the outgrowth cannot be attributed to tracks previously laid down in the tissue through which the axons elongate, nor can the pattern be attributed to a biochemical affinity established by previous contact between epithelial cells and neurons. Thus, the interpretation

of directed

outgrowth

in

Organ

Control

of Nerve

Growth

155

putial and salivary glands are derived from epithelium near the interface between ectodermal and endodermal epithelia; their development is dimorphic (i.e., both show sex-dependent structural differences); and the two glands are androgensensitive in the adults (e.g., male pheromone production by the preputial gland and NGF accumulation by the salivary gland are sensitive to testosterone levels). A further correlation between the two glands is indicated by the fact that salivary epithelial morphogenesis is supported by preputial gland mesenchyme, as judged by intraocular grafts (Cunha, 1972).

this system avoids the difficulties encountered in dealing with regeneration. While Norr’s recent study (1973) of the differentiation of sympathetic neurons from neural crest cells does concern a of Growth nonregenerating system, it does not reveal Directionality any pattern of nerve growth. Significantly, In all cases of salivary epithelium stimuthat differentiation is dependent on in- lation of nerves, the outgrowth of axons teractions with several tissues and NGF of was in the direction of the epithelium. itself is not sufficient to support differentiCultures of whole explants fixed and ation. stained at various times within the first As expected of a parasympathetic gan- day of culture did not show an initial glion, the development of axons from the random outgrowth of axons or axon bunsubmandibular ganglion and the accumudles. Rather, there was a definite directionlation of AChE activity was neither en- ality in the early growth (Coughlin, 1975). hanced by the presence of NGF in the A similar directionality of axon elongamedium, nor depressed by the antiserum tion can be inferred from the results with to NGF. Parasympathetic ganglia have for tissue recombinations. This is seen both in some time been considered to be insensicis- and transfilter recombinations. Most tive to the effects of NGF and Anti-NGF striking are the results of those experi(Levi-Montalcini and Angeletti, 1968). ments where epithelium was first recombined with cap mesoderm, allowed to conSpecificity of Effect dense for several hours, and ganglion-conThe capacity to stimulate axon out- taining mesenchyme added later. In these, growth from the submandibular ganglion is axons elongate as a bundle through up to restricted, although it is not completely 0.5 mm of mesenchyme to reach the epithespecific to salivary epithelium. Of the other lium and then distribute themselves actissues tested, only the preputial gland was cording to the pattern of epithelial morfound to elicit outgrowth from the ganglion phogenesis (as in Fig. 12). Not only do such and was able to compete with the salivary results indicate an initial, epitheliumepithelium in “attracting” axons. The re- dependent directionality in the outgrowth, sults of these experiments do not show but they also strongly suggest that the whether the active element of the preputial outgrowth in normal glands in uiuo and in gland is epithelial or mesenchymal (or vitro is not due to selective adhesion of both), but it should be noted that there are axons with the epithelium. several similarities between preputial and Both visual observation of stained matesalivary gland development: Both the pre- rial and radioassay for AChE indicate that

156

DEVELOPMENTAL

BIOLOGY

there is enhancement of ganglionic growth and nondirectional axonal extension in the presence of lung, other tissues, or medium enriched with horse serum or embryo extract. In these cases the ganglion stains more densely and appears larger than in controls; axons also seem to be somewhat more numerous. Whether this is due to proliferation of cells, an increase in protein synthesis or some other mechanism is not known, though horse serum in the medium favors greater accumulation of protein in the cultures than does fetal calf serum. Chemotuxis

vs. Differential

Adhesivity

Of the several different theories of guidance of axonal growth, the two most favored are (1) chemotaxis, that is, the directed growth of nerve fibers along a concentration gradient toward the source of some diffusible substance (so defined by Jacobson, 1970, p. 142), and (2) differential adhesivity, that is, outgrowth of nerve fibers oriented by selective adhesion of the axons to the substrate over which they pass. The marked influence on the outgrowth of nerve fibers by variations in the substrate has been demonstrated by LuduEna (1973) and more recently by Letourneau and Wessells (1974) and Letourneau (personal communication). However, the results of the transfilter recombinations in this study as well as the directed growth through up to 0.5 mm of randomly oriented mesenchyme seem difficult to interpret on the basis of selective adhesivity. A chemotactic interpretation of the results seems much more plausible. The limiting distance over which the epithelial influence was effective (0.5 mm) approximates the theoretical limit (1 mm) over which an effective concentration gradient could be set up in a developing organism in a matter of hours (Crick, 1970). But, although the transfilter results obtainable even with 0.1 pm filters and the directed growth over a distance of up to 0.5 mm argue for the influence of some diffusi-

VOLUME

43, 1975

ble substance, compelling evidence will require the demonstration of such a molecule. In preliminary experiments, crude homogenates of embryonic salivary glands have not stimulated nerve outgrowth (unpublished results). Further work is in progress to elucidate the mechanism by which the epithelium stimulates nerve outgrowth. I am grateful to Dr. Norman K. Wessells for advice, encouragement and support during the course of this work and for helpful suggestions and criticism in the preparation of this manuscript. I also thank Pam Oatis for assistance in several experiments and Kathy Falk for help in preparing the illustrations. This work was supported by USPHS Grant No. HD-04708 to Dr. Wessells and a National Science Foundation predoctoral fellowship to the author. REFERENCES AT~ARDI, D. G., and SPERRY, R. W. (1963). Preferential selection of central pathways by regenerating optic fibers. Erp. Neural. 7, 46-64. AT~ARDI, E. G., LEVI-M• NTALCINI, R., WENGER, B. S., and ANGELLETTI, P. U. (1965). Submaxillary gland of mouse: effects of a fraction on tissues of mesoderma1 origin in vitro. Science 150, 1307-1309. BLUME, A., GILBERT, F., WILSON, S., FARBER, J., ROSENBERG, R., and NIRENBERG, M. (1970). Regulation of acetylcholinesterase in Neuroblastoma cells.

Proc. Nat. Acad. Sci. U.S.A. 67, 786-792. BORGHESE, E. (1950). Explantation experiments on the influence of the connective tissue capsul on the development of the epithelial part of the submandibular gland of Mus musculus. J. Anat. London 84, 303-318. CHAMLEY, J. H., GALLER, I., and BURNSTOCK, G. (1973). Selective growth of sympathetic nerve fibers to explants of normally densely innervated autonomic effector organs in tissue culture. Deuelop. Biol. 31, 362-379. CHEN, J. S., and LEVI-M• NTALCINI, R. (1970). Axonal growth from insect neurons in glia-free cultures.

Proc. Nat. Acad. Sci. U.S.A. 66, 32-39. COUGHLIN, M. D. (1975). Early development of parasympathetic nerves in the mouse submandibular gland. Deuelop. Biol. 43,123-139. CRAIN, S. M. (1968). Development of functional neuromuscular connections between separate explants of fetal mammalian tissue after maturation in culture. Anat. Reu. 160, 466. CRICK, F. (1970). Diffusion in embryogenesis. Nature

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277-300. SESHAN, K. R., and LEVI-M• NTALCINI, R. (1973). Neuronal properties of nymphal and adult insect neurosecretory cells in uitro. Science 182, 291-293. SILBERSTEIN, S. D., JOHNSON, D. G., JACOBOWITZ, D. M., and KOPIN, I. J. (1971). Sympathetic reinnervation of the rat iris in organ culture. Proc. Nat. Acad. Sci. U.S.A. 68, 1121-1124. SINGER, M. (1952). The influence of the nerve in regeneration of the amphibian extremity. Quart. Reu. Biol. 27, 169-200. SNELL, R. S., and GARRETT, J. R. (1958). The effect of postganglionic sympathectomy on the histochemical appearances of cholinesterase in the nerves supplying the submandibular and sublingual glands of the rat. Z. Zellforsch. 48,201-214. SPOONER, B. S., and WESSELLS, N. K. (1970). Mammalian lung development: Interaction in primordium formation and bronchial morpbogenesis. J. Enp. Zool. 175, 445-454. TADERERA, J. V. (1967). Control of lung differentiation in uitro. Deuelop. Biol. 16, 489-512. WARTIOVAARA, J., LEHTONEN, E., NORDLING, S., and

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Williams and Wilkins, Baltimore, MD. WILSON, S. H., SCHRIER, B. K., FARBER, J. L., THOMPSON, E. J., ROSENBERG, R. N., BLUME, A. J., and NIRENBERG, M. W. (1972). Markers for gene expression in cultured cells from the nervous system. J. Biol. &em. 19, 3159-3169. YAMADA, K. M., SPOONER, B. S., and WESSELLS, N. K. (1971). Ultrastructure and function of growth cones and axons of cultured nerve cells. J. Cell Biol. 49, 614-635.