Neuron,

Vol. 6, 101-111,

January,

1991, Copyright

0 1991 by Cell Press

Gap Junctional Communication during Neuromuscular Junction Francesca Allen*

and Anne Warner

Department of Anatomy and University College London Cower Street London, WCIE 6BT England

Developmental

Biology

Summary We have tested whether gap junctions form between nerve and muscle during their initial contact, before establishing the chemical synapse. Embryonic Xenopus stage 18-20 myotomes and neural tubes were perme abilized with DMSO to load appropriate reagents, dissociated, and cocultured. When myotomes, loaded with Lucifer yellow, were cocultured with unlabeled neural tube cells, 23% of the neurons contained dye after 24 hr. Affinity-purified gap junction antibodies loaded into myocytes or neurons reduced neuronal labeling significantly to 5%. [sH]uridine nucleotide transfer was observed in both directions between myocytes and neurons. Again gap junction antibodies substantially reduced recipient label. In all cases preimmune IgCs did not reduce transfer. When acetylcholine receptor clustering was examined in cultures containing gap junction antibodies, no difference in the number of neuronally induced AChR clusters was observed. This suggests that the cluster-inducing signal between nerve and muscle does not pass through gap junctions. Introduction The vertebrate neuromuscular junction forms as a result of intimate interactions between two specialized cells. The arrival of a competent neuron at a suitable myocyte triggers a cascade of events at the putative synapse. Synapse specific molecules (e.g., acetylcholinesterase [Betz and Sakmann, 1973; McMahan et al., 19781, heparin sulphate proteoglycans [Anderson and Cohen, 1977; Anderson and Fambrough, 19831, s-laminin [Hunter et al., 19891) are inserted into the local basal lamina, and acetylcholine receptors (AChRs; Anderson et al., 1977) and related subsynaptic components (e.g., 43 kd [St. John et al., 1982],51 kd [Burden, 19821, 58 kd [Froehner, 19841, 270 kd [Burden et al., 19831, actin [Hall et al., 19811, vinculin [Bloch and Geiger, 19801, a-actinin and filamin [Bloch and Hall, 19831) cluster at the junction site. In the nerve terminal synaptic vesicles gather in response to a signal from the muscle. The mechanisms underlying formation of the neuromuscular junction are poorly understood. In Xeno*Present address: Ludwig tauld Building, 91 Riding gland.

Institute for Cancer Research, CourHouse Street, London, WIP BBT, En-

Formation

pus both AChR clustering and acetylcholinesterase localization are independent of either neuronal activity (Davey and Cohen, 1986) or muscle contraction (Anderson et al., 1977; Moody-Corbett and Cohen, 1982; Cohen et al., 1984). However, a number of neuronally derived factors have influence on these events. Extracts from the chick spinal cord (Cohen and Fischbath, 1977) and a neuroblastoma cell line NC108 (Bauer et al., 1981) cause AChR up regulation and redistribution (Christian et al., 1978), respectively, whereas other factors have both attributes (e.g., Jesse11 et al., 1979; Markelonis et al., 1982; Podleski et al., 1978). In regenerating neuromuscular junctions s-laminin (Hunter et al., 1989), attached to the local basal lamina, is sufficient to direct both the neuronand the synapse-specific components of the muscle back to the original synaptic site (Marshall et al., ,1977; Burden et al., 1979; McMahan and Slater, 1984). However, it is not known whether this molecule plays a role during initial synapse development. Extensive investigations on the cues that guide neurons to their specific target muscles suggest that neurons may be directed by a mixtureof substrate molecules and chemotaxis. Thus, N-CAM has been found to be present transiently on embryonic mouse myocytes during the period of nerve contact (Rieger et al., 1985), and it has been proposed that muscle extracts may direct neuron growth (McCaig, 1986). Despite this progress, the primary trigger that initiates the cascade of events prior to synapse formation has yet to be identified. One possibility is that the neuron and myocyteform gap junctions beforechemical transmission begins. This could provide a pathway for transfer of such a triggering factor. There have been reports suggesting that during synapse formation, the neuron can form transient gap junctions with the myocyte. Fischbach (1972) picked up a few brief instances of electrical coupling between chick neurons and myocytes in culture, and very small gap junction-like plaques have been revealed between young neuron and myocyte contacts in Xenopus cultures by freeze fracture (Peng et al., 1980). In chick cultures both electrical and dye coupling were reported between motoneurons and leg myoblasts (Bonner and Martin, 1984, J. Cell Biol., abstract). This ability has recently been correlated with developmental stage, occurring only when myoblasts were derived from embryos between stages 24 and 29 (Bonner, 1989). The relevance to in vivo events was not certain because of the long culture interval before assessment of transfer. If gap junctional communication is to be considered as a candidate mechanism for initiation and control of some of these primary events, or as a putative step in cell recognition, it is important to establish that exchange between neurons and myocytes occurs through gap junctions. We have used dye coupling

Figure

1. Xenopus

Cultures

(16 hr) of Mixed

Myotomes

and Neural

Tubes

Demonstrating

The tissue was soaked in a solution containing a high molecular weight (156.9K) and D). Intracellular staining is visible in (D) but not in (B). (A and C) Bright-field;

and nucleotide transfer to test the ability of Xenopus neurons and myocytes developing in culture to communicate directly with each other. The advantage of Xenopus over the chick is that primary differentiation in culture is extremely rapid, following the same time course in vitro and in vivo, and the myoblasts do not go through a proliferation-fusion cycle before differentiation. Antibodies raised against gap junction protein

are

observed

used

to determine

takes

place

whether

through

any

gap

direct

transfer

junctions.

the Effectiveness

of DMSO

Permeabilization

FITC dextran alone (A and B) or with (B and D) fluorescence. Bar, 50 pm.

5% DMSO

(C

ability of undifferentiated neural tube and myotomal cells todifferentiate. In culturesof untreated material, 61% (median; number of cultures [N] = 18; number of fields [n] = 129) of the cells differentiated into myocytes, with 12% forming neurons (N = 32; n = 255). Cultures of cells previously treated with DMSO were no different (myocytes = 63%; N = 43; n = 314; control versus DMSO, P = 0.23; median test with Yates correction for 1 degree of freedom; neurons = 12%; N = 29; n = 137). The total number of differentiating cells also was unaffected.

Results Dimethyl Sulfoxide loads large Molecules into Cells Figure 1 shows that when cells were treated with dimethyl sulfoxide (DMSO) containing FITC Dextrans of 156.9K, about the size of an IgG molecule, the dextran gained access to the cytoplasm. Figure ID illustrates the bright, particulate fluorescence achieved in the presence of DMSO; Figure IB shows that in the absence of DMSO the cells fail to take up this large molecule. DMSO treatment did not reduce the subsequent

Lucifer Yellow Transfers from Myocytes to Neurons Figure 2A shows the percentage of myocytes labeled with Lucifer yellow after DMSO permeabilization (169 fieldstaken from 18cultures).Themedianofthedistribution lay at 80%, indicating that bulk loading with DMSO was generally efficient. However, a high proportion of myocytes labeled correlated with high intensity of label. Conversely, the proportion of cells containing dye could be as low as 42%. Cultures prepared on the same occasion showed equivalent loading levels. Analysis of neuronal label was confined to

Gap Junctions 103

between

Myocytes

and Neurons

were found to contain 3B show part of a field processes contacting

LaI g 20 B Lucifer Yellow and ,’ I Reagents Loaded 10

r-J-7

1

4

30

40

% of Hyocytes Figure DMSO

I

Immunogenic

2. The Uptake Permeabilization

50

60 Labelled

of

Lucifer

I

70 with Yellow

80 Lucifer

90

100

Yellow

by Myocytes

during

Arrows indicate medians. (A) Myocytes loaded with Lucifer yellow alone, median 80%. (B) Myocytes loaded with Lucifer yellow and immunogenic reagents(antibodiesorpreimmune IgCs), median 83%. Abscissae: number of myocytes labeled with Lucifer yellow as a percentage of the total number of myocytes in each field. Ordinates: number of fields (n).

in which morethan labeled, since low levels reduced the chances of yellow uptake efficiency rent loading with IgGs myocytes labeled). When cells dissociated were plated together myocytes and cocultured

cultures

50% of the myocyteswere of Lucifer yellow in myocytes detecting any transfer. Lucifer was not affected by concur(Figure 2B; median = 83% of from unlabeled neural tubes with Lucifer yellow-labeled for 24 hr, many neurons

Lucifer yellow. Figures 3A and containing three neurons with and running across the myo-

cyte. In Figure 3B Lucifer yellow can be seen in the neuronal cell bodies. By the time the photo was taken, much of the myocyte label had moved out of the cytoplasm into vesicles, which are only partially visible on the right of this figure. The label within the recipient neurons is light compared with the donor myocytes, as would be expected from the small areas of contact between neuron and myocyte. A frequency distribution for the percentage of differentiated neurons containing dye is plotted in Figure 4A. The median of the distribution lies at 23% (N = 18; n = 116), though in some fields 50% of neurons were labeled. This implies that substantial transfer of dye occurs between myocytes and neurons. It is possible that single, dissociated cells that come intocontact immediatelyafter platingtransientlyform gap junctions with their neighbors and transfer dye before differentiation. Could transfer between two cells destined to form a neuron and a myocyte account for these results? Since it is not possible to identify the future fate of undifferentiated cells, we assessed the likelihood of random transfer by determining the frequency with which labeled (myotome) and unlabeled (neural tube) cells were found touching each other during the first hr after plating. Of the mixed population, 47% contained Lucifer yellow (median; N = 6; n = 60) and 53% were unlabeled. Of these unlabeled cells (which includes future neurons), 5.5% were touching a cell containing Lucifer yellow. It follows that 5.5% of the neuronal cells were potentially able toacquireLuciferyeIlow bytransferpriortodifferentiation. Since 23% of neurons contained Lucifer yellow and differentiation is already underway1 hr after plat-

Figure 3. A Field of Cultured cytes and Neurons Showing to Neurons and Localization

Xenopus MyoDye Transfer of AChRs

Stage 18-20 Xenopus myocytes were examined 24 hr after plating. The myotomes had been loaded with Lucifer yellow prior to plating with untreated, dissociated neural tubes. (A) Phase image showing a group of 3 neuronal cell bodies (n) whose axons contact a muscle mass (m, only partially illustrated) in several places (arrows). (B) Fluorescence image of the same neurons showing the presence of dye in the neuronal cell bodies; most of the myocyte label is in vacuoles, off the right edge of the photo. (C) Fluorescence image of the same cell group after fixation and staining with rhodamine-conjugated a-bungarotoxin. Bright lines of AChR clusters can be detected on the myocytes, along points of neuronal contact. Bar, 50 pm.

Neuron 104

12 %

of

2L

Neurones Lucrfer

36

40

Labelled

with

al., 1990) and myocytes and neurons withdraw from the cell cycle before differentiation, maximal block of junctional communication is probably maintained for the whole of the culture period (up to 24 hr). Purified preimmune IgGs were used as a control. Figure 46 shows that the ability of neurons to receive dye was not affected by the presence of preimmune IgGs in either the neurons or the myocytes (median = 19; N = 5; n = 38; compare Figures 4A and 46; 0.5 > P > 0.1). Figure 4C shows the frequency distribution for the percentage of neurons containing Lucifer yellow when gap junction antibody was loaded into myocytes along with Lucifer yellow. The frequency with which dye was detected in neurons was reduced substantially, with a median of 5% (N = 9; n = 76; compare Figures 4B and 4C; P < 0.001). When antibody was present in the recipient neurons (Figure 4D), the neuronal label fell to 3% (N = 6; n = 42), not significantly different from that seen with antibody in the myocytes (compare Figures 4C and 4D; 0.5 > P > 0.1). Thus, neuronal label is reduced significantlywhen antibody is present in either cell type. Taken together, in controls 22% of the neurons were labeled (N = 23; n = 154), compared with only 4% (N = 15; n = 118; P < 0.0001) when donor or recipient contained antibody. This suggests that transfer of dye from myocytes to neurons is occurring through gap junctions, formed between the embryonic neurons and myocytes during contact in culture.

Yellow

Figure 4. The Percentage of Neurons low by Transfer from Myocytes

Labeled

with

Lucifer

Yel-

Dye was introduced into the myotomes by DMSO permeabilization. Gap junction antibody or preimmune IgGs were loaded into either the myotomes or the neural tubes. Arrows indicate medians. Neuronal label when (A) myotomeswere loaded with Lucifer yellow alone, median = 23%; (B) myotomes or neurons were loaded with preimmune IgGs, median = 19%; (C) myocytes were loaded with gap junction antibody, median = 5%; (D) neural tube cells were loaded with gap junction antibody, median = 3%.

ing, the majority of dye transfer after differentiation. Furthermore, in neural crest-derived pigment from the neural tube, confirming to differentiation was negligible.

must have occurred dye was never found cells, which alsoarise that transfer prior

Transfer Is Blocked by Gap junction Antibodies To test whether this transfer occurred through gap junctions, affinity-purified polyclonal antibodies raised against rat liver gap junction protein were loaded into either donor myocytes or recipient neurons. These antibodies inhibit transfer through gap junctions in, for example, Xenopus embryos (Warner et al., 1984) and remain able to block gap junctional communication for 16-24 hr (Allen et al., 1990). Since cell division appears to be the main factor controlling the time for which the antibody maintains a block of cell-to-ceil communication (Warner and Gurdon, 1987; Allen et

Transfer of 3H-Labeled Nucleotide Can Both Directions To examine gap junctional communication

Occur

in from

neu-

rons to myocytes and quantify further the extent of any transfer, myotomes or neural tubes were loaded with [3H]uridine, which was trapped inside the cell by uridine kinase conversion to its nucleotide, to act as donors in metabolic cooperation. Gap junction antibodies or preimmune IgGs were loaded either into donors or recipients. After coculturing the labeled and unlabeled cells for 24 hr, the distribution of 3H-labeled nucleotide was detected as RNA by autoradiography. An example of a culture in which neurons acted as donors is shown in Figure 5A. A heavily labeled neuron is in contact with a myocyte, which has a higher level of labeling than the surrounding dish. Figure 6 shows the level of RNA label, derived from incorporation of loaded nucleotide, in cultures treated with DMSO. In Figures 6A, 6B, and 6C myocytes acted as donors; Figures 6D, 6E, and 6F show cultures in which neural tube cells were loaded. Both donor populations (Figures 6A and 6D) were heavily labeled, with a median grain count of 26 grains per area (gpa, see Experimental Procedures) over myocyte donors (A) and 45 gpa over neuron donors (D). Neuronal recipients (Figure 6B) showed a spread of label, with the median at 6 gpa, significantly higher than the background over the dish (compare Figures 66 and 66; P < 0.001). Myocyte recipients (Figure 6E) contained 6 gpa, again significantly higher than background (Fig-

Gap Junctions 105

Figure

between

Myocytes

5. Autoradiographs

(A) Culture preimmune into RNA. (B) Culture myocytes.

of Cultured

in which the neurons igGs. Abundant silver in which Although

and Neurons

Stage

18-20

Xenopus

Myocytes

(n) had been loaded with [3H]uridine grains can be seen over the myocyte,

neurons had been loaded with several heavily labeled neurons

and

gap junction antibodies (n) are in close contact,

ure 6F, 1 gpa; Figure 6E versus Figure 6F, P < 0.001). The low dish background in regions adjacent to the cells, which gives high sensitivity for detecting transfer, was achieved by low loading levels of radioactivity and extensivewashing before plating. Label over noncontacting recipients was not used to provide background because 15%-20% of the neurons were preferentially lost during fixation, precluding reliable identification of cells that had been isolated throughout the culture period. These results demonstrate transfer of nucleotides from myocyte to neuron, as found with Lucifer yellow. Furthermore, they show that this exchange is reciprocal, with movement from neuron to myocyte occurring with equal facility.

Neurons,

24 hr after

Plating

prior to dissociating and plating with myocytes indicating the presence of 3H-labeled nucleotide in addition to pH]uridine few silver grains are present

loaded with incorporated

prior to plating with untreated over the myocyte. Bar, 20 urn.

Gap Junction Antibodies Inhibit Transfer from Either Myocytes or Neurons The contribution of gap junctional communication to the transfer observed between myocytes and neurons and vice versa was determined by loading affinity-purified gap junction antibodies into either myotomes or neural tubes. Preimmune IgGs were loaded in parallel to provide controls. Figure 7 shows the results of these experiments in terms of the label detected over the relevant recipient cell populations. Thus Figure 7A shows neuronal label in cultures in which myotomal cells acted as donors, and Figure 76 shows label over myocytes when neural tube cells acted as donors. In each case panels a and b compare label over recipients when donor cells contained preimmune IgCs

NeLlrOn 106

Myocytes . .

Neurones

as Donors

-,LU 1

Myocytes

Figure 6. Metabolic Cooperation in Mixed Xenopus Cultures between Donor Myocytes or Donor Neurons and the Appropriate Recipient Cells after 24 hr

as Donors

2o

The donor cell populations were loaded with pH]uridine in the presence of DMSO. Arrows indicate medians. (A and D) Label over the donor cell populations: (A) myocytes, median = 26; (D) neurons, median = 45. (B and E) Label over the recipient cell populations: (B) neurons, median = 6: (E) myocytes, median = 6. (C and F) Background label over the dish in each treatment, median, 1 for both.

E

I‘““’

Myocytes

L.

F 120

Dish

Label

Dish

9c

Label

60

Grains

per

area

(gpa)

(panel a) or gap junction antibodies (panel b); panels c and d give results with preimmune or immune reagents, respectively, in the recipient population. With myocytes acting as donors (Figure 7A) and pre-

A: Recipient

Neuronal

Immunogenic Donor

1

30

i

Pre-immune

40

B: Recipient

Label

Donor

Neurones

Pre-immune

40

Pre-immune

30

0

I

200

d

b

90

80

3o

IL-

10

b

c

a

20

90

Myocytes

50

40

30.

in

Recipient

50

80

Pre-immune

20 10 0 28C

d

90

80

70

Gap Junction Antibody

or recipients (panels a at a median of 5 gpa, controls treated with 6B; 0.5 > P > 0.1). The

Label

Reagents

Neurones

c

-

Myocyte

Immunogenic

in

Recipient

a 40

Reagents

Myocytes

immune IgCs in either donors and c), neuronal labeling was insignificantly different from DMSO alone (compare Figure

Gap Junction Antibody

30

” 3.

Gap Junction Antibody

20 10 0 0

Grains Figure

7. Gap

Junction

per

area

Antibodies

10

20

(gpa) Reduce

the

Level

of Silver

Grains

30 40 Grains over

50

per

70

0

area

the Recipient

20

30

40

(gpa) Populations

Silver grains over the recipient populations in mixed Xenopus cultures showing the 3H-iabeled nucleotide distribution. (A) Myocyte donors, neurons as recipients. (B) Neuronal donors, myocytes as recipients. In both (A) and (B), the left two panels (a and b) show results with immunogenic reagents loaded into the donor population by DMSO permeablization. In the right two panels (c and d) the immunogenic reagents were loaded into the recipient cells. The top row (a and c) shows results of loading preimmune IgGs; the bottom row (b and d) shows the results of loading gap junction antibodies. Arrows indicate medians. (A) Neurons: (a) median = 5, n = 59; (b) median = 2, n = 47; (c) median 5, n = 35; (d) median = 1, n = 78 (see Experimental Procedures). (B) Myocytes: (a) median = 4, n = 90; (b) median = 1, n = 61; (c) median = 4, n = 61; (d) median = 1, n = 64 (see Experimental Procedures). Compare top row preimmune IgG distributions with bottom row gap junction antibody distributions.

Gap Junctions 107

between

Myocytes

and Neurons

inclusion of gap junction antibodies substantially and significantly reduced label over the neuronal recipients. When antibodies were present in donor myocytes (Figure 7A, panel b), only 2 gpa were detected over neurons and the spread was small (antibody versus preimmune, P < 0.0001). With antibody in the recipient neuronal population (Figure 7A, panel d), neuronal label was only 1 gpa, at dish background, again significantly lower than the preimmune control (P < 0.001). The greater sensitivity of the metabolic cooperation revealed that the antibodies were significantly more effective when present in neurons than in myocytes (P < 0.001). Figure 7B shows the reciprocal experiments in which neurons acted as donors. When preimmune IgGs were present in donor (panel a) or recipient (panel c), myocytes were labeled at 4 gpa (N = 151; n = 607), slightly lower than labeling in DMSO alone (compare Figure 6E). Myocyte label decreased to 1 gpa when gap junction antibodies were present (panels b and d), both distributions were significantly lower than that in preimmune controls (P < 0.001). Again, theantibodieswereslightlymoreeffectivewhen present in neurons (0.05 > P > 0.02). An example of a culture in which neuronal donors were loaded with gap junction antibody is illustrated in Figure 5B. Although the myocyte is contacted by several heavily labeled neurons, its level of label is indistinguishable from that of the surrounding dish background. For comparison with the results obtained with Lucifer yellow, we estimated the percentage of neurons in cultures that received nucleotide through gap junctions formed with myocyte donors. First, for each experiment, the background distribution of nucleotide over the dish was subtracted from the neuronal recipient distributions. This figure was then divided by the number of labeled neurons and expressed as a percentage of the total. These estimates suggested that in control cultures 67% of the neurons contained label above background levels, compared with a maximum of 10% of neurons from cultures loaded with gapjunction antibodies. The higher level of neuronal label revealed by metabolic cooperation experiments com-

Table

1. Frequency

of AChR

Hot

Spots

in Mixed

Xenopus

Cultures,

pared with the Lucifer yellow transfer is best explained by the greater sensitivity of the metabolic cooperation technique. Similarly, in cultures with neurons acting as donors, about 65% of the recipient myocytes were labeled in the controls, compared with only 12% of myocytes when gap junction antibody was present. The conclusion from these experiments is that the majority of transfer between neuron and myocyte occurs through gap junctions and that this gap junctional exchange is reciprocal. Does Gap Junctional Communication Play a Functional Role in Synapse Development? The construction of the neuromuscular junction during development requires the coordinated assembly of a number of different elements. As a first step in the analysis of the importance of gap junctional communication between neuron and myocyte in this process, we tested whether gap junctions were involved in the induction of neuronally induced AChR clustering. AChRs were labeled by the application of fluorescently conjugated a-bungarotoxin to living cultures. The cultures were set up as before, and the number and distribution of AChR clusters were compared between cultures in which all the cells had been loaded either with preimmune IgGs or with gap junction antibodies. Receptor clusters were first visible as “hot spots” 12 hr after plating and increased in size and number over the subsequent 12 hr. Comparisons were made after 24 hr in culture. An example of neuronally induced hot spots in a control culture is shown in Figure 3C. Labeled AChRs are clearly visible where the neurites course along the edge and over the myocyte. Table 1 shows the frequency with which hot spots were found, either associated with neuronal contacts or in isolation, in cultures that had been loaded with preimmune IgGs or gap junction antibodies prior to plating. There was no detectable significant difference between any of the categories studied. In particular, the percentage of myocytes that were contacted by neurons without inducing hot spots was very similar (preimmune, 8.9%; gap junction antibody, 9.7%), as was the frequency of uninduced spontaneous hot

with

and

without

Preimmune Myocytes Myoctyes Isolated Myocytes Myocytes Number Number Number

contacted by neurons, but no HS myocytes with HS, or with neurons elsewhere, i.e., uninduced without neurons or HS contacted by neurons with associated HS, i.e., induced of field+ of myocytesc of neurons

HS indicates hot spots. a Preimmune lg.3 versus gap junction antibodies. b Data were taken from 12 cultures of each condition, c All myocytes were counted; only neurons contacting

on two separate myocytes were

8.9 26.8 44.5 23.0 74 2996 999

occasions. included.

Gap IgGs f%)

Junction

Antibodies

Gap Junction Antibodies Myocytes (%)

P Value”

9.7 25.2 42.5 22.7 75 3157 1068

0.45 0.94 0.23 0.59

Figure 8. Field of Cultured Stage 18-20 Xenopus Myocytes and Neurons, Fixed 24 hr after Plating and Stained with Rhodamine-Conjugated a-Bungarotoxin The cells had been loaded with gap junction antibodies prior to dissociation. (A) Phase image of a myocyte (m) being crossed by an axon (arrows). (8) Fluorescence image showing aggregates of AChRs where the axon contacts a myocyte (white arrow). A more diffuse area of AChRs, which are not contacted by an axon, can be seen on the left of the picture. Bar, 20 pm.

spots. Notably, the number of myocytes with hot spots induced at the point of neuronal contactwas no different. In both cases about 70% of the neurons contacting myocytes (isolated neurons were not counted) induced hot spots at the point of contact. An example of hot spots associated with neurites where the cells had been loaded with gap junction antibody is shown in Figure 8. Taken together, the results suggest that the incidence of hot spots was not affected when myocytes or neurons were prevented from forming functional gap junctions, either with their own cell type, or at neuron-myocyte contacts (Mann-Whitney U-test). Discussion The main conclusion differentiated myocytes tions with each other cules when they first upon two assumptions. through gap junctions ferentiation has occurred. communication can be

from

these experiments is that neurons form gap juncand can exchange small molecontact. This conclusion rests First, that substantial transfer does not take place before difSecond, that gap junctional determined by preventing celland

to-cell transfer of dye and nucleotideswith affinity-purified polyclonal antibodies to gap junction protein. Although transfer between undifferentiated cells cannot be totally excluded, it is unlikely to be the major cause of the neuronal labeling with Lucifer yel-

low observed when unlabeled neural tube cells are cocultured with labeled myotomal cells. The opportunity for dye transfer between labeled and unlabeled cells prior to differentiation was insufficient to account for the proportion of neurons that contained Lucifer yellow. Neural tube-derived, nonneuronal cells, such as pigment ceils, were never labeled. We conclude that the observed transfer takes place between myocytes and neurons during early contacts established after primary differentiation. The exchange of radioactively labeled nucleotides took place under the same conditions, implying that the metabolic cooperation reflects exchange between newly differentiated cells also. The case for a specific block of gap junctional communication by the affinity-purified polyclonal antibodies to gap junction protein used in these experiments is now substantial. Inhibition of dye transfer and electrical coupling has been observed in such diverse systems as Xenopusembryos (Warner et al., 1984; WarnerandGurdon,1987),mouseembryos(Leeetal.,1987), Hydra (Fraser et al., 1987), and chick limb bud mesenthyme (Allen et al., 1990). In all cases preimmune IgGs were ineffective. DMSO permeabilization allowed FITC dextrans with a molecular weight of 156.9K to enter myocytes, confirming that this procedure gives access to the cytoplasm of large molecules equivalent in size to IgGs (see also Allen et al., 1990). DMSO treatment had no effect on differentiation.

Gap Junctions

between

Myocytes

and Neurons

109

Cells that had been loaded with gap junction antibodies showed a highly significant reduction in either dye transfer or metabolic cooperation when compared with DMSO-treated controls or cells loaded with preimmune IgCs. The antibody was effective whether it was loaded into neuron or myocyte, donor or recipient. Some transfer always remained. This probably re fleets variability in antibody loading efficiency, since even Lucifer yellow uptake varied from occasion to occasion, although the possibility that some transfer occurs by a non-gap junctional route cannot be excluded. In general, the antibodies were moreeffective in preventing transfer when they were present in the neuronal population. Presumably the effective concentration of antibody was higher in the relatively small neuronal cells than in the large myocytes, although this could refiect an increased affinity of the antibodies for the neuronal aspect of the gap junctions. The evidence supporting Fischbach’s (1972) suggestion that neurons and myocytes might form gap junctions at the time when they first contact during neuromuscular junction formation is increasing. Since there is no evidence to suggest that electrical transmission exists along side chemical transmission at the neuromuscular junction, this gap junctional communication must be transient. However, all the instances so far reported come from experiments in culture, and firm evidence that gap junctions are established between neuron and myocyte in vivo is still lacking. In Bonner’s (1989) experiments dye transfer and electrical coupling were observed in chick myoblasts, in which an extensive proliferation-fusion cycle precedes differentiation, necessitating a relatively long culture period; thus, the possiblitythat direct communication between neuron and myocyte is restricted to culture cannot be dismissed. In Xenopus the parallel between in vitro and in vivo events is closer. Differentiation in culture is rapid and has the same time course in both situations (e.g., Messenger and Warner, 1979). Comparisons of neuromuscular junction formation show that the culture condition faithfully reproduces in vivo events (e.g., Anderson and Cohen, 1977). The myocytes differentiate as mononuclear cells, without an intervening burst of proliferation. In the present experiments both neurons and myocytes underwent primary differentiation in culture, removing concerns that some form of regeneration might intervene. We made a number of attempts to confirm our results in vivo that were unsuccessful. This is perhaps not surprising, since neuromuscular junctions form sequentially in a rostro-caudal direction so that at any one moment only a small number of neuromuscular junctions will be in the very early stages. The myotomal muscle cells are coupled to each other, and it may be necessary for a pioneer neuron to make only a single gap junctional contact. It may prove very difficult to demonstrate functional transfer between neurons and myocytes in vivo. We estimated from 3H-labeled nucleotide transfer

that about two-thirds of neurons and myocytes took part in metabolic cooperation. Is this a limit of the experimental design, or does it reflect the different abilities of the subclasses of cells present? Cohen and Weldon (1980) found that only 70% of embryonic Xenopus spinal cord neurons were able to form neuromuscular junctions in culture, a finding backed up by our results that 70% of contacting neurons induce hot spots. It is possible that only this proportion of neurons is capable of gap junctional coupling with myocytes. The functional importance of the direct intercellular exchange that takes place through these gap junctions remains to be demonstrated. It could form part of the intimate recognition process that is one of the initial steps in establishment of this peripheral synapse. Bonner (1989) has suggested that, in the chick system, gap junctions may allow a myoblast maturation factor to be transferred from neuron to myoblast, as the presenceof neurons influences myoblast differentiation. However, this possibility can be properly assessed only by determining whether specifically blocking gap junctional communication between neurons and myoblasts prevents myoblast differentiation. We used gap junction antibodies to investigate whethercouplingmight beatriggerinitiatingtheclustering of AChRs under the neuron terminal. The results did not suggest a pivotal role for direct cell-to-cell communication in this particular process. A lack of effect on AChRs should not be taken as evidence against an important functional role for gap junctions, since neuromuscular junction formation is probably a multi-step process, and it is difficult to assay the success or failure of the initial recognition step. In the light of increasing evidence for similarity between formation of the neuromuscular junction and synapses between neurons, it seems likely that the transient formation of gap junctions will prove to be a general feature of early steps in the generation of synapses. Experimental

Procedures

Embryos were obtained by injecting adult Xenopus laevis with chorionic gonadotrophin (Pregnyl, Organon Ltd.; Chorulon, Intervet Laboratories Ltd.; males, 300 IUlml; females 450 IU/ml). All chemicals were obtained from Sigma Ltd. unless otherwise stated. Tissue Culture Stage 18-20 Xenopus laevis embryos (Nieuwkoop and Faber, 1956) werewashed in sterile Ringer’s solution (120 mM NaCI,2.54 mM KCI, 2.0 mM CaCb, 2.0 mM Tris-HCI [pH 7.4]), and the jelly and vitelline membranes were removed. The head and belly were cut away, and the remaining dorsal structures were treated in 1 mglml collagenase in a modified Ringer’s solution (100 mM NaCI, 2.5 mM KCI, 2.0 mM CaC&, 2.0 mM MgCI,, 5.0 mM NaHCOp [pH 7.41) for 20 min to facilitate separation of the neural tube and myotomal muscles. After loading either or both tissues with a tracer and/or antibody using a DMSO loading technique (see below), myotomes and neural tubes were recombined in calcium- and magnesium-free Ringer’s solution with 1.0 mM ECTA (pH 8.0) for 5 min and then mechanically dissociated into single cells using a fine glass pipette. Material from 3 embryos was

plated out evenly in a 35 mm tissue culture dish (Falcon; Nunc) in culture medium (modified Ringer’s solution, 10% fetal calf serum [GIBCO], 350 IU/ml penicillin/streptomycin), and thecultures was maintained at 22OC (Messenger and Warner, 1979). DMSO loading The technique of loading cells with macromolecules by permeabilization with DMSO has previously been used for chick cells (see Allen et al., 1990) and was modified from H. Bode and S. Fraser (see Fraser et al., 1987). Embryonic Xenopus cells were bulk loaded with the fluorescent dye Lucifer yellow, [3H]uridine, FITC dextran, antibodies, and preimmune IgGs. Two microliters of the loading substance dissolved in distilled H,O (20% Lucifer yellow, 25% FITC dextran) or buffer (IgGs) was added to a 5% DMSO solution in Ringer’s solution (total volume 10 ~1) in which the tissue was soaked for 1 hr. Lucifer yellow was loaded into myotomes only. After washing, the neural tubes and myotomes were reunited in 1 mM EGTA, dissociated, and plated out as described above. A polyclonal antibody raised against gap junction protein, previously shown to block gap junctional communication in Xenopus embryos (Warner et al., 1984), was loaded either into the recipient tissue or concurrently with the tracer into the donor cell type. The cultures were analyzed as described below. As a control for antibody specificity, purified mixed IgGs separated from the serum of the same rabbit before immunization were loaded into the cells in place of the antibody. The antibodies were generously provided by N. B. Cilula, and a full description of their production has already been published (Warner et al., 1984; Fraser et al., 1987). Autoradiography Myotomes or neural tubes were loaded with 200 bCi/ml [?H]uridine by DMSO permeabilization, before culturing as described above. In some experiments gap junction antibodies (or preimmune IgGs) were loaded simultaneously into one or the other cell type. The cells were plated into nonradioactive medium. After 18-24 hr, the cell numbers were counted (see Culture Analysis), and the cultures were fixed (3% glutaraldehyde, 30 min), washed three times with Ringer’s solution, dehydrated, and dried. The cultures were coated with G5 llford Nuclear Research emulsion (Ilford Ltd.; diluted I:1 in sterile distilled H20, 350 IUI ml penicillin/streptomycin, 45OC) and exposed for 4-6 weeks. The emulsion was processed in Kodak D-19 developer (22OC, 2 min), washed, fixed in Hypam (Ilford; diluted 1 + 4, 2 min); washed (30 min), and mounted in glycerin jelly (Peacock, 1950) with size 0 glass coverslips (Chance Propper Ltd.). Culture Analysis Cultureswereanalyzed blind afteran 18-24 hr incubation. Differentiation begins about 1 hr after plating and continues over the rest of the culture period in parallel with events in vivo (Messenger and Warner, 1979). To ensure that the DMSO had not adversely affected differentiation, ten random fields were counted under a 10x objective for the number of neurons, myocytes, and total cells present. The percentage of each cell type was compared between cultures undergoing different treatments, using the median test (Freund et al., 1960). Dye Transfer Analysis Culturesloadedwith Luciferyellowwereobserved underalOOx PlanapoLeitzwaterimmersionobjectiveattached toazeisscompound phase microscope (filter set 05) equipped for epifluorescence. Each culture was scanned in ten horizontal sweeps (fields), and every neuron and myocyte was scored for dye. The percentage of labeled cells in each cell type in each culture was calculated, and the results were compared using the MannWhitney U-test. Estimate of Possible Transfer between Undifferentiated Cells Cultures were set up from dissociated Lucifer yellow-loaded myotomes and unlabeled neural tubes. Within 1 hr of plating, the cultures were scored under epifluorescence for the total number of cells in each field of a 40x objective, the percentage

ofthosecellsthatweredyelabeled,andthenumberofunlabeled cells that were contacting labeled cells. The proportion of neurons that may have been able to receive label at this stage, prior to differentiation, was then estimated. Computer Analysis Autoradiographs were analyzed using a 100x oil immersion objective (Neofluar, Zeiss, Federal Republic of Germany) on a Zeiss compound phase microscope attached by a video camera to a visual image processor (Sight Systems Ltd.). A full description of this computer analysis is given by Allen et al. (1990). Briefly, two video images of each field were taken at different focal planes, corrected for uneven light levels, and summed. A binary image was superimposed, and the silver grains were then picked out as white by setting an appropriate gray level threshold. A preset box (35.4 pm*) was moved over cells or dish, and the white area within it was measured and expressed as the number of grains per area (gpa) using a calibration curve. Grain densities above 50 gpa could not be resolved (i.e., totally white) and were scored as 50 gpa. On average, 45 myocytes and 58 neurons (as recipients, 20 neurons as donors) were measured per culture. Grain densities over neurons, myocytes, and the neighboring dish were plotted as frequency histograms and compared using the median test. AChR Studies Myocyte and neuronal cultures were set up from stage 21 embryos, with both cell types loaded with either gap junction antibodyorpreimmune IgCs, butwithouttracers.Theywerestained live with rhodamine-conjugated a-bungarotoxin (kind gift of Tim Baldwin, diluted I:200 in modified Ringer’s solution with 0.5% fetal calf serum on live cultures) for 90 min before being washed three times in Ringer’s solution and fixed 24 hr after plating (3% glutaraldehyde in Ringer’s solution). Cultures were mounted with size 0 coverslips with Cityfluor antifade mountant and sealed with nail varnish. Hot spot analysis was carried out with a 63x Planapo Zeiss oil immersion objective using epifluorescence UV light and a rhodamine(510-560nm)filteronaZeisscompound phasemicroscope. Dishes were traversed in parallel sweeps, and each myocyte was checked for the presence of a neuron, any hot spots indicating AChR clusters, and the dependence of the clusters on the presence of the neuron. Results from cultures containing preimmune IgG or gap junction antibody-loaded cells were compared using the Mann-Whitney U-test. Acknowledgments This work was supported by grants from the Royal Society (A. W.) and the Medical Research Council. F. A. was initially supported byanMRCstudentshipandsubsequentlybytheMRC grant. We are indebted to N. B. Cilula (NIH grant GM 37904), who generously provided the affinity-purified antibodies to gap junction protein that made this work possible. Our thanks also to Tim Baldwin, who kindly gave us the rhodamine-conjugated a-bungarotoxin. Some of these results were included in a thesis submitted to the University of London in partial fulfillment of the requirements for the Ph. D. (Allen, 1988). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

13, 1990; revised

October

15,199O.

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Gap junctional communication during neuromuscular junction formation.

We have tested whether gap junctions form between nerve and muscle during their initial contact, before establishing the chemical synapse. Embryonic X...
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