seminars in CELL BIOLOGY Vol 3, 1992: pp 81-91

Gap junctions in development-a perspective Anne Warner The status of the gap junction as a pathway for cellular interactions during development is reviewed. Current evidence suggests thatgapjunctionsplay an important part in ensuring normal development, although theprecise role ofgapjunctional communication remains to be defined. Communication through gap junctions acts alongside cellular interactions achieved by the release ofgrowthfactors during embryogenesis. Differences between groups of developing cells may bereflected in, and possibly controlled by, alterations in the selectivity of the gap junctions. It seems likely that gap junctional communication is involved in the control of embryonic patterning rather than phenotypic differentiation.

mental biology, since they held out the excinng prospect of identifying the pathway for cellular interactions during development. The universal presence of gap junctional communication between all cells in early embryos has been amply confirmed in the 25 years that have passed since the original observations. The catalogue extends from invertebrates such as the sea urchin, starfish and ascidian, through vertebrates such as the frog, fish and chick up to the mammals such as the mouse. The timing of the first appearance of junctional communication varies somewhat between species; in some, for example the mouse, it is delayed until some cleavages have passed. In others, for example the amphibian embryo, junctional communication is present from the two cell stage onwards. The significance of this variation is not known, but could reflect those developmental times when important interactions take place through the pathway provided by gap junctions. Thus it is increasingly clear that some aspects of embryogenesis depend on the unequal distribution of molecul es within the fertilized egg, that become partitioned into specific regions of the embryo by cleavage (see e.g. ref 4). Junctional communication may only become appropriate. at times when the consequences of this molecular distribution need to be modulated. It is striking that gap junctional communication is usually in place when other evidence shows that cellular interactions are occurring. Potter et al were fully aware of the potential importance of their observations. They point out "the most striking possibility is that embryogenesis is controlled, at least in part, by such means . . . through the passage of substances from one cytoplasm to the next" . They go on : "However in order for differences between cells to become established and maintained, the communication between cells must be selective". These two quotations encapsulate the major issues in trying to elucidate the possible role of gap junctional communication during development. In this brief review I take these two quotations as my starting point for a discussion of current understanding of the problem. A parallel is frequently drawn between the properties of embryonic cells and cells that have been

Key words: gap junction I development I embryonic patterning I embryogenesis I cell-cell interactions

IN 1966 POTIER et all showed that cells of the early squid embryo were connected to each other by low electrical resistance connections, which provided a preferential pathway for the movement of small ions from cell to cell. Additionally they noted that the larger molecule used to mark the location of their injecting microelectrode occasionally passed from cell to cell also . They drew the important conclusion that all cells of the early squid embryo, regardless of eventual developmental fate, were interconnected to each other by this low resistance pathway. Potter etal speculated that the connections between the embryonic cells were of a similar nature to those found between inexcitable cells such as glia? and epithelial cells.I Furthermore, they noted that these direct connections were lost as organogenesis proceeded and that the disappearance of low resistance junctions was not uniform . We now know that such electrical coupling reflects the presence of the intercellular structure, the gap junction, between embryonic cells. It had long been recognized that extensive cellular interactions are a key feature of embryogenesis and these findings generated tremendous interest in develop-

From the Department ofAnatomy & Developmental Biology, University College London, Gower Street, London WC1 E 6BT, UK ©1992 Academic Press Ltd 1043·46821921010081 + 11$5.0010 81

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transformed to become cancerous. There is a substantial literature covering the possible role(s) of gap junctional communication in growth control, establishment of the cancerous state and in maintaining normal cellular behaviour. I have excluded consideration of such issues from this review, which is limited strictly to events occurring during embryonic development.

Embryogenesis is controlled by the passage of substances from one cell to the next The idea that specific molecules, often referred to as morphogens, might control embryogenesis is a well established concept in developmental biology, extending back to the early part of this century. Many embryological experiments have found ready ·explanation in terms of a gradient(s) of a substance or cellular property that provides embryonic cells with developmental cues and is interpreted to generate the appropriate cellular response. This was formalized in the concept of 'Positional information' by Wolpert.f A number of plausible theoretic · models have been formulated to provide a framework for the interpretation of experimental results. A particularly useful discussion of such models can be found in Slack." Many of them are based on a' diffusional gradient, with various modifications introduced to take account of the complexities of embryonic development (e.g. reaction-diffusion models''), They have in common the need for information to be transmitted from one cell to its neighbours. None of them have detailed requirements for the mechanism of cell to cell transfer, but most suppose 'the existence of molecules that can loosely · be termed as morphogens. Clearly the pathway provided by gap junctions has the potential to provide the mechanism for transferring such morphogens. But what do we know about these embryological morphogens and might they plausibly go through gap junctions? It is realistic to say that we have very little idea of the nature of natural morphogens. In Hydra, a small molecule, which possesses properties to be expected of a morphogen, is involved in head inhibition. Recently retinoic acid has found popularity as a potential morphogen because of its ability to mimic natural signalling regions such as the zone of polarizing activity (ZPA) in the chick limb. Retinoic acid is highly lipid soluble and would not need to move through gap junctions. However its current status

A. Warner

as a morphogen is uncertain. But there is evidence (e.g. ref7) that retinoic acid can affect gap junction permeability. Thus, since we know virtually nothing about natural morphogens, the possibility that some of them might move through gap junctions remains eminently plausible. If we take Hydra as our example, then molecules acting as morphogens could be hydrophilic molecules of about 500 in molecular weight. Will gap junctions between embryonic cells allow molecules of this size to move through them? The answer is almost certainly yes. Even though the range of probes available to explore the permeation properties of gap junctions is relatively restricted, there is good evidence in. most of the embryos examined that lucifer yellow (molecular weight 457) and fluorescein (molecular weight 332) can move from cell to cell through at least some, if not all (see later), the gap junctions found between embryonic cells. The upper limit of the size range has not been explored in any detail. Potter et all occasionally noted transfer of niagara sky blue (993) to more than one cell, suggesting that embryonic junctions can, under the appropriate circumstances, transfer even larger molecules. The main draw-back to these studies is that they rely on non-physiological molecules, whose intrinsic behaviour may not match normal cellular components, introduced into the cell for the purpose of probing junctional properties. The technique of metabolic cooperations which allows the movement from cell to cell of small, radio-labelled, naturally occurring nucleotides to be tracked through their incorporation into RNA and DNA, has not been transferred successfully from the culture situation to intact embryos because of the technical difficulties associated with nucleoside leakage into the restricted extracellular space between cells. However since studies on cultured cells using both dye transfer and metabolic cooperation techniques have given much the same answers in terms of the size of molecules that can permeate gap junctions, it is most likely, although not certain, that evidence derived from the study of non-physiological, fluorescent molecules applies also to physiologically available molecules of the same size. Thus we are left with the somewhat unsatisfactory situation that we know very little about morphogens, but they may be small and of a size that should move through gap junctions between embryonic cells. Whether the gap junctional pathway is the normal method for cell to cell transfer of such molecules remains to be demonstrated. Thus even after 25 years of endeavour, there has been little progress

Gapjunctions in development

in identifying any molecules that might move through gap junctions to control development. A more general approach to answering the question whether substances move through gap junctions to control embryogenesis is to examine whether there is evidence to show that gap junctional communication is necessary for normal development. Do gap junctions between groups of embryonic cells with different eventual fates disappear at a time that is correlated with divergence of fate? Do perturbations of gap junctional communication have important consequences for embryogenesis? There have been few systematic studies of _the time course of the abolition of gap junctional communication in relation to determination of fate; the scattered evidence does not suggest a close correlation. In the ovary, oocyte and follicle cells are connected by gap junctions; this communication is reduced prior to ovulation and eventually disappears before oocyte shedding. Potter et all showed that groups of cells in the squid embryo lose their junctional communication with others as differentiation progresses, at times that varied for different systems. Gap junctions between retinal cells disappear at about the time when axial polarity of the. eye bud is determined.? Prior to somite formation, gap junctional communication disappears between cells destined to become part of adjacent somites.!" However, gap junctional communication between lateral ectoderm cells, destined to form epidermis, and neural plate cells, destined to form the central nervous system, was retained until the neural tube closed, some time after the separation of developmental fate. 11 Trophectoderm and inner cell mass of the early mammalian embryo are still coupled well into the blastocyst stage, after their divergence onto different pathways.Jf In general gap junctional communication disappears after determination of future fate. This could have a relatively trivial explanation. The disappearance of gap junctions between groups of cells may not need to be terminated rapidly once the relevant interactions are complete and loss through junction turn-over may be sufficiently fast. Alter-natively complete loss of ionic coupling may precede physical separation of two groups of cells (as in somite formation and neural tube closure) and junctional changes related to specification may be achieved by alterations in the discrimination of the gap junction. For development purposes it could be sufficient to shut off transfer of molecules larger than small ions,

83 with ionic communication remaining to provide a signal that there is a near neighbour (see later). However once one turns to perturbation experiments, where gap junctional communication has been abolished experimentally either ahead of time or inappropriately, a rather different picture begins to emerge. Here the evidence for an important role for gap junctions in embryogenesis is strong, even though it is not yet clear exactly how junctional communication plays a part or what molecules are moving through them. The agents that can be used for perturbation experiments are few. This is because reagents that influence the permeability of gap junctions also are likely to have effects on cell metabolism. Thus raising intracellular ionic calcium will close down gap junctions, 13 but manipulating Ca, will affect many other cellular processes and separation of the effects of closing gap junctions from other consequences of altering intracellular calcium is well nigh impossible. Similarly, gap junctions are highly sensitive to alterations in intracellular pH.H,15 But again changes in pHj will have multiple consequences for the cell. Gap junction permeation also can depend on the voltage applied across the junction, as noted for amphibian embryonic cells, 16 and the classical rectifying electrical synapse displays strong voltage dependence.I? However the very fact that cells are electrically coupled mitigates against the generation of large differences in membrane potential between adjacent cells and manipulation of potential differences between cells is not a feasible method for testing the consequences of perturbing junctional communication. Antibodies against gap junction protein have so far proved more useful as tools for testing whether gap junctions play an important functional role during development. A number of workers have successfully raised antibodies that can identify gap junction proteins on immuno-blots and decorate gap junctions in sections specifically, both at the light and EM level. Most work has been directed towards the 32K connexin protein, originally described in liver,18-21 although antibodies to more recently identified connexins are now available (connexin 43 22; connexin 2623). Immunogen preparation and antibody purification has varied. Monoclonal antibodies (e.g. refs 24,25) and peptide antibodies to the amino terminus region 26,27 also have been described. Of these antibodies a number have been reported to block gap junctional communication. 20,27,28 In all these studies nonspecific IgGs and antibodies specific for other proteins were without effect on junctional communication.

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As yet, only the polyclonal antibody described by Warner et ai 20 has been used for developmental studies. This particular antibody was raised by N.B. Gilula using rat liver eluted 32K protein as the immunogen, with antibody purification against eluted protein also. It happened to recognize awide range of gap junction proteins, isolated from vertebrate and invertebrate preparations. It does not seem to be species (excluding arthropod gap junctions) or tissue specific, either in its immunoblotting or immuno-labeIIing characteristics and inhibits gap junctional communication in an equivalently' wide range of preparations (Hydra 29 ; amphibian2o ,30; chick 31; mouse32 ; BRL cells20) . This implies that the antibody is multi-epitope with at least one important epitope that is highly conserved between members of the gap junction protein family. The functional block is unlikely to be the simple consequence of screening by large IgGs since monovalent Fab fragments are equally effective. 20 Injection of these antibodies into one cell of the amphibian embryo at the 8-cell stage blocks junctional communication between the progeny of the injected cell. 20 The block is maintained up to the middle to late gastrula stage 30 and antibody can be recognized within the progeny of the injected cell after the neural tube has closed (N.J. Messenger and A.E. Warner, unpublished) implying that cells containing antibody do not die. There is no interruption or slowing in the cell cycle after antibody injection and.prior to organogenesis the communication incompetent progeny of the antibody injected cell cannot be distinguished visually from their communication competent neighbours. The injected cell was destined to give rise to the right hand anterior part of the nervous system and some of the somites. Antibody injected embryos developed with a range of patterning abnormalities in which structures derived from the communication incompetent region were either completely missing or of the wrong size and in the wrong place on the antero-posterior axis. The patterning disturbance was particularly noticeable in the brain, with forebrain extending back to the level of the eye. Patterning disturbances were equally evident when vegetal pole cells of the early embryo were injected (Warner et al, cited in ref 33). In these cases there appeared to be no dorso-ventral axis, yet a full range of differentiated phenotypes were present. Thus preventing gap junctional communication had little or no effect on the differentiation of phenotype, but major effects on the generation of pattern.

A.

~Varner

A similar conclusion may be drawn from experiments that examined particular elements of the embryonic patterning process. In Hydra, Fraser et ai 29 looked at the ability of a grafted head to suppress regeneration of a new head in Hydra. When the head is removed from Hydra, a new head wiII regenerate in its place. But if the head is replaced by a head grafted from another animal, head regeneration in the host is inhibited. This is supposed to arise from transmission of a head inhibiting morphogen from graft to host. Fraser et ai 29 compared the frequency of regeneration of a head at .the graft / host border in control hosts (preimmune IgGs) and hosts that had beenloaded with gap junction antibodies using a DMSO bulk loading technique. They showed that graft and host would establish gap junctional communication between them relatively rapidly, but where the host contained gap junction antibody the appearance ofjunctional communication was delayed for an interval that encompassed the time for the head inhibitor to have its effect. When hosts were loaded with gap junction antibodies and prevented from establishing junctional communication with the graft, a significant number of animals developed secondary heads at the host / graft margin. This implies that gap junctional communication is involved in normal transmission of the head inhibitor and, indeed, that it is an essential part of the mechanism by which head inhibition occurs. In the chick limb bud, Allen et ai 31 tested whether communication through gap junctions might be involved in the induction of additional digits in a host by grafted cells from the zone of polarizing activity (ZPA), which lies at the posterior margin of the limb bud. The antibody maintained a block of cell-cell communication for 16-24 h and did not affect cell division. When both grafted ZPA and adjacent anterior mesenchyme were rendered communication incompetent by bulk loading with gap junction antibody, the ability of the ZPA to specify additional digits was reduced significantly. These experiments pin-pointed heterologous gap junctions between polarizing posterior mesenchyme cells and responding anterior mesenchyme cells as crucial to the patterning process. In the mouse, inhibiting communication through gap junctions has lead to some interesting conclusions.P Previously it had been supposed that communication through gap junctions was not involved in the process of compaction, which occurs at the 8-cell stage when the cells increase intercellular

Gap junctions in development

contacts-they literally compact down onto each other. At this time the cells become polarized, begin to form tight junctions at their outer edges34 and communicate through gap junctions.P An antibody to uvomorulin, a component of the tight junction, would induce embryos to decompact, but did not affect gap junctional communication.V' It was, therefore, surprising that when antibodies were used to inhibit gap junctional communication this caused decompaction of the 'communication incompetent cell(s), while communicating cells remained in the compacted state. These results suggested that while the early stages of compaction may not require junctional communication, gap junctions are important for the maintenance of the compacted state. Since decompaction requires that the developing tight junction at the outer edges of compacting cells be disrupted also, there may be a link between the two junctional types. A similar conclusion can be drawn from a completely different set of experiments which did not rely on antibodies. Ref 36 examined gap junctional communication in embryos generated by a cross between females of the DDK strain, with males of an alien strain. This cross is known to generate defective progeny, 90-95 % of which are destined to decompact during the morula and blastocyst stages and fail to form normal blastocysts. A substantial proportion of DDK / C3H zygotes had poor gap junctional communication compared with the normal progeny of a DDK / DDK cross. Furthermore if gap junctional communication was improved by raising intracellular pH for 4-6 h before the defect became apparent, a substantial proportion of the genetically defective embryos maintained compaction and went on to form normal blastocysts. The conclusion can be drawn that gap junctional communication probably is essential for the maintenance of the compacted state. Antibodies to gap junction protein also have been able to define at least one interaction that apparently does not require gap junctional communication. In the amphibian embryo, the appearance of mRNAs for cardiac actin provides a sensitive indicator for the induction of somitic mesoderm in the animal pole region by vegetal pole cells.37 Warner and Gurdon-? examined whether this indicator of mesoderm induction no longer appeared when explants in which either inducing vegetal pole cells or responding animal pole cells were rendered communication incompetent by injection of antibodies to gap junction protein. The outcome was quite clear.

85 Induction of the mesodermal phenotype was not affected by the inhibition of gap junctional communication between endoderm and ectoderm. We can now put this result together with those in which embryos were left intact after injection of antibody into the vegetal pole (ref 33; see above). In the intact embryo there was no effect on mesodermal phenotype. Embryos that could not communicate through gap junctions established between vegetal and animal pole cells possessed the normal complement of mesodermal phenotypes. Nevertheless patterning was disturbed severely. Taken together, these experiments suggest that during mesoderm induction in the amphibian embryo the induction of phenotype proceeds through a route that is different from that which ensures normal patterning. Patterning requires gap junctional communication. Induction of phenotype does not. This conclusion fits well with other evidence suggesting that induction of phenotype is controlled by growth factors such as the activins. 38 The use of antibodies as tools to explore the functional role of gap junctions is an approach that requires caution. The crucial question relates to the specificity of the antibodies and the possibility that consequences of antibody treatment might relate to non-specific, possibly toxic effects. The perfect control, which would be a non-blocking, but localizing, antibody to gap junction protein, is not yet available. All those who have demonstrated a communication block by antibodies to gap junction protein showed that. equivalent amounts of preimmune IgGs were ineffective and that antibodies specific for other molecules were also without effect. On the time· scale appropriate for examining developmental effects it is more difficult to be absolutely certain that cumulative effects of low toxicity do not contribute to the results. However this concern can be reduced substantially by checking that the presence of gap junction antibody does not have effects on parameters such as cell cycle time, which provides a sensitive indicator of low levels of toxicity, and discarding batches of antibody that have such side-effects. This was done routinely for all the work quoted here. In the chick, there was. no evidence that cell cycle time was altered in cells containing gap junction antibody.U In the Xenopus embryo cell division proceeds rapidly and variations in cell cycle time can be recognized as a clone of cells of different size from the remainder. Cells injected with gap junction antibody at ' the 8-cell stage continued to divide at the same rate as cells derived from their uninjected siblings throughout the period

86 that the antibody remained effective functionally and beyond. 2o,30 Experiments showing that the presence of gap junction antibodies have no consequences provide additional confirmation that non-specific toxic effects do not complicate interpretation. Thus the absence of any effect on induction of mesodermal .phenotype,30 a normal time-course of primary differentiation of nerve and muscle cells in culture'? and the absence of any effect on the induction of digits by the ZPA in the chick limb bud when only anterior mesenchyme or only ZPA cells contained antibody-" all provide important evidence against non-specific effects of gap junction antibodies. As other blocking antibodies become available it will be important to see how they influence development. The overall evidence from these experiments is that gap junctional communication is important during embryogenesis because it contributes to patterning processes. Thus we may conclude that Potter etai's supposition that communication through gap junctions is important for development is well substantiated.' However we still have a long way to go before we can identify the molecules that move through junctions and .the precise mechanism by which gap junctions contribute.

For differences between cells to become established and maintained, communication between cells must be selective The number of identified gap junction genes is growing rapidly and gap junction proteins are now recognized .as a family both with substantial homology and regions of non-homology between members. This supports; in general, the accumulating evidence from work on developing systems that gap junctions within an embryonic system do not all have the same properties. There are a number of situations where a difference in gap junction properties is associated closely with developmental boundaries and developmental events. The most striking example has come from work on the epidermis oflarval stages of segmented insects. The grafting experiments of Locket? showed that each segment behaves as an autonomous developmental unit. Locke pointed out that experimentally each segment behaved as if there was a gradient of some property(ics) that was high at one end of the segment and low at the other, and was repeated from segment to segment. Furthermore each segment is derived from a small group of founder cells set aside

A. Warner

early in development whose progeny do not cross the segment boundary and form a developmental compartment. 41 One major question was whether variations in gap junctional communication within and between segments might be part of the mechanism that controlled the developmental role of the segment boundary. Early experiments42,43 found that ionic coupling was present between all cells in the epidermis and that there was no interruption at the segment border. However, when the movement of molecules larger than small ions, such as lucifer yellow (LV), was examined, it became clear that gap junctions between cells on either side of the segment border are not the same as elsewhere in the epidermis.. When LY was injected into a cell lying in the middle of the segment it spread freely to all its neighbours. However when a cell lying at the border was injected, dye did not spread to cells on the opposite side. 44,45 The number of gap junctions between cells lying on either side of the segment border and between cells in the same segment is the same46 so poor transfer of LY cannot be explained simply by lower numbers of junctions at the border. In Warner and Lawrence's experiments (in Oncopeltus) the barrier lay precisely at the border, while in BIennerhassett and Caveney's experiments (in Tenebrio) there was evidence for a line of cells with reduced junction permeability lying at the border. But the crucial point is that gap junction properties change at a precisely defined developmental boundary. Unfortunately the gap junction between cells in arthropods seems to be constructed from a protein(s) that differs from those found in non-arthropod systems and no clear candidate for arthropod gap junction protein(s) has yet emerged. This has meant that possible roles for gap junctional communication in development have not been illuminated by the genetic approaches that have led to great insights into other aspects of development in Drosophila. The ability of some embryonic junctions to discriminate against LY appears to be widespread and restrictions on the spread of LY through gap junctions which nevertheless allow exchange of small ions have been reported in a number of embryos. In the marine snail Patella there are marked restrictions in LY transfer that appear early and seem to define developmentally related groups of cells.t? In the starfish embryo electrical coupling is established well before any dye transfer is apparent.48 In Fundulus, Bennett et al 49 failed to see fluorescein transfer, although a later study yielded the opposite result, with transfer of both fluorescein and LY. 50

Gap junctions in development

In the mouse embryo it has been reported that dye transfer between inner cell mass and trophectoderm cells fails prior to implantation, even though electrical coupling remains.F In the' wing disc of Drosophila groups of cells have been found to be linked by junctions that transfer LY, with transfer to electrically coupled surrounding cells being restricted. 51,52 Such domains of LY transfer within regions that are extensively coupled electrically have been called 'communication compartments' although their relationship to development compartments defined on other criteria, such as those in the insect segment, and their developmental significance remains to be demonstrated (see ref 52). Transient junctional communication is a feature of later development with, for example, nerve and muscle probably establishing gap junctions between them prior to formation 'of the chemical synapse. 39,53 Insect pioneer neurones form short-lived, dye passing junctions with guidepost cells during navigation to their targets. 54 Thus the case for differences between gap junctions in developing systems seems strong, but in the instances quoted so far, the purpose of this discrimination, indeed the purpose ofjunctional communication itself, is not clear. However in the amphibian embryo some clarity is beginning to emerge, which should provide a basis for more focussed experiments. The presence of ionic communication between all cells of the early amphibian embryo is well established. 55-57 As in most embryos, the situation with regard to dye transfer has been more equivocal. Slack and Palmer-s did not observe fluorescein to move between cells in the Xenopus embryo. However, the key to the situation began to emerge once dye transfer was linked to cell position, and therefore developmental fate, within the embryo.59,60 The ability of LY to move through gap junctions in the amphibian embryo is determined by the position of the cell in relation to the future dorso-ventral axis of the embryo. Cells in future dorsal regions exchange LY both more extensively and more frequently than cells in future ventral regions. The situation has been investigated most completely at the 32-cell stage, but the relationship holds from the 16-cell stage to the mid-blastula stage. 60 At the 32-cell stage a limited, but nevertheless clear, selectivity sequence was determined for the dorsalmost and ventral-most cells in the animal pole. In ventral cells the sequence ran: dicyanoargentate (a small ion) > lead EDTA ~ LY = fluorescein. This

87 approximates well with size being the major determinant. In dorsal cells the sequence was not the same: dicyanoargentate> LY ~ lead EDTA ~ fluorescein. This sequence does not match size. It is interesting that the best indicator of the dorsal! ventral difference is LY, with the dorsal-most cell transferring LYon average in 70-90 % of tests, while the ventral-most cell transferred LYon only 6 % of trials, cells in intermediate regions showed intermediate levels of transfer. 59,60 Fluorescein always transferred rather little (20% between dorsal cells) but nevertheless, like LY, moved even less frequently between ventral cells. The difference between dorsal and ventral regions for LY transfer has been confirmed.61,62 In themselves these results simply add to the circumstantial evidence that gap junctional communication is important during development. The hope that this information might lead, in the long term, to greater understanding of what gap junctions are doing during embryogenesis stems from experiments in which the organization of the embryo is manipulated experimentally, because such manipulations lead to correlated, and predictable, changes in gap junction permeation. Experimental embryologists have longbeen aware that the dorso-ventral axis of the amphibian embryo can be altered: V.V. irradiation during the first two thirds of the first cleavage cycle interrupts the re-organization of cellular constituents that follow fertilization and causes the embryo to develop without dorsal structures. Conversely treatment of early cleavage stage embryos with lithium chloride induces regions that would normally be ventral to become dorsal and the embryo becomes entirely dorsal (e.g. ref63). Although these experimental manipulations are carried out at very early stages, their morphological consequences do not become apparent until after. gastrulation. However when the properties of gap junctions in embryos at the 32-cell stage are examined in either V.V. irradiated or lithium treated embryos, it is clear that a change in gap junction properties precedes alterations in the dorso-ventral axis and predicts the outcome of such experiments.P! In embryos destined to be totally dorsal, LY transfer in putative ventral regions rises to match that of dorsal regions. In V. V. irradiated embryos, destined to be ventral, L Y transfer throughout the animal pole at the 32-cell stage falls to that characteristic of ventral regions. The potential of an approach in which polarity is manipulated and gap junctional communication alters is exemplified by even more recent experiments

88 which tested the developmental consequences of ectopic expression of members of the wnl gene family. Injection of mRNAs for wnl genes, be they mouse, Xenopus or Drosophila homologues, at the one cell stage generates embryos, in which putative ventral regions have been converted to express dorsal characteristics (ref64; Colman, personal communication). Furthermore, after injection of mouse or Xenopus wnl gene mRNAs, LY transfer in previously ventral regions has shifted to the dorsal pattern by the 32-cell stage. 62 Although the evidence is not yet complete, it appears that either wnl homologues or modified constructs that do not induce double dorsal embryos also do not influence transfer through gap junctions, reinforcing the view that the two events are linked. Thus perturbing gap junctional communication induces patterning abnormalities (see above) while perturbing patterning alters gap junctional communication. While we are still a long way from understanding precisely how these two events are linked and what role is played by the changes in gap junction properties, we have reached a point where real progress with both issues should now be possible. Returning to Potter et al'sl original speculation that the need for cells to become different should be accompanied by differences in gap junction properties within an embryo, the accumulating evidence strongly suggests that this was correct.

Developmental changes in expression patterns of gap junction proteins The recognition that gap junction proteins are members of a large, and increasing, family is beginning to generate studies' in which the time course of appearance and disappearance of gap junction proteins is charted during development. It will be some while before it will be possible to interpret the changing patterns ofmRNA expression and protein distribution in a way that makes functional sense. However studies on the functional topology of gap junction proteins and better understanding of the way in which sequence controls permeability properties, should, in the future, make it possible to deduce the functional consequences for development of patterns of expression of the various gap junction proteins. In this section therefore I outline briefly the results from those studies that have so far appeared. An important caveat when looking at such results is that in the majority of cases it is not known whether gap junctions identified

A. TVarner

immunocytochemically are functionally operative and, in those cases where more than one gap junction protein gene is expressed in the same group of cells, we do not yet know the functional significance. Neither do we have any information on the possible role of proteins that might be associated with gap junctions and contribute to the control and modulation of junctional permeability. The state of phosphorylation of junction proteins may also prove to be important. Gimlich et al 65 examined the appearance of mRNAs for gap junction proteins during development of the Xenopus embryo. Oocytes contained mRNAs for an al connexin, which shares homology with the adult al connexin 43, but was not present in the fertilized egg. Early in development, prior to gastrulation, they identified maternal mRNAs for a protein called connexin 38 (or (2). At these stages this appeared to be the only source of gap junction protein. a2 mRNAs disappeared during gastrulation and were superseded by PI mRNAs, homologous with the adult liver PI connexin 32, which increased during development of the tadpole. The PI mRNA was abundant in adult lung, liver, intestines and stomach. In the mouse embryo, prior to implantation gap junctions are constructed from connexin 43. 66 ,67 This conclusion is based on mRNA expression and immunocytochemical labeling of gap junctions. So far there has been no indication of expression of other gap junction genes, although the difficulty of working with the small amounts of material available from early mouse embryos makes it hard to be absolutely certain that connexin 43 is the only gene expressed prior to implantation. As the mammalian embryo develops, a complex sequence of changing patterns of expression of gap junction gene products ensues. Risek and Gilula68 in a detailed study using immunocytochemistry and northern analysis, charted the appearance and disappearance of three gap junction proteins (connexin 43 (al), connexin 32 (PI) and connexin 26 (P2) in tissues of the developing rat embryo, the placenta and extra-embryonic membranes from implantation through to term. It is inappropriate to give extensive description of these findings here, and the interested reader should refer to the original paper. One of the most interesting observations was that tissues often expressed more than one gap junction protein, although never more than two. The combinations also showed a pattern in which al (connexin 43) and PI (connexin 32) were never found together, but

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each frequently was present along with {32 (connexin 26). The spatio-tcmporal differences were often celltype specific, and showed differential regulation of individual gap junction proteins in tissues where two proteins were expressed. Our understanding of the functional differences between the different members of the gap junction family is, as yet, too incomplete to allow these interesting results to be interpreted . As things stand the interpretation of studies of the changing patterns of expression of gap junction protein during development will have to wait until our knowledge of the way in which functional properties are linked to sequence, and how gap junction proteins interact with each other when they are expressed side by side in the same cell, has risen above its present rudimentary level. The issue of possible accessory proteins needs resolution also. However such studies will form an invaluable basis for future understanding of the way in which gap junctions contribute to developmental processes.

Conclusions As we stand 25 years on from Potter et at's seminal paper we can see great progress in some areas while in others hard information is distressingly sparse. It seems right to conclude that their major predictions have been borne out by subsequent experiment: gap junctions do play an important part in ensuring normal development. Communication through the direct intercellul ar pathway mediated by gap junction acts alongside cellular interactions achieved by the release of growth factors and their subsequent binding to membrane receptors to coordinate embryogenesis. The evidence suggests that gap junctional communication is involved in patterning rather than phenotypic differentiation, which .seems more likely to be controlled by cellular responses to growth factors, where interactions between a variety of second messenger mechanisms may determine the particular outcome. Evidence for variation in gap junction properties between cells in specific regions of an embryo is accumulating. It is very unlikely that developmental control is achieved by the gross tool of the presence or absence of junctional communication. It is much more probable that subtle alterations in junction properties, possibly operating at the level of molecules larger than small ions, provide delimiters for developmental variation. However the absence of evidence so far to indicate differences in the ionic transfer

properties of gap junctions within a developing embryo should not be taken as proof that ionic transfer mechanisms are not important. The analysis of ionic movement through gap junctions is not possible in the intact embryo and requires application of electrophysiological techniques such as double whole cell recording in pairs of cells taken from particular regions before the contribution of the transfer of small ions between cells can be assessed. The identification of a number of 'm aster' genes in Drosophila and the realization that their homologues are widely distributed in other species has barely touched the central issues related to cellular interactions during development. For such analysis to illuminate our understanding of the contribution of gap junctional communication we need, as a matter of urgency, to identify the proteins involved in construction of gap junctions in arthropods. The lack of progress with identification of developmental signalling molecules transmitted through gap junctions is disappointing, and contrasts starkly with recent advances in understanding of the role of specific growth factors, particularly in the amphibian embryo. This reflects, in part, the general difficulty experienced with the identification of molecules that might act as embryological morphogens and could mean that morphogens are common cellular components, hijacked to act as a morphogen during a particular time in development. If this is indeed the case then morphogen identification will continue to pose difficulties. We have moved a long way in the past 25 years. Progress has been steady rather than spectacular and particular elements of the problem of understanding how cellular interactions through the direct cell to cell pathway mediated by gap junctions still remain largely unsolved . The accumulation of evidence should provide a springboard for future progress, which will depend increasingly on the integration of information from molecular and cellular approaches in order to build up a composite view of how different mechanisms contribute to the evolution of an organism from the fertiliz ed egg.

Acknowledgements I thank the Royal Society for their support. Work in this laboratory was made possible by grants from the Medical Research Council and the \Vellcome Trust.

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