The Transmission of Morphogenetic Signals from Amphibian Mesoderm to Ectoderm in Primary Induction

Reviews The Transmissionof Morphogenetic Signals from Amphibian Mesoderm to Ectoderm in Primary Induction S. TOIVONEN1*, D. TARIN’, and L. SAXEN3

’ The Department

of Zoology, University of Helsinki, Finland and

’ Department of Histoualhologv, -_ Royal Postgraduate Medical School, London, U.K. and ~

Third Department of -Pathology, University i f Helsinki, Finland

Received March 1975

In the 1920s it was realised from the experimental work of Spemann’s group and many others that the initiation of nervous system formation in the ectoderm of the intact vertebrate embryo depends upon action exerted by the underlying mesoderm. The process is referred to as primary embryonic induction, to distinguish it from subsequent inductive phenomena in other organs which are referred to as “secondary”. The mechanism by which the signal is transmitted from one tissue to the other has been the subject of much investigation. By 1932 it was shown [ I ] that not only living mesoderm but also killed tissues are able to cause neural and other types of differentiation in competent amphibian ect0dermti.e. ectoderm which is young enough to respond to such inductive stimuli) and it became assumed that inductive action was chemically mediated. In the same connection Mangold published his finding that a piece of agar gel which had been in contact with neural plate tissue was subsequently able to induce neural differentiation in a new piece of competent ectoderm. This demonstration that inductive activity could apparently be conferred on non-cellular material corroborated the idea that inductive signals are transmitted by the passage of chemical agents. This concept was further developed by Dalcq [61 and by Needham [221who compared induction to virus,infection of the cells. Brachet has used a modification of the concept as a working hypothesis since 1940 131. He and his co-workers have suggested that the inductive stimulus is transmitted by transfer of microsome-like particles from the inductor to the ectoderm and that the active factor may be a nucleoprotein [4,51. The postulated diffusibility of the inductive agents has since however been the subject of much controversy and debate and an alternative theory of the mechanism of induction was presented by Paul Weiss [44,451. He argued that as contact, or at least a close relationship,

* Requests for reprints to S . Toivonen. Differentiation 5, 49-55

(1976) -

0 by Springer-Verlag 1976

between the inductive cells and the reacting tissue is essential in many systems, the process may be effected by interaction between the surfaces of the two cell types. In his own words “an attraction of key molecules to the new contact area is followed by orientated absorption, the building-on of orientated chains of molecules and consequent redispositionofthe chemical system ofthe cell” [451. However, Weiss did comment that different systems may depend on different mechanisms and warned that it is not wise to generalise [461. In the years that have passed, neither of these apparently self-sufficient and mutually exclusive theories of transmission of inductive information has completely triumphed over the other, But since 1950 much evidence has been obtained experimentally on the question of whether embryonic induction is accomplished by actual diffusion of chemical factors as against interaction between the surfaces of different groups of cells and the findings support the interpretation that the mechanism varies in different organ systems (see below). In fact a third proposal, that of “matrix interaction”, has been proposed by Grobstein [ 101to explain inductive effects. This postulates that interaction between molecules of restricted mobility associated with the cell surface coat or extracellular matrix is the basis for transfer of information. Four main types of experiments have been employed to investigate the transmission of stimuli from the inducing to the responding tissue: 1) Using labelled inductors or immunological methods to detect and follow any transfer of substances from the inductors to the ectoderm. 2) Testing the inductive capacity of cell-free extracts either by using the classical implantation and “sandwich” methods or by culture of competent ectoderm in “conditioned” solution. 3) The use of electron microscopy, histochemical methods and enzyme treatments to examine the interface between the interacting tissues and to assess the develop-

50 mental consequences of removing materials found in this zone. 4) Separation of the inducing and reacting tissues by means of interposing membranes of different types. Let us first estimate thevalue ofMangold's experiment with agar gel which originally led him to present the idea of diffusion of the active factor mentioned above. To the best of our knowledge nobody has been able to obtain positive results on repeating his experiments. One of us (S.T.) has also tried but obtained only negative results. It is therefore suggested that Mangold's positive result may have been caused by contamination; some cell detritus possibly having remained on the agar and caused the reaction. Nevertheless, evidence obtained subsequently by others using different techniques (see later), supports the conclusion that soluble factors can bring about neural differentiation in competent ectoderm and that such agents are active at least in the initiation of neural induction in vivo.

Experiments with Radioactive Tracers

Several attempts have been made to use radioactive labelling techniques to establish whether transmission of the inductive stimulus depends on the passage of specific molecules from the inducing to the responding tissue. (See for example Ficq IS], Waddington 1411, and Kuusi [ 15, 161. In general, the principle of the experiment was to label the inductor (which in some cases was living tissue and in others was killed by various methods) and combine it with unlabelled ectoderm for varying periods of time after which a search was made for the presence of radioactivity in the responding tissue. In all of these experiments, labelled material was found in the cytoplasm of the reacting cells, but did not provide direct evidence that the labelled substances were conjugated with active macromolecules in the recipient. It is also possible to argue, especially in those experiments using labelled killed inductors, that thelabel passed from theinductor to the responding tissue by nonspecific diffusion and that the original labelled chemicals eventually accumulated there in sufficient quantity to be detected. In order to overcome some of these objections Vainio et al. [421 designed the experiment described below. It was based on the knowledge obtained from earlier studies that HeLa cells grown in culture media containing fresh human serum are, after alcohol treatment, potent inductors of both neural and mesodermal (e.g. muscle, notochord) structures but that the latter activity can be abolished by heating the cells to 70" C. Equal proportions of heated and unheated HeLa cells were mixed to produce two preparations in each of which only one of these cell types was

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labelled (with C14 algal protein hydrolysate). It was assumed that the HeLa cells originally contained both neuralising and mesodermalising agents and that the mesodermalising principle is inactivated in the heated cells. Thus it was hoped to obtain two inductor preparations with the same inductive action but with the mesodermalising principle being labelled in only one of them. Following this, comparative counts of the radioactivity of the neural versus the mesodermal structures induced were made to obtain a picture of the specificity of incorporation. AS expected, in both series, after the cell mixtures had been implanted in amphibian (Triturus) gastrulae, both neural and mesodermal structures were induced. Autoradiographic studies of histological sections showed that in both series the labelling was heavier in the induced secondary formations than in the comparable structures of the host. This was expected, because the secondary structures were nearest to the inductor, but the finding that there was no difference in the labelling of the secondary structures in the two series was somewhat unexpected. The authors reasoned that, if the label had been specifically transmitted to the structures induced, the mesodermal structures in the series implanted with the supposedly labelled mesodermalising principle (i.e. with labelled untreated cells) should have been more heavily labelled than those in the series in which the heat-treated cells were labelled. However, this was not so and it was concluded that radioactivity had therefore not necessarily been conjugatedwith thepostulated inductive principles, but had reached the structures non-specifically by diffusion of the labelled chemical. Accordingly, it was judged that the use of radioactive tracers might not be ideal to investigate the transmission of inductive effects. Experiments Using Immunofluorescence

An alternative means of investigating whether the transfer of chemical agents from the inducing to the responding tissue is essential for primary induction was provided by the use of immunofluorescence techniques 1421. The experiments utilised the sandwich technique in which an inductor (in this case guinea-pig bone marrow) is enclosed in an envelope of ectoderm. Normally this results in the induction of mesodermal structures in the ectoderma1 envelope, if the alcohol treated marrow is left in situ. The removal of the marrow from the sandwiches after 1 h results in failure of any differentiation in the ectoderm on subsequent culture 1371; after 2 h, the ectoderm partly differentiates to mesodermal structures and after 4- 12 h association, the results are almost the same as in the control from which the inductor was not removed.

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When sections of such sandwiches were treated with antisera to guinea-pig bone marrow, raised in rabbits and conjugated with fluorescent isothiocyanate, the bone marrow tissue in the centre exhibited yellow fluorescence. In sandwiches from which the inductor was removed after one hour a few light granules were seen in the cytoplasm of the ectodermal cells nearest to the inductor tissue. The appearances suggested that the fluorescent particles were attached to the yolk patelets and demonstrated that some macromolecules originating from the bone marrow had penetrated into the reacting cells. After 6 h association between the killed bone marrow and the ectodermal envelope, fluorescent granules were seen, frequently extending as far as into the second row ofectodermal cells. Thus, the antigenicity does not remain on the surface of the marrow cells or at the interface between marrow and ectoderm, but has gone into the cytoplasm of the latter. Vainio et al. [421 concluded from this information that “the transfer of large-molecular (antigenic) material does occur from the inductor to the reacting cells, and that the conditions necessary for the passage of inductive information in this form. do in fact exist”.

in fact under the “minimal mass” for normal development of such complex structures. A better method for studying the effects of cell free extracts was developed by Tiedemann and his group [2]. They cultured larger pieces of competent Triturus ectoderm in hanging drop preparations in which curling of the ectoderm was prevented by placing a filter membrane on the outer side of the ectoderm and pressing it slightly towards the cover slip. When the culture medium was “conditioned” by chick embryo extracts, or a potent fraction thereof, prior to introducing the ectoderm, definite differentiation to notochord, striated muscle fibres and neural formations was obtained in this system. This shows conclusively that it is possible to produce certain inductive effects with cell free extracts in the absence of any inductive tissue. Yet, all efforts to isolate such active fractions from the normal inductor tissue(s) have failed. Therefore, despite the evidence that inductive phenomena can be mediated by chemical transmission, it does not establish that the inductive processes involved in co-ordinating development in vivo are all dependent on the diffusion of soluble chemicals.

Experiments with Cell Free Extracts

Observations on the Interface between the Dorsal Mesoderm and Ectoderm during Neural Induction

Evidence suggesting that among the macromolecules transferred from one tissue to the other there are some which areinductively active has been obtained by studying the effects of cell free extracts on tissues in culture. One of the main difficulties in culturing isolated competent ectoderm in conditioned media (i.e. ones enriched with extracts of inductive tissue) is that it curls very rapidly and forms a vesicle with the outer surface of the ectoderm outermost. The ectodermal surface coat which covers this aspect of the tissue is known to be very impermeable and can be penetrated only by small molecules. Isolated sheets of ectoderm are therefore unsuitable for experiments on induction unless special techniques are employed. Niu and Twitty 1211 avoided this handicap by using such small pieces of ectoderm that they did not have any tendency to curl. They cultured these in droplets of media in which normal inductive tissue had previously been cultured and reported differentiation of neural cells, pigment cells and myoblasts in a relativelyhigh percentage oftheexplants. In Niu’s later series [ 19,201, using extracts from different mammalian organs, various types of cell arrangements were noted, for instance tubule-like aggregates or neuroid tissues. Nevertheless, the conditions in these cultures seemed to be unfavourable for differentiation of organotypic formations. The most likely explanation for this is that the number of cells in the explants was too small, being

Recent improvements in microscope techniques have permitted a critical examination of the zone of apposition between the mesoderm and the ectoderm with a view to ascertaining whether there are any special features associated with the process of neural induction. It has been reported 113, 32,33,35, 36,431, that in the early gastrula quantities of small granules begin to accumulate in the extracellular spaces and particularly at the boundary between the dorsal mesoderm and ectoderm. The granules are small and regular approximating in size to that of animal ribosomes and frequently form paracrystalline arrangements. They persist until the neural plate has formed and then gradually decline in quantity, being replaced in the dorsal half of the embryo by a meshwork of fine extracellular filaments. These invest the notochord and paraxial mesoderm and so lie between such structures and the presumptive neuroepithelium 1331. Histochemical and enzyme digestion studies [341provide strong circumstantial evidence favouring the interpretation that the extracellular granules contain at least some RNA and are probably ribosomes. The enzyme digestion studies indicate that the extracellular filaments are mainly composed of glycosaminoglycans. In the living embryo the granules are extremely labile and can be lysed by a number of simple procedures. The

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filaments however are considerably more stable and, ofthe various methods tried, seemed to be removed only by the local injection of hyaluronidase. Embryos in which these interfacial materials have been destroyed develop quite normally and there is no disturbance of neural induction f 341. For practical purposes, therefore, one can assume that the extracellular materials found in the zone of apposition between the dorsal mesoderm and presumptive neuroepithelium are not essential mediators in the induction of the central nervous system.

Experiments with Interposed Membranes

The first attempts to investigate the mechanism of transmission of induction by insertion of different kinds of membranes between inducing and reacting tissues produced mainly negative results. Thus in Holtfreter’s [ 121 experiments on neural induction, interposition of vitelline membrane between the mesoderm and ectoderm was associated with failure of differentiation in the latter. Also in Brachet’s I51 experiments using porous membranes, the slight response in the ectoderm was interpreted by him to be caused perhaps by non-specific irritation by the filter. At first, therefore, the surface interaction concept of Weiss seemed the most plausible mechanism of induction. The first clear results challenging this hypothesis were obtained by Grobstein in a series of experiments beginning in 1953 [91. This work showed that inductive interactions between epithelium and mesenchyme, promoting duct and acinus formation in the salivary gland [9l and tubule formation in the metanephrogenic mesenchyme, can take place across a Millipore filter without demonstrable cytoplasmic contact [ 101(see later). Subsequently similar observations were made on many other secondary inductive systems in various developing organs(see Kratochwil [ 141 and Saxen [27] for full consideration of these reports). Saxen 1251 utilised the interposed Millipore filter method to study the transmission of primary induction and developed a somewhat modified technique to overcome the problems posed by the exceptional delicacy of early amphibian embryonic tissues. The thickness of the filter was 25 microns and the pore size 0.8 microns. The dorsal lip of the blastopore of Triturus embryos was used as inductor and competent gastrular ectoderm from the roof of the blastocoel as the reacting tissue. The tissues were cultured transfilter for one day and the ectoderm subsequently separated from the mesoderm and cultured alone for ten days. It was found that inductive action was able to traverse the porous membrane and the structures induced were mostly forebrain though in two experiments hindbrain with ear vesicles was formed. Later ultrastructural

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studies [241of similartransfilter cultures didnot reveal any cytoplasmic processes penetrating the pores of the filter from either the ectoderm or the mesenchyme. These findings indicated that inductive action is transmitted without any direct contacts between inducing and responding cells and seemed to show that Weiss’s model of induction is not valid in primary induction 1261. However, since the spaces in Millipore filters (which were the only suitable filters then obtainable) are not regular-sized straight channels but rather an irregular meshwork or maze, it was not possible to exclude with certainty the possibility of contact being achieved between tortuous processes penetrating the filter from each side. Recently, new filters made by bombardment of polycarbonate membrane with charged particles in a nuclear reactor and subsequent chemical etching have become available. These are traversed by regular straight channels resembling bullet tracks and the pore size and pore density have been established by measurements with scanning electron microscopy [471. It was found that the inductive stimulus was able to cross Nucleporem fdters of any pore size in the range tested, as judged by neural development in the overlying ectoderm on subsequent culture. There was no differentiation in the ectoderm when cellophane was interposed betweenit andthemesoderm,or whenit was culturedonits own with nothing on the other side of the Nucleporen membrane. We decided to use these Nucleporee filters in a re-investigation of whether direct cytoplasmic contact between the interacting tissues is a possible alternative mode of transmission. The details of the experiments are published elsewhere [40l but a brief account is given to facilitate the development of our conclusions. The technique of transfilter culture was similar to that developed by Saxen [251. The dorsal lip of the blastopore was used as an inductor and competent gastrular ectoderm from the roof of the blastocoele as the responding tissue. The pore sizes of the interposedmembranes used in these experiments were 0.1, 0.2, 0.6, 3.0 and 8.0pm. In the parallel histological and electron microscopical studies of the intact transfilter combinations, we did not find any cytoplasmic processes entering the channels traversing the filters. The absence of cell processes in the pores of our filters is a well substantiated observation, based on a large sample, but of course we cannot state that processes never enter the pores in this example of inductive interaction. It can only be said on the basis of this sample that if they do it must be a rare event. It is therefore concluded that neural development can be initiated in competent early gastrular ectoderm by an inductive stimulus transmitted from the underlying meso-

Morphogenetic Signals in Primary Induction

derm without demonstrable cytoplasmic contact between the two tissues. It is important to emphasise that the results we have cited above apply strictly to the initiation of neural development in uncommitted gastrular ectoderm. Our very recent results (unpublished) suggest that there are substantial changes in the relationships between these tissues in the later stages of primary induction, when the transformation of part of the neural plate to successively more caudal parts of the central nervous system takes place. These experiments used Nuclepore@ filters of 0.4 ym pore size with dorsal mesoderm of the archenteric roof on the one side and the neural plate of the neurula (stage 13) on the other. When isolation of the neural plate tissue from the filter was attempted it was found to be very firmly adherent and had to be mechanically separated using the operating needle. Histological examination of such neural plate tissues cultured for ten days after isolation showed that they had in general differentiated to form forebrain structures but in some examples there were also formations resembling hindbrains with ear vesicles suggesting the activity of a spino-caudal regionalising influence. Electron microscopical studies on intact transfilter cultures of these older tissues showed definite cytoplasmic processespenetrating the pores from the mesodermal side, the cells of the neuro-epithelium having a flat basal surface in contact with the membrane. There is ample evidence [30,391 that the regionalisationof the neural plate to form successively more caudal parts of the CNS (e.g. hindbrain and spinal cord) depends on continuing inductive influences from the underlying mesoderm. Thus, the observation of cellular processes traversing the filter suggests that the interactions at this stage inneural development are similar to those in kidney tubulogenesis and may require direct cytoplasmic contact for transmission of the stimulus. If this is so, one might ask why the transformation of the CNS towards more caudal characteristics was not more commonly seen in these transfilter cultures. In seeking to explain this it is pertinent to recall earlier observations [381, which showed that the competence for such transformation lasts for only ten hours. According to present knowledge this is too short a time to guarantee the formation of cytoplasmic bridges across the standard 10 pm thick filter and the low incidence of spino-caudal inductions is therefore understandable. Comparison of Neural Induction with Other Inductive Systems

Comparison of this state of knowledge about primary neural induction with recently obtained information on other inductive systems in development makes it possible

53 to draw inferences about the general properties of inductive phenomena. The induction of tubule formation in the mammalian kidney is a system which has been extensivelyinvestigated in aneffort to identifythe modeoftransmission. It has been shown that transfilter induction of kidney tubules in metanephrogenic mesenchyme taken from 12-day mouse embryos can be accomplished by portions of the spinal cord [ 101.Without any inductoron theother sideofthefilterthe mesenchyme remains un-differentiated on subsequent culture. It has been shown that inductive action from a piece of spinal cord will trigger (determine) tubulogenesis in about 24 hrs when the Millipore filter is about 25 microns thick I 10,231. However this estimate includes time for recovery of the tissue from the explantation procedure, and response to the stimulus, as well as the actual transmission of the signal across the filter. By interposing more than 1 filter between the tissues and measuring the corresponding delay in the passage of the signal Nordling et af. [231 provided a measure of the speed oftransmission itself. This speed was extremely slow, of the order of 2 wm/h, and did not obey the rules of chemical diffusion. On the basis of electron microscopical studies on transfilter cultures using Millipore membranes Grobstein and Dalton [ l l l were of- the opinion that there is no necessity for cytoplasmic contact between the different groups of cells in this inductive system. Grobstein [I01 later formulated the concept that the transmission of the signal is effected by interaction between the extracellular matrices associated with each of the tissues. Recently however S a x k and co-workers I 17,471re-investigated the problem, applying the original technique of Grobstein but using Millipore filters in one group of cultures and Nuclepore@ filters in the other. Their results showed a good correlation between the penetration of cytoplasmic processes into the filter and the passage of inductive signals, thus suggesting the importance of the intercellular contacts formed between the tissues on either side of the filter. In Nuclepore@ filters assemblies in which the pore size was 0.5 microns or greater, the transfilter cytoplasmic bridges could be seen with the light microscope. With the electron microscope it was still possible to demonstrate intercellular contact across the NucleporeB filter in pore sizes down to 0.2 pm. However the processes were not observed in filters with pore sizes of 0.15 pm and it has been established by this group that, in contrast to our results in primary neural induction, induction of kidney tubules cannot take place across such small pores. There are also other differences between the induction of the nervous system and that of kidney tubulogenesis. For instance kidney tubulogenesis cannot be induced by

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killed tissues as is the case in primary induction (see above). Another important distinction is that so-called assimilatory transmission (or homoiogenetic induction) is possible in the induction of the nervous system but not in the induction of kidney tubules. Thus Mangold and Spemann 1181 showed that a neural plate can be induced in uncommitted gastrular ectoderm using neuralised neuroepithelium as inductor and this has recently been confirmed by Deuchar [71. In contrast Sax& and Saksela [281showed that metanephrogenic mesenchyme which has been induced to form kidney tubules by transfilter culture with spinal cord is not itself capable of inducing tubular differentiation in fresh uncommitted kidney mesenchyme. Such combinations of induced and uninduced mesenchyme were arranged so that the partners were obtained from different strains of mice, one of which carried a marker chromosome. It was thus possible to establish with certainty that tubules form only from the mesenchyme which had been in transfilter contact with the spinal cord. Thus, the induction of tubulogenesis in the mammalian kidney differs from primary neural induction in amphibiansin three crucial aspects: primary induction can be brought about by killed tissues, but this is not so for tubulogenesis; embryonic neural tissue can exercise homoiogenetic induction but embryonic renal tissue cannot; and intercellular contacts between inducing and responding tissues seem not to be essential in the initiationofneural induction whereas they seem to be necessary for the induction of kidney tubules. It is important to emphasise that what is now said about the lack of requirement for cell contacts in primary neural induction concerns only the action of the primary trigger, that is to say the penetration ofthe neuralising principle which causes the determination of the neural plate. As remarked above, preliminary studies on the later stages of primary induction, in which regionalisation of the neural plate occurs, show that cytoplasmic contacts can be made between the two tissues and may have a developmental function. Observations indicating yet another type of transmission of inductive activity have been reported by Slavkin [3 11 in studies of the epithelial-mesenchymal interactions in tooth development in the rabbit. He described membranecoatedvesicles of 500 to 100,000 A diameter containing granular material in the intercellular matrix, the so-called progenitor mantle dentine, between the inner enamel epithelium and the sheet of odontoblasts. The electron density of the vesicles disappears following RNAse treatment indicating that they contain at least some RNA and Slavkin has suggested that these “matrix vesicles” are involved in the transmission of inductive information from one tissue to the other.

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Conclusions

The present state of knowledge about neural induction is therefore as follows: there is a transfer of macromolecules from the inducing to the responding tissue demonstrable by autoradiographic and fluorescent antibody-tracing methods. The experiments with cell free extracts demonstrate that there are macromolecules carrying aninductive principle which is soluble and diffusible. This raises the intriguing question of how the action of this agent in vivo is so localised and precise. Although the whole of the gastrular ectoderm is capable of responding to it, the boundaries of the developing nervous system are always sharp and distinct. Studies with interposed membrane between the inducing and reacting cells reveal that direct membrane to membrane contact is not required, at least for the initiation of neural induction, but may be for the later stages, in which regionalisation of the neural plate occurs. Comparison of these observations with available information on other inductive systems in development indicates that, although such phenomena possess features in common, the mode of transmission of the inductive stimulus is not uniform. Acknowledgements. The authors wish to thank the following Organisations for the generous support they have provided for different aspectsofthis work: TheRoyal Society,London, TheNuffieldFoundation, The Sigrid Jusklius Foundation, The Finnish Science Research Council, Japan Society for Promotion of Science.

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