1. Experiments were undertaken to investigate the degree to which the underlying mesenchyme influences the differentiation of an epithelium. Isolated epidermis of the 5-day chick embryo was explanted in vitro in contact with three different types of mesenchyme from chick embryos of the same age. These mesenchymes were derived from the gizzard, proventriculus, and heart. In a control series epidermis was re-implanted on limb mesenchyme. In each combination the epidermis behaved in a different and characteristic way.

  2. The cellular processes by which an isolated epithelium becomes established on mesenchyme in vitro are described.

  3. In the control series, epidermis re-implanted on limb mesenchyme keratinized normally, but in contact with cartilage it degenerated.

  4. On gizzard mesenchyme epidermis is prevented from keratinizing and is induced to secrete mucus and sometimes to become ciliated.

  5. On proventriculus mesenchyme epidermis is initially prevented from keratinizing and secretes mucus, but after about 7 days it reverts to its normal differentiation and keratinizes.

  6. Expiants of heart mesenchyme become subdivided into two regions: a central myoblastic area surrounded by a zone of fibroblastic outgrowth. On the central region of myoblasts the epidermis is prevented from keratinizing and spreads to a single-layered squamous epithelium. On heart fibroblasts, however, the epidermis keratinizes more densely than in the controls.

  7. The f our types of mesenchyme differ in the nature of the intercellular material that they contain, and in the amount of epithelial spreading they permit.

  8. The various effects of the mesenchyme are probably exerted specifically by the fibroblasts, which arrange themselves in contact with the epithelium in most types of culture.

  9. These epithelio-mesenchymal interactions are discussed in relation to the initiation and maintenance of embryonic and adult differentiation. It is suggested that the intercellular material of the mesenchyme may participate in the mechanisms underlying these reactions.

That the mesenchyme may determine the differentiation of epithelial derivatives has been demonstrated in a wide range of embryonic organs, e.g. limb-bud (Zwilling, 1956; Saunders, Gasseling, & Gfeller, 1958), feather-germs (Sengel, 1956), preen gland (Gomot, 1958), salivary gland (Borghese, 1950; Grobstein, 1953), and thymus (Auerbach, 1960). These inductions probably act through various mechanisms, but a feature that is common to many of them is the relatively brief duration of the mesenchymal stimulus, e.g. the mesenchymal induction of feather-germs in the chick lasts for half a day only. This brevity of action is also characteristic of the classic inductions, e.g. of the neural plate or lens.

The work reported here (and briefly elsewhere, McLoughlin 1961 a, b,) indicates that besides such brief inductions there are more prolonged epitheliomesenchymal interactions which maintain the normal growth and differentiation of tissues throughout embryonic life, and possibly in adult life also.

The existence of such persistent influences is suggested by histological considerations (Kingsbury, Allen, & Rotheram, 1953; Sylvèn, 1950; Ozzello & Speer, 1958) and by various experimental data. For example, certain embryonic epithelia fail to grow when cultivated in the absence of mesenchyme, and it has been found (McLoughlin, 1961a) that the isolated epidermis from 5-day chick embryos keratinizes completely under these conditions. From these results it would seem that in the embryo the mesenchyme must exert a continuous action on certain epithelia, without which the epithelial cells cannot survive and multiply.

Sobel (1958) showed that the epithelium of the pituitary rudiment from an 8-day chick embryo can neither grow nor differentiate in the absence of connective tissue, but is able to do both when perichondrial fibroblasts from the epiphyses of embryonic long-bones are added to the culture; in this rudiment, therefore, epithelial growth and differentiation are controlled by contact with cells of a particular type, i.e. perichondrial fibroblasts, and it is immaterial whether these come from the normal site, i.e. the chondrocranium, or from the limb-bones. Auerbach (1960) separated the epithelium of the embryonic thymus (mouse) from its own mesenchyme and combined it with the mesenchyme of other organs, such as lung and submandibular gland, and concludes that ‘three types of specificity seem indicated : 1. Mesenchyme derived from a variety of embryonic rudiments induces growth and morphogenesis in thymus epithelium. 2. The presence of thymic mesenchyme leads to immediate growth and morphogenesis whereas heterogeneous mesenchyme first causes rounding of the epithelium, morphogenesis and growth representing a distinct second phase of the response; and 3, the second phase of epithelial response to heterogeneous mesenchyme varies characteristically with the source of mesenchyme used. ‘

Thus it may be generally true that each type of mesenchyme has a characteristic influence on epithelia with which it is in contact. In order to investigate this question further, isolated epidermis was grown on various types of mesenchyme to see whether its differentiation would be modified by the cells with which it was combined. The results showed that each type of mesenchyme produced a characteristic effect on the implanted epidermis.

Preparation of tissue

Usually 2 or 3 embryos were dissected in each experiment. The hind limb-buds, the stomach (consisting of proventriculus and gizzard), and the heart were removed and placed in Tyrode’s solution with a few drops of embryo extract added (about 5 drops/ml.). The epidermis was removed from the hind limbbuds with trypsin as previously described (McLoughlin, 1961a), and transferred to 1:1 horse-serum and Tyrode’s solution. It was marked with carbon particles so that later on in culture, it could be distinguished from any fragments of gastric epithelium which might accidentally have remained attached to the mesenchyme on which it was to be implanted. Soon after isolation each epidermal fragment curled up with the basal cells inwards. The limb mesenchyme from which the epidermis had been removed was placed in a separate dish containing a mixture of equal parts of horse-serum and Tyrode’s solution.

Each stomach was divided into proventriculus and gizzard. These were treated separately with trypsin to remove the lining epithelium, which was usually discarded. The epithelium-free mesenchyme of each proventriculus was then usually divided into two, and that of each gizzard into three pieces, and these were also placed in horse-serum diluted 1:1 with Tyrode’s solution. The auricles were removed from the heart, and the ventricles cut into pieces corresponding in size with the fragments of gastric mesenchyme. Trypsin treatment is not necessary in the preparation of cardiac mesenchyme, but in some experiments the heart fragments were treated in the same way as limb and gastric mesenchyme in order to provide a control for any influence tryptic digestion might have on the mesenchymal factors that affect the implantation and differentiation of the epidermis; epidermis behaved in the same way on heart mesenchyme whether the heart had or had not been treated with trypsin.

Four groups of combined cultures were made, as shown in Table 1.

Table 1.

Number of cultures of epidermis on different types of mesenchyme

Number of cultures of epidermis on different types of mesenchyme
Number of cultures of epidermis on different types of mesenchyme

Culture method

To prepare a combined culture, a fragment of epidermis and a piece of the appropriate mesenchyme were placed together in a drop of horse-serum Tyrode on a Maximow double coverslip. The epidermis, bearing a few carbon particles, was unrolled with fine, blunted glass needles, and floated basal side down over the mesenchymal fragment. The horse-serum/Tyrode was then withdrawn with a fine pipette so that the unrolled epidermis sank down and became draped over the mesenchyme, with which it established close contact. A drop of embryo extract was then mixed in a Petri dish with a drop of plasma, approximately in the proportion of 1 part of extract to 2 parts of plasma, and this medium was spread round the combined tissues. Both mesenchyme and epidermis are nonadhesive after treatment with trypsin, so it was difficult to add the permanent medium without disturbing the explant: the best way was to spread the medium in a ring round the combined tissues and allow it to converge simultaneously from all directions. In 1–2 minutes the plasma clotted and held the explanted tissues closely together. Each Maximow double coverslip was then sealed on to a depression slide and incubated at 38° C.

In preparing these cultures, it was essential to unroll the epidermis and place it with the basal layer in contact with the mesenchyme; if placed peridermal side down, the epithelium fails to become implanted, indicating an interesting difference in the capacities of the basal and peridermal cells even at this early stage. In the proventriculus and gizzard cultures the epidermis was usually placed in contact with the mucosal surface of the mesenchyme. Sometimes, however, this was not possible, and the epidermis became implanted on the cut or serosal surface, but this did not affect the response of the epidermis to the mesenchyme.

The medium was replenished every 2nd day and the cultures were transplanted every 4th day, so that the tissues could retract and remain suitably thick for histological examination.

Histological methods

Expiants were fixed daily, from 1 to 16 days in vitro, in Zenker’s solution with 3 per cent, acetic acid or in Carnoy’s mixture (ethanol 6 parts, chloroform 3 parts, acetic acid 1 part). While in absolute alcohol they were removed from the coverslip with a razor, then embedded in paraffin and serial sections 7 p in thickness were cut at right angles to the coverslip. In addition to the usual staining methods such as Azan, iron haematoxylin, or Alcian blue (1 per cent, in distilled water, 10 seconds), the periodic-acid/Schiff technique (PAS) and basic dyes such as Toluidine blue (0-1 per cent.) and Azure A (0-01 per cent.) were used for the identification of mucopolysaccharides. Trevan’s method (personal communication) was found particularly useful. This consists of an initial treatment with Alcian blue at pH 3 to pick out acid mucopolysaccharides, followed by PAS which stains the remaining neutral mucopolysaccharides red. Mercury orange was also used to stain specifically for disulphide and sulphydryl groups (Bennett & Watts, 1958).

Initial interaction of epidermis with mesenchyme

During the first 24 hours the reaction of isolated epidermis to the mesenchyme on which it is explanted is similar in all composite cultures (except epidermis on heart myoblasts). This reaction culminates in the establishment of a normal relationship between epidermal and mesenchyme cells, and in the re-establishment of an oriented epithelial structure in the epidermis. The following is a general description of this process and any individual variations will be described in the appropriate section.

The epidermis behaves for the first 24 hours as if it were isolated, i.e. it loses its 2-layered arrangement and rounds up into a disorganized nodule in which the living cells become separated from each other, and there are many degenerations (McLoughlin, 1961a). At the point where basal cells are in contact with the mesenchyme, however, they become oriented towards it in an orderly way to form a single columnar epithelial layer (Plate 1, fig. 2). The orientation is transmitted, through fine filaments by which the cells make contact with each other, to cells farther within the epithelial nodule, so that these in turn move towards the point of attachment and become arranged in several rows above the first established layer.

Some cells of the second epidermal layer, and even of the third and fourth, send long processes containing a thick tonofibril down between the cells of the basal layer to make contact with the mesenchyme (Plate 1, fig. 3, P); sometimes this tonofibril is arranged in a fine spiral (Plate 1, fig. 4, T), and sometimes two or three cells send entwined processes which jointly penetrate the basal layer (Plate 1, fig. 3, PP). These cytoplasmic extensions resemble the spiral filaments of Herxheimer in normal skin: they end in minute branched feet, which give the impression of holding the cell anchored to the early basement membrane (Plate 1, fig. 5). The whole cell then seems to pull itself down by means of its process and squeezes in between the basal cells, so that it also becomes established on the basement membrane. To provide room for the incoming cells, the reconstructed epidermal sheet spreads gradually over the mesenchyme and the original epidermal nodule is thus depleted as its cells move downwards to make contact with the mesenchyme. This sequence of events often continues till the nodule disappears and a 2-layered epidermis has been reconstructed, after 48 hours in vitro (Plate 1, fig. 6).

At the point of contact with the epidermis, the mesenchyme soon forms a thin layer of mucopolysaccharide-containing, PAS-positive material, which represents a newly formed basement membrane. This becomes clearer as the epidermis spreads, so that, by the time it is fully established, a distinct basement membrane is present. The similarity between this initial reaction of epidermis to whole mesenchyme, and its response to isolated intercellular matrix of the mesenchyme (McLoughlin, 1961,a,c), suggests that the reaction by which epidermis becomes established on the basement membrane is not primarily to the fibroblasts of the mesenchyme, but to. the intercellular material they produce. The relation between epidermis and basement membrane described here recalls that observed in the amphibian tadpole in the electron microscope by Weiss (1958); there also the basement membrane is responsible for polarizing the epidermis, and the epidermis in turn organizes the basement membrane.

In all tissues except heart myoblasts, by the 4th day a distinct layer of fibroblasts can always be seen surrounding the epidermis, although the latter was initially implanted on tissue containing intermingled fibroblasts, myoblasts, and other cells. This indicates not only that epidermis and fibroblasts attract each other, but also suggests that the different and characteristic effects of the various mesenchymes on epidermal differentiation which will be described in the following sections, are the result of specific properties of the fibroblasts in the different mesenchymes.

The question arises of whether the newly formed basal layer and periderm in the reconstituted epithelium correspond to the original basal and peridermal layers. The following circumstantial evidence suggests identity of the original and reconstituted layers.

As stated above, the periderm will not become implanted on mesenchyme, so that epidermis will only ‘take’ if planted basal side down; it would be surprising, therefore, if peridermal cells take part in the formation of a new basal layer. Moreover, it has been noted (McLoughlin, 1961b) that the original peridermal cells display an early phagocytic activity that is not shown by the basal cells. Since considerable degeneration occurs during the first 12 hours after the epidermis is explanted, the peridermal cells become distended with phagocytosed cellular debris and thus can be distinguished. In the 24-hour-old epidermal plaque, the cells that become oriented against the mesenchyme contain no visible degenerate material, while those that are left as an upper layer contain phagocytosed fragments, indicating that they probably represent the original peridermal elements. This view receives further support from the fact that the carbon particles added to the explanted epidermis as markers, are usually found in the peridermal cells of the reconstructed epidermis.

At 48 hours the basement membrane is very distinct, and the epidermis usually lies upon 3-4 layers of fibroblasts, which are forming collagen fibrils.

Epidermis on limb-bud mesenchyme

Isolated epidermis recombined with normal limb-bud mesenchyme forms a normal squamous keratinizing epithelium.

The normal limb-bud mesenchyme

In histological sections of the normal limb-bud of the 5-day chick, differentiation is fairly advanced in the most proximal region but diminishes in degree towards the tip where the cells are apparently undifferentiated. At all levels there is a central core of cartilage or procartilage cells surrounded by myoblasts, which in turn are enclosed by a layer of early fibroblasts.

Cultures

All regions of the limb-bud mesenchyme, except the most distal part, differentiate to produce a central cartilaginous nodule or group of nodules with radiating bundles of multinucleated embryonic muscle fibres containing striated myofibrils. Many fibroblasts mingle with the muscle fibres and spread more peripherally. The most distal region of the limb-bud usually gives fibroblasts only; a small cartilaginous nodule may also appear, but muscle fibres are absent.

Forty-eight hours after the explantation, together, of epidermis and mesenchyme, there is plentiful mesenchymal outgrowth, and the epithelium appears as a translucent plaque, indicating that a normal relationship has been established between the two tissues. After 3 days the epidermal plaque has usually extended a little; it rarely spreads farther after this stage, but usually rounds up to form a cyst, basal side outwards, and keratinizing inwards. The first signs of keratinization appear at about 5 days, and by 7 days the epidermis has usually formed a cyst lined with opaque keratin and surrounded by a condensed coat of dermal fibroblasts and their collagen fibrils (Plate 1, fig. 1). This appearance, with increasing deposition of keratin, is maintained throughout the culture period. Often the epidermis that comes to lie on top of cartilage develops very poorly, and may actually degenerate. The dermal fibroblasts form many collagen fibrils coated with a mucopolysaccharide material that always stains red (for neutral mucopolysaccharides) with Trevan’s method. In histological sections it can be seen that the newly formed dermis associated with the explanted epidermis thickens and the number of cells increases faster than in fibroblastic areas elsewhere in the culture (Plate 2, fig. 7). It is not known whether the epidermis achieves this by promoting mitosis in the dermis, or by trapping passing fibroblasts.

After 2 days in vitro the epidermis may be either a simple 2-layered epithelium, or an early stratified squamous epithelium. In most cultures it is beginning to roll up to form a cyst, and occasionally this process is already completed. From this point onwards most of the epidermal fragments differentiate normally, but those in contact with cartilage do not.

Four types of epidermal behaviour have been observed: normal keratinization, failure to thrive in contact with cartilage, cyst formation, and the secretion of fluid by newly formed cysts.

Normal keratinization

By the 3rd to 5th day the epidermis has already become a stratified squamous epithelium : between the 5th and 7th days keratin begins to appear and the stratum corneum grows progressively thicker. The differentiation of epidermis recombined with limb mesenchyme (see Table 2) is usually indistinguishable from that of undisturbed epidermis, and, indeed, sections of these explants illustrate the normal process of keratinization very clearly (Plate 2, fig. 7).

Table 2.

Differentiation of epidermis recombined with limb mesenchyme

Differentiation of epidermis recombined with limb mesenchyme
Differentiation of epidermis recombined with limb mesenchyme
Epidermis on cartilage

Though the epidermis initially becomes implanted normally on perichondrial fibroblasts, it develops very poorly. The basal layer becomes disorganized, and the epithelium tends to spread; mitoses are rare, and differentiation is slow. Finally, the epidermis either degenerates completely or else keratinizes down to and including the basal layer. In early stages (up to 4 days), the cartilage often seems to suffer from the proximity of the epidermis; less matrix is deposited than elsewhere and many chondroblasts may become filled with iron-haematoxylin-staining droplets and degenerate. Some normally keratizing epidermal cysts lie with one end in contact with the perichondrium of a cartilaginous nodule, and this small region differentiates abnormally (Plate 2, fig. 8). This localized response to the perichondrium on the part of an otherwise normal cyst provides a particularly clear illustration of the incompatibility of the two tissues. It is suggested that cartilage is not capable of providing a normal basement-membrane for the epidermis. Of a total of 80 control cultures, 66 differentiated normally, while 14 came to lie on cartilage and with which they were incompatible (see Table 2).

Cysts

These may be formed in young cultures in one of two ways. An entire epidermal plaque may early become enclosed by fibroblasts so that a basal layer is formed all round the epidermal nodule. A central space forms in the plaque by the extreme vacuolar distension of a single cell (such distension is common in dying ‘disoriented’ epidermal cells). This space becomes surrounded by flattened peridermal elements applied to the thin cytoplasmic wall of the distended cell, and any remaining epidermal cells become oriented between the central lumen and the surrounding basal layer to form a stratified epithelium. The cytoplasm of the inmost distended cell finally becomes indistinguishable from the distal surfaces of the peridermal cells.

Alternatively, a cyst may be formed from an open epidermal sheet when the initial spreading movement has been halted. Dermal fibroblasts migrate over the edge of the sheet and move on to the surface of the epidermis, drawing the edges of the sheet with them : this causes a general inrolling around the edges of the epidermis. Fibroblasts on top of the sheet converge from all sides towards the centre, in much the same way in which a bag is closed by pulling a drawstring; the epidermis is pulled in their wake, and finally the inrolled margins fuse to form the upper side of an epithelial cyst which keratinizes inwards. Usually both these processes occur simultaneously in the same culture, so that several cysts are formed which may later fuse.

Secretion of fluid

When cysts are formed very early, i.e. about the 2nd day in vitro, the epidermis is still in a simple 2-layered state and appears to be able to produce fluid as long as it remains so. These cysts become distended with fluid, and consequently the epidermis preserves its primitive 2-layered structure for an unusually long time. A total of 12 such cysts were noted: no trace of mucus has ever been seen in them, and after about 5 days very flattened squamous layers always begin to appear. As keratinization begins (Plate 2, fig. 8) fluid is reabsorbed, and the once distended vesicles shrink and become filled with keratin. This observation that the simple 2-layered epidermis secretes fluid, suggests that it may be responsible for forming amniotic fluid in the chick embryo. The differentiation of epidermis on limb mesenchyme is summarized in Table 2.

Epidermis on gizzard mesenchyme

Epidermis implanted on gizzard mesenchyme fails to keratinize, but forms mucus and may become ciliated.

The normal gizzard

At 5 days this is a thick-walled organ lined with a pseudostratified columnar epithelium which secretes mucus that stains blue for acid mucopolysaccharides with Erevan’s method. The mesenchyme contains fibroblasts whose matrix stains slightly for acid mucopolysaccharides, numerous young smooth myoblasts, and scattered intramural ganglia with tracts of unmyelinated nerve fibres accompanied by Schwann cells. Surrounding the whole organ is the serosal lining which is usually underlain by a thin layer of fibroblasts.

Many simple glands arise in the lining of the gizzard. At 8 days their secretion stains blue for acid mucopolysaccharides with Trevan’s method, but at 17 days it is undergoing transformation to the adult horny lining, which is described by Aitken (1958). This secretion is produced as a fluid, but as it moves into the lumen of the gizzard it condenses to the characteristic horny material, which is resistant to digestion with hot 5 per cent. KOH. Near the secretory cells it stains for acid mucopolysaccharides, but farther out in the lumen it stains for neutral mucopolysaccharides only. Mercury orange staining shows that it is very rich in SH and S—S groups, so perhaps it hardens by the oxidation of sulphydryl groups to disulphide bonds.

There seem to be fewer fibroblasts per area in gastric than in dermal mesenchyme and the connective tissue they form contains finer bundles of collagen fibrils that lie in a more abundant matrix, which stains faintly for acid mucopolysaccharides. The myoblasts differentiate into elongated smooth muscle fibres, and are interwoven into a powerful muscular mass. Small autonomic ganglia interconnected by tracts of unmyelinated nerve fibres are scattered among these muscle fibres.

Cultures

Five-day gizzard mesenchyme explanted in vitro differentiates into groups of smooth muscle fibres interlaced with fibroblasts and penetrated by ramifying bundles of unmyelinated nerve fibres connecting numerous small autonomic ganglia. Vascular spaces are present. Peristalsis is occasionally observed between the 3rd and 6th day in vitro.

Epidermis explanted together with gizzard mesenchyme has usually become completely fused with the latter by the 2nd day, and can often be distinguished as a clear plaque. In contrast with its behaviour on limb mesenchyme, where it rolls up to form cysts, the epidermis shows a tendency to spread uncontrollably on gizzard mesenchyme : this may continue for up to 8 days, so that the epidermis often forms a hillock inside which the mesenchyme collects. As the epidermis continues to envelop the mesenchyme, this hillock gradually becomes pedunculate, finally detaches itself from the rest of the culture on the cover-glass, and may float freely as an isolated sphere of mesenchyme covered with epidermis. This tendency for the epithelium to extend its area of contact with the mesenchyme is characteristic of explants from any part of the digestive tract (Walker, personal communication). Thus epidermis on gizzard mesenchyme strikingly resembles gastric epithelium in this respect.

This behaviour, though interesting in itself, caused technical difficulties since cultures enveloped by epithelium provide poor material for observations on the differentiation of the epidermis. In later experiments, therefore, efforts were made to promote the formation of cysts by placing the isolated epidermis in a groove in the mesenchyme at the time of explantation. Under these conditions the epidermis forms a plaque surrounded by mesenchyme, and by the 3rd day a cyst usually develops by cavitation within the solid plaque, as described for epidermis on limb mesenchyme.

On the following days the cyst secretes fluid with which it is increasingly distended; its outline becomes refractile, which was found to indicate mucus secretion, and individual cells are seen to contain droplets of refractile material (Plate 2, fig. 9). Serial daily photographs show that the amount of fluid contained in such a cyst continues to increase until about the 9th day in culture. Occasionally these cysts collapse before the end of the culture period, owing to their extreme dilation.

Histological observations show that during the first 24 hours, epidermis explanted on gizzard mesenchyme establishes itself in the manner described in the first section. It responds more rapidly to gizzard than to limb mesenchyme, so that by the end of the 1st day more cells are seated on the basement membrane than in the limb-bud cultures, and by the 2nd day a simple 2-layered epithelium has always been reconstructed. This consists of a cubical basal layer covered with a cubical peridermal layer which contains most of the marking carbon particles. Occasionally a little mucus, staining with Alcian blue, is already present on the epithelial surface, and by the 3rd day this is always unmistakable as a distinct but very thin line.

As on limb mesenchyme, the epidermis is always found to lie upon fibroblasts. These form a basement membrane within the first 2 days, and this stains as a neutral mucopolysaccharide with Trevan’s method, but the intercellular material and matrix surrounding the collagen fibrils in the mesenchyme always stains partly or entirely as an acid mucopolysaccharide.

The differentiation of epidermis in cysts will be described first, since it is the most frequent type of development. As stated above, epidermis on gizzard mesenchyme produces much fluid, and one advantage of the arrangement of the epidermis in cysts is that the fluid is collected so that its amount can be assessed visually. The production of fluid usually begins on about the 3rd day, and observations of living cultures show that it may continue to increase until about the 9th day : fluid production appears to continue as long as the epidermis remains a simple 2-layered epithelium.

During the first 6 days the epidermis is arranged in a double columnar layer (Plate 2, fig. 10). The peridermal cells show distinct terminal bars and produce small quantities of mucus, but do not form typical goblet cells: the basal cells assume a tall columnar arrangement. A typical single-layered columnar epithelium is never formed, and the cells remain recognizably epidermal although their differentiation is much modified.

Basal cells on gizzard mesenchyme have a swollen nucleus with a very large nucleolus and clear cytoplasm with relatively few thin tonofibrils; thus they contrast with the basal cells of normal epidermis, which have smaller nuclei, basophilic cytoplasm, and abundant tonofibrils. Spiral filaments of Herxheimer are absent from epidermis on gizzard; intercellular bridges occur, but the cells are usually in such close contact that the bridges remain inconspicuous. The differences from the normal in the content and arrangement of tonofibrils are best seen in whole mounts of epithelial sheets. After 8 days a few layers of cells may be produced between the basal layer and the periderm; these may assume a squamous shape (Plate 2, fig. 11), but never show any sign of keratinization. Mucus secretion begins on about the 3rd day and increases slowly to about the 7th day (Plate 2, fig. 11). Cilia are occasionally observed on peridermal cells only, in scattered areas of the cyst wall.

Between the 12th and 14th day these distended cysts often collapse. Mitoses are usually not seen after about the 9th day, and subsequently part of a cyst may become eroded owing to degeneration. The changes in the thickly heapedup epidermis of such a collapsed cyst are of interest.

Immediately after the collapse, the peridermal cells which have been carried into the centre of the cyst form a peculiar secretion, sometimes in large quantities. This secretion may stain blue with Trevan’s method, where it lies in contact with the cells, but red for neutral mucopolysaccharides in the lumen of the cyst. It shrinks on fixation and stains strongly with Mercury orange for SH and S—S groups. In all these respects it resembles the normal secretion of the gizzard, but the meaning of this resemblance is doubtful, since a similar material is produced under some circumstances by epidermis on proventriculus and heart, and rarely even on limb mesenchyme (McLoughlin, 1961b). It seems to be formed instead of trichohyalin, the characteristic intracellular product of peridermal differentiation, when the epidermis keratinizes very rapidly beneath the periderm.

Many epidermal cells become heaped together in the centre of the cyst below the peridermal cells. Some of these, which are far removed from contact with the mesenchyme, follow their normal fate and form keratin. (In Table 3 the four explants in which keratin appeared were of this type.) This indicates that the gizzard mesenchyme exerts a continuous influence on epidermal differentiation, but that the influence extends only over a limited range; its effects are reversible, for when it is removed the epidermis can keratinize.

Table 3.

Differentiation of epidermis implanted on gizzard mesenchyme

Differentiation of epidermis implanted on gizzard mesenchyme
Differentiation of epidermis implanted on gizzard mesenchyme

In these collapsed cysts, the cells of basal origin, which no longer have a peridermal covering, often form characteristic goblet cells (Plate 2, fig. 12). From this it is clear that, although mucus secretion in these cultures is usually performed by the periderm as the most distal layer of the epithelium, the basal cells are sufficiently transformed to secrete mucus themselves. After about 11 days, some of the epidermal cysts begin to degenerate.

Though sheets occur less often than cysts (12 sheets to 35 cysts) they were sufficiently numerous to provide a parallel with each stage of differentiation of the cystic epidermis. The comparison is valuable since in open sheets the factor of distension with fluid and the subsequent tension, which might affect the differentiation of the epithelium, is eliminated. The differentiation of epidermal sheets on gizzard does not differ in any essential way from that in cysts. Ciliation of the peridermal layer (Plate 2, fig. 13) was found more often in open sheets than in cysts, i.e. in 8 out of 12 sheets as compared with 3 out of 35 cysts. The epithelium may become stratified, but usually shows no sign of a squamous structure and never forms keratin (Plate 2, fig. 13).

The differentiation of epidermis on gizzard mesenchyme is summarized in Table 3. It is to be noted that some cultures both secreted mucus and bore cilia. Thus gizzard mesenchyme influences epidermis to approach the form of a cuboidal epithelium, to secrete mucus, and to develop cilia on the peridermal layer.

The possible role of vitamin A in the response of epidermis to gizzard mesenchyme

The similarity between the responses of epidermis to excess vitamin A (Fell & Mellanby, 1953), and to gizzard mesenchyme suggest a common cause, e.g. the gizzard might store vitamin A and thus influence the epithelium to secrete mucus. Dr. Moore of the Dunn Nutritional Laboratory, Cambridge, kindly assayed stomachs from 5and 13-day embryos by the method of Carr & Price (1926) for their vitamin A content. No vitamin was found, nor could any be seen in frozen sections under the ultra-violet microscope; this excludes the presence of a very high concentration in the stomach. Although a permissive amount of vitamin A is probably necessary for the epidermis to secrete mucus in response to gizzard, a high concentration is unlikely to be a primary cause of this response.

Gizzard epithelium on limb mesenchyme

In order to see whether dermal fibroblasts would have the converse effect on gizzard epithelium, six fragments of gizzard epithelium were explanted on to limb mesenchyme. The gastric epithelium became established and surrounded by a thick coat of dermal fibroblasts, but its differentiation remained unaltered; it persisted as a columnar, mucus-secreting epithelium (Plate 3, fig. 14). Instead of spreading uncontrollably, however, as it does on its own gizzard mesenchyme, on limb mesenchyme it became rounded up to form compact cysts which produced their secretion inwards. Thus we have the following set of paired observations:

From this it was concluded that the spreading behaviour of an epithelium on a mesenchyme is not primarily a property of the epithelium, but may be determined by the mesenchyme.

Epidermis on proventriculus mesenchyme

In contact with proventriculus mesenchyme, epidermis initially reacts in the same way as it does to gizzard mesenchyme, i.e. by becoming a mucus-secreting epithelium; but after about 7 days it reverts to its characteristic differentiation and keratinizes normally and abundantly.

The normal proventriculus

This is a tubular dilation of the gastric end of the oesophagus, and is demarcated from the gizzard by a slight constriction. In histological detail it closely resembles the gizzard; the matrix laid down by the fibroblasts stains lightly blue with Trevan’s method.

At 7 days, elevated rings with a central depression begin to appear in the proventricular lining, and at 8 days the depressions have already deepened to form tubular diverticula into the mesenchymatous wall of the organ. By 17 days the wall of the proventriculus contains a mass of compound diverticula whose alveoli are lined by simple tubular glands which produce a material rich in SH and S—S groups that resembles the secretion of the gizzard.

Cultures

The differentiation of proventriculus mesenchyme in vitro is indistinguishable from that of gizzard mesenchyme. As with epidermis on gizzard mesenchyme, efforts were made to promote the formation of cysts.

The behaviour of epidermis on proventriculus mesenchyme resembles that on gizzard mesenchyme for the first 4 days, i.e. thin-walled cysts are formed with a refractile lining indicating mucus secretion (Plate 3, fig. 15a). After 4 days these cysts are seen to accumulate less fluid than cysts of epidermis on gizzard mesenchyme, and by the 7th day they usually begin to keratinize and simultaneously lose what fluid they contain (Plate 3, fig. 15b). Keratinization usually begins in the region of the epidermis that is farthest from the centre of the culture, and progresses rapidly from the 7th day onwards.

The histological study of cultures fixed from day 1 to day 14 confirms that epidermis on proventriculus mesenchyme initially behaves in every detail as it does on gizzard mesenchyme; the construction of a 2-layered epithelium which immediately begins to secrete small quantities of mucus (Plate 3, fig. 16) and the continued production of mucus up to about 6 days are identical in the two types of composite cultures, but with the proventriculus the production of fluid and mucus stops at about 6 days. The epidermis then becomes stratified and squamous (Plate 3, fig. 17), the fluid in the cyst is reabsorbed or lost, and normal keratinization proceeds very rapidly (Plate 3, fig. 18); in some older cultures it even involves the basal layer so that no living epidermal cells remain. The keratin carries the peridermal lining of the young cyst, as a row of cubical cells, into the centre of the cyst. As the epidermis thickens below them, the peridermal cells often produce an abundant secretion of the neutral mucopolysaccharidecontaining material rich in SH and S—S groups that is formed by peridermal cells under similar circumstances on limb and gizzard mesenchyme. Soon after keratin appears, these cells degenerate.

The results of explantation of epidermis on proventriculus mesenchyme are summarized in Table 4, from which the transition from a mucus-secreting to a keratinizing epithelium at about 7 days emerges clearly. All the explants listed in which keratin was formed, contained a central group of cuboidal peridermal cells together with traces of mucus, as an indication of their history (see Plate 3, fig. 15).

Table 4.

Epidermis on proventriculus mesenchyme

Epidermis on proventriculus mesenchyme
Epidermis on proventriculus mesenchyme

These results suggest that the proventricular mesenchyme may contain the same factor as the gizzard mesenchyme, which influences the epidermis to differentiate into a cuboidal, mucus-secreting epithelium, but that it is present in a smaller quantity than in the gizzard. To investigate this possibility, the differentiation of six epidermal fragments each on an entire proventriculus was compared with the differentiation of six similar fragments each on a very small portion of gizzard. All six cultures of epidermis on proventriculus secreted mucus initially, halted, and then keratinized, whereas all the explants of epidermis on gizzard failed to keratinize and continued to form mucus. Thus the epidermis appears to respond to differences between the cells of the two organs, rather than to differences in the absolute amount of a stored active substance.

Epidermis on heart mesenchyme

Epidermis in contact with heart mesenchyme spreads to a single-layered epithelium on myoblasts, but rapidly keratinizes on fibroblasts.

The normal chick heart

The heart of a 5-day chick embryo is a well-differentiated, actively functioning organ. The ventricles consist predominantly of a spongy meshwork of musclecells with a layer of young myoblasts immediately within the epicardium; the folded endocardial surface is lined by flattened endothelial cells. In the valvular and septal regions of the ventricle there is a mass of fibroblasts, with an abundant matrix rich in acid mucopolysaccharides.

Cultures

Expiants of chick heart produce a plentiful outgrowth within the first 2 days, and the culture becomes subdivided into two regions—a central area consisting mainly of myoblasts which continue to beat for 10 to 11 days, and a peripheral region containing most of the fibroblasts. Epidermis on heart mesenchyme behaves differently in contact with these two regions.

Epidermis which establishes contact with the myoblastic region is detectable as a dark nodule on the 1st day of culture, but by the 2nd day it has always spread widely and become translucent; by the 3rd day it is so thin that it is no longer distinguishable. When the cultures are cut out to be transplanted on the 4th day, retraction of the explant often reveals the formation of an extremely thin-walled fluid-filled cyst, which was previously so extended that it was undetectable (Plate 4, fig. 19). Where a strand of epidermis runs across the centre of a cyst, out of contact with the myoblasts (Plate 4, fig. 19), it thickens and begins to keratinize normally. Such cysts may continue unchanged throughout the culture period.

When the epidermis becomes implanted in the peripheral fibroblastic region, it seems never to spread beyond its first point of attachment, which nearly always remains small. On the 2nd day it thickens, and begins to keratinize. The majority of the epidermal explants come to lie partly on the myoblastic and partly on the fibroblastic region, and differentiate accordingly. Occasionally, an epidermal explant which initially appears to be developing as a thin-walled cyst in the myoblastic region, draws together and forms a thick nodule of keratin, apparently surrounded by myoblasts; histological sections show, however, that the keratinizing epidermis is surrounded by a thin layer of fibroblasts which have migrated in between the myoblasts and the epidermal lining of the cyst.

The two regions of the heart explants can easily be distinguished in histological sections. With Azan and Trevan’s method, most of the collagen is found in the peripheral zone of outgrowth, indicating the presence of the majority of the fibroblasts; only a few remain in the myoblastic area. The matrix formed by heart fibroblasts in vitro stains from mauve to blue with Trevan’s method, and is strongly metachromatic with Azure A, i.e. it contains acid mucopolysaccharides.

From the moment of contact, epidermis on heart mesenchyme reacts in very different ways to the two types of cells with which it is confronted. (1) Heart fibroblasts strongly inhibit spreading of the epidermis, which consequently does not reconstruct to form a 2-layered epithelium but acquires a stratified squamous structure by the 2nd day. As the epidermal cells keratinize the periderm frequently secretes a large quantity of neutral mucoprotein material. The basement membrane stains for neutral mucopolysaccharides with Trevan’s method, although the matrix produced by the fibroblasts generally contains a preponderance of acid mucopolysaccharides. Tangential sections show that tonofibrils are more numerous and closely packed than in epidermis on limb mesenchyme and the keratin formed is correspondingly more compact (Plate 4, fig. 20). After about 11 days the epidermis often becomes completely keratinized, down to and including the basal layer. (2) On myoblasts the epidermis spreads from the beginning as a single sheet of flattened cells which glide swiftly over the myoblastic region and may cover it entirely by 48 hours. When the spreading epidermal cells make contact with the surrounding fibroblasts, their progress is immediately halted. They pile up to form a stratified squamous epithelium around the myoblastic/fibroblastic boundary, and peridermal cells and cells of basal origin once more become distinguishable (Plate 4, fig. 21).

Sometimes the epidermis becomes completely surrounded by myoblasts and develops into a clear fluid-filled cyst, but more often the epidermal cells are too numerous to be all accommodated in a single squamous layer on top of the myoblastic region, and spread until some pile up on the fibroblastic zone. Thus epidermis on heart mesenchyme often lies partly on myoblasts and partly on fibroblasts.

Epidermal cells in contact with heart myoblasts retain their squamous shape throughout the culture period of 16 days (Plate 4, figs. 22 a, b). The basement membrane, if one is present, has become too thin to be seen. This single-layered squamous epithelium is so flattened that it resembles the endothelial lining of the heart, but it is known to be of epidermal, not endothelial, origin for the following reasons : (i) no similar cysts or sheets are found in cultures of heart mesenchyme alone; (ii) such sheets have been observed emerging from implanted epidermal fragments on the 1st day in culture; (iii) the single-layered epithelium sometimes carries carbon markings; (iv) where a cyst lies partly in the myoblastic and partly in the fibroblastic region, the cells in contact with the myoblasts remain flattened throughout the culture period, but an abrupt transition to a stratified squamous keratinizing epithelium occurs at the boundary of the myoblastic and fibroblastic regions; and (v) when a thin-walled cyst collapses and its epithelial lining becomes piled up, keratin is formed.

The question arises of whether the beating of heart myoblasts affects the differentiation of epidermis on heart mesenchyme. To answer this question, the beating was inhibited in 16 cultures by growing them in medium in which the proportion of embryo extract to plasma was 1:1, instead of the standard proportions of 1: 2. The beating of heart myoblasts was inhibited from the 2nd day. This inhibition is probably due to the elevated level of potassium in the medium, since embryo extract is enriched by the intracellular K+ of broken embryonic cells. Histological sections showed that up to about the 8th day the epidermis in these cultures differentiated exactly as in those that had not been prevented from beating; thereafter the immobilized muscle fibres began to degenerate, and fibroblasts migrated between them and the epidermis which simultaneously became transformed from a single squamous layer to a stratified squamous, keratinizing epithelium. The characteristic responses of epidermis to heart myoblasts and fibroblasts are therefore not the result of the regular beating of the myoblasts in culture, but are direct responses to the different properties of the two types of mesenchyme cells.

Table 5 summarizes the differentiation of epidermis on heart mesenchyme.

Table 5.

Differentiation of epidermis on heart mesenchyme

Differentiation of epidermis on heart mesenchyme
Differentiation of epidermis on heart mesenchyme

Since epidermis keratinizes in isolation (McLoughlin, 1961a) it is clear that contact with the various types of heterotypic mesenchyme, whose effects have been described, diverted the epithelium from its determined fate.

The response of epidermis to gizzard mesenchyme closely resembles its reaction to excess vitamin A, but no connexion has been found between the two stimuli. That such similar responses follow upon two apparently unrelated stimuli suggests that the epidermis has a limited repertory of forms of differentiation, and that its reaction to any of a wide range of stimuli is restricted to one or other of the various possibilities open to it. Except on heart myoblasts, the epithelium remains recognizably epidermal, e.g. intercellular bridges and tonofibrils persist, though they may be much modified. It is interesting that several types of mesenchyme evoke an appropriate response in the implant, i.e. each modifies the differentiation of the epidermis so that it comes to resemble the epithelium that normally clothes that mesenchyme. For instance, gizzard mesenchyme influences epidermis to resemble, as far as possible, the early, mucus-secreting gizzard epithelium; proventriculus mesenchyme has a similar, but weaker effect; and finally, heart myoblasts induce the epidermis to spread almost as thinly as the normal endocardial lining. An exception to this generalization is provided by heart fibroblasts, which are not normally in relation with any epithelium but cause epidermis to keratinize even more heavily than usual. Epidermis cannot establish itself on cartilage and withdraws from chondrifying tissues as does capillary endothelium (Clark & Clark, 1942).

An appropriate influence of mesenchyme on an epithelium was also found by Moscona (1961) in combined cultures of epidermis on mesenchyme from the oviduct of the 19-day chick embryo. When the explants were treated with oestradiole benzoate, the epidermis was transformed into a typical columnar mucus-secreting epithelium. Isolated epidermis treated with the oestrogen did not secrete mucus, so this effect seems to be mediated through the mesenchyme.

The range of mesenchymal tissues that have been found to exert characteristic effects on epidermal differentiation, permits the generalization that in mesenchyme cells specific factors are permanently present which can influence the differentiation of neighbouring epithelia, and which may be important in embryonic development. The interest of the results reported here is thought to lie less in the exact nature of the epidermal response than in the detection, through the epidermal response, of these normal factors in the mesenchyme.

Several observations suggest that the influence of the mesenchyme on the epithelium is reversible, e.g. when a cyst of epidermis in gizzard or heart myoblasts collapses and the epithelium piles up thickly so that it is spatially somewhat removed from the mesenchyme, it keratinizes; also, proventriculus influences the epidermis first in one way and then in another. This indicates that the mesenchymal influences discussed here must act continuously in order to maintain the modified differentiation of the epidermis. Such continuity of the mesenchymal action, and the reversibility of its effect, contrasts with the classical concept that ‘normally, of course, the tissues cease to be plastic as soon as they have undergone the inductive action of an organiser’ (Huxley & de Beer, 1934). Although the individual tissue interactions described in this paper clearly play no part in normal development (they occur between types of tissue that normally are never in contact), they demonstrate two important histogenetic principles : firstly, that mesenchymal factors are probably of widespread significance in influencing and maintaining the differentiation of overlying epithelia; and secondly, that, since the differentiation of the early embryonic epidermis of the chick is susceptible to modification by such factors, the maintenance as well as the appearance of epidermal derivatives may depend on the underlying mesenchyme. The interesting possibility is also raised that wherever cells of different types come into contact they may always influence each others’ differentiation.

Mesenchyme can influence not only the differentiation but also the growth of overlying epithelia. In the preceding paper (McLoughlin, 1961a) it was noted that many embryonic epithelia are unable to undergo mitosis when cultured in the absence of mesenchyme cells, i.e. that contact with mesenchyme is of general and basic importance to developing epithelia. When epidermis is replaced on limb mesenchyme a normally dividing basal layer is re-established and persists throughout the culture period. By contrast, on cartilage, heart fibroblasts and proventriculus, the ability to divide is restored to the epithelium at first but gradually the mitotic rate declines, and towards the end of the culture period the epidermis sometimes keratinizes down to and including the basal layer. This failure of several heterotypic mesenchymes to support mitosis consistently, indicates that only the dermal influence is correctly adapted to the epidermis. Similarly, Drew (1923) found that when he combined epithelia and fibroblasts of different origins in culture one of the tissues usually failed to survive.

The characteristic effects that each type of mesenchyme exerts on the epidermis are thought to be exercised particularly by the fibroblasts, since these congregate specifically around the epithelium in all composite cultures (except in epidermis on heart myoblasts). It is clear from existing data that fibroblasts and their products differ sufficiently in various tissues to provide a possible basis for the morphogenic effects described. In dermal mesenchyme in vitro, collagen fibrils coarse and coated with neutral mucopolysaccharide are abundant, while proventriculus, gizzard, and heart mesenchyme contain fine collagen fibrils which often lie in a matrix rich in acid mucopolysaccharides. It is well known that in the adult animal the connective tissues of different regions exhibit variations in the proportions of cells, fibres, and matrix as well as in the chemical nature of their mucopolysaccharides (Meyer & Rapport, 1951; Dische, Danilczenko, & Zelmenis, 1958) and in their behaviour in vitro (Parker, 1932).

Several considerations suggest that the fibroblasts exert their characteristic effects on epithelial differentiation by means of the different intercellular materials that they produce, and, more specifically, by means of the basement membrane. Firstly, intercellular material always intervenes between fibroblasts and epithelium, and since it is known to vary from tissue to tissue it may well provide the means whereby fibroblasts exercise their characteristic effects. Wilde’s (1960) demonstration that ‘extracellular material’ is able to influence differentiation in early amphibian embryos lends support to this idea. Secondly, the reaction of isolated epidermis to cell-free intercellular material (McLoughlin, 1961 c), and its similar response towhole mesenchyme, suggest that the basement membrane is formed from mesenchymal intercellular material under the influence of the epidermis and that it affects the orientation of the epithelium. Thirdly, the basement membrane varies with the underlying connective tissue, for it is not visible beneath epidermis on cartilage or heart myoblasts and must therefore be either altered or absent. On myoblasts, the epidermis is both oriented and capable of survival, which implies that a membrane is present though too thin to be seen. That cartilage, on the other hand, fails to supply a satisfactory basement membrane, is suggested by the disorientation and degeneration of epidermis implanted on it, and also by some results of Trinkaus (1961). He found that disaggregated cells from the epithelial pigment layer of the chick retina, when mixed with perichondrium, remain as isolated disoriented units, whereas in combination with kidney they form typical epithelial tubules. It is important to remark that the term ‘basement membrane’ is often used to describe different structures in electron and light microscopy. In this paper it is used in the light microscopist’s sense of a lamella, lying between epithelium and mesenchyme, that stains red with the PAS method and precipitates silver, i.e. that contains mucopolysaccharide and fine collagen fibrils (reticulin). It always stains for neutral mucopolysaccharides whatever the staining reaction of the general matrix. Ozzello & Speer (1958) found acid mucopolysaccharides in the basement membrane of the human mammary gland only at sites of carcinomatous invasion.

Finally, the different effects of gastric, limb, and heart fibroblasts on the spreading of implanted epidermis suggest that the basement membranes they provide differ, in spite of their similar appearance in the light microscope. Proventriculus and gizzard promote epithelial spreading, limb permits it, and heart fibroblasts strongly inhibit it; thus the ability of the basal cells to glide over their basement membrane varies according to the type of mesenchyme with which they are associated. Experiments in which gizzard epithelium was implanted on limb mesenchyme (p. 397) show that this is a result of differences in the basement membrane rather than in the cells themselves.

There is a correlation between the degree to which each mesenchyme inhibits spreading and the ability of the epidermis to keratinize on that mesenchyme, e.g. heart fibroblasts strongly inhibit spreading and promote keratinization, whereas gizzard promotes spreading and inhibits keratinization. That this connexion between the spreading and differentiation of the epithelium must be something more subtle than a purely mechanical effect is shown by two observations. Firstly, epidermis on gizzard mesenchyme may become as thickened as in keratinizing control cultures, and yet continues to secrete mucus (cf. Plate 2, fig. 13); and secondly, the cells even of the epidermal basal layer differ when placed on the various mesenchymes, e.g. on gizzard the basal cells contain few tonofibrils, on limb tonofibrils are plentiful, and on heart fibroblasts the basal cells are packed with them.

The possibility that epithelio-mesenchymal interactions continue into adult life, and their relation to pathology, have been discussed briefly elsewhere (McLoughlin, 1960). Recently, Lasfargues, Murray, & Moore (1960) have found that the mouse mammary carcinoma agent will not multiply in cultures of pure mammary gland epithelium, but grows in explants of mammary epithelium and stroma together. As this observation was made on adult tissue, it indicates that epithelio-mesenchymal interactions persist in the adult. This conclusion is further supported by the observation of Dawe (1960) that in mice the polyoma virus causes carcinomata of organs, such as salivary gland and thymus, where epithelio-mesenchymal interactions during development are either known or suspected.

L’importance des facteurs du mésenchyme dans la différenciation de l’épiderme de Poulet

II. Modification de la différenciation épidermique par contact avec différents types de mésenchymes

  1. Des expériences ont été entreprises pour savoir jusqu’à quel degré le mésenchyme sous-jacent influence la différenciation d’un épithélium. De l’épiderme d’embryon de Poulet de 5 jours a été associé, en culture in vitro, à 3 types différents de mésenchymes embryonnaires de Poulet de même âge. Ces mésenchymes proviennent du gésier, du proventricule et du cœur. Dans des séries témoins, l’épiderme a été réimplanté sur du mésenchyme de membre. L’épiderme se comporte d’une manière différente et caractéristique selon la combinaison réalisée.

  2. On décrit les processus cellulaires suivant lesquels l’épithélium isolé s’établit sur le mésenchyme, in vitro.

  3. Dans les séries témoins l’épiderme se kératinise normalement s’il est réimplanté sur du mésenchyme de membre, mais il dégénère au contact de cartilage.

  4. Le mésenchyme de gésier empêche l’épiderme associé de se kératiniser et l’induit à sécréter du mucus, et quelquefois à devenir cilié.

  5. Le mésenchyme de pro ventricule empêche d’abord l’épiderme associé de se kératiniser et le fait sécréter du mucus, mais, après 7 jours, cet épiderme retourne à sa différenciation normale et se kératinise.

  6. Des explants de mésenchyme cardiaque différencient deux zones : une aire centrale myoblastique qu’entoure une zone de croissance fibroblastique. La région centrale des myoblastes empêche l’épiderme de se kératiniser; celui-ci s’étale en un épithélium squameux formé d’une seule assise de cellules. Sur les fibroblastes de cœur, par contre, l’épiderme se kératinise d’une manière plus dense que chez les témoins.

  7. Les 4 types de mésenchyme diffèrent par la nature de leur matériel intercellulaire, et par la propriété qu’ils ont de permettre l’étalement épithélial en quantités différentes.

  8. Les fibroblastes qui s’organisent au contact de l’épithélium dans la plupart des types de culture, sont probablement responsables des différents effets du mésenchyme.

  9. La discussion porte sur les interactions épithélium-mésenchyme relatives au départ et au maintien de la différenciation embryonnaire et adulte. On suggère que le matériel intercellulaire du mésenchyme puisse participer aux mécanismes qui sont à la base de ces réactions.

Grateful thanks are due to Dr. H. B. Fell, F.R.S., Director of the Strangeways Research Laboratory, and to Dr. W. Jacobson for their continued help and many stimulating discussions throughout the course of this work. The author would also like to acknowledge with gratitude the receipt of a Research Scholarship and expenses from the Medical Research Council.

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Plate 1

Fig. 1. Living culture of epidermis on limb mesenchyme, 7 days in vitro. The epidermis has formed a keratinizing cyst (C). Cartilage black. Swathes of myoblasts are visible. ×24.

Fig. 2. Epidermis on limb mesenchyme after 24 hours in vitro. The epidermal nodule is surrounded by phagocytic peridermal cells (P). Basal cells (B) become oriented in contact with the mesenchyme. See also fig. 4. Meyer’s acid haemalum and Alcian blue. × 240.

Fig. 3. Epidermal cells situated above the basal layer send processes (P) down to the basement membrane. On the left, 2 cells send down entwined processes (PP). Iron haematoylin. × 1440.

Fig. 4. High-power view of part of the same section as in fig. 2. The processes of more distal cells sometimes contain a spiral filament (T). Meyer’s acid haemalum and Alcian blue. × 860.

Fig. 5. Processes of distal cells terminate in small feet (F) on the basement membrane (BM) which is cut somewhat obliquely. Iron haematoxylin. × 1440.

Fig. 6. The normal 2-layered structure of the epidermis is reconstructed after 48 hours in vitro. Iron haematoxylin. ×850.

Plate 1

Fig. 1. Living culture of epidermis on limb mesenchyme, 7 days in vitro. The epidermis has formed a keratinizing cyst (C). Cartilage black. Swathes of myoblasts are visible. ×24.

Fig. 2. Epidermis on limb mesenchyme after 24 hours in vitro. The epidermal nodule is surrounded by phagocytic peridermal cells (P). Basal cells (B) become oriented in contact with the mesenchyme. See also fig. 4. Meyer’s acid haemalum and Alcian blue. × 240.

Fig. 3. Epidermal cells situated above the basal layer send processes (P) down to the basement membrane. On the left, 2 cells send down entwined processes (PP). Iron haematoylin. × 1440.

Fig. 4. High-power view of part of the same section as in fig. 2. The processes of more distal cells sometimes contain a spiral filament (T). Meyer’s acid haemalum and Alcian blue. × 860.

Fig. 5. Processes of distal cells terminate in small feet (F) on the basement membrane (BM) which is cut somewhat obliquely. Iron haematoxylin. × 1440.

Fig. 6. The normal 2-layered structure of the epidermis is reconstructed after 48 hours in vitro. Iron haematoxylin. ×850.

Plate 2

Fig. 7. Typical keratinization in a cyst of epidermis on limb mesenchyme after 7 days in vitro. Note that the fibroblasts have been selectively attracted to the epidermis. Trevan’s method. × 210.

Fig. 8. Fluid-filled cyst of epidermis on limb mesenchyme after 12 days in vitro. Keratinization in such cysts is delayed but normal. On the left a small region of the epidermis is in contact with perichondrium, which causes a localized degeneration. Azan. × 250.

Fig. 9. Living culture of epidermis on gizzard mesenchyme after 5 days in vitro. The refractile lining of the epithelial cyst (C) is due to mucus secretion. Cords of epidermis extend into the medium. Note the large autonomic ganglion (G) with radiating bundles of nerve fibres. The dark object obscuring the lower right-hand part of the cyst is an artefact. × 24.

Fig. 10. Epidermis on gizzard mesenchyme after 5 days in vitro forms a 2-layered columnar epik thelium. A little mucus (M) is visible as a dark lining in the upper part of the lumen. Iron haematoxylin. × 228.

Fig. 11. Part of a distended cyst of epidermis on gizzard mesenchyme after 12 days in vitro. Mucus formed by the innermost epidermal layer stains darkly blue-green with Alcian blue. × 750.

Fig. 12. Part of a collapsed cyst of epidermis on gizzard mesenchyme after 11 days in vitro. Degenerating peridermal cells appear in the upper part of the picture; below them the thickened epidermis contains 4 goblet cells (G) of basal origin. Mesenchyme (M) in the lowest part of the picture. Meyer’s acid haemalum and Alcian blue. × 750.

Fig. 13. Epidermis arranged as a sheet on gizzard mesenchyme has become ciliated. Note that the epidermis is 4-cell-layers thick, but shows no sign of keratinizing at 6 days in vitro. Azan. × 1000.

Plate 2

Fig. 7. Typical keratinization in a cyst of epidermis on limb mesenchyme after 7 days in vitro. Note that the fibroblasts have been selectively attracted to the epidermis. Trevan’s method. × 210.

Fig. 8. Fluid-filled cyst of epidermis on limb mesenchyme after 12 days in vitro. Keratinization in such cysts is delayed but normal. On the left a small region of the epidermis is in contact with perichondrium, which causes a localized degeneration. Azan. × 250.

Fig. 9. Living culture of epidermis on gizzard mesenchyme after 5 days in vitro. The refractile lining of the epithelial cyst (C) is due to mucus secretion. Cords of epidermis extend into the medium. Note the large autonomic ganglion (G) with radiating bundles of nerve fibres. The dark object obscuring the lower right-hand part of the cyst is an artefact. × 24.

Fig. 10. Epidermis on gizzard mesenchyme after 5 days in vitro forms a 2-layered columnar epik thelium. A little mucus (M) is visible as a dark lining in the upper part of the lumen. Iron haematoxylin. × 228.

Fig. 11. Part of a distended cyst of epidermis on gizzard mesenchyme after 12 days in vitro. Mucus formed by the innermost epidermal layer stains darkly blue-green with Alcian blue. × 750.

Fig. 12. Part of a collapsed cyst of epidermis on gizzard mesenchyme after 11 days in vitro. Degenerating peridermal cells appear in the upper part of the picture; below them the thickened epidermis contains 4 goblet cells (G) of basal origin. Mesenchyme (M) in the lowest part of the picture. Meyer’s acid haemalum and Alcian blue. × 750.

Fig. 13. Epidermis arranged as a sheet on gizzard mesenchyme has become ciliated. Note that the epidermis is 4-cell-layers thick, but shows no sign of keratinizing at 6 days in vitro. Azan. × 1000.

Plate 3

Fig. 14. Gizzard epithelium on limb mesenchyme after 7 days in vitro continues to secrete mucus, i.e. its differentiation is unaltered, but the limb mesenchyme causes it to roll up into a cyst instead of spreading in a sheet, as gastric epithelium usually does. Note the nodule of cartilage in the upper right-hand comer. Meyer’s acid haemalum and mucicarmine. × 140.

Fig. 15. Living cultures of epidermis on proventriculus mesenchyme, a, after 5 days in vitro the epidermis has formed a cyst (c) which secretes mucus and fluid, b, after 8 days in vitro the epidermis thickens to begin forming keratin (K). × 24.

Fig. 16. Epidermis on proventriculus mesenchyme after 2 days in vitro has reconstructed into a 2layered epithelium arranged as a cyst around a flattened lumen (L). The inner peridermal cells carry the marker carbon particles (P), and are seated on a single layer of darker basal cells (B). A little mucus (M) is already visible on the surface of the peridermal cells. Meyer’s acid haemalum and mucicarmine. × 270.

Fig. 17. Epidermis on proventriculus mesenchyme after 7 days in vitro thickens prior to keratinizing. Meyer’s acid haemalum and Alcian blue. × 270.

Fig. 18. Part of a culture of epidermis on proventriculus mesenchyme, 10 days in vitro. The epidermis is keratinizing normally, and the previously secreted mucus (M) is carried into the centre of the cyst. Iron haematoxylin. × 750.

Plate 3

Fig. 14. Gizzard epithelium on limb mesenchyme after 7 days in vitro continues to secrete mucus, i.e. its differentiation is unaltered, but the limb mesenchyme causes it to roll up into a cyst instead of spreading in a sheet, as gastric epithelium usually does. Note the nodule of cartilage in the upper right-hand comer. Meyer’s acid haemalum and mucicarmine. × 140.

Fig. 15. Living cultures of epidermis on proventriculus mesenchyme, a, after 5 days in vitro the epidermis has formed a cyst (c) which secretes mucus and fluid, b, after 8 days in vitro the epidermis thickens to begin forming keratin (K). × 24.

Fig. 16. Epidermis on proventriculus mesenchyme after 2 days in vitro has reconstructed into a 2layered epithelium arranged as a cyst around a flattened lumen (L). The inner peridermal cells carry the marker carbon particles (P), and are seated on a single layer of darker basal cells (B). A little mucus (M) is already visible on the surface of the peridermal cells. Meyer’s acid haemalum and mucicarmine. × 270.

Fig. 17. Epidermis on proventriculus mesenchyme after 7 days in vitro thickens prior to keratinizing. Meyer’s acid haemalum and Alcian blue. × 270.

Fig. 18. Part of a culture of epidermis on proventriculus mesenchyme, 10 days in vitro. The epidermis is keratinizing normally, and the previously secreted mucus (M) is carried into the centre of the cyst. Iron haematoxylin. × 750.

Plate 4

Fig. 19. Living culture of epidermis on heart myoblasts (M) after 6 days in vitro. The epithelium forms a thin-walled, fluid-filled cyst (C). An epidermal strand stretching across the cyst, out of contact with the myoblasts, is keratinizing (three dark areas); this interpretation was confirmed histologically. × 24.

Fig. 20. An epidermal cyst on heart fibroblasts after 8 days in vitro forms compact keratin. The boundary of the myoblastic region (M) appears on the left. Azan. × 180.

Fig. 21. Section through the two ends of a sheet of epidermis on heart mesenchyme, 2 days in vitro. The epidermis has spread thinly over the entire myoblastic region (M), but at the boundary with the fibroblastic region (F) it is reflected and piles up to form a stratified squamous epithelium. Iron haematoxylin. × 230.

Fig. 22a. Epidermis on heart myoblasts, 8 days in vitro, remains as a single squamous layer. Azan staining shows the absence of fibroblasts in the lower wall. × 180.

Fig. 22b. High-power view of the lower left part of fig. 22a, showing the single layer of flattened epidermal cells, whose nuclei (A) appear darker than those of the surrounding myoblasts. A mitosis (M) indicates that the epidermis is still healthy. Azan. × 820.

Plate 1, fig. 3; Plate 2, figs. 7 and 11; and Plate 4, figs. 20 and 22a are reprinted from ‘Biological Approaches to Cancer Chemotherapy’ by kind permission of the publishers, Academic Press Inc. (London) Ltd.

Plate 4

Fig. 19. Living culture of epidermis on heart myoblasts (M) after 6 days in vitro. The epithelium forms a thin-walled, fluid-filled cyst (C). An epidermal strand stretching across the cyst, out of contact with the myoblasts, is keratinizing (three dark areas); this interpretation was confirmed histologically. × 24.

Fig. 20. An epidermal cyst on heart fibroblasts after 8 days in vitro forms compact keratin. The boundary of the myoblastic region (M) appears on the left. Azan. × 180.

Fig. 21. Section through the two ends of a sheet of epidermis on heart mesenchyme, 2 days in vitro. The epidermis has spread thinly over the entire myoblastic region (M), but at the boundary with the fibroblastic region (F) it is reflected and piles up to form a stratified squamous epithelium. Iron haematoxylin. × 230.

Fig. 22a. Epidermis on heart myoblasts, 8 days in vitro, remains as a single squamous layer. Azan staining shows the absence of fibroblasts in the lower wall. × 180.

Fig. 22b. High-power view of the lower left part of fig. 22a, showing the single layer of flattened epidermal cells, whose nuclei (A) appear darker than those of the surrounding myoblasts. A mitosis (M) indicates that the epidermis is still healthy. Azan. × 820.

Plate 1, fig. 3; Plate 2, figs. 7 and 11; and Plate 4, figs. 20 and 22a are reprinted from ‘Biological Approaches to Cancer Chemotherapy’ by kind permission of the publishers, Academic Press Inc. (London) Ltd.