Cell interactions in the developing somite: in vitro comparisons between amputated (am/am) and normal mouse embryos

In a number of previous studies we have examined the role of abnormal cell behaviour in the development of the mutant mouse amputated (Flint, 1977a, b; Flint & Ede, 1978a, b and Flint, Ede, Wilby & Proctor, 1978), concentrating on in vivo aspects of morphogenesis. In these, interpretation of embryonic cell behaviour was based upon a series of micrographs taken from TEM sections or from SEM preparations. In this paper we report observations on living mutant and normal embryonic cells in in vitro culture, using, as the main tool, time lapse cinémicrography. The cells chosen for study were those leaving explants prepared from somites of 9 ·5-day mice, and our observations confirm and amplify our interpretations of the mutant cell behaviour in vivo. The basis of this altered cell behaviour is further investigated in an SEM analysis of cell-cell contacts in the cultured cells.

The amputated gene is kept on a background of a C3H/101 hybrid intercross. Details of matings for the production of embryos of known age can be found in Flint & Ede (1978 a). All pregnant females were opened under sterile conditions, 9 ·5 days after noting a copulation plug. Each female was killed by cervical dislocation and dipped, for external sterilisation, in a solution of cetrimide (2 %) and sodium hypochlorite (2 %) in water (Bleby, 1972). Unless otherwise stated, all the solutions used in the preparation of the tissue cultures described below were warmed to 37 ·5 °C.

The technique for preparation of mouse somites for culture is a modification of that described by Cooper (1965). Each conceptus (decidua plus embryo) was dissected out into fresh Tyrode’s solution, and transferred to a solution of horse serum plus Tyrode’s (1:1, v/v) where the embryos were removed. Embryos with 18 somites were chosen and taken through the explant preparation as pairs of normal and amputated littermates. All the results were recorded as littermate pairs and the statistical analysis takes this into account. Any littermate pairs not prepared for explants were fixed for histological examination.

The embryos were treated in the following way. Any ventral trunk tissue up to the level of the somites was dissected away with fine forceps and pipetted off with a Pasteur pipette. The caudal half of the body was then cut off and placed in the well of a cavity slide in a solution of 3 % trypsin (DIFCO, 1:250) in calcium- and magnesium-free Tyrode’s solution (CMF) for one minute at room temperature. After gently aspirating the tissue two or three times through the mouth of a Pasteur pipette to loosen the epidermis the trunk was transferred to a drop of horse serum and Tyrode’s. It was then possible to remove the remaining epidermis and cleanly dissect the somites from the neural tube and notochord with finely sharpened tungsten needles. In every case the last four somites alone were dissected free from both sides of the trunk. These somites were then transferred quickly to a drop of 0 ·25 % trypsin in CMF for one minute at room temperature. It was found that if this second exposure to trypsin was omitted the explants would not later adhere to the culture dish (in a trial run, 1 out of 20 adhered if this step was omitted, 20 out of 20 if included). The somites were then transferred to a fresh drop of horse serum and Tyrode’s and chopped up into six to eight pieces, which were then dropped into culture medium to form the explants and cultured at 37 ·5 °C in 5 % CO₂: 95 % air. The medium was Eagle’s Minimal Essential Medium (MEM) plus medium supplements of non-essential amino acids, vitamins, sodium bicaibonate buffer and 10 % foetal calf serum (FCS). Antibiotics were also added to the medium to a final concentration of 0 ·02 % streptomycin and 0 ·012 % benzyl penicillin.

The cultures were incubated for 12 h while the explants adhered to the substrate and cells migrated centrifugally from the explant edge. Filming of each culture lasted a further 11 h and 7 min. Cultures were filmed as littermate derived pairs of normal and amputated somite explants on two Wild inverted microscopes connected by a comparison tube. If the cultures had been filmed sequentially the second culture, at the start of filming, would have been 11 h older than the first and it is possible that ageing may cause changes of cell motility. Abercrombie & Heaysman (1953) could detect no effect of culture age on heart fibroblast movement after 5 h of culture but Martz (1973) could not exclude an age-dependent effect reducing 3T3 motility over much longer periods in culture. It was therefore decided that filming of pairs would remove a possible and unnecessary bias. Only one pair of littermate-derived cultures was chosen per experiment for filming. Other culture pairs were left in the incubator and later fixed (see below).

Filmed cultures were maintained on the micioscope stage in a hot room at 37 ·5 °C. Each petri dish was held in a small perspex and glass chamber which was gassed with 5 % CO₂. Illumination was either by phase or optical shadow casting (Hlinka & Sanders, 1970). Fields for filming were chosen at random within the margin of the moving cell front. A lapse rate of one frame in 10 sec was chosen and controlled by a Payard-Wild Variometer. Twenty culture pairs were filmed in this way.

After filming the cultures were returned to the incubator and left to allow further spreading of the explant. After a total of 44 h of culture all the explants, including those not filmed, were fixed following the technique of Revel & Wolken (1973).

Petri dish bases were coated with carbon under vacuum at an angle of 60° in a Speedivac Coating Unit (Edwards High Vacuum Ltd).

It was not possible to dry the cultures by the critical-point technique because the solvents used attacked the plastic culture dishes. Glass coverslips were not used for the sake of uniformity in the culture of filmed cells and the other cultures. The results of air drying were critically compared with living cells observed under optical shadow casting (see below). The drying technique employed did not appear to cause any significant distortion or cracking. Revel & Wolken (1973) compared the scanning EM appearance of rapidly dried tissue cultured cells with critical-point preparations of similar cultures. They found that both techniques gave very good results, though slightly more shrinkage occurred in cells not dried by the critical-point method. Each piece of plastic with an explant was cut out from the petri dish base and mounted on a 12 mm aluminium stub with colloidal silver (Polaron) and uniformly coated with 500 nm of gold in a Polaron sputter coater. The explants were examined with a Cambridge S600 scanning electron microscope in the Anatomy Department of Glasgow University at accelerating voltages of 15 kV.

The films were analysed with a Specto Mark III motion analysis projector. Film was projected onto tracing paper on a glass-topped table via a mirror placed under the table at an angle of 45°. Cells were chosen at random at the beginning of each film and the position of the nucleolus marked. The new position of the nucleolus was marked every fifty frames (8-33 min) unless the cell either left the field, entered mitosis or lost its adhesion with the substrate (usually prior to mitosis). If any of these events occurred a new cell was chosen. If the nucleolus did not move after 50 frames, the length of time it remained stationary was recorded in units of 50 frames (rest length). If a cell moved, the length of each step was measured for each 50-frame unit (step-length). The cell density in each measured frame was also recorded.

Of the 20 pairs of cultures filmed it was possible to analyse 18 normal cultures and 19 amputated cultures. 158 normal cells were followed (an average of 8 ·78 ± 2 ·24 (s.D.) cells per film) and 151 amputated cells (an average of 7 ·95 ± 2 ·27 cells per film). Normal cells were followed on average for paths of 57 ·76 ± 23 ·99 μm (2 ·12 ± 0 ·88 h), and amputated cells for 42 ·14 ± 28 ·35 μm (2 ·19 ± 1 ·47 h). Normal cells moved an average number of 12 ·16 ±5 ·05 steps, and amputated cells 8 ·28 ± 5 ·57 steps in this time.

The explant consists of a central piece of somite which flattens slowly as cells migrate away from its edge onto the substrate. Cells were bipolar or multipolar, with a higher proportion of bipolar cells in normal cultures, and movement was normally in the direction of the most active lamellipodium in each cell (Fig. 1). Movement also occurred when cells in contact by extended filopodia were hauled together by contraction of these filopodia. Within explant borders in normal cultures cells tended to move away from one another, leaving large spaces between cells. The filopodia connecting them became stretched over quite long distances but they did not form permanent contacts, and soon broke away. By comparison, in the amputated cultures, cell contacts were maintained over much longer periods. Cells leaving the edge of the central solid explant remained close together, with extensive marginal cell-cell contact, and near the margin of the explant border, cells were far less dispersed than in normal cultures. Gaps eventually formed between cells so that the explant border came to consist of a series of interconnected clumps rather than a continuous cell sheet. This was in marked contrast to the loosely dispersed pattern of cells within normal explant borders (Fig. 1).

Optical shadow casting gives a true three-dimensional relief image of the cell surface (Hlinka & Sanders, 1970) so that the living cells in Fig. 1. may legitimately be compared with scanning electron micrographs of cultured cells. The chief difference is that these cells in SEM appear to be flatter than the living cells, indicating that the external structure of the cell has collapsed to some extent during drying. But details of cell contact and morphology are much more highly resolved in scanning electron micrographs than by optical shadow-casting.

The much longer filopodial connections between normal cells are clearly shown in Fig. 2. Normal cells have a much more elongated, often bipolar, appearance and are separated more widely than amputated cells, which form characteristic clumps. The extent of cell margin in contact between adjacent cells is much less in normal than in amputated cultures (Fig. 2a, b). Three types of cell contact were observed: (1) the apposition of one cell surface against another without much deformation of the cell. Only very small areas of this type of contact can be seen in normal cultures, for example in the lower right corner of Fig. 2 a, but very large areas of this kind of contact can be seen anywhere in the amputated cultures (Fig. 2b). (2) Fairly wide and tapering filopodia. These are found in both normal and amputated cultures, but they are usually much longer in normal cultures (compare Fig. 2 a, b). (3) Thin filopodia of uniform diameter (microspikes). These are much longer in normal cultures but short microspikes are much more numerous in amputated cultures. In general there is a greater area of cell contact in amputated cultures.

There are also differences between mutant and normal at the points of cell contact. When one normal cell makes contact with another, the actively moving lamellipodium or filopod can move a considerable way over or under the surface of the other cell. In amputated cultures filopodia hardly overlap or underlap the other cell at all (Fig. 2 c, d), though the number of amputated filopodial contacts can be so great as to form a mesh work between neighbouring cells (Fig. 2d).

Measurements on fixed and carbon-coated cultures indicated no significant difference in the average cell density of normal and amputated explant borders. A clear linear regression emerged when cell density was plotted against distance from the explant centre in both normal and amputated explants (Fig. 3), and no difference was found between amputated and normal regressions (see legend to Fig. 3). This indicates that cell density within the explant border is inversely related to distance from the explant centre and that there is no difference of cell density between normal and amputated explants at any point in the explant border.

Three parameters were assessed in measuring the cell paths from time-lapse films:

1. Cell speed.

2. Length of time the cell was at rest.

3. Length of each step taken by the cell when moving in the chosen interval of 8 ·33 min.

This follows the method of analysis of Ede & Flint (1975 b). Additional measurements were taken of the cell density in the film frame over the period of path measurement. Cell density varied only slightly over this period of path measurement in each film, as cells entered or left the field.

Cell speed (μm/hour) was calculated both for distance taken as the sum of the lengths of all the steps making up each cell’s path and for distance ‘as the crow flies’, i.e. the linear distance between the cell’s first and last positions. In the first case there was no change of cell speed with cell density in either amputated or normal cultures by Bartlett’s three group method^* (Sokal & Rohlf, 1969, p. 481). Normal cells moved significantly faster than amputated cells (Table 1). In the second case, of cell speed ‘as the crow flies’, there was again no change of cell speed with cell density by Bartlett’s three group method in amputated or normal cultures, and no difference between normal or amputated average speeds (Table 1).

Time at rest was measured in two ways: as a percentage of the whole time a cell was observed and as the average length of time in minutes a cell stayed at rest. There was no change in amputated or normal cultures in the average length of rest with cell density in either case (analysed by Bartlett’s three group method). Normal cells spend on average significantly less time at rest when this is calculated as a percentage of the whole time the cell was observed (Table 1), or as average length of each rest taken in minutes (Table 1). Clearly mutant cells pause more often and for longer periods than do normal cells.

There is no change of average step length with cell density in all cultures by Bartlett’s three group method nor is there any significant difference between amputated and normal (Table 1).

In both normal and amputated cultures, cells migrating from the explant are at first confluent but tend to disperse the further they are from the explant centre. The pattern of cells within the explant border suggests that whereas normal cells disperse individually, amputated cells disperse as small clumps, i.e. as groups of cells which remain in contact over long periods of observation in time-lapse films. In amputated the cells are strongly bound together by large numbers of adhesive contacts, including short filopodia in large numbers, sometimes amounting to a meshwork, preventing their moving apart. In normal cultures the few and relatively extensible filopodia allow the cells they connect to move apart. The smaller degree of overlapping and underlapping by filopodia and lamellipodia in amputated cultures suggests that the stronger adhesions tend to immobilise moving cell projections more rapidly than the weaker adhesions formed by normal cells.

There is a striking similarity between these cultured somite cells and amputated mesenchyme cells observed in the embryo in the sclerotome (Flint & Ede, 1978 a), the facial mesenchyme (Flint, 1977 a; Flint & Ede, 1978b) and in the palate (Flint, 1980). In all these places amputated cells clump together, far greater areas of cell contact are observed between amputated cells than between normal cells, and filopodia tend to mesh together into knots as they do in the cultures of amputated cells (Fig. 3d).

Taking the age of each culture when fixed as 44 h, and the average width of the explant border in normal and amputated cultures as 408 μm (480 μm average explant radius, less 15 % for the explant itself) the rate of advance of the cell border margin is 9 ·3 μm per hour. This is in very close agreement with the measurement of cell speed for amputated and normal cells ‘as the crow flies’ (11 ·0 μm per hour). Cell speed as measured in this way is therefore approximately equivalent to the rate of radial migration of cells within the explant border. Normal and amputated explant cultures expand at the same rate, but it is the details of individual cell movement within the border that differentiate between the two.

Normal cells move significantly further, over the whole path, in an hour than amputated cells (Table 1), because they spend less time at rest. This follows from the looser, more dispersed arrangement of cells in the normal explant border and from the normal cells forming weaker contacts when they meet other cells, so that the resumption of cell movement after contact inhibition is not delayed.

Rather similar observations on cell movement have been made in other mutants in which morphogenesis is disturbed by abnormalities of cell-cell contact behaviour. In the chick mutant talpid³ Ede & Flint (1975 b) found that cells migrating from primary explants of talpid³ chick wing mesenchyme, moved more slowly than normal chick cells because they spent more time at rest. This was correlated with other measurements showing that talpid³ cell-cell adhesions in rotation reaggregation experiments were stronger than normal cell adhesions (Ede & Flint, 1975 a). The talpid³ cell, like the amputated cell, appears to form more extensive and stronger adhesions than normal cells, inhibiting cell movement in culture, but not to such a marked degree, so that clumping is not visible in vivo (Ede & Flint, 1975 b). Another example is the t⁹ mutant of the mouse, in which Spiegelman & Bennett (1974) found that the movement of cells through the primitive streak of t⁹/t⁹ embryos was retarded because the ingressed cells formed extensive contacts and because of this, tended not to disperse as a primary mesenchyme. Yanigasawa & Fujimoto (1977) in a study of t⁹ cell aggregation in vitro have been able to confirm that the more extensive cell contacts observed indicate stronger adhesions between mutant cells.

In each of these examples a good case has been made for the effects upon cell contact relations, and consequently on cell behaviour, being the basic cause of morphogenetic abnormalities produced by the mutant genes - in amputated, abnormalities of somitogenesis (Flint et al. 1978), facial development (Flint & Ede, 1978 b) and cleft palate (Flint, 1980); in t⁹, abnormalities of early development and axis formation; in talpid³, abnormalities of the developing limbs (Ede & Flint, 1975a, b) and the somites (Ms in preparation). In all of them abnormalities of cell movement and cell adhesion have been demonstrated in in vitro culture, but it would be too simple to say merely that in all of them the cells are abnormally sticky and relatively immobile. If this implied that the abnormalities at the cellular level were identical it would be difficult to understand why their effects on morphogenesis are different. But this is not the case; e.g. in their behaviour and morphology amputated and talpid³ cells have quite distinct characteristics, and this would explain why their effects upon morphogenesis are not the same. Bellairs, Sanders & Portch (1980) have shown how subtly the behavioural properties of chick embryo cells alter with time in the course of mesodermal differentiation, with each type of mesoderm exhibiting characteristic patterns of cellular behaviour in vitro. Alterations in these behavioural properties produced by mutant genes will be equally subtle and diverse, and through them we may ultimately expect to discover a complex dynamic programme of gene-controlled cell behaviour patterns underlying all of the morphogenic aspects of embryogenesis.

The authors wish to thank the Science Research Council for financial support.