Measurements of the cell perimeter, cell-cell contact number and length and of the thickness of the surface coat were performed in limb buds of mouse embryos during the process of cell condensation of the chondrogenic cell mass. This study starts with measurements of ‘non-limb’ mesenchyme.of day-9 embryos and ends with the young cartilage of day 13. It is shown that until day 12 all cells in the limb bud are interconnected by cell contacts of the gap-junction type. Contact number and contact length increase during development, but increase about twice as much between central, prechondric cells as between peripheral or distal cells. Later in development, chondroblasts lose their contacts almost completely, whereas between peripheral cells, the amount of cell contacts drops to the initial value of mesenchymal cells. The cell size decreases during chondrogenesis. A decrease in the thickness of the surface coat of the cells during the whole differentiation period is shown. It may be assumed that this ‘wave of cell contacts’ is the last step for the initiation of the chondrogenic differentiation process.

The development of the limb skeleton starts with the formation of condensations of mesenchymal cells in the prospective chondrogenic areas (Fell, 1935; Searls & Janners, 1969; Summerbell, 1976; Cairns, 1977). This process of blastema formation is not yet fully understood. A local increase in cell proliferation can be excluded (Hornbruch & Wolpert, 1970; Janners & Searls, 1970; Cairns, 1977), but there are some indications concerning the participation of cell movements and changes of cell adhesion and intercellular matrix (Toole, Jackson & Gross, 1972; Ede & Flint, 1975; Ede, Flint, Wilby & Colquhoun, 1977; Holmes & Trelstad, 1980).

Polarization of mesenchymal cells towards the centre of the condensation were shown by Trelstad (1977) and Ede & Wilby (1981). The importance of cell adhesion and slight cell movements was indicated by comparison of chondrogenesis in normal and talpid3 chick embryos (Ede & Agerback, 1968; Ede, Flint & Teague, 1975; Ede et al. 1977). A decrease in intercellular spaces by action of cellular hyaluronidase may be also involved in the condensation process (Toole, 1972, 1973; Zimmermann, 1981).

The morphological changes during blastema formation are well known in the chick embryo. In prospective chondrogenic regions, the loose mesenchyme condenses, the width of the intercellular spaces decreases and an enhanced formation of cell-cell contacts occurs (Searls, Hilfer & Mirow, 1972; Summerbell, Lewis & Wolpert, 1973; Thorogood & Hinchliffe, 1975; Kelly & Fallon, 1978).

Similar events are described in the development of the skeletal blastema in mammals (Jurand, 1965; Neubert, Merker & Tapken, 1974; Borck, 1977). Participation of cell movements (Holmes & Trelstad, 1977, 1980) and cell adhesion (Duke & Elmer, 1978, 1979) was shown.

Although the importance of the formation of cell contacts during blastema formation in the limb (Searls et al. 1972; Kelly & Fallon, 1978) as well as in other organs (Trelstad, Hay & Revel, 1967; De Haan & Sachs, 1972; Bunge et al. 1979; Gilula, 1980; Loewenstein, 1980) has been pointed out, no quantitative estimations are available. Regarding cell adhesion, quantitative measurements of the cell surface coat in the condensing mesenchymal cells are also lacking.

In this study, cell size, quantity of cell contacts and the thickness of the cell surface coat in limb skeletal development of mouse embryos were measured. The whole developmental period was covered starting with ‘non-limb’ mesenchyme at day 9 of development until early chondrogenesis at day 13.

Upper limb buds of mouse embryos of days 10, 11, 12 and 13 (mouse strain NMRI, day 0 = day of conception) were removed and fixed immediately. Body segments of day-9 embryos in the prospective limb-forming region (somites 8–11) were excised and fixed. For this study, embryos of three to four different litters were used, and two to three embryos per litter were examined.

Fixation and embedding procedure

Fixation was performed in 2 % glutaraldehyde (Ferak, Berlin) plus 1 % tannic acid (E. Merck, Darmstadt) in 0.1 M-phosphate buffer, pH 7·2, for 1 h at room temperature. After rinsing, post-fixation was done in 1 % OsO4 in 0·1 M-phosphate buffer, pH 7·2, for about 1 h at +4 °C. After rinsing thoroughly, the expiants were dehydrated in ethanol and embedded in Epon or Mikropal (Ferak, Berlin).

Electron microscopy

Different parts of the limbs were prepared for electron microscopy after orientation on semithin sections. From day-9 embryos, the lateral body wall between somatopleura and the outer epithelium was used. From day-10 embryos, the whole limbs could be sectioned horizontally in the medial plane. The apical ectodermal ridge served as reference point. Electron microscopic pictures were taken in distal regions under the apical ectodermal ridge, in central regions and the periphery. The limbs of day-11 and -12 embryos were divided into a distal and a proximal part. From the distal parts, pictures were taken under the apical ectodermal ridge and around the marginal sinus; from the proximal part, in the centre in the area of the prospective humerus, and from the periphery, under the lateral basement membrane (Fig. 1). In day-13 limbs, only the proximal/central part (humerus) and the peripheral regions were examined.

Fig. 1.

Regions measured for cell circumference, contact number and contact length, and surface coat thickness. (1) Distal; (2) central; (3) peripheral. Example showing a day-11 limb.

Fig. 1.

Regions measured for cell circumference, contact number and contact length, and surface coat thickness. (1) Distal; (2) central; (3) peripheral. Example showing a day-11 limb.

Electron microscopic pictures were taken at primary magnifications of 2000 and 40000. Measurements of the cell perimeter and of contact number and contact length were done on enlargements of 6000, measurements of the surface coat on enlargements of 120000.

Measurements of the cell perimeter and the contact length

Cell perimeter, contact length and the number of contacts of each measured cell were performed with an Interactive Image Analysing System IBAS 1 (Kontron, Echingen, München). In this system, lines of the measured structure are covered by a contact pen manually. The position data of x and y are computed and processed in an integrated microprocessor. Histograms, single data, mean and standard deviations were printed out. One cell contact length was defined from one intercellular space to the next.

Measurements of the cell surface coat

The thickness of the surface coat was estimated on pictures of a magnification of 120000 by using a measuring magnifier. In each litter series, 40 positions were measured in every region. Only exact cross sections through the cell membrane with well-recognizable bilayer structure were used.

Statistical analysis

For statistical analysis, a PDP-11/34A computer (digital equipment, Minnesota) was used with a RSX-1 IM operating system. Mean and standard deviations of the surface coat thickness were calculated for each litter series separately to show the variance in the different series. The /-test was used for testing the significance of differences between the data of different stages and regions.

Mean and standard deviations of cell perimeter were printed by the IBAS. The /-test was performed with a PDP-11/34A computer. Since the data of the contact length are not normally distributed, the t test could not be used for statistical analysis of the data. Therefore, the single data printed by the IBAS were fed into the PDP-11/34A. The Mann-Whitney-Wilcoxon test for the difference between two populations was performed using the statistical program ‘minitab’, release 81·1 (Copyright Penn. State University 1981, University of Toledo).

Between the outer epithelium and the somatopleura in the presumptive limb-forming region of day-9 mouse embryos, the mesenchymal cells are almost completely separated by wide intercellular spaces. The cells exhibit an irregular cell surface, and many cell processes cross the intercellular space. Only a few focal cell—cell contacts are visible (Fig. 2 a). Most of the contacts can be considered as gap junctions (Fig. 2 b). The mean perimeter of a cell amounts to 30 μm, a single contact exhibits a mean length of about 1.0 μm, and about 2·3 contacts per cell are present (Table 1). The histogram of the contact length shows a relatively high amount of very short cell contacts (Fig. 7).

Fig. 2.

2(a). Loose mesenchyme between the somatopleura and the epithelium of the body wall in the prospective limb-forming region (between somites 8 and 11) of day-9 mouse embryos. Irregularly shaped cells show many cell processes and some cell-cell contacts (➡).×6000; bar = 1 μm. (b) High magnification of a cell-cell contact of the loose mesenchymal cells reveals the typical morphology of a gap junction, × 120000; bar = 100 nm. (c) The surface coat of the mesenchymal cells in the prospective limb-forming region of day-9 mouse embryos exhibits a thickness of about 8 nm (➡). Some filamentous structures extend to up to 80 nm into the extracellular space (▸).× 120000; bar = 100 nm,

Fig. 2.

2(a). Loose mesenchyme between the somatopleura and the epithelium of the body wall in the prospective limb-forming region (between somites 8 and 11) of day-9 mouse embryos. Irregularly shaped cells show many cell processes and some cell-cell contacts (➡).×6000; bar = 1 μm. (b) High magnification of a cell-cell contact of the loose mesenchymal cells reveals the typical morphology of a gap junction, × 120000; bar = 100 nm. (c) The surface coat of the mesenchymal cells in the prospective limb-forming region of day-9 mouse embryos exhibits a thickness of about 8 nm (➡). Some filamentous structures extend to up to 80 nm into the extracellular space (▸).× 120000; bar = 100 nm,

Table 1.

Measurements of cell perimeter and cell contact number and length on cells in the limb anlage of different stages

Measurements of cell perimeter and cell contact number and length on cells in the limb anlage of different stages
Measurements of cell perimeter and cell contact number and length on cells in the limb anlage of different stages

The surface coat of these cells has a thickness of about 8 nm (Fig. 2 c, Table 2). Very often, larger structures of up to 80 nm extend beyond the dense surface layer.

Table 2.

Thickness of the cell surface coat on cells in the limb buds of mouse embryos of different stages

Thickness of the cell surface coat on cells in the limb buds of mouse embryos of different stages
Thickness of the cell surface coat on cells in the limb buds of mouse embryos of different stages

At day 10, upper limb buds of about 0.5 mm length have formed, whereas the lower limb buds become just recognizable. The mesenchyme in the upper limb anlage is very uniform. The cells are interconnected by small cell contacts, irregularly shaped and separated by large intercellular spaces, such as the mesenchymal cells at day 9. Measurements of the cell perimeter in different areas of the limb bud (distal, central, peripheral) show no differences. The portion of the cell membrane involved in cell contacts is about 9 % and quite similar in all regions (Table 1).

The lengths of the cell contacts show significant differences. In the central part of the limb, the cell contacts exhibit a length of about 1.4μm (mean = 1.4 μm, median = 1·0μm), which is significantly longer than in the distal and peripheral areas (Table 1). On the other hand, the number of cell contacts is only about 2 per cell in the central parts, 2·4 in the distal and 3·2 in the peripheral parts. Therefore, the percentage of cell contacts is not different in the measured areas. A tendency of increasing contact length is also demonstrated in the histograms (Fig. 7).

In all measured regions, the surface coat of the cells is quite similar and the thickness comes to 6 nm (Fig. 3 a, Table 2). Some structures are present at the cell surface, which extend by more than 50 nm into the extracellular space. Sometimes cell contacts which are about to form are detectable. These structures have a regular cell-cell distance of about 20 nm ; the space is filled by electron-dense material. At one end of such wide contacts, the membranes of the adjacent cells come together to form a gap junction (Fig. 3 b).

Fig. 3.

(a). A surface coat of about 6nm thickness (➡) is detectable on the cell membrane of day-10 mesenchymal cells. Some structures extend to up to 50 nm (▸) into the extracellular space, × 120000; bar = 100nm. (b) A developing gap junction is shown in this micrograph. A regular intercellular space of about 20 nm, filled with electron-dense material, and a forming gap junction at one end (➡) are visible, × 120000; bar = 100 nm.

Fig. 3.

(a). A surface coat of about 6nm thickness (➡) is detectable on the cell membrane of day-10 mesenchymal cells. Some structures extend to up to 50 nm (▸) into the extracellular space, × 120000; bar = 100nm. (b) A developing gap junction is shown in this micrograph. A regular intercellular space of about 20 nm, filled with electron-dense material, and a forming gap junction at one end (➡) are visible, × 120000; bar = 100 nm.

In the limb buds of day-11 mouse embryos, the first formation of cell condensation occurs in the region of the prospective humerus. Measurements of the cell contacts in the three different regions reveal a high increase in both the number of contacts per cell as well as in the mean (or median) length of a contact. This results in a very high percentage of cell membranes involved in cell contacts.

Cells in the peripheral and the distal areas show a similar behaviour. The cells are close together, but considerable intercellular spaces are present (Fig. 4 a). The number of cell processes seems to be diminished; this is indicated in the peripheral cells by the decrease of the cell perimeter (P < 0 ·01, significant against the mesenchymal cells of day 9, day 10 and central or distal regions of day 11) (Table 1).

In the central parts, the percentage of contacts has increased to more than 60%, the cells are very close together and only a small intercellular space is visible (Fig. 4b). All the contacts between the cells are typical gap junctions with an electron-dense space of about 2 nm between the two outer lamellae of the adjacent cells (Fig. 4c). In the contacts, small distensions cut the junction into regular sections of 100–150 nm. This dimension is probably the ‘real length’ of a gap junction, but in the measurements done here, one cell contact is defined from one intercellular space to the next.

Fig. 4.

(a). Mesenchymal cells in the distal region of day-11 mouse limb buds. Similar morphology can be shown in peripheral parts. Cell density has increased and cells are interconnected by gap junctions (➡). ×6000; bar = 1 μm. (b). Cells in the central region of day-11 mouse limb buds exhibit a maximal condensation density. The intercellular space (✖) has diminished. Cells are interconnected by long cell contacts (➡). × 6000; bar = 1μm. (c). High magnification of the cell contacts reveals the typical morphology of gap junctions, × 120000; bar = 100 nm. (d). The surface coat of the cells in all measured regions exhibits a thickness of about 6 nm (➡). Only delicate structures extend into the extracellular space (▸ ). × 120000 bar = 100 nm.

Fig. 4.

(a). Mesenchymal cells in the distal region of day-11 mouse limb buds. Similar morphology can be shown in peripheral parts. Cell density has increased and cells are interconnected by gap junctions (➡). ×6000; bar = 1 μm. (b). Cells in the central region of day-11 mouse limb buds exhibit a maximal condensation density. The intercellular space (✖) has diminished. Cells are interconnected by long cell contacts (➡). × 6000; bar = 1μm. (c). High magnification of the cell contacts reveals the typical morphology of gap junctions, × 120000; bar = 100 nm. (d). The surface coat of the cells in all measured regions exhibits a thickness of about 6 nm (➡). Only delicate structures extend into the extracellular space (▸ ). × 120000 bar = 100 nm.

No clear-cut changes in the surface coat are recognizable. The cells in all estimated areas exhibit a surface coat of about 6 nm thickness (Fig. 4d, Table 2).

In the limb buds of day-12 mouse embryos, no distinct differences were detected between the distal and the peripheral regions (Table 1). Again the organization of the tissue rather resembles that of the mesenchyme. On the other hand, about 47 % of the membranes of the cells in the central part (humerus) are involved in contacts. The cells are still close together, but a dilatation of the intercellular space is visible (Fig. 5 a). In the intercellular space as well as in the cells, some myelin-like membrane structures are detectable (Fig. 5 a). At higher magnification, these structures look like coiled gap junctions (Fig. 5 b). This possibly reflects the, dis-assembly of cell contacts.

Fig. 5.

(a). In the central region of day-12 mouse limb buds, the intercellular space shows little enlargement. Most of the cells are yet interconnected by cell contacts (➡). Myelin-like figures are detectable in the cells as well as in the intercellular space (▸). x6000; bar = 1 μm. (b) The morphology of a myelin-like structure shown at high magnification resembles that of coiled gap junctions, × 120000; bar =100 nm. (c) The thickness of the surface coat of all cells in the central region of day-12 limb buds is reduced to about 4nm. × 120000; bar = 100 nm.

Fig. 5.

(a). In the central region of day-12 mouse limb buds, the intercellular space shows little enlargement. Most of the cells are yet interconnected by cell contacts (➡). Myelin-like figures are detectable in the cells as well as in the intercellular space (▸). x6000; bar = 1 μm. (b) The morphology of a myelin-like structure shown at high magnification resembles that of coiled gap junctions, × 120000; bar =100 nm. (c) The thickness of the surface coat of all cells in the central region of day-12 limb buds is reduced to about 4nm. × 120000; bar = 100 nm.

The thickness of the surface coat of the cells in all three areas is now diminished (Fig. 5d). Measurements reveal a thickness of about 4 nm (Table 2).

In the central region (humerus) of the limb of day-13 mouse embryo, chondrogenesis has started. The cells show the typical morphology of young chondroblasts (Fig. 6a). The perimeter of a cell has further decreased significantly (P > 0·01) to a mean of 21 μm. Only very few cell contacts are detectable: 0-85 contacts per cell are counted. The intercellular space has widened and is filled with the typical chondrogenic matrix. The surface coat of these cells is only very thin; the thickness has further decreased to about 2nm (Fig. 6 b, Table 2).

Fig. 6.

(a) Chondroblasts in the central part of day-13 mouse limb buds are separated by wide intercellular spaces filled with collagen filaments. Only very few cell contacts are present (➡).× 6000; bar = 1μm. (b) The surface coat of such chondroblasts is only very thin. Extracellular proteoglycan granules are present (➡) near the cell membrane, × 120000; bar = 100 nm.

Fig. 6.

(a) Chondroblasts in the central part of day-13 mouse limb buds are separated by wide intercellular spaces filled with collagen filaments. Only very few cell contacts are present (➡).× 6000; bar = 1μm. (b) The surface coat of such chondroblasts is only very thin. Extracellular proteoglycan granules are present (➡) near the cell membrane, × 120000; bar = 100 nm.

The cells in the distal region are difficult to define. Distally to the measured central area the anlagen of forearm, wrist and hand are localized. They are now advanced in development, so is the central part at day 12. Cells lying directly under the distal epithelium resemble peripheral cells. Since the apical ectodermal ridge is now absent, no clear-cut position finding was possible, so that measurements in this area were renounced.

Perimeter, contact length and contact number as well as the morphology of the peripheral cells resemble those of young mesenchymal cells at day 9 or 10 (Table 1). Also the thickness of the surface coat comes to 5 ·m, a value similar to that of the cells at day 10 (Table 2).

The contact length of the cells in different areas and at different stages are demonstrated in the histograms of Fig. 7. It is shown that the distribution of the contact length differs characteristically. Before cell condensation occurs (day 9 and 10), most of the cell contacts are very short, but during the condensation process and at the onset of chondrogenesis (day 11 and 12), longer contacts are detectable. These alterations in the distribution pattern are more pronounced in the central and distal regions than in the periphery. During chondrogenesis on day 13, the short contacts are present again in the central and in the peripheral areas.

The percentage of cell membranes involved in cell-cell contacts are plotted against the stage in Fig. 8. It is shown that in the peripheral and distal regions cell contact behaviour is identical. It starts from about 9 % at day 10, reaching a maximum of about 35 % at day 11 and decreases to the 5 % level at day 13. The cells in the central part also start at the 10 % level at day 10. Then, however, they reach a maximum at day 11 with more than 60% and decline to less than 1 % at day 13, when the cells are differentiated. That ‘wave of cell—cell contact’ therefore is highest in the cells of the central region, which results in chondrogenic differentiation, and is lower in the cells of the distal and peripheral parts of the limb, where the cells do not undergo chondrogenic development.

Fig. 7.

Histogram of contact length in different regions and at different stages of mouse limb buds. Each section on the abscissa represents a contact length of 0·5 μm. Note the relative preponderance of short contacts between the cells in limb buds of days 9, 10 and day 13. During the cell condensation process at days 11 and 12 the number of longer cell contacts increases, but more in the central and distal parts than in the periphery.

Fig. 7.

Histogram of contact length in different regions and at different stages of mouse limb buds. Each section on the abscissa represents a contact length of 0·5 μm. Note the relative preponderance of short contacts between the cells in limb buds of days 9, 10 and day 13. During the cell condensation process at days 11 and 12 the number of longer cell contacts increases, but more in the central and distal parts than in the periphery.

Fig. 8.

Percentage of cell membranes involved in cell contacts of mouse limb buds of different stages. ▴ = Central region (humerus); • = distal region; ◼ = peripheral region. The increase in the percentage of contact-participating cell membranes is highest during the process of cell condensation between days 11 and 12 of development. The increase is much higher in the central parts where chondrogenesis is to start than in the distal and peripheral regions which show the same course. When at day 13 chondrogenesis has begun, the contact in these (central) parts comes to less than 1 %, whereas in the peripheral parts, the initial values of days 9 and 10 are measurable.

Fig. 8.

Percentage of cell membranes involved in cell contacts of mouse limb buds of different stages. ▴ = Central region (humerus); • = distal region; ◼ = peripheral region. The increase in the percentage of contact-participating cell membranes is highest during the process of cell condensation between days 11 and 12 of development. The increase is much higher in the central parts where chondrogenesis is to start than in the distal and peripheral regions which show the same course. When at day 13 chondrogenesis has begun, the contact in these (central) parts comes to less than 1 %, whereas in the peripheral parts, the initial values of days 9 and 10 are measurable.

Measurements of cell perimeter and cell contacts together with electron microscopic findings on the cells in the limb bud at different stages of development allow a more detailed description of chondrogenic differentiation in the limb. The results can be summarized as follows:

  1. From the earliest stage of development until day 12 all the cells in the limb are more or less interconnected by cell-cell contacts of the gap-junction type.

  2. During the cell condensation process, cell contacts increase in all regions of the limb, but maximal formation of cell contacts occurs in the central parts, which will further develop into cartilage.

  3. After completion of the condensation process, chondrogenesis starts in the central parts, whereas the cells in the periphery revert to mesenchymal characteristics. The chondroblasts lose their contacts almost completely.

  4. The changes in contact behaviour are accompanied by a reduction of the cell perimeter. The central cells lose their processes resulting in almost round chondroblasts. Peripheral cells also reduce their processes but they become recognizable again once the condensation process has been completed.

  5. The condensation process and the beginning of chondrogenesis are accompanied by a reduction of the cell surface coat. While the chondroblasts lose their surface coat almost completely, it is retained by the peripheral cells.

The process of cartilage development starting from the undifferentiated mesenchyme is therefore describable in terms of increased cell contacts, reduced cell circumference and diminished surface coat. At the time of cell condensation, all the cells present in the limb anlage are involved in these alterations, but the greatest changes occur in presumptive chondrogenic cells. All the other cells (an exception are blood capillaries, which are not considered in this study) show a concomitant reaction with the same characteristics, but of a much lower degree.

Cell condensation is always demonstrable just before chondrogenesis (Fell, 1935; Searls & Janners, 1969; Summerbell, 1976; Cairns, 1977). Some cell activities, such as cell movements and cell adhesion, are involved in the condensation process (Ede & Agerbak, 1968; Ede et al. 1977; Duke & Elmer, 1979; Holmes & Trelstad, 1980). Occurrence of cell contacts in such cell condensations has been reported (Gould, Day & Wolpert, 1972; Searls et al. 1972; Thorogood & Hinchliffe, 1975; Borck, 1977). It is, however, not possible to differentiate between ‘active’ and ‘inactive’ gap junctions electron microscopically. Some of the junctions shown here may be open, others may be closed. Nothing is known about the relevance of cell contacts in chondrogenesis. On the other hand, in vitro studies have shown that a certain number of mesenchymal cells and cell contacts is necessary for chondrogenesis (Merker, Zimmerman & Grund-mann 1980). Isolated mesenchymal cells are able to undergo chondrogenesis only under special culture conditions, permitting high cell densities (Kelly, Barker, Crissman & Henderson 1973; Dienstman, Biehl, Holtzer & Holtzer 1974; Goel & Jurand, 1975; Solursh, Ahrens & Reifer, 1978). This may indicate the necessity for a certain extent of cell-cell communication. In limb buds of chick embryos stage 22–24, Kelly & Fallon (1978) using freeze-etch replicas, found 8 to 12 cell contacts per 100 cells. Although the importance of cell-cell contacts has been discussed by these authors, the number of contacts per cell seems to be too little for coupling. In limb buds of day-10 mouse embryos which are similar to stage 22–24, two to three contacts per cell were detectable (Table 1).

Another aspect extensively studied by Toole and co-workers should be mentioned. When, as shown here, the number of cell contacts increases in the limb bud during cell condensation, the intercellular space has to decrease. The resulting space around the cell condensation has to be filled by new cells. This is possible because the mitotic index decreases only in the condensed cell mass, where the cell density has reached its maximum, while cell proliferation proceeds in the distal and peripheral regions (Hornbruch & Wolpert, 1970; Janners & Searls, 1970). Toole and co-workers have shown an increase in the activity of hyaluronidase in the limb bud just before and during cell condensation (Toole & Gross, 1971 ; Toole, 1972; 1973), leading to a digestion of the hyaluronic acid-rich matrix. Our studies on hyaluronidase have shown a complete disappearance of the intercellular space followed by a very dense cell packing in the whole limb bud after treatment with hyaluronidase (Zimmermann, 1981). If, according to Toole’s predictions, the presumptive chondrogenic cells produce hyaluronidase to form a condensed cell mass, the activity of the enzyme may not be strictly limited in the central core, but it should also act to a lesser extent via diffusion in distal and peripheral regions. This may explain the occurrence of a ‘wave of cell-cell contacts’ in the whole limb bud.

Muscle blastemata are also present in the limb bud. They are formed at about day 12 in the proximal region of the limb, perhaps also during the ‘wave of cell-cell contacts’. Our measurements were very distinctly restricted to the central region of the humerus anlage and distally to just under the apical ectodermal ridge, mostly around the marginal sinus. We are sure that in these regions no muscle blastemata have been measured. Regarding the peripheral areas, muscle blastemata are expectable by day 12 the earliest in the region between the central cell condensation and the periphery. Measurements were done very carefully in the tissue just under the basement membrane. Furthermore, a control measurement was performed in the distal/peripheral region, where muscle blastemata are not yet present at day 12. Here, we found data similar to those of both the distal and the peripheral region. Hence we are certain that no significant amount of muscle blastema was measured.

This work was supported by grants of the Deutsche Forschungsgemejnschaft awarded to Sonderforschungsbereich 29 - ‘Embryonale Entwicklung und Differenzierung - Embryonal- Pharmakologie’ Our thanks are due to Mrs Heidi Somogyi and Mrs Heidi Krüger for their excellent assistance in doing the electron microscopic sections and to Prof H.-J. Merker for his helpful, advice in preparation of this manuscript. The translation assistance of Mrs Barbara Steyn is gratefully acknowledged.

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