cell contac ant d surface coat alterations of limb-bud ... · cell contac ant d surface coat...

/ . Embryol. exp. Morph. Vol. 72, pp. 1-18, 1982 \

Printed in Great Britain © Company of Biologists Limited 1982

Cell contact and surface coat alterationsof limb-bud mesenchymal cells during

differentiation

By BERND ZIMMERMANN1, EVAMARIA SCHARLACHAND ROMAN KAATZ2

From the Institut fur Toxikologie und Embryonal-Pharmakologie,Freie Universitdt, Berlin

SUMMARY

Measurements of the cell perimeter, cell-cell contact number and length and of the thick-ness of the surface coat were performed in limb buds of mouse embryos during the process ofcell 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 isshown that until day 12 all cells in the limb bud are interconnected by cell contacts of thegap-junction type. Contact number and contact length increase during development, butincrease about twice as much between central, prechondric cells as between peripheral ordistal cells. Later in development, chondroblasts lose their contacts almost completely,whereas between peripheral cells, the amount of cell contacts drops to the initial value ofmesenchymal cells. The cell size decreases during chondrogenesis. A decrease in thethickness 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 thechondrogenic differentiation process.

INTRODUCTION

The development of the limb skeleton starts with the formation of conden-sations of mesenchymal cells in the prospective chondrogenic areas (Fell,1935; Searls & Janners, 1969; Summerbell, 1976; Cairns, 1977). This processof blastema formation is not yet fully understood. A local increase in cellproliferation can be excluded (Hornbruch & Wolpert, 1970; Janners & Searls,1970; Cairns, 1977), but there are some indications concerning the participationof 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 condensationwere shown by Trelstad (1977) and Ede & Wilby (1981). The importance of cell

1 Author's address: Institut fur Toxikologie und Embryonal-Pharmakologie, Freie Univer-sitat, Berlin, Garystr. 9, D-1000 Berlin 33, FRG.

2 Author's address: Anatomisches Insititut, Freie Universitat Berlin, Garystr. 9, D-1000Berlin 33, FRG.

2 B. ZIMMERMANN, E. SCHARLACH AND R. KAATZadhesion and slight cell movements was indicated by comparison of chon-drogenesis in normal and talpid3 chick embryos (Ede & Agerback, 1968; Ede,Flint & Teague, 1975; Ede et al. 1977). A decrease in intercellular spaces byaction of cellular hyaluronidase may be also involved in the condensationprocess (Toole, 1972, 1973; Zimmermann, 1981).

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

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

Although the importance of the formation of cell contacts during blastemaformation in the limb (Searls et al. 1972; Kelly & Fallon, 1978) as well as inother organs (Trelstad, Hay & Revel, 1967; De Haan & Sachs, 1972; Bungeet al. 1979; Gilula, 1980; Loewenstein, 1980) has been pointed out, no quantita-tive estimations are available. Regarding cell adhesion, quantitative measure-ments of the cell surface coat in the condensing mesenchymal cells are alsolacking.

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

MATERIALS AND METHODS

Upper limb buds of mouse embryos of days 10, 11, 12 and 13 (mouse strainNMRI, 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 fourdifferent 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 hat room temperature. After rinsing, post-fixation was done in 1 % OsO4 in0-1 M-phosphate buffer, pH 7-2, for about 1 h at +4 °C. After rinsing thoroughly,the explants were dehydrated in ethanol and embedded in Epon or Mikropal(Ferak, Berlin).

Cell contact and surface coat alterations

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

Electron microscopy

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

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

Measurements of the cell perimeter and the contact length

Cell perimeter, contact length and the number of contacts of each measuredcell were performed with an Interactive Image Analysing System IBAS 1(Kontron, Echingen, Miinchen). In this system, lines of the measured structureare covered by a contact pen manually. The position data of x and y are com-puted and processed in an integrated microprocessor. Histograms, single data,mean and standard deviations were printed out. One cell contact length wasdefined 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 magnifica-tion of 120000 by using a measuring magnifier. In each litter series, 40 positions

4 B. ZIMMERMANN, E. SCHARLACH AND R. KAATZwere measured in every region. Only exact cross sections through the cellmembrane with well-recognizable bilayer structure were used.

Statistical analysis

For statistical analysis, a PDP-11/34A computer (digital equipment, Minne-sota) was used with a RSX-11M operating system. Mean and standard deviationsof the surface coat thickness were calculated for each litter series separately toshow the variance in the different series. The /-test was used for testing thesignificance of differences between the data of different stages and regions.

Mean and standard deviations of cell perimeter were printed by the IB AS.The Mest was performed with a PDP-11/34A computer. Since the data of thecontact length are not normally distributed, the t test could not be used forstatistical analysis of the data. Therefore, the single data printed by the IBASwere fed into the PDP-11/34A. The Mann-Whitney-Wilcoxon test for thedifference between two populations was performed using the statistical pro-gram 'minitab', release 81-1 (Copyright Penn. State University 1981,University of Toledo).

RESULTS

Between the outer epithelium and the somatopleura in the presumptivelimb-forming region of day-9 mouse embryos, the mesenchymal cells arealmost completely separated by wide intercellular spaces. The cells exhibit anirregular 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 contactscan be considered as gap junctions (Fig. 2b). The mean perimeter of a cellamounts to 30 /im, a single contact exhibits a mean length of about 1.0 /*m,and about 2-3 contacts per cell are present (Table 1). The histogram of thecontact length shows a relatively high amount of very short cell contacts(Fig. 7).

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

At day 10, upper limb buds of about 0.5 mm length have formed, whereasthe lower limb buds become just recognizable. The mesenchyme in the upper

Fig. 2(a). Loose mesenchyme between the somatopleura and the epithelium of thebody wall in the prospective limb-forming region (between somites 8 and 11)of day-9 mouse embryos. Irregularly shaped cells show many cell processes andsome cell-cell contacts (-*•). x6000; bar = 1 /im. (b) High magnification of acell-cell contact of the loose mesenchymal cells reveals the typical morphology of agap junction, x 120000; bar = 100 nm. (c) The surface coat of the mesenchymalcells in the prospective limb-forming region of day-9 mouse embryos exhibits athickness of about 8 nm (-*•). Some filamentous structures extend to up to 80 nminto the extracellular space ( • ) . x 120000; bar = 100 nm,

Cell contact and surface coat alterations 5

2(b) >*f?w&SF**

6 B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ

Table 1. Measurements of cell perimeter and cell contact number and length oncells in the limb anlage of different stages

Stage/region1

910 d10 c10 pl i dl i eU p

12 d12c12 p12 dp8

13c13p

Cell perimeter

X2

30283028

302925

23262423

2128

S.D.3

9-87-88-36-6

7-57-66-0

4-35-86-04-3

4-07-1

N*

40452625

513350

4839

10343

3834

Length of cell contacts

( X

1-091-091-430-80

2-153-101-60

1-402-611-701-40

0-580-92

>S.D.

101-01-00-55

1-472-051-15

1-152-601-501 0 5

0-450-78

N

931105379

266195256

158182327133

3275

M 5

0-720-721-040-58

1-782-511-34

0-921-921-101-22

0-430-65

Contacts per cell

No.

2-322-442-043-15

5-225-895-15

3-304-683-183 1 0

0-852-20

Length6

2-562-662-922-52

11-2018-308-15

4-6112-205-404-34

0-492-02

%7

8-59-59-89 0

35-463032-6

20047-022-51 9 0

2-37-2

1 Stage: day of embryonic development; region: d = distal, c = central, p = peripheral.2 x = mean (jim)3 S.D. = standard deviations.4 Number of cells or contacts resp. measured.5 M = median (/«n); since the values of contact length do not show a normal distribution

(see histograms Fig. 7), the median is necessary for statistical analyses (see 'statistics' inMaterials and Methods).

6 Total contact length per cell is a calculated value of the number of cell contacts multi-plied by the mean contact length.

7 % of contacts describes the percentage of cell membranes of one cell involved in cellcontacts.

8 dp = distal-peripheral, measurements in a region between distal and peripheral, servingas a control of reproducibility, reveals the correspondence to the values of 12 d and 12 p.

limb anlage is very uniform. The cells are interconnected by small cell contacts,irregularly shaped and separated by large intercellular spaces, such as themesenchymal cells at day 9. Measurements of the cell perimeter in differentareas of the limb bud (distal, central, peripheral) show no differences. Theportion of the cell membrane involved in cell contacts is about 9 % and quitesimilar in all regions (Table 1).

The lengths of the cell contacts show significant differences. In the centralpart of the limb, the cell contacts exhibit a length of about 1-4 fim (mean =1-4/an, median = 1-0 fim), which is significantly longer than in the distal andperipheral areas (Table 1). On the other hand, the number of cell contacts isonly about 2 per cell in the central parts, 2-4 in the distal and 3-2 in the peri-pheral parts. Therefore, the percentage of cell contacts is not different in themeasured areas. A tendency of increasing contact length is also demonstratedin the histograms (Fig. 7).


Table 2. Thickness of the cell surface coat on cells in the limb buds of mouseembryos of different stages

Stage/region

Day 9

Day 10

Day 11

Day 12

Day 13

Distal

—

5-6±l-06-2±M5-4 + 0-8

6-3 ±1-35-6 + 0-97-0±l-8

3-4 ±0-74-9±l-04-2 ±0-8

—

Central

9-8 + 1-97-1 ±1-68-3±l-8

6-4+1-46-2±H

6-5 ±1-65-4 ±0-96-1 ±1-1

4-0 ±0-93-3 ±0-73-7 + 0-74-1 ±1-0

1-8 + 0-42-1 ±0-61-5 + 0-5

Peripheral

—

5-2±l-06-7 ±1-4

6-0 ±1-05-5 ±0-86-2 ±0-9

4-9+1-15-5+1-13-9 ±0-8

5-1 + 1-43-7±l-05-4+1-2

Data are given in nm.Measurements were done on micrographs of a magnification of 120000.Mean and standard deviations of different series are shown (N of each series = 40).Measurements were done only at exact cross sections of the cell membrane.

In all measured regions, the surface coat of the cells is quite similar and thethickness comes to 6 nm (Fig. 3 a, Table 2). Some structures are present at thecell surface, which extend by more than 50 nm into the extracellular space.Sometimes cell contacts which are about to form are detectable. These structureshave 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 ad-jacent cells come together to form a gap junction (Fig. 3b).

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

Cells in the peripheral and the distal areas show a similar behaviour. The cellsare close together, but considerable intercellular spaces are present (Fig. 4a).The number of cell processes seems to be diminished; this is indicated in theperipheral cells by the decrease of the cell perimeter (P < 001, significantagainst the mesenchymal cells of day 9, day 10 and central or distal regions ofday 11) (Table 1).

8 B. ZIMMERMANN, E. SCHARLACH AND R. KAATZIn 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 isvisible (Fig. 4 b). All the contacts between the cells are typical gap junctionswith an electron-dense space of about 2 nm between the two outer lamellaeof the adjacent cells (Fig. 4c). In the contacts, small distensions cut the junctioninto regular sections of 100-150 nm. This dimension is probably the 'reallength' of a gap junction, but in the measurements done here, one cell contactis defined from one intercellular space to the next.

No clear-cut changes in the surface coat are recognizable. The cells in allestimated 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 weredetected between the distal and the peripheral regions (Table 1). Again theorganization of the tissue rather resembles that of the mesenchyme. On theother hand, about 47 % of the membranes of the cells in the central part (hu-merus) are involved in contacts. The cells are still close together, but a dilatationof the intercellular space is visible (Fig. 5 a). In the intercellular space as wellas in the cells, sdme 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.

The thickness of the surface coat of the cells in all three areas is now dimini-shed (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, chon-drogenesis has started. The cells show the typical morphology of young chon-droblasts (Fig. 6 a). The perimeter of a cell has further decreased significantly(P > 0-01) to a mean of 21 /im. Only very few cell contacts are detectable:0-85 contacts per cell are counted. The intercellular space has widened and isfilled with the typical chondrogenic matrix. The surface coat of these cells isonly very thin; the thickness has further decreased to about 2nm (Fig. 6b,Table 2).

The cells in the distal region are difficult to define. Distally to the measuredcentral area the anlagen of forearm, wrist and hand are localized. They are nowadvanced in development, so is the central part at day 12. Cells lying directlyunder the distal epithelium resemble peripheral cells. Since the apical ecto-dermal ridge is now absent, no clear-cut position finding was possible, so thatmeasurements in this area were renounced.

Perimeter, contact length and contact number as well as the morphology ofthe 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 /tm, a value similarto that of the cells at day 10 (Table 2).

The contact length of the cells in different areas and at different stages aredemonstrated in the histograms of Fig. 7. It is shown that the distribution of thecontact length differs characteristically. Before cell condensation occurs (day9 and 10), most of the cell contacts are very short, but during the condensation


Fig. 3(o). A surface coat of about 6nm thickness (-*•) is detectable on the cellmembrane of day-10 mesenchymal cells. Some structures extend to up to 50 nm( • ) into the extracellular space, x 120000; bar = 100nm. (b) A developing gapjunction 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 ( ^ ) arevisible, x 120000; bar = 100 nm.

Cell contact and surface coat alterations 11process and at the onset of chondrogenesis (day 11 and 12), longer contacts aredetectable. These alterations in the distribution pattern are more pronouncedin the central and distal regions than in the periphery. During chondrogenesison day 13, the short contacts are present again in the central and in the peri-pheral areas.

The percentage of cell membranes involved in cell-cell contacts are plottedagainst the stage in Fig. 8. It is shown that in the peripheral and distal regionscell contact behaviour is identical. It starts from about 9 % at day 10, reachinga 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, how-ever, they reach a maximum at day 11 with more than 60% and decline toless than 1 % at day 13, when the cells are differentiated. That 'wave of cell-cellcontact' therefore is highest in the cells of the central region, which results inchondrogenic differentiation, and is lower in the cells of the distal and peri-pheral parts of the limb, where the cells do not undergo chondrogenic develop-ment.

DISCUSSION

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

(1) From the earliest stage of development until day 12 all the cells in thelimb are more or less interconnected by cell-cell contacts of the gap-junctiontype.

(2) During the cell condensation process, cell contacts increase in all regionsof 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 inthe central parts, whereas the cells in the periphery revert to mesenchymalcharacteristics. The chondroblasts lose their contacts almost completely.

Fig. 4(a). Mesenchymal cells in the distal region of day-11 mouse limb buds. Similarmorphology can be shown in peripheral parts. Cell density has increased and cellsare interconnected by gap junctions (-*•). x6000; bar = 1 ftm. (b). Cells in thecentral region of day-11 mouse limb buds exhibit a maximal condensation density.The intercellular space (X) has diminished. Cells are interconnected by long cellcontacts (-*•). x 6000; bar = 1/tm. (c). High magnification of the cell contactsreveals the typical morphology of gap junctions, x 120000; bar = 100 nm. (d).The surface coat of the cells in all measured regions exhibits a thickness of about6 nm (-•). Only delicate structures extend into the extracellular space ( • ) . x 120000bar = 100 nm.


Fig. 6 (a) Chondroblasts in the central part of day-13 mouse limb buds are separatedby wide intercellular spaces filled with collagen filaments. Only very few cell contactsare present (-*•). x 6000; bar = 1 /im. (b) The surface coat of such chondroblastsis only very thin. Extracellular proteoglycan granules are present (-•) near the cellmembrane, x 120000; bar = 100 nm.

Fig. 5 (a). In the central region of day-12 mouse limb buds, the intercellular spaceshows 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 intercellularspace ( • ) . x6000; bar = 1 /tm. (b) The morphology of a myelin-like structureshown at high magnification resembles that of coiled gap junctions, x 120000;bar =100 nm. (c) The thickness of the surface coat of all cells in the centralregion of day-12 limb buds is reduced to about 4nm. x 120000; bar = 100 nm.


40-

20

40

20-

8 40-

20

40-

20

40'

20

Day 9

Day 10

Day 11

Day 12

Day 13

Distal Central Peripheral

Fig. 7. Histogram of contact length in different regions and at different stagesof mouse limb buds. Each section on the abscissa represents a contact length of0-5 /tm. Note the relative preponderance of short contacts between the cells inlimb buds of days 9, 10 and day 13. During the cell condensation process at days11 and 12 the number of longer cell contacts increases, but more in the central anddistal parts than in the periphery.

(4) The changes in contact behaviour are accompanied by a reduction of thecell perimeter. The central cells lose their processes resulting in almost roundchondroblasts. Peripheral cells also reduce their processes but they becomerecognizable again once the condensation process has been completed.

(5) The condensation process and the beginning of chondrogenesis areaccompanied by a reduction of the cell surface coat. While the chondroblastslose their surface coat almost completely, it is retained by the peripheralcells.


70 T

15

10-

13

Day of development

Fig. 8. Percentage of cell membranes involved in cell contacts of mouse limb budsof different stages. A = Central region (humerus); • = distal region; • =peripheral region. The increase in the percentage of contact-participating cellmembranes is highest during the process of cell condensation between days 11 and 12of development. The increase is much higher in the central parts where chondrogene-sis 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) partscomes to less than 1 %, whereas in the peripheral parts, the initial values of days9 and 10 are measurable.

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

Cell condensation is always demonstrable just before chondrogenesis (Fell,1935; Searls & Janners, 1969; Summerbell, 1976; Cairns, 1977). Some cellactivities, such as cell movements and cell adhesion, are involved in the con-densation process (Ede & Agerbak, 1968; Ede et al 1977; Duke & Elmer, 1979;Holmes & Trelstad, 1980). Occurrence of cell contacts in such cell condensationshas been reported (Gould, Day & Wolpert, 1972; Searls et al 1972; Thorogood

16 B. ZIMMERMANN, E. SC&ARLACH AND R. KAATZ

& Hinchliffe, 1975; Borck, 1977). It is, however, not possible to differentiatebetween 'active' and 'inactive' gap junctions electron microscopically. Some ofthe junctions shown here may be open, others may be closed. Nothing is knownabout the relevance of cell contacts in chondrogenesis. On the other hand,in vitro studies have shown that a certain number of mesenchymal cells andcell contacts is necessary for chondrogenesis (Merker, Zimmerman & Grund-mann 1980). Isolated mesenchymal cells are able to undergo chondrogenesisonly under special culture conditions, permitting high cell densities (Kelly,Barker, Crissman & Henderson 1973; Dienstman, Biehl, Holtzer & Holtzer1974; Goel & Jurand, 1975; Solursh, Ahrens & Reifer, 1978). This may indicatethe necessity for a certain extent of cell-cell communication. In limb buds ofchick 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-cellcontacts has been discussed by these authors, the number of contacts per cellseems to be too little for coupling. In limb buds of day-10 mouse embryoswhich 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 bementioned. When, as shown here, the number of cell contacts increases in thelimb bud during cell condensation, the intercellular space has to decrease. Theresulting space around the cell condensation has to be filled by new cells. Thisis possible because the mitotic index decreases only in the condensed cell mass,where the cell density has reached its maximum, while cell proliferation pro-ceeds in the distal and peripheral regions (Hornbruch & Wolpert, 1970; Janners& Searls, 1970). Toole and co-workers have shown an increase in the activityof hyaluronidase in the limb bud just before and during cell condensation(Toole & Gross, 1971; Toole, 1972; 1973), leading to a digestion of the hyalu-ronic acid-rich matrix. Our studies on hyaluronidase have shown a completedisappearance of the intercellular space followed by a very dense cell packingin the whole limb bud after treatment with hyaluronidase (Zimmermann, 1981).If, according to Toole's predictions, the presumptive chondrogenic cellsproduce hyaluronidase to form a condensed cell mass, the activity of theenzyme may not be strictly limited in the central core, but it should also act to alesser extent via diffusion in distal and peripheral regions. This may explainthe 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 atabout day 12 in the proximal region of the limb, perhaps also during the 'waveof cell-cell contacts'. Our measurements were very distinctly restricted to thecentral region of the humerus anlage and distally to just under the apical ecto-dermal ridge, mostly around the marginal sinus. We are sure that in theseregions no muscle blastemata have been measured. Regarding the peripheralareas, muscle blastemata are expectable by day 12 the earliest in the regionbetween the central cell condensation and the periphery. Measurements were


done very carefully in the tissue just under the basement membrane. Further-more, a control measurement was performed in the distal/peripheral region,where muscle blastemata are not yet present at day 12. Here, we found datasimilar to those of both the distal and the peripheral region. Hence we arecertain that no significant amount of muscle blastema was measured.

This work was supported by grants of the Deutsche Forschungsgemejnschaft awarded toSonderforschungsbereich 29 - 'Embryonale Entwicklung und Differenzierung - Embryonal-Pharmakologie'

Our thanks are due to Mrs Heidi Somogyi and Mrs Heidi Kriiger for their excellent assist-ance 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 isgratefully acknowledged.

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BUNGE, R., GLASER, L., LIEBERMAN, M., RABEN, D., SALZER, J., WHITTENBERGER, B. &WOOLSEY, T. (1979). Growth control by cell to cell contact. / . Supramol. Struct. 11,175-187.

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EDE, D. A. & FLINT, O. P. (1975). Cell movement and adhesion in the developing chick wingbud: studies on cultured mesenchyme cells from normal and talpid3 mutant embryos.J.Cell Sci. 18, 301-313.

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(Received 24 March 1982, revised 4 June 1982)

cell contac ant d surface coat alterations of limb-bud ... · cell contac ant d surface coat...

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