Anat. Embryol. 153, 95-104 (1978)
Anatomy
and Embryology 9 by Springer-Verlag1978
The Development of Alkaline Phosphatase Activity in Limb Buds of Mouse Embryos in vitro and Its Relation to Chondrogenesis* Bernd Zimmermann Anatomisches Institut der Freien Universit~itBerlin (Project: Prof. Dr. H.-J. Merker),K6nigin-LuiseStr. 15, D-1000 Berlin 33
Summary. The development of alkaline phosphatase (aPh) activity and chondrogenesis were studied in the limb buds of mouse embryos (day 11 p.c.) that had been grown in an organ culture. During a 12-day culture period an increase in aPh activity to more than 40 mU/limb bud was measured from day 2 in vitro onward. Depending on the time of application, aPh formation can be inhibited by certain substances. Cytosine-arabinoside inhibits aPh activity when the substance is added on day 2, 3, or 4. Chondrogenesis, on the other hand, is affected on days I, 2, and 3 and to a lesser degree on day 4. Actinomycin D interferes with aPh activity after its addition on day 1, 2, 3, or 4. Chondrogenesis is only inhibited when the drug is applied on the 1st, 2nd, or to a lesser degree on the 3rd day. Cycloheximide inhibits aPh formation on all days of treatment, but to a lesser degree on days 5 and 6; chondrogenesis is most influenced on days 2, 3, and 4. On day 6 of the culture period, aPh activity can be demonstrated histochemically only in the region of humerus and proximal parts of radius and ulna. Alterations in the distal cartilage skeleton, therefore, do not influence the activity data. A prerequisite for an increase in aPh activity is cartilage growth in the proximal part of the limb buds and subsequent induction of a perichondral cell population to proliferation and differentiation. Key words: Limb buds - Alkaline phosphatase - Chondrogenesis - Organ culture.
Introduction In an in vitro system the undifferentiated limb buds of mouse embryos (day 11 p.c.) develop cartilage anlagen of humerus, radius, ulna, metacarpals, and phalanges (Aydelotte and Kochhar, 1972; Neubert et al., 1974; Merker, 1975). Since in these cultures electron-microscopically amorphous calcification foci could be demonstrated after a 6 to 12-day culture period (Merker, 1975), it seemed interesting to study the development of aPh activity. Alkaline phosphomonoesterase (E.C. 3.1.3.1) * This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich29--Embryonal-Pharmakologie.
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can be demonstrated in all physiologic calcification processes (Robinson, 1923; M a c F a r l a n e et al., 1934; Lorch, 1947 and 1949; Pritchard, 1952; Cabrini, 1961; Teller et al., 1971). Investigations on aPh in vitro h a v e so far only been performed on pre-formed cartilage (Teaford and White, 1964), not, however, starting from the undifferentiated blastema. In order to gain more insight into the dependency o f aPh synthesis in this system on D N A , R N A , and protein synthesis as well as on the developmental stage of the limb bud and o f the cartilage growth, we studied the increase in aPh activity under the influence o f inhibitors (cytosine-arabinoside, actinomycin D, and cycloheximide). The action of cytosine-arabinoside inhibits D N A synthesis, whereas R N A and protein synthesis are only slightly influenced b y this c o m p o u n d (Silagi, 1965; K i m and Eidinoff, 1965; C h u and Fischer, 1968; Inagaki et al., 1969; G r a h a m and Whitmore, 1970). A c t i n o m y c i n D mainly impedes R N A synthesis a n d thus protein synthesis. However, after higher doses D N A synthesis is also affected (Reich et al., 1961; Hurwitz et al., 1962; Goldberg et al., 1962; Perry, 1963; K i m et al., 1968). Cycloheximide in low doses inhibits protein synthesis. W h e n given at higher doses it also partially inhibits D N A synthesis. Thus, most enzymatic activities including those o f R N A and D N A synthesis, are diminished (Bennett et al., 1964; Siegel and Sisler, 1964; K i m et al., 1968; J a c k s o n and Studzinski, 1968).
Materials and Methods The upper limb buds of 11-day-old mouse embryos (40-45 somite pairs, strain NMRI, day 0 = day of conception) were cultured according to the method of Aydelotte and Kochhar (1972) in an organ culture (Trowell, 1959) on a membrane filter SW 11303 (Sartorius, pore width 1.2/an) at 37 ~ C in a water-saturated atmosphere with 5% CO 2. Medium: BGJ (Biggers et al., 1961) plus 25% fetal calf serum, 150 stg/ml ascorbic acid, and 50 I.U. of both streptomycin and penicillin (Gibco/Biocult). The limb buds were cultured for 12 days to determine the increase in aPh activity during the culture period. On every 3rd day the medium was changed. Eight limb buds were taken daily from the cultures and frozen at -28 C until further processing. The investigation of the effect the inhibitors exerted on aPh formation was performed after a culture period of 6 days. From culture day 1 to 6, the substances were added on only one of the days, then the cultures were rinsed in medium and further cultured in a new medium until day 6. The drugs (Serva, Heidelberg, Germany) were added to the culture at the following concentrations: Cytosine-arabinoside: Actinomycin D: Cycloheximide:
1.00 #g/mI medium 0.01/tg/ml medium 1.00/zg/ml medium
APh activity was determined in every limb bud following homogenization in a glycine buffer at pH 10.5. Na-nitrophenyl phosphate served as the substrate at a final concentration of 5.5 mM (BiochemicaTest-Combination; Boehringer-Mannheim, Germany). After 30 min incubation at 37 ~ C the reaction was stopped by adding 10 ml 0.02 N NaOH. Released p-nitrophenol was measured spectrophotometrically at 405 nm. Cartilage anlagen can be demonstrated after fixation in formaldehyde solution plus methylene blue and subsequent clearing. The "cleared" preparations were evaluated with the structure analyzer Quantimet (Metals Research, Imanco) with the help of which the dimension of certain planes can easily be determined. The histochemical demonstration of aPh was carried out on frozen sections using Na-flglycerophosphate as the substrate. Significancewas tested using the Student's t-test.
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Results
1. The Development of Alkaline Phosphatase Activity during the Culture Period The uncultured limb bud of 11-day-old mouse embryos has an average aPh activity of 0.3 mU/limb bud. After 24 hr in culture the activity per limb bud had not changed. After a 2-day culture period the activity had increased to 0.5 mU/limb bud, and after 3 days to 1.0 mU/limb bud. In the semilogarithmic presentation, the activity increase proceeds linearly to about 8 mU/limb bud until day 6 of the culture period. Afterward, a slight decrease can be detected. From day 10 to day 12, the activity only increases from 32 to 40 mU/limb bud (Fig. 1). The localization of aPh activity after histochemical preparation is demonstrated in Figure 2, which shows a 6-day-old limb bud culture. The enzyme activity can clearly be seen in the perichondral region of the humerus and in proximal parts of the radius and ulna where later desmal osteogenesis occurs.
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Fig. 1 DevelopmentofaPh activity in embryonicmouse limb buds (day 11 p.c.) in vitro. Ordinate: aPh activity (log mU/limb bud). Abscissa: days of the culture period, Data of four experiments with eight single values each: Two experiments day 0 to 8 (O . . . . O, 9 0) and two experimentsday 8 to 12 (s . . . . A, i A)
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Fig. 2A and B Histochemical demonstration of aPh in the limb bud blastema after a 6-day culture period. (A) Localization of the activity in the perichondrium of humerus (H), radius (R), and ulna (U). x 30. (B) Localization of the activity in the perichondrium with only a very low activity in the cartilage ceils, x360
2, Alkaline Phosphatase Activity and the Degree of Chondrogenesis under the Influence of Cytosine-A rabinoside, A etinomycin D, and Cycloheximide The drugs were added to the cultures for 24 hr between days 1 and 6 of the culture period. Figure 3 shows aPh activity and the degree of chondrogenesis. After a 6-day culture period, aPh activity reached control values when 1/ag/ml cytosine-arabinoside was added on day 1, 5, or 6. Only after treatment on day 2, 3, or 4 could a highly significant decrease in aPh activity (approx. 80%) be observed (Fig. 31). Chondrogenesis, however, was inhibited by approx. 80% after treatment with 1 pg cytosine-arabinoside on day 1 as well as on days 2 and 3, but treatment on day 4 caused an approx. 50% inhibition of chondrogenesis. N o effects could be observed after treatment on days 5 and 6. Only the distal parts of the cartilage skeleton were
Alkaline Phosphatase in Mouse Limb Bud Organ Culture
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Fig. 3A-C Alkaline phsophatase activity ~ 9 and degree of chondrogenesis [] . . . . . . [] in embryonic mouse limb buds, which had been cultured for 6 days, after application of 1/~g/ml cytosinearabinoside (A), 0.01 #g/ml actinomycin D (B), and 1/lg/ml cycloheximide (C). Ordinate: percent of control. Abscissa: day of application. Mean values _+SD influenced, the humerus and the proximal parts of radius and ulna being fully developed (Fig. 4B). After treatment with 0.01 #g/ml actinomycin D on day 1 of the culture period, no aPh activity could be measured 6 days later. When actinomycin D was added on days 2, 3, or 4, aPh activities of 30-50% of the control values could be demonstrated. Treatment on day 5 or 6 did not show any difference to the control (Fig. 3B). Chondrogenesis following actinomycin D application on day 1 was inhibited by approx. 90%, while after treatment on day 2 only an approx. 40% inhibition could be observed. Treatment on day 3, 4, or 6 had no significant influence on the degree of chondrogenesis (Fig. 3B). "Cleared" preparations showed an inhibition of chondrogenesis in the region of humerus and proximal parts of radius and ulna after actinomycin D application on days 1 and 2 (Fig. 4C). One/Lg-ml cycloheximide generally diminished aPh activity. After application between days 1 and 5, an approx. 85% inhibition was measured. When the substance was added on_day 6 the inhibition decreased to 60% (Fig. 3C). Chondrogenesis was similarly inhibited, though not as drastically. Here, too, the maximum inhibition of ~_70% was attained after treatment on day 2, 3, or 4. Treatment on day 1 inhibited
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Fig. 4A-D "Cleared" preparations (methyleneblue). A = control. B = cytosine-arabinoside(1/zg/rnl, day l). C = actinomycinD (0.01 ag/ml, day 1). D = cycloheximide(1 ag/mt, day 1)
chondrogenesis by only ~40%. Addition of cycloheximide on day 5 or 6 had, however, no influence on chondrogenesis (Fig. 3C). Examination of the "cleared" preparations showed that cartilage growth decreased, depending on the time of drug application, in both the proximal (days 1 and 2) and distal skeletal parts (days 3 and 4) (Fig. 4D).
Discussion In this paper we have attempted to show a relationship between chondrogenesis and alkaline phosphatase activity. However, several methodologic points must be borne in mind. The enzyme or enzyme mixture called alkaline phosphatase hydrolyses pnitrophenol phosphate under the conditions described in 'Materials and Methods' to
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p-nitrophenol and PPi. It cannot be excluded that besides the enzyme alkaline phosphatase (E.C. 3.1.3.1), unspecific phosphatases, e.g., ATPases or pyrophosphatases, are also determined (M~kinen and Knuuttila, 1972). Day 6 of the in vitro treatment was chosen as the time of evaluation, since at this stage chondrogenesis is still easy to follow. With this system the peak of chondrogenesis is reached on day 8, but at that time it is no longer easy to differentiate between the various cartilage anlagen. In our in vitro system, aPh activity in the limb buds starts to increase on day 2 of the culture period. On day 1 in vitro necroses can be demonstrated in the blastema (Merker, i975), which must be interpreted as adaptation phenomena. But as early as during day 1 of the culture period, many cells are performing D N A synthesis, as autoradiographic investigations have shown (Herken, 1975). Moreover, from day 1 of the culture period onward, collagen synthesis can be detected (Neubert et al., 1974; Barrach et al., 1975). Hence, DNA and protein synthesis can be demonstrated as early as day 1 of the culture period, so that the absence of measurable aPh activity on day 1 is not to be attributed to an inhibition caused by adaptation processes, but to the lack of cartilage tissue and competent cells. Inhibition of D N A synthesis in the limb bud culture caused by cytosine-arabinoside shows that a decrease in aPh activity only occurs when this substance is added on day 1 of the culture period (Herken, 1975); DNA synthesis inhibition on day 1 causes a decrease in the cartilage size but no drop in aPh activity. In interpreting this discrepancy, the morphologic findings must also be considered. Simultaneous automatic measurement of all cartilage pieces of the limb bud does not allow any conclusions to be drawn on a localization-dependent effect of the treatment. Here the proximodistal maturation gradient that is also clearly pronounced in vitro has to be considered. Cytosine-arabinoside application on day 1 in vitro does not suppress the formation of proximal cartilage (scapula, humerus, and proximal parts of the radius and ulna). At the beginning of the culture period the cells of these cartilage anlagen have already gone through the necessary mitoses and are about to start differentiation. The formation of the proximal cartilage parts can, therefore, be influenced only via an inhibition of the RNA and protein synthesis with compounds such as actinomycin D or cycloheximide (Zimmermann et al., 1975; Neubert et al., 1974). Cytosine-arabinoside treatment on day 1, however, inhibits only the development of the distal parts of radius and ulna, carpus, metacarpus, and phalanges, but after a 6 day culture period these distal cartilage parts do not reach the developmental stage that is characterized by formation of perichondral aPh. Thus considerably less cartilage is formed when cytosine-arabinoside is present, but the aPh activity remains unaltered. The decrease in enzyme activity after cytosine-arabinoside application on day 2, 3, or 4 can be explained by the mitosisinhibiting effect this substance exerts on the cell population that synthesizes aPh, i.e., the perichondral cells. On these days the "critical mitoses" proceed (Holtzer and Abbott, 1968; Bischoff and Holtzer, 1969), but on days 5 and 6 these mitoses have occurred and thus treatment is ineffectual. RNA synthesis inhibition after actinomycin D application on day 5 or 6 of the culture period has no measurable influence on aPh activity. This may be attributed to large amounts of enzyme present at this time or due to a long half-life period of the aPh-specific m-RNA. If, however, RNA synthesis is inhibited on day 2, 3, or 4, i.e., on the days of the highest synthesis rate of the enzyme, aPh activity is only 30-50% of the control values. Moreover, inhibition of RNA synthesis and, therefore, protein synthesis are also likely to influence the mitoses of the enzyme-producing cell
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population. It must also be considered, however, that the dose of actinomycin D used does not cause a 100% inhibition of RNA synthesis, as actinomycin D is stoichiometrically bound to DNA (Cerami et al., 1967; Ebstein, 1967). Inhibition of aPh activity after actinomycin D application on day 1 indicates the importance of proximal cartilage anlagen for the induction of enzyme activity. "Cleared" preparations have shown that the proximal parts of the cartilage skeleton are not formed (Zimmermann et al., 1975), but the enzyme is produced in the perichondrium of these cartilage anlagen. While chondrogenesis is no longer significantly influenced by actinomycin D on day 3 of the culture period, a decrease in the influence on aPh activity is seen only from day 5 onward. This difference of two days shows that the differentiation of the blastema to cartilage starts before the differentiation to aPh activity. Other investigators, too, suggest that aPh formation depends on preceding cartilage differentiation (Pritchard, 1952; Cabrini, 1961). It is, therefore, possible that cartilage or certain alterations of the cartilage (development of hypertrophic cartilage ceils and mineralization of cartilage matrix) have an inducing effect on aPh formation or differentiation of the responsive cells. Cycloheximide inhibits the synthesis of the enzymes on all days of the culture period. The slight increase in aPh activity after treatment on day 5 or 6 might represent the degree of aPh activity at the beginning of treatment, i.e., after 4 and 5 days in culture. This interpretation would, however, presuppose a long half-life period of the enzyme, in the order of 48 hr or more. From histochemical investigations it is known that aPh is a very stable enzyme, retaining enzymatic activity through fixation and even embedding in paraffin. A certain stability of this enzyme in vitro is, therefore, quite conceivable. The fact that after application of cycloheximide on day 1 a low, yet significant, aPh activity could be measured shows that it had not been possible to inhibit induction completely. According to the data obtained after treatment on day 2, 3, or 4, however, a cycloheximide concentration of 1/~g/ml is sufficient for a total protein synthesis inhibition. However, after cycloheximide application on day 1, approx. 70% of the cartilage is still formed. It would appear, therefore, that application of cycloheximide on day 1 inhibits the formation of the cartilage matrix in the proximal region, while the competent eeUs for cartilage formation are still present. Chondrogenesis, therefore, occurs after the inhibiting effect has ceased, i.e., it is delayed. Thus on day 6, proximal cartilage anlagen can again be demonstrated, but the full degree of aPh formation has not yet been reached. These results indicate that the formation and induction of aPh-producing cells only occurs during the culture period after formation of the cartilage. Before these aPh-producing cells can develop, the necessary cell cycles (critical mitoses) must occur. This induction is characterized by cycloheximide-inhibitable RNA and protein syntheses. Here the question arises whether, apart from a delayed cartilage formation, we are not dealing with cell populations of different sensitivities. Both processes, chondrogenesis in the proximal parts of the limb bud and enzyme formation, are therefore somewhat deferred and can thus show different behavior and kinetics toward inhibitors. References Aydelotte, M.B., Kochhar, D.M.: Development of mouse limb buds in organ cultures. Chondrogenesis in the presence of a proline analogue, L-azetidine-2-carboxylic acid. Dev. Biol. 28, 191-201 (1972)
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Barrach, H.-J., Rautenberg, M., Tapken, S., Neubert, D.: Some biochemical characteristics of mouse timb buds differentiating in organ culture. In: 'New approaches to the evaluation of abnormal embryonic development' (D. Neubert, and H.-J. Merker, eds.) pp. 114-132. Stuttgart: Georg Thieme 1975 Bennett, L.L., Smithers, D., Ward, C.T.: Inhibition of DNA synthesis in mammalian ceils by actidione. Biochim. Biophys. Acta 87, 60--69 (1964) Biggers, J.D., Gwatkin, R.B.L., Heyner, S.: Growth of embryonic avian and mammalian tibiae on a relatively simple chemically defined medium. Exp. C ell Res. 25, 41-58 (1961) Bischoff, R., Holtzer, H.: Mitosis and the process of differentiation of myogenic cells in vitro. J. Cell Biol. 41, 188-200 (1969) Cabrini, R.L.: Histochemistry of ossification. Int. Rev. C ytol. 11, 283-306 (1961) Cerami, A., Reich, E., Ward, D.C., Goldberg, I.H.: The interaction of actinomycin with DNA: Requirement for the 2-amino group of purines. Proc. Natl. Acad. Sci. USA 57, 1036-1042 (1967) Chu, M,Y., Fischer, G.A.: Effects of cytosine arabinoside on the cell viability and uptake of deoxypyrimidine nucleosides in L 5178 Y ceils. Biochem. Pharmacol. 17, 741-751 (1968) Ebstein, B.S.: Tritiated actinomycin D as a cytochemical label for small amounts of DNA. J. Cell Biol. 35, 709-713 (1967) Goldberg, I.H., Rabinowitz, M., Reich, E.: Basis of actinomycin action. I. DNA Binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc. Natl. Acad. ScL USA 48, 20942101 (1962) Graham, F.L., Whitmore, G.F.: The effect of 1-fl-D-arabinofuranosylcytosine on growth, viability and DNA-synthesis of mouse L-cells. Cancer Res. 30, 2627-2635 (1970) Herken, R.: Autoradiographic investigations with 3H-thymidine in limb bud culture. In: New approaches to the evaluation of abnormal embryonic development (D. Neubert and H.-J. Merker, eds.) pp. 200-212. Georg Thieme, Stuttgart, 1975 Holtzer, H., Abbott, J.: Oscillation of the chondrogenetic phenotype in vitro. In: Stability of the differentiated state (Springer Berlin-Heidelberg-New York: H. Ursprung, ed.) 1968 Hurwitz, J., Furth, J.J., Malamy, M., Alexander, M.: The role of deoxyribonucleic acid in ribonucleic acid synthesis. III. The inhibition of the enzymatic synthesis of ribonucleic acid and deoxyribonucleic acid by actinomycin D and profiavine. Proc. Natl. Acad. Sci. USA 48, 1222-1230 (1962) Inagaki, A., Nakamura, T., Wakisaka, G.: Studies on the mechanism of action of 1-fl-Darabinofuranosylcytosine as an inhibitor of DNA synthesis in human leukemic ieukocytes. Cancer Res. 29, 2169-2176 (1969) Jackson, L.G., Studzinski, G.T.: Autoradiographic studies of the effects of inhibitors of protein synthesis on RNA synthesis in HeLa cells. Exp. Cell Res. 52, 408-418 (1968) Kim, J.H., Eidinoff, M.L.: Action of 1-fl-D-arabinofuranosylcytosine on the nucleic acid metabolism and viability of HeLa cells. Cancer Res. 25, 698-702 (1965) Kim, J.H., Gelbard, A.S., Perez, A.G.: Inhibition of DNA synthesis by actinomycin D and cycloheximide in synchronized HeLa cells. Exp. Cell. Res. 53, 478-487 (1968) Lorch, I.J.: Localization of alkaline phosphatase in mammalian bones. Q. J. Microsc. Sci. 88, 367-381 (1947) Lorch, LJ.: The distribution of alkaline phosphatase in relation to calcification in Scyliorinus Canicula. Q. J. Microsc. Sci. 90, 381-390 (I949) MacFarlane, M.G., Brown-Patterson, L.M., Robinson, R.: The phosphatase activity of animal tissue. Biochem. J. 28, 720-724 (1934) M/ikinent K.K., Knuuttila, M.L.: Concerning the similarities between three alkaline phosphatases of human foetal parietal bones. C alcif. Tissue Res. 9, 28-38 (i 972 Merker, H.-J.: Significance of the limb bud culture system for investigations of teratogenic mechanisms. In: New approaches to the evaluation of abnormal embryonic development (D. Neubert and H.-J. Merker, eds.) pp. 161-199 Stuttgart: Georg Thieme, 1975 Neubert, D., Merker, H.-J., Tapken, S.: Comparative studies on the prenatal development of mouse extremities in vivo and in organ culture. Naunyn Schmiedebergs Arch. Pharmakol. 286, 251-270 (1974) Neubert, D., Tapken, S., Merker, H.-J.: Inductions of skeletal malformations in organ cultures of mammalian embryonic tissues. Naunyn Schmiedebergs Arch. Pharmakol. 286, 271-282 (1974) Perry, R.P.: Selective effects of actinomycin D on the intracellular distribution of R N A synthesis in tissue cuPJre cells. Exp. Cell Res. 29, 400-406 (1963) Pritchard, J.J.: A cytological and histochemical study of bone and cartilage formation in the rat. J. Anat. 86, 259-277 (1952)
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Reich, E., Franklin, R., Shatkin, A., Tatum, E.: Effects of actinomycin D on cellular nucleic acid synthesis and virus production. Science 134, 556-557 (1961) Robinson, R.: The possible significance of hexosephosphoric esters in ossification. Biochem. J. 17, 286293 (1923) Siegel, M.R., Sisler, H.D.: Site of action of cycloheximide in cells of Saccharomyces pastorianus. II. The nature of inhibition of protein synthesis in a cell free system. Biochim. Biophys. Acta 87, 83-89 (1964) Silagi, S.: Metabolism of 1-/~-D-arabinofuranosylcytosine in L ceils. Cancer Res. 25, 1446-1453 (1965) Teaford, M.E., White, A.A.: Alkaline phosphatase and osteogenesis in vitro. Proc. Soc. Exp. Biol. Med. 117, 541-546 (1964) Teller, C., Merker, H.-J., G(inter, Th.: Uber das Verhalten und die Lokalisation der alkalischen Phosphatase wiihrend der Skelettentwicklung bei Rattenf6ten. Histochemie 27, 193-204 (197t) Trowetl, O.A.: The culture of mature organs in a synthetic medium. Exp. Cell Res. 16, 118-147 (1959) Zimmermann, B., Neubert, D., Bachmann, D., Merker, H.-J.: Induction of skeletal malformations in "organ cultures of mouse limb buds. Experentia 31, 227-228 (1975) Received November 30, 1977