1. The effects of actinomycin D upon the induction of tubulogenesis in mouse metanephrogenic mesenchyme by dorsal spinal cord have been studied. A drug concentration of 0 · 05 μg./c.c. was found to be optimal for inhibiting differentiation without being lethal for the cells.

  2. Treatment of both spinal cord and mesenchyme, or of mesenchyme only, for 30 min. just prior to recombination, inhibited tubulogenesis. Treatment of the spinal cord (inducer) only, either just prior to or up to 8 hr. before recombina-tion had no effect.

  3. Treatment of the spinal cord and mesenchyme for 30 min. with 0 · 05 μg./c.c. actinomycin at any time up to 20 hr. after recombination inhibited tubulogenesis. If 24 or more hours elapsed between recombination and treatment with the drug, however, differentiation could no longer be inhibited.

  4. In all cases exposure to actinomycin reduced tissue viability, but both spinal cord and liver (dummy inducer) protected the mesenchyme and increased cell viability in the latter, measured by neutral red uptake.

  5. In the discussion the significance of the change in the effect of exposure to actinomycin between 20 and 24 hr. after recombination is considered with reference both to other studies of this time period in the mesenchyme induction system, and to analogous results achieved with actinomycin in other systems.

Actinomycin Disapolypeptide antibiotic, which combines with the guanosine residues of DNA and is reported to inhibit, in very low concentrations, DNA-dependent RNA synthesis (Goldberg et al., 1962; Hurwitz et al., 1962; Reich et al., 1962). It thus provides a tool to study the possible role of DNA-dependent RNA synthesis in embryonic induction and differentiation. The effect of actinomycin on embryonic differentiation has been studied by several investigators. Most of this work has involved studies of early differentiation in am-phibian embryos either whole (Brachet & Denis, 1963; Wallace & Elsdale, 1963; Flickinger, 1963), in explants (Denis, 1963), or disaggregated (Wallace & Elsdale, 1963; Toivonen et al., 1964). In all cases it was found that actinomycin inhibited differentiation although different tissues were not affected identically (Denis, 1963; Flickinger, 1963; Wallace & Elsdale, 1963). It was also noted that actino-mycin penetrated cells poorly and that inhibition was sometimes dependent on whether or not the antibiotic got in (Denis, 1963; Gross & Cousineau, 1963; Wallace & Elsdale, 1963; Toivonen et al., 1964). In both amphibians (Wallace & Elsdale, 1963; Toivonen et al., 1964) and sea urchins (Gross & Cousineau, 1963), although actinomycin inhibited development, the cells remained viable. Gross and Cousineau demonstrated that development in sea urchin eggs ceased, along with uptake of uridine C14, but that cell division and early protein synthesis were unaffected.

Temporal differences in the effects of actinomycin were suggested by the work of Flickinger (1963) in Rana pipiens, and Klein & Pierro (1963) with chick embryos. They showed that actinomycin, applied to 11–13 somite chick embryos, inhibited differentiation and decreased DNA, RNA and protein nitrogen con-tents in these portions of the embryo which had not differentiated prior to ex-posure, while not affecting significantly those portions which had. Additionally, Tuchmann-Duplessis & Mercier-Parot (1960) injected actinomycin intraperi-toneally to pregnant rats, and produced teratogenic effects only when the injections were on days 7 or 9, although resorption of foetuses could be induced with both earlier and later treatment.

It was felt desirable to extend these experiments to the interacting system of foetal mouse metanephrogenic mesenchyme and dorsal thoracic spinal cord because this inductive system is perhaps better characterized and more standard-ized than any other known (Grobstein, 1955, 1956). The mesenchyme-spinal cord recombination is a limited system, and both its developmental and temporal sequences have been thoroughly elucidated. It is certain that induction of tissue differentiation is involved, and it is possible to treat the inducing (spinal cord) and responding (mesenchyme) tissues independently. Accordingly, both spinal cord and mesenchyme were treated with actinomycin D, either separately or together, and the effects of the antibiotic upon subsequent tissue survival and differentiation were studied.

Organ cultures

The kidney rudiments were aseptically removed from 12-day foetuses of random bred Swiss strain mice. The ureteric bud and metanephrogenic mesenchyme were separated by gentle mechanical manipulation in 0 · 02 per cent, versene (ethylenediaminetetra acetic acid). From five to seven mesenchyme pieces were grouped together and placed on a TA Millipore filter disc on a Trowell-type metal screen (Trowell, 1954), at the medium gas interface. A fragment of dorsal thoracic spinal cord, also from the foetuses, was placed on the filter disc to serve as inductor, and the mesenchyme pieces were placed closely around the spinal cord.

The culture medium consisted of Eagle’s basal medium in Earle’s balanced salt solution, 10 per cent, inactivated foetal calf serum, and 0 · 0194 M sodium bicarbonate. The explants were incubated for 65 or 87 – 90 hr. at 37° C. in 5 per cent. CO2 in air.

Histology

The tissue, together with the filter, was removed from the metal screen and fixed in Carnoy’s for 20 min., dried, embedded in paraffin, serially sectioned and stained with haemalum-eosin.

Treatment with actinomycin

The interacting system of metanephrogenic mesenchyme and dorsal spinal cord was tested by exposure to actinomycin D in several ways.

  1. Both mesenchyme pieces and spinal cord were exposed, separately, to varying concentrations of actinomycin for 30 min. prior to recombination. They were then washed for 5 min. in medium, transferred to the filter disc and incubated in fresh medium.

  2. Either the mesenchyme or the spinal cord, but not both, was exposed to actinomycin for 30 min. prior to recombination, washed and recombined as above.

  3. Only the spinal cord was exposed to actinomycin, 0 · 05 μg./c.c. for 30 min at varying times prior to recombination with the mesenchyme. It was then washed, placed on the filter and screen, and kept at 37° C. in 5 per cent, of CO2. Upon completion of the pre-incubation time, the mesenchyme pieces were placed around the cord. To control each experiment, spinal cord untreated with actinomycin was pre-incubated the same length of time.

  4. Both mesenchyme and spinal cord were exposed to’actinomycin, 0 · 05 μg./c.c. for 30 min. at varying times after recombination. The original medium was removed from the culture dish and replaced by temperature- and pH-preadjusted actinomycin-containing medium. After exposure, the actinomycin was removed and replaced with a wash medium. This was removed after 5 min., and replaced with fresh cultivation medium. Total time of incubation remained 87– 90 hr.

Viability determinations

The viability of the tissue treated with actinomycin was tested as follows. Groups of ten mesenchyme pieces were incubated for 20 hr., either alone or with spinal cord (inducer) or liver (dummy inducer) (Vainio, 1964) in a transfilter set-up (Grobstein, 1956). Similar groups of mesenchyme were treated with 0· 05 μg./c.c. actinomycin D for 30 min. prior to incubation. After 20 hr. the mesenchyme pieces were scraped off the filters, trypsinized and stained with neutral red (Vainio, 1964).

Controls

At 65 hr., groups of mesenchymal cells had condensed and become closely packed, with the outermost cells appearing elongated and spindle-shaped. Numerous tubule anlagen, at different stages of development, were also visible. By 87– 90 hr. the tubules had become elongated and often U-shaped (see Plate, Fig. A), in accordance with the normal maturation sequence for this system described by Rapóla et al. (1963).

Actinomycin treatment

(1) Treatment of mesenchyme and spinal cord with Actinomycin prior to recom-bination

The results of this experiment are summarized in Table 1 and illustrated in Figs. B-E of the Plate. The therapeutic range of the antibiotic is very limited. Concentrations lower than 0 · 04 μg./c.c. fail appreciably to inhibit differentiation, while concentrations greater than 0 · 05 μg./c.c. are lethal to the cells. In all cases there was less damage to the spinal cord than to the mesenchymal tissue. Further-more, differentiated mesenchyme survived better than undifferentiated mesen-chymal tissue.

Table 1.

Results of exposure of mesenchyme and spinal cord to actinomycin D for 30 min. just prior to recombination

Results of exposure of mesenchyme and spinal cord to actinomycin D for 30 min. just prior to recombination
Results of exposure of mesenchyme and spinal cord to actinomycin D for 30 min. just prior to recombination

(2) Treatment of either mesenchyme or spinal cord, but not both, just prior to recombination

The mesenchyme was pretreated with 0 · 0075 and 0 · 05 μg./c.c. actinomycin. The lower concentration affected neither viability nor differentia-tion. At 0 · 05 μg./c.c., much of the mesenchyme was necrotic, and no differentia-tion was seen. Table 2 gives the results of pre-treating the spinal cord. Treatment did not inhibit differentiation in any case, and retarded it only when the con-centration of the antibiotic was sufficiently high to kill a great portion of the cord cells, as in one case at 0 · 06 μg./c.c.

Table 2.

Results of exposure of spinal cord to actinomycin D for 30 min. just prior to recombination

Results of exposure of spinal cord to actinomycin D for 30 min. just prior to recombination
Results of exposure of spinal cord to actinomycin D for 30 min. just prior to recombination

(3) Treatment of spinal cord with 0 · 05 μg./c.c. actinomycin at various times prior to recombination

The spinal cord was exposed at 2, 4, 6 or 8 hr. before recombination. Both control and actinomycin -treated explants showed the normal differentiation pattern after 87 – 90 hr. incubation, with elongated and U-shaped tubules present.

(4) Treatment of mesenchyme and spinal cord with 0 · 05 μ g./c.c. Actinomycin after recombination

The results of this series are given in Table 3. There is a distinct difference in the effect produced by actinomycin treatment before and after 24 hr. of incubation. When the recombination was treated before it had incubated 24 hr., differentiation was either inhibited or greatly retarded in all cases. At and after 24 hr. of incubation, however, exposure to actinomycin failed to inhibit differentiation significantly. Also, those recombinations treated after 24 hr. in culture showed much better viability than those treated before 24 hr.

Table 3.

Results of exposure of mesenchyme and spinal cord to recombination D, 0 · 05 μ g./c.c., for 30 min. at different times after recombination (total incubation time 87 – 90 hr.)

Results of exposure of mesenchyme and spinal cord to recombination D, 0 · 05 μ g./c.c., for 30 min. at different times after recombination (total incubation time 87 – 90 hr.)
Results of exposure of mesenchyme and spinal cord to recombination D, 0 · 05 μ g./c.c., for 30 min. at different times after recombination (total incubation time 87 – 90 hr.)

Viability studies

In all cases exposure to actinomycin reduced tissue viability after 20 hr. However, spinal cord and liver both exerted a protective effect on the mesenchyme pieces. According to the number of cells which picked up neutral red, only 41 per cent, of the mesenchymal cells were viable 20 hr. after exposure to actino-mycin. Cells not treated with the drug showed 75 per cent, viability after the same incubation period. The presence of spinal cord or liver increased the survival rate of the mesenchyme pieces exposed to actinomycin to 62 per cent., but did not affect mesenchymal cells which had not been treated (Table 4).

Table 4.

Results of viability studies on mesenchymal explants after 20 hr. in vitro cultivation

Results of viability studies on mesenchymal explants after 20 hr. in vitro cultivation
Results of viability studies on mesenchymal explants after 20 hr. in vitro cultivation

Up to the present time, the induction of tubulogenesis in mouse metanephro-genic mesenchyme is defined by morphological events only. In the present studies, therefore, only such criteria could be used to determine the optimal inhibitory concentration of actinomycin D. In all series, additionally, the results of the experiments were read well after the antibiotic was used (up to 88 hr. later), since tubulogenesis occurred in the controls only after 30 to 40 hr. of incubation (Rapóla et al., 1963), and only then could the effect of the drug upon tubule formation be estimated. Those concentrations chosen by us as optimal for inhibiting tubulogenesis had little effect on cell viability, determined by neutral red, during the first 6 to 8 hr. after exposure (unpublished results). At 20 hr., the viability was less than in untreated cells but greater than in mesenchyme pieces exposed to the drug and subsequently incubated without spinal cord or liver. We conclude that, in the concentrations used, although viability was somewhat decreased, the effects of actinomycin could hardly be attributed to toxicity alone. At present it appears that the concentration of actinomycin which seems to inhibit tubulogenesis closely corresponds to the concentrations reported by other investigators to have an effect on RNA synthesis in mammalian cells (Reich et al., 1962; Perry, 1962, 1963).

Our studies indicate that between 20 and 24 hr. of induction there is a distinct change in the effect of actinomycin D upon tubulogenesis. If mesenchymal explants are treated after 20 hr. contact with spinal cord, their differentiation to tubules is inhibited. If they are treated after 24 hr. contact, normal differentiation occurs. This time period is also critical in transfilter induction of the mesenchyme, and some 26 to 30 hr. contact is required before the inductive stimulus become irreversible (Grobstein, 1961, 1964). These results correlate with those obtained by Wessells (1964) in his studies of the development of mouse pancreatic tissue. He found that, after an inital sensitivity, an actinomycin -insensitive exocrine cell population developed between 48 and 72 hr. of culture. In chick embryos, as noted above, if 11 – 13 somite embryos are exposed to actinomycin D, only those portions of the axial skeleton which are elaborated from mesenchyme still unsegmented at the time of exposure are prevented from differentiating (Klein & Pierro, 1963). It appears that some significant changes occur at certain critical time periods (24 hr. for mouse metanephrogenic mesenchyme), and that these changes tend to produce irreversible differentiation in the system. When they occur, actinomycin D can no longer inhibit differentiation.

Another type of experiment also suggests the importance of the period between 20 and 25 hr. in the mesenchyme induction system. Incorporation studies with H3-uridine indicate that RNA synthesis in the mesenchymal explants decreases during the first 20 hr. of in vitro transfilter contact with spinal cord. After this there is a rapid increase in the incorporation of H3-uridine, indicating increased RNA synthesis. In non-induced mesenchyme pieces this increase is much less or non-existent (Vainio, 1964). It seems tempting to speculate that the observed increase in RNA synthesis in induced mesenchymes is connected to the stabiliza-tion of a new differentiating stage, and that it is by inhibiting this synthesis that actinomycin inhibit tubulogenesis.

Other possible explanations for actinomycin inhibition also exist. One concerns the penetration of the drug, which in some other systems has been shown to play a decisive rôle (Toivonen et al., 1964). One could suggest that the mesenchymal cells which form pre-tubular condensates are less accessible to the drug which thus cannot influence their nucleic acid synthesis. It is also possible that the effect of actinomycin is merely a toxic one, and that differentia-tion only increases the cells’ ability to withstand the drug’s toxicity. However, if liver is used as a heterogenous inductor and viability after actinomycin treat-ment is determined by neutral red, mesenchyme pieces exposed to liver survive just as well as those exposed to spinal cord, while those without any other tissue have a considerably poorer survival rate.

In the study of the mechanism of actinomycin action in mammalian cells, some confusion has been created by the employment of a large variety of con-centrations. Thus high concentrations of the drug (5 μg./c.c.) seem to inhibit the nuclear incorporation of uridine instantly (Scherrer et al., 1963). Most of the uridine incorporated prior to the application of the drug goes to the ribosomal RNA. One-third of the label, however, goes to the acid-soluble form, showing that this part could be an intranuclear messenger RNA. These studies lead one to conclude that high concentrations of actinomycin blocks intranuclear messenger as well as pre-ribosomal RNA. Low concentrations of actinomycin, on the other hand, seem preferentially to block pre-ribosomal (nucleolar) incorporation while allowing the rest of the rapid nuclear incorporation to continue (Perry, 1962, 1963). Whether this implies that low concentrations of actinomycin attack ribosomal cistrons in preference to others is far from clear at the moment. In this connexion, it is relevant to note that different enzyme induction systems in B. subtilis have varying susceptibility to actinomycin D (Pollock, 1963). It is attractive to speculate that the drug might affect differentia-tion through its action on polysome formation (Penham et al., 1963). It is not worth while discussing further the mechanism by which actinomycin D interferes with tubulogenesis, however, until more details of the action of the drug on DNA-dependent RNA synthesis are known.

Etudes sur la tubulogénèse dans le rein. I. L’effet de l’actinomycine D sur l’induction des tubules

  1. Les effets de l’actinomycine D sur la tubulogénèse dans le mesenchyme métanéphrogène de la Souris ont été étudiés, l’inducteur étant la moelle épinière dorsale. La concentration optimale de ce produit qui soit capable d’inhiber la différenciation sans être létale pour les cellules s’est trouvée être de 0,05 Mgm. par ml.

  2. En traitant pendant 30 min., juste avant la recombinaison, soit le seul mésenchyme, soit à la fois celui-ci et le fragment de moelle épinière, on inhibe la tubulogénèse. Aucun effet n’est obtenu si le traitement porte sur le seul inducteur (la moelle épinière) que ce soit just avant sa juxtaposition au mésenchyme ou 8 h. plus tôt.

  3. Traiter la moelle épinière et le mésenchyme pendant 30 min. avec 0,05 Mgr. l’actinomycine par ml., et ce à tout moment compris dans les 20 premières heures après la recombinaison, a inhibé la tubulogénèse. Si toutefois 24 h. ou plus s’étaient écoulées entre la recombinaison et le traitement par l’antibiotique, il n’était plus possible d’empêcher la différenciation de se produire.

  4. Dans tous les cas, l’exposition à l’ actinomycine a réduit la viabilité du tissu, mais la moelle épinière a protégé le mésenchyme et accru sa viabilité, que l’on évaluait par l’affinité pour le rouge neutre. Le foie exercé la même action protectrice, alors qu’il n’est pas inducteur.

  5. La discussion a porté sur la signification du changement constaté dans l’effet qu’a l’exposition à l’ actinomycine lorsque le délai après la recombinaison passe de 20 à 24 h. Ce fait est considéré en fonction d’autres études portant sur le dispositif d’induction du mésenchyme envisagé à la même période. Il est examiné aussi en relation avec des résultats analogues obtenus avec l’ actinomycine dans d’autres expériences d’induction.

This investigation was supported by grants from National Cancer Institute, National Insti-tutes of Health, U.S. Public HealthService(CA05347), andfrom the Sigrid J usélius Foundation.

Bracket
,
J.
&
Denis
,
H.
(
1963
).
Effects of Actinomycin D on morphogenesis
.
Nature, Lond
.
198
,
205
6
.
Denis
,
H.
(
1963
).
Effet de l’ Actinomycine sur la différenciation nerveuse de l’ectoblast chez les embryons d’amphibien
.
Exp. Cell Res
.
30
,
613
15
.
Flickinger
,
R. A.
(
1963
).
Actinomycin D effects in frog embryos: Evidence for sequential synthesis of DNA-dependent RNA
.
Science
,
141
,
1063
4
.
Goldberg
,
I. H.
,
Rabinowitz
,
M.
&
Reich
,
E.
(
1962
).
Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin
.
Proc, nat. Acad. Sci., Wash
.
48
,
2094
101
.
Grobstein
,
C.
(
1955
).
Inductive interaction in the development of the mouse metanephros
.
J. exp. Zool
.
130
,
319
40
.
Grobstein
,
C.
(
1956
).
Trans-filter induction in tubules in mouse metanephrogenic mesenchyme
.
Exp. Cell Res
.
10
,
424
40
.
Grobstein
,
C.
(
1961
).
Passage of radioactivity into a membrane filter from spinal cord preincubated with tritiated amino acids or nucleosides. In Actes du Colloques International sur La Culture Organotypique
.
Associations et Dissociations d’Organes en Culture in vitro
(ed.
Et.
Wolff
), pp. 169-82. Paris: Editions du Centre National de la Recherche Scientifique.
Grobstein
,
C.
(
1964
).
Cytodifferentiation and its controls
.
Science
,
143
,
643
50
.
Gross
,
P. R.
&
Cousineau
,
G. H.
(
1963
).
Effects of Actinomycin D on macromolecule synthesis and early development in sea urchin eggs
.
Biochem. biophys. Res. Commun
.
10
,
321
6
.
Hurwitz
,
J.
,
Furth
,
J. J.
,
Malamy
,
M.
&
Alexander
,
M.
(
1962
).
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 proflavin
.
Proc, nat. Acad. Sci., Wash
.
48
,
1222
30
.
Klein
,
N. W.
&
Pierro
,
L. J.
(
1963
).
Actinomycin D: Specific inhibitory effects on the explanted chick embryo
.
Science
,
142
,
967
9
.
Penham
,
S.
,
Scherrer
,
K.
,
Becker
,
Y.
&
Darnell
,
J. E.
(
1963
).
Polyribosomes in normal and poliovirus-infected HeLa cells and their relationship to messenger-RNA
.
Proc. nat. Acad. Sci., Wash
.
49
,
654
62
.
Perry
,
R. P.
(
1962
).
The cellular sites of synthesis of ribosomal and 4S RNA
.
Proc. nat. Acad. Sci., Wash
.
48
,
2179
86
.
Perry
,
R. P.
(
1963
).
Selective effects of Actinomycin D on the intracellular distribution of RNA synthesis in tissue culture cells
.
Exp. Cell Res
.
29
,
400
6
.
Pollock
,
M. R.
(
1963
).
The differential effect of Actinomycin D on the biosynthesis of enzymes in Bacillus subtilis and Bacillus cereus
.
Biochem. Biophys. Acta
,
76
,
80
93
.
Rapola
,
J.
,
Vainio
,
T.
&
Saxén
,
L.
(
1963
).
Viral susceptibility and embryonic differentiation. IV. An attempt to correlate viral susceptibility with the metabolism and proliferation in embryonic tissues. J
.
Embryol. exp. Morph
.
11
,
757
64
.
Reich
,
E.
,
Franklin
,
R. M.
,
Shatkin
,
A. J.
&
Tatum
,
E. L.
(
1962
).
Action of Actinomycin D on animal cells and viruses
.
Proc. nat. Acad. Sci., Wash
.
48
,
1238
45
.
Scherrer
,
K.
,
Latham
,
H.
&
Darnell
,
J. E.
(
1963
).
Demonstration of an unstable RNA and of a precursor of ribosomal RNA in HeLa cells
.
Proc. nat. Acad. Sci. Wash
.
49
,
240
8
.
Toivonen
,
S.
,
Vainio
,
T.
&
Saxén
,
L.
(
1964
).
The effect of actinomycin D on primary embryonic induction
.
Revue suisse Zool
.
71
,
139
45
.
Trowell
,
O. A.
(
1954
).
A modified technique for organ culture in vitro
.
Exp. Cell Res
.
6
,
246
8
.
Tuchmann-Duplessis
,
H.
&
Mercier-Parot
,
L.
(
1960
).
The teratogenic action of the antibiotic Actinomycin D
. In
CIBA Foundation Symposium on Congenital Malformations
(eds.
G. E. W.
Wolstenholme
and
C. M.
O’connor
), pp.
115
28
.
London
:
J. & A. Churchill, Ltd
.
Vainio
,
T.
(
1964
).
On the mechanism of cytodifferentiation (to be published)
.
Wallace
,
H.
&
Elsdale
,
T. R.
(
1963
).
Effects of Actinomycin D on amphibian development
.
Acta Embryol. Morph. exp
.
6
,
275
82
.
Wessels
,
N. K.
(
1964
).
Acquisition of Actinomycin D insensitivity during differentiation of pancreas exorcine cells
.
Devel. Biol
.
9
,
92
114
.

Figs. A-C. Cultures of mouse metanephrogenic mesenchyme and dorsal spinal cord after 87 – 90 hr. of cultivation. Control (Fig. A) and culture exposed to 0 · 02 μg./c.c. actinomycin D for 30 min. prior to recombination (Fig. B) both show advanced tubulogenesis and very little cell death. Culture treated with 0 · 05 μg./c.c. actinomycin (Fig. C) for 30 min. prior to recombination shows many pycnotic nuclei. Haemalum-eosin. × 150.

Figs. D and E. Cultures of mouse metanephrogenic mesenchyme and dorsal spinal cord exposed to actinomycin D, 0 05 μg./c.c. after recombination and cultivated for a total of 78 · 90 hr. The explant in Fig. D was exposed for 30 min. 20 hr. after recombination, shows one condensation only, with many pycnotic nuclei and a necrotic area near the condensation. The explant in Fig. E was exposed for 30 min. 24 hr. after recombination, shows advanced tubulogenesis, very little cell death. Haemalum-eosin. × 150.

Figs. A-C. Cultures of mouse metanephrogenic mesenchyme and dorsal spinal cord after 87 – 90 hr. of cultivation. Control (Fig. A) and culture exposed to 0 · 02 μg./c.c. actinomycin D for 30 min. prior to recombination (Fig. B) both show advanced tubulogenesis and very little cell death. Culture treated with 0 · 05 μg./c.c. actinomycin (Fig. C) for 30 min. prior to recombination shows many pycnotic nuclei. Haemalum-eosin. × 150.

Figs. D and E. Cultures of mouse metanephrogenic mesenchyme and dorsal spinal cord exposed to actinomycin D, 0 05 μg./c.c. after recombination and cultivated for a total of 78 · 90 hr. The explant in Fig. D was exposed for 30 min. 20 hr. after recombination, shows one condensation only, with many pycnotic nuclei and a necrotic area near the condensation. The explant in Fig. E was exposed for 30 min. 24 hr. after recombination, shows advanced tubulogenesis, very little cell death. Haemalum-eosin. × 150.