a re-examination of succinic dehydrogenase activity...

J. Cell Sci. 15, 17-29 (1974) 17Printed in Great Britain

A RE-EXAMINATION OF SUCCINIC

DEHYDROGENASE ACTIVITY AND ITS

ASSOCIATION WITH CELL DEATH IN THE

INTERDIGIT OF THE CHICK FOOT

J. F. FALLON, R. F. BRUCKER AND CYNTHIA M. HARRISDepartments of Anatomy and Physiological Chemistry, University of WisconsinMedical School, Madison, Wisconsin 53706, U.S.A.

SUMMARY

Massive cell death, which accompanies the freeing of the digits, occurs in the interdigits of thedeveloping chick foot during stages 30-35. The possibility was reexamined that mitochondrialsuccinic dehydrogenase activity disappears in the interdigital cells of the chick foot 24 h beforecell mortality. Using standard histochemical means it was found that succinic dehydrogenaseactivity was demonstrable in the interdigital cell population up to and including the time whencell death begins. This was confirmed quantitatively employing biochemical methods whichindicated that succinic-cytochrome c reductase specific activity was approximately the same inhomogenates of digital and interdigital tissues harvested just prior to the death of interdigitalcells. Further, NADH-cytochrome c reductase specific activity was comparable in digital andinterdigital tissue homogenates. These data make it unlikely that ' biochemical degeneration'associated with alterations in aerobic energy metabolism occurs in the interdigital cells beforedeath as has been suggested by other workers previously. The methods used by these authorsto demonstrate succinic dehydrogenase were repeated on chick feet in our laboratories and analternative interpretation of the results is offered.

INTRODUCTION

Mesodermal cell deaths occur at precise developmental times and in predictablelocations during the morphogenesis of avian (Saunders & Fallon, 1967) and mamma-lian limbs (Milaire, 1965; Ballard & Holt, 1968). This phenomenon has been mostextensively studied in the chick embryo, where maps have been constructed ofregions of cell death in both the wing (Saunders, Gasseling & Saunders, 1962) andleg (Saunders, unpublished observations; Saunders & Fallon, 1967). Generally,cell deaths occur in a predictable sequence along the pre- and postaxial superficialmesoderm of developing limb buds, climaxing in a massive interdigital degenera-tion accompanying the freeing of the digits. Dying or dead cells are phagocytosed byneighbouring cells.

Experimental studies using the zone of necrosis localized at the postaxial junctionof the wing bud and body wall (the posterior necrotic zone) at developmental stage 24(Hamburger & Hamilton, 1951), indicate that these cells are 'programmed' to die earlyin their history (Saunders et al. 1962; Fallon & Saunders, 1968). However, there havebeen few detailed studies on changing patterns of macromolecular syntheses inprospectively dying cells. It is clear that these cells do not synthesize degradative

2 C E L 15

18 J.F. Fallon, R. F. Brucker and C. M. Harris

enzymes which eventually autolyse the cell (Dawd & Hinchliffe, 1971; Fallon & Rasch,in preparation). Rather, these enzymes appear only after cell death has occurred andthe phagocytic process has been initiated. Thus, it is reasonable to conclude that thedegradative enzymes are synthesized exclusively by phagocytic cells. Preliminarycommunications have appeared on changing patterns of DNA (Held & Saunders,1965) and protein synthesis (Pollak & Fallon, 1972) in prospectively degenerating cellsof the chick wing bud posterior necrotic zone. These studies suggest that the synthesesof both of these species of macromolecules are depressed prior to frank necrosis.

Using a histochemical technique, on whole feet, Hammar & Mottet (1971) haverecently reported a progressive decrease in succinic dehydrogenase (SDH) activity ininterdigital tissue of the chick foot during stages 25 through 27. This loss continuedduring development so that SDH activity was completely absent at least 24-36 h(stage 28) prior to the peak of interdigital cell death (stage 32). In contradistinction,digital cells showed no diminution of SDH activity during this same time interval.Thus, a 'biochemical degeneration' was claimed to have occurred in interdigital cellsprior to death. Further, it was suggested that this loss in activity was at least one of thefactors leading to the death of interdigital cells.

The far-reaching implications (e.g. see Cooper, 1973) of the report of Hammarand Mottet justified the repetition and extension of their work in an independentlaboratory. Further, the observations of these workers were of particular interest tothis laboratory because of previous work with a mitochondrial specific stain, JanusGreen-B (JGB) (Menkes & Deleanu, 1964; Saunders & Fallon, 1967; Fallon, 1972a, b).Briefly, injection of small amounts of this dye into the amniotic fluid at 6-5 days ofincubation was found to prevent normal cell death in chick embryo interdigits result-ing in a soft tissue syndactyly. In vitro experiments with JGB indicate that this agentmodifies the oxidation of succinate and NADH by mitochondria as a consequence ofthe interaction of this compound with flavine-containing components of the mito-chondrial electron transport chain (Harmon & Feigelson, 1952; Cooperstein & Lazarow,

1953)-Therefore, with these considerations in mind, we undertook a histochemical analysis

of succinic dehydrogenase activity in chick embryo interdigital tissue. We decided toapproach the problem using different methods from those employed by Hammar &Mottet (1971). Instead of incubating whole limbs, fresh frozen sections were used forhistochemical localization of succinic dehydrogenase activity. This was done to permitmore complete and uniform penetration of components of the reaction medium thanmight occur using intact feet. Further, we have used biochemical methods on pooleddigit and interdigital tissues to check quantitatively the qualitative results of thehistochemical studies. Finally, we have repeated the analysis for SDH activity withthe methods used by Hammar & Mottet (1971).

Succinic dehydrogenase and cell death 19

MATERIALS AND METHODS

White Leghorn chick embryos from medium size eggs (46-53 g per egg) incubated at 37 °C,were used exclusively in this study.

Fig. 1 is a schematic representation of the mitochondrial electron-transport chain and thoseportions of the chain involved in the histochemical and biochemical assays employed in thisstudy.

NADH

\ e© eo ee es e° e3 e ° .A I >-fpN >ubiquinone »~cyt.b »-cyt c, >-cyt.c >-cyt.aa3—-4U-O,

V I II 'NAD |e° KCN

FormazanA| Formazan

NBT !

Succinate-

Malonate

to NBT1 i u - '• *

. . . Ubiqumone •-• i eo

ee t e S , -Succinate * - fps~ ' e°

cyt.c

e© QQ ,... Ubiqumone »-cyt. b »-cyt.c,

eoSuccinate »- fps

Fig. 1. A, schematic representation of flow of electrons, e°, through the mitochondrialelectron transport chain (ETC) coupled to NADH and succinate. B, representation ofportion of ETC involved in succinic dehydrogenase (SDH) histochemical assay. Brokenarrows indicate artificial electron acceptor pathway. Nitro blue tetrazolium (NBT) maybe used alone as the artificial electron acceptor, or phenazine methosulphate (PMS)may be used to enhance the reaction, with NBT as the final artificial electron acceptor.It is likely (Farber & Bueding, 1956) that NBT is not reduced by direct interaction withSDH but by interaction with a second component, ubiquinone (Wottenberg & Leong,i960; Horwitz, Benitez & Bray, 1967). Phenazine methosulphate, on the other hand,probably interacts directly with SDH and subsequently reduces the NBT (Farber &Bueding, 1956; Singer, Kearney & Bernath, 1956; Horwitz et al. 1967; Singer, 1968).c, representation of portion of ETC involved in succinate-cytochrome c reductasespectrophotometric assay. Broken arrow indicates pathway to exogenous cytochromec. fpN, NAD linked flavoprotein (dehydrogenase); fp3, succinate-linked flavoprotein(dehydrogenase); II, inhibitory sites.

Histochemical procedures

Fresh limbs of stage 29 and 30 were washed briefly in phosphate-buffered saline and frozenin Ames O.C.T. Compound, cut at 10 or 20 /tm on an IEC Cryostat and affixed to clean coverslips. The sections were immediately placed in either the complete reaction mixture or a controlmixture at 37 °C (see below).

2 o J. F. Fallon, R. F. Brucker and C. M. Harris

The reaction mixture for succinic dehydrogenase activity localization (see Fig. i B) was thatrecommended by Pearse (1960, p. 910). Equal volumes of 0 2 M phosphate buffer (pH 7-6) and0 2 M sodium succinate were mixed and an aqueous solution of nitro blue tetrazolium (SigmaChemical Company, lot number 40C-5430) was added to a final concentration of 1 mg per ml inthe reaction medium. To enhance the reaction, 2 mg of fresh phenazine methosulphate wasadded to 50 ml of the reaction mixture (cf. Chayen, Bitensky, Butcher & Poulter, 1969, pp. 194and 199).

Two control reaction mixtures were used. In the first, 0-05 M sodium malonate (a competitiveinhibitor of succinic dehydrogenase) was present in the reaction medium (cf. Chayen et al. 1969,p. 198). When this control was used, tissue sections were preincubated in buffer plus malonateat 37 °C for 15 min to remove endogenous substrate and permit malonate to permeate tissues ineffective concentrations. In the second control, sodium succinate was eliminated from thereaction mixture. When this control was used, sections were pre-incubated in buffer at 37 °Cfor 15 min to reduce the level of endogenous substrate.

Tissue preparation for chemical assay

Tissue samples were collected by microdissection, stored briefly in Sorensen's phosphate-buffer saline (087 % NaCl, 005 M Na2HPO4, and 0-05 M NaH2PO4, pH 74) , placed in approxi-mately 4 vol. of a homogenization buffer containing: sucrose (0-25 M), histidine (100 ITIM), andethyleneglycol-bis-(/?-amino-ethyl ether) N, JV'-tetra-acetic acid (EGTA) (02 mM) at pH 7 5 .Tissue homogenization was accomplished with a loose-fitting teflon pestle and Potter-Elvehjemtype homogenizer. Three strokes of the pestle at 550 rev/min were sufficient to homogenizethe tissue effectively. The homogenate was then filtered through 4 layers of buffer-washedgauze. All operations were carried out at 4 °C.

Chemical assay

Succinic dehydrogenase-cytochrome c reductase activity (see Fig. ic) was assayed in thefollowing manner: a ioo-/tl aliquot of filtrate was added to a pH 7 5 reaction medium (2-7 ml)containing: Tris (50 mM), KCN (03 mM), EDTA (01 mM), sodium succinate (5 mM) andallowed to equilibrate for 6 min at 30 °C. KCN is included in the medium to inhibit the terminalstep in the electron-transport chain, thus permitting assay of the reduction of exogenous cyto-chrome c. The reaction was initiated at zero time by the addition of cytochrome c (250 fi\ of1-5 mM stock solution). The reaction was followed with a Gilford 2000 recording spectrophoto-meter by measuring the change in absorbance at 550 nm due to the reduction of cytochrome c.Rotenone-sensitive, i.e. mitochondrial, NADH cytochrome c reductase activity (in the presenceand absence of 2 fig of rotenone) was determined using an identical reaction medium exceptthat 015 mM NADH was included instead of sodium succinate. Enzyme activity was expressedas /tmol cytochrome c reduced/min/mg protein or fig DNA at 30 CC. A millimolar extinctioncoefficient of 176 was used in this study (cf. Ernster, Siekevitz & Palade, 1962). An enzymeblank was run and corrections made for endogenous activity.

Protein was assayed by the modified Lowry technique described by Hartree (1972). DNAwas determined by means of Burton's (1956) modification of the diphenylamine method.

Hammar and Mottet methodology

Whole feet of stage 29, 30 and 31 chick embryos were washed in cold Earle's solution andimmediately placed in the complete reaction mixture, as per the method of Hammar & Mottet(1971), or control reaction mixture. Since Hammar and Mottet did not mention a control, thosecontrols employed in the histochemical procedure which are described above were used in thisinstance as well. Dissected limbs were incubated at 37 °C while rocking gently (3-25 oscillationsper min) on a Bellco rocker platform. Limbs were removed from the incubation at 30, 60, 90and 120 min and treated in one of two ways: fixed in 10 % formalin, dehydrated in ethanol andembedded in paraffin for histological study; or fixed in 10 % formalin, dehydrated and embeddedin Epon as a permanent record; these whole mounts were photographed (see Figs. 9-11). Somelimbs from each group were washed in Earle's solution, to remove free NBT before fixation andembedding, but this had no effect on the distribution of formazan.


RESULTS

Histochemical localization of inter digital SDH activity in frozen sections

Forty-five experimental and 20 control limbs were analysed using the histochemicalprocedures described in Materials and Methods. It was found that reaction time of15 min was optimal in interdigital cells at both stages 29 and 30 when phenazinemethosulphate (PMS) was included in the reaction mixture. However, a 30-minreaction time was optimal when PMS was omitted. In either case, discrete, round,light-purple formazan deposits could be seen in the cytoplasm of the interdigital cells.This was true at stage 29 (Fig. 3) when no degeneration could be seen in any of theinterdigits and at stage 30 when necrosis had begun in the deep region of the inter-digit between digits 3 and 4. This is the first area of interdigital degeneration and isillustrated in Fig. 4. Several phagocytic cells in the interdigit have begun ingestingdead (or dying) cells. The cells which surround this small focus of degeneration are notlacking in enzyme activity. It is stressed that all of these cells are destined to die withina few hours.

The control sections incubated in complete reaction medium plus malonate (acompetitive inhibitor of SDH) or without substrate were negative, in that no reactionproduct was formed (Fig. 5).

Comparison of succinic-cytochrome c reductase specific activity in the digit and theinterdigit

When conflicting results using histochemical procedures are obtained in differentlaboratories, quantitative biochemical assays often help to resolve the differences. Tothis end we asked the question, what is the difference in succinate oxidation in the digit

Fig. 2. Drawing of stage 29 to 30 chick foot. The areas within the V-shaped brokenlines represent the interdigits (id) which were dissected out and pooled in ice-coldbuffered saline. The digital areas (d), up to the broken line which transects the limb,were pooled and stored in ice-cold buffered saline. The region (x) to the left of thebroken line was discarded.

and the interdigit of the chick foot ? Digits and interdigits from stage 29 and 30 weredissected (Fig. 2), pooled separately, and prepared as described in Materials andMethods for chemical assay. Five separate preparations, each composed of tissues

22 J. F. Fallon, R. F. Brucker and C. M. Harris

collected from about 10 dozen embryos, were assayed as described in Materials andMethods for determination of succinic cytochrome c reductase activity, and totalprotein.

Succinic cytochrome c reductase activity was found to be present as the interdigitalcells approached death. Furthermore, no significant difference (P = 0-05) was ob-served in the specific activity of succinic cytochrome c reductase in comparisons ofpooled interdigits and pooled digits of stages 29 and 30. As can be seen from Table 1,the activities were 0-207 + 0-009 /*rnol cytochrome c reduced/min/mg protein for theinterdigit and 0-179 + 0-024/tmol cytochrome c reduced/min/mg protein for the digit.

In 2 independent assays specific activities of succinate cytochrome c reductase werecalculated on the basis of micrograms of DNA. Each experiment was assayed inquadruplicate and the specific activities of the digital and interdigital tissues werefound to be comparable.

Table 1. Succinate and NADH-cytochrome c reductase activity of interdigital anddigital tissues of the chick foot*

Rotenone-sensitiveSuccinate-cytochrome c NADH-cytochrome c NADH-cytochrome c

reductasef reductasej reductase§

Interdigit 0207 + 0009 1-130 ±0-082 0080 ± 0009

Digit 0-179 + 0-024 1-31010-057 0-08710-012

* Activity expressed as /wnol cytochrome c reduced/min/mg filtrate protein. Values repre-sent the means + S.D.

-f Data represent 5 separate experiments, each run in triplicate.% The data represent specific activity of the tissue homogenates of 3 separate experiments

each run in quadruplicate.§ The data represent specific activity attributable to mitochondrial inner membrane deter-

mined for tissue homogenates in +•

SDH activity using whole limbsWhen fresh whole feet of stages 29-31 were placed in the reaction mixture used by

Hammar & Mottet (1971) there was a gradual accumulation of blue-black reactionproduct which generally followed the pattern of digital cartilage condensations (seeFig. 9). In 6 experiments, consisting of 8 feet per trial, the reaction was found to bevariable, in that the time required to outline the digital pattern with reaction productvaried from foot to foot. Generally, 60-90 min were optimal to demonstrate the digitalcartilage condensations. However, controls which lacked substrate or contained malo-nate showed exactly the same pattern of black reaction product (Fig. 10). Further, withincubation intervals of 90-120 min the entire foot, digit and interdigit, turned com-pletely black (see Fig. 11).

Histological examination of experimental and control feet, incubated for 60-90 min,showed a blue-black crystalline precipitate which was predominantly in, but notlimited to, the area of the digital condensations (see Figs. 6, 8). Furthermore, thisprecipitate appeared at least in part to be extracellular (see Fig. 8). No discrete, round,light-purple reaction product could be found in the paraffin sectioned material (com-pare Figs. 7 and 8).


Rotenone-sensitive NADH-cytochrome c reductase specific activity

The concept of biochemical degeneration is an important one and linking it withaerobic energy metabolism is intriguing. We examined whether the cells of theinterdigit of stage 29-30 showed reduction or a lack of rotenone-sensitive NADH-cytochrome c reductase activity when compared with the digit (see Table 1 for rote-none-sensitive and insensitive activities). Three separate preparations of about 10dozen pooled digits and interdigits were assayed with no apparent difference inspecific activity between the digits and interdigits being detected. Specifically, as canbe seen from Table 1 the activities were 0-080 + 0-009 /*mol cytochrome c reduced/min/mg protein for the interdigit and 0-087 + 0-012 /tmol for the digit.

DISCUSSION

The results reported in this paper demonstrate by using standard histochemicaltechniques on unfixed frozen sections of the developing chick foot, that succinicdehydrogenase activity can be detected in interdigital cells through the time when thesecells die. Moreover the specific activity of succinic cytochrome c reductase is notsignificantly different in the interdigit and digit immediately before interdigital celldeath (stage 29-30). This demonstrates that succinate oxidation probably does notdiminish to extinction before the cells die. Further, if the digit is considered as astandard or reference tissue, it is unlikely that there is any loss in specific activitybefore death. In addition, rotenone-sensitive i.e. mitochondrial, NADH-cytochromec reductase specific activity is maintained at comparable levels in digital and inter-digital cells at least until just prior (stage 29-30) to the period of maximal interdigitalcell degeneration. These observations make it very unlikely that there is a mito-chondrial, aerobic energy associated, 'biochemical degeneration' occurring in theinterdigital cells before they show overt signs of necrosis.

This conclusion is in direct opposition to that drawn by Hammar and Mottet usingthe same material but a different method. When using the method of these authors wewere able to reproduce the results reported in their paper, i.e. formazan deposition inthe digits and not in the interdigital areas of whole feet. However, it should be notedthat very long incubation times were required in order to obtain these results. Thisprobably is due to very poor penetration of the highly polar nitro blue tetrazoliuminto whole limbs. Moreover from our results it appears that when whole limbs areincubated in reaction medium to demonstrate SDH activity, the developing cartilageof the digits contains a material able to reduce nitro blue tetrazolium to a formazan,which is deposited in a blue-black crystalline form. We believe it is the differentialdeposition of this precipitate which Hammar & Mottet (1971) demonstrated.

Our interpretation is reinforced by the following. Whole feet incubated in controlreaction mixtures, which lack substrate or contain malonate, show exactly the samedistribution of this blue-black formazan. In addition, the same crystalline depositionwill occur in the interdigit if the incubation interval is sufficient (90 min or more)leading to a more uniform distribution of reaction product. Thus we believe their


interpretation of these crystals as mitochondrial SDH activity is incorrect. Novikoff(1959a, b; also see Matula & Paterson, 1971; Neuwirtova & Setkova, 1973) has pointedout that nitro blue tetrazolium will form a crystalline precipitate in some situationsand that such crystalline depositions have been mistaken for mitochondria in the past.This artifact is invariably blue-black in colour, whereas true enzyme activity is demon-strated by a light blue-purple coloured formazan deposition in a discrete round form(Novikoff, personal communication of 1973).

Briefly summarized, our results demonstrate that SDH activity cannot be detectedusing intact chick feet incubated in the Hammar and Mottet reaction medium. On theother hand, succinate oxidation is readily demonstrated in frozen sections of chickfeet or in interdigit and digit homogenates, indicating that mitochondria in thesetissues do have the capacity to oxidize succinate, that the associated enzymes arefunctional, and are present in the interdigital cells at stages 29 to 30 in amounts atleast comparable to those of digital cells at the same developmental stages.

At the present time there is no unequivocal evidence for ' biochemical degeneration'preceding overt necrosis in the cells which die during normal chick limb development.However, biochemical changes must occur in these cells before they show overt signsof degeneration. Clearly, these changes must be elucidated in order that some insightbe gained into how the original programming for death occurs.

This work was supported by grant GB-40506 from the National Science Foundation, by theGraduate School Research Committee of the University of Wisconsin, by General ResearchSupport Funds to the University of Wisconsin Medical School from the National Institutes ofHealth and by an institutional grant to the University of Wisconsin from the American CancerSociety.

We are grateful to Drs David B. Slautterback, Philip P. Cohen, Ellen M. Rasch, Allen W.Clark, Steven E. Kornguth and Mr Richard Pollak for their constructive review of this manu-script.

We wish to thank Mrs Debra Reierson and Miss Bonnie Wallace for typing the manuscript,Mr Richard Pollak for help with the tissue dissections and Dr Roger Danke for carrying out theDNA determinations.

REFERENCES

BALLARD, K. J. & HOLT, S. J. (1968). Cytological and cytochemical studies on cell death anddigestion in the foetal rat foot: the role of macrophages and hydrolytic enzymes. J. Cell Sci.3, 245-262.

BURTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reactionfor the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323.

CHAYEN, J., BITENSKY, L., BUTCHER, R. & POULTER, L. (1969). A Guide to Practical Histo-chemistry. Philadelphia: Lippincott.

COOPER, E. H. (1973). The biology of cell death in tumours. Cell Tiss. Kinetics 6, 87-95.COOPERSTEIN, S. J. & LAZAROW, A. (1953). Studies on the mechanism of Janus Green B staining

of mitochondria. III . Reduction of Janus Green B by isolated enzyme systems. Expl Cell Res.5, 82-97.

DAWD, D. S. & HINCHLIFFE, J. R. (1971). Cell death in the opaque patch in the central mesen-chyme of the developing chick limb: a cytological, cytochemical and electron microscopicanalysis. J. Embryol. exp. Morph. 26, 401—424.

ERNSTER, L., SIEKEVITZ, P. & PALADE, G. E. (1962). Enzyme-structure relationships in theendoplasmic reticulum of rat liver. J. Cell Biol. 15, 541-562.

FALLON, J. F. (1972a). The effects of Janus Green B on the fate and morphology of the pro-spectively necrotic interdigital mesoderm of the chick foot. Teratology 5, 254.


FALLON, J. F. (19726). The morphology and fate of the apical ectodermal ridge in the normaland Janus Green B treated chick foot. Am. Zool. 12, 701-702.

FALLON, J. F. & SAUNDERS, J. W. JR. (1968). In vitro analysis of the control of cell death in azone of prospective necrosis from the chick wing bud. Devi Biol. 18, 553-570.

FARBER, E. & BUEDING, E. (1956). Histochemical localization of specific oxidative enzymes. V.The dissociation of succinic dehydrogenase from carriers by lipase and the specific histo-chemical localization of the dehydrogenase with phenazine methosulfate and tetrazolium.J. Histochem. Cytochem. 4, 357-362.

HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in development of thechick embryo. J. Morph. 88, 49-92.

HAMMAR, S. P. & MOTTET, N. K. (1971). Tetrazolium salt and electron-microscopic studies ofcellular degeneration and necrosis in the interdigital areas of the developing chick limb.J. CellSci. 8, 229-251.

HARMON, J. W. & FEIGELSON, M. (1952). Studies on mitochondria. V. The relationship ofstructure and oxidative phosphorylation in mitochondria of heart muscle. Expl Cell Res. 3,5O9-525-

HARTREE, E. F. (1972). Determination of protein: a modification of the Lowry method thatgives a linear photometric response. Analyt. Biochem. 48, 422-427.

HELD, W. A. & SAUNDERS, J. W., JR. (1965). Incorporation of tritiated thymidine by nuclei in azone of prospectively necrotic cells in the wing bud of the normal chick embryo. Am. Zool. 5,214.

HORWITZ, C. A., BENITEZ, L. & BRAY, M. (1967). The effect of coenzyme Q on the histochemicalsuccinic tetrazolium reductase reaction: a histochemical study. J. Histochem. Cytochem. 15,216-224.

MATULA, G. & PATERSON, P. Y. (1971). Spontaneous in vitro reduction of nitro blue tetra-zolium by neutrophils of adult patients with bacterial infection. New Eng. J. Med. 285,3II-3I7-

MENKES, B. & DELEANU, M. (1964). Leg differentiation and experimental syndactyly in chickembryo. Part II . Experimental syndactyly in chick embryo. Rev. roum. Embryol. Cytol. 1,69-77.

MILAIRE, J. (1965). Aspects of limb morphogenesis in mammals. In Organogenesis (ed. R. L.DeHaan & H. Ursprung), pp. 283-300. New York: Holt, Rinehart and Winston.

NEUWIRTOVA, R. & SETKOVA, O. (1973). NBT and cell clumping. New Eng. J. Med. 288, 970.NOVIKOFF, A. B. (1959a). Enzyme cytochemistry: pitfalls in the current use of tetrazolium

techniques. J. Histochem. Cytochem. 7, 301-302.NOVIKOFF, A. B. (19596). The intracellular localization of chemical constituents. In Analytical

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{Received 12 July 1973)


Fig. 3. io-/tm frozen section through a stage 29 chick foot, incubated for 15 min in thecomplete reaction mixture plus phenazine methosulphate. Small round dots indicatesuccinic dehydrogenase activity in the cells of the digit (d) and in the cells of the inter-digit (id), x 384.Fig. 4. io-/*m frozen section through a stage 30 interdigit between digits 3 and 4,incubated for 30 min in the complete reaction mixture. This region is the first inter-digital region to show cell death. Approximately 12 macrophages can be seen in thismicrograph (2 are circled). It is not known why the macrophage contents are dark. Thecells which surround this small focus of degeneration will die within hours. It is clearfrom the amount of reaction product in these surrounding cells that they do not lacksuccinic dehydrogenase activity, x 384.Fig. 5. io-/*m frozen section through a stage 29 chick foot incubated for 30 min in thecontrol reaction medium which lacked substrate. The diaphragm of the microscopesubstage condenser was closed, effectively making a smaller numerical aperture, in orderto enhance the scatter image due to diffraction at the cell boundaries. This section isnegative for reaction product in both the digit (d) and the interdigit (id), x 384.Fig. 6. 6-/tm paraffin section of a stage 29 chick foot. The whole foot was incubated inthe Hammar and Mottet medium for 60 min, fixed in formalin, dehydrated, embeddedand sectioned. Crystal-shaped deposits are clearly visible in the digit area and a few(arrows) are beginning to form in the interdigit. The general area of this frontalsection is indicated by the box on a different limb in Fig. 9. x 384.


id

id

\V.»i


Fig. 7. High magnification of stage 29 interdigit of Fig. 3, to show discrete, roundreaction product indicating succinic dehydrogenase activity. Compare with Fig. 8.x 840.Fig. 8. High magnification of stage 29 digit of Fig. 6 to show artifactual crystallinereaction product, at least some of which is extracellular. Compare with Fig. 7. x 840.Fig. 9. Stage 30 chick foot, incubated for 90 min in Hammar and Mottet medium.Reaction product is limited to the digital cartilage condensations with the interdigitremaining clear. Box indicates a comparable area of a different limb shown in Fig. 6.x 20.

Fig. 10. Stage 29+ chick foot, incubated for 90 min in control Hammar and Mottetmedium without substrate. Distribution of reaction product is approximately thatseen in Fig. 9. x 20.Fig. 11. Stage 29 + chick foot incubated in Hammar and Mottet medium for 90 min.Reaction product is seen throughout the foot plate, digit and interdigit. This figureunderscores the variability of the amount of reaction product laid down per givenincubation time. It is pointed out that usually between 90 and 120 min were requiredto obtain complete blackening of the digital plate, x 20.

Siiccinic dehydrogenase and cell death

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a re-examination of succinic dehydrogenase activity...

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