differentiation and the reaction of rat embryos to radiation

DIFFERENTIATION AND THE EEACTION O F RAT EXBRYOS TO RADIATION

JAMES G. WILSONZ Department of A n a t m ~ y , College of Medicine, Unitrersity

of Cincinnati, Cincinnati, Ohio

ONE FIGURE

The earlier studies on the effects of ionizing radiations on developing mammalian embryos have been adequately re- viewed by Warkany and Schraffenberger ( '47) and Russell ('50). The present report will deal not so much with the specific effects of irradiation as with changes in the pattern of response when embryos at different stages in differentiation are exposed under standard conditions. A study of this type must be as nearly quantitative as possible, which requires that sensitive criteria be set up as a basis f o r evaluating the responses. Previous experiments (Wilson and Karr, '51 ; Wilson, Jordan, and Brent, '53 ; Wilson, Brent, and Jordan, '53) have shown that suitable criteria were: (1) the rate of intrauterine mortality, ( 2 ) the degree of intrauterine growth retardation, and (3) the number and types of developmental malformations induced.

To obtain quantitative data it was also necessary to attempt control of several possible sources of error that are en- countered when embryos are irradiated in conjunction with whole-body or abdominal irradiation of the mother. F o r this purpose a method was devised whereby selected embryos could be exposed to X rays without irradiation of the mother

Based in part on work performed under contract with the United States Atomic Energy Commission a t the TJniversity of Rochester Atomic Energy Project.

The collaboration of Dr. John W. Karr, Dr. Robert L. Brent, and Mr. H. Charles Jordan in the cxperinients on which this report it based is gratefully acknowledged.

11

12 JAMES G. WILSON

or the remaining embryos. It was possible to prevent fluc- tuations in the depth dosage resulting from the intervention of maternal tissues of varying thickness and density by ex- posing the implantation sites directly, through an abdominal incision in the mother. The possibility of affecting the embryo secondarily by some primary reaction to irradiation on the part of the mother was reduced by shielding all maternal

I l T H DAY 20 + PAIRS OF SOMITES NEURALTUBECLOSED IOTH DAY OPTIC VESICLE FORMED OTlC VESICLE CLOSED 3 PAIRS AORTIC ARCHES HEART, PRIMITIVE 4-CHAMBERED GUT TRACT COMPLETE LARYNGOTRACHEAL GROOVE HEPATIC DIVERTICULUM EARLY MESONE PHROS FORELIMB BUD

6 t PAIRS OF SOMITES NEURALTUBE BEGINNING CLOSURE OPTIC EVAGIMATION I PAIR AORTIC ARCHES HEART, SIMPLE TUBE FOREGUT, HIND GUT FORMING

Fig. 1 Schematic representation of the stages of differentiation existing early o n the eighth, ninth, tenth, and eleventh days of gestation in the rat. On the eiglith day the embryo may be considered essentially an undifferentiated mass of cells. Although tlic surface of the embryonic cylinder is covered by primitive yolk endoderm, thr significance of this layer in fu ture derelopmmt is nncertain. T t is probahle that many if not all rells u.it,lrin the embryonic mass arc multi- potential a t this stage. On the ninth day the three primary germ layers, ectoderm, nie~oderm, and endoderm, are readily identified. The polarity of the embryo has been indicated by the primitive node and streak, hut organ formation has not begun. On the tenth (lay organ formation is under way in the nervous, cardiovascular, and digestive systems and the somatic mesoderm is beginning segmenta- tion. By the eleventh day some degrer of organ formation is in progress in almost all majnr systems and is morlerately advanced in the cardiovascular and digestive systems. (The elevcnth-day embryo is too large and complex to be rcpresented in the scheme used here.)

REACTION O F RAT EMBRYOS TO RADIATION 13

tissue except the segment of uterus containing the selected implantation sites. The shielding of some embryos in each pregnancy made available nonirradiated “litter-mate” controls for the precise evaluation of growth and developmental changes in the exposed embryos. Exact rates of mortality were determined by comparing the number of embryos present after any postirradiation interval with the original number observed at the time of operation.

The stages of differentiation selected for such a study are of particular importance. They should represent consecutive steps in development, each characterized by easily recognized morphological features. Such a series of stepwise stages occurs between the eighth and the eleventh days of gestation in the rat (fig. 1). On the eighth day differentiation has not yet begun, at least is not apparent, f o r the embryo consists merely of a randomly arranged mass of blastomercs at the embryonic pole of the egg cylinder. By the ninth day the first major step in differentiation has occurred, in that the three primary germ layers have formed, but differentiation of these toward organ formation has not begun. The tenth day is characterized by the beginning of organogenesis in certain systems, particularly the nervous, cardiovascular, and digestive systems. On the eleventh day organogenesis of some degree is in progress in virtually all major systems and is moderately advanced in several. Accordingly, embryos have been exposed to X rays at these specific stages because they encompass the initiation as well as the first important steps in differentiation.

METHOD

The details of the technique by which selected embryos were exposed to direct irradiation have been discussed and illustrated elsewhere (Wilson and Karr, 51), and only the basic procedure need be repeated here. Gestation was considered to have begun at 9 A.M. of the morning on which sperm were found in the vaginal smear of female rats allowed to breed during the preceding night. The products of conception were regarded as 1 day of age 24 hours after sperm were

14 J A M E S G . WILSON

found. All subsequent procedures referable to the age of the embryos (exposure to X rays and termination of pregnancy) were also carried out at approximately 9: 00 A.M. in order that timing might be recorded in uniform daily units.

On the eighth, ninth, tenth, or eleventh day of gestation female rats of the TVistar albino strain were anesthetized with nembutal and the ventral abdominal wall opened by a midline incision. One uterine horn was brought to the surface of the incision, and lead plates were then arranged around and underneath two to five of the implantation sites in such a manner as to shield completely all remaining embryos and all material tissues except the segment of uterus containing the unsliielded sites. After exposure of the unshielded embryos, these were promptly restored to their position in the abdominal cavity. The second uterine horn was then brought to the surface of the incision and a similar number of implantation sites carried through the same manipulations, except that the latter Tvere not irradiated. Before this horn was restored to the abdominal cavity and the incision closed, the number and position of all implantation sites in both uterine horns were noted in a diagram, to facilitate later identification of irradiated and nonirradiated embryos.

A dose of 12.5, 25, 50, 100, 200, 400, or 600 r (in air) was given at a single exposure to the unshielded embryos. A therapeutic X-ray machine calibrated with a standardized Victoreen r-meter was used. The beam was that produced by the following factors : 85 kvp, 10 nia, and 1.0 mm of aluminum added filter. Further characteristics were : half-value layer of 2.0mm of aluminum, and 1.0cm depth dose equivalent to the air dose. The target-object distance varied between 10 and 20cm, and the duration of exposure never exceeded 2 minutes. The distribution of irradiated embryos with respect to dosage is presented in table 1.

After postirradiation intervals of one or more days, or early on the day of expected delivery, the mothers TTTere killed by decapitation and both uterine horns inspected in situ for


resorbed implantation sites. The irradiated and control embryos were identified while still in utero (using the diagram made at the time of exposure) and the entire uterus then removed and fixed in Bouin's fluid. After being transferred to 70% alcohol all embryos were dissected from the uterus, weighed, and examined externally under low power magnification. The irradiated embryos together with one or two of their nonirradiated siblings were serially sectioned and stained with hematoxylin and eosin for microscopic study.

TABLE 1

Distribl*tion of irradiated embryos 6 s t o day of treatment , dosage, and number of nonirradiated siblings

NUNBER O F IRPI.4DIATED AND CONTXOL EICURYOS' IN EAC'H DOSAGE GROUP

DAY 12.5 r 25 r 50 r 100 r 200 r 400 r 600 r _ _ _ ~ ~ - - - ~ Irr. Con. Irr. Con. lrr. Con. Irr. Con. lrr. Con. Irr. Con. Irr. Con.

8th 13 13 27 34 28 32 27 23 15 24 9th 17 23 49 76 178 162 43 53 10 7

10th 30 54 62 102 56 75 19 26 11th 4 6 18 26 40 61 25 42 14 17

OBSERVATIONS

The observations presented here on the effect of irradiation at the eighth, ninth, and tenth days of gestation are based on data published previously (Wilson and Karr, '51 ; Wilson, Jordan, and Brent, '53 ; Wilson, Brent, and Jordan, ' 5 3 ) . In order to obtain more meaningful comparisons, however, some of the earlier data have been regrouped. Further- more, since the emphasis in thc present report is on over-all effects, the observations made on the first postirradiation day have been oniittcd from all groups because it has been found that typical effects frequently do not appear within so short an interval. The observations dealing with the effects of treatment on the eleventh day have not been previously published.

Mortal i ty. The rates of intrauterine mortality after various doses of X rays given on the eighth, ninth, tenth, o r

16 JAMES G. WILSON

eleventh day are preseiited in table 2. After irradiation on the ninth, tenth, o r eleventh day the mortality increased as the dosage increased ; but this dose-response relation ~ 7 a s r i o t a i~1~arent in the cahe of eightli-daq- irradiation. I n the latter instaiice the nioi.tality la te w a s not significantly in- cr'c'a~ed above that of litter-rnatc control.; until the dosage w a s raiqed to 2000 r, hy which dosage all exposed embryos -were killed within D days. Thus, eighth-day embryos seem ahle to withstand the lethal effects of X irradiation up to a poiiit, lmt l iqond this, akx.uptly they lxeonie totally suscep- ti1)le.

hth !I 8 0 0 13 100 iitll ir il I 1 96 74 100

li i t l l 14 11 2.3 47 100 11th 6 i) 11 3 3 92 100

It i\ also apparent f rom the data in table 2 that as the w y c of the embi*yo at the time of exposure advances, susceptibility to tlie lethal acrion of S rays tends to decrease. Wllcreas 200 r was sufficient to kill all eighth-day enibryos within 3 days, 400 r was required to kill ninth- or tenth-day c.i i i l) i .p: , and GOO r to kill elevciitli-day embryos within a com1)arable period. Actnall;v, smaller doses than those indicated v-crc found to eanse toial or near-total mortality after ninth-, teiitli-, or eleventh-day treatment if the postirradia- tioii interval w-c:.e lcngtliened to 5 days or more. Two hun- drcd 1-oentgen units f o r ninth- and tenth-day embryos and 400 I' f o r eleventh-day embryo? should perhaps be regarded a.: the doses that ultiniately kill all exposed animals. V i t h lower doses the rates of mortality were also lower. Again a decreasing susceptibilitp \\-as iioted as the age of the embryo at the time of esp)o.:iirc atlvaiiced beyond the ninth day.

REACTION OF I ~ A T EmmPos TO RADIATION 17

ri i l ike these differences in the degree of response, shown by embryos a t the ninth, tenth, and eleventh days, the eighth-day embryo exhibited a diff ererit pattern of response. Mortality did not increase in a graded fashion as the dosage increased, hut suddenly became coinplete at 200 r.

€letardntio.n of growth . I n table 3 the deficiency in weight of irradiated embryos, as compared with their rionirradiated sib liiigs, is expressed as percentage retardation. Since such data for animals at diff ereiit postirradiation periods (from

TABLE 3

Retardation of growt7i 172 sztrttcang, t i radmlcd embi yos expressed as irienn 7cetqht of controls -mean wczqht o f trratl.

= percentage retardatton mean wtight of controls

DAY O F [RR*4DI 4TlO;U

8th 8th 9th 9th

10th 10th 11th 11th

POSTII<RAD. DAY

5 7 4 6 3 3

4 5

UOSY: ( r j

12.5 2 9 50 100 200

1 1 2 18 30 5 6 5 14

< I 1 1 7 6 10 1 7 27"

0 6 "3 0 14 % a

2 1 3 a 9

Only oiie aniinal surviving.

thtl second day to term) cannot be readily combined, data foi. two representative postirradiation days are presented. Despite the variability of these figure?, it is clear that ap- propriate doses of radiation caused considerable retardation in the rate of growth of surviving embryos. The degree of retardation tended to increase with the dosage, although not in direct proportion. I n most instances when retardation exceeded 307: the rate of mortality increased sharply. It is noteworthy that, uiilike the observations on mortality, the relation between retardation and increased dosage was as evident after eighth-day irradiation as after treatment at any of the older ages.

18 JAMES G. WILSON

The dosage required to cause a comparable percentage of retardation became higher as embryos of older age were irradiated. This is suggested by the data in table 3, but is more graphically shown by comparing directly the values obtained at the same interval (5 days) after irradiation with the same dosage (100 r ) on different days of gestation, as represented in table 4. Then, it may be concluded that from the eighth through the eleventh days the embryo becomes incrc a sing1 y resist ant to the grow th-r e t arding influence of X rays.

TABLE 4

Pcrcentag? retardat ion 5 days after irradiation with 105 r -~ ~~~

Day of irradiation 8th 9th 10th 11th Percentage retardation 30 20 a 14 2

a See Xilson, Jord:in, and Brent, ' 5 3 .

TABLE 5

T y p c s and pcrcenta.ge znc idmce o f malformations produced by irradiation o n drfferent days of gpstation

DOSK ( T i

2 5 50 100 200 DAY

8th None None

9th Eye, 6 Eye, 72; Brain, 9 ; spinal cord, 3

10th

11th

Eye, 1.1

None

Eye, 9 0 ; brain, 41; spinal cord, 27; heart, 20 ; face, 14 ; situs inrersus, 13 ; arotic arcs, 10 ; urinary, 5

Eye, 76 ; urinary, 11 ; brain, 3

None

(No survivors)

Eye, 100; brain 7 8 ; spinal cord, 67; situs inversis, 5 3 ; heart, 2 2 ; face, i i ; aortic arch, 11

Eye, 94; feet, 3 3 ; brain, 19 ; urinal v,

11; aortic arch, 1 I

Eye, 100 ; urinary, 7 7 ; brain, 5 4 ; spinal cord, 31; nor t ic arch, 2 3 ; ear. 2 3 ; tail, 23; lipart, 15 ; jaw, 15: fwt , 7


XaZf ornzations. Embryos of different ages displayed a striking difference in sensitivity to the teratogenic influence of X rays (table 5). No malformations were produced by any dosage given to the eighth-day embryo. This is in sharp contrast to the ninth-day embryo which was found to be highly sensitive in this respect. h great variety of malformations was produced by exposure to 100r o r 200r, and a dose as small as 25 r occasionally caused anomalous development of the eye. The tenth-day embryo was somewhat less sensitive but, nevertheless, did exhibit maldevelopment of several organs after treatment with 100 or 200r. By the eleventh day the rat embryo was still more resistant to the teratogenic action of X rays. Malformations were not produced by any of the doses used which were less than 200r. Undoubtedly, doses intermediate between 100 r and 200 r would have been effective, but the significant fact is that IOOr, which caused a high incidence of malformation after ninth- or tenth-day irradiation, had no effect on developmental processes at the eleventh day.

Whenever malformations occurred the eye was always the organ most frequently affected (table 5 ) . I n the lower range of effective dosage it was the only site of abnormality. After ninth-day irradiation the brain and spinal cord also were often anomalous but were less frequently affected after treatment on the tenth or eleventh day. Following tenth-day exposure the feet ranked next to the eye in order of frequency, and following eleventh-day treatment the urinary tract was the second most common site of malformation. Al- though the order of incidence varied, it should be noted that the same organs tended to be affected to some degree, whether exposure occurred on the ninth, tenth, or eleventh day.

Even though the same organs were often malformed by irradiation on different days, the types of malformation were not always the same. The prevalent types of ocular anomaly after either ninth- or tenth-day treatment were anophthalmia and extreme distortion of the optic cup, but eleventh-day treatment most often caused coloboma (persistent choroidal

20 J A M E S 0. WIISON

fissure). The brain mas differently affected by irradiation on all 3 days: on the ninth day, an a r ray of brain defects conse- quent to fusion of the hrain wall with the ectoderni; on the tenth day, hypoplasia and inequalities of growth in various parts of the forebrain; and on the eleventh day, eversion of the choroid plexus and other minor distortions. The kidneys were affected 1))- agenesis of one or both organs after ninth- day exposure, by fusion (horseshoe kidney) after tenth-day exposure, and by both types after eleventh-day exposure. Situs inversus, on the other hand, occurred only after irradiation on the ninth day, a t which age it was induced with considerable frequency. The feet were often malformed after treatnient on the tenth day but not at all by that on the ninth daj7 and only occasionally by that on the eleventh day. It is evident that the same organ may remain susceptible to the teratogenic action of irradiation for several days, but the specific type of defect produced is likely to change radically with increasing embryonic age a t the time of irradiation.

C0RIME:NT

The foregoing observations have shown that the capacity of the rat embryo to react to irradiation undergoes a qualitative change between the eighth and the ninth days of gestation and a quantitative change from the ninth through the eleventh day. Exposure of embryos at each of the older ages (ninth, tenth, and eleventh days) resulted in similar patterns of response: the rate of mortalitv, the amount of growth retardation, and the incidence of malformation.: all tended to increase as dosage increased. The major difference was in the degree of the response. ,411 the effects were more pronounced after irradiation on the ninth than on the tenth day, and more so on the tcnth than the eleventh day. Treat- ment on the eighth day, however, did not cause malformations at any dosage level, and the rate of mortality did not show a graded increase as dosage increased. This appears to con- stitute a different pattern of response from that observed when older embryos were treated.

ILEACTIOS O F RAT EAIRRTOS TO RADIATION 21

The differences in reaction at the various ages are undoubtedly related to tlie state of differentiation existing in the embryo at the time of irradiation. It should be recalled that differentiation had not visibly begun on the eighth day; on the ninth day it had progressed to the first stage, namely, formation of the germ layers ; on the tenth day active organogenesis had started in a few systems; and by the eleventh day organogenesis was moderately advanced in several systems (fig. 1). The manner in which the susceptibility to irradiation was influenced by the degree of differentiation is unknown. I t has been often generalized that susceptihility to irradiation tends to be inversely related to the degree of differentiation. The decline in sensitivity observed as differentiation progressed from the ninth to the eleventh day in tlie present experiment is in accordaiice with this generaliza- tion. An exception to the rule, however, is the more limited response of the eighth-day as compared with the ninth-, tenth-, and eleventh-day embryos. It appears, therefore, that the diversity of reaction cannot be explained simply in terms of greater or lesser degrees of differentiation.

In view of the particular susceptihility to irradiation of cells undergoing rapid mitosis, the possibility that changes in mitotic rate could account fo r tlie cliaiiging reactions of the embryos at various ages must be considered. Xitotic counts mere not attempted, but it was a matter of direct observation that cell division was frequent and uniformly distributed throughout the undifferentiated eighth-day embryo. A similar frequency and distribution was noted within the three germ layers of the ninth-day embryo. On the tenth and eleventh days, homerer, cell division became relatively more concentrated in the regions where active organogenesis was in progress. Although these observations are crude, they are, nevertheless, sufficient to indicate that mitotic rate cannot be closely correlated with the results obtained in these experiments. For example, on the eighth arid ninth days frequency and distribution of mitosis were similar but the reactions of embryos at these ages were strikingly different. On the other

22 J A M E S 0. WILSOK

hand, mitoses became increasinglv localized in particular tissues between the ninth and eleventh days, yet the reaction of embryos of these ages ivere basically similar, differing only in degree. Xany tissues, such as somites and digestive tract, undergoing rapid proliferation at this time were never affected by malformation. Other structures that mere not even recognizable as primordia (true of all organs on ninth day), hence, not specifically proliferating at the time of irradiation, were often later affected by malformation.

Since differentiation occurs primarily at the cellular level, a more adequate interprctation may be reached by considering the various biological reactions to irradiation shown by rapidly proliferating cells. It is generally agreed that proliferating cells, such as comprised these embryos, may show any of three such reactions: (1) death may occur without further cell division, (2) mitosis may be temporarily inhibited but later resumed at an approximately normal rate, or (3) subtle genic alterations (somatic mutations) may occur, but not be maniiest until later by faulty differentiation of descend- ant cells.

The thresholds of sensitivity of these three reactions need not necessarily remain in a set ratio, nor need they change at the same rate as differentiation progresses. The change observed in the embryonic response a t different ages can be explained by postulating that, as differentiation began and proceeded, the component cells of the embryos changed their propensity to show one or the other of the cellular reactions enumerated above. For example, the only observed effect o f irradiation on the eighth day mas smallness of size, except when the dose was raised to 200 r, a t which level total mortality occurred. The simplest interpretation is that all cells which were “hi t” ow affected in any manner either died or lost the capacity to proceed with differentiation. The elimina- tion of a moderate number of these totally undifferentiated blastomeres would result in nothing more than a setback in the developmental schedule equivalent to the time required to replace the missing cells. This alone, or perhaps in com-

R E A C T I O N O F RAT EMBRYOS TO RADIATION 23

bination with some mitotic delay in the surviving cells, could account for the retardation later observed. Such destruction of blastomeres presumably could be tolerated up to a critical number, with no consequence other than generalized growth retardation ; but destruction beyond this hypothetical point might be expected to cause embryonic death. The absence of an increase in mortality after all smaller doses and the total mortality after 200r are results compatible with such an explanation. Malformations could not occur if all cells were undifferentiated, since a prerequisite f o r later maldevelopment mould be damage to or loss of cells with predetermined potentialities.

By the ninth day differentiation has begun and no longer are all cells in the embryonic body alike. In addition to hav- ing acquired various developmental potentialities, it is proba- ble that different groups of cells have also become variously susceptible to irradiation. Very likely many cells in the ninth- day embryo, as in the eighth-day embryo, were promptly killed by the irradiation, but the destruction of cells already induced to form specific parts of the developing body could be less well tolerated than the loss of totally indifferent cells. This destruction of predetermined cells would lead to tissue defiviencies in later development (one type of malformation) or, if cell death were prevalent in regions of critical poten- tiality for development, death of the entire embryo would be the consequence. The latter would in part account for the graded mortality rate as dosage increased. It is possible, however, that some affected cells in the ninth-day embryo were not killed, but survived and continued to proliferate more or less normally until some critical event in organ formation was reached. The malformations which later developed in the ninth-day embryo may include some derived from such cells that survived irradiation but bore altered developmental potentialities. As indicated, malformations can result from the deletion of some or all of the cells already induced to form a particular organ, but this mechanism is thought to entail specific deficiency of tissues or parts in the abnormal

24 JAMES G. WILSON

organ. Scveral of the observed malformations, such as coloboma, double aortic arch, transposition of cardiac vessels, cryptorchid testes, situs inversus, etc., did not appear to in- volve such deficiencies. They could be considered to have resulted from aberrant developmental processes initiated by certain of the cells that survived irradiation but suffered genic alterations which only later became evident in defective organ formation. It is also possible that some such “somatic niutatioris ” had lethal effects and thus secondarily contributed to the increased mortality among the irradiated embryos.

On the tenth day differentiation had progressed to the point that overt organogenesis was present in certain systems. Despite the relatively great developmental advance, the tenth- day embryo reacted in a similar nianner as did the ninth-day embryo, the only significant difference being that a higher dosage was usually required to elicit a comparable response. This may be taken as an indication that the susceptibility of the cells to the various radiation-induced effects was also similar, but that the threshold of reactivity to such effects had risen. By the eleventh day diff erentiatioii had reached the stage that organogenesis was moderately advanced in most of the niajor systems. Nevertheless, the basic response to irradiation was again essentially the same as at the ninth and tenth days, except that the eleventh-day embryo was even less sensitive to a given dose than the tenth-day embryo. Thus the reaction of the rat embryo to irradiation between the ninth and eleventh days of gestation remains qualitatively the same, but during the period sensitivity to the various radiation-induced effects diminishes as differentiation proceeds.

The qualitative change which occurs between the eighth and ninth days of gestation has been shown to coincide with the beginninq of cellular differentiation. Although no observations were made on embryos younger than 8 days, it is assunied that they, being equally undifferentiated, would react in a similar fashion as did the eighth-day embryo. As regards the occurrence of malformations, this assumption

REACTION O F RAT EMBRYOS T O RADIATION 2 .j

receives support from the results of other investigators. Rus- sell ('50) observed that the irradiation of mouse embryos prior to day 64 did not cause maldevelopment. If due allow- ance is made for the relatively faster developmental schedule of the mouse (approximately 24 hours), the change in susceptibility is reasonably close to that observed in the rat. Likewise, Hicks ('53) was unable to find malformatioiis in young rats irradiated during the first 8 days of gestation, although exposure on the ninth day and subsequently caused a variety of anomalies of the nervous system. Job et al,. ( ' 3 5 ) also observed a change in the susceptibility of rat embryos to maldevelopment induced by radiations, but these awthors indicated that the change occurred between the seventh and eighth days. Regardless of the precise time at which it occurs, however, all available evidence indicates that the reactions of both rat and mouse embryos are somewhat different during the first third and during the subsequent parts of the gestation period. The present experiment has defined this difference, not only in terms of the incidence of malformations, but also in terms of the rate of niortality and the degree of growth retardation resulting from irradiation in either period. These differences in reaction have been attributed to changing susceptibilities to the various biological effects of irradiation as the embryo passes from the stage of total undiff erentiation to that of beginning differentiation.

SUMMARY

Rat embryos on the eighth, ninth, tenth, or eleventh day of gestation were exposed to doses of 12.5-6001- directly through an abdominal incision in the mother. Lead plates were arranged so that selected embryos could be irradiated while the mother and remaining embryos were shielded. The irradiated and nonirradiated embryos were removed one to several days later, or at term, and compared as to weight, the presence of malformations, and the rate of intrauterine mortality.

26 J A M E S G. WILSON

Exposure on the eighth day had a qualitatively different effect on subsequent development than did similar treatment on the ninth, tenth, or eleventh day. The only residual effect after doses less than the totally lethal one (200r) on the eighth day was retardation of growth. Irradiation at the older ages, however, resulted in increases in the number of malformations and the rate of mortality,. as well as in retardation of growth, as the dosage increased. This difference in the pattt.rn of response is attributed to a change in the susceptibility of the cells to the various biological effects of irradiation, as the embryo passes from the undifferentiated state on the eighth day into the early stages of differentiation which begin on the ninth day.

Embryos exposed on the ninth, tenth, o r eleventh day all displayed a similar pattern of response, but sensitivity to thc various radiation-induced effects diminished as the embryo grew older. To produce a comparable response a higher dose was required on the tenth than on the ninth day, and li l~e~visc, a higher dose on the eleventh than on the tenth day. These quantitative differences mere ascribed to an increasing threshold of sensitivity to the same radiation-induced effects as differciitiation proceeds, once it has started.

DIXCTT8610.N

Clicririnnn HAMRLTRGER : TVe are confronted immediately with the formidable problem of teratogenesis. I should like to focus ~7our attention on one aspect: To what extent can the radiation effects be attributed to interference with mitotic activity? During this discussion it occurred to me that we should pay considerable attention to other components of embryonic development, such as : morphogenetic movements, inductions and segregations, that is, the orderly apportioning of materials. For instance, it seems to me by no means obvi- ous that such abnormalities as anophthalmia or microphthal- mia are due to the loss of cells in the eye anlage. They could be due as well to the abnormal apportioning of medullary plate material, or they could result from an inhibition of the

REACTION O F RAT EMBRYOS T O RADIATION 27

evagination of the optic vesicle, or from abnormal action of the medullary plate inductor, that is, the subjacent mesoderm.

WILSON: First I shall comment on your suggestion that changes in mitotic activity might be involved in producing these results. We did make some crude observations on mitotic rates, enough to indicate that the observed differences in embryonic reaction cannot be attributed to differences in mitotic rate at the time of irradiation. The ninth-day embryo which had begun differentiation showed about the same frequency and about the same uniform distribution of mitosis as did the undifferentiated eighth-day embryo. Yet, you will recall that the large qualitative difference in response to irradiation occurred between the eighth and ninth days of gestation. On the other hand, mitotic activity became more and more localized in regions of active organogenesis on the tenth and eleventh days, but the reaction of the embryo at these ages was only quantitatively different from that on the ninth day. Furthermore, the primordia of the digestive tract showed great mitotic activity a t all times, but the digestive tract was not affected by malformations. There was nothing in these appraisals of mitotic activity to indicate that either the rate or distribution of mitosis was responsible for the type of response shown by the embryo.

GENNARO: As excellent as this technique is, it does not eliminate the possibility that there is some effect produced through interruption of the normal physiology of the placenta, perhaps through the decreased oxygenation or impaired nutrition of the embryo.

Have any vitamin A or carotene determinations been carried out on the embryonic tissue itself because of the high incidence of optic deformation?

WILSON : Regarding the apparent similarity between the ocular malformations that were produced by X rays and those produced by vitamin A deficiency, with which I am quite familiar, I can assure you that they are not the same histologically. They are similar only in that on the eleventh day we did get coloboma, and coloboma is a common occur-

2s JAMES G. WILSON

rcnce after vitamin A deficiency; but even the types of coloboma mere different.

The coloboma seen after vitamin A deficiency is characterized riot only by the usual persistent clioroidal fissure but also by foldings eversion, and cysts of the retina; and of course there is always the associated postlenticular fibro- plasia which map be called the hallmark of vitamin A deficiency. I feel that the vague similarity does not justify bringing vitamin A deficiency into the picture. No carotene determinations were made.

Your question about the placenta is good and one that needs to be looked into. Fixed placental material is available but has not yet been subjected to critical study.

GR~-EKWALD : The mechanisrn by ~ d i i c h these malformations are produced is obvioi is l~ a matter of conjecture. I am not quite sure that abnormal differentiation of cells is involved here, hccause even if we have fused kidneys, the tissues are still ~iormal kidney tissues. I suspect that we are dealing here with the change in the pattern of growth. I do not think thore is too much evidence for ahnornial differentiations.

I should like to make another suggestion. Within a day, development in a rat proceeds very much as can be seen by comparing the eighth-, ninth-, arid tenth-day embryos. I -\\.oiider wliethcr it mild he a good idea, if this is feasible, to amputate the top of one of the horns of the uterus and, a t tlie time of the operation, to have tlie embryos fixed in order to Im0117 exactly at what stage you operated.

\T'II,SOS : As Dr. Gruenwald suggests, tlie fused kidneys which we observed seemcd to contain normal renal tissue as far as one could tell by examination nnder low and moderate powers of magnification. TTcl did not do cytological studies. T did not intend to imply that there had been abnormal differentiation within tlic kidney primordia. Bly suggestion was that perhaps such inalformatioris as these - malforniations \vliich are not in any way preformctl or simulated in prior development - arc perhaps the result of somatic mut a t ' ions. Thc malformation^ could have resulted from deviations in

REACTION OF RAT EMBRYOS TO RADIATION 29

the developniental pattern of the whole region, due to some basic alteration in the chromosomal or genic makeup of pre- cursor cells. The developiiiental deviations could consist of abnormal patterns of growth, as you have suggested, but possibly also of abnormal differentiation somewhere in the caudal end of the body, as I have suggested.

Yes, I think it mould be possible to remove an embryo at the time of operation to determine the exact stage of development.

BRUNST: What is your explanation of some of the numbers in your slides dealing with inortality after irradiation, such as 8 days, 0% ; 9 days, 11;4 ; 10 days, 147; ; 11 days, 0% - all after a dosage of 50 r ? It is not clear to me.

Another question: Are the sizes of cells in the irradiated tissue normal? I ask hecause many observations show that the size of cells in irradiated tissue is not normal. Are these inalforrnations the result of dainago to the differentiation process or the result of irihibitioiis of growth and cell damage ?

I was glad to hear about these types of defects in the de- velopineiit of eyes; this is in accordance with my data. In the matter of development of the retina after irradiation, did you observe sortie inalformation in the retina aid, particularly, in the rods aiid coiies layer7 Of course there are some papers which descrilied the special sensitivity of the retiiia. Is this true according to your material?

I think it is very interesting to investigate the effect of local irradiation with your method. Thin lead strips can be uscd to protect part of the embryo, aiid in such a way the effect of X irradiation inay be investigated.

TTILSON: I think you have taken the data out of context, hence the apparent inconsistency. l lortali tg rates in rat embryos are highly variable, a s you noted from the control data. Rather than concentrate on a single dosage or age group one has to compare over-all effects when a graduated series of doses or several age groups are used. That is what I tried to do.

30 J A M E S G. WILSON

Eegarding your second question, I have no data on the size of cells in irradiated as compared with nonirradiated animals. Likewise, I am unable to answer your question on the detailed structure of the retina.

I shall not attempt to repeat all my speculations on the manner in which the malformations were brought about but shall simply say that I feel that both abnormal differentiation as well as abnormal growth patterns may have been involved.

WEISS: As to the question of mitosis, of course we know, more or less, that the process of cell division falls into two phases - the synthesis of protoplasm (increase of the cell) and the triggering off of the act of division. Is there perhaps a difference in the periods before and after 8 days so that in the earlier period there would be something corresponding more nearly to the act of cleavage, where previously syn- thesized material is broken up into cells ; whereas, only during the later period the process of increased synthesis would be involved7 Might this not explain this rather sharp cut between the two processes?

Then I have a purely terminological question in regard to what Dr. Gruenwald said. A fused kidney is described as a teratoma. That only means we are perhaps looking at the more conspicuous, but not at the crucial, part of the system, because the fused kidney itself may have nothing wrong with it. The effect may be on the surrounding tissues. I think it is entirely illegitimate to pick out arbitrarily, so to speak, one part of the system, in this case the kidney, and focus on it just because it stains better or because it is more conspicuous, and imply that it is the primary site of action. The primary defect may be in the surroundings. I n that case kidney fusion is merely a secondary consequence.

A third point, there was this remarkable falling off of sensitivity to teratogenic effects with age. Is that perhaps merely a technical problem correlated with the increased mortality? Since there is increased mortality with age, by that time those embryos which did survive were less likely to


shon- tciratogenic effects because if they had been defective they would have died and been listed among the dead ones. Therefore the question arises whether this is merely an illu- sion of the figures.

WILSON : You have suggested the interesting possibility that the change in type of embryonic reaction between the eighth and ninth days may be related to a change in the synthesizing activities of the cells. I can think of nothinq in my data that would speak against it. I do not agree with your further suggestion that the falling off of the embryo’s sensitivity to the teratogenic effect of irradiation was due to an increase in mortality as the embryos grew older. Rlor- tality did not increase in older embryos. Actually, sensitivity to the lethal effect of irradiation diminished with age almost as rapidly as did sensitivity to the teratogenic effect.

BUTLER: I am interested especially in the remarks about the relation effects of irradiation on cell proliferation and on differentiation, since well before the advent of the atomic age, I was interested in the effect of radiation on various aspects of growth. I t would be especially interesting if, in this type of work on mammals, individual organs or individual areas were chosen for considerable detailed study of the effect of radiation on differentiation. In this type of study we have found that irradiation of the limb of an am- phibian embryo, just a t the time the limb area is being determined and differentiation is about to become evident, will not greatly interfere with the development of a limb. But the limb which develops will not be a normal limb; differentiation of certain limb structures will be interfered with. A limb of considerable size may develop, but be completely de- void of digits. We are dealing, in other words, with the influence of radiation on morphogenetic activity.

Similar experiments can be done on limb regeneration. When a urodele limb is amputated a blastema forms and regeneration ordinarily progresses rapidly. By irradiating a regenerating limb one can govern, so to speak, the type of morphogenetic expression. For example, by irradiating at

32 JAMES G. WILSON

the proper time with proper dosage, a two-digit hand, in- stead of a three-digit or four-digit hand can be produced. It is possible in such experiments to permit mitotic activity and increase in cell number, but at the same time rather pre- cisely to govern morphogenesis and differentiation. It would be very interesting and instructive if experiments of such a nature could be carried through on organs of the mammalian embryo-such organs as the eye, for example.

I n Dr. Wilson's results we noted that in the mammal irradiation was done at such a time as to prevent the develop- merit of the forelimb, and yet in the same embryo a fairly normal hind limb developed. The forelimb in the normal sequence of events is established earlier than the hind limb.

KIMBALL: I think there are great difficulties, from the genetic point of view, in the somatic mutation hypothesis. Viable somatic mutations should be induced at all stages of development, not only after the eighth day. Furthermore, the mutations should be scattered a t random among the cells. Cell-lethal chromosome aberrations might be considered as a cause, but not somatic mutation in the usual sense.

VILSON: Many of the questions seem to hinge around my conjecture regarding somatic mutations as possibly being involved in producing some of the malformations. That is my naive way of looking a t it. I find it difficult to see how all of the effects can be attributed simply to cell death or to slowed mitotic rates. Since I am neither a geneticist nor an experimental embryologist, I am really not competent to dis- cuss the matter critically.

HESTON: I am, of course, interested in the comparison of these abnormalities that occur following treatment, with those that occur without treatment and have a genetic basis - in particular, the absence of the kidney. I n the highly inbred strain A x C 9935 (Ir ish) approximately 35% of the animals lack one kidney, or in some cases, have a poorly developed kidney. It would appear that this is another character the incidence of which is established in the inbred strain by the genetic constitution of the strain.

REACTION O F BAT EXBRYOS TO RADIATION 33

But in this particular condition, riot only is the kidney missing but also various portions of the genital tract. I n the females, the corresponding horn of the uterus or a proxi- mal portion of the horn, or the ovarian capsule may be missing, whereas in the male, the corresponding epididymis or vas deferens may be missing. In your treated animals was the absence of the kidney accompanied by such abnormalities of the genital tract?

WILSON : Yes, associated with the renal anomalies there occasionally mere defects in other urogenital organs. Males with agenesis of one kidney usually had cryptorchid testes and faulty or absent male ducts on the side of the missing kidney. Aside from this, however, I do not recall other situa- tions in which renal anomaly mas regularly accompanied by defects in the genital system.

CARTER: The suggestion that somatic mutation may play any but a very minor role in the induction of embryonic abnormalities seems open to question, at least until more is known about the developmental mechanisms involved. Horse- shoe kidney may be taken as a case in point. The mechanism by which it developed in Dr. Wilson’s rats is unknown; but the development of a similar condition has been investigated in a genetically abnormal mouse strain. There it was found to be the remote result of an abnormality of the umbilical arteries, which led to fusion of the kidneys through mechani- cal interference with renal migration. It must at least be borne in mind, therefore, that the organ in which an abnormality is seen is not necessarily that in which the first change occurred.

WILSON: I agree that the structure showing the greatest deviation from normal is not necessarily the one first affected. You are right in saying that the mechanism by which the horseshoe kidneys developed is unknown, but in studying these I paid particular attention to the matter of the umbilical arteries. There was no indication of abnormal size, location or proximity of these vessels after the kidneys had fused, but irradiated embryos have not been studied at ages

34 J A M E S G . WILSOTS

when the kidneys were in process of passing between the umbilical arteries.

L. RUSSELL: I should like to take issue against interpreting these abnormalities by the production of somatic mutations (and thus add my criticism to Dr. I<imball's). First of all, it is necessary to assume directed niutations in order to give similar abnormalities in different irradiated animals. Such an assuniption is against the known facts of radiation-induced mutation.

Secondly, it is not possible to account by gene mutation f o r the frequency with which abnormalities follow irradiation of embryos. Even taking niammalian germinal mutation rates, which apparently are higher than Drosophila rates (Russell, %I), and assuming the same rates in somatic cells, 200 r would give about two mutations per body cell. With the averagc degree of dominance being presumably quite low, only a very minute fraction of the cells would thus be affected in the diploid somatic tissues.

I believe that it is possible to postulate death of cells as the primary cellular effect of radiation in embryos. Such a primary change would touch off exceedingly complex chains of development and it is not necessary to assume that the finally observed abnormality is in the nature of a deficiency of tissuc. These points are discussed in more detail in my paper (This Supplement).

I should also like to comnient on the general question of mitotic rate studies. I believe it would be most important to coinpare rates of different regions of an embryo at any given stage and correlate the findings with abnormalities observed to follow irradiation of that stage. On the other hand, it seems to me that only little could be gained from looking for possible over-all differences between an eighth- and a ninth- day embryo, as Dr. Wilson has suggested. The response of the former seems certainly to be due to the great regulatory powers of the early mammalian embryo and correspondh closely to the mouse results for days 3 to 5$ postconception (Russell, '50).


Finally, I should like to answer Dr. Brunst’s question about the feasibility of irradiating only selected parts of embryos. Raynaud and Frilley, in France, have directed a very thin pencil X ray to the head region, particularly to the developing pituitary of rat embryos.

EDDS: Since the issue of possible differences in mitotic rate has been raised, it is important to draw attention to the work of Corliss ( ’ 5 3 ) . His observations of the mitotic activity in the rat embryo during the ninth day of development bear on the results Dr. Wilson has reported. Corliss finds that during the ninth day, there is a progressive increase in the number of dividing cells per unit volume of tissue. The increase is small during the early part of the day but quite pronounced during the second half of the day. Mitotic activity is not differentially localized in any one region, however, but is uniformly distributed through the embryo.

WAELSCH: It is very interesting that Dr. Wilson’s ir- radiations of rats on the eighth day of pregnancy produce either complete lethality, i.e., death of the embryo, or new- borns which are perfectly normal. Dr. Wilson, have you looked at those embryos just before or after death; and did they perhaps appear abnormal in any way just before they diedt It is really not too surprising that those embryos which do survive appear perfectly normal; the reason f o r it is that the rat embryo at that stage is probably still in a condition in which its regulatory power is strong enough to overcome the damaging effect of the X rays in all its developmental systems, in contrast to later stages when regulatory power has heconie considerably weaker.

Have you looked at any of the stages of the developing nervous system preceding the appearance of exencephaly and did I understand you to say that the brain was fused with the overlying ectoderm? I wonder whether the neural folds do close before this condition, i.e., exencephaly arises, or whether they never close, or whether there has not been a chance to look a t embryos before the abnormality appears?

36 JAMES G. WILSON

FILSON: As to your question about dead embryos, whenever the embryo was still in a sufficiently good state of preser- vation to make any sort of histologic study possible, we did section and study it. I n such eighth-day embryos we have found nothing that we could identify as a malformation. Your suggestion that the reparative power of the eighth-day embryo might be superior to that of the older embryos is certainly a possibility.

The neural folds were seen to be closed in all early embryos that were examined. Frequently on the second, third, and fourth days postirradiation, however, a closed neural tube was seen to be fused with the overlying ectoderm. Thus we feel sure that the defects in the brain and the spinal cord were not related to primary failure of closure of the neural tube, but were somehow the result of fusion between ectoderm and neural tube.

HICKS: I n answer to those last two questions, we have given 400 r to eighth-day embryos in zctero and usually found them to be necrotic some hours later. I shall show in my paper (This Supplement) that in later embryos selective necrosis occurs following irradiation; e.g., on the ninth day the an- terior neural folds are vulnerable.

LITERATURE CITED

CORLISS, C. E. A study of mitotic activity in the early ra t embryo. J. Exp. Zool., 162: 193-227.

ITIcKS, S. P. 1953 Developrncntal nialformations produced by radiation. Am. J. Roentgenol., 6 9 : 272-293.

JOB, T. T., G. J. LEIBOLD, AND H. A. FITZMAURICE 1933 Biological effects of roentgen rays. The determination of critical periods in mammalian development with X-rays. Am. J . Anat., 5 6 : 97-117.

X-ray induced dcvelopmental ahnormalities in the mouse and their use in the analysis of embryological patterns. I. External and gross visceral changes. J. Exp. Zool., 124: 515-602.

Symp. Qiiant. Eiol., 1G: 327-336.

in rats by roentgen rays. Am. J. Roentgenol., 5 7 : 455-463.

1953

RUSSFLL, L. B. 1950

RUSSELL, W. L. 1951 X-ray-induced mutations in mice. Cold Spring Harbor

WARKANW, J., AND E. SCHRAFFF~NBERGER 1947 Congenital malformations induced

R E A C T I O N O F RAT EMBRYOS TO RADIATION 37

WILSON, J. G., R. L. BRENT, A N D H. C. JORDAN 1953 Differentiation as a de- terminant of the reaction of rat embryos t o X-irradiation. Proc. Soc. Esp. Biol. 85 Med., 88: 67-70.

Effects of irradiation on embryonic development. 11. X-rays on the 9th day of gestation in the rat . Am. J. Anat., 92: 153-188.

WILSON, J. G., AND J. W. KARR 1951 Effects of irradiation on embryonic development. I. X-rays on the 10th day of gestation in the rat. Am. J. Anat., 88: 1-34.

WILSON, J. G., H. C. JORDIAN, AND R. L. BRENT 1953

differentiation and the reaction of rat embryos to radiation

Documents