VARIATION I N MOUSE EMBRYOS O F 8 DAYS GESTATION

EZRA ALLEN AND E. C. MACDOWELL Curnegie Institution of Washington, Department of Genetics,

Cold Spring Harbor, Long Island, New Pork

TWO PLATES (FIFTEEN FIGURES)

INTRODUCTION

In the mouse the primitive streak generally appears near the close of the seventh day of gestation (Sobotta, '11; Mac- Dowell, Allen and MacDowell, '27). The eighth is consequently the first day in the history of the embryo proper. This paper records data on the variation in size and number of somites at the end of the eighth day.

When weighing embryos of triple hybrid mice for the study of prenatal growth (MacDowell, Allen and MacDowell, '27), individuals of only two 8-day litters were weighed separately. The other 8-day litters were weighed in groups. The variation in litter weight is shown in figure 1 of the paper referred to. The average weight in grams of eleven litters consisting of ninety-three embryos was 0.00008. In one of the litters weighed by individuals, the weights in grams varied from 0.00002 to 0.00025 ; in the other litter, from 0.00005 to 0.0004. In the pres- ent paper are shown photographs of several litters of 8-day Bagg-albino mice, which confirm the possibility of wide varia- tion in mice embryos at this age. To how great an extent the early differences in size and degree of development may be compensated fo r in the later developmental processes is at present conjectural. On account of its relatively small size the mouse embryo offers excellent opportunity for study of the histology and cytolo,gy of the various organs, yet much remains to be discovered of these conditions at various stages in the history.

165

166 EZRA ALLEN AND E. C. MACDOWELL

MATERIAL AND METHODS

The material for the data recorded in this paper came from the MacDowell colony at the Department of Genetics of the Carnegie Institution of Washington, a t Cold Spring Harbor‘, New York. The data for the number of somites are from ob- servations made on the triple hybrid mice employed in the study of prenatal growth (MacDowell, Allen and NacDowell, ’27). The photographs are of white mice of the Bagg-albino strain. All the embryos were timed within an hour of insemina- tion. Two males were placed with six females. A t the expira- tion of an hour the males were removed and the females examined. Those which showed vaginal plugs were isolated in separate cages until killed for embryos. The time of sacri- fice was based on the end of the hour during which the males and females were together.

The method of preparing the embryos for photographing was to kill the mother by ether or by breaking the neck by quick pressure with the operator’s thumb on the cervical re- gion. The uteri were immediately removed to Locke’s solution in a low Stender dish, the bottom of which was covered with black wax. Under a low-power binocular microscope the uterine horns were turned ventral side up, stretched slightly, and pinned to the wax with the niesometrium flattened. The uterine muscles on the exposed surface were then carefully cut away from the decidual capsules. A shallow incision was made in the capsule wall to help admit the fixative, a small amount of which was immediately applied directly to the cap- sules by pipette. Then, by aid of needles and iridectomy scis- sors, the exposed side of the capsule n7as dissected away, additional fixative being pipetted on during the dissection (fig. 5). When the embryo was uncovered the mixture of Locke’s solution and fixative was replaced by pure fixative. In some cases the photographs were taken before the fixative was finally added. In others, if Bouin’s or B-15 fixative had been used, the uteri were left about 1 hour in the fixative and then run up to 5076 alcohol, after which tlie dissection was com- pleted. The decidual tissue dissects more easily in 50% than

VARIATION I N MOUSE EMBRYOS 167

95 I 31 10 I 10

in 70% alcohol. After photographing at the desired magnifica- tion the uteri, with the embryos in situ, were stored in separate dishes, or the embryos removed for sectioning.

The pliotographs chosen for reproduction represent only a few of the litters which showed marked variation. A litter at 8s days gestation is introduced for comparison in size (fig. 6).

It is difficult to fix and dehydrate embryos of this age with- out some distortion. Of those reproduced, figures 4 to 6 and 11 to 15 show the least-practically none. The gradual addition

4

TABLE 1

Variat ion in nwmber of somiles in ninety-fioe embryos ilk nine l i t ters of triple hybrid mice a t end of 8 days of gestation

c

e: e

5 - 8-2 8-3 8-4 8-5 8-6 8-7 8-8 8-9

8-10 9 -

1

NUMBBR OF S031ITES PER EMBRYO

2

4

-

5

2 2

2 2

2

1 2 T 1

7 -

8

-

1

- 1 -

of fixative to Locke’s solution as described above usually re- sults in less distortion than if the embryo is completely uncovered in Locke’s solution, removed from the capsule, and then placed in the fixative.

The method used for the triple hybrid embryos recorded in table 1 was to take one capsule at a time from the uterus and remove the embryo under Locke’s solution as described in the paper by MacDowell, Allen and RIacDowell ( ’27). Meanwhile the uterus remained in the body of the mother until all em- bryos had been removed. Sornite determination was made at

168 EZRA ALLEN AXD E. C. MACDOWELL

once under a binocular dissecting microscope while the embryo was still alive.

For purposes of record, the embryos were numbered by posi- tion in the uterine horn. Thus R-1 refers to the embryo nearest the vagina in the right horn. L-2, to the second embryo in the left horn.

OBSERVATIONS

In some litters the embryos in both horns of the uterus, whether equal in number or not, were about the same size. I n some, as in figure 5, they were larger in one horn. I n others, variations were almost the same in each horn (figs. 1 and 2). Position in horn, number in horn, or number in litter seems to have no influence on size or degree of development.

I n the litter shown in figure 5, variation ranges from the primitive groove stage (R-2) to the 4-somite stage (L-1 and R-5); the other embryos vary between these two extremes. 33-4 is in about the same stage as the two embryos shown en- larged in figure 7-early head fold. L-2 and L-3 are a little farther along than R-4, but with no somites. L-2 is shown enlarged in figure 8. R-3 and R-6 are slightly farther advanced than R-4, but not so fa r advanced as R-5 and L-I.

I n figures 1-3, no embryo has reached the somite stage. Among the Ragg-albino strain, none at this age was found with more than three somites. The somite variation in the triple hybrid stock is from one to eight, based on ninety-five embryos (table 1). Of the normal embryos, thirty-one had no somites ; their development varied from the primitive groove to the head-fold stage. Only one embryo had eight somites. The number of embryos is too small to establish a mean num- ber of somites.

No other data concerning stage of development are consid- cred in this paper. The photographs indicate that gross size is an approximate index of somite number. By the end of another 12 hours (84 days of gestation), the somites may vary from five to fourteen (one litter).

A type of variation was found in which one capsule might contain more than one embryo, each with its own yolk sac

VARIATION I N MOUSE EMBRYOS 169

(fig. 3, L-8 and 9, and fig. 13). Generally in such cases one embryo was smaller than the other. The two shown in figure 13 were the nearest in size of any found at this age. Among older embryos, relatively few cases of two or more embryos in a capsule were found. As these were generally of nearly the same size, it was concluded that if two embryos in one capsule develop unequally the smaller one does not survive. Some capsules have been sectioned which externally appeared to have only one embryo but in reality two were present, one of which might have stopped its growth very shortly after implantation. One case showed two embryos at different stages of development, one at a right angle to the other. Various types of malformation have also been observed in embryos of 8 days gestation as well as in earlier stages.

LITERATURE CITED

MACDOWELL, E. C., EZRA ALLEN AND C. G. MACDOWELL 1927 The prenatal growth of the mouse. J. Gen. Physiol., vol. 11, pp. 57-70.

SOBOTTA, J. Die Entwicklung des Eies der Maus vom ersten Auftreten des Mesoderms a n bis zur Ausbildung der Einbryoiialanlage und dem Auf- treten der Allantois. Arch. mikr. Anat., vol. 78, pp. 271-352.

1911

EXPLANATION OF PLATES

R and TJ refer t o right and left uterine horns. Numbering of embryos begins with the one next the ragina.

PLATE 1

EXPLAXATION OF FIGURES

Ventral views of right and left uterine horns after removal of ventral half of capsules. I n some figures only one horn of a given uterus is shown. 911 embryos are from the Bagg-albino strain. X 4.

Figures 1-5, &day embryos. Figure 6, 84-day embryo. 1 and 2 Series 8-5. 3 Left horn of 8-8. 4 Lef t horn of 8-1. 5 6

Right and left horns of 8-7. Left horn of 8+-1, with one partially dissected embryo, R-1.

170

VARIATION IN MOUSE EMBRPOS EZR.4 ALLEX A I l ) E. C. 31AP D O W E I ~ l ~

171

PT2A’l’E 2

ESPI,ANATION OF FlGUHES

Individual e m b r p s froiii litters (wit,li one ercrpt ion) slio\vn in plate 1. x 12.

I

8 9

10 11 12 13 14 15

Series no. 8 8,

8-7 8-8 8-1 8-7 8 i 8-2 8;-l 8:-1

rmhryos L-6 and i (coiiipare fig. 3 ) . L-2 (compare fig. 5). L-2 (tonipare fig. 3 ) . I,-? and 3 (compare fig. 4) . H-6 (coinpnre fig. 5 ) . I,-l (roinpare f ig . 5 ) . I A and 6 (not slio\~ii i n plate 1). L-4 (cwnpare fig. 6 ) . IA (coinpare fig. 6) .