A minute deficiency in linkage group II of the mouse, involving the short-ear locus and at least one other functional unit, was found to be lethal by day 8 after coitus when homozygous. The earliest time at which mutants could be detected was 7 days 16 h after coitus. Histologically, the mutant embryos showed overgrowth of ectoderm and trophoblastic giant cells. The mesoderm was almost entirely lacking and the mutant embryos were smaller than their normal litter-mates.

It was concluded that the mutant was first expressed morphologically late on day 7 after coitus and that its primary effect was to stimulate growth of extra-embryonic ectoderm.

In the course of the radiation experiments on mice conducted at this laboratory by W. L. Russell, over 200 mutations have been generated in the dilute short-ear region of linkage group II. More than 100 of these mutations have been used recently by L. B. Russell (1970) to construct a complementation map of this region. An interesting result of this work is the discovery of complementing embryonic lethals. Among several independent short-ear lethals that yield complex complementation patterns is the mutant 5RD300H, which L. B. Russell (1971) concludes to be a minute deficiency involving the short-ear locus and at least one other functional lethal unit, l4. The lethal mutant, 5RD300H (hereafter designated se1), yields viable short-eared young when it is combined with several independent dilute short-ear deficiencies that are preimplantation lethals (L. B. Russell, unpublished) or with certain other short-ear lethal mutants (L. B. Russell, 1971). It is therefore of interest to determine the time and mechanism of action of se1. A preliminary report of this work has appeared earlier (Dunn, 1970).

The lethal is maintained by intercrossing + se1/d+ mice (where d = dilute, and + = wild type). The stock is tested periodically to assure that se1 has not been lost through recombination. All mice used in these experiments were segregants of the above cross.

Embryos of known ages were obtained by caging males and females of the desired genotypes and checking the females each morning for vaginal plugs. The day a plug was found was designated day 0. Hour designations were only approximate, being calculated from the middle of the dark period, which is correlated to the time of ovulation (Braden, 1957).

Pregnant females were killed at the indicated times by cervical dislocation and their uteri were removed. Nine days after coitus, embryos were scored as (1) living if a heartbeat was seen, (2) having died after day 8 if the embryos had completed rotation but had no heartbeat, or (3) having died before day 8 if an egg cylinder stage or no embryo was found in the decidua. Eight days after coitus, implants were scored as living if a well developed embryo was present, or dead if only an egg cylinder or no embryo was found.

Histological preparations were made using the technique of Fekete, Bartholomew & Snell (1940) with minor modifications. The embryos were sectioned at 10 μm and stained with hematoxylin and eosin.

Table 1 gives the results of the examination of uterine contents on day 9 after coitus. In all four types of matings, embryonic death is high after day 8. Only in the +se1/d+ × +se1/d+ mating is there a high death-rate prior to day 8.

Table 1.

Survival of implants to day 9 after coitus

Survival of implants to day 9 after coitus
Survival of implants to day 9 after coitus

Table 2 shows the survival of implants to day 8 after coitus. Only one control mating was examined since all three controls had given similar results on day 9. The difference in survival between the two matings is highly significant, so it is concluded that the mutants were dying prior to day 8.

Table 2.

Survival of implants to day 8 after coitus

Survival of implants to day 8 after coitus
Survival of implants to day 8 after coitus

Embryos ranging in age from 7 days 9 h to 8 days 9 h were examined histologically. Table 3 gives a summary of the classification of these embryos using the criteria described below. The data conform reasonably well to the expected 3 normal/1 mutant ratio.

Table 3.

Characterization of embryos from

+se1d+×+se1d+
matings

Characterization of embryos from +se1d+×+se1d+ matings
Characterization of embryos from +se1d+×+se1d+ matings

At 7 days 9 h only three of 34 embryos examined were identified as mutants. Furthermore, these three mutants were similar in developmental stage to mutants examined at 7 days 16 h. Two of the 7 day 9 h mutants occurred in the same litter, and their litter-mates were classified as normal early 7 day embryos. This variability in developmental stage within the litter also occurred between normal litter-mates at all ages. Therefore, variation within the litter in development of embryos is apparently a characteristic of the stock of mice used and not a result of the mutation. Ignoring the three ‘advanced’ mutants, no embryo was morphologically mutant at 7 days 9 h. Nor could size be used as a criterion for recognizing mutants at this age. The orientation of 18 of the 34 embryos examined at 7 days 9 h was such that measurements could be made of their midsagittal length. These data do not show a bimodal distribution as would be expected if the growth of the mutants were already affected at this age.

Mutant embryos could first be consistently identified at 7 days 16 h. The mutants were smaller than the normal embryos and lacked mesoderm which is normally present in a well defined layer by this age (compare Figs. 1 and 2). There were also indications that the extra-embryonic ectoderm and giant cells were beginning to proliferate abnormally.

Fig. 1.

Sagittal section of presumed mutant (sel/sel) embryo at 7 days 16 h. This embryo has been compressed during histological preparation. The extra-embryonic ectoderm (a) has begun to proliferate abnormally and the endoderm (b) is folded. × 125.

Fig. 1.

Sagittal section of presumed mutant (sel/sel) embryo at 7 days 16 h. This embryo has been compressed during histological preparation. The extra-embryonic ectoderm (a) has begun to proliferate abnormally and the endoderm (b) is folded. × 125.

Fig. 2.

Sagittal section of a normal ( + /+) embryo at 7 days 16 h. Mesoderm (m) is present in large amounts, whereas it is absent from mutant embryos. × 125.

Fig. 2.

Sagittal section of a normal ( + /+) embryo at 7 days 16 h. Mesoderm (m) is present in large amounts, whereas it is absent from mutant embryos. × 125.

The entire ectopiacental cavity and exocoelom were filled with ectoderm-like cells by 7 days 20 h. The number of giant cells had increased considerably (Fig. 3). Both embryonic and extra-embryonic ectodermal cells may have been proliferating, since many mitotic figures were seen in both.

Fig. 3.

Sagittal section of presumed mutant embryo at 7 days 20 h. Ectodermal cells (a) have filled all but the amniotic cavity of the embryo. Numerous giant cells (gc) are present. ×125.

Fig. 3.

Sagittal section of presumed mutant embryo at 7 days 20 h. Ectodermal cells (a) have filled all but the amniotic cavity of the embryo. Numerous giant cells (gc) are present. ×125.

By 8 days 0 h only the most ventral portion of the amniotic cavity was not filled with ectoderm-like cells. This latter cavity was completely filled with cells by 8 days 9 h (Fig. 4) ; the mutants at this age were therefore masses of ectodermal cells surrounded by histologically normal endoderm. Mitotic figures could still be seen in the ectodermal mass, indicating that the mutants were still alive at this time. Their overall dimensions were approximately those of a normal embryo at early day 7. A portion of a normal embryo at 8 days 9 h is shown in Fig. 5 to illustrate the typical developmental changes which occur during this time period.

Fig. 4.

Presumed mutant embryo at 8 days 9 h. The embryo is a solid mass of ectodermal cells (a) surrounded by morphologically normal endoderm (b). Extensive hemorrhage (h) is present. This embryo appears smaller than the other mutant embryos because of the angle of sectioning. × 125.

Fig. 4.

Presumed mutant embryo at 8 days 9 h. The embryo is a solid mass of ectodermal cells (a) surrounded by morphologically normal endoderm (b). Extensive hemorrhage (h) is present. This embryo appears smaller than the other mutant embryos because of the angle of sectioning. × 125.

Fig. 5.

Section transverse to neural fold of a normal embryo at 8 days 9 h showing neural groove (ng), epimyocardium (em), and blood islands (bi) in the yolk sac. × 125.

Fig. 5.

Section transverse to neural fold of a normal embryo at 8 days 9 h showing neural groove (ng), epimyocardium (em), and blood islands (bi) in the yolk sac. × 125.

On the basis of these observations, the general characteristics of presumed mutant embryos can be summarized as follows: (1) extensive proliferation of extra-embryonic ectoderm, (2) nearly total absence of mesoderm, (3) folding of the embryonic endoderm at the ventral end of the embryo, (4) increased numbers of giant cells and extensive hemorrhage around the embryo, and (5) reduced size relative to normal.

The primary defect of se1 is apparently in the extra-embryonic ectoderm (which includes the trophectoderm). The genetic material missing is evidently involved in the control of proliferation of this tissue. The massive hemorrhage seen in deciduae containing mutant embryos is probably the result of overproliferation of giant cells, which have been shown to arise, at least in part, from extra-embryonic ectoderm (Fawcett, Wislocki & Waldo, 1947). These cells may be involved in the breakdown of the decidual tissue (Alden, 1948). Overproliferation of giant cells would then be expected to cause excessive destruction of uterine tissue and extensive hemorrhage.

The virtual absence of mesoderm in the mutants is most simply explained as a consequence of the overgrowth of the extra-embryonic ectoderm. Several mechanisms for this phenomenon could be postulated, but the present data do not permit any conclusions as to which is the most likely. Since se1 is a deficiency, however, the possibility cannot be excluded that an additional functional unit is involved which controls mesoderm formation. That the lack of mesoderm, which presumably would be lethal if it were the only defect in an embryo, is not due to the absence of the se locus in these mice is shown by the fact that the combination of se1 with other deficiencies which overlap se1 only at se yields viable young (L. B. Russell, 1971).

It does not seem likely that the morphology of the mutant embryo is due to abnormal differentiation of mesoderm. This can be shown by comparing the development of normal and mutant embryos. Normally, mesoderm first appears between the ectoderm and endoderm at the junction of the embryonic and extraembryonic tissues ; it then proliferates as a thin layer of cells between the endodermal and ectodermal layers. In the mutant embryos, however, there is proliferation of cells beginning in the region of the ectoplacental cone, an area which normally never produces mesoderm. Furthermore, few cells are seen between the embryonic ectoderm and endoderm of the mutant embryos. On the basis of these facts, the interpretation of the development of mutants given here seems to be the most likely, even though it is based on topographical characterization of the cell types involved rather than any intrinsic differences between the germ layers.

The folding of the endoderm observed at all the stages examined is interpreted to be the result of normal growth of this germ layer. Since little mesoderm is formed, and since the size of the embryo is reduced, the normal mechanical forces which would cause the endoderm to develop as a smooth layer are absent and the excess endoderm therefore collapses upon itself. This conclusion is further supported by the fact that both the distal and proximal endoderm appear histologically normal and contain normal numbers of mitoses.

The time of expression of se1 has not been exactly determined. The typical mutant syndrome is not seen before 7 days 16 h, with the exceptions noted above. It does not seem likely that use of shorter and more precise time intervals would offer any additional information. The variability in developmental stage within litters, minimally estimated to be 6 h for these mice, sets a limit to the precision which could be obtained with this type of analysis. It may be concluded on the basis of the 7 day 9 h data, however, that se1 is not expressed morphologically until the latter half of day 7 after coitus.

I wish to thank Dr L. B. Russell for supplying the initial stock of mice for these experiments and for much helpful discussion. I also thank Mr N. L. A. Cacheiro for help in preparing the figures. This research was supported by USPHS Predoctoral Fellowship 5-F01-45095-02 and by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.

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