/ . Embryol. exp. Morph. Vol. 27, 1, pp. 147-154, 1972 147

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Embryological effects of a minute deficiency inlinkage group II of the mouse

By G. R. DUNN1

From The University of Tennessee-Oak Ridge Graduate School ofBiomedical Sciences and the Biology Division,

Oak Ridge National Laboratory

SUMMARY

A minute deficiency in linkage group II of the mouse, involving the short-ear locus and atleast 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. Histo-logically, the mutant embryos showed overgrowth of ectoderm and trophoblastic giant cells.The mesoderm was almost entirely lacking and the mutant embryos were smaller than theirnormal litter-mates.

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

INTRODUCTION

In the course of the radiation experiments on mice conducted at this labora-tory by W. L. Russell, over 200 mutations have been generated in the diluteshort-ear region of linkage group II. More than 100 of these mutations havebeen used recently by L. B. Russell (1970) to construct a complementation mapof this region. An interesting result of this work is the discovery of complement-ing embryonic lethals. Among several independent short-ear lethals that yieldcomplex complementation patterns is the mutant 5RD300H, which L. B. Russell(1971) concludes to be a minute deficiency involving the short-ear locus and atleast one other functional lethal unit, /4. The lethal mutant, 5RD300H (hereafterdesignated sel), yields viable short-eared young when it is combined with severalindependent dilute short-ear deficiencies that are preimplantation lethals (L. B.Russell, unpublished) or with certain other short-ear lethal mutants (L. B.Russell, 1971).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 ofaction of se1. A preliminary report of this work has appeared earlier (Dunn,1970).

1 Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh EH9, U.K.

148 G. R. DUNN

MATERIALS AND METHODS

The lethal is maintained by intercrossing + sel/d+ mice (where d = dilute,and + = wild type). The stock is tested periodically to assure that sex has notbeen lost through recombination. All mice used in these experiments weresegregants of the above cross.

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

Pregnant females were killed at the indicated times by cervical dislocationand 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 hadcompleted rotation but had no heartbeat, or (3) having died before day 8 if anegg cylinder stage or no embryo was found in the decidua. Eight days aftercoitus, implants were scored as living if a well developed embryo was present, ordead if only an egg cylinder or no embryo was found.

Histological preparations were made using the technique of Fekete, Bartholo-mew & Snell (1940) with minor modifications. The embryos were sectioned at10 /-cm and stained with hematoxylin and eosin.

Table 1. Survival of implants to day 9 after coitus

No. of implants

Mating*No. of $$examined Alive

Diedearlyt

Diedlatej Total

+ sel +sel

• x •

d+ d++ sel d+

xd+ d+d+ +sel

d+_ d+d+Xd+

20

20

20

20

* Matings are indicated as $ xX After day 8 after coitus.

69(47-0)§ 39(26-5) 39(26-5) 147

105(70-5) 12(8-1) 32(21-4) 149

111(70-2) 7(4-4) 40(25-4) 158

116(73-0) 14(8-8) 29(18-2) 159

t Before day 8 after coitus.§ Numbers in parentheses are % of total.

RESULTS

Table 1 gives the results of the examination of uterine contents on day 9 aftercoitus. In all four types of matings, embryonic death is high after day 8. Onlyin the +seljd+ x +seljd+ mating is there a high death-rate prior to day 8.

Embryology of'se1 149

Table 2 shows the survival of implants to day 8 after coitus. Only one controlmating 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 isconcluded that the mutants were dying prior to day 8.

Embryos ranging in age from 7 days 9 h to 8 days 9 h were examined histologi-cally. Table 3 gives a summary of the classification of these embryos using thecriteria described below. The data conform reasonably well to the expected3 normal/1 mutant ratio.

Table 2. Survival of implants to day 8 after coitus

No. of implants

No. of $?Mating* examined Alive Dead Total

+ se' + se'—,— x—— 20 120 (69-8)t 52(30-2) 172a+ d+

-j— x-— 20 132(86-8) 20(13-2) 152

X2 = 13-6; d.f. = 1;P < 001.* Matings are indicated as $ x <$.t Numbers in parentheses are % of total.

Table 3. Characterization of embryos

from —z— x —— matings

No. of implants

Age*

7 + 97 + 167 + 208 + 08 + 9

No. of $?examined

44213

r

Normal

261995

13

Presumedmutant

34736

Abnormal!

52000

Total

3425168

19

* Days + hours after coitus.| These embryos had neither normal nor typical mutant morphology and are presumed

to be those which died due to other causes during this period.

At 7 days 9 h only three of 34 embryos examined were identified as mutants.Furthermore, these three mutants were similar in developmental stage tomutants examined at 7 days 16 h. Two of the 7 day 9 h mutants occurred in thesame litter, and their litter-mates were classified as normal early 7 day embryos.This variability in developmental stage within the litter also occurred betweennormal litter-mates at all ages. Therefore, variation within the litter in develop-ment of embryos is apparently a characteristic of the stock of mice used and

150 G. R. DUNN

Fig. 1. Sagittal section of presumed mutant (sel/sel) embryo at 7 days 16 h. Thisembryo has been compressed during histological preparation. The extra-embryonicectoderm (a) has begun to proliferate abnormally and the endoderm (b) is folded.xl25.Fig. 2. Sagittal section of a normal ( + / + ) embryo at 7 days 16 h. Mesoderm (m) ispresent in large amounts, whereas it is absent from mutant embryos, x 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, x 125.

Embryology o/se1 151not a result of the mutation. Ignoring the three 'advanced' mutants, no embryowas morphologically mutant at 7 days 9 h. Nor could size be used as a criterionfor recognizing mutants at this age. The orientation of 18 of the 34 embryos

Fig. 4. Presumed mutant embryo at 8 days 9 h. The embryo is a solid mass of ecto-dermal cells (a) surrounded by morphologically normal endoderm (b). Extensivehemorrhage (h) is present. This embryo appears smaller than the other mutantembryos because of the angle of sectioning, x 125.Fig. 5. Section transverse to neural fold of a normal embryo at 8 days 9 h showingneural groove (ng), epimyocardium (em), and blood islands (bi) in the yolk sac. x 125.

152 G.R.DUNN

examined at 7 days 9 h was such that measurements could be made of theirmidsagittal length. These data do not show a bimodal distribution as would beexpected if the growth of the mutants were already affected at this age.

Mutant embryos could first be consistently identified at 7 days 16 h. Themutants were smaller than the normal embryos and lacked mesoderm which isnormally 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 cellswere beginning to proliferate abnormally.

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

By 8 days 0 h only the most ventral portion of the amniotic cavity was notfilled with ectoderm-like cells. This latter cavity was completely filled with cellsby 8 days 9 h (Fig. 4); the mutants at this age were therefore masses of ecto-dermal cells surrounded by histologically normal endoderm. Mitotic figurescould still be seen in the ectodermal mass, indicating that the mutants were stillalive at this time. Their overall dimensions were approximately those of a nor-mal embryo at early day 7. A portion of a normal embryo at 8 days 9 h is shownin Fig. 5 to illustrate the typical developmental changes which occur during thistime period.

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

DISCUSSION

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

The virtual absence of mesoderm in the mutants is most simply explained as aconsequence of the overgrowth of the extra-embryonic ectoderm. Severalmechanisms for this phenomenon could be postulated, but the present data donot 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

Embryology o/se1 153is 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 notdue to the absence of the se locus in these mice is shown by the fact that thecombination of se1 with other deficiencies which overlap se1 only at se yieldsviable young (L. B. Russell, 1971).

It does not seem likely that the morphology of the mutant embryo is due toabnormal differentiation of mesoderm. This can be shown by comparing thedevelopment of normal and mutant embryos. Normally, mesoderm first appearsbetween the ectoderm and endoderm at the junction of the embryonic and extra-embryonic tissues; it then proliferates as a thin layer of cells between the endo-dermal and ectodermal layers. In the mutant embryos, however, there is pro-liferation of cells beginning in the region of the ectoplacental cone, an areawhich normally never produces mesoderm. Furthermore, few cells are seenbetween the embryonic ectoderm and endoderm of the mutant embryos. On thebasis of these facts, the interpretation of the development of mutants given hereseems to be the most likely, even though it is based on topographical charac-terization of the cell types involved rather than any intrinsic differences betweenthe germ layers.

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

The time of expression of se1 has not been exactly determined. The typicalmutant syndrome is not seen before 7 days 16 h, with the exceptions notedabove. It does not seem likely that use of shorter and more precise time intervalswould offer any additional information. The variability in developmental stagewithin litters, minimally estimated to be 6 h for these mice, sets a limit tothe precision which could be obtained with this type of analysis. It may beconcluded on the basis of the 7 day 9 h data, however, that se1 is not expressedmorphologically 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 experimentsand for much helpful discussion. I also thank Mr N. L. A. Cacheiro for help in preparing thefigures. This research was supported by USPHS Predoctoral Fellowship 5-F01-45095-02 andby the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.

154 G.R.DUNN

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trophoblast giant cells. Am. J. Anat. 83, 143-181.BRADEN, A. W. H. (1957). The relationship of the diurnal light cycle and the time of ovulation

in mice. /. exp. Zool. 34, 177-188.DUNN, G. R. (1970). Embryological effect of a short-ear lethal allele in the mouse. Genetics,

Princeton 64, s 17—s 18.FAWCETT, D. W., WISLOCKI, G. B. & WALDO, C. M. (1947). The development of the mouse

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FEKETE, E., BARTHOLOMEW, O. & SNELL, G. D. (1940). A technique for the preparation ofsections of early mouse embryos. Anat. Rec. 76, 441-447.

RUSSELL, L. B. (1971). Definition of functional units in a small chromosomal segment of themouse and its use in interpreting the nature of radiation-induced mutations. Mutat. Res.11, 107-123.

{Manuscript received 18 March 1971)