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Steel (Sl) mutation in mice: Identification of mutant embryos early in development
DEVELOPMENTAL
BIOLOGY
49,
Steel (SI) Mutation
300-303 (19%)
in Mice: Identification
of Mutant
Embryos
Early in
Development DAVID H. K. Department
of Pathology,
CHUI, BRIAN V. Faculty of Medicine,
LOYER, AND ELIZABETH S. RUSSELL
McMaster
Jackson Laboratory, Accepted
University, Hamilton,
Ontario, Canada, and
Bar Harbor, Maine
October 20, 1975
A method is described for the reliable identification of Steel mutant mouse embryos in segregating litters as early as Day 11 of gestation based on the color of hair developed in embryonic skin explants. INTRODUCTION
Mice afflicted with two mutant alleles at the steel (5’1) locus in linkage group IV have anemia, white hair, and sterility. The fetal hepatic erythropoiesis in these mutant mice is abnormal, and anemia can be detected by Day 13 of gestation (Chui and Russell, 1974; Chui and Loyer, 1975). Attempts to study the establishment of erythropoiesis in fetal livers under the influence of mutant SI genes have been severely hampered by the inability to identify mutant embryos in segregating litters during earlier gestational stages. In the present study, evidence is presented that the color of hair developed in embryonic skin explants, which have been grafted to adult testes of histocompatible recipient mice, can form the basis for reliable identification of SIISld embryos in segregating litters as early as Day 11 of gestation. MATERIALS AND METHODS
Animals. Adult mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Most embryos were derived from matings between WC/&J - Sl/+ x C57BLKJ - Sld/ + . Day 11 embryos were obtained from matings between WC/F&J - Sll+ x WC/&J - Sll+ or WC/ReJ-+I + x WC/ReJ- +/+. Female mice were examined daily for vaginal plugs. The morning a plug was found was designated Day 0 of gestation.
Embryos and tissues. Embryos were removed and placed in 90% Waymouth medium (MB 752/l; Microbiological Associates, Inc., Bethesda, Md.) and 10% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.). The genotype of Day 13 or older embryos were determined on the basis of mean cell volume of circulating fetal erythrocytes, as previously described in detail (Chui and Loyer, 1975). Embryonic skin (1 mm’) was removed by a pair of fine scissors. From each embryo of Day 12 or older, two pieces of skin were removed from the ventral portion of the embryo between the fore- and hindlimbs. On Day 11, four pieces of skin were removed, two from the dorsal region of the embryo between the levels of the fore- and hindlimbs and another two from the dorsal region just anterior to the level of the forelimbs. These pieces of skin were kept in cold Waymouth medium until transplantation. Grafting of embryonic skin. Adult WCBGFl-+/+, Sl/+, or Sldl+ male mice were used as the recipients. In some experiments, WC/ReJ- +/+ or SZ/+ adult male mice also served as the recipients. Mice were anesthesized with Avertin given intraperitoneally. A small incision was made on the capsule of the exposed testis, and with a fine-tipped Pasteur pipet, each piece of embryonic skin was introduced into a different testis. Subse-
300 Copyright0
1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF
301
NOTES
quently testes were replaced in their proper anatomical positions, and the abdominal incision was closed with surgical silk and wound clips. Two to three weeks later, recipient animals were sacrificed and their testes were removed, fixed in 6% form01 saline, dehydrated in graded alcohol, and finally cleared by methyl salicylate. The embryonic skin explants were then examined under a stereomicroscope, and the color of the hair that had developed in the grafts was noted.
bryos can be positively identified by the hair color in their skin explants. On Day 12 of gestation, 90 embryos from 12 litters were obtained from Sll+ x Sl dl + matings. Of 59 donors that produced hair in both skin grafts, 41 embryos had only black hair in both skin grafts, indicating that these were either heterozygous or wild-type embryos, and 12 had white hair in both explants, indicating that the genotype of the donors was SlISl” (Table 2). In 33 Day-11 embryos, at least three skin explants from each embryo grew hair (Table 3). In 14 embryos obtained from +/ + x +/+ matings, all but one grew mostly black hair (Table 4). However, the one exception was an embryo that had only white hair in all four of its explants. In 19 embryos obtained from Sll+ x Sll+ matings, there were four that grew only white hair in their skin explants suggesting that these were mutant embryos. The other 15 embryos had either only black hairs or some black and some white hairs in their explants, indicating that these were either heterozygous or wild type embryos. In one Day-11 embryo obtained from SZ/ + x Sl/+ mating three skin explants grew white hair while only one explant grew black hair, thus indicating that the geno-
RESULTS
Seventy-six embryos from 10 litters of Days 13-17 of gestation were obtained from SZ/+ x SZd/ + matings (Table 1). Genotypes of these fetuses were assigned based on hematological criteria (Chui and Loyer, 1975). Skin explants from all mutant embryos grew only white hair, with the exception of one embryo, which failed to have any hair growth in either of its two skin explants. Explants from heterozygous or wild-type embryos developed only black hair. In one other Day-15 litter, a product of +I+ x +I+, all eight grafts from four embryos grew black hair. These results confirm that on Days 13-17, mutant em-
TABLE 1 DISTRIBUTION
Genotype of embryo
Sli+
Number of Embryos
OF DAYS
13-17
EMBRYOS
BASED ON HAIR
COLOR OF GRAFTS
Number of Embryos with Hair color of one graft:
White
White
No Hair
Black
Black
Black
Hair color of another graft:
White
No hair
No hair
White
No hair
Black
3 13 0
13 27 0
+I+
16
0
0
0
0
or Sldl+
40 20
0 16
0 3
0 1
0 0
SliSld
TABLE 2 DISTRIBUTION
Hair color of one graft: Hair color of another graft: Number of embryos
OF 12-DAY
EMBRYOS
BASED ON HAIR
COLOR OF GRAFTS
White
White
No hair
Black
Black
Black
White 12
No hair 10
No hair 1
White 6
No hair 20
Black 41
302
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE GRAFTS Mating
Number of litters
+I+ x +/+ SW+ x Sl/+
Mating
+I+ Sl/+
x +/+ x Sl/+
11-DAY
92 104
4
White hair only
White and black hair
1 4
0 6
SKIN
Grafts with hair recovered
23 26
OF DAY 11 EMBRYOS BASED COLOR OF GRAFTS”
1976
3 EMBRYONIC
Number of embryos
5 5 TABLE
DISTRIBUTION
OF
49,
ON HAIR
FE? hair 13 7
a Four pieces of skin obtained from each Day 11 embryo were grafted. Those embryos, each of which had at least three successful grafts with hair growth, were tabulated. White hair only, those embryos each of which had either four grafts with white hair (referred to as 4W) or three grafts with white hair and another graft without hair (referred to as 3W); white and black hair, those embryos each of which had either three grafts with white hair and one graft with black hair (referred to as 3WlB), ZWIB, ZWZB, or lW2B; mostly black hair, those embryos each of which had lW3B, 3B, or 4B.
type of this embryo was either heterozygous or possibly wild type. This observation thus demonstrated the need to perform at least four grafts from each Day-11 embryo for genotype determination. In skin explants obtained from embryos of Days 13 or older, only 12% of the grafts were either not recovered or if recovered found to have no hair growth (Table 1). On Day 12, the failure rate was up to 18% (Table 21, and by Day 11, 26% failed to grow hair (Table 3). DISCUSSION
The present investigation shows that from Days 13 to 17, embryos which have been determined to be SIISld on the basis of hematological criteria (Chui and Loyer, 1975) also have demonstrable abnormality in their skin as shown by the growth of
Embryos with 3-4 grafts with hair recovered
(No.)
(8)
(No.)
(%)
66 79
72 76
14 19
61 73
pigment-free hair in their skin explants. At the same gestational stage, germ cells are absent in these mutant embryos (Stevens, 1967). On Days 11 and 12 of gestation, skin grafts from approximately 25% of embryos derived from matings between SZ heterozygotes grow hair which is totally pigment-free, indicating that the donors are steel mutant embryos. On the other hand, grafts from the remaining 75% of embryos produce black hair, suggesting that the donors are either heterozygous or wildtype embryos. Therefore, the color of hair developed from embryonic skin grafts can be used to identify mutant embryos in segregating litters at these early stages of fetal development. In one Day-11 embryo, a product of +/+ x +/+ mating, all four skin grafts developed white hair (Table 4). This observation is consistent with the hypothesis that on Day 11 during normal mouse development, melanoblasts have not yet migrated to populate all of the embryonic skin (Rawles, 1947). Therefore, the present method of genotyping fetuses on Day 11 can almost certainly identify all mutant embryos, but does not exclude the possibility of misidentifying an occasional normal or heterozygous embryo as being mutant. In this study, 1 out of 14 +/+ embryos (or 7%) would have been classified as S&S1 based on the hair color in skin explants (Table 4). The steel mutation in mice is a useful model in the analysis of cell-cell interaction during mammalian embryogenesis. The present investigation makes it feasi-
BRIEF NOTES
303
REFERENCES ble to study the early developmental events under the influence of mutant SZ CHUI, D. H. K., and RUSSELL,E. S. (1974). Fetal erythropoiesis in steel mutant mice. I. A morphogenes, especially with regard to fetal helogical study of erythroid cell development in fetal patic erythropoiesis, at the stage of gestaliver. Develop. Biol. 40, 256-269. tion considerably earlier than previously CHUI, D. H. K., and LOYER, B. V. (1975). Fetal possible. erythropoiesis in steel mutant mice. II. HaemoWe gratefully acknowledge the technical assistance of Mr. F. Krestynski. This investigation was supported by research grants from the Medical Research Council of Canada (Nos. DG-65 and MA5004). Dr. Chui is a MRC Scholar. Part of this work was done while D.H.K.C. was a Visiting Investigator at the Jackson Laboratory.
poietic stem cells in fetal livers during development. Brit. J. Huemat. 29, 553-565. RAWLES,M. E. (1947). Origin in pigment cells from the neural crest in the mouse embryo. Physiol. Zod. 20, 246-266. STEVENS,L. C. (1967). Origin of testicular teratomas from primordial germ cells in mice. J. Nat. Cancer Inst. 38, 549552.
BIOLOGY
49,
Steel (SI) Mutation
300-303 (19%)
in Mice: Identification
of Mutant
Embryos
Early in
Development DAVID H. K. Department
of Pathology,
CHUI, BRIAN V. Faculty of Medicine,
LOYER, AND ELIZABETH S. RUSSELL
McMaster
Jackson Laboratory, Accepted
University, Hamilton,
Ontario, Canada, and
Bar Harbor, Maine
October 20, 1975
A method is described for the reliable identification of Steel mutant mouse embryos in segregating litters as early as Day 11 of gestation based on the color of hair developed in embryonic skin explants. INTRODUCTION
Mice afflicted with two mutant alleles at the steel (5’1) locus in linkage group IV have anemia, white hair, and sterility. The fetal hepatic erythropoiesis in these mutant mice is abnormal, and anemia can be detected by Day 13 of gestation (Chui and Russell, 1974; Chui and Loyer, 1975). Attempts to study the establishment of erythropoiesis in fetal livers under the influence of mutant SI genes have been severely hampered by the inability to identify mutant embryos in segregating litters during earlier gestational stages. In the present study, evidence is presented that the color of hair developed in embryonic skin explants, which have been grafted to adult testes of histocompatible recipient mice, can form the basis for reliable identification of SIISld embryos in segregating litters as early as Day 11 of gestation. MATERIALS AND METHODS
Animals. Adult mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Most embryos were derived from matings between WC/&J - Sl/+ x C57BLKJ - Sld/ + . Day 11 embryos were obtained from matings between WC/F&J - Sll+ x WC/&J - Sll+ or WC/ReJ-+I + x WC/ReJ- +/+. Female mice were examined daily for vaginal plugs. The morning a plug was found was designated Day 0 of gestation.
Embryos and tissues. Embryos were removed and placed in 90% Waymouth medium (MB 752/l; Microbiological Associates, Inc., Bethesda, Md.) and 10% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.). The genotype of Day 13 or older embryos were determined on the basis of mean cell volume of circulating fetal erythrocytes, as previously described in detail (Chui and Loyer, 1975). Embryonic skin (1 mm’) was removed by a pair of fine scissors. From each embryo of Day 12 or older, two pieces of skin were removed from the ventral portion of the embryo between the fore- and hindlimbs. On Day 11, four pieces of skin were removed, two from the dorsal region of the embryo between the levels of the fore- and hindlimbs and another two from the dorsal region just anterior to the level of the forelimbs. These pieces of skin were kept in cold Waymouth medium until transplantation. Grafting of embryonic skin. Adult WCBGFl-+/+, Sl/+, or Sldl+ male mice were used as the recipients. In some experiments, WC/ReJ- +/+ or SZ/+ adult male mice also served as the recipients. Mice were anesthesized with Avertin given intraperitoneally. A small incision was made on the capsule of the exposed testis, and with a fine-tipped Pasteur pipet, each piece of embryonic skin was introduced into a different testis. Subse-
300 Copyright0
1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF
301
NOTES
quently testes were replaced in their proper anatomical positions, and the abdominal incision was closed with surgical silk and wound clips. Two to three weeks later, recipient animals were sacrificed and their testes were removed, fixed in 6% form01 saline, dehydrated in graded alcohol, and finally cleared by methyl salicylate. The embryonic skin explants were then examined under a stereomicroscope, and the color of the hair that had developed in the grafts was noted.
bryos can be positively identified by the hair color in their skin explants. On Day 12 of gestation, 90 embryos from 12 litters were obtained from Sll+ x Sl dl + matings. Of 59 donors that produced hair in both skin grafts, 41 embryos had only black hair in both skin grafts, indicating that these were either heterozygous or wild-type embryos, and 12 had white hair in both explants, indicating that the genotype of the donors was SlISl” (Table 2). In 33 Day-11 embryos, at least three skin explants from each embryo grew hair (Table 3). In 14 embryos obtained from +/ + x +/+ matings, all but one grew mostly black hair (Table 4). However, the one exception was an embryo that had only white hair in all four of its explants. In 19 embryos obtained from Sll+ x Sll+ matings, there were four that grew only white hair in their skin explants suggesting that these were mutant embryos. The other 15 embryos had either only black hairs or some black and some white hairs in their explants, indicating that these were either heterozygous or wild type embryos. In one Day-11 embryo obtained from SZ/ + x Sl/+ mating three skin explants grew white hair while only one explant grew black hair, thus indicating that the geno-
RESULTS
Seventy-six embryos from 10 litters of Days 13-17 of gestation were obtained from SZ/+ x SZd/ + matings (Table 1). Genotypes of these fetuses were assigned based on hematological criteria (Chui and Loyer, 1975). Skin explants from all mutant embryos grew only white hair, with the exception of one embryo, which failed to have any hair growth in either of its two skin explants. Explants from heterozygous or wild-type embryos developed only black hair. In one other Day-15 litter, a product of +I+ x +I+, all eight grafts from four embryos grew black hair. These results confirm that on Days 13-17, mutant em-
TABLE 1 DISTRIBUTION
Genotype of embryo
Sli+
Number of Embryos
OF DAYS
13-17
EMBRYOS
BASED ON HAIR
COLOR OF GRAFTS
Number of Embryos with Hair color of one graft:
White
White
No Hair
Black
Black
Black
Hair color of another graft:
White
No hair
No hair
White
No hair
Black
3 13 0
13 27 0
+I+
16
0
0
0
0
or Sldl+
40 20
0 16
0 3
0 1
0 0
SliSld
TABLE 2 DISTRIBUTION
Hair color of one graft: Hair color of another graft: Number of embryos
OF 12-DAY
EMBRYOS
BASED ON HAIR
COLOR OF GRAFTS
White
White
No hair
Black
Black
Black
White 12
No hair 10
No hair 1
White 6
No hair 20
Black 41
302
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE GRAFTS Mating
Number of litters
+I+ x +/+ SW+ x Sl/+
Mating
+I+ Sl/+
x +/+ x Sl/+
11-DAY
92 104
4
White hair only
White and black hair
1 4
0 6
SKIN
Grafts with hair recovered
23 26
OF DAY 11 EMBRYOS BASED COLOR OF GRAFTS”
1976
3 EMBRYONIC
Number of embryos
5 5 TABLE
DISTRIBUTION
OF
49,
ON HAIR
FE? hair 13 7
a Four pieces of skin obtained from each Day 11 embryo were grafted. Those embryos, each of which had at least three successful grafts with hair growth, were tabulated. White hair only, those embryos each of which had either four grafts with white hair (referred to as 4W) or three grafts with white hair and another graft without hair (referred to as 3W); white and black hair, those embryos each of which had either three grafts with white hair and one graft with black hair (referred to as 3WlB), ZWIB, ZWZB, or lW2B; mostly black hair, those embryos each of which had lW3B, 3B, or 4B.
type of this embryo was either heterozygous or possibly wild type. This observation thus demonstrated the need to perform at least four grafts from each Day-11 embryo for genotype determination. In skin explants obtained from embryos of Days 13 or older, only 12% of the grafts were either not recovered or if recovered found to have no hair growth (Table 1). On Day 12, the failure rate was up to 18% (Table 21, and by Day 11, 26% failed to grow hair (Table 3). DISCUSSION
The present investigation shows that from Days 13 to 17, embryos which have been determined to be SIISld on the basis of hematological criteria (Chui and Loyer, 1975) also have demonstrable abnormality in their skin as shown by the growth of
Embryos with 3-4 grafts with hair recovered
(No.)
(8)
(No.)
(%)
66 79
72 76
14 19
61 73
pigment-free hair in their skin explants. At the same gestational stage, germ cells are absent in these mutant embryos (Stevens, 1967). On Days 11 and 12 of gestation, skin grafts from approximately 25% of embryos derived from matings between SZ heterozygotes grow hair which is totally pigment-free, indicating that the donors are steel mutant embryos. On the other hand, grafts from the remaining 75% of embryos produce black hair, suggesting that the donors are either heterozygous or wildtype embryos. Therefore, the color of hair developed from embryonic skin grafts can be used to identify mutant embryos in segregating litters at these early stages of fetal development. In one Day-11 embryo, a product of +/+ x +/+ mating, all four skin grafts developed white hair (Table 4). This observation is consistent with the hypothesis that on Day 11 during normal mouse development, melanoblasts have not yet migrated to populate all of the embryonic skin (Rawles, 1947). Therefore, the present method of genotyping fetuses on Day 11 can almost certainly identify all mutant embryos, but does not exclude the possibility of misidentifying an occasional normal or heterozygous embryo as being mutant. In this study, 1 out of 14 +/+ embryos (or 7%) would have been classified as S&S1 based on the hair color in skin explants (Table 4). The steel mutation in mice is a useful model in the analysis of cell-cell interaction during mammalian embryogenesis. The present investigation makes it feasi-
BRIEF NOTES
303
REFERENCES ble to study the early developmental events under the influence of mutant SZ CHUI, D. H. K., and RUSSELL,E. S. (1974). Fetal erythropoiesis in steel mutant mice. I. A morphogenes, especially with regard to fetal helogical study of erythroid cell development in fetal patic erythropoiesis, at the stage of gestaliver. Develop. Biol. 40, 256-269. tion considerably earlier than previously CHUI, D. H. K., and LOYER, B. V. (1975). Fetal possible. erythropoiesis in steel mutant mice. II. HaemoWe gratefully acknowledge the technical assistance of Mr. F. Krestynski. This investigation was supported by research grants from the Medical Research Council of Canada (Nos. DG-65 and MA5004). Dr. Chui is a MRC Scholar. Part of this work was done while D.H.K.C. was a Visiting Investigator at the Jackson Laboratory.
poietic stem cells in fetal livers during development. Brit. J. Huemat. 29, 553-565. RAWLES,M. E. (1947). Origin in pigment cells from the neural crest in the mouse embryo. Physiol. Zod. 20, 246-266. STEVENS,L. C. (1967). Origin of testicular teratomas from primordial germ cells in mice. J. Nat. Cancer Inst. 38, 549552.