Targeted disruption of the mouse mHR23B gene.
The mouse mHR23B locus was isolated and partly characterized. Deletion of exon III not only removes the sequence encoding amino acid residues 148 to 228 of the mHR23B protein but also results in a frameshift and accordingly is expected to give rise to a null allele (Fig. ). Following transfection of ES cell line E14, properly targeted heterozygous ES cells were obtained at a frequency of 21% (Fig. ). Two independent ES clones for which the absence of visible chromosomal abnormalities and additional randomly integrated constructs had been verified (data not shown) were used for blastocyst injections. Germ line transmission was obtained for both clones. Heterozygous offspring from matings between chimeric males and C57BL/6 female mice were intercrossed in order to generate homozygous mutant mHR23B animals (Fig. ). In parallel, these matings served to isolate MEFs of different genotypes from day 13.5 embryos. The effect of targeted disruption on the expression of the mHR23B gene was analyzed in MEFs: neither mHR23B mRNA nor mHR23B protein could be detected in mHR23B−/− cells by RNA and immunoblot analyses, respectively (Fig. and ). We conclude that we have created mHR23B null mutants. Homozygous mouse mutants and cell lines from the two independent ES transformants yielded identical results in all subsequent studies, indicating that the findings reported below are not due to uncontrolled events that might have occurred in one targeted ES clone but are the result of mHR23B inactivation.
FIG. 1. Targeted disruption of the mHR23B gene by homologous recombination. (A) Genomic organization and disruption strategy for mHR23B showing the gene, the targeting construct, and the targeted mHR23B allele. Exon III is deleted, and the upstream mouse sequence (more ...) mHR23B-deficient cells are NER proficient.
In view of the role of S. cerevisiae
RAD23 in NER and the tight interaction of mHR23B with XPC (26
), we examined cellular survival of wild-type, heterozygous, and homozygous mHR23B
mutant MEFs after exposure to increasing UV doses. Unexpectedly, UV survival of mHR23B+/−
cells appeared indistinguishable from that of the wild-type cells (Fig. ).Moreover, mHR23B-
deficient MEFs show normal DNA repair synthesis (UDS [Fig. ]) and recovery of RNA synthesis after UV exposure (Fig. ), indicating that neither GG-NER nor TC-NER subpathways were affected. Also, in other respects (e.g., morphology, growth rate, etc.), mHR23B-
deficient MEFs behaved normally. Assuming that a total mHR23
inactivation would result in a DNA repair deficiency, as in S. cerevisiae
), these data suggest that mHR23A can fully substitute for mHR23B, at least for its function in NER, not only in vitro but also in vivo.
FIG. 2. Repair characteristics of mHR23B-deficient MEFs. (A) UV survival curves of mHR23B+/+, mHR23B+/−, and mHR23B−/− fibroblast lines. Identical results were obtained with three other cell lines of mHR23B+/+ (more ...) mHR23B deficiency causes impaired embryonic development and intrauterine death.
When (phenotypically normal) heterozygous animals were crossed to produce mHR23B-deficient mice, the targeted mHR23B allele was found to segregate at a ratio far below that expected by Mendelian inheritance (~10-fold) (a total of 671 animals analyzed) (Table ), suggesting that a lack of mHR23B protein causes intrauterine and/or perinatal death. Since mHR23B-deficient MEF lines were obtained at an almost Mendelian ratio (data not shown), lethal events must occur after day 13.5 of gestation (E13.5). Analysis of a large series of embryos at different stages of development revealed a near twofold reduction in the number of viable mHR23B-deficient embryos between E13.5 and E15.5 (Table ). Since at E18.5, 50% of the mHR23B-deficient embryos were still alive (and the litters that were born contained only 10% of the expected numbers of knockout animals), 80% of the remaining embryos are assumed to have died immediately prior to, during, or shortly after birth, which is consistent with the observed high number of dead newborn mHR23B−/− mice.
Intrauterine and perinatal lethality of mHR23B−/− mutants at different embryonic and fetal stages
mHR23B-deficient embryos (E13.5 to E19.5) showed clear signs of growth retardation and were readily recognized by a marked reduction in body size (Fig. ). This is reflected by a reduced body weight, which becomes more pronounced towards term (data not shown). All mHR23B−/− embryos alive at stages E13.5 to E19.5 appeared pale, whereas major blood vessels were not clearly visible (Fig. and ). This suggests that vascularization and/or blood supply was poor. In addition, large numbers of mHR23B−/− embryos showed edema (Fig. ). Also, embryos with interstitial bleeding throughout the body were found (Fig. ). Moreover, in numerous mHR23B−/− embryos (E13.5 to E19.5), the eyelids were not closed and the mouth was widely open, which is a characteristic feature of maceration following embryonic death. Eyelid closure and fusion normally occur between E15.5 and E16.5 of mouse embryonic development. A wide open mouth normally indicates micrognathia or cleft lip resulting from retarded growth of the mandible.
FIG. 3. Developmental impairment in mHR23B−/− embryos at E15.5. mHR23B+/+ (A and D) and mHR23B−/− (B, C, E, and F) embryos with (A to C) and without (D to F) amniotic or chorionic plates and placentas. The embryo (more ...)
Preliminary histopathological examination of living mHR23B-deficient E15.5 and E18.5 embryos revealed no obvious abnormal architecture of vital organs and tissues, and the reduction in organ weight appeared proportional to the reduction in total body weight. However, in one mHR23B-deficient E18.5 embryo, we observed an open secondary palate (cleft palate) resulting from imperfect closure of the palatal shelves of the maxilla (in normally developing embryos, this is completed at E16; data not shown). Taken together, these data indicate that the mHR23B protein is required for proper embryonic development and that mHR23A cannot substitute or can substitute only partly for this function.
Inactivation of mHR23B causes a placental defect.
In line with the pale appearance of a large number of mHR23B
-deficient embryos, the placentas of mHR23B−/−
mutants at stage E18.5 (n
3) appeared pale and smaller compared to mHR23B+/+
placentas. Histological examination revealed poor vascularization of mutant placentas, as evident from the reduced number of fetal blood vessels in the labyrinth (Fig. and ).Transmission electron microscopy of mHR23B−/−
= 3) revealed swollen trophoblastic cells (Fig. and ). In addition, altered morphology of the vascular basement membrane of mHR23B−/−
placentas was observed (Fig. ). The vascular basement membrane of mHR23B−/−
placentas was darker and thicker than that of wild-type placentas, which might affect the exchange of gases and transport of nutrients and waste products.
FIG. 4. Placental defect in mHR23B−/− embryos at E18.5. Histological 1-μm-thick sections (A and B) and transmission electron micrographs (C and D) of mHR23B+/+ (A and C) and mHR23B−/− (B and D) placentas. (more ...) mHR23B-deficient mice show retarded growth and facial dysmorphology.
Despite the pronounced impact of the mHR23B deficiency on embryonic development, about one-tenth of the expected number of homozygous mutant mice was found alive in litters born from heterozygous breeding couples (Table ). Like mHR23B−/− embryos, newborn homozygous mutant mice showed a marked reduction in body size and are readily distinguishable from their heterozygous and wild-type littermates (Fig. ).After the mice were weaned, we did not notice any further weight loss of mHR23B−/− animals. As evident from body weight measurements, mHR23B−/− mice display retarded growth, particularly in the last days before weaning (day 21). Up to 7 weeks after birth, the average body weight of homozygous mutant males (n = 4) and females (n = 2) was still approximately 50% lower than that of wild-type (n = 3) and heterozygous (n = 8) littermates (Fig. ). This situation remained throughout life (body weight at 3 months shown in Fig. ). Vital organs were proportionally reduced in size (data not shown). Adult mHR23B−/− males and females (up to 1 year and older) lacked fatty tissues, while excessive fat was observed in the abdominal cavity of wild-type mice. However, histopathological examination of the vital organs, sciatic nerve, and skeletal muscle from adult mHR23B-deficient mice (n = 4) failed to reveal any obvious abnormality (data not shown).
FIG. 5. Growth retardation in mHR23B−/− mice. (A) A 19-day-old wild-type male mouse (brown) and its smaller mHR23B−/− (black) littermate. (B) Growth curve of mHR23B mice. Data for mHR23B+/+ (n = 3 [males]) (more ...)
All mHR23B-deficient mice and embryonic stages analyzed from E16.5 on showed facial dysmorphology. The nose had a blunted shape rather than the tapered appearance characteristic for rodents due to hypoplasia of the maxilla and mandible (Fig. and ). In addition, more than one-third of the mHR23B-deficient mice developed so-called elephant teeth (overgrown teeth). The cause of death at <0.5 to 1 year of age may be secondary to the poor overall condition of the mutant mice. However, we did not observe cancer thus far.
mHR23B-deficient mice have ocular pathology.
Some 7 to 10 days after birth, mHR23B-deficient mice started to develop eye pathology, characterized by excessive eye fluid and swelling of the eyelids. Animals refrained from opening their eyes widely. Probably as a result of the continuously drenched eyes, mHR23B-deficient mice showed excessive washing activities. These features persisted into adulthood with frequent signs of inflammation in eyelids (Fig. ) that could not be treated by application of eye ointment (Terramycin containing oxytetracycline-polymyxin). In addition, a large number of mHR23B-deficient animals seemed to suffer from itching, as evident from extensive scratching, which was not restricted to the head region but also involved the ears and the neck line. Some mHR23B-deficient mice had opaque eyes (data not shown).
Histological analysis of mHR23B-deficient mice (n = 6) confirmed that the mice had conjunctivitis. In addition, in the corners of the eyes of mHR23B-deficient mice, inflammation cells containing polymorphic nuclei (neutrophils) were observed possibly due to infections (data not shown). A clear cause of the wet eyes was not detected. The tear-producing glands showed no overt abnormalities. However, in one mHR23B-deficient mouse, the number of conjunctival goblet cells that produce the mucous layer of the tear film was determined (n = 2) and found to be reduced. The drainage of the tears was checked in one mHR23B-deficient animal and appeared normal. Moreover, we failed to observe any abnormalities in other parts of the eye, such as the retina (data not shown).
To examine the possibility of any inflammatory disease, the ratio of immunoglobulin A (IgA), IgG, and IgM and the white blood cell counts of adult mHR23B-deficient mice (n = 5) were determined, but abnormalities were not found, indicating that the immunological system is not compromised (data not shown).
Defective spermatogenesis in mHR23B−/− male mice.
Attempts to use mHR23B−/− males in breeding protocols with either mHR23B−/− or wild-type female mice did not result in pregnancies. Since mHR23B−/− males show mating activity (as evident from the presence of copulatory plugs in the female mice), their inability to produce progeny appears not to be related to either reduced body size (which may affect physical sexual performance) or hormonal disturbances. Inspection of the reproductive organs of adult mHR23B−/− males (3.5 to 7 months of age; n = 11) disclosed a disproportionate reduction in the size of the testes (Fig. ).Whereas the weight of all internal organs was proportional to the twofold reduction in body weight, the weight of mHR23B−/− testes was reduced by about sevenfold. The weight of mHR23B−/− epididymides and seminal vesicles was reduced by about twofold (Fig. ).
FIG. 6. The growth of the testes of mHR23B−/− males is retarded. (A) Macrograph of adult wild-type (left) and mHR23B−/− (right) testes. Besides reduction in size, blood vessels are not clearly visible in mHR23B−/− (more ...)
Morphology of seminal vesicles of mHR23B-
deficient males (n
= 2) showed no abnormalities. However, histological examination of the testes of mHR23B-
deficient males revealed seminiferous tubules with a small diameter and relatively abundant interstitial tissue in all animals analyzed (n
= 11) (Fig. ).Most striking is the total absence of spermatogenesis, which is in line with the absence of sperm cells in the epididymis (Fig. ). In the tubules, Sertoli cells appear to be the predominant cell type (Fig. and ). In the center of most tubules, we observed a concentration of cells, which are typical of Sertoli cell clusters, which represent Sertoli cells detached from the basal membrane of the seminiferous tubule (Fig. and ). Such clusters were not observed in wild-type seminiferous tubules, containing all stages of spermatogenesis (Fig. and ). Release of Sertoli cells from the basal membrane and clustering in the lumen has been observed in other male mouse sterility models, particularly in the older animals (32
FIG. 7. mHR23B−/− males show impaired spermatogenesis and dysfunction of Sertoli cells. (A to F) Histological examination of 91-day-old caput epididymis (A and B) and testis (C to F) from wild-type (A, C, and E) and mHR23B−/− mice (more ...)
The impairment of spermatogenesis in adult mHR23B-deficient mice might reflect a primary defect, resulting in a block at an early or later phase of spermatogenesis. To study this in more detail, a histological analysis was performed on the testes of 15-day-old mHR23B−/− animals, when spermatogenesis is normally initiated. Morphology of the testis of a mHR23B-deficient mouse revealed no initiation of spermatogenesis compared to normal initiation of spermatogenesis, with presence of pachytene spermatocytes, in the testis of a wild-type mouse. The majority of the tubules of the testis of a 15-day-old mHR23B-deficient mouse showed a Sertoli-cell-only phenotype, although a few tubules contained some spermatogonia (Fig. and ). Histological examination of an E15.5 mHR23B-deficient male revealed normal urogenital morphology of Müllerian duct regression and Wolffian duct development, indicating testicular production of anti-Müllerian hormone (AMH) by Sertoli cells and testosterone by the interstitial Leydig cells. Although testicular histology of E15.5 mHR23B-deficient animals displayed normal Sertoli cells, the number of gonocytes (originating from primordial germ cells) seemed to be reduced compared to the testes of wild-type mice (Fig. ).
FIG. 8. mHR23B−/− embryos at E15.5 show impaired development of gonocytes. Histological analysis of the tubules of E15.5 testis from wild-type (A) and mHR23B−/− (B) mutants. Sertoli cell nuclei are indicated by black arrowheads. (more ...) mHR23B−/− females exhibit reduced fertility.
In contrast to mHR23B homozygous mutant males, mHR23B−/− females (n = 5) were fertile. However, compared to mHR23B+/+ or mHR23B+/− females, fertility of mHR23B-deficient females (n = 5) was clearly reduced. Copulatory plugs were found after interbreeding with wild-type or mHR23B+/− males, but litters born from mHR23B-deficient females were consistently smaller than normal (only one or two pups/litter). Histology of mHR23B−/− ovaries (n = 3) showed a full spectrum of follicular development including Graafian follicles and corpora lutea, indicating normal endocrine regulation of ovarian function by follicle-stimulating hormone and luteinizing hormone (data not shown).
Additional findings in mHR23B−/− mice.
A large proportion of mHR23B-deficient mice frequently suffered from inflamed or swollen anuses in parallel with ulcers, resulting from rectal prolapse. Also, the mice had soft oily feces, which may point to an intestinal malfunctioning. The skin of mHR23B-deficient mice (n = 16) appeared thinner than that of wild-type mice. However, the skin of mHR23B-deficient mice (n = 4) showed no overt abnormalities compared with that of wild-type mice (mHR23B+/−; n = 2). In addition, the amount of subcutaneous fat in mHR23B−/− mice was comparable to that of wild-type mice (data not shown). Finally, a few mHR23B-deficient mice showed abnormal behavior, like jumping (n = 3) and circling or waltzing (n = 1), although definite conclusions await analysis using several behavioral tests with a larger number of animals.