Pattern of skeletal malformations produced by Dominant hemimelia (Dh)

The hindlimb malformations in adult mice heterozygous for the dominant gene Dominant hemimelia (Dh) and +/+ littermates were characterized in skeletons that had been fixed, stained, and cleared. When the tibia was shortened, the deficiency was always an absence of the distal portion, and never the proximal portion. Although tibial hemimelia has been well documented in Dh mice, this study demonstrated a distinctive pattern of shortening of the tibia. Measurements of the length of the tibia (relative to the length of the humerus) showed only three patterns of shortening of the tibia (i.e., mild, moderate, and severe), rather than a continuous spectrum of shortening from mild to complete absence. The hindlimb malformation of Dh/+ mice occurred in association with a reduced number (five) of lumbar vertebrae. The interrelationship of the hindlimb malformations and the reduction in the vertebral number suggests a relationship between the development of the axial skeleton and the abnormal limb.     

Enhanced susceptibility of mouse embryos heterozygous for oligosyndactyly (Os/+) to mitomycin C-induced skeletal abnormalities

The mouse mutation, oligosyndactyly (Os), results in syndactyly, muscle anomalies, and deficiency of nephrons in heterozygous animals and early embryonic lethality in homozygotes. Since the homozygous lethality results from mitotic arrest with intact spindles at the time of implantation, we have hypothesized that the heterozygous manifestations may result from impairment of cell proliferation in regions with high proliferative rates. To test this hypothesis, Os/+ and +/+ mouse embryos at 6.5 days of gestation were exposed to mitomycin C (MMC), an agent that causes a high degree of embryonic cell death which is "compensated" for by a period of rapid cell proliferation. 17.9% of MMC-treated +/+ fetuses had fused vertebrae, a significant increase over untreated fetuses, and this frequency was further increased to 33.6% in MMC-treated Os/+ fetuses. Saline treated Os/+ and +/+ fetuses showed the same low rate (0-3%) of vertebral fusion. These results indicate that Os/+ embryos have an increased sensitivity to the vertebral fusion-inducing effect of MMC at 6.5 days of gestation, a finding compatible with the hypothesis that rapid cell proliferation may be impaired in Os/+ embryos and fetuses.     

The paralogous Hox genes Hoxa10 and Hoxd10 interact to pattern the mouse hindlimb peripheral nervous system and skeleton

The most 5' mouse Hoxa and Hoxd genes, which occupy positions 9-13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. We have examined the effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrated impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation were also observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb.     

A comparison of Babesia infections in intact,surgically splenectomised,and congenitally asplenic (Dh/+) mice

Infections with Babesia rodhaini and B. microti were studied in congenitally asplenic (Dh/+) mice, surgically splenectomised mice and intact mice. Mice without spleens were more susceptible to infections than intact mice, but Dh/+ mice were less susceptible than surgically splenectomised mice, indicating that some functional splenic activity had been taken over by other tissues in Dh/+ mice. It is suggested that this functional activity may be mediated by natural killer (NK) cells, and that Dh/+ mice could prove of value in the study of babesiosis in general and NK activity in particular.Male mice were more susceptible to infection than females.     

Identification of Dh/+ and Dh/Dh embryos through close linkage of Dh and peptidase-3

The close linkage between the genes Dominant hemimelia (Dh) and peptidase-3 (Pep-3) has been determined in 65 informative matings with the recombination frequency of 3.8%. Progeny testing showed that nonpenetrance does occur in Dh/+ adults. The presence of the "slow" and "fast" variants of Pep-3 can be determined in homogenates of kidney tissue as well as in a portion of the day 10 and 11 embryos. In a litter of embryos born to an informative mating, those which are Dh/Dh, Dh/+, and +/+ can be distinguished by the presence of the Pep-3 allele known to be in coupling with the Dh gene. This technique makes it possible to identify and to study the limb malformations and other phenotypic effects of Dh during their development and before the limb deformity is visible.     

Prevention of polydactyly manifestation in Polydactyly Nagoya (Pdn) mice by administration of cytosine arabinoside during pregnancy

Male mice heterozygous for the dominant polydactyly gene Pdn (Polydactyly Nagoya) were crossed with normal or heterozygous females of the same strain. Pregnant females were treated with 5 mg/kg of cytosine arabinoside (Ara-C) on day 12 of gestation. The offspring were removed on day 18 of gestation and examined for external malformations, and the fore- and hindlimbs were examined by means of bone- and cartilage-stained cleared specimens. In +/+ x Pdn/+ matings, Pdn/+ fetuses, bearing preaxial polydactyly of the distal phalangeal type in the hindlimb and deformity of the 1st digit in the forelimb, were obtained in about 50% of the nontreated group. In treated fetuses, however, the incidence of polydactyly and deformity of the 1st digit decreased to 1.4 and 10.1%, respectively. Nontreated Pdn/Pdn fetuses exhibited preaxial polydactyly of the duplicated or triplicated metacarpal/metatarsal type both in the fore- and hindlimbs. In the treated Pdn/Pdn fetuses, the number of preaxial extra digits decreased in both limbs. Some hindlimbs of the treated Pdn/Pdn fetuses exhibited five metatarsals, normally. In the vitally stained specimens at 6 and 24 hours after injection of Ara-C, preaxial marginal necrotic zones (fMI) were observed in almost all of the treated embryos from +/+ x Pdn/+ matings. However, approximately half of the embryos did not exhibit fMI in the nontreated control group at the same stage. Those embryos deficient in fMI were regarded as Pdn/+. These findings indicated that a subteratogenic dose of Ara-C prevented the genetic expression of polydactyly in almost all Pdn/+ and some cases of Pdn/Pdn mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Congenital malformations in rats with the semilethal mutation fused pulmonary lobes

It has been demonstrated that an autosomal recessive gene, fused pulmonary lobes (fpl), causes fusion of the right pulmonary lobes with several associated malformations and a high incidence of death in homozygous newborns (Aoyama et al. Teratology 1988; 37:159-166). The aim of the present study was to investigate whether the deaths of fpl/fpl newborns were caused by functional abnormalities of the malformed lung or other associated malformations. Day-20 fpl/fpl and fpl/+ fetuses were weighed and examined for gross abnormalities. The lungs of selected fetuses were further examined for histological abnormalities. A wide variety of associated external, visceral, and skeletal anomalies as well as relatively lower body weights than those of phenotypically normal fpl/+ littermates were observed in the fpl/fpl fetuses. The associated anomalies consisted of hematomas and/or subcutaneous hemorrhages in the head, truncus and limbs, eyelid anomalies, CNS defects, lobation anomalies of the liver, hypoplasia of the spleen, partial absence of the skull bones, and dorsi- or ventriflexion of the phalanges of the limbs. Among them, CNS defects and partial absence of the skull bones were considered to be possible causes of newborn deaths. However, the incidence of these malformations was approximately 10% and was lower than the neonatal mortality, which had been estimated to be approximately 50% in the previous study (Aoyama et al. Teratology 1988; 37:159-166). The lungs of fpl/fpl fetuses consistently had hypoplasia of the intermediate lobe and fusion of the right pulmonary lobes. No histological changes suggesting postnatal respiratory insufficiency were found in the lungs of day-20 fpl/fpl fetuses, and the cause of newborn death remains unclear.     

terra is a left-right asymmetry gene required for left-right synchronization of the segmentation clock

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.     

Effects of hyperthermia and boric acid on skeletal development in rat embryos

BACKGROUND: The individual effects of boric acid (BA) and hyperthermia on the development of the axial skeleton have been reported previously. Both cause an increased incidence of axial skeletal defects including a decrease in the total number of ribs and vertebrae. Because of the similarity in the effects of the two agents, we examined their interaction when given in combination to pregnant rats on gestational day (GD) 10. METHODS: Dams were treated on GD 10 with BA (0, 250, or 500 mg/kg) and hyperthermia (37, 41, or 42 degrees C) and allowed to deliver their pups. Doses of BA were based on results from a dose-finding study. Litters were evaluated on postnatal days (PND) 1 and 3 for number, gender, and weight of pups. On PND3, pups were examined externally and viscerally, and double-stained for skeletal evaluation. RESULTS: A dose-dependent, statistically significant increase in fetal skeletal defects was seen on PND 3 with BA or hyperthermia alone with even greater effects when given in combination. Defects included rib and vertebral fusions, split vertebral centra in the thoracic and lumbar areas, and a decrease in the total number of ribs and vertebrae. CONCLUSIONS: The increased incidence of skeletal defects resulting from combined exposure to hyperthermia and BA was additive for segmentation defects and synergistic for the reduction in numbers of vertebrae.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Slj/+ and +/+ mice. The Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic 'missing' colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal 'niches' for colony formation. However, 35% of these niches is defective in its proliferative support.     

Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Slj/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU-end) formation following sublethal doses of gamma-radiation compared with their normal +/+ littermates, which is likely to be due to the low pre-irradiation CFU-S content of the Slj/+ spleen. CFU-S in these congenic mice do not differ in their sensitivity to gamma-irradiation or stem cell-activating factor. While injection of +/+ mice with 10 micrograms of lipopolysaccharide-W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Slj/+ mice was only slightly increased. Irradiation induced a similar dose-related reduction in the numbers of CFU-S in the spleen and femora of LPS-injected Slj/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU-end formation and higher numbers of CFU-S and nucleated cells in the Slj/+ spleens compared to LPS-injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Slj/+ mice with respect to the splenic and femoral 59Fe-incorporation and the femoral CFU-S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU-S to the CFU-S numbers and endogenous colony formation in at least the Slj/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential anti-radiation drugs.     

Axial and appendicular skeletal transformations, ligament alterations, and motor neuron loss in Hoxc10 mutants

Vertebrate Hox genes regulate many aspects of embryonic body plan development and patterning. In particular, Hox genes have been shown to regulate regional patterning of the axial and appendicular skeleton and of the central nervous system. We have identified patterning defects resulting from the targeted mutation of Hoxc10, a member of the Hox10 paralogous family. Hoxc10 mutant mice have skeletal transformations in thoracic, lumbar, and sacral vertebrae and in the pelvis, along with alterations in the bones and ligaments of the hindlimbs. These results suggest that Hoxc10, along with other members of the Hox10 paralogous gene family, regulates vertebral identity at the transition from thoracic to lumbar and lumbar to sacral regions. Our results also suggest a general role for Hoxc10 in regulating chondrogenesis and osteogenesis in the hindlimb, along with a specific role in shaping femoral architecture. In addition, mutant mice have a reduction in lumbar motor neurons and a change in locomotor behavior. These results suggest a role for Hoxc10 in generating or maintaining the normal complement of lumbar motor neurons.     

Deterioration of bone quality in the tibia and fibula in growing mice during skeletal unloading: gender-related differences

Skeletal unloading causes bone loss in both men and women; however, only a few studies have been performed on the effects of gender differences on bone quality during skeletal unloading. Moreover, although the fibula also plays an important role in load bearing and ankle stability, the effects of unloading on the fibula have been rarely investigated. The present study aimed to investigate the effects of skeletal unloading on bone quality of the tibia and fibula in growing animals and to determine whether differences existed between genders. Six-week-old female and male mice were randomly allocated into two groups. The right hindlimb of each mouse in the skeletal unloading group was subjected to sciatic neurectomy. After two weeks of skeletal unloading, the structural characteristics of the tibia and fibula in both genders were worsened. In addition, the bone mineralization density distribution (MDD) of the tibia and fibula in both genders were altered. However, the magnitude of deterioration and alteration of the MDD in the bones of females were larger than in those of males. These results demonstrate that skeletal unloading diminishes bone quality in the tibia and fibula, leading to an increase in bone fracture risks, particularly in females.     

Asymmetry of the transcallosal responses in the parietal cortex of kittens in the 1st month of life

In acute experiments on kittens the process of formation of asymmetry of transcallosal responses (TCR) was studied in multiple leads from symmetrical points of the parietal cortex. By the early positive-negative TCR complex, vanishing as a result of callosotomy, predominance of positive components in the right hemisphere was found in 2-7 days kittens, whereas in 8-24 days animals the left hemisphere dominated by both phases of responses. By the late TCR component preserved after section of the callosal body, left-hemispheric asymmetry was found in the elder group of kittens; it was absent in the younger animals. TCR asymmetry in the parietal cortex depended on the sex of the animals. With their age its inversion and enhancement took place. This process is based on the increase of TCR amplitude in the left hemisphere, with no increase in the right hemisphere.     

Characteristics of the Cfu-S Population In Mice Carrying the SlJ Allele

Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosen-sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Sl^J and in their normal littermates. Sl^J/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU-s of Sl^J/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. the seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Sl^J/+ recipients. However, in irradiated SI^J/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Sl^J/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Sl^J/+ mice, but significantly increased in Sl/Sl^d mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Sl^J/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro y irradiation. Femurs and spleens of both Sl^J/+ and +/+ origin were implanted into both Sl^J/+ and +/+ hosts. Six weeks later the Sl^J/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Sl^J/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under ‘steady-state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of Sl^J/+ mice segregate them from Sl/Sl^d mice, i.e. a deficient splenic CFU-s content, normal seeding fractions ‘f’ of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the Sl^J trait may be a useful extension of the current Sl/Sl^d model for exploration of hereditary defects in haematopoietic stroma.     

Maternal treatment with teratogen causes congenital malformations in mouse embryos

Heterozygotes for the tail-short (Ts) mutant gene in the Balb/c strain have minor skeletal defects and a short, kinky tail. If heterozygote Ts/+ mothers are mated with normal-tail +/+ males and are treated with teratogenic doses of trypan blue on the eighth day of pregnancy, the mutant F1 heterozygotes develop exencephaly, folded neural tube and spina bifida significantly more often than non-mutants. This is indicative of gene-teratogen interaction, with the Ts gene increasing the embryo's susceptibility to trypan blue-induced neural tube defects.     

Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.     

Increased susceptibility to 6-aminonicotinamide-induced cleft lip of heterozygote Dancer mice

Dancer heterozygotes (Dc/+) very rarely have cleft lip and show a dancing behaviour due to inner ear defects while homozygotes (Dc/Dc) have cleft lip. Males of the two genotypes Dc/+ and +/+ were mated to C3H strain and R stock females and Dc/+ males to Dc/+ females. On day 10/8 of gestation females were treated with 6-aminonicotinamide (6AN) at either 19 mg/kg or 28.5 mg/kg followed 3 h later by a protective dose of nicotinamide. Controls were untreated. Both 6AN treatments caused a significant increase in cleft lip to between 25% and 29% for crosses of Dc/+ males to C3H and R females whereas crosses with +/+ males gave 0% cleft lip. In the controls the cleft lip frequency was: for Dc/+ X Dc/+ 14%, Dc/+ X C3H 1.4%, and for the other three crosses 0%. The four crosses given the high dose of 6AN and the +/+ X C3H and Dc/+ X R cross at the low dose showed significantly increased resorption rates to between 23% and 47% over the control rates of from 5% to 11%. The presence of the Dc gene increased the susceptibility to cleft lip caused by 6AN.