Disordered vertebral and rib morphology in pudgy mice. Structural relationships to human scoliosis.

Normal and abnormal vertebral development have been studied over the past 200 years at increasing levels of resolution as techniques for biological investigation have improved. Disordered development of the axial skeleton from the early embryonic period on leads to structurally malformed vertebrae and intervertebral discs and ribs causing the severe deformities of scoliosis, kyphosis, and kyphoscoliosis. Developmental malformation of the axial skeleton therefore has led to considerable biological and clinical interest. This work will detail our studies on the structural deformities of the vertebral column and adjacent ribs in the pudgy mouse [1] caused by mutations in the delta-like 3 (Dll3) gene of the Notch family [2]. While gene abnormalities in the pudgy mouse have been outlined, there has been no in-depth assessment of the histopathology of the pudgy vertebral and rib abnormalities that this study will provide. In addition, although congenital scoliosis has been recognized as a clinical problem since the mid-nineteenth century (1800s) [3] and accurately defined by radiography since the early twentieth century (1900s) [4-6], there have been few detailed histopathologic studies of human cases. We will also relate our histopathologic findings in the pudgy mouse to the histopathology of human vertebral and rib malformations in clinical cases of congenital scoliosis, one of which we defined in detail previously [7].

A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins.

The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease.

Pathbase and the MPATH ontology. Community resources for mouse histopathology.

Pathbase, the database of mouse histopathology images, was developed as a resource to provide free access to representative images of lesions in background and mutant strains of laboratory mice. When utilized with diagnostic workups or phenotyping of mutant mice, it can provide a "virtual second opinion" for those working without access to groups of experienced pathologists. This is a community resource, and it facilitates the sharing of expertise and data among members of the pathology community worldwide. MPATH-the mouse pathology ontology-was developed alongside Pathbase for the annotation of images and now represents an important resource for the coding of diagnoses, permitting sophisticated data retrieval and computational analysis of mouse phenotypes. In this article, the structure and use of MPATH is discussed, along with current and future challenges for the coding of mutant mouse phenotypes.

Cerebellum-related characteristics of Scn8a-mutant mice.

Among ten sodium channel alpha-subunit genes mapped in human and mouse genomes, the SCN8A gene is primarily expressed in neurons and glia. Mice with two types of Scn8a null mutations--Scn8a ( med ) and Scn8a ( medTg )--live for only 21-24 days, but those with incomplete mutations-Scn8a ( medJ ) and Scn8a ( medJo )--and those with knockout of Scn8a only in cerebellar Purkinje cells live to adult age. We review here previous work on cerebellum and related regions of Scn8a mutant mice and include some newer immunohistochemical and microchemical results. The resurgent sodium current that underlies the repeated firing of Purkinje cells is reduced in Scn8a mutant and knockout mice. Purkinje cells of mutant mice have greatly reduced spontaneous activity, as do the analogous cartwheel cells of the dorsal cochlear nucleus. Up-regulation of GABA(A) receptors in regions to which Purkinje cells project may partially compensate for their decreased activity in the mutant mice. The somata of cerebellar Purkinje cells of Scn8a ( medJ ) and Scn8a ( medJo ) mice, as revealed by PEP-19 immunoreaction, are slightly smaller than normal, and their axons, especially in Scn8a ( medJo ) mice, sometimes show enlargements similar to those in other types of mutant mice. Density of GABA-like immunoreactivity is decreased in Purkinje somata and regions of termination in deep cerebellar and vestibular nuclei of Scn8a ( medJ ) mice, but measured GABA concentration is not significantly reduced in microdissected samples of these regions. The concentrations of taurine and glutamine are significantly increased in cerebellar-related regions of Scn8a ( medJ ) mice, possibly suggesting up-regulation of glial amino acid metabolism.

Short-limbed dwarfism: slw is a new allele of Npr2 causing chondrodysplasia.

Short-limbed dwarfism (SLW) is a new mutant mouse characterized by a dwarf phenotype with markedly short body, limbs, and tail. In the present study, we investigated the skeletal phenotypes of the SLW mouse and determined the chromosomal localization to identify the gene responsible for the phenotypes (slw). Skeletal preparations stained with alcian blue and alizarin red revealed that longitudinal growth of the extremities of the affected (slw/slw) mice was significantly reduced in comparison with that of normal mice, whereas the positions and numbers of skeletal elements were normal. Histological examination of tibial growth plates of the affected mice showed that the numbers of proliferating and hypertrophic chondrocytes were obviously diminished. These phenotypes resembled to those of human chondrodysplasias caused by defective chondrocyte proliferation and differentiation. We mapped the slw locus on an 11.7-cM interval of the proximal region of mouse chromosome 4 by linkage analysis. Furthermore, allelism test using Npr2(cn) locus, a mutant allele of Npr2 gene encoding a natriuretic peptide receptor B, revealed that slw locus is an allele of the Npr2 gene. These results suggest that the dwarf phenotype of the SLW mouse is caused by the disturbed endochondral ossification, and a mutation in the Npr2 gene is expected to be responsible for the phenotypes of the SLW mouse.

Behavioral phenotypes of Disc1 missense mutations in mice.

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.

Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection.

Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P < 5 x 10(-10)), with the HMI hybrid greatest in both body and placental weights. In an application study using these F1 offspring, we analyzed their mitochondrial DNA using intersubspecific polymorphisms and found the consistent disappearance of sperm mitochondrial DNA in the F1 progeny. In a second series of experiments, we generated F1 blastocysts by injecting MSM spermatozoa into C57BL/6 oocytes and used them to generate hybrid ES cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.

Comparison of contractile responses in isolated mouse aorta and pulmonary artery: Influence of strain and sex.

Transgenic mice are often used to study the physiologic role of a known gene. The design of experiments with transgenic mice usually does not take into account strain and sex differences, at least in isolated vessels. Therefore, we have compared the contractile response of isolated aortae and isolated pulmonary arteries of male and female mice of different strains (CD1, BL6, and DBA). Contractile stimulation was achieved by depolarization due to KCl, alpha1-adrenoceptor stimulation by phenylephrine and inhibition of protein phosphatase activity by cantharidin. In isolated aorta, strain-specific differences in contractility and sex-specific differences could be observed. The concentration of phenylephrine (PE) inducing half maximal contraction (EC50) was different between aortae from DBA male mice and the other strains tested. Phasic contractions of isolated aortic rings due to PE were seen in all mice except DBA male. In isolated pulmonary arteries, strain-specific differences and sex-specific differences could be observed. The EC50-values of PE were not different between all groups. Phasic contractions due to PE were only seen in pulmonary arteries from CD1 male and BL6 female. In conclusion, strain- and sex-specific differences should be considered in selecting mice used for transgenesis or gene targeting experiments.

Canalization and developmental stability in the Brachyrrhine mouse.

The semi-dominant Br mutation affects presphenoid growth, producing the facial retrognathism and globular neurocranial vault that characterize heterozygotes. We analysed the impact of this mutation on skull shape, comparing heterozygotes to wildtype mice, to determine if the effects are skull-wide or confined to the sphenoid region targeted by the mutation. In addition, we examined patterns of variability of shape for the skull as a whole and for three regions (basicranium, face and neurocranium). We found that the Br mice differed significantly from wildtype mice in skull shape in all three regions as well as in the shape of the skull as a whole. However, the significant increases in variance and fluctuating asymmetry were found only in the basicranium of mutant mice. These results suggest that the mutation has a significant effect on the underlying developmental architecture of the skull, which produces an increase in phenotypic variability that is localized to the anatomical region in which the mean phenotype is most dramatically affected. These results suggest that the same developmental mechanisms that produce the change in phenotypic mean also produce the change in variance.

A mutation in stratifin is responsible for the repeated epilation (Er) phenotype in mice.

Stratifin (Sfn, also called 14-3-3sigma) is highly expressed in differentiating epidermis and mediates cell cycle arrest. Sfn is repressed in cancer, but its function during development is uncharacterized. We identified an insertion mutation in the gene Sfn in repeated epilation (Er) mutant mice by positional cloning. Er/+ mice expressed a truncated Sfn protein, which probably contributes to the defects in Er/Er and Er/+ epidermis and to cancer development in Er/+ mice.

Development of the inner ear in Splotch mutant mice.

The Splotch mouse, a Pax 3 mutation, represents a model of Waardenburg syndrome I. We show that the homozygous Splotch mutation (Sp(2H)) is associated with severe defects that prevent the formation of the cochlea and vestibulo-cochlear ganglion. To clarify the role of Pax 3 in inner ear formation, we examined the expression of polysialic acid (PSA) associated with neural cell adhesion molecule (NCAM). In accordance with the occurrence of phenotypic abnormalities, PSA NCAM was expressed early in otocyst development in the otic epithelium and the vestibulo-cochlear anlage. During the period of vestibular and cochlear ganglia formation, PSA NCAM expression was decreased. In the late phase of embryonic development, the expression of calcium binding proteins (S100) in the vestibulo-cochlear ganglion was also decreased. Minor differences in S100 immunostaining were found postnatally between the cochleas of heterozygous and wild type animals.

A genetic basis for the "Adonis" phenotype of low adiposity and strong bones.

Toll receptors in Drosophila contribute to host defense and establish the body plan. Mammalian homologues of Toll, the Toll-like receptors (TLRs), are thought to function only in host defense. Here, we report that mice harboring mutations in TLR4 or in CD14, a co-receptor for TLR4, have an "ideal" body plan consisting of increased bone mineral content, density, and size as well as decreased body fat. These mutant mice live long lives, have normal activity and fertility, and show no evidence of infection. Unlike many strains of caged wild-type mice, they do not become obese. Although all mice continue to gain body fat, bone content, and overall weight, the difference in bone content and body fat between mutant and wild-type mice increases with age. Thus, defects in TLR4/CD14 complex generate an "Adonis" phenotype, characterized by this ideal body type, and this function could potentially be exploited for the treatment of osteoporosis and obesity.

New mutant mouse with skeletal deformities caused by mutation in delta like 3 (Dll3) gene.

We have established a new mouse strain with vertebral deformities caused by an autosomal single recessive mutation (oma). The mutant mice showed short trunk and short and kinky tail. The skeletal preparations of newborn and prenatal mice showed disorganized vertebrae and numerous vertebral and rib fusions which are thought to be caused by patterning defects at the stage of somitegenesis. Linkage analysis localized the oma locus on the proximal region of mouse chromosome 7 close to Dll3 gene. Dll3 is the gene involved in the Notch signaling pathway and null-mutation of the gene has been reported to cause vertebral deformities. The phenotypic similarity between oma and Dll3 null-mutant mice suggests that the causative gene for the oma mutant is the Dll3 gene. We, therefore, investigated the nucleotide sequence of the Dll3 gene of the oma mouse and found a single nucleotide substitution of G to T which causes missense mutation of glycine to cysteine at codon 409. Since the amino acid substitution is a nonconservative amino acid substitution at the conserved portion of the Dll3 protein, and the substitution is specific to the mutant mice, we concluded that the nucleotide substitution of the Dll3 gene is responsible for the skeletal deformities of the oma mouse.

Circling mouse, a spontaneous mutant in the inner ear.

A spontaneous mutant was established in the ICR mouse strain. The affected mice became hyperactive at about 7 days of age, and then showed circling behavior. The body weight decreased significantly 2 weeks after birth, and developmental defects were revealed in the middle ear, cochlea, cochlear nerve, and semicircular canal areas. The mutation was inherited by an autosomal single recessive gene and is referred to as cir.

Early development and degeneration of vestibular hair cells in bronx waltzer mutant mice.

In bronx waltzer mouse mutants, inner hair cells die at an early stage in their development, from around 17.5 days of gestation onwards. In contrast, outer hair cells appear to develop normally. Vestibular hair cells also degenerate, but the earliest signs of vestibular abnormalities have not yet been described. We looked at prenatal and early postnatal stages of vestibular development by scanning electron microscopy in the mutants, and established that vestibular hair cells (types I and II) never reach beyond the middle stages of differentiation (at least up to P2) and instead show signs of degeneration. Thus, it appears that the bronx waltzer gene product is required for the continued survival and differentiation of inner and vestibular hair cells past a set point in their development.

Genetic sources of individual differences in the cerebellum.

The highly regular anatomy of the cerebellum that results from myriad genetic, environmental, and stochastic events during pre- and postnatal development is nonetheless quantitatively very different among individuals. Understanding the sources of these individual differences represents an immense challenge to those interested in the cerebellum. Here we highlight the use of new methods to dissect individual differences to their genetic sources by reviewing quantitative trait locus mapping efforts in the mouse model system. We further suggest and illustrate how to combine these methods with other modern genetic techniques to accelerate our understanding. Finally, we embed these methods in a hypothetical line of cerebellar research to illustrate the vast potential of combining complex trait analysis with a systems neuroscience perspective.

Evaluating mutant mice: anatomic pathology.

As the human and mouse genome projects approach their goals, initiatives in functional genomics are advancing. When the nucleotide sequences are available, identification of gene functions will assume even greater importance. Determination of gene products and their proximal biochemical functions provide a part of the picture, but determination of their functions in the context of the whole organism is the ultimate goal. The manipulated mouse genome has become accepted as a model for understanding the genetic basis of human conditions and diseases. Consequently, biomedical research institutions have seen significant increases in the use of mice since the early 1980s, and these increases are largely attributable to the use of genetically modified mice. The role of comparative pathology in research on mutant mouse models of disease is increasing in response to these trends. Evaluation and phenotypic characterization of mutant mice, via clinical and anatomic pathology techniques, will be an important component of functional genomics initiatives.

Evidence for intrinsic development of olfactory structures in Pax-6 mutant mice.

It has been reported that the arrival of primary olfactory axons is required to induce the development of the olfactory bulb (OB). On the other hand, the Sey(Neu)/Sey(Neu) mutant mouse (Small eye) has been previously described as a model for the absence of olfactory bulbs, owing to the lack of olfactory epithelium (OE). In the present report, we take advantage of this mutant and study a neural structure in the rostral pole of the telencephalon that phenotypically resembles the prospective OB. We named this formation olfactory bulb-like structure (OBLS). We also report the occurrence, in the mutants, of small epithelial vesicles in the malformed craneofacial pits, resembling an atrophic OE, although a mature olfactory nerve was not identified. Axonal tracing, birthdating, immunohistochemistry, and in situ hybridization using antibodies and probes expressed in the olfactory system, indicated that two distinct structures observed in the OBLS correspond to the main and accessory olfactory bulbs of the control mouse. We propose that the OBLS has developed independently of the external influences exerted by the olfactory nerve. The presence of a prospective OB in the mutants, without intervening olfactory fibers, suggests that intrinsic factors could define brain territories even in absence of the proper afferent innervation. The intrinsic mechanisms and environmental cues in the telencephalon could be sufficient to promote axonogenesis in the projection neurons of the OB and guide their axons in a lateral prospective tract, in the absence of olfactory axons.

Pads and flexion creases on the plantar surface of hammertoe mutant mouse (Hm).

The purpose of the present work was to determine the effects of the hereditary malformation of Hammertoe mutant mice (gene symbol Hm) on the surrounding morphological structures and, specifically, on the volar pads, i.e., the sites of the epidermal ridge patterns (dermatoglyphics). The hindlimbs of the wild-type (+/+) Hammertoe mice show no anomalies and their major pad and flexion crease configurations correspond to those of normal mice. The heterozygous (Hm/+) and homozygous (Hm/Hm) mice display a fusion of the interdigital tissues involving all digits with the exception of digit I. In Hm/Hm mice, this webbing extends to the distal phalanx and the markedly flexed digits form a shape resembling a hammer. In Hm/+ mice, the interdigital webbing does not extend as far and the digits show moderate flexion compared to those of Hm/Hm mice. Both Hm/Hm and Hm/+ have a rudimentary extra digit in the postaxial area of the hindlimbs. The ventral volar skin of the flexed digits is incompletely developed. The more posterior digits show the more severe camptodactyly. These aberrant configurations are related to the abnormal occurrence of the programmed cell death (PCD) in the interdigital zones II-IV and the proximal part of the postaxial margin during hindlimb development. They are limited to the pads on the plantar surface of the postaxial area; the preaxial area is not affected. As a result of a severe camptodactyly of digit V, its volar skin is shifted into the distal portion of the hypothenar area. This shifting affects the number, size, and location of the pads, especially of the hypothenar pad, resulting in varying pad configurations, such as a displacement of the distal and proximal components of the hypothenar pad, or a fusion of the two components of the hypothenar pad, leading to a reduced final pad number. These pad modifications are induced by the postaxial plantar surface shifting proximally and are not affected by the presence of an extra rudimentary digit. The pad modifications in Hammertoe mice with webbed digits and postaxial polydactyly resemble closely those of the previously studied mice with genetic preaxial polydactyly.

Establishment and characterization of the MSKR inbred strain originated from Japanese wild mice (Mus musculus molossinus).

A new inbred strain, MSKR, originated from Japanese wild mice was established in April, 1998. The MSKR mice were 60% of the C57BL/6N inbred mice in the 60-day body weight. Tail length/head-body length and hind-foot length/head-body length of the MSKR mice were significantly smaller than those of the C57BL/6N mice (0.896 vs 1.061, 0.189 vs 0.204), but ear length/head-body length of the MSKR mice was significantly larger than that of the C57BL/6N mice (0.143 vs 0.137). The age of the first parturition and size of the first litter were 63.20 +/- 2.71 days and 6.20 +/- 0.37, respectively, at the 20th and 22nd inbreeding generations. Genetic characterization of the MSKR strain was performed using 34 microsatellite markers, 29 biochemical markers, 9 immunogenetic markers, 3 coat color markers, and mitochondrial DNA RFLP-haplotypes. The result indicated that this newly established inbred strain has some different gene constitution from already known molossinus and common laboratory strains.