Targeted inactivation of dipeptidyl peptidase 9 enzymatic activity causes mouse neonate lethality.

Dipeptidyl Peptidase (DPP) 4 and related dipeptidyl peptidases are emerging as current and potential therapeutic targets. DPP9 is an intracellular protease that is regulated by redox status and by SUMO1. DPP9 can influence antigen processing, epidermal growth factor (EGF)-mediated signaling and tumor biology. We made the first gene knock-in (gki) mouse with a serine to alanine point mutation at the DPP9 active site (S729A). Weaned heterozygote DPP9 (wt/S729A) pups from 110 intercrosses were indistinguishable from wild-type littermates. No homozygote DPP9 (S729A/S729A) weaned mice were detected. DPP9 (S729A/S729A) homozygote embryos, which were morphologically indistinguishable from their wild-type littermate embryos at embryonic day (ED) 12.5 to ED 17.5, were born live but these neonates died within 8 to 24 hours of birth. All neonates suckled and contained milk spots and were of similar body weight. No gender differences were seen. No histological or DPP9 immunostaining pattern differences were seen between genotypes in embryos and neonates. Mouse embryonic fibroblasts (MEFs) from DPP9 (S729A/S729A) ED13.5 embryos and neonate DPP9 (S729A/S729A) mouse livers collected within 6 hours after birth had levels of DPP9 protein and DPP9-related proteases that were similar to wild-type but had less DPP9/DPP8-derived activity. These data confirmed the absence of DPP9 enzymatic activity due to the presence of the serine to alanine mutation and no compensation from related proteases. These novel findings suggest that DPP9 enzymatic activity is essential for early neonatal survival in mice.

Aspects of achondroplasia in the skulls of dwarf transgenic mice: a cephalometric study.

Achondroplasia, the most common short-limbed dwarfism in humans, results from a single nucleotide substitution in the gene for fibroblast growth factor receptor 3 (FGFR3). FGFR3 regulates bone growth in part via the mitogen-activated protein kinase pathway (MAPK). To examine the role of this pathway in chondrocyte differentiation, a transgenic mouse was generated that expresses a constitutively active mutant of MEK1 in chondrocytes and exhibits dwarfing characteristics typical of human achondroplasia, i.e., shortened axial and appendicular skeletons, mid-facial hypoplasia, and dome-shaped cranium. In this study, cephalometrics of the MEK1 mutant skulls were assessed to determine if the MEK1 mice are a good model of achondroplasia. Skull length, arc of the cranial vault, and area, maximum and minimum diameters of the brain case were measured on digitized radiographs of skulls of MEK1 and control mice. Cranial base and nasal bone length and foramen magnum diameter were measured on midsagittal micro-CT sections. Data were normalized by dividing by the cube root of each animal's weight. Transgenic mice exhibited a domed skull, deficient midface, and (relatively) prognathic mandible and had a shorter cranial base and nasal bone than the wild-type. Skull length was significantly less in transgenic mice, but cranial arc was significantly greater. The brain case was larger and more circular and minimum diameter of the brain case was significantly greater in transgenic mice. The foramen magnum was displaced anteriorly but not narrowed. MEK1 mouse cephalometrics confirm these mice as a model for achondroplasia, demonstrating that the MAP kinase signaling pathway is involved in FGF signaling in skeletal development.

Axonal transport, tau protein, and neurodegeneration in Alzheimer's disease.

The molecular causes and the genetic and environmental modifying factors of the sporadic form of Alzheimer's disease (AD) remain elusive. Extrapolating from the known mutations that cause the rare familial forms and from the typical post-mortem pathological lesions in all AD patients--e.g., amyloid plaques and neurofibrillary tangles (NFTs)-the evident molecular candidates are amyloid precursor protein (APP), presenilin, and tau protein. To include ApoE4 as the only certain genetic modifier known leaves us to face the challenge of implementing these very different molecules into an evident pathological partnership. In more than one respect, the proposition of disturbed axonal transport appears attractive with more details becoming available on APP processing and microtubular transport and also of the pathology in the model systems--e.g., transgenic mice expressing APP or protein tau. Conversely, the resistance of APP-transgenic mice with full-blown amyloid pathology to also develop tau-related neurofibrillar pathology is a major challenge for this hypothesis. From the most relevant data discussed here, we conclude that the postulate of disturbed axonal transport as the primary event in AD is difficult to defend. On the other hand, failing axonal transport appears to be of major importance in the later stages in AD, by further compromising tau protein, APP metabolism, and synaptic functioning. Protein tau may thus be the central "executer" in the chain of events leading from amyloid neurotoxicity to tau hyperphosphorylation, microtubular destabilization, disturbed axonal transport, and synaptic failure to neurodegeneration. In order to identify normal physiological processes and novel pathological targets, definition is needed--in molecular detail--of the complex mechanisms involved.

Retinal organization in the bcl-2-overexpressing transgenic mouse.

Naturally occurring cell death is believed to play a major role during the development of the nervous system in the establishment of neuronal architecture. Here we study the effects of cell death inhibition by using a transgenic mouse in which the powerful antiapoptotic gene bcl-2 is expressed in neurons. The retina of this mouse reveals that the general neuronal plan has been maintained. However, bcl-2 overexpression leads to altered frequencies of the major cell types in the retina. Thus, it is possible to estimate cell-type-specific rates of apoptosis by observing the increases in numbers of cells in the bcl-2-overexpressing transgenic mouse.

To die or not to die, does it change the function? Behavior of transgenic mice reveals a role for developmental cell death.

In humans, perturbations in the developmental neuronal death leading to an excess of neurons could be associated with developmental neuropsychiatric disorders. Hu-bcl-2 transgenic mice appear to be a valuable tool to study the functional role of developmental programmed cell death. Indeed, the over-expression of the anti-apoptotic gene bcl-2 decreases developmental neuronal death and Hu-bcl-2 mice present supernumerary neurons in several brain regions. A detailed behavioral analysis of these mice revealed selective deficits. Hu-bcl-2 mice have normal vision, general activity and motor skills. Only the most complex behavior like anxiety and learning abilities are impaired in these mice.

EAAT1 and EAAT2 immunoreactivity in transgenic mice with a G93A mutant SOD1 gene.

Immunocytochemical and quantitative analyses were used to correlate the localisation of excitatory amino acid transporter proteins EAAT1, EAAT2 with time in spinal motoneurones of presymptomatic and symptomatic mice with the G93A mutant SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. EAAT1 and EAAT2 immunoreactivity was well-preserved in the gray matter in both controls and transgenic mice at all ages, and there was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between controls and transgenic mice. These findings suggest that EAAT1 and EAAT2 may not play a pivotal role in the degeneration of motoneurons in this animal model.

Survival and plasticity of basal forebrain cholinergic systems in mice transgenic for presenilin-1 and amyloid precursor protein mutant genes.

The basalo-cortical cholinergic system was characterized in mice expressing mutant human genes for presenilin-1 (PS1), amyloid precursor protein (APP), and combined PS/APP. Dual immunocytochemistry for ChAT and A beta revealed swollen cholinergic processes within cortical plaques in both APP and PS/APP brains by 12 months, suggesting aberrant sprouting or redistribution of cholinergic processes in response to amyloid deposition. At 8 months, cortical and subcortical ChAT activity was normal (PS/APP) or elevated (PS, APP frontal cortex), while cholinergic cell counts (nBM/SI) and receptor binding were unchanged. ChAT mRNA was up-regulated in the nBM/SI of all three transgenic lines at 8 months. The data indicate that the basal forebrain cholinergic system does not degenerate in mice expressing AD-related transgenes, even in mice with extreme amyloid load. The

Oligodendroglial tau filament formation in transgenic mice expressing G272V tau.

Genetic evidence indicates that several mutations in tau, including G272V, are linked to frontotemporal dementia with parkinsonism. We expressed this mutation in mouse brains by combining a prion protein promoter-driven expression system with an autoregulatory transactivator loop that resulted in high expression of human G272V tau in neurons and in oligodendrocytes. We show that G272V tau can form filaments in murine oligodendrocytes. Electron microscopy established that the filaments were either straight or had a twisted structure; these were 17-20 nm wide and had a periodicity of approximately 75 nm. Filament formation was associated with tau phosphorylation at distinct sites, including the AT8 epitope 202/205 in vivo. Immunogold electron microscopy of sarcosyl-extracted spinal cords from G272V transgenic mice using phosphorylation-dependent antibodies AT8 or AT100 identified several sparsely gold-labelled 6-nm filaments. In the spinal cord, fibrillary inclusions were also identified by thioflavin-S fluorescent microscopy in oligodendrocytes and motor neurons. These results establish that expression of the G272V mutation in mice causes oligodendroglial fibrillary lesions that are similar to those seen in human tauopathies.

Dopamine deficiency in mice.

Dopamine is the principal neurotransmitter that mediates a wide range of brain functions, including locomotion, emotion, learning, and neuroendocrine modulation. To clarify the role of dopamine during postnatal development, it is useful to have mutant mice genetically deleting dopamine. In this paper, we describe the mice lacking expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme of catecholamine biosynthetic pathway, in the dopaminergic neuronal type. In these mice, TH expression in noradrenergic and adrenergic cells was restored. Lack of TH expression in dopaminergic neurons resulted in a marked reduction of dopamine accumulation. This led to multiple behavioral abnormalities at the juvenile stage, which were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine-induced hyperactivity, cataleptic behavior, and defect in active avoidance learning. In contrast, development of pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained in spite of the reduced dopamine synthesis. Our findings provide genetic evidence that dopamine is essential for controlling spontaneous and voluntary movement and emotional learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways.

Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice.

Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.

Progressive formation of inclusions in the striatum and hippocampus of mice transgenic for the human Huntington's disease mutation.

The significance of neuronal intranuclear inclusions (NIIs) and extranuclear inclusions (ENNIs) in the brains of patients with polyglutamine repeat diseases and transgenic mice modelling these diseases is hotly debated. We examined inclusions in the brains of mice transgenic for the human Huntington's disease mutation and found that their size, number and location varied markedly with age and neuronal phenotype. In striatum and hippocampus particularly, inclusions appeared at different times in different cell types. Further, the mechanism of formation of inclusions appears to be complex, with several distinct phases. These include a precipitous formation of NIIs followed by NII growth, and the concomitant formation ENNIs. While the timing of appearance of NIIs and ENNIs parallels the cognitive and motor decline of the mice, the precise role of NIIs and ENNIs is unknown. It has been variously suggested that NIIs may be deleterious, benign or beneficial. However, our data allows the possibility that each of these is possible, and suggest also that the role of inclusions changes with time. The precipitous formation of NIIs may play a protective role by removing polyglutamine, while the subsequent growth of NIIs may be deleterious, since it would allow other proteins to be sequestered into inclusions. The formation of ENNIs in neurites and synapses is also more likely to have deleterious than beneficial consequences for a cell. Thus, our study suggests that the relationship between inclusion formation and neurological dysfunction depends not only upon the phenotype of the neurons involved, but also upon the molecular composition and the subcellular localisation of the inclusions.

Methotrexate-resistant form of dihydrofolate reductase protects transgenic murine embryos from teratogenic effects of methotrexate.

Methotrexate, a potent inhibitor of the ubiquitously expressed enzyme dihydrofolate reductase, induces limb and facial anomalies that resemble vascular disruptions in their evolution and final outcome. Previous studies suggest that inhibition of dihydrofolate reductase is responsible for methotrexate-induced embryopathy, although specific sites of methotrexate activity have not been well defined. In this report, we show that constitutive expression of a methotrexate-resistant form of dihydrofolate reductase in transgenic embryos and their placentas ameliorates methotrexate teratogenicity. However, expression of the transgene in maternal tissues had no significant protective effect. The results confirm the role of dihydrofolate reductase inhibition in the pathogenesis of methotrexate-induced birth defects and provide a foundation for future studies of targeted transgene expression in select embryonic or placental cell populations.

Overexpression of mouse follistatin causes reproductive defects in transgenic mice.

Follistatin is an activin-binding protein that can act as an activin antagonist in vitro. Follistatin also binds heparin sulfate proteoglycans and may function as a reservoir for activins in vivo. In the mouse, follistatin mRNA is first detected in the deciduum on embryonic day 5.5 and later in the developing hindbrain, somites, vibrissae, teeth, epidermis, and muscle. We have previously shown that follistatin-deficient mice have numerous embryonic defects including shiny, taut skin, growth retardation, and cleft palate leading to death within hours of birth. To further define the roles of follistatin during mammalian reproduction and development, we created gain-of-function mutant mice in which mouse follistatin is overexpressed. The mouse metallothionein (MT)-I promoter was placed upstream of the six-exon mouse follistatin (FS) gene. To distinguish wild-type and transgenic follistatin mRNA, the 3'-untranslated region of the mouse follistatin gene was replaced with the SV40 untranslated and polyA sequences. Three male and two female founder transgenic mice were produced, were fertile, and transmitted the transgene to offspring. Northern blot analysis demonstrated that the transgene mRNA was expressed at varying levels in the livers of offspring from four of five of the transgenic lines and was expressed in the testes in all five lines. In MT-FS line 4, which had the highest expression of the transgene mRNA in the liver, the transgene transcripts were also present in multiple other tissues. Phenotypically, the MT-FS transgenic lines had defects in the testis, ovary, and hair. Mice from MT-FS lines 7 and 10 had slightly decreased testis size, whereas mice from lines 4, 5, and 9 had much smaller testes and shiny, somewhat irregular, fur. Histological analysis of the adult testes from line 5 and 9 males showed variable degrees of Leydig cell hyperplasia, an arrest of spermatogenesis, and seminiferous tubular degeneration leading to infertility. Female transgenic mice from lines 4 and 9 had thin uteri and small ovaries due to a block in folliculogenesis at various stages. Many of the line 9 female mice eventually became infertile, and all of the line 4 female mice were infertile. Suppressed serum FSH levels were seen in only the line 4 transgenic male and female mice, the line with widespread expression of the transgene. Serum FSH levels were not significantly different in gonadectomized wild-type and line 5 transgenic male mice despite high levels of the follistatin transgene mRNA in the liver of these transgenic mice. These results suggest that follistatin exerts its effects at the levels of the gonads and pituitary as a local regulator of activin and possibly other transforming growth factor-beta family members.

TGF-beta receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis.

TGF-beta signaling is mediated through two types of serine/threonin kinase-containing receptors, type I (TGF-betaRI) and type II (TGF-betaRII), which form a heteromeric complex. In this signaling complex, ligand binding TGF-betaRII phosphorylates and thereby activates the TGF-betaRI to signal downstream pathways. To determine the role of TGF-betaRII in embryogenesis, we have generated a TGF-betaRII gene (Tgfbr2) knockout mouse line. The heterozygous Tgfbr2 knockout mice are developmentally normal. The homozygous Tgfbr2 mutation causes defects in the yolk sac hematopoiesis and vasculogenesis, resulting in an embryonic lethality around 10.5 days of gestation. This phenotype is indistinguishable from the previously reported embryonic lethality by the homozygous TGF-beta1 gene (Tgfb1) null mutation. In addition, we have generated chimeric mice using a Tgfbr2 (-/-) embryonic stem cell line. Some chimeric mice showed several types of congenital anomalies, suggesting that TGF-beta II is important for normal development in a variety of organs.

Axial homeosis and appendicular skeleton defects in mice with a targeted disruption of hoxd-11.

Using gene targeting, we have created mice with a disruption in the homeobox-containing gene hoxd-11. Homozygous mutants are viable and the only outwardly apparent abnormality is male infertility. Skeletons of mutant mice show a homeotic transformation that repatterns the sacrum such that each vertebra adopts the structure of the next most anterior vertebra. Defects are also seen in the bones of the limb, including regional malformations at the distal end of the forelimb affecting the length and structure of phalanges and metacarpals, inappropriate fusions between wrist bones, and defects at the most distal end in the long bones of the radius and ulna. The phenotypes show both incomplete penetrance and variable expressivity. In contrast to the defects observed in the vertebral column, the phenotypes in the appendicular skeleton do not resemble homeotic transformations, but rather regional malformations in the shapes, length and segmentation of bones. Our results are discussed in the context of two other recent gene targeting studies involving the paralogous gene hoxa-11 and another member of the Hox D locus, hoxd-13. The position of these limb deformities reflects the temporal and structural colinearity of the Hox genes, such that inactivation of 3' genes has a more proximal phenotypic boundary (affecting both the zeugopod and autopod of the limb) than that of the more 5' genes (affecting only the autopod). Taken together, these observations suggest an important role for Hox genes in controlling localized growth of those cells that contribute to forming the appendicular skeleton.

Malformation and degeneration in the inner ear of mos transgenic mice.

Transgenic mice carrying the mos proto-oncogene linked to a retroviral transcriptional control sequence display behavioral abnormalities including circling, hyperactivity, head tilt, and bobbing. Axonal degeneration, neuronal chromatolysis, spongiform encephalopathy, gliosis, and inflammatory infiltrates are reportedly found in the central nervous systems of all mutants with the behavioral traits. Hearing was tested by means of broadband free-field rarefaction clicks with auditory brain stem response recorded between vertex and mouth electrodes. No detectable auditory response was elicited in transgenic animals, in contrast to five distinct positive peaks observed in littermate control animals. Light microscopic survey of temporal bone histopathology in mutants revealed extensive degeneration of the organ of Corti with loss of hair cells in all cochlear turns and loss of supporting cells and atrophy of spiral ganglion cells. The spiral limbus was deformed, with replacement of the usual convexity of the superior surface by a flattened trough configuration. Hair cells of the vestibular end organs appeared normal. Pathologic alteration in levels of mos transgene RNA appears to have a direct effect on the structural integrity of the inner ear.

Microtia as an autosomal dominant mutation in a transgenic mouse line: a possible animal model of branchial arch anomalies.

Microtia was found in a transgenic mouse 643 and all offspring with microtia had the transgene. No anomalies, other than occasional low set ear and abnormal biting, were identified in other tissues and organs. In the developmental analysis, on the 9th and 10th days of gestation, hypoplasia of the second branchial arch was observed, while various kinds of malformed hillocks were noted on the 12th day. All of these anomalous embryos were transgenic. Histologically, hemorrhage and subsequent phagocytosis were noted at the second branchial arch. Left sided anomalies were predominant and in bilaterally defective ones asymmetry existed. These findings closely resembled to those in experimental animals with a phenocopy of the first and second branchial arch syndrome in humans. Since all other transgenic mouse lines with the same transgene as 643 appeared normal, this dysmorphic phenotype may be caused by an insertional mutation of a host gene, although inappropriate expression of the transgene should be examined further as a possible cause. These results suggest that this transgenic mouse line 643 may be useful as an animal model of branchial arch anomalies in humans.

Phenotypic characterization of the transgenic mouse insertional mutation, legless.

In this report, we describe the dysmorphologic phenotype associated with the transgenic insertional mutation legless. This autosomal recessive, perinatally lethal mutation results in an interesting pleiotropic array of congenital malformations. The phenotype of the legless mutation in homozygous perinatal mutants is compared to wild-type nontransgenic and heterozygous siblings. Skeletal, craniofacial, and visceral malformations are characterized. We have observed by skeletal analysis a consistent loss of distal hindlimb structures, as well as the loss of distal forelimb structures with a predilection for the preaxial side of the developing forelimb. Craniofacial malformations commonly observed appear to represent a range of severity of affect, with the mildest manifestation evident as apparently shallow lateral clefts of the upper lip and mild midfacial clefts accompanied by clefts of the secondary palate. At the severe end of the spectrum, the midline clefts of the face (and secondary palate) are very wide, with obvious accompanying frontonasal encephaloceles and overt lateral clefts of the upper lip. Examination of the mutant brain has demonstrated marked defects in the anterior structures, particularly the olfactory lobes and cerebrum, in greater than 90% of the brains studied. Observation of the internal viscera has identified transposition of thoracic and abdominal organs in approximately 50% of the mutant offspring. The limb, head, and visceral defects were not observed in the wild-type nontransgenic or heterozygous siblings. Transgenic insertional mutations leading to congenital malformations are useful because the transgene sequence may serve as a tag to facilitate molecular retrieval. Analysis of the flanking DNA sequences will allow the identification of the interrupted gene. A complete description of the mutant phenotype will assist in the understanding of this genetic locus.