A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Allelic heterogeneity contributes to variability in ocular dysgenesis, myopathy and brain malformations caused by Col4a1 and Col4a2 mutations.

Collagen type IV alpha 1 and 2 (COL4A1 and COL4A2) are present in nearly all basement membranes. COL4A1 and COL4A2 mutations are pleiotropic, affecting multiple organ systems to differing degrees, and both genetic-context and environmental factors influence this variable expressivity. Here, we report important phenotypic and molecular differences in an allelic series of Col4a1 and Col4a2 mutant mice that are on a uniform genetic background. We evaluated three organs commonly affected by COL4A1 and COL4A2 mutations and discovered allelic heterogeneity in the penetrance and severity of ocular dysgenesis, myopathy and brain malformations. Similarly, we show allelic heterogeneity in COL4A1 and COL4A2 biosynthesis. While most mutations that we examined caused increased intracellular and decreased extracellular COL4A1 and COL4A2, we identified three mutations with distinct biosynthetic signatures. Reduced temperature or presence of 4-phenylbutyrate ameliorated biosynthetic defects in primary cell lines derived from mutant mice. Together, our data demonstrate the effects and clinical implications of allelic heterogeneity in Col4a1- and Col4a2-related diseases. Understanding allelic differences will be valuable for increasing prognostic accuracy and for the development of therapeutic interventions that consider the nature of the molecular cause in patients with COL4A1 and COL4A2 mutations.

COL4A2 mutations impair COL4A1 and COL4A2 secretion and cause hemorrhagic stroke.

Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

A Rb1 promoter variant with reduced activity contributes to osteosarcoma susceptibility in irradiated mice.

BACKGROUND: Syndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility. METHODS: Tumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay. RESULTS: A common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation. CONCLUSION: This is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant transformation of bone cells after radiation exposure.

Standardized, systemic phenotypic analysis of Slc12a1I299F mutant mice.

BACKGROUND: Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. RESULTS: To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. CONCLUSIONS: In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

The Opdc missense mutation of Pax2 has a milder than loss-of-function phenotype.

Renal-coloboma syndrome, also known as papillorenal syndrome, is an autosomal dominant human disorder in which optic disc coloboma is associated with kidney abnormalities. Mutations in the paired domain transcription factor PAX2 have been found to be the underlying cause of this disease. Disease severity varies between patients, and in some cases, renal hypoplasia has been found in the absence of any retinal defects. Here we report an N-ethyl-N-nitrosourea-induced mouse mutation, Opdc, which is an isoleucinetothreonine missense mutation, I40T, in the first α-helix of the Pax2 paired domain. The mutant protein binds target DNA sequences less strongly than the wild-type protein and acts poorly to transactivate target promoters in culture. The phenotypic consequence of this mutation on the development of the eye and ear is similar to that reported for null alleles of Pax2. However, in homozygotes, cerebellar development is normal on a genetic background in which loss of Pax2 results in failure of cerebellar formation. Moreover, there is a genetic background effect on the heterozygous phenotype such that on some strain backgrounds, kidney development is unaffected. Opdc is the first hypomorphic mutation reported for Pax2 that differs in phenotype from loss-of-function mutations. These results suggest that PAX2 is a strong candidate gene for cases in which human patients have optic disc coloboma not associated with renal dysplasia.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology.

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.

New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.

Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.

GDNF-induced osteopontin from Müller glial cells promotes photoreceptor survival in the Pde6brd1 mouse model of retinal degeneration.

Glial cell line-derived neurotrophic factor (GDNF) enhances the survival of a variety of neurons, including photoreceptors (PR) in the retina. In contrast to most other GDNF receptive neurons, GDNF does, however, not exert its neuroprotective activity directly on PR neurons but transmits it indirectly by inducing expression of yet unknown neurotrophic factors in retinal Müller glial (RMG) cells. Genome-wide differential transcriptome analyses of GDNF-treated mouse retinas revealed 30 GDNF-induced transcripts containing a total of six genes coding for secreted molecules. Among them was (OPN), a secreted glycoprotein which was expressed in mouse RMG and secreted from primary mouse RMG in culture. Furthermore, OPN secretion was significantly upregulated on GDNF treatment of primary RMG. To validate, whether OPN could qualify as a neuroprotective factor for PR, we evaluated its potential neurotrophic activity on isolated PR in vitro as well as on retinal explants from the retinal degeneration 1 (Pde6brd1) mouse mutant. OPN exerted a significant, positive survival effect on primary porcine PR cells in a concentration-dependent manner and induced activation of PI3K/Akt pro-survival pathway. Moreover, in retinal explant cultures from Pde6brd1 mice, OPN significantly reduced the percentage of apoptotic cells to levels comparable with that observed in explants from wild-type mice and led to survival of significantly more PR in long-term retinal explant cultures. Our findings suggest that RMG-derived OPN is a novel candidate protein that transmits part of the GDNF-induced neuroprotective activity of RMG to PR cells.

Relative roles of the different Pax6 domains for pancreatic alpha cell development.

BACKGROUND: The transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6-/- knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing alpha-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of alpha-cells, is affected by selective inactivation of either one of the three major domains of Pax6. RESULTS: The Pax6Aey18 mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6-/- knockout mice with a near complete absence of glucagon-positive alpha-cells (0-4 cells/section; < or =1% of wt), reduced beta-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive epsilon-cells was expanded. In Pax6Sey-Neu mutants, which lack the transactivation domain, alpha-and beta-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax64Neu and Pax6132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax64Neu homozygotes showed a reduction of alpha- and beta-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6132-14Neu mutant strain. CONCLUSIONS: We show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic alpha-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for alpha-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.

Microphthalmia, parkinsonism, and enhanced nociception in Pitx3 ( 416insG ) mice.

A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ( ak/+ ) mice confirmed eyl as a new allele of Pitx3 (Pitx3 ( eyl )). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ( eyl/eyl ) mutants expressed ophthalmological and brain defects similar to Pitx3 ( ak/ak ) mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ( eyl/eyl ) mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ( eyl ) mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.

Mutation in Bmpr1b Leads to Optic Disc Coloboma and Ventral Retinal Gliosis in Mice.

Purpose: The clinical phenotype of retinal gliosis occurs in different forms; here, we characterize one novel genetic feature, (i.e., signaling via BMP-receptor 1b). Methods: Mouse mutants were generated within a recessive ENU mutagenesis screen; the underlying mutation was identified by linkage analysis and Sanger sequencing. The eye phenotype was characterized by fundoscopy, optical coherence tomography, optokinetic drum, electroretinography, and visual evoked potentials, by histology, immunohistology, and electron-microscopy. Results: The mutation affects intron 10 of the Bmpr1b gene, which is causative for skipping of exon 10. The expression levels of pSMAD1/5/8 were reduced in the mutant retina. The loss of BMPR1B-mediated signaling leads to optic nerve coloboma, gliosis in the optic nerve head and ventral retina, defective optic nerve axons, and irregular retinal vessels. The ventral retinal gliosis is proliferative and hypertrophic, which is concomitant with neuronal delamination and the reduction of retinal ganglion cells (RGCs); it is dominated by activated astrocytes overexpressing PAX2 and SOX2 but not PAX6, indicating that they may retain properties of gliogenic precursor cells. The expression pattern of PAX2 in the optic nerve head and ventral retina is altered during embryonic development. These events finally result in reduced electrical transmission of the retina and optic nerve and significantly reduced visual acuity. Conclusions: Our study demonstrates that BMPR1B is necessary for the development of the optic nerve and ventral retina. This study could also indicate a new mechanism in the formation of retinal gliosis; it opens new routes for its treatment eventually preventing scar formation in the retina.

Relationship of Pax6 activity levels to the extent of eye development in the mouse, Mus musculus.

In this study we extend the mouse Pax6 mutant allelic series to include a homozygous and hemizygous viable hypomorph allele. The Pax6(132-14Neu) allele is a Phe272Ile missense mutation within the third helix of the homeodomain. The mutant Pax6 homeodomain shows greatly reduced binding activity to the P3 DNA binding target. Glucagon-promoter activation by the entire mutant Pax6 product of a reporter gene driven by the G1 paired and homeodomain DNA binding target was slightly increased. We constructed mutant Pax6 genotypes such that Pax6 activity ranged between 100 and 0% and show that the extent of eye development is progressively reduced as Pax6 activity decreased. Two apparent thresholds identify three groups in which the extent of eye development abruptly shifted from complete eye at the highest levels of Pax6 to a rudimentary eye at intermediate levels of Pax6 to very early termination of eye development at the lowest levels of Pax6. Of the two Pax6-positive regions that participate in eye development, the surface ectoderm, which develops into the lens vesicle and the cornea, is more sensitive to reduced levels of Pax6 activity than the optic vesicle, which develops into the inner and outer retinal layers.

Systemic first-line phenotyping.

With the completion of the mouse genome sequence an essential task for biomedical sciences in the twenty-first century will be the generation and functional analysis of mouse models for every gene in the mammalian genome. More than 30,000 mutations in ES cells will be engineered and thousands of mouse disease models will become available over the coming years by the collaborative effort of the International Mouse Knockout Consortium. In order to realize the full value of the mouse models proper characterization, archiving and dissemination of mouse disease models to the research community have to be performed. Phenotyping centers (mouse clinics) provide the necessary capacity, broad expertise, equipment, and infrastructure to carry out large-scale systemic first-line phenotyping. Using the example of the German Mouse Clinic (GMC) we will introduce the reader to the different aspects of the organization of a mouse clinic and present selected methods used in first-line phenotyping.

Pleiotropic effects in Eya3 knockout mice.

BACKGROUND: In Drosophila, mutations in the gene eyes absent (eya) lead to severe defects in eye development. The functions of its mammalian orthologs Eya1-4 are only partially understood and no mouse model exists for Eya3. Therefore, we characterized the phenotype of a new Eya3 knockout mouse mutant. RESULTS: Expression analysis of Eya3 by in-situ hybridizations and beta-Gal-staining of Eya3 mutant mice revealed abundant expression of the gene throughout development, e.g. in brain, eyes, heart, somites and limbs suggesting pleiotropic effects of the mutated gene. A similar complex expression pattern was observed also in zebrafish embryos. The phenotype of young adult Eya3 mouse mutants was systematically analyzed within the German Mouse Clinic. There was no obvious defect in the eyes, ears and kidneys of Eya3 mutant mice. Homozygous mutants displayed decreased bone mineral content and shorter body length. In the lung, the tidal volume at rest was decreased, and electrocardiography showed increased JT- and PQ intervals as well as decreased QRS amplitude. Behavioral analysis of the mutants demonstrated a mild increase in exploratory behavior, but decreased locomotor activity and reduced muscle strength. Analysis of differential gene expression revealed 110 regulated genes in heart and brain. Using real-time PCR, we confirmed Nup155 being down regulated in both organs. CONCLUSION: The loss of Eya3 in the mouse has no apparent effect on eye development. The wide-spread expression of Eya3 in mouse and zebrafish embryos is in contrast to the restricted expression pattern in Xenopus embryos. The loss of Eya3 in mice leads to a broad spectrum of minor physiological changes. Among them, the mutant mice move less than the wild-type mice and, together with the effects on respiratory, muscle and heart function, the mutation might lead to more severe effects when the mice become older. Therefore, future investigations of Eya3 function should focus on aging mice.

"Sighted C3H" mice--a tool for analysing the influence of vision on mouse behaviour?

It is unclear what role vision plays in guiding mouse behaviour, since the mouse eye is of comparably low optical quality, and mice are considered to rely primarily on other senses. All C3H substrains are homozygous for the Pde6b(rd1) mutation and get blind by weaning age. To study the impact of the Pde6b(rd1) mutation on mouse behaviour and physiology, sighted C3H (C3H.Pde6b+) and normal C3H/HeH mice were phenotyped for different aspects. We confirmed retinal degeneration 1 in C3H/HeH mice, and the presence of a morphologically normal retina as well as visual ability in C3H.Pde6b+ mice. However, C3H.Pde6b+ mice showed an abnormal retinal function in the electroretinogram response, indicating that their vision was not normal as expected. C3H.Pde6b+ mice showed reduced latencies for several behaviours without any further alterations in these behaviours in comparison to C3H/HeH mice, suggesting that visual ability, although impaired, enables earlier usage of the behavioural repertoire in a novel environment, but does not lead to increased activity levels. These results emphasize the importance of comprehensive behavioural and physiological phenotyping.

Novel allele of crybb2 in the mouse and its expression in the brain.

PURPOSE: O377 was identified as a new dominant cataract mutation in mice after radiation experiments. The purpose of this study was to genetically characterize the mutation and to analyze its biological consequences. METHODS: Linkage analysis of the O377 mouse mutant was performed; candidate genes including Crybb2 were sequenced. The authors analyzed eyes and brains of the mutants by histology and the expression domains of Crybb2 by in situ hybridization and immunohistochemistry. RNA was isolated from whole brains of heterozygous and homozygous O377 mutants, and differential expression arrays were performed. All studies were compared with age- and strain-matched wild-type mice. RESULTS: The mutation was mapped to chromosome 5 and characterized as an A-->T substitution at the end of intron 5 of the Crybb2 gene. It led to alternative splicing with a 57-bp insertion in the mRNA and to 19 additional amino acids in the protein. In the brain, betaB2-crystallin was expressed in the cerebellum, olfactory bulb, cerebral cortex, and hippocampus. The only morphologic difference in the brain is the increased number of Purkinje cells in the cerebellum of homozygous strain-matched mutants. Differential expression analysis revealed the upregulation of calpain-3 in the brain of homozygous mutants, which was confirmed by quantitative real-time PCR. CONCLUSIONS: These results confirm the third allele of Crybb2 in the mouse that also affected exon 6 and the fourth Greek key motif. Moreover, expression analysis of Crybb2 identified for the first time distinct regions of expression in the brain, and the differential expression analysis points to the participation of Ca2+ in the corresponding pathologic processes.

Type IV procollagen missense mutations associated with defects of the eye, vascular stability, the brain, kidney function and embryonic or postnatal viability in the mouse, Mus musculus: an extension of the Col4a1 allelic series and the identification of the first two Col4a2 mutant alleles.

The basement membrane is important for proper tissue development, stability, and physiology. Major components of the basement membrane include laminins and type IV collagens. The type IV procollagens Col4a1 and Col4a2 form the heterotrimer [alpha1(IV)]2[alpha2(IV)], which is ubiquitously expressed in basement membranes during early developmental stages. We present the genetic, molecular, and phenotypic characterization of nine Col4a1 and three Col4a2 missense mutations recovered in random mutagenesis experiments in the mouse. Heterozygous carriers express defects in the eye, the brain, kidney function, vascular stability, and viability. Homozygotes do not survive beyond the second trimester. Ten mutations result in amino acid substitutions at nine conserved Gly sites within the collagenous domain, one mutation is in the carboxy-terminal noncollagenous domain, and one mutation is in the signal peptide sequence and is predicted to disrupt the signal peptide cleavage site. Patients with COL4A2 mutations have still not been identified. We suggest that the spontaneous intraorbital hemorrhages observed in the mouse are a clinically relevant phenotype with a relatively high predictive value to identify carriers of COL4A1 or COL4A2 mutations.

Molecular interaction between projection neuron precursors and invading interneurons via stromal-derived factor 1 (CXCL12)/CXCR4 signaling in the cortical subventricular zone/intermediate zone.

Most cortical interneurons are generated in the subpallial ganglionic eminences and migrate tangentially to their final destinations in the neocortex. Within the cortex, interneurons follow mainly stereotype routes in the subventricular zone/intermediate zone (SVZ/IZ) and in the marginal zone. It has been suggested that interactions between invading interneurons and locally generated projection neurons are implicated in the temporal and spatial regulation of the invasion process. However, so far experimental evidence for such interactions is lacking. We show here that the chemokine stromal-derived factor 1 (SDF-1; CXCL12) is expressed in the main invasion route for cortical interneurons in the SVZ/IZ. Most SDF-1-positive cells are proliferating and express the homeodomain transcription factors Cux1 and Cux2. Using MASH-1 mutant mice in concert with the interneuron marker DLX, we exclude that interneurons themselves produce the chemokine in an autocrine manner. We conclude that the SDF-1-expressing cell population represents the precursors of projection neurons during their transition and amplification in the SVZ/IZ. Using mice lacking the SDF-1 receptor CXCR4 or Pax6, we demonstrate that SDF-1 expression in the cortical SVZ/IZ is essential for recognition of this pathway by interneurons. These results represent the first evidence for a molecular interaction between precursors of projection neurons and invading interneurons during corticogenesis.

Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters.

PURPOSE: To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS: Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS: The new mutations--ADD4802, Aey11, and Aey18--were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C-->T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G-->A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G-->A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters. CONCLUSIONS: Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.