An L314Q mutation in Map3k1 gene results in failure of eyelid fusion in the N-ethyl-N-nitrosourea-induced mutant line

In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with “eye open at birth” (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1^L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1^L314Q mouse may be associated with the reduced level of p-JNK and c-Jun.     

A Mouse Model for Osseous Heteroplasia

GNAS/Gnas encodes G_sα that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G_sα. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G_sα. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G_sα deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G_sα and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G_sα signalling pathways play a vital role in repressing ectopic bone formation.     

High Prevalence of Posterior Polymorphous Corneal Dystrophy in the Czech Republic; Linkage Disequilibrium Mapping and Dating an Ancestral Mutation

Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1–12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64–133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene, ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.     

A Trans-Acting Protein Effect Causes Severe Eye Malformation in the Mp Mouse

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1^Mp) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 ^Mp) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1–2646, exons 1–62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2^Mp forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM – known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp “worse-than-null” eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.     

Progesterone Antagonist Therapy in a Pelizaeus-Merzbacher Mouse Model

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disease, characterized by ataxia, intellectual disability, epilepsy, and premature death. In the majority of cases, PMD is caused by duplication of PLP1 that is expressed in myelinating oligodendrocytes. Despite detailed knowledge of PLP1, there is presently no curative therapy for PMD. We used a Plp1 transgenic PMD mouse model to test the therapeutic effect of Lonaprisan, an antagonist of the nuclear progesterone receptor, in lowering Plp1 mRNA overexpression. We applied placebo-controlled Lonaprisan therapy to PMD mice for 10 weeks and performed the grid slip analysis to assess the clinical phenotype. Additionally, mRNA expression and protein accumulation as well as histological analysis of the central nervous system were performed. Although Plp1 mRNA levels are increased 1.8-fold in PMD mice compared to wild-type controls, daily Lonaprisan treatment reduced overexpression at the RNA level to about 1.5-fold, which was sufficient to significantly improve the poor motor phenotype. Electron microscopy confirmed a 25% increase in the number of myelinated axons in the corticospinal tract when compared to untreated PMD mice. Microarray analysis revealed the upregulation of proapoptotic genes in PMD mice that could be partially rescued by Lonaprisan treatment, which also reduced microgliosis, astrogliosis, and lymphocyte infiltration.     

Repeated Evolution of Inactive Pseudonucleases in a Fungal Branch of the Dis3/RNase II Family of Nucleases

The RNase II family of 3′–5′ exoribonucleases is present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like “pseudonucleases.” The latter function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. However, the evolutionary origins of these pseudonucleases are unknown: What sequence of events led to their novel function, and when did these events occur? Here, we show how RNase II pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. In contrast, N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the nonnuclease function requires these regions. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid “titan” growth stages. This phenotype of C. neoformans Ssd1/Dis3L2 deletion is consistent with those of inactive fungal pseudonucleases, yet the protein retains an active site sequence signature. We propose that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, whereas the RNase activity was lost repeatedly in independent lineages.     

Insights on Foxn1 Biological Significance and Usages of the “Nude” Mouse in Studies of T-Lymphopoiesis

Mutation in the “nude” gene, i.e. the FoxN1 gene, induces a hairless phenotype and a rudimentary thymus gland in mice (nude mouse) and humans (T-cell related primary immunodeficiency). Conventional FoxN1 gene knockout and transgenic mouse models have been generated for studies of FoxN1 gene function related to skin and immune diseases, and for cancer models. It appeared that FoxN1''s role was fully understood and the nude mouse model was fully utilized. However, in recent years, with the development of inducible gene knockout/knockin mouse models with the loxP-Cre(ER^T) and diphtheria toxin receptor-induced cell abolished systems, it appears that the complete repertoire of FoxN1''s roles and deep-going usage of nude mouse model in immune function studies have just begun. Here we summarize the research progress made by several recent works studying the role of FoxN1 in the thymus and utilizing nude and “second (conditional) nude” mouse models for studies of T-cell development and function. We also raise questions and propose further consideration of FoxN1 functions and utilizing this mouse model for immune function studies.     

α-Actinin-4-Mediated FSGS: An Inherited Kidney Disease Caused by an Aggregated and Rapidly Degraded Cytoskeletal Protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in theα-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant α-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant α-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a “knockin” mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant α-actinin-4, mediated, at least in part, by the ubiquitin–proteasome pathway. We correlate these findings with studies of α-actinin-4 expression in human samples. “Knockin” mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 “knockin” and “knockout” mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

bfb,a Novel ENU-Induced blebs Mutant Resulting from a Missense Mutation in Fras1

Fras1 is an extracellular matrix associated protein with essential roles in adhesion of epithelia and mesenchyme during early embryonic development. The adhesive function of Fras1 is achieved through interaction with a group of related proteins, Frem 1–3, and a cytoplasmic adaptor protein Grip1. Mutation of each of these proteins results in characteristic epithelial blistering and have therefore become known as “blebs” proteins. Human Fraser syndrome presents with a similar phenotype and the blebs mice have been instrumental in identification of the genetic basis of Fraser syndrome. We have identified a new ENU-induced blebs allele resulting from a novel missense mutation in Fras1. The resulting mouse strain, blood filled blisters (bfb), presents with a classic blebs phenotype but does not exhibit embryonic lethality typical of other blebs mutants and in addition, we report novel palate and sternal defects. Analysis of the bfb phenotype confirms the presence of epithelial-mesenchymal adhesion defects but also supports the emerging role of blebs proteins in regulating signalling during organogenesis. The bfb strain provides new opportunities to investigate the role of Fras1 in development.     

A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2^P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2^+/− mice. The Phd2^P294R/+-associated erythrocytosis is reversed in a Hif2a^+/−, but not a Hif1a^+/− background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.     

Schlemm's Canal Is a Unique Vessel with a Combination of Blood Vascular and Lymphatic Phenotypes that Forms by a Novel Developmental Process

Schlemm''s canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name “canalogenesis.” Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision.     

First Autonomous Bio-Optical Profiling Float in the Gulf of Mexico Reveals Dynamic Biogeochemistry in Deep Waters

Profiling floats equipped with bio-optical sensors well complement ship-based and satellite ocean color measurements by providing highly-resolved time-series data on the vertical structure of biogeochemical processes in oceanic waters. This is the first study to employ an autonomous profiling (APEX) float in the Gulf of Mexico for measuring spatiotemporal variability in bio-optics and hydrography. During the 17-month deployment (July 2011 to December 2012), the float mission collected profiles of temperature, salinity, chlorophyll fluorescence, particulate backscattering (b_bp), and colored dissolved organic matter (CDOM) fluorescence from the ocean surface to a depth of 1,500 m. Biogeochemical variability was characterized by distinct depth trends and local “hot spots”, including impacts from mesoscale processes associated with each of the water masses sampled, from ambient deep waters over the Florida Plain, into the Loop Current, up the Florida Canyon, and eventually into the Florida Straits. A deep chlorophyll maximum (DCM) occurred between 30 and 120 m, with the DCM depth significantly related to the unique density layer ρ = 1023.6 (R² = 0.62). Particulate backscattering, b_bp, demonstrated multiple peaks throughout the water column, including from phytoplankton, deep scattering layers, and resuspension. The bio-optical relationship developed between b_bp and chlorophyll (R² = 0.49) was compared to a global relationship and could significantly improve regional ocean-color algorithms. Photooxidation and autochthonous production contributed to CDOM distributions in the upper water column, whereas in deep water, CDOM behaved as a semi-conservative tracer of water masses, demonstrating a tight relationship with density (R² = 0.87). In the wake of the Deepwater Horizon oil spill, this research lends support to the use of autonomous drifting profilers as a powerful tool for consideration in the design of an expanded and integrated observing network for the Gulf of Mexico.     

M tuberculosis in the Adjuvant Modulates Time of Appearance of CNS-Specific Effector T Cells in the Spleen through a Polymorphic Site of TLR2

DC deliver information regulating trafficking of effector T cells along T-cell priming. However, the role of pathogen-derived motives in the regulation of movement of T cells has not been studied. We hereinafter report that amount of M tuberculosis in the adjuvant modulates relocation of PLP139-151 specific T cells. In the presence of a low dose of M tuberculosis in the adjuvant, T cells (detected by CDR3 BV-BJ spectratyping, the so-called “immunoscope”) mostly reach the spleen by day 28 after immunization (“late relocation”) in the SJL strain, whereas T cells reach the spleen by d 14 with a high dose of M tuberculosis (“early relocation”). The C57Bl/6 background confers a dominant “early relocation” phenotype to F1 (SJL×C57Bl/6) mice, allowing early relocation of T cells in the presence of low dose M tuberculosis. A single non-synonymous polymorphism of TLR2 is responsible for “early/late” relocation phenotype. Egress of T lymphocytes is regulated by TLR2 expressed on T cells. Thus, pathogens engaging TLR2 on T cells regulate directly T-cell trafficking, and polymorphisms of TLR2 condition T-cell trafficking upon a limiting concentration of ligand.