Mechanosensing through talin 1 contributes to tissue mechanical homeostasis.

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

Evolutionary innovation in conserved regulatory elements across the mammalian tree of life.

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Evolution of enhancer sequences can alter target gene expression without causing detrimental misexpression in other contexts. It has long been thought that this modularity allows evolutionary changes in enhancers to escape pleiotropic constraints, which is especially important for evolutionary constrained developmental patterning genes. However, there is still little data supporting this hypothesis. Here we identified signatures of accelerated evolution in conserved enhancer elements across the mammalian phylogeny. We found that pleiotropic genes involved in gene regulatory and developmental processes were enriched for accelerated sequence evolution within their enhancer elements. These genes were associated with an excess number of enhancers compared to other genes, and due to this they exhibit a substantial degree of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. We studied one acceleration event in depth and found that its sequence evolution led to the emergence of a new enhancer activity domain that may be involved in the evolution of digit reduction in hoofed mammals. Our results provide tangible evidence that enhancer evolution has been a frequent contributor to modifications involving constrained developmental signaling genes in mammals.

Mutation of key signaling regulators of cerebrovascular development in vein of Galen malformations.

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and most severe of congenital brain arteriovenous malformations, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (2042.5-fold, p = 4.79 x 10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (17.5-fold, p = 1.22 x 10-5), which cooperates with p120 RasGAP to regulate vascular development. Additional probands had damaging variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomic analysis defined developing endothelial cells as a likely spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant (Phe867Leu) exhibited disrupted developmental angiogenesis and impaired hierarchical development of arterial-capillary-venous networks, but only in the presence of a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have implications for patients and their families.

Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities.

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.

CpG island turnover events predict evolutionary changes in enhancer activity.

Genetic changes that modify the function of transcriptional enhancers have been linked to the evolution of biological diversity across species. Multiple studies have focused on the role of nucleotide substitutions, transposition, and insertions and deletions in altering enhancer function. Here we show that turnover of CpG islands (CGIs), which contribute to enhancer activation, is broadly associated with changes in enhancer activity across mammals, including humans. We integrated maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and found that CGI content in enhancers was strongly associated with increased histone modification levels. CGIs showed widespread turnover across species and species-specific CGIs were strongly enriched for enhancers exhibiting species-specific activity across all tissues and species we examined. Genes associated with enhancers with species-specific CGIs showed concordant biases in their expression, supporting that CGI turnover contributes to gene regulatory innovation. Our results also implicate CGI turnover in the evolution of Human Gain Enhancers (HGEs), which show increased activity in human embryonic development and may have contributed to the evolution of uniquely human traits. Using a humanized mouse model, we show that a highly conserved HGE with a large CGI absent from the mouse ortholog shows increased activity at the human CGI in the humanized mouse diencephalon. Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals.

Genetic dysregulation of an endothelial Ras signaling network in vein of Galen malformations.

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and severe congenital brain arteriovenous malformation, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP ( RASA1 ) harbored a genome-wide significant burden of loss-of-function de novo variants (p=4.79×10 -7 ). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 ( EPHB4 ) (p=1.22×10 -5 ), which cooperates with p120 RasGAP to limit Ras activation. Other probands had pathogenic variants in ACVRL1 , NOTCH1 , ITGB1 , and PTPN11 . ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomics defined developing endothelial cells as a key spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant exhibited constitutive endothelial Ras/ERK/MAPK activation and impaired hierarchical development of angiogenesis-regulated arterial-capillary-venous networks, but only when carrying a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have clinical implications.

Loss of Serum Glucocorticoid-Inducible Kinase 1 SGK1 Worsens Malabsorption and Diarrhea in Microvillus Inclusion Disease (MVID).

Microvillus inclusion disease (MVID), a lethal congenital diarrheal disease, results from loss of function mutations in the apical actin motor myosin VB (MYO5B). How loss of MYO5B leads to both malabsorption and fluid secretion is not well understood. Serum glucocorticoid-inducible kinase 1 (SGK1) regulates intestinal carbohydrate and ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR). We hypothesized that loss of SGK1 could reduce CFTR fluid secretion and MVID diarrhea. Using CRISPR-Cas9 approaches, we generated R26CreER;MYO5Bf/f conditional single knockout (cMYO5BKO) and R26CreER;MYO5Bf/f;SGK1f/f double knockout (cSGK1/MYO5B-DKO) mice. Tamoxifen-treated cMYO5BKO mice resulted in characteristic features of human MVID including severe diarrhea, microvillus inclusions (MIs) in enterocytes, defective apical traffic, and depolarization of transporters. However, apical CFTR distribution was preserved in crypts and depolarized in villus enterocytes, and CFTR high expresser (CHE) cells were observed. cMYO5BKO mice displayed increased phosphorylation of SGK1, PDK1, and the PDK1 target PKCι in the intestine. Surprisingly, tamoxifen-treated cSGK1/MYO5B-DKO mice displayed more severe diarrhea than cMYO5BKO, with preservation of apical CFTR and CHE cells, greater fecal glucose and reduced SGLT1 and GLUT2 in the intestine. We conclude that loss of SGK1 worsens carbohydrate malabsorption and diarrhea in MVID.

A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

OBJECTIVE: To determine if single-nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model. METHODS: Human exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting. Wildtype mice and mice heterozygous and homozygous for the MSH6 variant were then monitored for 2 years for the development of autoimmune phenotypes, including the presence of high levels of antinuclear antibodies (ANA). Additionally, somatic hypermutation frequencies and spectra of the intronic region downstream of the VH J558-rearranged JH4 immunoglobulin gene was characterized from Peyer's patches. RESULTS: Based on the human exome chip data, the MSH6 variant (rs63750897, p.Ser503Cys) is enriched among patients with SLE versus controls after we corrected for ancestry (odds ratio = 8.39, P = 0.0398). Mice homozygous for the MSH6 variant (Msh6S502C/S502C ) harbor significantly increased levels of ANA. Additionally, the Msh6S502C/S502C mice display a significant increase in the infiltration of CD68+ cells (a marker for monocytes and macrophages) into the lung alveolar space as well as apoptotic cells. Furthermore, characterization of somatic hypermutation in these mice reveals an increase in the DNA polymerase η mutational signature. CONCLUSION: An MSH6 mutation that is enriched in humans diagnosed with lupus was identified. Mice harboring this Msh6 mutation develop increased autoantibodies and an inflammatory lung disease. These results suggest that the human MSH6 variant is linked to the development of SLE.

Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome.

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1hM1560V/+ ).

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h-/- ). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h-/- mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h-/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Rho-GTPase Activating Protein myosin MYO9A identified as a novel candidate gene for monogenic focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a podocytopathy leading to kidney failure, whose molecular cause frequently remains unresolved. Here, we describe a rare MYO9A loss of function nonsense heterozygous mutation (p.Arg701∗) as a possible contributor to disease in a sibling pair with familial FSGS/proteinuria. MYO9A variants of uncertain significance were identified by whole exome sequencing in a cohort of 94 biopsy proven patients with FSGS. MYO9A is an unconventional myosin with a Rho-GAP domain that controls epithelial cell junction assembly, crosslinks and bundles actin and deactivates the small GTPase protein encoded by the RHOA gene. RhoA activity is associated with cytoskeleton regulation of actin stress fiber formation and actomyosin contractility. Myo9A was detected in mouse and human podocytes in vitro and in vivo. Knockin mice carrying the p.Arg701∗MYO9A (Myo9AR701X) generated by gene editing developed proteinuria, podocyte effacement and FSGS. Kidneys and podocytes from Myo9AR701X/+ mutant mice revealed Myo9A haploinsufficiency, increased RhoA activity, decreased Myo9A-actin-calmodulin interaction, impaired podocyte attachment and migration. Our results indicate that Myo9A is a novel component of the podocyte cytoskeletal apparatus that regulates RhoA activity and podocyte function. Thus, Myo9AR701X/+ knock-in mice recapitulate the proband FSGS phenotype, demonstrate that p.R701X Myo9A is an FSGS-causing mutation in mice and suggest that heterozygous loss-of-function MYO9A mutations may cause a novel form of human autosomal dominant FSGS. Hence, identification of MYO9A pathogenic variants in additional individuals with familial or sporadic FSGS is needed to ascertain the gene contribution to disease.

A transgenic inducible GFP extracellular-vesicle reporter (TIGER) mouse illuminates neonatal cortical astrocytes as a source of immunomodulatory extracellular vesicles.

Extracellular vesicles (EVs) are cellular derived particles found throughout the body in nearly all tissues and bodily fluids. EVs contain biological molecules including small RNAs and protein. EVs are proposed to be transferred between cells, notably, cells of the immune system. Tools that allow for in vivo EV labeling while retaining the ability to resolve cellular sources and timing of release are required for a full understanding of EV functions. Fluorescent EV fusion proteins are useful for the study of EV biogenesis, release, and identification of EV cellular recipients. Among the most plentiful and frequently identified EV proteins is CD9, a tetraspanin protein. A transgenic mouse containing a CRE-recombinase inducible CAG promoter driven CD9 protein fused to Turbo-GFP derived from the copepod Pontellina plumata was generated as an EV reporter. The transgenic inducible GFP EV reporter (TIGER) mouse was electroporated with CAG-CRE plasmids or crossed with tamoxifen inducible CAG-CRE-ERT2 or nestin-CRE-ERT2 mice. CD9-GFP labeled cells included glutamine synthetase and glial fibrillary acidic protein positive astrocytes. Cortical astrocytes released ~136 nm EVs that contained CD9. Intraventricular injected EVs were taken up by CD11b/IBA1 positive microglia surrounding the lateral ventricles. Neonatal electroporation and shRNA mediated knockdown of Rab27a in dorsal subventricular zone NSCs and astrocytes increased the number of CD11b/IBA1 positive rounded microglia. Neonatal astrocyte EVs had a unique small RNA signature comprised of morphogenic miRNAs that induce microglia cytokine release. The results from this study demonstrate that inducible CD9-GFP mice will provide the EV community with a tool that allows for EV labeling in a cell-type specific manner while simultaneously allowing in vivo experimentation and provides evidence that EVs are required immunomodulators of the developing nervous system.

Specific Disruption of Abca1 Targeting Largely Mimics the Effects of miR-33 Knockout on Macrophage Cholesterol Efflux and Atherosclerotic Plaque Development.

RATIONALE: Inhibition of miR-33 reduces atherosclerotic plaque burden, but miR-33 deficient mice are predisposed to the development of obesity and metabolic dysfunction. The proatherogenic effects of miR-33 are thought to be in large part because of its repression of macrophage cholesterol efflux, through targeting of Abca1 (ATP-binding cassette subfamily A member 1). However, targeting of other factors may also be required for the beneficial effects of miR-33, and currently available approaches have not allowed researchers to determine the specific impact of individual miRNA target interactions in vivo. OBJECTIVE: In this work, we sought to determine how specific disruption of Abca1 targeting by miR-33 impacts macrophage cholesterol efflux and atherosclerotic plaque formation in vivo. METHODS AND RESULTS: We have generated a novel mouse model with specific point mutations in the miR-33 binding sites of the Abca1 3'untranslated region, which prevents targeting by miR-33. Abca1 binding site mutant ( Abca1BSM) mice had increased hepatic ABCA1 expression but did not show any differences in body weight or metabolic function after high fat diet feeding. Macrophages from Abca1BSM mice also had increased ABCA1 expression, as well as enhanced cholesterol efflux and reduced foam cell formation. Moreover, LDLR (low-density lipoprotein receptor) deficient animals transplanted with bone marrow from Abca1BSM mice had reduced atherosclerotic plaque formation, similar to mice transplanted with bone marrow from miR-33 knockout mice. CONCLUSION: Although the more pronounced phenotype of miR-33 deficient animals suggests that other targets may also play an important role, our data clearly demonstrate that repression of ABCA1 is primarily responsible for the proatherogenic effects of miR-33. This work shows for the first time that disruption of a single miRNA/target interaction can be sufficient to mimic the effects of miRNA deficiency on complex physiological phenotypes in vivo and provides an approach by which to assess the impact of individual miRNA targets.

Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development.

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of Pitx1, which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between Pitx1 and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in Pitx1 regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, Pitx1 expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the Pitx1 promoter and potentially redundant enhancers. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1-/- mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.

Impaired LRP6-TCF7L2 Activity Enhances Smooth Muscle Cell Plasticity and Causes Coronary Artery Disease.

Mutations in Wnt-signaling coreceptor LRP6 have been linked to coronary artery disease (CAD) by unknown mechanisms. Here, we show that reduced LRP6 activity in LRP6(R611C) mice promotes loss of vascular smooth muscle cell (VSMC) differentiation, leading to aortic medial hyperplasia. Carotid injury augmented these effects and led to partial to total vascular obstruction. LRP6(R611C) mice on high-fat diet displayed dramatic obstructive CAD and exhibited an accelerated atherosclerotic burden on LDLR knockout background. Mechanistically, impaired LRP6 activity leads to enhanced non-canonical Wnt signaling, culminating in diminished TCF7L2 and increased Sp1-dependent activation of PDGF signaling. Wnt3a administration to LRP6(R611C) mice improved LRP6 activity, led to TCF7L2-dependent VSMC differentiation, and rescued post-carotid-injury neointima formation. These findings demonstrate the critical role of intact Wnt signaling in the vessel wall, establish a causal link between impaired LRP6/TCF7L2 activities and arterial disease, and identify Wnt signaling as a therapeutic target against CAD.

Kinesin 5B (KIF5B) is required for progression through female meiosis and proper chromosomal segregation in mitotic cells.

The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.

Dynamic migration and cell-cell interactions of early reprogramming revealed by high-resolution time-lapse imaging.

Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multicolor time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as 5 minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells that emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies, and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct two-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate that E-cadherin is required for proper cellular interactions from an early stage of reprogramming, including the two-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.

Y265C DNA polymerase beta knockin mice survive past birth and accumulate base excision repair intermediate substrates.

DNA is susceptible to damage by a wide variety of chemical agents that are generated either as byproducts of cellular metabolism or exposure to man-made and harmful environments. Therefore, to maintain genomic integrity, having reliable DNA repair systems is important. DNA polymerase ß is known to be a key player in the base excision repair pathway, and mice devoid of DNA polymerase beta do not live beyond a few hours after birth. In this study, we characterized mice harboring an impaired pol ß variant. This Y265C pol ß variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C pol ß are born at normal Mendelian ratios. However, they are small, and 60% die within a few hours after birth. Slow proliferation and significantly increased levels of cell death are observed in many organs of the E14 homozygous embryos compared with WT littermates. Mouse embryo fibroblasts prepared from the Y265C pol ß embryos proliferate at a rate slower than WT cells and exhibit a gap-filling deficiency during base excision repair. As a result of this, chromosomal aberrations and single- and double-strand breaks are present at significantly higher levels in the homozygous mutant versus WT mouse embryo fibroblasts. This is study in mice is unique in that two enzymatic activities of pol ß have been separated; the data clearly demonstrate that the DNA polymerase activity of pol ß is essential for survival and genome stability.

Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage.

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.