Deficits In Proliferation Research Articles

Prolactin Stimulates Precursor Cells in the Adult Mouse Hippocampus

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.

A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.

Treatment of depressive‐like behaviour in Huntington's disease mice by chronic sertraline and exercise

Depression is the most common psychiatric disorder in Huntington's disease (HD) patients. Women are more prone to develop depression and such susceptibility might be related to 5-hydroxytryptaminergic (serotonergic) dysregulation. We performed tests of depression-related behaviours on female R6/1 HD mice that had been chronically treated with sertraline or provided with running-wheels. Functional assessments of 5-HT(1A) and 5-HT(2A) receptors were performed by measuring behavioural and physiological responses following administration of specific agonists, in combination with analysis of hippocampal gene expression. Finally we assessed the effect of exercise on hippocampal cell proliferation. Female HD mice recorded increased immobility time in the forced-swimming test, reduced saccharin preference and a hyperthermic response to stress compared with wild-type animals. These alterations were improved by chronic sertraline treatment. Wheel-running also resulted in similar improvements with the exception of saccharin preference but failed to correct the hippocampal cell proliferation deficits displayed by HD mice. The benefits of sertraline treatment and exercise involved altered 5-HT(1A) autoreceptor function, as demonstrated by modulation of the exaggerated 8-OH-DPAT-induced hypothermia exhibited by female HD mice. On the other hand, sertraline treatment was unable to restore the reduced 5-HT(1A) and 5-HT(2) heteroceptor function observed in HD animals. We report for the first time a crucial role for 5-HT(1A) autoreceptor function in mediating the sex-specific depressive-like phenotype of female R6/1 HD mice. Our data further support a differential effect of chronic sertraline treatment and exercise on hippocampal cell proliferation despite common behavioural benefits.

Exposure to N-Ethyl-N-Nitrosourea in Adult Mice Alters Structural and Functional Integrity of Neurogenic Sites

BackgroundPrevious studies have shown that prenatal exposure to the mutagen N-ethyl-N-nitrosourea (ENU), a N-nitroso compound (NOC) found in the environment, disrupts developmental neurogenesis and alters memory formation. Previously, we showed that postnatal ENU treatment induced lasting deficits in proliferation of neural progenitors in the subventricular zone (SVZ), the main neurogenic region in the adult mouse brain. The present study is aimed to examine, in mice exposed to ENU, both the structural features of adult neurogenic sites, incorporating the dentate gyrus (DG), and the behavioral performance in tasks sensitive to manipulations of adult neurogenesis.Methodology/Principal Findings2-month old mice received 5 doses of ENU and were sacrificed 45 days after treatment. Then, an ultrastructural analysis of the SVZ and DG was performed to determine cellular composition in these regions, confirming a significant alteration. After bromodeoxyuridine injections, an S-phase exogenous marker, the immunohistochemical analysis revealed a deficit in proliferation and a decreased recruitment of newly generated cells in neurogenic areas of ENU-treated animals. Behavioral effects were also detected after ENU-exposure, observing impairment in odor discrimination task (habituation-dishabituation test) and a deficit in spatial memory (Barnes maze performance), two functions primarily related to the SVZ and the DG regions, respectively.Conclusions/SignificanceThe results demonstrate that postnatal exposure to ENU produces severe disruption of adult neurogenesis in the SVZ and DG, as well as strong behavioral impairments. These findings highlight the potential risk of environmental NOC-exposure for the development of neural and behavioral deficits.

Impaired osteoblastogenesis potential of progenitor cells in skeletal unloading is associated with alterations in angiogenic and energy metabolism profile

Skeletal unloading provokes bone loss. These bone alterations have been shown to be associated with impairment of osteoblastic activity. In the present study, we evaluated the effect of skeletal unloading on bone marrow progenitor cells, for exploration of the underlying mechanism. Wistar rats were randomized to be either hindlimb unloaded for 9 days or to act as controls. Micro-CT was used to detect tibial trabecular architecture changes in response to skeletal unloading. Microgravity conditions for 9 days resulted in a decreased number and an increased spacing of the bone trabeculae in the proximal tibia. The proliferative capacity of the femoral bone marrow samples was assessed (fibroblast-colony-forming assay). By using qPCR, the expression of selected markers of vascularization (Vegfa; Hif1a; Angpt1), energy metabolism (Prkaa2; Mtor), bone formation (Runx2; Alp; Bglap; Bmp2; Bmp4; Bmp7) and bone resorption (Acp5; Tnfsf11; Tnfrsf11b) in these bone marrow suspensions was measured. We demonstrated a striking decrease in the number of fibroblastic progenitors in response to hindlimb unloading. This deficit in proliferation was shown to be accompanied by altered hindlimb perfusion and cellular energy homeostasis. Ex vivo culture assays of the bone marrow-derived progenitor cells screened for osteogenic (Runx2; Alp; Bglap) and adipogenic (Pparg; Fabp4) differentiation alterations in response to microgravity. Induced progenitor cells from unloaded rats showed a delay in osteogenic differentiation and impaired adipogenic differentiation compared to control. The data of this multi-level approach demonstrate that skeletal unloading significantly affects the bone tissue and its metabolism at the progenitor stage. The molecular expressions of the bone marrow population support a role of cellular metabolic stresses in skeletal alterations induced by inactivity.

Combination Effects of Sodium Butyrate and Pyridoxine Treatment on Cell Proliferation and Neuroblast Differentiation in the Dentate Gyrus of d-Galactose-Induced Aging Model Mice

We previously reported that sodium butyrate (SB), a histone deacetylase inhibitor, robustly increased pyridoxine-induced cell proliferation and neuroblast differentiation in the dentate gyrus of the adult mouse. In this study, we investigated the effects of treatment with SB combined with pyridoxine on cell proliferation and neuroblast differentiation in the dentate gyrus of a mouse model of aging induced by D: -galactose (D: -gal). D: -gal was administered to 20-week-old male mice (D: -gal mice) for 10 weeks to induce changes that resemble natural aging in animals. Seven weeks after D: -gal (100 mg/kg) treatment, vehicle (physiological saline; D: -gal-vehicle mice) and SB (300 mg/kg) combined with pyridoxine (Pyr; 350 mg/kg) were administered to the mice (D: -gal-Pyr-SB mice) for 3 weeks. Escape latency under water maze in the D: -gal mice was longer than that in the control mice. In the D: -gal-Pyr-SB mice, escape latency was similar to that in the control mice. In the D: -gal mice, many cells in the granule cell layer of the dentate gyrus showed pyknosis and condensation of the cytoplasm. However, in the D: -gal-Pyr-SB mice, such cellular changes were rarely found. Furthermore, the D: -gal mice showed a great reduction in cell proliferation (Ki67-positive cells) and neuroblast differentiation (doublecortin-positive neuroblasts) in the dentate gyrus compared to control mice. However, in the D: -gal-Pyr-SB mice, cell proliferation and neuroblast differentiation were markedly increased in the dentate gyrus. Furthermore, the administration of pyridoxine with sodium butyrate significantly increased Ser133-phosphorylated cyclic AMP response element binding protein in the dentate gyrus. These results indicate that the combination treatment of Pyr with SB in D: -gal mice ameliorated the D: -gal-induced reduction in cell proliferation, neuroblast differentiation, and memory deficits.

Interaction of p24alpha2 with gamma-secretase complex attenuates gamma-cleavage of APP

impairments, suggesting that deficits in neurogenesis may play a role in the disease or contribute to neuropathology. To address this, in this study we examined possible molecular link(s) between neurogenic signaling and FAD proteins.Methods:To determine the significance of APP cleavage by a-secretase and the role of sAPPa in adult neurogenesis we inhibited a-secretase activity in neural progenitor cells using metalloproteinase inhibitor. To determine the role of PS1 in adult neurogenesis we developed a siRNA-expressing lentiviral vector system for the silencing of PS1 in the neurogenic microenvironments in the adult mouse brain. Results: We show that inhibition of a-secretase decreases neural progenitor cell proliferation without affecting their differentiation, and that this decrease can be rescued by sAPPa. In addition, sAPPa can rescue proliferation deficits of neural progenitor cells derived from APP knockout mice. We further show that knocking down PS1 expression in the neurogenic niches of the adult brain enhances cell cycle exit of neural progenitor cell and induces their differentiation. Neural progenitor cells expressing reduced levels of PS1 could differentiate into neurons and glia. Conclusions: This study suggests that sAPPa and PS1 regulate neural progenitor cell maintenance in the adult brain. The results of these studies imply that FAD-linked mutations may alter neurogenic processes in Alzheimer’s disease.

Sprouty genes prevent excessive FGF signalling in multiple cell types throughout development of the cerebellum.

Fibroblast growth factors (FGFs) and regulators of the FGF signalling pathway are expressed in several cell types within the cerebellum throughout its development. Although much is known about the function of this pathway during the establishment of the cerebellar territory during early embryogenesis, the role of this pathway during later developmental stages is still poorly understood. Here, we investigated the function of sprouty genes (Spry1, Spry2 and Spry4), which encode feedback antagonists of FGF signalling, during cerebellar development in the mouse. Simultaneous deletion of more than one of these genes resulted in a number of defects, including mediolateral expansion of the cerebellar vermis, reduced thickness of the granule cell layer and abnormal foliation. Analysis of cerebellar development revealed that the anterior cerebellar neuroepithelium in the early embryonic cerebellum was expanded and that granule cell proliferation during late embryogenesis and early postnatal development was reduced. We show that the granule cell proliferation deficit correlated with reduced sonic hedgehog (SHH) expression and signalling. A reduction in Fgfr1 dosage during development rescued these defects, confirming that the abnormalities are due to excess FGF signalling. Our data indicate that sprouty acts both cell autonomously in granule cell precursors and non-cell autonomously to regulate granule cell number. Taken together, our data demonstrate that FGF signalling levels have to be tightly controlled throughout cerebellar development in order to maintain the normal development of multiple cell types.

Gfer inhibits Jab1-mediated degradation of p27kip1to restrict proliferation of hematopoietic stem cells

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.

Programmed death-1 receptor and interleukin-10 in liver transplant recipients at high risk for late cytomegalovirus disease

Despite significant advances in antiviral treatment, solid organ transplant (SOT) recipients remain at heightened risk for developing late cytomegalovirus (CMV) disease. Elevated inhibitory immune signaling suggests a state of immune impairment in SOT recipients, who do not control CMV infection and develop severe clinical symptoms after discontinuation of antiviral prophylaxis. We longitudinally monitored the negative immune modulator programmed death (PD)-1 receptor on both CD4 and CD8 T cells, co-expressing the CD137 surface marker of recent activation, in a liver transplant cohort. Liver recipients who progressed to CMV disease expressed elevated levels of PD-1 on CD137(+) CD4 and CD8 T cells, following stimulation with either full-length peptide libraries or CMV lysate. This novel approach, applicable to a multitude of human leukocyte antigen types, enhances the usefulness of the PD-1 measurements as a clinical strategy to predict late CMV disease. In parallel, we detected an increased level of the immunosuppressive cytokine interleukin (IL)-10, in plasma of liver recipients diagnosed with CMV disease. CMV-specific T cells were still functional when both PD-1 and IL-10 were upregulated; however they showed a marked proliferation deficit, which may limit their ability to contain viremia and lead to CMV disease. Our preliminary observations support further investigation of dual monitoring of PD-1 and IL-10, as potential immune markers of CMV disease.

Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease

Our previous analyses showed that allopregnanolone (APalpha) significantly increased proliferation of rodent and human neural progenitor cells in vitro. In this study, we investigated the efficacy of APalpha to promote neurogenesis in the hippocampal subgranular zone (SGZ), to reverse learning and memory deficits in 3-month-old male triple transgenic mouse model of Alzheimer's (3xTgAD) and the correlation between APalpha-induced neural progenitor cell survival and memory function in 3xTgAD mice. Neural progenitor cell proliferation was determined by unbiased stereological analysis of BrdU incorporation and survival determined by FACS for BrdU+ cells. Learning and memory function was assessed using the hippocampal-dependent trace eye-blink conditioning paradigm. At 3 months, basal level of BrdU+ cells in the SGZ of 3xTgAD mice was significantly lower relative to non-Tg mice, despite the lack of evident AD pathology. APalpha significantly increased, in a dose-dependent manner, BrdU+ cells in SGZ in 3xTgAD mice and restored SGZ proliferation to normal magnitude. As with the deficit in proliferation, 3xTgAD mice exhibited deficits in learning and memory. APalpha reversed the cognitive deficits to restore learning and memory performance to the level of normal non-Tg mice. In 3xTgAD mice, APalpha-induced survival of neural progenitors was significantly correlated with APalpha-induced memory performance. These findings suggest that early neurogenic deficits, which were evident before immunodetectable Abeta, may contribute to the cognitive phenotype of AD, and that APalpha could serve as a regenerative therapeutic to prevent or delay neurogenic and cognitive deficits associated with mild cognitive impairment and Alzheimer's disease.

Thyroid hormone receptor β mutation causes severe impairment of cerebellar development

Cerebellar development on the postnatal period is mainly characterized by cellular proliferation in the external granular layer (EGL) followed by migration of granular cells in the molecular layer through the Bergmann glia (BG) fibers in order to form the granular layer in the adult. All these events are drastically affected by thyroid hormones (TH), which actions are mainly mediated by alpha (TRα) and beta (TRβ) nuclear receptor isoforms. Here, we analyzed the effects of a natural human mutation (337T) in the TRβ locus, which impairs T3 binding to its receptor, on the mouse cerebellum ontogenesis. We report that target inactivation of TRβ-TH binding leads to a smaller cerebellum area characterized by impaired lamination and foliation. Further, TRβ mutant mice presented severe deficits in proliferation of granular precursors, arborization of Purkinje cells and organization of BG fibers. Together, our data suggest that the action of TH via TRβ regulates important events of cerebellar ontogenesis contributing to a better understanding of some neuroendocrine disorders. Further, our data correlate TRβ with cerebellar foliation, and provide, for the first time, evidence of a receptor-mediated mechanism underlying TH actions on this event.

The chemotherapy agent, thioTEPA, yields long-term impairment of hippocampal cell proliferation and memory deficits but not depression-related behaviors in mice

ThioTEPA is a chemotherapeutic agent used in the treatment of cancers, and more recently has been proposed as a component of high-dose therapy for young patients with recurrent malignant brain tumors. We previously demonstrated a significant dose-dependent reduction of cell proliferation in the dentate gyrus of the hippocampus in mice immediately following a 3-day regiment of thioTEPA. The aim of this study was to evaluate the long-term effects of thioTEPA treatment on hippocampal cell proliferation and potential effects on memory deficit or depression-related behavior in C57BL/6J mice. A 3-day regimen of thioTEPA (10 mg/kg/d, i.p.) yielded a significant reduction in cell proliferation immediately after treatment as assessed by BrdU incorporation, and none of the labeled progeny that initially survived the treatment were detectable one week later. Following a 3-week rebound in proliferation following treatment, a significant deficit in proliferation reappeared and persisted for at least 21 weeks following treatment. ThioTEPA-treated mice subjected to an object recognition test 1, 2, 3, 4, 8, 12, 20 or 30 weeks following treatment demonstrated significant memory deficits at 12 and 20 weeks. Mice demonstrated a similar deficit in an object placement test when tested 20 weeks following thioTEPA treatment. However, no observable effects on performance in the Porsolt forced swim test or the tail suspension test were observed in thioTEPA-treated mice. Together, these studies suggest that cumulative long-term negative effects of thioTEPA treatment on proliferation of new cells in the dentate gyrus may contribute to cognitive impairments associated with its use in the treatment of cancer.

Proliferation deficits and gene expression dysregulation in Down's syndrome (Ts1Cje) neural progenitor cells cultured from neurospheres

Down's syndrome neurophenotypes are characterized by mental retardation and a decreased brain volume. To identify whether deficits in proliferation could be responsible for this phenotype, neural progenitor cells were isolated from the developing E14 neocortex of Down's syndrome partial trisomy Ts1Cje mice and euploid (WT) littermates and grown as neurospheres. Ts1Cje neural progenitors proliferated at a slower rate, because of a longer cell cycle, and a greater number of cells were positive for glial fibrillary acidic protein. An increase in cell death was also noted. Gene expression profiles of neural progenitor cells from Ts1Cje and WT showed that 54% of triploid genes had expression ratios (Ts1Cje/WT) significantly greater than the expected diploid gene ratio of 1.0. Some diploid genes associated with proliferation, differentiation, and glial function were dysregulated. Interestingly, proliferation and gene expression dysregulation detected in the Ts1Cje mice did not require overexpression of the chromosome 21 genes amyloid precursor protein (App) and soluble superoxide dismutase 1 (Sod1).

Cutting Edge: The PTPN22 Allelic Variant Associated with Autoimmunity Impairs B Cell Signaling

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp. A missense mutation changing residue 1858 from cytosine to thymidine (1858C/T) is associated with multiple autoimmune disorders. Studies have demonstrated that Lyp has an inhibitory effect on TCR signaling; however, the presence of autoantibodies in all of the diseases associated with the 1858T variant and recent evidence that Ca(2+) flux is altered in B cells of 1858T carriers indicate a role for Lyp in B cell signaling. In this study we show that B cell signal transduction is impaired in individuals who express the variant. This defect in signaling is characterized by a deficit in proliferation, a decrease in phosphorylation of key signaling proteins, and is reversed by inhibition of Lyp. These findings suggest that the PTPN22 1858T variant alters BCR signaling and implicate B cells in the mechanism by which the PTPN22 1858T variant contributes to autoimmunity.

Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos

BackgroundThe Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation.Methodology/Principal FindingsDepletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5′ sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545–558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3.Conclusions/SignificanceThese results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Interaction of Gata4 and Gata6 with Tbx5 is critical for normal cardiac development

Congenital heart disease is the most common type of birth defect with an incidence of 1%. Previously, we described a point mutation in GATA4 that segregated with cardiac defects in a family with autosomal dominant disease. The mutation (G296S) exhibited biochemical deficits and disrupted a novel interaction between Gata4 and Tbx5. To determine if Gata4 and Tbx5 genetically interact in vivo, we generated mice heterozygous for both alleles. We found that nearly 100% of mice heterozygous for Gata4 and Tbx5 were embryonic or neonatal lethal and had complete atrioventricular (AV) septal defects with a single AV valve and myocardial thinning. Consistent with this phenotype, Gata4 and Tbx5 are co-expressed in the developing endocardial cushions and myocardium. In mutant embryos, cardiomyocyte proliferation deficits were identified compatible with the myocardial hypoplasia. Similar to Gata4, Gata6 and Tbx5 are co-expressed in the embryonic heart, and the transcription factors synergistically activate the atrial natiuretic factor promoter. We demonstrate a genetic interaction between Gata6 and Tbx5 with an incompletely penetrant phenotype of neonatal lethality and thin myocardium. Gene expression analyses were performed on both sets of compound heterozygotes and demonstrated downregulation of α-myosin heavy chain only in Gata4/Tbx5 heterozygotes. These findings highlight the unique genetic interactions of Gata4 and Gata6 with Tbx5 for normal cardiac morphogenesis in vivo.

The TNF-Family Receptor DR3 is Essential for Diverse T Cell-Mediated Inflammatory Diseases

DR3 (TRAMP, LARD, WSL-1, TNFRSF25) is a death-domain-containing tumor necrosis factor (TNF)-family receptor primarily expressed on T cells. TL1A, the TNF-family ligand for DR3, can costimulate T cells, but the physiological function of TL1A-DR3 interactions in immune responses is not known. Using DR3-deficient mice, we identified DR3 as the receptor responsible for TL1A-induced T cell costimulation and dendritic cells as the likely source for TL1A during T cell activation. Despite its role in costimulation, DR3 was not required for in vivo T cell priming, for polarization into T helper 1 (Th1), Th2, or Th17 effector cell subtypes, or for effective control of infection with Toxoplasma gondii. Instead, DR3 expression was required on T cells for immunopathology, local T cell accumulation, and cytokine production in Experimental Autoimmune Encephalomyelitis (EAE) and allergic lung inflammation, disease models that depend on distinct effector T cell subsets. DR3 could be an attractive therapeutic target for T cell-mediated autoimmune and allergic diseases.

The PTPN22 allelic variant associated with autoimmunity impairs B cell signaling

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp in humans. A missense mutation in the gene, changing residue 1858 from a cytosine to a thymidine (1858 C/T), is associated with the development of multiple autoimmune disorders. The mechanism by which the 1858T variant contributes to the development of autoimmunity remains elusive. Although studies have demonstrated that Lyp has an inhibitory effect on T cell receptor mediated signaling pathways its role in B cell signaling has not been examined. In this study, we examine the impact of the 1858T variant on B lymphocyte function. B lymphocytes from healthy subjects expressing the variant allele were compared to those of healthy subjects without the variant. When B cells were stimulated via the B‐cell receptor, we observed impaired BCR signal transduction among subjects possessing the variant allele. This defect in signaling is characterized by a deficit in proliferation, diminished calcium mobilization and decreased phosphorylation of key signaling proteins including Syk, which we further demonstrates interacts with Lyp. These findings suggest that the PTPN22 1858T variant alters BCR signaling and implicates B cells in the mechanism by which the PTPN22 1858T variant contributes to autoimmunity. This work was supported by funds from the Juvenile Diabetic Research Foundation (the Center for Translational Research at Benaroya Research Institute).