Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to ...incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene’s transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon–intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN’s artificial intron opens up engineering modalities for the generation of multifunctional alleles.
Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent ...developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.
Abstract
Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by ...global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.
Significance Hypothalamic agouti-related peptide (AGRP) neurons control food intake and body weight. G protein-coupled receptor 17 (GPR17) was recently shown to be expressed in these neurons and ...controls their activity, thereby reducing body weight and food intake in mice. In the current study, we demonstrate that Gpr17 -deficient mice have normal hypothalamic and circulating AGRP levels. Body weight, food intake, and glucose homeostasis appear normal in the GPR17-deficient mice. The current data do not validate GPR17 as a therapeutic target for obesity or type 2 diabetes.
G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 ( Gpr17 ⁻/⁻) have similar food intake and body weight compared with their wild-type littermates. Gpr17 ⁻/⁻ mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.
Angiogenesis is largely driven by motile endothelial tip-cells capable of invading avascular tissue domains and enabling new vessel formation. Highly responsive to Vascular Endothelial ...Growth-Factor-A (VEGFA), endothelial tip-cells also suppress angiogenic sprouting in adjacent stalk cells, and thus have been a primary therapeutic focus in addressing neovascular pathologies. Surprisingly, however, there remains a paucity of specific endothelial tip-cell markers. Here, we employ transcriptional profiling and a
lacZ
reporter allele to identify
Kcne3
as an early and selective endothelial tip-cell marker in multiple angiogenic contexts. In development,
Kcne3
expression initiates during early phases of angiogenesis (E9) and remains specific to endothelial tip-cells, often adjacent to regions expressing VEGFA. Consistently,
Kcne3
activation is highly responsive to exogenous VEGFA but maintains tip-cell specificity throughout normal retinal angiogenesis. We also demonstrate endothelial tip-cell selectivity of
Kcne3
in several injury and tumor models. Together, our data show that
Kcne3
is a unique marker of sprouting angiogenic tip-cells and offers new opportunities for investigating and targeting this cell type.
The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; ...however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct. Summary Sentence ADAM6, a testis-specific protein, is required for sperm ascent into the oviduct and its deletion results in deficits in both sperm–sperm and sperm–extracellular matrix associations.
The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the ...olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.
The Eph family of receptor tyrosine kinases plays important roles in neural development. Previous studies have implicated Eph receptors and their ligands, the ephrins, in neuronal migration, axon ...bundling and guidance to specific targets, dendritic spine formation and neural plasticity. However, specific contributions of EphA5 and EphA6 receptors to the regulation of neuronal cell morphology have not been well studied.
Here we show that deletion of EphA5 and EphA6 results in abnormal Golgi staining patterns of cells in the brain, and abnormal spine morphology.
These observations suggest novel functions of these Eph receptors in the regulation of neuronal and spine structure in brain development and function.
Development of the vascular system depends on the highly coordinated actions of a variety of angiogenic regulators. Several of these regulators are members of the tyrosine kinase superfamily, ...including VEGF receptors and angiopoietin receptors, Tie1 and Tie2. Tyrosine kinase signaling is counter-regulated by the activity of tyrosine phosphatases, including vascular endothelial protein tyrosine phosphatase (VE-PTP), which has previously been shown to modulate Tie2 activity. We generated mice in which VE-PTP is replaced with a reporter gene. We confirm that VE-PTP is expressed in endothelium and also show that VE-PTP is highly expressed in the developing outflow tract of the heart and later is expressed in developing heart valves. Vasculogenesis occurs normally in mice lacking VE-PTP; however, angiogenesis is abnormal. Angiogenic defects in VE-PTP-null mice were most pronounced in the yolk sac and include a complete failure to elaborate the primitive vascular scaffold into higher-order branched arteries, veins, and capillaries. VE-PTP continues to be expressed into adulthood in the vasculature and heart valves, suggesting later roles in vascular development or homeostasis. VE-PTP is also expressed in the vasculature of growing tumors, suggesting that VE-PTP may be a new potential target for angiogenic therapies.
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