Allelic Series Of Mice Research Articles

An increased copy number of glycine decarboxylase (GLDC) associated with psychosis reduces extracellular glycine and impairs NMDA receptor function.

Glycine is an obligatory co-agonist at excitatory NMDA receptors in the brain, especially in the dentate gyrus, which has been postulated to be crucial for the development of psychotic associations and memories with psychotic content. Drugs modulating glycine levels are in clinical development for improving cognition in schizophrenia. However, the functional relevance of the regulation of glycine metabolism by endogenous enzymes is unclear. Using a chromosome-engineered allelic series in mice, we report that a triplication of the gene encoding the glycine-catabolizing enzyme glycine decarboxylase (GLDC) - as found on a small supernumerary marker chromosome in patients with psychosis - reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) and suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in schizophrenia-like behaviors which are in part known to be dependent on the activity of the dentate gyrus, e.g., prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results demonstrate that Gldc negatively regulates long-term synaptic plasticity in the dentate gyrus in mice, suggesting that an increase in GLDC copy number possibly contributes to the development of psychosis in humans.

Correction to “Generation of an OMgp allelic series in mice”

Correction to “Generation of an <scp><i>OMgp</i></scp> allelic series in mice”

OR24-1 Structure-Function Analysis of Human OTX2 Variants Defines Required Region of C-Terminus for Pituitary, Eye, and Craniofacial Development

OTX2 is a homeobox transcription factor important for eye, craniofacial and midline development. Whole gene deletions and heterozygous mutations in OTX2 cause variable anomalies with incomplete penetrance, and most cases present with craniofacial / ocular abnormalities, and hypopituitarism, including either isolated or combined pituitary hormone deficiency. Patients with variants of unknown significance in OTX2 pose a dilemma for medical diagnosis. OTX2 is highly conserved evolutionarily: variable craniofacial defects are observed in heterozygous Otx2 null mice and zebrafish with knockdown of otx2b together with other genes. Thus, mouse and zebrafish offer opportunities for functional analysis of human patient variants. A patient diagnosed with combined pituitary hormone deficiency, including deficiencies in GH, ACTH, FSH, and LH, with no obvious ocular or craniofacial abnormalities, had an intriguing, rare, likely deleterious OTX2H230L/+ variant in a predicted protein-protein interaction domain within the C-terminus. We tested the functional significance of the variant allele. No differences in the transactivation properties were observed in cell culture transfections. To assess function in a more physiological context, we generated an Otx2 allelic series in mice using CRISPR/Cas9. This included nested C-terminal deletions, frameshifts, the OTX2H230L/+ allele and other missense mutations. Surprisingly, all Otx2 variants after codon 220 were tolerated; no gross ocular, craniofacial, or pituitary growth insufficiency phenotypes were noted. In contrast, ocular phenotypes were 100% penetrant in Otx2L219Pfs*17/+ mice, including unilateral or bilateral anophthalmia or microphthalmia. Given the time and cost of generating mouse models of human disease, we explored the utility of zebrafish to assess patient variants in OTX2. Although zebrafish have two otx2 genes, previous studies showed that morpholino knockdown of otx2b caused craniofacial defects that mimic those of human patients if an unrelated gene in the pathway was also knocked down. We sought to characterize the phenotypic consequences of a complete loss of function variant in zebrafish because it could offer a cleaner, more robust system for assessment of human variants than the use of multiple, gene-specific morpholinos. Zebrafish homozygous for an essential splice site mutation, otx2bc.507-2A>C (otx2bhu3625) died at 11 days post fertilization. Preliminary data suggest mutants have reduced growth hormone transcripts at day 5, and assessment of zebrafish mutant vision and eye morphology is underway. The mouse studies suggest that the patient OTX2H230L/+ variant is likely tolerated, as are other variants and deletions C-terminal to position 220 of OTX2. Analysis of zebrafish genetic loss of function mutants suggest they offer an avenue for assessing the functional significance of human OTX2 variants.

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1.

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.

Mouse and Human CRKL Is Dosage Sensitive for Cardiac Outflow Tract Formation

The human chromosome 22q11.2 region is susceptible to rearrangements during meiosis leading to velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) characterized by conotruncal heart defects (CTDs) and other congenital anomalies. The majority of individuals have a 3 Mb deletion whose proximal region contains the presumed disease-associated gene TBX1 (T-box 1). Although a small subset have proximal nested deletions including TBX1, individuals with distal deletions that exclude TBX1 have also been identified. The deletions are flanked by low-copy repeats (LCR22A, B, C, D). We describe cardiac phenotypes in 25 individuals with atypical distal nested deletions within the 3 Mb region that do not include TBX1 including 20 with LCR22B to LCR22D deletions and 5 with nested LCR22C to LCR22D deletions. Together with previous reports, 12 of 37 (32%) with LCR22B-D deletions and 5 of 34 (15%) individuals with LCR22C-D deletions had CTDs including tetralogy of Fallot. In the absence of TBX1, we hypothesized that CRKL (Crk-like), mapping to the LCR22C-D region, might contribute to the cardiac phenotype in these individuals. We created an allelic series in mice of Crkl, including a hypomorphic allele, to test for gene expression effects on phenotype. We found that the spectrum of heart defects depends on Crkl expression, occurring with analogous malformations to that in human individuals, suggesting that haploinsufficiency of CRKL could be responsible for the etiology of CTDs in individuals with nested distal deletions and might act as a genetic modifier of individuals with the typical 3 Mb deletion.

Abstract PR02: Myc-Ras cooperation can overwhelm tumor suppressive mechanisms within lung adenocarcinomas

Abstract The ability of Myc and Ras to cooperate during tumorigenesis was first observed over thirty years ago. Numerous mechanisms have been implicated in their collaboration. For instance, Myc can act to circumvent Ras-induced growth arrest. Alternatively, Ras has been shown to prevent Myc-induced apoptosis by activating the anti-apoptotic kinase Akt. Early experiments in primary rodent cells demonstrated that neither Ras nor Myc alone was sufficient for transformation, whereas when expressed together, transformation occurred. However, these transformation events were rare, suggesting additional co-operating mutations were also required. These and other investigations ultimately led to the concept of ‘intrinsic tumor suppression’, whereby the effects of activation of an individual oncogene is limited by their dual potential to invoke cell cycle arrest or apoptosis, which limit their other oncogenic functions. The dogma suggests that cancers only arise in oncogene expressing cells when anti-apoptotic/cell arrest mutations are acquired and the disruption of these pathways leads to net proliferation. There is increasing evidence that the activation of intrinsic tumor suppressive mechanisms depend on increased oncogenic flux resulting from changes in the level of oncogene expression. For example, high expression of oncogenic Ras results in premature growth arrest in MEFs, whereas an endogenous level does not. To address this phenomenon with regards to Myc, we previously generated a novel knock-in mouse (R26c-MycER) in which the c-MycERTAM protein was inserted into the relatively ubiquitous, albeit low expressing Rosa26 locus. In contrast to high levels of Myc expressed in classical transgenic models, low-level c-Myc expression did not trigger intrinsic tumor suppression mechanisms in most tissues, suggesting that low levels of oncogenic Myc may be a more efficient initiator of oncogenesis than over expressed Myc, as it might not require cooperating loss of tumor suppressive pathways. We have now created a R26CAG-c-MycER allele that expresses a 5-10 fold higher constitutive expression of MycERTAM than the R26c-MycER allele. Together with R26c-MycER mice we have been able to generate an allelic series of mice that allow the control, in a spatial and temporal fashion, the activity of de-regulated c-Myc at low, medium and high levels. When we sporadically activated c-MycERTAM within the lung epithelial cells, only very few high-level expressing c-MycERTAM and none of the low-level mice developed tumors within 18 weeks suggesting Myc alone is insufficient for tumorigenesis irrespective of oncogenic level. However, in the presence of an endogenous level of oncogenic K-RasG12D, lung adenomas are evident in mice expressing both low and high levels of c-Myc. Mice expressing the highest levels of c-Myc developed adenocarcinomas with extremely short latency and have a greatly reduced survival when compared to mice with lower levels of c-Myc. As expected, low level c-Myc did not activate intrinsic tumor suppression via the Arf/p53 pathways within K-Ras driven lesions and these tumors grew faster than lesions with oncogenic K-Ras alone. However, tumors expressing high levels of c-Myc did engage a number of key tumor suppressors and apoptosis increased as a function of c-Myc level, despite the continued growth and extremely short latency of the adenomas/adenocarcinomas in these mice. Thus, when combined with oncogenic K-Ras, high c-Myc cooperation results in rapid tumorigenesis whereby tumors are still able to grow prolifically despite the apparent activation of the surveillance mechanisms. Further analysis is currently underway to determine the specific survival advantage oncogenic K-Ras offers within this system. This abstract is also presented as Poster B20. Citation Format: Catherine H. Wilson, Deborah L. Burkhart, Jinyang Li, Trevor D. Littlewood, Gerard I. Evan. Myc-Ras cooperation can overwhelm tumor suppressive mechanisms within lung adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR02. doi: 10.1158/1557-3125.RASONC14-PR02

Retinaldehyde dehydrogenase enzymes regulate colon enteric nervous system structure and function

The enteric nervous system (ENS) forms from the neural crest-derived precursors that colonize the bowel before differentiating into a network of neurons and glia that control intestinal function. Retinoids are essential for normal ENS development, but the role of retinoic acid (RA) metabolism in development remains incompletely understood. Because RA is produced locally in the tissues where it acts by stimulating RAR and RXR receptors, RA signaling during development is absolutely dependent on the rate of RA synthesis and degradation. RA is produced by three different enzymes called retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3) that are all expressed in the developing bowel. To determine the relative importance of these enzymes for ENS development, we analyzed whole mount preparations of adult (8–12-week old) myenteric and submucosal plexus stained with NADPH diaphorase (neurons and neurites), anti-TuJ1 (neurons and neurites), anti-HuC/HuD (neurons), and anti-S100β (glia) in an allelic series of mice with mutations in Raldh1, Raldh2, and Raldh3. We found that Raldh1−/−, Raldh2+/−, Raldh3+/− (R1KOR2HetR3Het) mutant mice had a reduced colon myenteric neuron density, reduced colon myenteric neuron to glia ratio, reduced colon submucosal neuron density, and increased colon myenteric fibers per neuron when compared to the wild type (WT; Raldh1WT, Raldh2WT, Raldh3WT) mice. These defects are unlikely to be due to defective ENS precursor migration since R1KOR2HetR3KO mice had increased enteric neuron progenitor migration into the distal colon compared to WT during development. RALDH mutant mice also have reduced contractility in the colon compared to WT mice. These data suggest that RALDH1, RALDH2 and RALDH3 each contribute to ENS development and function.

Novel Tools to Dissect the Dynamic Regulation of TCR Signaling by the Kinase Csk and the Phosphatase CD45

Although the biochemical events induced by T-cell receptor (TCR) triggering have been well studied, both the mediators and function of basal signaling in T cells remain poorly understood. Furthermore, the precise mechanisms by which MHC-peptide interaction with the TCR disrupt the basal equilibrium to induce downstream signaling are also unclear. Here we describe novel approaches to understand the basal state of T cells and the mechanisms of TCR triggering by perturbing regulation of the Src family kinases (SFKs). The SFKs are critical proximal mediators of TCR signaling that are in turn tightly regulated by the tyrosine kinase Csk and the receptor-like tyrosine phosphatase CD45. We have developed a small-molecule analog-sensitive allele of Csk and an allelic series of mice in which expression of CD45 is varied across a broad range. Our studies have unmasked contributions of Csk and CD45 to maintain the basal state of T cells and also suggest that dynamic regulation of Csk may be involved in TCR triggering.

RNF4 is required for DNA double-strand break repair in vivo

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.

Alterations in TCF7L2 expression define its role as a key regulator of glucose metabolism

Genome-wide association studies (GWAS) have consistently implicated noncoding variation within the TCF7L2 locus with type 2 diabetes (T2D) risk. While this locus represents the strongest genetic determinant for T2D risk in humans, it remains unclear how these noncoding variants affect disease etiology. To test the hypothesis that the T2D-associated interval harbors cis-regulatory elements controlling TCF7L2 expression, we conducted in vivo transgenic reporter assays to characterize the TCF7L2 regulatory landscape. We found that the 92-kb genomic interval associated with T2D harbors long-range enhancers regulating various aspects of the spatial-temporal expression patterns of TCF7L2, including expression in tissues involved in the control of glucose homeostasis. By selectively deleting this interval, we establish a critical role for these enhancers in robust TCF7L2 expression. To further determine whether variation in Tcf7l2 expression may lead to diabetes, we developed a Tcf7l2 copy-number allelic series in mice. We show that a null Tcf7l2 allele leads, in a dose-dependent manner, to lower glycemic profiles. Tcf7l2 null mice also display enhanced glucose tolerance coupled to significantly lowered insulin levels, suggesting that these mice are protected against T2D. Confirming these observations, transgenic mice harboring multiple Tcf7l2 copies and overexpressing this gene display reciprocal phenotypes, including glucose intolerance. These results directly demonstrate that Tcf7l2 plays a role in regulating glucose tolerance, suggesting that overexpression of this gene is associated with increased risk of T2D. These data highlight the role of enhancer elements as mediators of T2D risk in humans, strengthening the evidence that variation in cis-regulatory elements may be a paradigm for genetic predispositions to common disease.

Generation of an OMgp allelic series in mice.

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin-derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type-specific roles of OMgp in CNS injury-induced axonal plasticity.

Pitx2 is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification.

Analysis of an allelic series in mice revealed that the Pitx2 homeobox gene is required at multiple stages of pituitary development. It is necessary for initiating expansion of Rathke's pouch and maintaining expression of the fetal-specific transcription factors Hesx1 and Prop1. At later stages Pitx2 is necessary for specification and expansion of the gonadotropes and Pit1 lineage within the ventral and caudomedial anterior pituitary. Mechanistically, this is due to the dependence of several critical lineage-specific transcription factors, Pit1, Gata2, Egr1 and Sf1, on a threshold level of PITX2. The related Pitx1 gene has a role in hormone gene transcription, and it is important late in ontogeny for the final expansion of the differentiated cell types. Pitx1 and Pitx2 have overlapping functions in the expansion of Rathke's pouch, revealing the sensitivity of pituitary organogenesis to the dosage of the PITX family. The model developed for PITX gene function in pituitary development provides a better understanding of the etiology of Rieger syndrome and may extend to other PITX-sensitive developmental processes.

Generation of allelic series in mice by gene targeting.

Generation of allelic series in mice by gene targeting.