Mouse Genetic Approaches Research Articles

Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein

Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility

The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients.

Halting renal fibrosis: an unexpected role for mTORC2 signaling

Slowly progressive renal fibrosis is the hallmark of chronic kidney disease, independent of its etiology. Transforming growth factor-β (TGF-β) has been found to be the main profibrotic renal cytokine. Li et al. identify mTORC2 as an important mediator of TGF-β signaling. Being upstream of the previously described profibrotic kinases AKT, SGK1, and PKC-α, mTORC2 represents a potentially interesting drug target. Yet, although the mouse genetic approach yielded convincing results, transition from bench to bedside will remain challenging.

Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity.

Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes. PIK3CA and P53 (also known as TP53) are the two most frequently mutated genes and are associated with different types of human breast cancers. The cellular origin and the mechanisms leading to PIK3CA-induced tumour heterogeneity remain unknown. Here we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and the impact of mutations in this gene on tumour heterogeneity. Surprisingly, oncogenic Pik3ca(H1047R) mutant expression at physiological levels in basal cells using keratin (K)5-CreER(T2) mice induced the formation of luminal oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive tumours, while its expression in luminal cells using K8-CReER(T2) mice gave rise to luminal ER(+)PR(+) tumours or basal-like ER(-)PR(-) tumours. Concomitant deletion of p53 and expression of Pik3ca(H1047R) accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca(H1047R) in unipotent basal cells gave rise to luminal-like cells, while its expression in unipotent luminal cells gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that underwent cell fate transition upon Pik3ca(H1047R) expression in unipotent progenitors demonstrated a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures characteristic of the different cell fate switches that occur upon Pik3ca(H1047R) expression in basal and luminal cells, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca(H1047R) activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation.

Identification of novel loci associated with respiratory disease by a forward genetic approach in mouse

Identification of novel loci associated with respiratory disease by a forward genetic approach in mouse

Transcription Factors GATA4 and HNF4A Control Distinct Aspects of Intestinal Homeostasis in Conjunction with Transcription Factor CDX2

Distinct groups of transcription factors (TFs) assemble at tissue-specific cis-regulatory sites, implying that different TF combinations may control different genes and cellular functions. Within such combinations, TFs that specify or maintain a lineage and are therefore considered master regulators may play a key role. Gene enhancers often attract these tissue-restricted TFs, as well as TFs that are expressed more broadly. However, the contributions of the individual TFs to combinatorial regulatory activity have not been examined critically in many cases in vivo. We address this question using a genetic approach in mice to inactivate the intestine-specifying and intestine-restricted factor CDX2 alone or in combination with its more broadly expressed partner factors, GATA4 and HNF4A. Compared with single mutants, each combination produced significantly greater defects and rapid lethality through distinct anomalies. Intestines lacking Gata4 and Cdx2 were deficient in crypt cell replication, whereas combined loss of Hnf4a and Cdx2 specifically impaired viability and maturation of villus enterocytes. Integrated analysis of TF binding and of transcripts affected in Hnf4a;Cdx2 compound-mutant intestines indicated that this TF pair controls genes required to construct the apical brush border and absorb nutrients, including dietary lipids. This study thus defines combinatorial TF activities, their specific requirements during tissue homeostasis, and modules of transcriptional targets in intestinal epithelial cells in vivo.

Interleukin-5–producing group 2 innate lymphoid cells control eosinophilia induced by interleukin-2 therapy

AbstractInterleukin (IL)-2 promotes regulatory T-cell development and function, and treatment with IL-2 is being tested as therapy for some autoimmune diseases. However, patients receiving IL-2 treatment also experience eosinophilia due to an unknown mechanism. Here, we show that patients receiving low-dose IL-2 have elevated levels of serum IL-5, and this correlates with their degree of eosinophilia. In mice, low-dose IL-2–anti-IL-2 antibody complexes drove group 2 innate lymphoid cells (ILC2) to produce IL-5 and proliferate. Using genetic approaches in mice, we demonstrate that activation of ILC2 was responsible for the eosinophilia observed with IL-2 therapy. These observations reveal a novel cellular network that is activated during IL-2 treatment. A better understanding of the cross talk between these cell populations may lead to more effective targeting of IL-2 to treat autoimmune disease.

KDM6 demethylase independent loss of histone H3 lysine 27 trimethylation during early embryonic development.

The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the Kdm6 gene family, Utx (Kdm6a, X-linked gene) and Jmjd3 (Kdm6b, autosomal gene). Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family.

Wise regulates bone deposition through genetic interactions with Lrp5.

In this study using genetic approaches in mouse we demonstrate that the secreted protein Wise plays essential roles in regulating early bone formation through its ability to modulate Wnt signaling via interactions with the Lrp5 co-receptor. In Wise−/− mutant mice we find an increase in the rate of osteoblast proliferation and a transient increase in bone mineral density. This change in proliferation is dependent upon Lrp5, as Wise;Lrp5 double mutants have normal bone mass. This suggests that Wise serves as a negative modulator of Wnt signaling in active osteoblasts. Wise and the closely related protein Sclerostin (Sost) are expressed in osteoblast cells during temporally distinct early and late phases in a manner consistent with the temporal onset of their respective increased bone density phenotypes. These data suggest that Wise and Sost may have common roles in regulating bone development through their ability to control the balance of Wnt signaling. We find that Wise is also required to potentiate proliferation in chondrocytes, serving as a potential positive modulator of Wnt activity. Our analyses demonstrate that Wise plays a key role in processes that control the number of osteoblasts and chondrocytes during bone homeostasis and provide important insight into mechanisms regulating the Wnt pathway during skeletal development.

C-kit+ cells minimally contribute cardiomyocytes to the heart.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level.

Genetic analysis of the Hippo signaling pathway in mammalian liver development and disease (210.2)

The Hippo signaling pathway is an emerging growth control and tumor suppressor pathway. Our laboratory has been using a genetic approach in mice to study the role of Hippo signaling in regulating liver progenitor cell growth and differentiation. Hippo signaling is dynamically modulated during liver development to ensure proper maturation of two major cell types of the liver: hepatocytes and biliary epithelial cells. In addition, Hippo signaling is required in the adult liver to maintain quiescence and to repress activation of resident liver stem cells. These and other recent findings from our laboratory will be discussed.

Genetic Evidence for the Adhesion Protein IgSF9/Dasm1 to Regulate Inhibitory Synapse Development Independent of its Intracellular Domain

Normal brain function requires balanced development of excitatory and inhibitory synapses. An imbalance in synaptic transmission underlies many brain disorders such as epilepsy, schizophrenia, and autism. Compared with excitatory synapses, relatively little is known about the molecular control of inhibitory synapse development. We used a genetic approach in mice to identify the Ig superfamily member IgSF9/Dasm1 as a candidate homophilic synaptic adhesion protein that regulates inhibitory synapse development. IgSF9 is expressed in pyramidal cells and subsets of interneurons in the CA1 region of hippocampus. Electrophysiological recordings of acute hippocampal slices revealed that genetic inactivation of the IgSF9 gene resulted in fewer functional inhibitory synapses; however, the strength of the remaining synapses was unaltered. These physiological abnormalities were correlated with decreased expression of inhibitory synapse markers in IgSF9(-/-) mice, providing anatomical evidence for a reduction in inhibitory synapse numbers, whereas excitatory synapse development was normal. Surprisingly, knock-in mice expressing a mutant isoform of IgSF9 lacking the entire cytoplasmic domain (IgSF9(ΔC/ΔC) mice) had no defects in inhibitory synapse development, providing genetic evidence that IgSF9 regulates synapse development via ectodomain interactions rather than acting itself as a signaling receptor. Further, we found that IgSF9 mediated homotypic binding and cell aggregation, but failed to induce synapse formation, suggesting that IgSF9 acts as a cell adhesion molecule (CAM) to maintain synapses. Juvenile IgSF9(-/-) mice exhibited increased seizure susceptibility indicative of an imbalance in synaptic excitation and inhibition. These results provide genetic evidence for a specific role of IgSF9 in inhibitory synapse development/maintenance, presumably by its CAM-like activity.

Dickkopf-3: a stubborn protector of cardiac hypertrophy

This editorial refers to ‘Dickkopf-3 attenuates pressure overload-induced cardiac remodelling’ by Y. Zhang et al. , pp. 35–45, this issue. In response to haemodynamic overload under pathological conditions such as hypertension, valvular heart disease, and myocardial infarction, the heart undergoes hypertrophic growth by increasing cell size and protein synthesis as well as changing the transcriptional programme in cardiac myocytes.1 Although cardiac hypertrophy is beneficial in the way that it reduces ventricular wall tension and maintains pump function, it promotes cardiac structural remodelling and dysfunction, and eventually leads to development of congestive heart failure, arrhythmia, and sudden death. At present, several drugs such as inhibitors of the renin–angiotensin–aldosterone system, β-adrenergic receptor blockers, and calcium channel blockers are clinically available for the management of hypertension, and these drugs have shown significant efficacy in preventing load-induced cardiac hypertrophy and remodelling. However, these pharmacological agents are currently of limited effectiveness, and further discovery and development of novel classes of cardioprotective drugs are in urgent need.2 For that purpose, it is important to elucidate the molecular mechanisms underlying the development of cardiac hypertrophy. In the article by Zhang et al. ,3 integrating genetic approaches in mice have shed light on Dickkopf-3 (DKK3) as a cardioprotective regulator of cardiac hypertrophy. DKK3 is a secreted glycoprotein of the Dickkopf family that typically antagonizes the Wnt/β-catenin signalling by interfering with Wnt co-receptors, low-density lipoprotein receptor-related protein and kremen.4 ‘Dickkopf’ is a German word for ‘big head’ or ‘stubborn’, and the protein …

Focal Contact and Hemidesmosomal Proteins in Keratinocyte Migration and Wound Repair

Significance: During wound healing of the skin, keratinocytes should move over while still adhering to their underlying matrix. Thus, mechanistic insights into the wound-healing process require an understanding of the forms and functions of keratinocyte matrix adhesions, specifically focal contacts and hemidesmosomes, and their components. Recent Advances: Although the structure and composition of focal contacts and hemidesmosomes are relatively well defined, the functions of their components are only now being delineated using mouse genetic models and knockdown approaches in cell culture systems. Remarkably, both focal contact and hemidesmosomal proteins appear involved in determining the speed and directional migration of epidermal cells by modulating several signal transduction pathways. Critical Issues: Although many publications are centered on focal contacts, their existence in tissues such as the skin is controversial. Nonetheless, focal contact proteins are central to mechanisms that regulate skin cell motility. Conversely, hemidesmosomes have been identified in intact skin but whether hemidesmosomal components play a positive regulatory function in keratinocyte motility remains debated in the field. Future Directions: Defective wound healing is a developing problem in the aged, hospitalized and diabetic populations. Hence, deriving new insights into the molecular roles of matrix adhesion proteins in wound healing is a prerequisite to the development of novel therapeutics to enhance tissue repair and regeneration.

Transcriptional regulation of chondrogenesis and articular cartilage destruction

In 20th century, cartilage researches were performed by in vitro culture system, and current trend of mouse genetic approaches makes a good progress in the studies of molecular mechanisms underlying chondrogenesis. Sox family and Runt family transcription factors play central roles in chondrogenesis and pathogenesis of osteoarthritis. In this review, we would like to introduce recent progress in understanding of molecular regulation of transcription factors in chondrogenesis and articular cartilage destruction.

Deletion of the Transforming Growth Factor β Receptor Type II Gene in Articular Chondrocytes Leads to a Progressive Osteoarthritis‐like Phenotype in Mice

While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. TGFβ receptor type II (TGFβRII)-conditional knockout (KO) (TGFβRII(Col2ER)) mice were generated by breeding TGFβRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)- and TGFβRII/ADAMTS-5-double-KO mice were generated and analyzed. Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA.

Abstract IA4: Deconstructing p53 pathways in vivo

Abstract The activation of the p53 protein by cellular stress signals is fundamental for tumor suppression but also promotes pathological states, such as provoking the side effects of genotoxic cancer therapies. To better understand the mechanisms of p53 action in different contexts, we have leveraged both mouse genetic and genomic approaches. First, we have used mouse genetics to define transcriptional programs involved in p53 function in different in vivo settings, specifically by generating a panel of p53 transcriptional activation domain mutant knock-in mouse strains. These include strains expressing p53 mutants in the first (p5325,26), second (p5353,54), or both transactivation domains (p5325,26,53,54). We have observed that p5325,26 is severely compromised for transactivation of most classical p53 target genes, but retains the ability to activate a subset of p53 targets, while p5325,26,53,54 lacks transactivation activity completely. Interestingly, although unable to induce apoptosis or cell cycle arrest in response to acute DNA damage signals, p5325,26 retains full activity in suppressing cancers of a wide range of types, indicating that robust transactivation of most canonical p53 targets is dispensable for tumor suppression. Importantly, as p5325,26 activates only a subset of p53-dependent genes, yet retains tumor suppressor activity, it has helped to define a small set of novel p53-inducible tumor suppression-associated genes, which we are currently analyzing in detail. Second, we have utilized genomic approaches to better understand p53 function. Using ChIP-sequencing and RNA-sequencing to analyze transcriptional programs in acute DNA damage-treated mouse embryo fibroblasts, our studies have revealed an extensive p53-regulated autophagy program that contributes to p53 responses. Together, these approaches will help better define the transcriptional networks important for p53 action in different settings. Citation Format: Colleen A. Brady, Daniela Kenzelmann Broz, Dadi Jiang, Stephano Spano Mello, Kathryn Bieging, Thomas M. Johnson, Leslie A. Jarvis, Margaret M. Kozak, Shashwati Basak, Laura D. Attardi. Deconstructing p53 pathways in vivo. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA4.

Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects

BackgroundAtopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency, with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype.ObjectiveWe sought to identify the matted mutant gene in mice and further define whether mutations in the human gene were associated with AD.MethodsA mouse genetics approach was used to separate the matted and Flg mutations to produce congenic single-mutant strains for genetic and immunologic analysis. Next-generation sequencing was used to identify the matted gene. Five independently recruited AD case collections were analyzed to define associations between single nucleotide polymorphisms (SNPs) in the human gene and AD.ResultsThe matted phenotype in flaky tail mice is due to a mutation in the Tmem79/Matt gene, with no expression of the encoded protein mattrin in the skin of mutant mice. Mattft mice spontaneously have dermatitis and atopy caused by a defective skin barrier, with mutant mice having systemic sensitization after cutaneous challenge with house dust mite allergens. Meta-analysis of 4,245 AD cases and 10,558 population-matched control subjects showed that a missense SNP, rs6694514, in the human MATT gene has a small but significant association with AD.ConclusionIn mice mutations in Matt cause a defective skin barrier and spontaneous dermatitis and atopy. A common SNP in MATT has an association with AD in human subjects.

Dissection of gene function at clonal level using mosaic analysis with double markers

MADM (Mosaic Analysis with Double Markers) technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cre recombinase/loxP-dependent interchromosomal mitotic recombination to reconstitute two split marker genes—green GFP and red tdTomato—and can label sparse clones of homozygous mutant cells in one color and wild-type cells in the other color in an otherwise unlabeled background. At present, major MADM applications include lineage tracing, single cell labeling, conditional knockouts in small populations of cells and induction of uniparental chromosome disomy to assess effects of genomic imprinting. MADM can be applied universally in the mouse with the sole limitation being the specificity of the promoter controlling Cre recombinase expression. Here I review recent developments and extensions of the MADM technique and give an overview of the major discoveries and progresses enabled by the implementation of the novel genetic MADM tools.

Gene dosage in mammals: characterization of haploid embryonic stem cells

Most animals are diploid but in several cases haploid genomes can support development to some extent. In contrast to fish, haploidy is not compatible with embryonic development in mammals. Albeit, haploid cells have been observed in egg cylinder stage mouse embryos the majority of surviving embryos becomes diploid. Differences between the development of mammals and fish include genomic imprinting and dosage compensation. Both processes are thought to contribute to the loss of haploid embryos. Haploid embryonic stem cells (ESCs) have recently been derived from mouse parthenogenetic embryos. These cells maintain key properties of mouse ESCs and might overcome existing limitations in developmental genetic approaches in mice. Haploid ESCs have been established through the activation of unfertilized oocytes from a variety of mouse strains and maintain a wide developmental potential in culture and in chimeric mice. Notably, parthenogenetic haploid ESCs possess robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. Contribution to the embryo correlates with an efficient gain of a diploid karyotype. We have also observed that differentiation in culture results in diploidization, which likely is the result of endoreduplication and not cell fusion. In contrast to differentiation into embryonic cell types a haploid karyotype is maintained under certain conditions during forced differentiation to extraembryonic cell fates. Exploring the differentiation potential of a haploid karyotype highlights developmental constraints imposed by evolutionary adaptations specific for the mammalian genome namely genomic imprinting and X chromosome inactivation. Our data would suggest that early embryos possess a remarkable ability to buffer disturbances of both epigenetic processes.