Gene-targeted Mice Research Articles

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Genetic Instruction of Megakaryocytes and Platelets Derived from Human Induced Pluripotent Stem Cells for Studies of Integrin Regulation.

Platelets are small, anucleate cells that play oversized roles in hemostasis, immunity, and inflammation. An important mediator of platelet function is integrin αIIbβ3, which is required for fibrinogen-dependent platelet aggregation during hemostasis. This platelet response is dependent on conformational changes in the integrin induced by "inside-out" biochemical signals that are triggered by platelet agonists. In turn, fibrinogen binding to αIIbβ3 initiates "outside-in" biochemical and mechanical signals that regulate the platelet cytoskeleton and help to promote full platelet aggregation and secretory responses. Without a nucleus, there is a limited range of experimental manipulations that are possible with human platelets to study the molecular basis of integrin signaling in these primary cells. Consequently, many studies of αIIbβ3 function use genetic approaches that rely on heterologous expression systems or platelets from gene-targeted mice, sometimes with uncertain applicability to human platelets. This chapter will detail a method for genetic manipulation of megakaryocytes and platelets derived from human induced pluripotent stem cells for molecular studies of αIIbβ3 signaling and for modeling of human platelet functions potentially relevant to hemostasis, immunity, and inflammation.

Annexin A7 is a critical regulator of Ca2+ mobilization and lipid metabolism during platelet activation and arterial thrombosis

Abstract Background Platelet activation after contact to subendothelial collagen following atherosclerotic plaque rupture can lead to arterial thrombosis with acute thrombotic vascular occlusion. Annexin A7 (AnxA7) is an intracellular Ca2+- and phospholipid-binding protein that participates in the regulation of prostaglandin production in inflammatory diseases, but also in cell survival and tumor growth. Objective In the present study, we aimed to determine the role of AnxA7 for platelet Ca2+ signaling and lipid metabolism in platelet activation and arterial thrombosis in gene-targeted mice lacking annexin A7 (Anxa7−/−). Results AnxA7 is strongly expressed in platelets of platelet-rich human coronary thrombi aspirated from patients with acute ST elevation myocardial infarction. Functionally, platelet aggregation and dense granule secretion were significantly abrogated in Anxa7−/− platelets as compared to wildtype platelets (Anxa7+/+) after activation with collagen or collagen-related peptide (CRP), a specific agonist of the major platelet collagen receptor glycoprotein VI (GPVI). Further, in vitro thrombus formation on a collagen-coated surface under high arterial shear rates was significantly diminished in Anxa7-deficient platelets, and thrombotic vascular occlusion after FeCl3-induced injury in vivo was blunted in Anxa7−/−bone marrow chimeric mice, but no prolongation of bleeding time was observed. Moreover, Anxa7−/− platelets showed a significant reduction of IP3 production due to an abolished phospholipase C (PLC) gamma2 phosphorylation resulting in an abolished increase of [Ca2+]i after platelet activation with CRP. Moreover, we could show by quantitative lipidomics analysis that annexin A7 critically affects platelet oxylipid metabolism following activation of GPVI-dependent platelet signalling since Anxa7−/− platelets showed a significant reduction of the bioactive metabolites thromboxane A2 and 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE) levels as well as significantly reduced levels of several other prostaglandins following stimulation with collagen or CRP. Finally, defective PLCgamma2 phosphorylation, IP1 production and blunted increase of [Ca2+]i in Anxa7−/− platelets could be rescued by exogenous addition of 12(S)-HETE indicating that AnxA7 is a critical regulator of the platelet oxygenase 12-lipoxygenase (12-LOX) in GPVI-dependent platelet Ca2+ signalling during arterial thrombosis following activation by collagen. Conclusions The present study reveals annexin A7 as a critical regulator of oxylipid metabolism and Ca2+ signaling in GPVI-dependent platelet activation. Anxa7-deficiency further results in decreased in vitro and in vivo thrombus formation, but does not affect bleeding time. In conclusion, annexin A7 plays an important role in platelet signaling during arterial thrombosis and thus, may reflect a promising target for novel antiplatelet strategies. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (Deutsche Forschungsgemeinschaft, DFG)

Abstract 5972: HLJ1 deficiency enhanced diethylnitrosamine-induced hepatocarcinogenesis in a gene targeting mouse model

Abstract Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related death worldwide. The conventional treatments such as surgical resection and liver transplantation are applicable to only a small proportion of patients and prolongation of survival is restricted. Therefore, it is urgent to develop cancer biomarkers for defining cancer risk, diagnosis, prognosis and even find new therapeutic targets. Human Liver DnaJ-Like Protein (HLJ1), which belongs to DnaJ heat shock protein family (HSP40) member B4 (DNAJB4), has been reported to act as a tumor suppressor in NSCLC, colorectal cancer, melanoma metastasis. However, the precise role of HLJ1 in liver cancer tumorigenesis and the progress of HCC carcinogenesis has not been elucidated yet. Thus, we investigate whether HLJ1 has tumor-suppressive or oncogenic role on DEN-induced liver cancer development in a mouse model. Wild-type (WT) and HLJ1 knockout (HLJ1−/−) mice are injected with 20 mg/kgbw DEN at postnatal day 15. Thereafter, 50 mg/kgbw DEN is administrated weekly to mice between age 4 to 12 weeks. Mice were sacrificed at age 30 and 36 weeks to assess the tumor count and size. In another group, mice are injected with DEN and age day 15 and then 500 ppm PB is administrated in drinking water until mice were sacrifice at age 52 weeks. The liver tissues are frozen and fixed for western blotting and IHC analysis. For cDNA microarray analysis, gene expression microarray samples were from 6-8 weeks wild-type and HLJ1−/− mice livers. When sacrificed at age 30 and 36 weeks, HLJ1−/− mice exhibited higher tumor multiplicity, serum ALT, AST levels, and larger macroscopic tumor nodules than WT mice. In DEN+PB treatment group, higher incidence rate can be observed in HLJ1−/− mice than in WT mice. This indicated HLJ1 protects liver from DEN-induced HCC carcinogenesis and knockout of HLJ1 confers to more severe HCC induction. Furthermore, p-STAT3 expression was higher in normal part of HLJ1−/− mice liver compared to that of WT mice liver, which suggest the role of HLJ1 in regulating p-STAT3 signaling in the early stage of tumor formation. Using IHC staining and western blotting to analyze HLJ1 expression level, we found half of the tumors express higher HLJ1 than adjacent normal part did, which suggested a tumor-promoting role of HLJ1 after tumor initiation and formation. In cDNA microarray analysis, network analysis of genes enriched in HCC map folder revealed involved objects JunB, c-Myc, p21 and Cyclin D1, which was further confirmed by quantitative PCR. p21 and Cyclin D1 were highly expressed in the liver of HLJ1−/− comparing to that of WT mice, which suggested HLJ1-related genes p21 and Cyclin D1 may have roles in HCC formation. Clinical information of 362 liver cancer patients from TCGA database exhibited that higher expression of HLJ1 in tumor part is correlated to lower disease-specific survival rate. It indicated that HLJ1 may be a tumor marker for HCC prognosis and clinical outcome prediction. It also suggested that HLJ1 may play an oncogenic role after tumor formation. In summary, HLJ1 has protective role against DEN-induced hepatocarcinogenesis through down-regulated p-STAT3 and cyclin D1 whereas it could also be an enhancer to promote tumor progression after tumor formation. Hence, HLJ1 could be a target for liver cancer therapy and a prognostic biomarker in the future. Citation Format: Wei-Jia Luo, Min-Hui Chien, Jeremy J. W. Chen, Sung-Liang Yu, Pan-Chyr Yang, Kang-Yi Su. HLJ1 deficiency enhanced diethylnitrosamine-induced hepatocarcinogenesis in a gene targeting mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5972.

SUMOylation of Enzymes and Ion Channels in Sensory Neurons Protects against Metabolic Dysfunction, Neuropathy, and Sensory Loss in Diabetes

Diabetic peripheral neuropathy (DPN) is a highly frequent and debilitating clinical complication of diabetes that lacks therapies. Cellular oxidative stress regulates post-translational modifications, including SUMOylation. Here, using unbiased screens, we identified key enzymes in metabolic pathways and ion channels as novel molecular targets of SUMOylation that critically regulated their activity. Sensory neurons of diabetic patients and diabetic mice demonstrated changes in the SUMOylation status of metabolic enzymes and ion channels. In support of this, profound metabolic dysfunction, accelerated neuropathology, and sensory loss were observed in diabetic gene-targeted mice selectively lacking the ability to SUMOylate proteins in peripheral sensory neurons. TRPV1 function was impaired by diabetes-induced de-SUMOylation as well as by metabolic imbalance elicited by de-SUMOylation of metabolic enzymes, facilitating diabetic sensory loss. Our results unexpectedly uncover an endogenous post-translational mechanism regulating diabetic neuropathy in patients and mouse models that protects against metabolic dysfunction, nerve damage, and altered sensory perception.

Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats

CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

Generation and characterization of Aldh3-Cre transgenic mice as a tool for conditional gene deletion in postnatal cornea

Conditional gene targeting in mice by means of Cre-loxP strategy represents a powerful approach to study mammalian gene function. This approach is however dependent on the availability of suitable strains of mice with a tissue or time restricted activity of the Cre recombinase. Here we describe Aldh3-Cre transgenic mice as a useful tool to conditionally delete genes in cornea, a specialized transparent tissue found on the anterior-most part of the eye, which acts as a protective barrier and contributes to the refractive power. Using a set of floxed alleles we demonstrate high Aldh3-Cre activity in corneal epithelial cells, corneal stroma and conjunctival epithelial cells at postnatal stages. Aldh3-Cre will thus be particularly beneficial for functional analysis of genes which are vital for postnatal development of cornea and conjunctiva.

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.

Photoactivatable Cre recombinase 3.0 for in vivo mouse applications

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.

The B-cell inhibitory receptor CD22 is a major factor in host resistance to Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a major human pathogen, causing pneumonia and sepsis. Genetic components strongly influence host responses to pneumococcal infections, but the responsible loci are unknown. We have previously identified a locus on mouse chromosome 7 from a susceptible mouse strain, CBA/Ca, to be crucial for pneumococcal infection. Here we identify a responsible gene, Cd22, which carries a point mutation in the CBA/Ca strain, leading to loss of CD22 on B cells. CBA/Ca mice and gene-targeted CD22-deficient mice on a C57BL/6 background are both similarly susceptible to pneumococcal infection, as shown by bacterial replication in the lungs, high bacteremia and early death. After bacterial infections, CD22-deficient mice had strongly reduced B cell populations in the lung, including GM-CSF producing, IgM secreting innate response activator B cells, which are crucial for protection. This study provides striking evidence that CD22 is crucial for protection during invasive pneumococcal disease.

Class I HDAC Differentially Regulates Blood Pressure and Renal Injury in Npr1 Gene‐targeted Male and Female Mutant Mice

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which plays a critical role in the regulation of blood pressure (BP) and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit pathophysiology of hypertension, kidney disease, and heart failure; however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to gain insight into the effect of class I‐specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on Npr1 gene expression and regulation of BP and renal injury in gene‐targeted mutant mice. We utilized 16‐ to 20‐weeks old male and female Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+), and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. All mice genotypes were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate day for 2‐weeks. The Western blot results showed that MGCD0103 significantly increased the renal NPRA protein levels in male and female mice compared with vehicle‐treated (olive oil) controls. After MGCD treatment, the renal GC‐A activity was significantly increased in both male and female mice. Male mice exhibited significantly higher systolic BP (SBP) than female mice and treatment with MGCD0103 decreased SBP in both 1‐copy male (106 ± 0.6 vs. vehicle‐treated, 129 ± 1.9; mmHg) and female (97 ± 2.2 vs. vehicle‐treated, 110 ± 2.1 mmHg, p < 0.001) mice. Renal HDAC activity was considerably higher (p < 0.05) in male mice than female mice; however, treatment with MGCD0103 attenuated (p < 0.05) HDAC activity by 40% in male and ~50% in female animals. Significantly higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD0103 treatment in male mice. The present results demonstrate that class I HDAC inhibitor, MGCD0103 upregulates NPRA expression in vivo, lowers SBP, and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a gender‐related manner.Support or Funding InformationThis work was supported by NIH grant HL062147.

Two distinct ubiquitin-binding motifs in A20 mediate its anti-inflammatory and cell-protective activities.

Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.

Targeting the Extrinsic Pathway of Hepatocyte Apoptosis Promotes Clearance of Plasmodium Liver Infection

Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite.

Is the Amiloride-Sensitive Na+ Channel in Taste Cells Really ENaC?

Among the 5 taste qualities, salt is the least understood. The receptors, their expression pattern in taste cells, and the transduction mechanisms for salt taste are still unclear. Previous studies have suggested that low concentrations of NaCl are detected by the amiloride-sensitive epithelial Na+ channel (ENaC), which in other systems requires assembly of 3 homologous subunits (α, β, and γ) to form a functional channel. However, a new study from Lossow and colleagues, published in this issue of Chemical Senses, challenges that hypothesis by examining expression levels of the 3 ENaC subunits in individual taste cells using gene-targeted mice in combination with immunohistochemistry and in situ hybridization. Results show a lack of colocalization of ENaC subunits in taste cells as well as expression of subunits in taste cells that show no amiloride sensitivity. These new results question the molecular identity of the amiloride-sensitive Na+ conductance in taste cells.

The essentials of developmental apoptosis.

Apoptotic cells are commonly observed in a broad range of tissues during mammalian embryonic and fetal development. Specific requirements and functions of programmed cell death were inferred from early observations. These inferences did not hold up to functional proof for a requirement of apoptosis for normal tissue development in all cases. In this review, we summarize how the appraisal of the importance of developmental apoptosis has changed over the years, in particular with detailed functional assessment, such as by using gene-targeted mice lacking essential initiators or mediators of apoptosis. In recent years, the essentials of developmental apoptosis have emerged. We hypothesize that apoptosis is predominantly required to balance cell proliferation. The two interdependent processes—cell proliferation and apoptosis—together more powerfully regulate tissue growth than does each process alone. We proposed that this ensures that tissues and cell populations attain the appropriate size that allows fusion in the body midline and retain the size of cavities once formed. In addition, a limited number of tissues, albeit not all previously proposed, rely on apoptosis for remodeling, chiefly aortic arch remodeling, elimination of supernumerary neurons, removal of vaginal septa, and removal of interdigital webs in the formation of hands and feet.

TYK2 in Tumor Immunosurveillance.

We review the history of the tyrosine kinase 2 (TYK2) as the founding member of the Janus kinase (JAK) family and outline its structure-function relation. Gene-targeted mice and hereditary defects of TYK2 in men have established the biological and pathological functions of TYK2 in innate and adaptive immune responses to infection and cancer and in (auto-)inflammation. We describe the architecture of the main cytokine receptor families associated with TYK2, which activate signal transducers and activators of transcription (STATs). We summarize the cytokine receptor activities with well characterized dependency on TYK2, the types of cells that respond to cytokines and TYK2 signaling-induced cytokine production. TYK2 may drive beneficial or detrimental activities, which we explain based on the concepts of tumor immunoediting and the cancer-immunity cycle in the tumor microenvironment. Finally, we summarize current knowledge of TYK2 functions in mouse models of tumor surveillance. The biology and biochemistry of JAKs, TYK2-dependent cytokines and cytokine signaling in tumor surveillance are well covered in recent reviews and the oncogenic properties of TYK2 are reviewed in the recent Special Issue ‘Targeting STAT3 and STAT5 in Cancer’ of Cancers.

Deletion of Histone Methyltransferase G9a Suppresses Mutant Kras-driven Pancreatic Carcinogenesis.

The entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model. We established pancreas-specific G9aflox/flox mice and crossed them with Ptf1aCre/; KrasG12D/+ (KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice. G9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes. Thus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.

Fgf8 genetic labeling reveals the early specification of vestibular hair cell type in mouse utricle.

FGF8 signaling plays diverse roles in inner ear development, acting at multiple stages from otic placode induction to cellular differentiation in the organ of Corti. As a secreted morphogen with diverse functions, Fgf8 expression is likely to be spatially restricted and temporally dynamic throughout inner ear development. We evaluated these characteristics using genetic labeling mediated by Fgf8mcm gene-targeted mice and determined that Fgf8 expression is a specific and early marker of Type-I vestibular hair cell identity. Fgf8mcm expression initiates at E11.5 in the future striolar region of the utricle, labeling hair cells following EdU birthdating, and demonstrates that sub-type identity is determined shortly after terminal mitosis. This early fate specification is not apparent using markers or morphological criteria that are not present before birth in the mouse. Although analyses of Fgf8 conditional knockout mice did not reveal developmental phenotypes, the restricted pattern of Fgf8 expression suggests that functionally redundant FGF ligands may contribute to vestibular hair cell differentiation and supports a developmental model in which Type-I and Type-II hair cells develop in parallel rather than from an intermediate precursor.

Infiltrating Myeloid Cells Drive Osteosarcoma Progression via GRM4 Regulation of IL23.

The glutamate metabotropic receptor 4 (GRM4) locus is linked to susceptibility to human osteosarcoma, through unknown mechanisms. We show that Grm4-/- gene-targeted mice demonstrate accelerated radiation-induced tumor development to an extent comparable with Rb1+/- mice. GRM4 is expressed in myeloid cells, selectively regulating expression of IL23 and the related cytokine IL12. Osteosarcoma-conditioned media induce myeloid cell Il23 expression in a GRM4-dependent fashion, while suppressing the related cytokine Il12. Both human and mouse osteosarcomas express an increased IL23:IL12 ratio, whereas higher IL23 expression is associated with worse survival in humans. Consistent with an oncogenic role, Il23 -/- mice are strikingly resistant to osteosarcoma development. Agonists of GRM4 or a neutralizing antibody to IL23 suppressed osteosarcoma growth in mice. These findings identify a novel, druggable myeloid suppressor pathway linking GRM4 to the proinflammatory IL23/IL12 axis. SIGNIFICANCE: Few novel systemic therapies targeting osteosarcoma have emerged in the last four decades. Using insights gained from a genome-wide association study and mouse modeling, we show that GRM4 plays a role in driving osteosarcoma via a non-cell-autonomous mechanism regulating IL23, opening new avenues for therapeutic intervention.See related commentary by Jones, p. 1484.This article is highlighted in the In This Issue feature, p. 1469.

Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at systems-level. Here, we isolated primary CD4+ T cells from 15 gene-targeted mice each expressing one tagged-form of a canonical protein of the TCR signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the signalosomes assembling around each of the tagged protein over 600 seconds of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction permitting to anticipate whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.