The mouse genetics toolkit: revealing function and mechanism

Large-scale projects are providing rapid global access to a wealth of mouse genetic resources to help discover disease genes and to manipulate their function

Sinonasal meningioma in a Siberian tiger (Panthera tigris altaica)

Meningiomas are the most common primary brain tumour in dogs and cats. However, whilst there are numerous reports of extracranial (spinal, orbital and sinonasal) meningiomas in the dog, there have only been a few case reports of spinal meningiomas, and no post-mortem confirmed orbital or sinonasal meningiomas in cats. In this report, a 20-year-old captive tiger (Panthera tigris altaica) with a history of chronic ocular inflammation resulting in enucleation, spontaneously developed tetanic convulsions (epileptic seizures) that over a 2-year period resulted in a gradually worsening condition and the animal was eventually euthanized. At autopsy, a focal, expansile, neoplastic mass was found in the caudal nasal cavity midline, abutting the cribriform plate and slightly compressing the calvarium. Histological analysis revealed nasal turbinates attached to a wellcircumscribed expansile multi-lobular mass consisting of interlacing whorls and streams of neoplastic cells supported by a variably fibrous to microcystic collagenous matrix displaying rare psammoma bodies. The diagnosis was sinonasal transitional meningioma. This is the first report of a captive wild felid with an extracranial meningioma, specifically a tiger with a sinonasal transitional meningioma.The Wellcome Trust.https://www.mdpi.com/journal/vetsciam2023Centre for Veterinary Wildlife StudiesParaclinical Science

Ectodomains of the LDL receptor-related proteins LRP1b and LRP4 have anchorage independent functions in vivo.

BACKGROUND: The low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD) is fully viable with no apparent phenotype. METHODS AND PRINCIPAL FINDINGS: Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and gamma-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain. CONCLUSIONS AND SIGNIFICANCE: Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b) or perinatally (Lrp4) lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby preserving signaling thresholds that are critical for embryonic development, as well as for the clear, but poorly understood role of LRP1b in cancer

Successful treatment of early cutaneous squamous cell carcinoma with hypofractionated radiation therapy in an African lion (Panthera leo)

Cutaneous squamous cell carcinoma (SCC) is a slow growing but locally invasive neoplasm, most commonly caused by prolonged exposure to ultraviolet (UV) radiation. Whilst SCC accounts for 15% of skin tumours in domesticated cats, cutaneous SCC in non-domesticated felids (apart from captive snow leopards) appears to be uncommon, with only three reports in the literature to date. In this report, a captive African lion (Panthera leo) presented with two ulcerative lesions on the nasal planum. Histopathology of the lesions revealed epidermal keratinocyte dysplasia and neoplastic basal- and supra-basal epithelial cells with dyskeratosis and evidence of basement membrane breaching and dermal invasion, consistent with a diagnosis of SCC. There was also evidence of laminar fibrosis and inflammation of the subjacent dermis suggesting that the SCC most likely resulted from UV-induced neoplastic transformation of the epidermal squamous epithelium following actinic keratosis. The lion was treated with hypofractionated radiation therapy and remained in remission until his death (euthanised 17 months later because of age-related chronic renal failure). This is the first report of cutaneous SCC in a lion with evidence of actinic damage and resolution after radiation therapy.L.v.d.W. was supported by the Wellcome Trust grant 20619.http://www.jsava.co.zaam2022Centre for Veterinary Wildlife StudiesCompanion Animal Clinical StudiesParaclinical SciencesProduction Animal Studie

ZNF265—a novel spliceosomal protein able to induce alternative splicing

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-β1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF35. Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265–EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain–containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery

Acute sensitivity of the oral mucosa to oncogenic K-ras.

Mouse models of cancer represent powerful tools for analysing the role of genetic alterations in carcinogenesis. Using a mouse model that allows tamoxifen-inducible somatic activation (by Cre-mediated recombination) of oncogenic K-ras(G12D) in a wide range of tissues, we observed hyperplasia of squamous epithelium located in moist or frequently abraded mucosa, with the most dramatic effects in the oral mucosa. This epithelium showed a sequence of squamous hyperplasia followed by squamous papilloma with dysplasia, in which some areas progressed to early invasive squamous cell carcinoma, within 14 days of widespread oncogenic K-ras activation. The marked proliferative response of the oral mucosa to K-ras(G12D) was most evident in the basal layers of the squamous epithelium of the outer lip with hair follicles and wet mucosal surface, with these cells staining positively for pAKT and cyclin D1, showing Ras/AKT pathway activation and increased proliferation with Ki-67 and EdU positivity. The stromal cells also showed gene activation by recombination and immunopositivity for pERK indicating K-Ras/ERK pathway activation, but without Ki-67 positivity or increase in stromal proliferation. The oral neoplasms showed changes in the expression pattern of cytokeratins (CK6 and CK13), similar to those observed in human oral tumours. Sporadic activation of the K-ras(G12D) allele (due to background spontaneous recombination in occasional cells) resulted in the development of benign oral squamous papillomas only showing a mild degree of dysplasia with no invasion. In summary, we show that oral mucosa is acutely sensitive to oncogenic K-ras, as widespread expression of activated K-ras in the murine oral mucosal squamous epithelium and underlying stroma can drive the oral squamous papilloma-carcinoma sequence

Somatic drivers of B-ALL in a model of ETV6-RUNX1; Pax5(+/-) leukemia.

BACKGROUND: B-cell precursor acute lymphoblastic leukemia (B-ALL) is amongst the leading causes of childhood cancer-related mortality. Its most common chromosomal aberration is the ETV6-RUNX1 fusion gene, with ~25% of ETV6-RUNX1 patients also carrying PAX5 alterations. METHODS: We have recreated this mutation background by inter-crossing Etv6-RUNX1 (Etv6 (RUNX1-SB)) and Pax5(+/-) mice and performed an in vivo analysis to find driver genes using Sleeping Beauty transposon-mediated mutagenesis and also exome sequencing. RESULTS: Combination of Etv6-RUNX1 and Pax5(+/-) alleles generated a transplantable B220 + CD19+ B-ALL with a significant disease incidence. RNA-seq analysis showed a gene expression pattern consistent with arrest at the pre-B stage. Analysis of the transposon common insertion sites identified genes involved in B-cell development (Zfp423) and the JAK/STAT signaling pathway (Jak1, Stat5 and Il2rb), while exome sequencing revealed somatic hotspot mutations in Jak1 and Jak3 at residues analogous to those mutated in human leukemias, and also mutation of Trp53. CONCLUSIONS: Powerful synergies exists in our model suggesting STAT pathway activation and mutation of Trp53 are potent drivers of B-ALL in the context of Etv6-RUNX1;Pax5(+/-)

Human T-Cell leukemia virus type 1 (HTLV- 1) tax requires CADM1/TSLC1 for inactivation of the NF-κB inhibitor A20 and constitutive NF-κB signaling

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells