INFRAFRONTIER quality principles in systemic phenotyping.

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.

Rapid in vivo analysis of mutant forms of the LAT adaptor using Pax5-Lat double-deficient pro-B cells.

Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.

Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies.

Laminopathies are a group of disorders caused by mutations in the LMNA gene encoding A-type lamins, components of the nuclear lamina. Three of these disorders affect specifically the skeletal and/or cardiac muscles, and their pathogenic mechanisms are still unknown. We chose the LMNA H222P missense mutation identified in a family with autosomal dominant Emery-Dreifuss muscular dystrophy, one of the striated muscle-specific laminopathies, to create a faithful mouse model of this type of laminopathy. The mutant mice exhibit overtly normal embryonic development and sexual maturity. At adulthood, male homozygous mice display reduced locomotion activity with abnormal stiff walking posture and all of them die by 9 months of age. As for cardiac phenotype, they develop chamber dilation and hypokinesia with conduction defects. These abnormal skeletal and cardiac features were also observed in the female homozygous mice but with a later-onset than in males. Histopathological analysis of the mice revealed muscle degeneration with fibrosis associated with dislocation of heterochromatin and activation of Smad signalling in heart and skeletal muscles. These results demonstrate that LmnaH222P/H222P mice represent a good model for studying laminopathies affecting striated muscles as they develop a dystrophic condition of both skeletal and cardiac muscles similar to the human diseases.

Linker for activation of T cells integrates positive and negative signaling in mast cells.

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcepsilonRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like alphabeta or gammadelta T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

LAT regulates gammadelta T cell homeostasis and differentiation.

LAT (linker for activation of T cells) is essential for T cell receptor signaling. Mice homozygous for a mutation of the three C-terminal LAT tyrosine residues showed a block in alphabeta T cell development and a partially impaired gammadelta T cell development. Without intentional immunization, they accumulated gammadelta T cells in the spleen and lymph nodes that chronically produced T helper type 2 cytokines in large amounts, and caused the maturation of plasma cells secreting immunoglobulin E (IgE) and IgG1. These effects are very similar to that triggered in the alphabeta lineage by a mutation involving a distinct LAT tyrosine. Thus, LAT is an essential regulator of T cell homeostasis and terminal differentiation.

Induction of T helper type 2 immunity by a point mutation in the LAT adaptor.

The transmembrane protein LAT (linker for activation of T cells) couples the T cell receptor (TCR) to downstream signaling effectors. Mice homozygous for a mutation of a single LAT tyrosine residue showed impeded T cell development. However, later they accumulated polyclonal helper T (TH) cells that chronically produced type 2 cytokines in large amounts. This exaggerated TH2 differentiation caused tissue eosinophilia and massive maturation of plasma cells secreting to immunoglobulins of the E and G1 isotypes. This paradoxical phenotype establishes an unanticipated inhibitory function for LAT that is critical for the differentiation and homeostasis of TH cells.