Journal:

Article Title: Intrinsic susceptibility of mouse trophoblasts to natural killer cell-mediated attack in vivo

doi: 10.1073/pnas.222652199

Figure Lengend Snippet: TSras2 tumor formation in Rag2−/−γc−/− mice but not Rag2−/− mice. (A) Allogeneic Rag2−/−γc−/− (12/12) and syngeneic FVB/N (7/8) mice developed tumors within 30–40 days; however, allogeneic Rag2−/− (1/10) and B6 (0/12) mice rarely developed tumors. Data represent the compilation of three independent experiments, each with two to four mice per genotype.

Article Snippet: C57BL/6 (B6) (H-2 b ), BALB/c (H-2 d ), FVB/N (H-2 q ), Rag2 −/− γ c −/− (on a mixed B6 × C57BL/10 background), and Rag2 −/− mice (B6 background) were obtained from Taconic Farms.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

doi: 10.1186/s12974-019-1530-4

Figure Lengend Snippet: Altered C3 complement protein expression in the Smarca5 cKO cerebellum of exercised and sedentary mice. Increases in mRNA transcripts coding for complement, complement-related proteins, and inflammation-related proteins in the Smarca5 cKO cerebellum, as indicated by RNAseq analysis ( a ). Fold changes are shown for the Smarca5 cKO groups (sedentary or exercised) relative to corresponding wild-type groups. qRT-PCR analysis confirmed the increases in C3, GFAP, USP18, and VGF ( b ), though the magnitudes of these increases varied from the RNAseq data set. Shown are the fold changes in the Smarca5 cKO cerebellum relative to wild-type littermates ( n = 3 in each of the four groups of wild-type exercised (run) or sedentary (sed), and mutant exercised or sedentary animals; differences relative to wild-type littermates are noted with ** p < 0.005 and *** p < 0.001). No increase was observed for the C3a receptor, C3aR. Protein analysis demonstrated a clear increase in GFAP expression in Smarca5 cKO cerebellum samples ( c ). C3 protein expression was also altered in the Smarca5 cKO cerebellum. The C3α chain was less prominent relative to the C3β chain in Smarca5 cKO cerebellum samples compared to wild-type samples. Blotting results are representative of similar results from four mice/group

Article Snippet: To examine the background strain differences of our mice, we sent tail DNA for SNP analysis against C57BL/6 N, FVB/N, and BALB/c reference strains (Taconic) from the following lines: Smarca5 f/f (C57BL/6) mice, Smarca5 f/f (C57BL/6 N; FVB/N mixed), and the resulting lines used to generate the mice for our experiments, namely Smarca5 +/− ;nestin-cre +/− ;C3aR +/− , and Smarca5 fl/fl ;C3aR1 −/− mice.

Techniques: Expressing, Quantitative RT-PCR, Mutagenesis

Journal: Journal of Neuroinflammation

Article Title: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

doi: 10.1186/s12974-019-1530-4

Figure Lengend Snippet: Altered C3 and VGF processing in the P10 Smarca5 cKO cerebellum. An N-terminal C3α chain sequence (shaded gray) found within the C3a peptide bears structural similarity to a C-terminal VGF sequence (shaded gray) within the TLQP-62 peptide ( a ). Alignment of the human (Homo), chimpanzee (Pan), and mouse (Mus) sequences are shown. C3 and VGF immunoblotting showed additional bands (small closed arrows), presumably corresponding to cleavage products, in Smarca5 cKO and Smarca5 cKO, C3aR KO double mutant (dKO) cerebellum samples ( b ). Open arrows in b indicated full-length C3α and VGF. VGF mRNA was upregulated ~ 2-fold in the P10 Smarca5 cKO and dKO cerebellum ( c ) (* p < 0.05, ** p < 0.005). C3 mRNA expression, however, was unchanged in the mutant cerebellum samples. d The common receptor for VGF and C3, C3aR, was detected only in Iba1 + macrophages and microglia (Additional file ). The specificity of the C3aR labeling was demonstrated by the lack of immunolabeling in the dKO cerebellum. Top images in each pair show the merge of Iba1 and C3aR labeling, and bottom images show C3aR labeling alone. Scale bar =50 μm and applies to all images

Article Snippet: To examine the background strain differences of our mice, we sent tail DNA for SNP analysis against C57BL/6 N, FVB/N, and BALB/c reference strains (Taconic) from the following lines: Smarca5 f/f (C57BL/6) mice, Smarca5 f/f (C57BL/6 N; FVB/N mixed), and the resulting lines used to generate the mice for our experiments, namely Smarca5 +/− ;nestin-cre +/− ;C3aR +/− , and Smarca5 fl/fl ;C3aR1 −/− mice.

Techniques: Sequencing, Western Blot, Mutagenesis, Expressing, Labeling, Immunolabeling

Journal: Journal of Neuroinflammation

Article Title: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

doi: 10.1186/s12974-019-1530-4

Figure Lengend Snippet: Morphologies of the P10 Smarca5 cKO and dKO cerebellum. P10 wild-type, C3aR KO, Smarca5 cKO, and dKO cerebellums were labeled to show Purkinje cells (calbindin) and astroglia (GFAP) in sections through the vermis. All sections are oriented with the anterior end towards the top. Large open arrows point to the Purkinje cell layer; small closed arrows point to the external granule cell layer (EGL), and small open arrows point to choroid plexus (ChP). Also labeled is the region of the deep cerebellar nuclei (DCN). Both the Smarca5 cKO and dKO cerebellums displayed more intense GFAP labelling; this labelling in the dKO was more prominent around the periphery. The dKO also displayed a more pronounced loss of EGL in comparison to the Smarca5 cKO single mutant. The C3aR single mutant did not have any apparent abnormalities in comparison to the wild-type sections. The scale bar in the middle panel = 500 μm, and applies to all images

Article Snippet: To examine the background strain differences of our mice, we sent tail DNA for SNP analysis against C57BL/6 N, FVB/N, and BALB/c reference strains (Taconic) from the following lines: Smarca5 f/f (C57BL/6) mice, Smarca5 f/f (C57BL/6 N; FVB/N mixed), and the resulting lines used to generate the mice for our experiments, namely Smarca5 +/− ;nestin-cre +/− ;C3aR +/− , and Smarca5 fl/fl ;C3aR1 −/− mice.

Techniques: Labeling, Mutagenesis

Journal: Journal of Neuroinflammation

Article Title: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

doi: 10.1186/s12974-019-1530-4

Figure Lengend Snippet: Loss of C3aR in the Smarca5 cKO cerebellum results in increased Bergmann glia disorganization. At P1, BLBP labeling in mutant cerebellums showed the cell bodies of the Bergmann glia to be mislocalized ( a ), being frequently positioned immediately adjacent to the EGL (arrows; the EGL is indicated with a dashed line). BLBP labeling was further altered in the dKO samples in that it was weak throughout the cerebellum, except within the EGL, where it labeled strongly. Iba1 + cells were also labeled to look for any correspondence in Bergmann glia disorganization and phagocyte localization, which was not observed. In P10 samples, the orderly array of Bergmann glia cell bodies within the Purkinje cell layer observed in control samples was mostly absent in both Smarca5 cKO and dKO samples. Both of these mutants also displayed increased GFAP labeling and aberrant arborization, which was worsened in the dKO sections. Within fissures of the cerebellum ( b ), the nearly absent EGL and strongly GFAP + and disorganized processes of the Bergmann glia in the P10 dKO contrasted with a more normal-appearing Smarca5 cKO single mutant. Calbindin labeling identified the Purkinje cells. c Transcript expression for GFAP was increased in both of the Smarca5 cKO and dKO cerebellums, being higher in the dKO mutants ( n = 4 mice/genotype; ** p < 0.005, *** p < 0.0001). Transcripts for the inflammatory cytokines IL6 and TNF were not increased (IL6, n = 4–5 mice/genotype; TNF, n = 5 mice/genotype). d Total GFAP protein was increased in both the Smarca5 cKO and dKO cerebellums relative to cerebellums from controls and C3aR KO mice. e Quantification of the GFAP/BLBP ratio from the immunoblots shown in D (** p < 0.01; *** p < 0.001). Images in a and b were reconstructions of optical sections. Scale bar in a = 40 μm, and applies to all panels; scale bar in b = 40 μm, and applies to both panels

Article Snippet: To examine the background strain differences of our mice, we sent tail DNA for SNP analysis against C57BL/6 N, FVB/N, and BALB/c reference strains (Taconic) from the following lines: Smarca5 f/f (C57BL/6) mice, Smarca5 f/f (C57BL/6 N; FVB/N mixed), and the resulting lines used to generate the mice for our experiments, namely Smarca5 +/− ;nestin-cre +/− ;C3aR +/− , and Smarca5 fl/fl ;C3aR1 −/− mice.

Techniques: Labeling, Mutagenesis, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

doi: 10.1186/s12974-019-1530-4

Figure Lengend Snippet: Loss of C3aR in the Smarca5 cKO cerebellum results in increased apoptosis in the EGL and an early invasion of phagocyte cells. P1 ( a ) and P10 ( b ) cerebellums from control and mutant mice showing granule neuron precursors (Pax6 + ) in the EGL and NeuN + granule neurons. At P10, the loss of Pax6 + cells from the EGL was observed in both the Smarca5 cKO and dKO cerebellum, and NeuN + cells in the IGL were sparse in these mutants in comparison to control and C3aR KO cerebellums. Within the EGL of Smarca5 cKO and dKO mutant mice, cleaved caspase 3 + (cCasp3) apoptotic cells and TUNEL + cells (arrows) were found in every section examined ( c ). These cCasp3 + cells in the EGL were occasionally observed within Iba1 + phagocyte cells (left-hand panel). d Iba1 + phagocytes were also observed in the EGL of Smarca5 cKO and dKO mutants at P10, though cCasp3 + cells were infrequent at this age. Most cCasp3 + cells observed at P10 were in the IGL (arrow, right-hand panel). The number of cCasp3 + and TUNEL + cells ( e ) was highest in the dKO mice ( n = 5 mice/genotype; error bars indicate standard error). However, only P1 dKO cerebellums showed a consistent, statistically significant increase in TUNEL labeling and cCasp3 + cells compared to Smarca5 cKO single mutants. At P10, TUNEL labeling was more variable; only the dKO cerebellums showed a statistically significant increase in TUNEL + cells relative to the non-mutant control group at this age. Both the Smarca5 cKO and dKO cerebellums had high numbers of phagocytes in the EGL at P10 ( f ), whereas the Smarca5 cKO mutants had less than half as many as the dKO at P1 ( n = 4 mice/genotype at each age; error bars indicate the standard error). * p < 0.05, ** p < 0.005, *** p < 0.001 in e , f . Scale bars = 100 μm in all image panels

Article Snippet: To examine the background strain differences of our mice, we sent tail DNA for SNP analysis against C57BL/6 N, FVB/N, and BALB/c reference strains (Taconic) from the following lines: Smarca5 f/f (C57BL/6) mice, Smarca5 f/f (C57BL/6 N; FVB/N mixed), and the resulting lines used to generate the mice for our experiments, namely Smarca5 +/− ;nestin-cre +/− ;C3aR +/− , and Smarca5 fl/fl ;C3aR1 −/− mice.

Techniques: Mutagenesis, TUNEL Assay, Labeling

Journal: Journal of Neuroinflammation

Article Title: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum

doi: 10.1186/s12974-019-1530-4

Figure Lengend Snippet: Increased MerTK expression in the Smarca5 cKO cerebellum is attenuated in the absence of C3aR. a qRT-PCR analysis indicated that MerTK was increased over 2-fold in the Smarca5 cKO cerebellum at P10 ( n = 5 mice/genotype), but not at P1 ( n = 3 mice/genotype). This increase was almost completely attenuated with the loss of C3aR in the dKO mice (* p < 0.05). Transcripts for two other proteins involved in efferocytosis, SR-B1 and MFG-E8 ( n = 5 mice/genotype), were not increased in any of the mice. b MerTK expression was observed almost exclusively on Iba1 + macrophages (arrowheads) and microglia (open arrows) in the mutant cerebellums. Most other labeling with the MerTK antibody was non-specific blood vessel (BV) labeling. Scale bar = 50 μm, and applies to all panels

Article Snippet: To examine the background strain differences of our mice, we sent tail DNA for SNP analysis against C57BL/6 N, FVB/N, and BALB/c reference strains (Taconic) from the following lines: Smarca5 f/f (C57BL/6) mice, Smarca5 f/f (C57BL/6 N; FVB/N mixed), and the resulting lines used to generate the mice for our experiments, namely Smarca5 +/− ;nestin-cre +/− ;C3aR +/− , and Smarca5 fl/fl ;C3aR1 −/− mice.

Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Labeling