Fig 1: Characterization of Spontaneous Obese Mice. (A, B) Some littermates displayed pronounced obesity and adiposity, which also manifested in increased mass of the liver, heart, kidney, and adipose tissue. (C, D) Obesity occurred between week 4 and 5 of age as a result of a marked increase in food intake. (E) Obesity was associated with hyperglycemia, hyperinsulinemia, increased leptin, but unaltered plasma NEFA concentrations. (F) In total, 36 nonsynonymous mutations were shared between two obese animals compared to one lean littermate, including 16 common mutations (occurring in >10 other species), 10 rare mutations (occurring in 2–5 other species), and 10 unique mutations (occurring in <2 other species). (G) The Cyfip2 gene appears to be a hotspot for mutations, including the S968F mutation and several others within the cytoplasmic fragile-X interacting, WAVE and Nap1 binding domains. Data expressed as mean ± SEM of results obtained from n = 3–10 for each genotype. *P < .05 versus lean (unpaired two-tailed Student’s t-test). Nap1, Nck-associated protein 1; NEFA, nonesterified fatty acids; WAVE, Wiskott–Aldrich syndrome protein–family verprolin-homologous protein.
Fig 2: Hypersensitivity of triggerhappyp400 Mutants Caused by cyfip2 Mutations and Rescued by Conditional Cyfip2-GFP Expression(A) Acoustic startle circuit. Acoustic nerve (VIII), posterior lateral line nerve (PLL), feedforward (FF) inhibitory, and excitatory spiral fiber (SF) neurons connect to the Mauthner cells (red).(B) Cyfip2 protein interaction domains (Abekhoukh and Bardoni, 2014; Pittman et al., 2010). triggerhappyp400 (cyfip2p400) mutants have a premature stop codon after 342 of 1,253 amino acids. The previously identified nevermind (cyfip2tr230b) mutation (Pittman et al., 2010) is shown.(C) Startle sensitivity curves of siblings and trans-heterozygous (trans-het) larvae from cyfip2p400/+ X cyfip2tr230b/+ crosses (n = 75 siblings, 34 trans-hets; mean ± SEM).(D) Startle sensitivity index in cyfip2p400 sibling and mutant larvae expressing Tg(hsp70:cyfip2-GFP). Larvae were given no heat shock or one 40-min heat shock at 30 hpf. Cyfip2-GFP fluorescence was largely restricted to the CNS and was visible 90 min after heat shock, peaked around 3 hr after heat shock, and was detectable at low levels 24 hr later (Figure S3C). Without a heat shock, cyfip2p400 mutants had increased startle sensitivity (***p < 0.001, Mann-Whitney test), whereas heat shock reduced the sensitivity of cyfip2 mutants with the transgene compared with those without it (**p < 0.01, Mann-Whitney test).(E) Startle sensitivity curves for fmr1 sibling (n = 62) and mutant larvae (fmr1hu2787/hu2787, n = 20) at 5 dpf (mean ± SEM).(F) Hindbrain expression of Cyfip2 in 5 dpf wild-type (cyfip2+/+) and mutant (cyfip2p400/p400) larvae using a Cyfip2 antibody (Ab). Membranes of VIII neurons are marked by Tg(SCP1:Gal4FF(y256Et)); Tg(UAS:gap43-citrine) and anti-GFP Ab. Dashed lines indicate the otic vesicles (OVs). Scale bar, 10 μm.
Fig 3: Differential cell-type-expression of CYFIP1 and CYFIP2 in the adult mouse hippocampus. (A) Bar graph showing the three cell types with the highest Cyfip1 expression levels in nine different regions of the adult mouse brain. The values were obtained from the DropViz database (http://dropviz.org/). (B) Bar graph showing the three cell types with the highest Cyfip2 expression levels in nine different regions of the adult mouse brain. Blue bar, non-neurons; red bar, neurons. (C) Bar graphs showing Cyfip1 (upper panel) and Cyfip2 (lower panel) expression levels across 17 different hippocampal cell types. (D) Confocal images of fluorescent immunohistochemistry (IHC) using CYFIP2 antibody in the brain sections from embryonic day 16.5 wild-type and Cyfip2-null mice. DAPI is a nuclear counterstain. Scale bar, 200 μm. (E) Confocal images of fluorescent IHC using CYFIP1, CYFIP2, Iba1, and NeuN antibodies in the adult mouse hippocampus. The right panels are high magnification images of the regions in dotted-line boxes in the left panels. DG, dentate gyrus; SR, stratum radiatum; SLM, stratum lacunosum and moleculare. Scale bars, 400 μm (left panel) and 40 μm (right panel). (F) Confocal images of fluorescent IHC using CYFIP1, CYFIP2, GFAP, and MBP antibodies in the adult mouse hippocampus. Scale bars, 400 μm (left panel), 40 μm (middle panel), and 10 μm (right panel).
Fig 4: SF Axon Terminal Activity Is Increased in cyfip2 Mutants(A) Maximum intensity projection of Tg(−6.7FRhcrtR:gal4VP16); Tg(UAS:GCaMP5), showing labeled SF neurons (green). M-cells (M) and other reticulospinal neurons were labeled with rhodamine dextran (magenta). Arrowheads indicate SF cell bodies, and asterisks mark SF axon terminals in the M-cell axon cap. Scale bar, 10 μm.(B) Representative pseudocolored images of baseline (F0) and peak fluorescence in SF axon terminals following a strong acoustic stimulus (13 dB; the color scale denotes fluorescence intensity; black, lowest; white, highest). Scale bar, 10 μm.(C) Averaged traces of SF terminal Ca2+ responses following low (−14 dB), medium (−12 dB), and strong (13 dB) acoustic stimuli (n = 42 responses from 10 siblings, blue line; n = 36 responses from 9 mutants, red line; mean ± SEM; *p < 0.05, Mann-Whitney test).(D) Scatterplot of the area under the curve for individual SF axon terminal Ca2+ responses (mean ± SD; **p = 0.0049, ***p = 0.0003, Mann-Whitney test).
Fig 5: VIII Nerve Excitatory Inputs to the Mauthner Cell Are Normal in cyfip2 Mutants(A) Diagram of the stimulating electrode (stimulus) adjacent to the OV posterior macula, the club-ending mixed synapse between VIII afferents and the M-cell, and the recording electrode (voltage/current [V/I]) on the M-cell.(B) Representative traces of M-cell synaptic responses after stimulation of VIII afferents in cyfip2+/+ (left) and cyfip2p400/p400 (right) larvae at 5 dpf. The stimulation artifact has been truncated for clarity, and the electrical and chemical components are indicated.(C and D) Mean amplitude of M-cell electrical (C) and chemical synaptic responses(D) ± SD (n = 8 siblings, 8 mutants; p = 0.78, 0.29, Mann-Whitney test).(E) Paired-pulse ratios were unaltered in cyfip2p400/p400 larvae (p = 0.76, Mann-Whitney test).
Supplier Page from Abcam for Anti-CYFIP2 antibody