Average duration of calls (±Std Dev.) for different categories of calls.

<p>There was no effect of genotype on duration of calls for any of those call subtypes.</p

Body weight comparison between WT and in <i>Fmr1</i>-KO mice: Body weights of pups were measured on post natal day eight.

<p>Black circles: WT littermates; Gray circles: <i>Fmr1</i>-KO mice. Error bar represents standard error of the mean (SE). The body weights of mouse pups were not significantly different between genotypes.</p

Carrier frequency and frequency modulation of vocalizations during the three minutes of isolation period from mother and littermates:

<p>A) Box plot showing the mean carrier frequency of flat calls (Diamonds) together with the 25^th to 75^th percentile (box height) and median (horizontal line). Triangles at the top and bottom indicate 99 and 1 percentile, respectively. Error bars represent standard error of mean (SE). Mean carrier frequency was calculated from first ten flat calls during isolation. Black outlines: WT littermate control mice; gray outlines: <i>Fmr1</i>-KO. B) Box plot as in A) but showing the mean range of frequency modulation of complex calls. Black outlines: WT littermate control mice; gray outlines: <i>Fmr1</i>-KO. Dashes (-) mark minimum and maximum values. The average carrier frequency of flat calls emitted by KO mice was higher (p<0.001) than in WT animals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044816#pone-0044816-g002" target="_blank">Figure 2A</a>). Call frequency modulation was significantly increased in <i>Fmr1</i>-KO mice (p = 0.004) compared to WT pups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044816#pone-0044816-g002" target="_blank">Figure 2B</a>).</p

Comparison of different categories of calls produced by WT and <i>Fmr1</i>-KO mice:

<p>A) Total number and B) The percentages of the different call categories within the genotype. Percentages were calculated in each genotype as: (number of calls in each category/total number of calls analyzed in each subject) * 100. The total number of total calls analyzed across all mice was: WT = 1399; KO = 1543 collected from 10 pups per genotype. The total number of call emissions and the number of calls within a category (including downward calls) were not significantly different between genotypes (A). However, analysis of the percentage of different call categorys revealed that the percentage of downward calls produced by the <i>Fmr1</i>-KO pups were significantly lower (p<0.001) compared to their WT littermates (B).</p

Duration of separation induced ultrasonic vocalizations.

<p>The average duration measurements were obtained from 10 mice of each genotype, averaging 10 calls of each type per mouse. Black bar: WT littermates; gray bar: <i>Fmr1</i>-KO mice. Error bars represent standard error of mean (SE). There was no significant difference in the duration of any call type between the genotypes.</p

Dab2IP GTPase Activating Protein Regulates Dendrite Development and Synapse Number in Cerebellum

<div><p>DOC-2/DAB-2 interacting protein (Dab2IP) is a GTPase activating protein that binds to Disabled-1, a cytosolic adapter protein involved in Reelin signaling and brain development. Dab2IP regulates PI3K-AKT signaling and is associated with metastatic prostate cancer, abdominal aortic aneurysms and coronary heart disease. To date, the physiological function of Dab2IP in the nervous system, where it is highly expressed, is relatively unknown. In this study, we generated a mouse model with a targeted disruption of <em>Dab2IP</em> using a retrovirus gene trap strategy. Unlike <em>reeler</em> mice, Dab2IP knock-down mice did not exhibit severe ataxia or cerebellar hypoplasia. However, Dab2IP deficiency produced a number of cerebellar abnormalities such as a delay in the development of Purkinje cell (PC) dendrites, a decrease in the parallel fiber synaptic marker VGluT1, and an increase in the climbing fiber synaptic marker VGluT2. These findings demonstrate for the first time that Dab2IP plays an important role in dendrite development and regulates the number of synapses in the cerebellum.</p> </div

Changes in parallel fiber and climbing fiber synaptic markers in Dab2IP KD mice.

<p>(A–D) Confocal images of VGluT1-labeled parallel fibers terminals (red) on PCs stained for Calbindin (green) in P30 control mice (A) or (C) Dab2IP KD littermates. B and D correspond to boxed areas in A and C. Single plane confocal images were used to determine the number of VGluT1 positive varicosities per 100 µm². (E) Quantitation of VGluT1 positive puncta in WT (N = 3, 360 observations) and Dab2IP KD (N = 3, 360 observations) littermates. **, p<0.01, student’s t-test. Scale bars: 50 µm (A, C), 5 µm (B, D). (F–K) Confocal images of VGluT2-labeled climbing fibers terminals (red) on PCs stained for Calbindin (green) in P30 WT (F–H) and Dab2IP KD littermates (I–K). Scale bars: 20 µm. (G, J) Quantitation of VGluT2 positive puncta in WT (N = 3, 171 observations) and Dab2IP KD (N = 3, 195 observations) along the entire length of the molecular layer (L) or in five equal segments from PC soma to the most distal part of the molecular layer (M). **, p<0.01, *, P<0.05, student’s t-test.</p

Cellular distribution of Dab2IP in P30 cerebellar Purkinje cells and granule cell layer.

<p>(A1–A3) Double immunofluorescent labeling of Dab2IP (green) and Calbindin (red) in Purkinje cell layer. (B1–B3) Double immunofluorescent labeling of Dab2IP (green) and GluR delta2 (red) in the molecular layer of the cerebellum. (C1–C3) Double immunofluorescent labeling of Dab2IP (green) and VGluT1 (red) in the granular layer of the cerebellum. (D1–D3) Double immunofluorescent labeling of Dab2IP (green) and VGluT2 (red) in the granular layer of the cerebellum. Scale bars: 5 µm.</p

Dab2IP KD strategy and validation.

<p>(A) Structure of the gene trap cassette and its insertion site in <i>Dab2IP</i>. (B) Domain structure of two Dab2IP isoforms found in mouse. The location of PCR primers specifically targeting the PH or GRD domains are indicated in green. The location of the epitope for the polyclonal anti-Dab2IP antisera used in this study is indicated in blue. (C) PCR genotyping assay for detection of the gene trap cassette (−/−) or WT locus (+/+). The location of the primers is indicated in A. (D) Immunoblot analysis of brain lysates from P30 WT (+/+) and Dab2IP KD (−/−) mice. (E) Quantitative RT-PCR using probes to either the GRD domain (common to all isoforms) or PH-domain. Values are presented as percent change in Dab2IP KD compared to WT controls. (F) Immunodetection of Dab2IP in sagittal cerebellar sections of P30 WT (F1–F3) and Dab2IP KD (F4–F6) littermates. ML, molecular layer; PCL, Purkinje cell layer; GL, granule layer. Scale bars, 50 µm (F1, F4); 20 µm (F2, F3, F5, F6).</p

Dab2IP expression in brain.

<p>(A) Immunohistochemical staining of sagittal brain section of P30 mouse using rabbit polyclonal antiserum specific to Dab2IP. Dab2IP is highly expressed throughout the brain. The distance of the sections from the midline of the cerebellum is ∼0.4 mm. (B) In the cerebellum, Dab2IP is expressed in granule cell layer, Purkinje cells bodies and dendrites and molecular layer. (C) Higher magnification of boxed area in B. GL, granule cell layer. ML, molecular layer. Scale bars: 250 µm (A), 100 µm (B), 25 µm (C).</p