Descriptive characteristics of the study samples.

<p>Descriptive characteristics of the study samples.</p

The strongest differentially abundant microbial taxa for ADHD cases (n = 19) versus healthy controls (n = 77), shown in the graphical Cytoscape visualization [32].

<p>Nodes represent taxa (node size represents average relative abundance, for both experimental groups combined), edges (dashed lines) link the different taxonomic levels. The weighed fold-change (node color) is calculated as the 2log of the ratio of the relative abundance between control and ADHD (0 = no difference between genotypes, 1 = twice as abundant in control, etcetera). In other words: yellow to red indicates an overrepresentation in control, hence an underrepresentation in ADHD, and vice versa for light- to dark blue. The significance (node border width) is expressed as the p-value of a Mann–Whitney <i>U</i> test, uncorrected for multiple comparisons.</p

The ADHD microbiome contains significantly increased levels of predicted cyclohexadienyl dehydratase (CDT; KEGG Ortholog K01713; EC:4.2.1.51), responsible for phenylalanine synthesis (Fig B in S1 Appendix).

<p>This analysis is based on functional predictions deriving from 16S profiles of the microbiome, as performed by PICRUSt [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183509#pone.0183509.ref033" target="_blank">33</a>]. Box plots represent the relative abundance of predicted CDT, with 5–95% percentile whiskers (dots represent outliers). The significance was tested with a non-parametric MWU (* p = 0.038), Bonferroni-corrected for 15 K numbers identified.</p

fMRI results.

<p><b>A</b>. Main effect of reward anticipation, cluster-level corrected at the whole-brain level (pFWE < 0.05). Color bars reflect T-values. <b>B</b>. Diagnosis effects in the anatomical region of interest (ROI) of the ventral striatum. <b>C</b>. Negative correlation of the microbiome function CDT (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183509#pone.0183509.g004" target="_blank">Fig 4</a></b>) with reward anticipation responses across the whole-brain (n = 28), intensity threshold at p < 0.001 uncorrected (T = 3.45). The clusters in bilateral ventral striatum (x = -11, y = 11, z = -9, cluster size = 8, p(FWE, cluster) = 0.024; x = 11, y = 6, z = -11, cluster size = 2, p(FWE, cluster) = 0.036) are significant after correcting for multiple comparisons across the search volume (cluster-level pFWE < 0.05, SVC), i.e. the anatomically defined ventral striatum shown in panel B. SVC = small volume correction. * indicates p < 0.05.</p

Microbiome sample, fMRI sample, and their overlap.

<p>Microbiome sample, fMRI sample, and their overlap.</p

Replication of the most significantly associated SNPs in genetically divergent populations.

<p>NA: Not available.</p

Association of rs3916441 with gray matter and white matter in genetically divergent populations.

<p>NA: Not available.</p

Spatiotemporal expression profiling of IL3 and its receptor in the developing mouse brain.

<p>(<b>a</b>) RT-PCR revealed the expression of IL3 and its receptor in developing mouse brain from E12.5 to adult. (<b>b</b>) Quantitative PCR showed that the expression of IL3 and its receptors peaks at P1–P4, a period with active neural proliferation and neurogenesis. Data are expressed as mean ± s.e.m. (n = 3). (<b>c–n</b>) Immunohistochemistry analysis indicated that IL3 and its receptor were expressed in the mouse brain. Co-expression of IL3 and IL3RA were detected (arrows in <b>l–n</b>), indicating the activation of IL3-mediated signaling pathways in the developing mouse brain. (<b>o–p</b>) IL3RA is expressed in radial glia (resides in ventricular zone and characterized by long radial processes, arrowhead in <b>o</b>) and migratory neurons (arrowheads in <b>p</b>). Ctx, cortex; VZ, ventricular zone. Scale bars, (c, d, e) 25 µm; O, 10 µm.</p

The Interleukin 3 Gene (IL3) Contributes to Human Brain Volume Variation by Regulating Proliferation and Survival of Neural Progenitors

<div><p>One of the most significant evolutionary changes underlying the highly developed cognitive abilities of humans is the greatly enlarged brain volume. In addition to being far greater than in most other species, the volume of the human brain exhibits extensive variation and distinct sexual dimorphism in the general population. However, little is known about the genetic mechanisms underlying normal variation as well as the observed sex difference in human brain volume. Here we show that interleukin-3 (IL3) is strongly associated with brain volume variation in four genetically divergent populations. We identified a sequence polymorphism (rs31480) in the IL3 promoter which alters the expression of IL3 by affecting the binding affinity of transcription factor SP1. Further analysis indicated that IL3 and its receptors are continuously expressed in the developing mouse brain, reaching highest levels at postnatal day 1–4. Furthermore, we found IL3 receptor alpha (IL3RA) was mainly expressed in neural progenitors and neurons, and IL3 could promote proliferation and survival of the neural progenitors. The expression level of IL3 thus played pivotal roles in the expansion and maintenance of the neural progenitor pool and the number of surviving neurons. Moreover, we found that IL3 activated both estrogen receptors, but estrogen didn’t directly regulate the expression of IL3. Our results demonstrate that genetic variation in the IL3 promoter regulates human brain volume and reveals novel roles of IL3 in regulating brain development.</p> </div