Sexual-dimorphism in human immune system aging.

Differences in immune function and responses contribute to health- and life-span disparities between sexes. However, the role of sex in immune system aging is not well understood. Here, we characterize peripheral blood mononuclear cells from 172 healthy adults 22-93 years of age using ATAC-seq, RNA-seq, and flow cytometry. These data reveal a shared epigenomic signature of aging including declining naïve T cell and increasing monocyte and cytotoxic cell functions. These changes are greater in magnitude in men and accompanied by a male-specific decline in B-cell specific loci. Age-related epigenomic changes first spike around late-thirties with similar timing and magnitude between sexes, whereas the second spike is earlier and stronger in men. Unexpectedly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity. Impact of age and sex on immune phenotypes can be visualized at https://immune-aging.jax.org to provide insights into future studies

Functional Dissection of the Neural Substrates for Sexual Behaviors in Drosophila melanogaster

The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ∼1000 GAL4 lines, using assays for general courtship, male–male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male–male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation

A Microbe Associated with Sleep Revealed by a Novel Systems Genetic Analysis of the Microbiome in Collaborative Cross Mice.

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice (Mus musculus) enables data-driven discovery of biological network components and mechanisms of host-microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of Odoribacter (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg-Dock7m +/+ Leprdb/J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by Odoribacter, treatment with antibiotics altered sleep in a genotype-dependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms

<em>doublesex</em> Functions Early and Late in Gustatory Sense Organ Development

<div><p>Somatic sexual dimorphisms outside of the nervous system in <em>Drosophila melanogaster</em> are largely controlled by the male- and female-specific Doublesex transcription factors (DSX^M and DSX^F, respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with <em>doublesex</em> (<em>dsx</em>) gene function, we focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. We show that DSX^M and DSX^F promote and repress GSO formation, respectively, and that their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. Our results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSX^F then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX.</p> </div

<i>dsx</i> regulates the number of foreleg GSOs.

<p>(A) The sex determination hierarchy directs the generation of sex-specific DSX and FRU isoforms. The 2∶2 ratio of <i>X</i> chromosomes to autosomes in females sets off a female-specific alternative RNA splicing cascade in which TRA directs splicing of <i>dsx</i> and <i>fru</i> transcripts into the female forms. The lack of TRA activity in males results in the production of male forms of these transcripts. (B–D) <i>poxn-GAL4</i> driving expression of <i>UAS-mCD8::GFP</i> in a (B) male and (C) female foreleg at 48 h APF. Tarsal segments T1–T5 are indicated. Note that there are more clusters of neurons labeled in the male than in the female in T1–T4. (D) Magnified view of two distinct GSOs. The GRNs (arrows) of each GSO project their dendrites into the base of their cognate bristle (arrowheads). (E) Quantitation of foreleg GSOs in T1–T4. <i>3XP3DsRed</i> was used to distinguish <i>XY</i> flies from <i>XX</i> flies in a <i>dsx</i>-deficient background where chromosomal sex could not otherwise be distinguished. All <i>XY</i> males had a sex chromosome genotype of <i>w</i>/<i>Y</i>. The genotype of the sex chromosomes of <i>dsx-</i>deficient chromosomal females was <i>w/w, 3XP3DsRed,</i> while all other females were <i>w</i>/<i>y w, 3XP3DsRed.</i> Genotype abbreviations: <i>dsx</i>⁺ (<i>UAS-mCD8::GFP</i>; <i>FRT82B dsx¹, poxn-GAL4/TM6B</i>). <i>dsx</i>⁻ (<i>UAS-mCD8::GFP</i>; <i>FRT82B dsx¹, poxn-GAL4/dsx^M+R13</i>). <i>dsx^D</i> (<i>UAS-mCD8::GFP</i>; <i>FRT82B dsx¹, poxn-GAL4/dsx^D</i>). <i>dsx</i>⁺ and <i>dsx^D</i> are siblings from the same cross. Error bars indicate SEM. P-values are for comparisons between the indicated <i>dsx</i> mutant and <i>dsx</i>⁺ of the same chromosomal sex. (*p = .07, **p<.0001, † p = .04, ‡ p = .14, Tukey multiple comparisons of means.).</p

DSX^M is present in the foreleg disc epithelium when AC accumulates in proneural clusters.

<p>(A–E) Male foreleg discs from the indicated time points of third instar larval development were stained for AC (green) and DSX^M (magenta). Merged images on right show overlap in white. (A and B) From 36–40 h 3I, DSX^M is present in a crescent within T1 and there is no overlap with AC. (C) At 44 h 3I, DSX^M signal increases across the epithelium of tarsal segments distal to T1 (i.e. toward disc center) and is present in some clusters of AC-positive cells (arrows). (D) At 48 h 3I, DSX^M is present in swaths of epithelial cells in T1–T4 and overlaps in these segments with subsets of the AC-positive cells that are proneural clusters (arrows). A candidate SOP with high levels of AC and DSX^M (barbed arrow) is seen in T2. (E) Magnified view of boxed region in (D). Candidate SOP in T2 (barbed arrow). (F) Same image as (E) with AC (green), DSX^M (red), and stained with DAPI (blue) to visualize all nuclei in the focal planes shown. All images are projections of only those focal planes that encompass the majority of DSX^M signal within the disc. Scale bars (A–D) 50 µm and (E and F) 10 µm.</p

DSX is present in SOP daughters of the foreleg disc.

<p>(A and B) DSX (magenta) is present across the tarsal segment epithelium in male discs at 0 h APF as well as in subsets of cells expressing <i>ase-lacZ</i> (green) in T5 (arrows) and T4 (boxed area). (B) Magnified view of boxed region in (A) shown as a partial projection. Daughters of a recently divided SOP (arrows). (C) DSX (magenta) is present in the tarsal segment epithelium in male discs at 6 h APF. DSX overlaps with <i>neur-lacZ</i> expression (green) in several cells across T1–T5 (arrowheads) and in a transverse row of cells in T1 that likely correspond to the sex comb bristle lineages (small arrows). (D) T2–T3 from a separate male leg disc at 6 h APF marked as per (C) with DSX (red) in right panel and DAPI-stained DNA (blue). For A–D, images on right are a merge of the left and middle images. Projection of multiple focal planes shown. Projection of multiple focal planes shown. Scale bars (A, C, and D) 25 µm and (B) 10 µm.</p

<i>dsx</i> regulates axonal morphology independent of GSO number.

<p>(A–F”) <i>fru^GAL4</i> driving <i>UAS-mCD8::GFP</i> (green) labels GRN cell bodies and axons. Forelegs of (A) control male (<i>UAS-mCD8::GFP/SM6; fru^GAL4/+</i> ), (B) male with feminized GRNs (<i>UAS-DSX^F/UAS-mCD8::GFP; fru^GAL4/+</i>), and (C) control female (<i>UAS-mCD8::GFP/SM6; fru^GAL4/+</i>). Cuticular autofluorescence (magenta). There is no difference in the number of FRU^M-positive GRN clusters between forelegs of the two male genotypes, while both have more than the female. (D–F”) VNC prothoracic neuromeres with labeled GRN projections (D, E, F; green in D”, E”, F”) and counterstained for DN-cadherin (D’, E’, F’; magenta in D”, E”, F”). Arrowheads indicate the VNC midline. (D–D”) GRNs cross the midline in control males, but feminized male GRNs do not cross (E–E”). (F–F”) GRNs also do not cross the midline in control females.</p

The sexually dimorphic number of foreleg GSOs is specified by 8 h APF.

<p>Male (A) and female (B) forelegs with <i>poxn-GAL4</i> driving <i>UAS-mCD8::GFP</i> (green) were stained for 22C10 (magenta) at 8 h APF. Merged images on right show overlap in yellow and DAPI-stained DNA (blue). Tarsal segment boundaries indicated with blue lines. Cells marked with 22C10 were classified based both on colocalization of <i>poxn-GAL4</i> and morphology of the cells or cell clusters: GSO lineage cells (magenta arrows); non-GSO cells that lack <i>poxn-GAL4</i> in T3 (dark blue arrows); non-GSO cells marked by <i>poxn-GAL4</i> but lacking GSO morphology in T4 (light blue arrows). Scale bars, 50 µm. (C) Averages and SEMs of quantitated GSO numbers in T2, T3, and T4 for both male (n = 8) and female (n = 9) forelegs at 8 h APF.</p

Other examples of 12A variants.

<p>(<b>A</b>) An example of the ectopic branch phenotype in T1 (compare with the typical T1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g002" target="_blank">Fig 2B</a> and the ectopic branch phenotype of T2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">Fig 3A</a>). All examples were bilaterally symmetrical with a fully formed commissure (arrowhead), in contrast to 12A in T2, which often exhibits bilateral asymmetry. (<b>B</b>) Example of ventral arch routing of the late-born 12A neurons in T2. Compare the branch on the left (arrowhead) with the unoccupied Neuroglian-stained tract on the right (arrow). (<b>C</b>) Transverse optical section of S3 in which 12A shows the T2 morphology (with unsplit bundles). Compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g006" target="_blank">Fig 6B</a> and the variants of 12A that are missing the intermediate bundle as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g006" target="_blank">6A</a>. (<b>D</b>) Transverse optical section of T1 in which the left bundle fails to split. (<b>E</b>) Two examples of animals with duplicated hemilineages. (<b>F</b>) One of two cases in which one hemilineage ectopically projected all of its neurites to the contralateral side, leaving one T1 hemisegment completely uninnervated by 12A and the other double innervated.</p