Mushroom body-specific profiling of gene expression identifies regulators of long-term memory in Drosophila

Memory formation is achieved by genetically tightly controlled molecular pathways that result in a change of synaptic strength and synapse organization. While for short- term memory traces rapidly acting biochemical pathways are in place, the formation of long-lasting memories requires changes in the transcriptional program of a cell. Although many genes involved in learning and memory formation have been identified, little is known about the genetic mechanisms required for changing the transcriptional program during different phases of long-term memory formation. With Drosophila melanogaster as a model system we profiled transcriptomic changes in the mushroom body, a memory center in the fly brain, at distinct time intervals during long- term memory formation using the targeted DamID technique. We describe the gene expression profiles during these phases and tested 33 selected candidate genes for deficits in long-term memory formation using RNAi knockdown. We identified 10 genes that enhance or decrease memory when knocked-down in the mushroom body. For vajk-1 and hacd1, the two strongest hits, we gained further support for their crucial role in learning and forgetting. These findings show that profiling gene expression changes in specific cell-types harboring memory traces provides a powerful entry point to identify new genes involved in learning and memory. The presented transcriptomic data may further be used as resource to study genes acting at different memory phases

Regulators of Long-Term Memory Revealed by Mushroom Body-Specific Gene Expression Profiling in Drosophila melanogaster

Memory formation is achieved by genetically tightly controlled molecular pathways that result in a change of synaptic strength and synapse organization. While for short- term memory traces, rapidly acting biochemical pathways are in place, the formation of long-lasting memories requires changes in the transcriptional program of a cell. Although many genes involved in learning and memory formation have been identified, little is known about the genetic mechanisms required for changing the transcriptional program during different phases of long-term memory (LTM) formation. With Drosophila melanogaster as a model system, we profiled transcriptomic changes in the mushroom body—a memory center in the fly brain—at distinct time intervals during appetitive olfactory LTM formation using the targeted DamID technique. We describe the gene expression profiles during these phases and tested 33 selected candidate genes for deficits in LTM formation using RNAi knockdown. We identified 10 genes that enhance or decrease memory when knocked-down in the mushroom body. For vajk-1 and hacd1—the two strongest hits—we gained further support for their crucial role in appetitive learning and forgetting. These findings show that profiling gene expression changes in specific cell-types harboring memory traces provides a powerful entry point to identify new genes involved in learning and memory. The presented transcriptomic data may further be used as resource to study genes acting at different memory phases

Xenacoelomorpha Survey Reveals That All 11 Animal Homeobox Gene Classes Were Present in the First Bilaterians

Homeodomain transcription factors are involved in many developmental processes across animals and have been linked to body plan evolution. Detailed classifications of these proteins identified 11 distinct classes of homeodomain proteins in animal genomes, each harboring specific sequence composition and protein domains. Although humans contain the full set of classes, Drosophila melanogaster and Caenorhabditis elegans each lack one specific class. Furthermore, representative previous analyses in sponges, ctenophores, and cnidarians could not identify several classes in those nonbilaterian metazoan taxa. Consequently, it is currently unknown when certain homeodomain protein classes first evolved during animal evolution. Here, we investigate representatives of the sister group to all remaining bilaterians, the Xenacoelomorpha. We analyzed three acoel, one nemertodermatid, and one Xenoturbella transcriptomes and identified their expressed homeodomain protein content. We report the identification of representatives of all 11 classes of animal homeodomain transcription factors in Xenacoelomorpha and we describe and classify their homeobox genes relative to the established animal homeodomain protein families. Our findings suggest that the genome of the last common ancestor of bilateria contained the full set of these gene classes, supporting the subsequent diversification of bilaterians

Germ Cell-Specific Targeting of DICER or DGCR8 Reveals a Novel Role for Endo-siRNAs in the Progression of Mammalian Spermatogenesis and Male Fertility

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

Supplemental Material for Widmer et al., 2018

Supplementary figures and tables for Widmer et al. 2018<br> <p>Figure S1. Pattern analysis.</p> <p>Figure S2. Gene expression difference between paired and unpaired of the identified hits. </p> <p>Table S1. Differentially regulated genes. </p><p>Table S2. Patterns of differentially expressed genes after training.</p> <p>Table S3. Results 48 h memory RNAi screen.</p> <p>Table S4. Predicted <i>mir-282</i> target genes. </p

Gene expression in chronic granulomatous disease and interferon-gamma receptor-deficient cells treated in vitro with interferon-gamma

Interferon-gamma (IFN-gamma) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN-gamma/interleukin (IL)-12 axis. Using transcriptome profiling (RNA-seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein-Barr virus-transformed B lymphocytes (B-EBV) cells from CGD patients, IFN-gamma receptor deficiency patients, and normal controls, treated in vitro with IFN-gamma for 48 hours. Our results show that IFN-gamma increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN-gamma-induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome-mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN-gamma also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre-mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl-transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN-gamma in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN-gamma treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN-gamma treatment. These data support the concept that IFN-gamma treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing

Reduction in testis size and near complete absence of mature spermatozoa in GC-Dcr1 and GC-Dgcr8 mutant mice.

<p>(A–C, M) At P60, testis weight showed a 55% and 50% reduction in GC-Dcr1 (n = 16) and GC-Dgcr8 mutants (n = 5) compared to control testes (n = 17). H&E staining of testis sections (D–F) revealed several defects in the architecture of the seminiferous epithelium, near complete absence of mature spermatozoa, and reduced tubular diameter (N) (Control: 170.9 µm²±1.7, GC-Dcr1 mut: 122.5 µm²±2.2, GC-Dgcr8 mut: 142.6 µm²±1.8). (G–I) Anti-H3K9me3 stained sex-body in round spermatids (RS). We observed a 60% reduction in the number of RS per tubule in GC-Dcr1 mutants and 55% reduction in GC-Dgcr8 mutants compared to control testes (P). (J–L) Anti-protamine revealed a 75% reduction in the number of elongated spermatids (ES) per tubule in GC-Dcr1 mutants and a 62% reduction in GC-Dgcr8 mutants compared to control testes (Q). DAPI (blue) was used for nuclear staining. (O) Epididymal sperm count analysis showed a 99% decrease in GC-Dcr1 mutants and 96% in GC-Dgcr8 mutants. TW: Testis Weight, BW: Body Weight, RS: Round Spermatids, ES: Elongated Spermatids. Results are mean ± SEM, * = p<0.05, ** = p<0.005, *** = p<0.0001 GC-Dcr1 mutant vs. control, GC-Dgcr8 mutant vs. control or GC-Dcr1 vs. GC-Dgcr8. Scale bars: 20 µm (D–L).</p

Impaired spermiogenesis leads to altered morphology of spermatozoa in both GC-Dcr1 and GC-Dgcr8 mutants at P60.

<p>Representative transmission electron micrographs from P60 control (A, D), GC-Dcr1 mutants (B,E) and GC-Dgcr8 mutants (C,F). In round spermatids (A–C), the acrosome is fragmented or asymmetric in both mutants (arrowheads). In elongated spermatids (D–F), nuclear shape (red arrows), chromatin condensation and the acrosome (arrowheads) are abnormal in both GC-Dcr1 and GC-Dgcr8 mutants. Note the presence of vacuoles within nuclei (blue arrows) in both mutants. Scale bar: 2 µm. H&E staining of epididymal sperm spreads of control (G) and mutant (H–L) adult mice. In contrast to control mice, spermatozoa of both mutant mice exhibited multiple defects of morphology, such as falciform head (H), intermediate head (I), round head (J), elongated head (K), pinhead (L) and abnormal midpiece (J). Scale bars: 10 µm. The histogram shows the percentage of spermatozoa in each category (M). Results are mean ± SEM (minimum n = 3/genotype), a = p<0.005 GC-Dcr1 mut vs. control, b = p<0.005 GC-Dgcr8 mut vs. control and c = p<0.005 GC-Dcr1 mut vs. GC-Dgcr8 mut.</p

Altered mRNA transcriptome in GC-Dcr1 spermatocytes.

<p>(A) Hierarchical clustering of the six sequenced samples. We calculated the distance between each pair of samples as 1 - <i>rho</i>, where <i>rho</i> was the Spearman correlation coefficient for the gene expression levels in the two samples. Clustering was performed using the <i>hclust</i> function in R and Ward's method. (B) Overview of the normalized gene expression data. Genes were grouped into 10 equally sized bins based on their combined expression in GC-Dcr1 and control spermatocytes. Highly expressed genes are shown on the right. Expression change was calculated as the difference between the log₂-transformed normalized expression values in GC-Dcr1 and control spermatocytes. (C) Comparison of expression change in genes targeted by highly and lowly expressed miRNAs (see text for details). Expression change was calculated as in (B).</p