Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.

The Zygotic Division Regulator ZAR1 Plays a Negative Role in Defense Against Botrytis cinerea in Arabidopsis

A phosphorylation/dephosphorylation cycle at tyrosine 428 of CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) plays an essential role in chitin triggered immunity in Arabidopsis thaliana. In this study, we used a differential peptide pull-down (PPD) assay to identify factors that could participate downstream of this cycle. We identified ZYGOTIC ARREST 1 (ZAR1) and showed that it interacts with CERK1 specifically when the tyrosine 428 (Y428) residue of CERK1 is dephosphorylated. ZAR1 was originally characterized as an integrator for calmodulin and G-protein signals to regulate zygotic division in Arabidopsis. Our current results established that ZAR1 also negatively contributed to defense against the fungus Botrytis cinerea and played a redundant role with its homolog ZAR2 in this process. The zar1-3 zar2-1 double mutant exhibited stronger resistance to B. cinerea compared with zar1-3 single mutant, zar2-1 single mutant, and wild-type plants. Moreover, the inducible expression of numerous defense response genes upon B. cinerea infection was increased in the zar1-3zar2-1 double mutant, consistent with a repressive role for ZAR proteins in the defense response. Therefore, our findings provided insight into the function of ZAR1 in multiple defenses and developmental regulation pathways.

VISTA as a ligand downregulates LPS-mediated inflammation in macrophages and neutrophils

V-domain immunoglobulin suppressor of T-cell activation (VISTA) has emerged as a unique immunoregulatory receptor on cells of the myeloid lineage. Agonizing VISTA on myeloid cells has recently been demonstrated to have a profound effect on dampening inflammatory responses. VISTA has been proposed to function both as a ligand and as a receptor. In this context, the role of VISTA as a ligand has been largely ignored. Using a high-avidity agonist of the VISTA receptor (VISTA-COMP), we investigated the effect of exogenous VISTA, as a ligand, on macrophages and neutrophil cellular pathways in an acute inflammatory setting. RNA sequencing analysis demonstrated that VISTA-COMP downregulates pro-inflammatory cytokines and chemokines and upregulates immunoregulatory genes in both LPS-stimulated macrophages and neutrophils ex vivo. Interestingly, unlike VISTA itself, the receptor is only expressed following LPS stimulation of these cell populations. Furthermore, the administration of VISTA-COMP attenuated the rise in circulating TNFα levels in LPS-treated mice in vivo. These results suggest that VISTA serves a redundant role on macrophages and neutrophils acting as both a ligand and a receptor in the context of an acute inflammatory event.

Activation of EGFR signaling by Tc-Vein and Tc-Spitz regulates the metamorphic transition in the red flour beetle Tribolium castaneum

Animal development relies on a sequence of specific stages that allow the formation of adult structures with a determined size. In general, juvenile stages are dedicated mainly to growth, whereas last stages are devoted predominantly to the maturation of adult structures. In holometabolous insects, metamorphosis marks the end of the growth period as the animals stops feeding and initiate the final differentiation of the tissues. This transition is controlled by the steroid hormone ecdysone produced in the prothoracic gland. In Drosophila melanogaster different signals have been shown to regulate the production of ecdysone, such as PTTH/Torso, TGFß and Egfr signaling. However, to which extent the roles of these signals are conserved remains unknown. Here, we study the role of Egfr signaling in post-embryonic development of the basal holometabolous beetle Tribolium castaneum. We show that Tc-Egfr and Tc-pointed are required to induced a proper larval-pupal transition through the control of the expression of ecdysone biosynthetic genes. Furthermore, we identified an additional Tc-Egfr ligand in the Tribolium genome, the neuregulin-like protein Tc-Vein (Tc-Vn), which contributes to induce larval-pupal transition together with Tc-Spitz (Tc-Spi). Interestingly, we found that in addition to the redundant role in the control of pupa formation, each ligand possesses different functions in organ morphogenesis. Whereas Tc-Spi acts as the main ligand in urogomphi and gin traps, Tc-Vn is required in wings and elytra. Altogether, our findings show that in Tribolium, post-embryonic Tc-Egfr signaling activation depends on the presence of two ligands and that its role in metamorphic transition is conserved in holometabolous insects.

A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.

Role of the Forkhead Transcription Factors Fd4 and Fd5 During Drosophila Leg Development

Appendage development requires the coordinated function of signaling pathways and transcription factors to pattern the leg along the three main axes: the antero-posterior (AP), proximo-distal (PD), and dorso-ventral (DV). The Drosophila leg DV axis is organized by two morphogens, Decapentaplegic (Dpp), and Wingless (Wg), which direct dorsal and ventral cell fates, respectively. However, how these signals regulate the differential expression of its target genes is mostly unknown. In this work, we found that two members of the Drosophila forkhead family of transcription factors, Fd4 and Fd5 (also known as fd96Ca and fd96Cb), are identically expressed in the ventro-lateral domain of the leg imaginal disc in response to Dpp signaling. Here, we analyze the expression regulation and function of these genes during leg development. We have generated specific mutant alleles for each gene and a double fd4/fd5 mutant chromosome to study their function during development. We highlight the redundant role of the fd4/fd5 genes during the formation of the sex comb, a male specific structure that appears in the ventro-lateral domain of the prothoracic leg.

MAGUKs are essential, but redundant, in long-term potentiation

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

The Early Postnatal Life: A Dynamic Period in Thymic Epithelial Cell Differentiation

The microenvironments formed by cortical (c) and medullary (m) thymic epithelial cells (TECs) play a non-redundant role in the generation of functionally diverse and self-tolerant T cells. The role of TECs during the first weeks of the murine postnatal life is particularly challenging due to the significant augment in T cell production. Here, we critically review recent studies centered on the timely coordination between the expansion and maturation of TECs during this period and their specialized role in T cell development and selection. We further discuss how aging impacts on the pool of TEC progenitors and maintenance of functionally thymic epithelial microenvironments, and the implications of these chances in the capacity of the thymus to sustain regular thymopoiesis throughout life.

A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses show that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.

Activation of EGFR Signaling by Tc-Vein and Tc-Spitz Regulates the Metamorphic Transition in the Red Flour Beetle Tribolium Castaneum

Animal development relies on a sequence of specific stages that allow the formation of adult structures with a determined size. In general, juvenile stages are dedicated mainly to growth, whereas last stages are devoted predominantly to the maturation of adult structures. In holometabolous insects, metamorphosis marks the end of the growth period as the animals stops feeding and initiate the final differentiation of the tissues. This transition is controlled by the steroid hormone ecdysone produced in the prothoracic gland. In Drosophila different signals have been shown to regulate the production of ecdysone, such as PTTH/Torso, TGFß and Egfr signaling. However, to which extent the role of these signals is conserved remains unknown. Here, we study the role of Egfr signaling in post-embryonic development of the basal holometabolous beetle Tribolium castaneum. We show that Tc-Egfr and Tc-pointed are required to induced a proper larval-pupal transition through the control of the expression of ecdysone biosynthetic genes. Furthermore, we identified an additional Tc-Egfr ligand in the Tribolium genome, the neuregulin-like protein Tc-Vein (Tc-Vn), which contributes to induce larval-pupal transition together with Tc-Spitz (Tc-Spi). Interestingly, we found that in addition to the redundant role in the control of pupa formation, each ligand possesses different functions in organ morphogenesis. Whereas Tc-Spi acts as the main ligand in urogomphi and gin traps, Tc-Vn is required in wings and elytra. Altogether, our findings show that in Tribolium, post-embryonic Tc-Egfr signaling activation depends on the presence of two ligands and that its role in metamorphic transition is conserved in holometabolous insects.