BODIPY-Based Analogue of the TREM2-Binding Molecular Adjuvant Sulfavant A, a Chemical Tool for Imaging and Tracking Biological Systems.

Recently, we described synthetic sulfolipids named Sulfavants as a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. The members of this family, Sulfavant A (1), Sulfavant R (2), and Sulfavant S (3), showed important effects on triggering receptor expressed on myeloid cells 2 (TREM2)-induced differentiation and maturation of human dendritic cells (hDC), through a novel cell mechanism underlying the regulation of the immune response. As these molecules are involved in biological TREM2-mediated processes crucial for cell survival, here, we report the synthesis and application of a fluorescent analogue of Sulfavant A bearing the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene moiety (Me4-BODIPY). The fluorescent derivative, named PB-SULF A (4), preserving the biological activity of Sulfavants, opens the way to chemical biology and cell biology experiments to better understand the interactions with cellular and in vivo organ targets and to improve our comprehension of complex molecular mechanisms underlying the not fully understood ligand-induced TREM2 activity.

Chronic Exposure to Chlorpyrifos Damages Thyroid Activity and Imbalances Hepatic Thyroid Hormones Signaling and Glucose Metabolism: Dependency of T3-FOXO1 Axis by Hyperglycemia.

Early life exposure to Endocrine Disruptor Chemicals (EDCs), such as the organophosphate pesticide Chlorpyrifos (CPF), affects the thyroid activity and dependent process, including the glucose metabolism. The damage of thyroid hormones (THs) as a mechanism of action of CPF is underestimated because the studies rarely consider that TH levels and signaling are customized peripherally. Here, we investigated the impairment of metabolism/signaling of THs and lipid/glucose metabolism in the livers of 6-month-old mice, developmentally and lifelong exposed to 0.1, 1, and 10 mg/kg/die CPF (F1) and their offspring similarly exposed (F2), analyzing the levels of transcripts of the enzymes involved in the metabolism of T3 (Dio1), lipids (Fasn, Acc1), and glucose (G6pase, Pck1). Both processes were altered only in F2 males, affected by hypothyroidism and by a systemic hyperglycemia linked to the activation of gluconeogenesis in mice exposed to 1 and 10 mg/kg/die CPF. Interestingly, we observed an increase in active FOXO1 protein due to a decrease in AKT phosphorylation, despite insulin signaling activation. Experiments in vitro revealed that chronic exposure to CPF affected glucose metabolism via the direct modulation of FOXO1 activity and T3 levels in hepatic cells. In conclusion, we described different sex and intergenerational effects of CPF exposure on the hepatic homeostasis of THs, their signaling, and, finally, glucose metabolism. The data points to FOXO1-T3-glucose signaling as a target of CPF in liver.

A Cross-Species Analysis Reveals Dysthyroidism of the Ovaries as a Common Trait of Premature Ovarian Aging.

Although the imbalance of circulating levels of Thyroid Hormones (THs) affects female fertility in vertebrates, its involvement in the promotion of Premature Ovarian Aging (POA) is debated. Therefore, altered synthesis of THs in both thyroid and ovary can be a trait of POA. We investigated the relationship between abnormal TH signaling, dysthyroidism, and POA in evolutionary distant vertebrates: from zebrafish to humans. Ovarian T3 signaling/metabolism was evaluated by measuring T3 levels, T3 responsive transcript, and protein levels along with transcripts governing T3 availability (deiodinases) and signaling (TH receptors) in distinct models of POA depending on genetic background and environmental exposures (e.g., diets, pesticides). Expression levels of well-known (Amh, Gdf9, and Inhibins) and novel (miR143/145 and Gas5) biomarkers of POA were assessed. Ovarian dysthyroidism was slightly influenced by genetics since very few differences were found between C57BL/6J and FVB/NJ females. However, diets exacerbated it in a strain-dependent manner. Similar findings were observed in zebrafish and mouse models of POA induced by developmental and long-life exposure to low-dose chlorpyrifos (CPF). Lastly, the T3 decrease in follicular fluids from women affected by diminished ovarian reserve, as well as of the transcripts modulating T3 signaling/availability in the cumulus cells, confirmed ovarian dysthyroidism as a common and evolutionary conserved trait of POA.

Short-term exposure to the simple polyphenolic compound gallic acid induces neuronal hyperactivity in zebrafish larvae.

A growing body of evidence suggests that the biological effects of polyphenols are not restricted to antioxidant activity, but they exert a wide range of modulatory effects on metabolic pathways, cellular signaling and gene expression. In this study, we tested the minimum safe concentration of gallic acid (GA) in 72 hpf zebrafish larvae in order to evaluate the effects on the central nervous system and the behavioral response. We showed that a short exposure (30 min) induces the depletion of the two main excitatory and inhibitory neurotransmitters, Glu and GABA, respectively, in the larval nervous system. The acute impairment of GABAergic-glutamatergic balance was paralleled by an increase of the fosab neuronal activity marker in specific brain areas, such as the forebrain, olfactory bulbs, pallial area, ventral midbrain, tegmentum, and the medulla oblongata ventral area. The neuronal excitation was mirrored by the increased cumulative motor response. The inhibition of the olfactory epithelium with brief cadmium exposition suggests a direct involvement of olfaction in the larvae response to GA. Our results demonstrate that a brief exposure to GA induces motoneuronal hyperexcitability in zebrafish. The behavioral response was probably elicited through the activation of an odorous, or chemical, stimulus. The specificity of the activated neuronal territories suggests the involvement of additional signaling pathways. Although the underlying molecular mechanisms remain to be elucidated, our data support the hypothesis that GA acts as an excitatory molecule, capable of inducing a specific nerve response. These results offer a new vision on potential effects of GA.

Into the bloom: Molecular response of pelagic tunicates to fluctuating food availability.

The planktonic tunicates appendicularians and thaliaceans are highly efficient filter feeders on a wide range of prey size including bacteria and have shorter generation times than any other marine grazers. These traits allow some tunicate species to reach high population densities and ensure their success in a favourable environment. However, there are still few studies focusing on which genes and gene pathways are associated with responses of pelagic tunicates to environmental variability. Herein, we present the effect of food availability increase on tunicate community and gene expression at the Marquesas Islands (South-East Pacific Ocean). By using data from the Tara Oceans expedition, we show that changes in phytoplankton density and composition trigger the success of a dominant larvacean species (an undescribed appendicularian). Transcriptional signature to the autotroph bloom suggests key functions in specific physiological processes, i.e., energy metabolism, muscle contraction, membrane trafficking, and proteostasis. The relative abundance of reverse transcription-related Pfams was lower at bloom conditions, suggesting a link with adaptive genetic diversity in tunicates in natural ecosystems. Downstream of the bloom, pelagic tunicates were outcompeted by copepods. Our work represents the first metaomics study of the biological effects of phytoplankton bloom on a key zooplankton taxon.

Immunological response to bacterial infection in a pelagic tunicate: Inflammation in the salp Thalia democratica.

Thaliaceans are pelagic tunicates that play a key role in trophic chains of the oceans. In the field of tunicate immunity, a notable gap is the lack of data on their inflammatory response. The common salp, Thalia democratica, possesses scant immunocytes, represented by a phagocytic line (hyaline amebocytes) and a mast cell-like line (granular cells). We aimed to provide the first investigation of defense reactions upon exposure to a large amount of bacteria (Bacillus clausii). We detected (i) bacterial phagocytosis by hyaline amebocytes, (ii) degradation of phagocytizing hyaline amebocytes in the tunic after transcellular diapedesis from the hemocoel, and (iii) release of heparin, histamine, and TNF-α by granular cells. Cell degranulation and phagocytosis occurred in epidermal cells lining the hemocoel, and an excess of mucus was observed in the post-branchial gut, causing a functional inhibition of cilia and microvilli. These findings indicate multi-step events comparable to an inflammation involving responses at both tissue and organismal levels.

Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny.

BACKGROUND: Thaliaceans is one of the understudied classes of the phylum Tunicata. In particular, their phylogenetic relationships remain an issue of debate. The overall pattern of serotonin (5-HT) distribution is an excellent biochemical trait to interpret internal relationships at order level. In the experiments reported here we compared serotonin-like immunoreactivity at different life cycle stages of two salpid, one doliolid, and one pyrosomatid species. This multi-species comparison provides new neuroanatomical data for better resolving the phylogeny of the class Thaliacea. RESULTS: Adults of all four examined thaliacean species exhibited serotonin-like immunoreactivity in neuronal and non-neuronal cell types, whose anatomical position with respect to the nervous system is consistently identifiable due to α-tubulin immunoreactivity. The results indicate an extensive pattern that is consistent with the presence of serotonin in cell bodies of variable morphology and position, with some variation within and among orders. Serotonin-like immunoreactivity was not found in immature forms such as blastozooids (Salpida), tadpole larvae (Doliolida) and young zooids (Pyrosomatida). CONCLUSIONS: Comparative anatomy of serotonin-like immunoreactivity in all three thaliacean clades has not been reported previously. These results are discussed with regard to studies of serotonin-like immunoreactivity in adult ascidians. Lack of serotonin-like immunoreactivity in the endostyle of Salpida and Doliolida compared to Pyrosomella verticillata might be the result of secondary loss of serotonin control over ciliary beating and mucus secretion. These data, when combined with other plesiomorphic characters, support the hypothesis that Pyrosomatida is basal to these clades within Phlebobranchiata and that Salpida and Doliolida constitute sister-groups.

Onecut Regulates Core Components of the Molecular Machinery for Neurotransmission in Photoreceptor Differentiation.

Photoreceptor cells (PRC) are neurons highly specialized for sensing light stimuli and have considerably diversified during evolution. The genetic mechanisms that underlie photoreceptor differentiation and accompanied the progressive increase in complexity and diversification of this sensory cell type are a matter of great interest in the field. A role of the homeodomain transcription factor Onecut (Oc) in photoreceptor cell formation is proposed throughout multicellular organisms. However, knowledge of the identity of the Oc downstream-acting factors that mediate specific tasks in the differentiation of the PRC remains limited. Here, we used transgenic perturbation of the Ciona robusta Oc protein to show its requirement for ciliary PRC differentiation. Then, transcriptome profiling between the trans-activation and trans-repression Oc phenotypes identified differentially expressed genes that are enriched in exocytosis, calcium homeostasis, and neurotransmission. Finally, comparison of RNA-Seq datasets in Ciona and mouse identifies a set of Oc downstream genes conserved between tunicates and vertebrates. The transcription factor Oc emerges as a key regulator of neurotransmission in retinal cell types.

Multi Species Analyses Reveal Testicular T3 Metabolism and Signalling as a Target of Environmental Pesticides.

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

Peripheral T3 signaling is the target of pesticides in zebrafish larvae and adult liver.

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in 'de novo lipogenesis' and ß-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages.

BACKGROUND: The concerted activity of Meis and Hoxa11 transcription factors is essential for the subdivision of tetrapod limbs into proximo-distal (PD) domains; however, little is know about the evolution of this patterning mechanism. Here, we aim to study the expression of meis and hoxa11 orthologues in the median and paired rayed fins of zebrafish and in the lobed fins of the Australian lungfish. RESULTS: First, a late phase of expression of meis1.1 and hoxa11b in zebrafish dorsal and anal fins relates with segmentation of endochondral elements in proximal and distal radials. Second, our zebrafish in situ hybridization results reveal spatial and temporal changes between pectoral and pelvic fins. Third, in situ analysis of meis1, meis3 and hoxa11 genes in Neoceratodus pectoral fins identifies decoupled domains of expression along the PD axis. CONCLUSIONS: Our data raise the possibility that the origin of stylopod and zeugopod lies much deeper in gnathostome evolution and that variation in meis and hoxa11 expression has played a substantial role in the transformation of appendage anatomy. Moreover, these observations provide evidence that the Meis/Hoxa11 profile considered a hallmark of stylopod/zeugopod patterning is present in Neoceratodus.

A comprehensive analysis of neurotrophins and neurotrophin tyrosine kinase receptors expression during development of zebrafish.

Neurotrophins (NTF) are a family of secreted nerve growth factors with affinity for tyrosine kinase (Ntrk) and p75 receptors. To fully understand the variety of developmental roles played by NTFs, it is critical to know when and where genes encoding individual ligands and receptors are transcribed. Identification of ntf and ntrk transcripts in zebrafish development remains to be fully characterized for further uncovering the potential function(s) of the NTF signal transduction pathway. Here, we conducted a systematic analysis of the expression profiles of four ntf and five ntrk genes during zebrafish development using whole-mount in situ hybridization. Our study unveils new expression domains in the developing embryo, confirms those previously known, and shows that ntf and ntrk genes have different degrees of cell- and tissue-type specificity. The unique and overlapping expression patterns here depicted indicate the coordination of the redundant and divergent functions of NTFs and represent valuable tools for deciphering the molecular pathways involved in the specification and function of embryonic cell types.