Blocking endogenous retinoic acid degradation induces oral tooth formation in zebrafish.

According to Dollo's Law of irreversibility in evolution, a lost structure is usually considered to be unable to reappear in evolution due to the accumulation over time of mutations in the genes required for its formation. Cypriniform fish are a classic model of evolutionary loss because, while they form fully operational teeth in the ventral posterior pharynx, unlike other teleosts, they do not possess oral teeth. Paleontological data show that Cypriniforms, a clade of teleost fish that includes the zebrafish, lost their oral teeth 50 to 100 Mya. In order to attempt to reverse oral tooth loss in zebrafish, we block the degradation of endogenous levels of retinoic acid (RA) using a specific inhibitor of the Cyp26 RA degrading enzymes. We demonstrate the inhibition of endogenous RA degradation is sufficient to restore oral tooth induction as marked by the re-appearance of expression of early dental mesenchyme and epithelium genes such as dlx2b and sp7 in the oral cavity. Furthermore, we show that these exogenously induced oral tooth germs are able to be at least partly calcified. Taken together, our data show that modifications of signaling pathways can have a significant effect on the reemergence of once-lost structures leading to experimentally induced reversibility of evolutionary tooth loss in cypriniforms.

Analysis of oxygen consumption rates in zebrafish reveals differences based on sex, age and physical activity recovery.

Introduction: Mitochondrial dysfunction is linked to a variety of human diseases. Understanding the dynamic alterations in mitochondrial respiration at various stages of development is important to our understanding of disease progression. Zebrafish provide a system for investigating mitochondrial function and alterations during different life stages. The purpose of this study was to investigate our ability to measure mitochondrial oxygen consumption rates in zebrafish embryos, larvae, and adults as an indicator of mitochondrial function. Methods: Basal respiration of entire zebrafish embryos (5 dpf), larvae (0.6-0.9 cm), young adults (3-month-old), and old adults (12-month-old) was measured using an Oroboros Oxygraph, with a stirrer speed of 26 rpm. For embryos and larvae, "leak" respiration (plus oligomycin), maximum respiration (plus uncoupler), non-mitochondrial respiration (plus inhibitors), and complex IV activity were also measured. To induce physical activity in adult fish, the stirrer speed was increased to 200 rpm. Results and Discussion: We demonstrate the ability to accurately measure respiration rates in zebrafish at various ages using the Oroboros Oxygraph. When comparing zebrafish embryos to larvae, embryos have a higher maximum respiration. Three-month-old zebrafish males have higher basal respiration than females, while 12-month-old zebrafish females exhibit greater rates of respiration than males and younger females. When the stirrer speed was increased, respiration rates decrease, but with differences depending on sex. This study demonstrates a simple and accessible method to assess zebrafish physiology by mitochondrial oxygen consumption measurements in an unmodified Oroboros Oxygraph. The method should facilitate studies to understand the intricate interplay between mitochondrial function, development, and aging.

Enhanced production of eicosanoids in plasma and activation of DNA damage pathways in PBMCs are correlated with the severity of ancestral COVID-19 infection.

Background: Many questions remain unanswered regarding the implication of lipid metabolites in severe SARS-CoV-2 infections. By re-analyzed sequencing data from the nasopharynx of a previously published cohort, we found that alox genes, involved in eicosanoid synthesis, were up-regulated in high WHO score patients, especially in goblet cells. Herein, we aimed to further understand the roles played by eicosanoids during severe SARS-CoV-2 infection. Methods and findings: We performed a total fatty acid panel on plasma and bulk RNA-seq analysis on peripheral blood mononuclear cells (PBMCs) collected from 10 infected and 10 uninfected patients. Univariate comparison of lipid metabolites revealed that lipid metabolites were increased in SARS-CoV-2 patients including the lipid mediators Arachidonic Acid (AA) and Eicosapentaenoic Acid (EPA). AA, EPA and the fatty acids Docosahexaenoic acid (DHA) and Docosapentaenoic acid (DPA), were positively correlated to WHO disease severity score. Transcriptomic analysis demonstrated that COVID-19 patients can be segregated based on WHO scores. Ontology, KEGG and Reactome analysis identified pathways enriched for genes related to innate immunity, interactions between lymphoid and nonlymphoid cells, interleukin signaling and, cell cycling pathways. Conclusions: Our study offers an association between nasopharynx mucosa eicosanoid genes expression, specific serum inflammatory lipids and, subsequent DNA damage pathways activation in PBMCs to severity of COVID-19 infection.

Excess glucose or fat differentially affects metabolism and appetite-related gene expression during zebrafish embryogenesis.

Zebrafish embryos use their yolk sac reserve as the sole nutrient source during embryogenesis. The two main forms of energy fuel can be found in the form of glucose or fat. Zebrafish embryos were exposed to glucose or injected with free fatty acid/Triacylglycerol (FFA/TAG) into the yolk sac at 24 hpf. At 72 hpf, glucose exposed or FFA/TAG injected had differential effects on gene expression in embryos, with fat activating lipolysis and ß-oxidation and glucose activating the insulin pathway. Bulk RNA-seq revealed that more gene expression was affected by glucose exposure compared to FFA/TAGs injection. Appetite-controlling genes were also differently affected by glucose exposure or FFA/TAG injections. Because the embryo did not yet feed itself at the time of our analysis, gene expression changes occurred in absence of actual hunger and revealed how the embryo manages its nutrient intake before active feeding.

The multi-level regulation of clownfish metamorphosis by thyroid hormones.

Most marine organisms have a biphasic life cycle during which pelagic larvae transform into radically different juveniles. In vertebrates, the role of thyroid hormones (THs) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrated analysis of metamorphosis of a marine teleost, the false clownfish (Amphiprion ocellaris). We show how THs coordinate a change in color vision as well as a major metabolic shift in energy production, highlighting how it orchestrates this transformation. By manipulating the activity of liver X regulator (LXR), a major regulator of metabolism, we also identify a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild, explaining how hormones coordinate energy needs with available resources during the life cycle.

Development of a Triple-Negative Breast Cancer Leptomeningeal Disease Model in Zebrafish.

Leptomeningeal disease occurs when cancer cells migrate into the ventricles of the brain and spinal cord and then colonize the meninges of the central nervous system. The triple-negative subtype of breast cancer often progresses toward leptomeningeal disease and has a poor prognosis because of limited treatment options. This is due, in part, to a lack of animal models with which to study leptomeningeal disease. Here, we developed a translucent zebrafish casper (roy-/-; nacre-/-) xenograft model of leptomeningeal disease in which fluorescent labeled MDA-MB-231 human triple-negative breast cancer cells are microinjected into the ventricles of zebrafish embryos and then tracked and measured using fluorescent microscopy and multimodal plate reader technology. We then used these techniques to measure tumor area, cell proliferation, and cell death in samples treated with the breast cancer drug doxorubicin and a vehicle control. We monitored MDA-MB-231 cell localization and tumor area, and showed that samples treated with doxorubicin exhibited decreased tumor area and proliferation and increased apoptosis compared to control samples.

Embryonic Hyperglycemia Delays the Development of Retinal Synapses in a Zebrafish Model.

Embryonic hyperglycemia negatively impacts retinal development, leading to abnormal visual behavior, altered timing of retinal progenitor differentiation, decreased numbers of retinal ganglion cells and Müller glia, and vascular leakage. Because synaptic disorganization is a prominent feature of many neurological diseases, the goal of the current work was to study the potential impact of hyperglycemia on retinal ribbon synapses during embryonic development. Our approach utilized reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence labeling to compare the transcription of synaptic proteins and their localization in hyperglycemic zebrafish embryos, respectively. Our data revealed that the maturity of synaptic ribbons was compromised in hyperglycemic zebrafish larvae, where altered ribeye expression coincided with the delay in establishing retinal ribbon synapses and an increase in the immature synaptic ribbons. Our results suggested that embryonic hyperglycemia disrupts retinal synapses by altering the development of the synaptic ribbon, which can lead to visual defects. Future studies using zebrafish models of hyperglycemia will allow us to study the underlying mechanisms of retinal synapse development.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.

Ecotoxic effects of microplastics and contaminated microplastics - Emerging evidence and perspective.

The high prevalence and persistence of microplastics (MPs) in pristine habitats along with their accumulation across environmental compartments globally, has become a matter of grave concern. The resilience conferred to MPs using the material engineering approaches for outperforming other materials has become key to the challenge that they now represent. The characteristics that make MPs hazardous are their micro to nano scale dimensions, surface varied wettability and often hydrophobicity, leading to non-biodegradability. In addition, MPs exhibit a strong tendency to bind to other contaminants along with the ability to sustain extreme chemical conditions thus increasing their residence time in the environment. Adsorption of these co-contaminants leads to modification in toxicity varying from additive, synergistic, and sometimes antagonistic, having consequences on flora, fauna, and ultimately the end of the food chain, human health. The resulting environmental fate and associated risks of MPs, therefore greatly depend upon their complex interactions with the co-contaminants and the nature of the environment in which they reside. Net outcomes of such complex interactions vary with core characteristics of MPs, the properties of co-contaminants and the abiotic factors, and are required to be better understood to minimize the inherent risks. Toxicity assays addressing these concerns should be ecologically relevant, assessing the impacts at different levels of biological organization to develop an environmental perspective. This review analyzed and evaluated 171 studies to present research status on MP toxicity. This analysis supported the identification and development of research gaps and recommended priority areas of research, accounting for disproportionate risks faced by different countries. An ecological perspective is also developed on the environmental toxicity of contaminated MPs in the light of multi-variant stressors and directions are provided to conduct an ecologically relevant risk assessment. The presented analyses will also serve as a foundation for developing environmentally appropriate remediation methods and evaluation frameworks.

Identification of novel lipid biomarkers in xmrk- and Myc-induced models of hepatocellular carcinoma in zebrafish.

BACKGROUND: Hepatocellular carcinoma (HCC) is the predominant form of liver cancer and is accompanied by complex dysregulation of lipids. Increasing evidence suggests that particular lipid species are associated with HCC progression. Here, we aimed to identify lipid biomarkers of HCC associated with the induction of two oncogenes, xmrk, a zebrafish homolog of the human epidermal growth factor receptor (EGFR), and Myc, a regulator of EGFR expression during HCC. METHODS: We induced HCC in transgenic xmrk, Myc, and xmrk/Myc zebrafish models. Liver specimens were histologically analyzed to characterize the HCC stage, Oil-Red-O stained to detect lipids, and liquid chromatography/mass spectrometry analyzed to assign and quantify lipid species. Quantitative real-time polymerase chain reaction was used to measure lipid metabolic gene expression in liver samples. Lipid species data was analyzed using univariate and multivariate logistic modeling to correlate lipid class levels with HCC progression. RESULTS: We found that induction of xmrk, Myc and xmrk/Myc caused different stages of HCC. Lipid deposition and class levels generally increased during tumor progression, but triglyceride levels decreased. Myc appears to control early HCC stage lipid species levels in double transgenics, whereas xmrk may take over this role in later stages. Lipid metabolic gene expression can be regulated by either xmrk, Myc, or both oncogenes. Our computational models showed that variations in total levels of several lipid classes are associated with HCC progression. CONCLUSIONS: These data indicate that xmrk and Myc can temporally regulate lipid species that may serve as effective biomarkers of HCC progression.

Xmrks the Spot: Fish Models for Investigating Epidermal Growth Factor Receptor Signaling in Cancer Research.

Studies conducted in several fish species, e.g., Xiphophorus hellerii (green swordtail) and Xiphophorus maculatus (southern platyfish) crosses, Oryzias latipes (medaka), and Danio rerio (zebrafish), have identified an oncogenic role for the receptor tyrosine kinase, Xmrk, a gene product closely related to the human epidermal growth factor receptor (EGFR), which is associated with a wide variety of pathological conditions, including cancer. Comparative analyses of Xmrk and EGFR signal transduction in melanoma have shown that both utilize STAT5 signaling to regulate apoptosis and cell proliferation, PI3K to modulate apoptosis, FAK to control migration, and the Ras/Raf/MEK/MAPK pathway to regulate cell survival, proliferation, and differentiation. Further, Xmrk and EGFR may also modulate similar chemokine, extracellular matrix, oxidative stress, and microRNA signaling pathways in melanoma. In hepatocellular carcinoma (HCC), Xmrk and EGFR signaling utilize STAT5 to regulate cell proliferation, and Xmrk may signal through PI3K and FasR to modulate apoptosis. At the same time, both activate the Ras/Raf/MEK/MAPK pathway to regulate cell proliferation and E-cadherin signaling. Xmrk models of melanoma have shown that inhibitors of PI3K and MEK have an anti-cancer effect, and in HCC, that the steroidal drug, adrenosterone, can prevent metastasis and recover E-cadherin expression, suggesting that fish Xmrk models can exploit similarities with EGFR signal transduction to identify and study new chemotherapeutic drugs.

Cisplatin and phenanthriplatin modulate long-noncoding RNA expression in A549 and IMR90 cells revealing regulation of microRNAs, Wnt/ß-catenin and TGF-ß signaling.

The monofunctional platinum(II) complex, phenanthriplatin, acts by blocking transcription, but its regulatory effects on long-noncoding RNAs (lncRNAs) have not been elucidated relative to traditional platinum-based chemotherapeutics, e.g., cisplatin. Here, we treated A549 non-small cell lung cancer and IMR90 lung fibroblast cells for 24 h with either cisplatin, phenanthriplatin or a solvent control, and then performed microarray analysis to identify regulated lncRNAs. RNA22 v2 microRNA software was subsequently used to identify microRNAs (miRNAs) that might be suppressed by the most regulated lncRNAs. We found that miR-25-5p, -30a-3p, -138-5p, -149-3p, -185-5p, -378j, -608, -650, -708-5p, -1253, -1254, -4458, and -4516, were predicted to target the cisplatin upregulated lncRNAs, IMMP2L-1, CBR3-1 and ATAD2B-5, and the phenanthriplatin downregulated lncRNAs, AGO2-1, COX7A1-2 and SLC26A3-1. Then, we used qRT-PCR to measure the expression of miR-25-5p, -378j, -4516 (A549) and miR-149-3p, -608, and -4458 (IMR90) to identify distinct signaling effects associated with cisplatin and phenanthriplatin. The signaling pathways associated with these miRNAs suggests that phenanthriplatin may modulate Wnt/ß-catenin and TGF-ß signaling through the MAPK/ERK and PTEN/AKT pathways differently than cisplatin. Further, as some of these miRNAs may be subject to dissimilar lncRNA targeting in A549 and IMR90 cells, the monofunctional complex may not cause toxicity in normal lung compared to cancer cells by acting through distinct lncRNA and miRNA networks.

The endocannabinoid system and retinoic acid signaling combine to influence bone growth.

Osteoporosis is an increasing burden on public health as the world-wide population ages and effective therapeutics are severely needed. Two pathways with high potential for osteoporosis treatment are the retinoic acid (RA) and endocannabinoid system (ECS) signaling pathways. We sought to elucidate the roles that these pathways play in bone development and maturation. Here, we use chemical treatments to modulate the RA and ECS pathways at distinct early, intermediate, and late times bone development in zebrafish. We further assessed osteoclast activity later in zebrafish and medaka. Finally, by combining sub-optimal doses of AR and ECS modulators, we show that enhancing RA signaling or reducing the ECS promote bone formation and decrease osteoclast abundance and activity. These data demonstrate that RA signaling and the ECS can be combined as sub-optimal doses to influence bone growth and may be key targets for potential therapeutics.

RNA-Seq Analysis of Cisplatin and the Monofunctional Platinum(II) Complex, Phenanthriplatin, in A549 Non-Small Cell Lung Cancer and IMR90 Lung Fibroblast Cell Lines.

Phenanthriplatin is a new monofunctional platinum(II) complex that binds only one strand of DNA and acts by blocking gene transcription, but its effect on gene regulation has not been characterized relative to the traditional platinum-based complex, cisplatin. A549 non-small cell lung cancer and IMR90 lung fibroblast cells were treated with cisplatin, phenanthriplatin, or a control and then their RNA transcripts were subjected to next generation sequencing analysis. DESeq2 and CuffDiff2 were used to identify up- and downregulated genes and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to identify pathways and functions. We found that phenanthriplatin may regulate the genes GPRC5a, TFF1, and TNFRSF10D, which act through p53 to control apoptosis, differently or to a greater extent than cisplatin, and that it, unlike cisplatin, could upregulate ATP5MD, a gene which signals through the Wnt/ß catenin pathway. Furthermore, phenanthriplatin caused unique or enhanced effects compared to cisplatin on genes regulating the cytoskeleton, cell migration, and proliferation, e.g., AGAP1, DIAPH2, GDF15, and THSD1 (p < 0.05; q < 0.05). Phenanthriplatin may modulate some oncogenes differently than cisplatin potentially leading to improved clinical outcome, but this monofunctional complex should be carefully matched with cancer gene data to be successfully applied in chemotherapy.

Retinoids in Embryonic Development.

Retinoids constitute a class of compounds chemically related to vitamin A [...].

Endothelin-1 in the pathophysiology of obesity and insulin resistance.

The association between plasma endothelin-1 (ET-1) and obesity has been documented for decades, yet the contribution of ET-1 to risk factors associated with obesity is not fully understood. In 1994, one of first papers to document this association also noted a positive correlation between plasma insulin and ET-1, suggesting a potential contribution of ET-1 to the development of insulin resistance. Both endogenous receptors for ET-1, ETA and ETB are present in all insulin-sensitive tissues including adipose, liver and muscle, and ET-1 actions within these tissues suggest that ET-1 may be playing a role in the pathogenesis of insulin resistance. Further, antagonists for ET-1 receptors are clinically approved making these sites attractive therapeutic targets. This review focuses on known mechanisms through which ET-1 affects plasma lipid profiles and insulin signalling in these metabolically important tissues and also identifies gaps in our understanding of ET-1 in obesity-related pathophysiology.

The protective effects of human milk-derived peptides on the pancreatic islet biology.

Several epidemiological studies support the protective role of breastfeeding in reducing the risk for type 1 diabetes. Human breast milk is the perfect nutrition for infants and contains many complex proteins, lipids and carbohydrates. In this study, we examined the physiological effects of human milk-derived opioid peptides, ß-casomorphins (BCM), and compared them with bovine-milk-derived opioid peptides on pancreatic hormone regulation and ß-cell regeneration. Exposure of wild-type zebrafish embryos to 50 µg/ml of human BCM-5 and -7 from 3 days post fertilisation until 6 days post fertilisation resulted in an increased insulin domain of expression while exposure to bovine BCM-5 and -7 significantly reduced the insulin domain of expression as analysed by whole-mount in situ hybridisation. These changes may be accounted for by reduced insulin expression or ß-cell number and were mitigated by the µ-opioid receptor antagonist, naloxone. The effect of BCM on ß-cell regeneration was assessed following ablation of ß-cells in Tg (ins: CFP-NTR) zebrafish from 3 days post fertilisation to 4 days post fertilisation, followed by exposure of bovine and human BCM-5 and -7 (50 µg/ml) from 4 days post fertilisation until 7 days post fertilisation. The regenerative capacity of ß-cells was not impeded following exposure to human BCM-5 and -7, whereas the capacity of ß-cells to regenerate following bovine BCM-5 and -7 exposure was reduced. Our data suggest that human BCM-5 and -7 may promote ß-cell development and enable the regeneration of ß-cells, while the bovine-milk-derived peptides, BCM-5 and -7, play an opposite role. These data may provide some biological explanation for the protective effect of breastfeeding on the development of type 1 diabetes.

The Vertebrate Tooth Row: Is It Initiated by a Single Organizing Tooth?

Teeth are one of the most fascinating innovations of vertebrates. Their diversity of shape, size, location, and number in vertebrates is astonishing. If the molecular mechanisms underlying the morphogenesis of individual teeth are now relatively well understood, thanks to the detailed experimental work that has been performed in model organisms (mainly mouse and zebrafish), the mechanisms that control the organization of the dentition are still a mystery. Mammals display simplified dentitions when compared to other vertebrates with only a single tooth row positioned in the anterior part of the mouth, whereas other vertebrates exhibit tooth rows in many locations. As proposed 60 years ago, tooth rows can be formed sequentially from an initiator tooth. Recent results in zebrafish have now largely confirmed this hypothesis. Here this observation is generalized upon and it is suggested that in most vertebrates tooth rows could form sequentially from a single initiator tooth.

Retinoic Acid Signaling and the Zebrafish Dentition During Development and Evolution.

Explaining how the extensive diversity in form of vertebrate teeth arose in evolution and the mechanisms by which teeth are made during embryogenesis are intertwined questions that can merit from a better understanding of the roles of retinoic acid (RA) in tooth development. Pioneering studies in rodents showed that dietary vitamin A (VA), and eventually RA (one of the major active metabolites of VA), are required for proper tooth formation and that dentin-forming odontoblast cells seem to be especially sensitive to changes in RA levels. Later, rodent studies further indicated that RA signaling interactions with other cell-signaling pathways are an important part of RA's actions in odontogenesis. Recent investigations employing zebrafish and other teleost fish continued this work in an evolutionary context, and specifically demonstrated that RA is required for the initiation of tooth development. RA is also sufficient in certain circumstances to induce de novo tooth formation. Both effects appear to involve cranial-neural crest cells, again suggesting that RA signaling has a particular influence on odontoblast development. These teleost studies have also highlighted both evolutionary conservation and change in how RA is employed during odontogenesis in different vertebrate lineages, and thus raises the possibility that developmental changes to RA signaling has led to some of the diversity of form seen across vertebrate dentitions. Future progress in this area will come at least in part from expanding the species examined to get a better picture of how often RA signaling has changed in evolution and how this relates to the evolution of dental form.