The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization (WISH). Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA-modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. In addition, RA treatment increased expression of CCAAT/enhancer-binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development.
The diversity of teeth patterns in actinopterygians is impressive with tooth rows in many locations in the oral and pharyngeal regions. The first-formed tooth has been hypothesized to serve as an initiator controlling the formation of the subsequent teeth. In zebrafish, the existence of the first tooth (named 4 V 1 ) is puzzling as its replacement is induced before the opening of the mouth. Functionally, it has been shown that 4 V 1 formation requires fibroblast growth factor (FGF) and retinoic acid (RA) signalling. Here, we show that the ablation of 4 V 1 prevents the development of the dental row demonstrating its dependency over it. If endogenous levels of FGF and RA are restored after 4 V 1 ablation, embryonic dentition starts again by de novo formation of a first tooth, followed by the dental row. Similarly, induction of anterior ectopic teeth induces subsequent tooth formation, demonstrating that the initiator tooth is necessary and sufficient for dental row formation, probably via FGF ligands released by 4 V 1 to induce the formation of subsequent teeth. Our results show that by modifying the formation of the initiator tooth it is possible to control the formation of a dental row. This could help to explain the diversity of tooth patterns observed in actinopterygians and more broadly, how diverse traits evolved through molecular fine-tuning.
Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed treatments for depression and, as a class of drugs, are among the most used medications in the world. Concern regarding possible effects of SSRI treatment on fetal development has arisen recently as studies have suggested a link between maternal SSRI use and an increase in birth defects such as persistent pulmonary hypertension, seizures and craniosynostosis. Furthermore, SSRI exposure in adults is associated with decreased bone mineral density and increased fracture risk, and serotonin receptors are expressed in human osteoblasts and osteoclasts. To determine possible effects of SSRI exposure on developing bone, we treated both zebrafish, during embryonic development, and human mesenchymal stem cells (MSCs), during differentiation into osteoblasts, with the two most prescribed SSRIs, citalopram and sertraline. SSRI treatment in zebrafish decreased bone mineralization, visualized by alizarin red staining and decreased the expression of mature osteoblast-specific markers during embryogenesis. Furthermore, we showed that this inhibition was not associated with increased apoptosis. In differentiating human MSCs, we observed a decrease in osteoblast activity that was associated with a decrease in expression of the osteoblast-specific genes Runx2, Sparc and Spp1, measured with quantitative real-time PCR (qRT-PCR). Similar to the developing zebrafish, no increase in expression of the apoptotic marker Caspase 3 was observed. Therefore, we propose that SSRIs inhibit bone development by affecting osteoblast maturation during embryonic development and MSC differentiation. These results highlight the need to further investigate the risks of SSRI use during pregnancy in exposing unborn babies to potential skeletal abnormalities.
The amyloid precursor protein (APP) generates a number of peptides when processed through different cleavage mechanisms, including the amyloid beta peptide that is implicated in the development of Alzheimer's disease. It is well established that APP via its cleaved peptides regulates aspects of neuronal metabolism. Emerging evidence suggests that amyloidogenic processing of APP can lead to altered systemic metabolism, similar to that observed in metabolic disease states. In the present study, we investigated the effect of APP deficiency on obesity-induced alterations in systemic metabolism. Compared with WT littermates, APP-deficient mice were resistant to diet-induced obesity, which was linked to higher energy expenditure and lipid oxidation throughout the dark phase and was associated with increased spontaneous physical activity. Consistent with this lean phenotype, APP-deficient mice fed a high-fat diet (HFD) had normal insulin tolerance. However, despite normal insulin action, these mice were glucose intolerant, similar to WT mice fed a HFD. This was associated with reduced plasma insulin in the early phase of the glucose tolerance test. Analysis of the pancreas showed that APP was required to maintain normal islet and β-cell mass under high fat feeding conditions. These studies show that, in addition to regulating aspects of neuronal metabolism, APP is an important regulator of whole body energy expenditure and glucose homeostasis under high fat feeding conditions.
We showed that direct exposure to metformin during embryogenesis does not increase insulin-expressing area but does increase beta-cell number. Further, we identified novel consequences of beta-cell ablation to alter the expression of other pancreatic hormones that were enhanced by metformin. Therefore, this study provides a greater understanding of the beta-cell development/regenerative processes and the effect of metformin, bringing us closer to identifying how to increase beta-cells in humans.
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