Zebrafish are now widely used to study skeletal development and bone-related diseases. To that end, understanding osteoblast differentiation and function, the expression of essential transcription factors, signaling molecules, and extracellular matrix proteins is crucial. We isolated Sp7-expressing osteoblasts from 4-day-old larvae using a fluorescent reporter. We identified two distinct subpopulations and characterized their specific transcriptome as well as their structural, regulatory, and signaling profile. Based on their differential expression in these subpopulations, we generated mutants for the extracellular matrix protein genes col10a1a and fbln1 to study their functions. The col10a1a^−/− mutant larvae display reduced chondrocranium size and decreased bone mineralization, while in adults a reduced vertebral thickness and tissue mineral density, and fusion of the caudal fin vertebrae were observed. In contrast, fbln1^−/− mutants showed an increased mineralization of cranial elements and a reduced ceratohyal angle in larvae, while in adults a significantly increased vertebral centra thickness, length, volume, surface area, and tissue mineral density was observed. In addition, absence of the opercle specifically on the right side was observed. Transcriptomic analysis reveals up-regulation of genes involved in collagen biosynthesis and down-regulation of Fgf8 signaling in fbln1^−/− mutants. Taken together, our results highlight the importance of bone extracellular matrix protein genes col10a1a and fbln1 in skeletal development and homeostasis.

Persistent organic pollutants (POPs) are posing major environmental and health threats due to their stability, ubiquity, and bioaccumulation. Most of the numerous studies of these compounds deal with single chemicals, although real exposures always consist of mixtures. Thus, using different tests, we screened the effects on zebrafish larvae caused by exposure to an environmentally relevant POP mixture. Our mixture consisted of 29 chemicals as found in the blood of a Scandinavian human population. Larvae exposed to this POP mix at realistic concentrations, or sub-mixtures thereof, presented growth retardation, edemas, retarded swim bladder inflation, hyperactive swimming behavior, and other striking malformations such as microphthalmia. The most deleterious compounds in the mixture belong to the per- and polyfluorinated acids class, although chlorinated and brominated compounds modulated the effects. Analyzing the changes in transcriptome caused by POP exposure, we observed an increase of insulin signaling and identified genes involved in brain and eye development, leading us to propose that the impaired function of the condensin I complex caused the observed eye defect. Our findings contribute to the understanding of POP mixtures, their consequences, and potential threats to human and animal populations, indicating that more mechanistic, monitoring, and long-term studies are imperative.

"Persistent organic pollutants (POPs)" have a plethora of deleterious effects on humans and the environment due to their bioaccumulative, persistent, and mimicking properties. Individually, each of these chemicals has been tested and its effects measured, however they are rather found as parts of complex mixtures of which we do not fully grasp the extent of their potential consequences. Here we studied the effects of realistic, environmentally relevant mixtures of 29 POPs on cartilage and bone development using zebrafish as a model species. We observed developmental issues in cartilage, in the form of diverse malformations such as micrognathia, reduced size of the Meckel’s and other structures. Also, mineralized bone formation was disrupted, hence impacting the overall development of the larvae at later life stages. Assessment of the transcriptome revealed disruption of nuclear receptor pathways, such as androgen, vitamin D, and retinoic acid, that may explain the mechanisms of action of the compounds within the tested mixtures. In addition, clustering of the compounds using their chemical signatures revealed structural similarities with the model chemicals vitamin D and retinoic acid that can explain the effects and/or enhancing the phenotypes we witnessed. Further mechanistic studies will be required to fully understand this kind of molecular interactions and their repercussions in organisms. Our results contribute to the already existing catalogue of deleterious effects caused by exposure to POPs and help to understand the potential consequences in at risk populations.

Endocrine disruptors are chemicals that have been in the spotlight for some time now. Their modulating action on endocrine signaling pathways made them a particularly interesting topic of research within the field of ecotoxicology. Traditionally, endocrine disrupting properties are studied using exposure to suspected chemicals. In recent years, a major breakthrough in biology has been the advent of targeted gene editing tools to directly assess the function of specific genes. Among these, the CRISPR/Cas9 method has accelerated progress across many disciplines in biology. This versatile tool allows to address antagonism differently, by directly inactivating the receptors targeted by endocrine disruptors. Here, we used the CRISPR/Cas9 method to knock out the different estrogen receptors in zebrafish and we assessed the potential effects this generates during development. We used a panel of biological tests generally used in zebrafish larvae to investigate exposure to compounds deemed as endocrine disrupting chemicals. We demonstrate that the absence of individual functional estrogen receptors (Esr1, Esr2b, or Gper1) does affect behavior, heart rate and overall development. Each mutant line was viable and could be grown to adulthood, the larvae tended to be morphologically grossly normal. A substantial fraction (70%) of the esr1 mutants presented severe craniofacial deformations, while the remaining 30% of esr1 mutants also had changes in behavior. esr2b mutants had significantly increased heart rate and significant impacts on craniofacial morphometrics. Finally, mutation of gper1 affected behavior, decreased standard length, and decreased bone mineralization as assessed in the opercle. Although the exact molecular mechanisms underlying these effects will require further investigations in the future, we added a new concept and new tools to explore and better understand the actions of the large group of endocrine disrupting chemicals found in our environment.

With over 350,000 chemicals and mixtures currently registered for production and use worldwide, around 83% of authorized chemicals lack adequate toxicity data, leaving the majority of chemicals poorly characterized. International agencies urge scientists to develop screening methods to explore, identify, and predict chemical hazards, supporting the prioritization of chemical risk assessment. Here, Tree Manifold Approximation and Projection (TMAP) were applied, with the aim of reducing the dimensionality of large toxicological dataset, providing the foundations to data imputation methods allowing to get an understanding of chemical modes of action. Specifically, TMAP was implemented using MHFP6 fingerprints and the NORMAN SusDat database, which contains over 100,000 compounds. To ensure that the TMAP layout preserves chemical structural similarity, a quantitative parameter optimization procedure was developed. The defined optimal parameter set allowed us to define the embeddings that preserves the most structural similarity among nearest connected neighbors, with similarity progressively decreasing as the distance between nodes increases. Leveraging this approach, a graph-based spatial imputation function was generated to obtain insights into the potential ecotoxicity mechanisms of data poor chemicals using physicochemical properties and CTD toxicogenomic data. The relevance and meaningfulness of TMAP chemical space was explored for Daphnia magna, Pimephales promelas and Algae. Chemical classes known to be structurally similar were found to be grouped together in the TMAP chemical space, while heterogeneous classes were found to be sparse. Data imputation allowed for the identification of known and potential chemical mechanisms of action. Indeed, acetylcholinesterase and transthyretin were confirmed as major mechanisms of action of organothiophosphate and brominated flame retardant toxicity in Daphnia magna and Pimephales promelas, respectively. Overall, transdisciplinary toxicological databases combined with TMAP, stand out as a computationally efficient and suitable method to explore and analyze large datasets, allowing for the inference of associations between chemical structures and chemical hazard identification and other potential applications of this hypotheses-generating tool.