Polystyrene nanoplastics disrupt glucose metabolism and cortisol levels with a possible link to behavioural changes in larval zebrafish

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Brun, N. R., van Hage, P., Hunting, E. R., Haramis, A. G., Vink, S. C., Vijver, M. G., Schaaf, M. J. M., & Tudorache, C. Polystyrene nanoplastics disrupt glucose metabolism and cortisol levels with a possible link to behavioural changes in larval zebrafish. Communications Biology, 2, (2019): 382, doi:10.1038/s42003-019-0629-6.Plastic nanoparticles originating from weathering plastic waste are emerging contaminants in aquatic environments, with unknown modes of action in aquatic organisms. Recent studies suggest that internalised nanoplastics may disrupt processes related to energy metabolism. Such disruption can be crucial for organisms during development and may ultimately lead to changes in behaviour. Here, we investigated the link between polystyrene nanoplastic (PSNP)-induced signalling events and behavioural changes. Larval zebrafish exhibited PSNP accumulation in the pancreas, which coincided with a decreased glucose level. By using hyperglycemic and glucocorticoid receptor (Gr) mutant larvae, we demonstrate that the PSNP-induced disruption in glucose homoeostasis coincided with increased cortisol secretion and hyperactivity in challenge phases. Our work sheds new light on a potential mechanism underlying nanoplastics toxicity in fish, suggesting that the adverse effect of PSNPs are at least in part mediated by Gr activation in response to disrupted glucose homeostasis, ultimately leading to aberrant locomotor activity.We thank Natalia Novik and Laurie Mans for technical assistance during glucose assay and in situ hybridisation, respectively, Rubén Marín-Juez for providing the ins riboprobe, and John J. Stegeman for his helpful comments on the manuscript. The research described in this work was supported by the Dutch research council NWO (MGV; 864.13.010)

The polycomb group protein ring1b/rnf2 is specifically required for craniofacial development

Polycomb group (PcG) genes are chromatin modifiers that mediate epigenetic silencing of target genes. PcG-mediated epigenetic silencing is implicated in embryonic development, stem cell plasticity, cell fate maintenance, cellular differentiation and cancer. However, analysis of the roles of PcG proteins in maintaining differentiation programs during vertebrate embryogenesis has been hampered due to the early embryonic lethality of several PcG knock-outs in the mouse. Here, we show that zebrafish Ring1b/Rnf2, the single E3 ubiquitin ligase in the Polycomb Repressive Complex 1, critically regulates the developmental program of craniofacial cell lineages. Zebrafish ring1b mutants display a severe craniofacial phenotype, which includes an almost complete absence of all cranial cartilage, bone and musculature. We show that Cranial Neural Crest (CNC)-derived cartilage precursors migrate correctly into the pharyngeal arches, but fail to differentiate into chondrocytes. This phenotype is specific for cartilage precursors, since other neural crest-derived cell lineages, including glia, neurons and chromatophores, are formed normally in ring1b mutants. Our results therefore reveal a critical and specific role for Ring1b in promoting the differentiation of cranial neural crest cells into chondrocytes. The molecular mechanisms underlying the pathogenesis of craniofacial abnormalities, which are among the most common genetic birth defects in humans, remain poorly understood. The zebrafish ring1b mutant provides a molecular model for investigating these mechanisms and may lead to the discovery of new treatments or preventions of craniofacial abnormalitie

<i>Ring1b</i> mutants lack almost all head cartilage elements.

<p>Lateral view of WT and <i>ring1b</i> live embryos at 72 hpf (A, B). Alcian-Blue stained head cartilages of WT (C, E, G and H) and <i>ring1b</i> (D, F, H and J) mutants at the indicated developmental points, ventral views. The paired trabeculae have elongated and fused posteriorly in WT embryos at 56 hpf (E) and by 72 hpf the elaborate cartilagenous skeleton of the head has been established (I). In contrast, no cartilage is visible in <i>ring1b</i> mutants except for two minute cartilage deposits at 72 hpf <i>ring1b</i> mutants (J: arrowheads). ch: ceratohyal; ep: ethmoid plate; hys: hyosymplectic; m: Meckel’s cartilage; pc: parachordal, pq: palatoquadrate; tc: trabeculae.</p

Apoptosis is slightly increased in the pharyngeal arch of <i>ring1b</i> mutants.

<p>Lateral views of WT and <i>ring1b</i> embryos at 36 hpf stained for TUNEL. In WT embryos two small clusters of TUNEL-positive apoptotic cells were detected in the pharyngeal arch region just posterior to the eye (A, arrows). These clusters appear to contain more apoptotic cells in the <i>ring1b</i> mutants (B, arrows). The arrowhead indicates the otic vesicle (ov). WT embryos at 36 hpf contain few apoptotic cells in the trunk (C), whereas there is an increase in overall apoptosis particularly in the trunk and the tail in <i>ring1b</i> mutants (D, arrows).</p

Role of phosphatidylinositol 5-phosphate 4-kinase α in zebrafish development

Phosphatidylinositol 5-phosphate 4-kinases (PIP4Ks) phosphorylate phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5-bisphosphate; their most likely function is the regulation of the levels of PI5P, a putative signalling intermediate. There are three mammalian PIP4Ks isoforms (α, β and γ), but their physiological roles remain poorly understood. In the present study, we identified the zebrafish orthologue (zPIP4Kα) of the high-activity human PIP4K α isoform and analyzed its role in embryonic development. RT-PCR analysis and whole-mount in situ hybridization experiments showed that zPIP4Kα is maternally expressed. At later embryonic stages, high PIP4Kα expression was detected in the head and the pectoral fins. Knockdown of zPIP4Kα by antisense morpholino oligonucleotides led to severe morphological abnormalities, including midbody winding defects at 48hpf. The abnormal phenotype could be rescued, at least in large part, by injection of human PIP4Kα mRNA. Our results reveal a key role for PIP4Kα and its activity in vertebrate tissue homeostasis and organ development.</p

Cranial musculature development is severely impaired in <i>ring1b</i> mutants.

<p>Ventral views of WT and <i>ring1b</i> embryos stained with the MF20 antibody (A-F). The anterior mandibularis (am) has not formed in <i>ring1b</i> mutants at 56 hpf (B), the sternohyoideus (sh) is reduced at 65 hpf (D) and at 72 hpf, cranial muscles are almost completely absent (F). hh: hyohyoideus; ih: interhyoideus; ima: intermandibularis anterioris; imp: intermandibularis posterioris.</p

Loss of endochondral and dermal ossification in <i>ring1b</i> mutants.

<p>Ventral (A–D, E, F) and lateral (A’–D’, E’,) views of <i>in situ</i> hybridizations with riboprobes against the indicated genes in WT and <i>ring1b</i> mutants at 68–72 hpf. In WT embryos, <i>runx2a</i> and <i>runx2b</i> are expressed in hypertrophic pharyngeal arch-derived chondrocytes, as well as in the dermal ossification centers of the operculum, parasphenoid and cleithrum. Weak expression is detected in pharyngeal arches and the parasphenoid of <i>ring1b</i> mutants (B, B’, D, D’). At 72 hpf, <i>col10a1</i> is expressed in developing dermal bones in WT embryos, but not in <i>ring1b</i> mutants (E–F). cl: cleithrum; de: dentary; h: hyoid; m: mandibular; mx: maxilla; pa: pharyngeal arches; pq: palatoquadrate; ps: parasphenoid, op: operculum cl: the cleithrum. Numbers indicate the respective pharyngeal arches.</p

De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine

Little is known about the signaling mechanisms that determine the highly regular patterning of the intestinal epithelium into crypts and villi. With the use of mouse models, we show that bone morphogenetic protein (BMP)-4 expression occurs exclusively in the intravillus mesenchyme. Villus epithelial cells respond to the BMP signal. Inhibition of BMP signaling by transgenic expression of noggin results in the formation of numerous ectopic crypt units perpendicular to the crypt-villus axis. These changes phenocopy the intestinal histopathology of patients with the cancer predisposition syndrome juvenile polyposis (JP), including the frequent occurrence of intraepithelial neoplasia. Many JP cases are known to harbor mutations in BMP pathway genes. These data indicate that intestinal BMP signaling represses de novo crypt formation and polyp growt