Fig 1.
Survival rate of zebrafish embryos after DMSO treatment.
Zebrafish embryos with 48 hs were treated with DMSO with concentrations ranging from 1 to 5% and survival rate was quantified after 24 hs and 48 hs of treatment. Note that nearly all embryos die after 48 hs of treatment with 5% of DMSO. Scale bars = 500 μm. Number of embryos analyzed per experimental group = 30.
Fig 2.
DMSO alters the morphology of zebrafish embryos.
Zebrafish embryos with 48 or 72 hpf were treated with DMSO with concentrations ranging from 1 to 5%. Bright field images were acquired after 24 hs of treatment and quantification of several morphological parameters was performed. Scale bar = 1 mm. Number of embryos analyzed per experimental group = 30.
Fig 3.
DMSO induces cardiac edema in zebrafish.
Zebrafish embryos at 48 hpf were treated with DMSO with concentrations ranging from 1 to 5% and cardiac edema and heart beating were quantified. Scale bars = 1 mm and 100 μm. Number of embryos analyzed per experimental group = 30.
Fig 4.
DMSO induced alterations in zebrafish notochord.
Zebrafish embryos with 48 hs were treated with DMSO with concentrations ranging from 1 to 5% and bright field images were acquired after 24 hs of treatment. Black arrows point to alterations in the notochord morphology of zebrafish treated with DMSO. Scale bar = 50 μm. Number of embryos analyzed per experimental group = 30.
Fig 5.
Zebrafish embryoswere observed under polarized and fluorescent microscopy. Embryos were labelled for F-actin with Phalloidin (red), for vinculin (green) and with DAPI (blue). Somites size and shape were altered after DMSO treatment, which can be observed by the birefringent images of embryo’s skeletal muscles (top middle images in the middle column). Scale bar = 1 mm. Number of embryos analyzed per experimental group = 30.
Fig 6.
DMSO treatment interferes with myofibril alignment.
Confocal analysis of zebrafish larvae at 48 hpf stained with the F-actin specific probe Phalloidin (red) and DAPI (blue). In control embryos, aligned striated myofibrils can be observed, whereas misaligned myofibrils are seen in 4% DMSO treated embryos. Scale bar = 50 μm. Number of embryos analyzed per experimental group = 30.
Fig 7.
DMSO induces a reduction in somite’s size.
Zebrafish embryos (A) were analyzed under Differential Interference Contrast microscopy (DIC) in a Disk Confocal microscope and somites (B) were measured in their middle line (C). Somites’ thickness significantly decreases after treatment with 2%, 3% and 4% DMSO (D). ns = non-significant. Number of embryos analyzed per experimental group = 30.
Fig 8.
Long term DMSO treatment does not induce an up-curved phenotype.
Zebrafish embryos were treated with DMSO for 6 days and no changes in body curvature were observed under bright field microscopy. Scale bar = 1 mm. Number of embryos analyzed per experimental group = 40.
Fig 9.
Long term DMSO treatment induces changes in fish swim bladder.
Zebrafish embryos were treated with DMSO for 6 days and embryos caudal length, body thickness and swim bladder size were measured (A-C). Embryos’ thickness increased while swim bladder size decreased after long term treatment. Images in D show the alterations induced by long term DMSO treatment in the swim bladder. Scale bars = 1 mm in C and 0.5 mm in D. Number of embryos analyzed per experimental group = 40.
Fig 10.
Long term DMSO treatment does not alter zebrafish somites.
Zebrafish embryos were treated with DMSO for 6 days and observed under polarized and fluorescent microscopy. Embryos were labelled for F-actin with Phalloidin (red), for vinculin (green) and with DAPI (blue). Somites’ size and shape were not altered after DMSO treatment, which can be observed by the birefringent images of embryo’s skeletal muscles. Scale bar = 1 mm. Number of embryos analyzed per experimental group = 40.
Table 1.
Effects of DMSO on the morphology and physiology of zebrafish embryos. DMSO can significantly, in a dose-dependent manner, alter the morphology and physiology of zebrafish embryos, including changes in embryo’s size and curvature, heart beating frequency, pericardium area, somite size and myofibril alignment, melanocyte size, and morphology of notochord and swim bladder. (*) only the swim bladder area was quantified in 7 dpf zebrafish larvae treated with 2% DMSO, while all other measurements were quantified in 72 hpf zebrafish embryos treated with 4% DMSO. CI = curvature index.
Fig 11.
Schematic representation of DMSO-induced changes in zebrafish embryos.
Our results show that 96% of zebrafish embryos were up-curved after treatment with 4% DMSO for 48 h and 98% with 5% DMSO for 24 and 48 h. The upward tail curvature phenotype is well-characterized for PDK2/ALK5 zebrafish mutants [17]. Mitochondrial dysfunction can alter ALK5 signaling pathway [18]. Our hypothesis is that DMSO alters plasma membrane permeability in ZF embryos cells, allowing DMSO to enter the cells and once inside them can induce water pore formation in mitochondria, leading to altered mitochondrial physiology and altered ALK5 signaling.