Modelling stripe formation in zebrafish: an agent-based approach

Zebrafish have distinctive black stripes and yellow interstripes that form owing to the interaction of different pigment cells. We present a two-population agent-based model for the development and regeneration of these stripes and interstripes informed by recent experimental results. Our model describes stripe pattern formation, laser ablation and mutations. We find that fish growth shortens the necessary scale for long-range interactions and that iridophores, a third type of pigment cell, help align stripes and interstripes.     

Visualizations of Mercury Methylation and Dynamic Transformations by In Vivo Imaging

Visualization of Hg(II) and MeHg in their native contexts is significant for examining mercury poisoning, while it is challenging because of indistinguishable fluorescent (FL) signals during FL imaging. Herein, visualizations of mercury methylation and dynamic transformations of Hg(II) and MeHg are achieved in living biological systems. Well distinguishable FL responses (blue emission for Hg(II), yellow emission for MeHg) are obtained by a double‐response FL probe (DPAHB) without any interference. As demonstrated by experimental and computational studies, the distinguishable signals are attributed to selective binding with DPAHB and different inhibition of excited‐state proton transfer. Through control tests for live‐dead markers, mercury methylation is demonstrated to be employed in living biological systems. Therefore, the methylation and dynamic transformations of both ions are monitored in zebrafish by imaging, and these results are confirmed by traditional high‐performance liquid chromatography‐based methods. The methylation of Hg(II) to MeHg, dynamic transformations and final accumulations of both species in zebrafish tissues are visualized successfully. This method is also convenient for fast evaluation of detoxification reagents. This is the first visualization of in vivo mercury methylation and dynamic transformation of both species and is effective for studying pathological processes in their native contexts.     

Searching for motifs in the behaviour of larval Drosophila melanogaster and Caenorhabditis elegans reveals continuity between behavioural states

We present a novel method for the unsupervised discovery of behavioural motifs in larval Drosophila melanogaster and Caenorhabditis elegans. A motif is defined as a particular sequence of postures that recurs frequently. The animal''s changing posture is represented by an eigenshape time series, and we look for motifs in this time series. To find motifs, the eigenshape time series is segmented, and the segments clustered using spline regression. Unlike previous approaches, our method can classify sequences of unequal duration as the same motif. The behavioural motifs are used as the basis of a probabilistic behavioural annotator, the eigenshape annotator (ESA). Probabilistic annotation avoids rigid threshold values and allows classification uncertainty to be quantified. We apply eigenshape annotation to both larval Drosophila and C. elegans and produce a good match to hand annotation of behavioural states. However, we find many behavioural events cannot be unambiguously classified. By comparing the results with ESA of an artificial agent''s behaviour, we argue that the ambiguity is due to greater continuity between behavioural states than is generally assumed for these organisms.     

A simple method allowing DIC imaging in conjunction with confocal microscopy

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique ( Barone-Nugent et al., 2002 ). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.     

The photoreceptive cells of the pineal gland in adult zebrafish (Danio rerio)

The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions.     

Bright Aggregation‐Induced Emission Dots for Dynamic Tracking and Grading of Patient‐Derived Xenografts in Zebrafish

Cancer prognosis will benefit from a scoring system that could grade malignant traits of patient‐derived cells by assessing their growth and metastasis in a living system. Specific tracking of patient‐derived cells requires labeling by contrast agents with good signal‐to‐noise ratio and no specific stain of host tissues. Towards this aim, aggregation‐induced emission (AIE) dots are developed for in vivo cancer tracking with emphasis on reproducible optimized formulation and specific fluorescent labeling of cells that enable enhanced spatial temporal resolution in vivo. The importance of energy‐dependent AIE dots uptake for patient‐derived cell labeling is emphasized to reveal their specific uptake by viable cancer cells. Using optically transparent zebrafish embryo, the ability is demonstrated to follow the engraftment of transplanted AIE dot labeled cells in zebrafish brains over one week. Cells detected outside the brain after 7 d are quantified as metastatic cells. Results from seven clinical samples demonstrate the utility of this methodology to differentiate low engraftment level of benign neoplasms from higher engraftment level and metastasis detected in malignant ovarian cancer specimens. Achieving clinically validated results supports the use of AIE dot labeled patient derived cells in zebrafish xenografts for future cancer prognosis.     

Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle–Cell Interactions

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real‐time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.     

强度超声对斑马鱼胚胎发育的影响

超声波的生物学效应一直深受人们关注,低强度超声对细胞的作用机理也不断地被人们所了解。为了研究超声对斑马鱼胚胎发育的影响,通过声学共振理论以及有限元仿真得到圆球形细胞及椭球形细胞的共振频率,并设计了一种低强度超声装置对斑马鱼胚胎进行刺激。通过正交试验设计研究了超声刺激频率、刺激时间和信号激励电压对鱼卵孵化指数的影响,实验结果表明：在频率为1.26 MHz,刺激时间为30 min,信号激励电压为3 Vpp条件下鱼卵孵化指数明显高于对照组,且最佳刺激频率与仿真计算的结果基本一致。由此可见,本研究前期的理论推导及仿真计算结果可信,也证明了适当条件的超声刺激可以加速斑马鱼胚胎细胞的生长。     

Hedgehog Antagonist Pyrimidine–Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation

One of the crucial regulators of embryonic patterning and tissue development is the Hedgehog‐glioma (Hh‐Gli) signalling pathway; its uncontrolled activation has been implicated in different types of cancer in adult tissues. Primary cilium is one of the important factors required for the activation of Hh signalling, as it brings the critical components together for key protein–protein interactions required for Hh pathway regulation. Most of the synthetic and natural small molecule modulators of the pathway primarily antagonise Smoothened (Smo) or other effectors like Hh ligand or Gli. Here, we report a previously described Hh antagonist, with a pyrimidine–indole hybrid (PIH) core structure, as an inhibitor of ciliogenesis. The compound is unique in its mode of action, as it shows perturbation of microtubule dynamics in both cell‐based assays and in vivo systems (zebrafish embryos). Further studies revealed that the probable targets are α‐tubulin and its acetylated form, found in the cytoplasm and primary cilia. PIH also showed axonal defasiculation in developing zebrafish embryos. We thus propose that PIH antagonises Hh signalling by repressing cilia biogenesis and disassembling α‐tubulin from its stabilised form.     

Robust Red Organic Nanoparticles for In Vivo Fluorescence Imaging of Cancer Cell Progression in Xenografted Zebrafish

Bright and red‐emissive organic nanoparticles (NPs) are demonstrated as promising for in vivo fluorescence imaging. However, most red organic dyes show greatly weakened or quenched emission in the aggregated state. In this work, a robust red fluorophore (t‐BPITBT‐TPE) with strong aggregate‐state photoluminescence and good biocompatibility is presented. The NPs comprised of t‐BPITBT‐TPE aggregates encapsulated within 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol) (DSPE‐mPEG) micelles exhibit a photoluminescence peak at 660 nm with a high fluorescence quantum yield of 32% in aqueous media. The NPs can be facilely charged by using the same polymeric matrix with different terminal groups, e.g., methoxy (DSPE‐mPEG), amine (DSPE‐PEG‐NH₂), or carboxymethyl (DSPE‐PEG‐COOH) groups. The biocompatibility, toxicity, circulation, and biodistribution of the NPs are assessed using the zebrafish model through whole embryo soaking and intravenous delivery. Furthermore, HeLa and MCF‐7 cells tagged with t‐BPITBT‐TPE in DSPE‐PEG‐NH₂‐TAT polymer NPs are xenografted into zebrafish larvae to successfully track the cancer cell proliferation and metastasis, demonstrating that these new NPs are efficient cancer cell trackers. In addition, the NPs also show good in vivo imaging ability toward 4T1 tumors in xenografted BALB/c mice.