Early Stage Zebrafish Embryos Research Articles

Non-fitting FLIM-FRET facilitates analysis of protein interactions in live zebrafish embryos.

Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos.

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

Ultrasmall Au-Ag Alloy Nanoparticles: Protein-Directed Synthesis, Biocompatibility, and X-ray Computed Tomography Imaging.

The ultrasmall sizes of nanoparticles have attracted significant attention for potential applications in the fields of catalysis and nanomedicine. Herein, we reported on the green preparation and X-ray computed tomography (CT) imaging of ultrasmall bimetallic bovine serum albumin-directed gold-silver (Au-Ag@BSA) nanoparticles (2-4 nm) using BSA as a stabilizing and template-directed agent. Further, the effects of synthesis conditions were systematically explored to prepare products by adjusting the different molar ratios of Au/Ag. The resulting Au-Ag@BSA nanoparticles exhibited the spherical shape, well-dispersed ability, as well as long-term room-temperature stability. The cytotoxicity effects of Au-Ag@BSA nanoparticles on A549 and MCF-7 cells were compared with those of individual Ag nanoparticles, and the results indicated a lower cytotoxicity effect by Au-Ag@BSA nanoparticles. Furthermore, the in vivo toxicity of Au-Ag@BSA nanoparticles was investigated in the early stage zebrafish embryos. The results indicate that there are not any obvious changes of survival and hatching percentages at multiple growth stages (4-120 hpf) even with a high level of Au-Ag@BSA nanoparticles (up to 80 mM), revealing good biocompatibility. Interestingly, a rational design of the Au/Ag molar ratio (3:2) surprisingly possessed enhanced CT performance compared to the performance of the Au nanoparticles and iohexol. Accordingly, this study highlights a new prospect in the green preparation of ultrasmall alloy nanomaterials with good biocompatibility and will be of great interest in developing CT contrast agent, catalyst, as well as drug delivery carrier.

Transplantation of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors into Early-Stage Zebrafish Embryos.

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.

Quantifying compressive forces between living cell layers and within tissues using elastic round microgels

Increasing evidence shows that mechanical stresses are critical in regulating cell functions, fate, and diseases. However, no methods exist that can quantify isotropic compressive stresses. Here we describe fluorescent nanoparticle-labeled, monodisperse elastic microspheres made of Arg-Gly-Asp-conjugated alginate hydrogels (elastic round microgels, ERMGs). We generate 3D displacements and calculate strains and tractions exerted on an ERMG. Average compressive tractions on an ERMG are 570 Pa within cell layers and 360 Pa in tumor-repopulating cell (TRC) colonies grown in 400-Pa matrices. 3D compressive tractions on a 1.4-kPa ERMG are applied by surrounding cells via endogenous actomyosin forces but not via mature focal adhesions. Compressive stresses are substantially heterogeneous on ERMGs within a uniform cell colony and do not increase with TRC colony sizes. Early-stage zebrafish embryos generate spatial and temporal differences in local normal and shear stresses. This ERMG method could be useful for quantifying stresses in vitro and in vivo.

Impact of Low-level Ionizing Radiation on Cell Death During Zebrafish Embryonic Development.

Ionizing radiation (IR) has been linked to multiple types of cellular responses, but its effects on developing organisms are still poorly understood. The authors investigated whether zebrafish embryos exhibit differential responses relative to IR dose and developmental age at time of exposure. Early-stage zebrafish embryos were exposed to different levels of gamma radiation and then, at varying points after irradiation, assayed for morphological defects and levels of cell death. To quantify in vivo cellular responses to low-dose IR exposure and explore how tissue-specific cell functions affect radiation response, apoptotic cells were counted in three regions: the tail, urogenital papilla, and left eye. The authors found that increased gamma radiation doses correlated with increased levels of apoptosis in the developing tail and eye, whereas cells of the urogenital papilla appeared to undergo apoptosis independently of radiation dose. This suggests that the linear-no-threshold model may not be appropriate in all contexts. Grouping embryos by age at IR exposure revealed that gamma radiation exposure resulted in higher levels of apoptosis in embryos irradiated at 2 d post fertilization (dpf), suggesting a radiosensitive stage of development. Moreover, levels of apoptosis were statistically influenced by days grown after irradiation, with embryos fixed at later stages showing more dramatic apoptotic responses to radiation exposure. This latency to effect suggests potential competition between DNA repair and apoptosis pathways, which may lead to the accumulation of apoptotic cells only after an initial lag period.

Repair Proteins and Transcription Factors Compete

DNA lesions repaired by base excision repair (BER) modulate CREB1 binding to its CRE consensus sequence negatively and positively.1 Only a G/U mispair lesion in the CRE CpG islet results in tighter binding.1 Since DNA lesions bind repair proteins, we investigated how CREB1 competes with repair glycosylases by in vitro and in vivo methods. We incubated 39-mer ds oligonucleotides containing normal or modified CRE with recombinant hCREB1 alone or with hUNG2 or hOGG1 and analyzed binding by electrophoretic mobility shift assay. We focused on CREB1 dimerization on CRE, as only the dimer activates transcription. BER glycosylases impacted dimerization depending on the glycosylase and which protein had first access to CRE. UNG2 reduced CREB1 dimerization when CRE contained a G/U mispair in the CpG islet, but only if UNG2 retained glycosylase enzymatic activity. If CREB1 had first access, CREB1 dimerization was hardly impacted but repair activity of UNG2 was diminished. OGG1 reduced CREB1 dimerization when CRE contained oG in the CpG islet, but only if OGG1 exposure followed CREB1. Physiological relevance was shown by microinjecting early-stage zebrafish embryos with the same oligonucleotides. As predicted, microinjection with unmodified CRE or lesion-containing CRE, but not scrambled-CRE, resulted in increased embryo death. Surviving embryos had more abnormalities after microinjection with the G/U mispair CRE, but not with the other oligonucleotides. We propose that exogenous CRE acts as a sink for endogenous CREB1 required for normal development, with a G/U mispair providing the more efficient sink. In sum, CREB1 and DNA glycosylases compete for damaged CRE, impacting transcriptional regulation. (Supported with funds from Aid for Cancer Research and Northeastern University) 1 Moore, et al. 2013, DNA Repair, Vol. 12

Smyd1b_tv1, a Key Regulator of Sarcomere Assembly, Is Localized on the M-Line of Skeletal Muscle Fibers

BackgroundSmyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos.Methodology/Principal FindingsTo better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1.Conclusion/SignificanceTogether, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1.

Smyd1b_tv1, a key regulator of sarcomere assembly, requires Ser225 for its sarcomeric localization

Smyd1b is a member of the Smyd family that contains the highly conserved SET and MYND domains. smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion in the SET domain. Smyd1b plays a key role in sarcomere assembly during myofibrillogenesis. Knockdown of smyd1b expression resulted in paralyzed zebrafish larvae with defective sarcomere assembly in skeletal muscles. To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in zebrafish embryos. Smyd1b_tv1 and Smyd1b_tv2 exhibited different patterns of subcellular localization during muscle development. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, Smyd1b_tv1 and Smyd1b_tv2 exhibited distinct subcellular localization in differentiated myofibers. Smyd1b_tv1 showed a strong sarcomeric localization compared with Smyd1b_tv2. The sarcomeric localization depended on Ser225 located within the Smyd1b-tv1-specific 13 aa insertion. S225A substitution abolished its sarcomeric localization. However, S225T substitution retained the sarcomeric localization of Smyd1b-tv1. Together, these data indicate that Smyd1b-tv1 may play a direct role in sarcomere assembly, and post-translational modification by phosphorylation may modulate Smyd1b_tv1 sarcomeric localization and function. Supported by a research grant MB-8716-08 from BARD.

Cyclodextrin/dextran based drug carriers for a controlled release of hydrophobic drugs in zebrafish embryos

Hydrogel-based drug carriers have been developed from biocompatible materials, cyclodextrin, dextran and poly(ethylene glycol) and were used in zebrafish embryos. Maleimide modified dextrans (Dex-mal) were functionalized with cyclodextrins and crosslinked to form a hydrogel using either per-6-thio-β-cyclodextrin (PSCD) or a combination of mono-6-thio-β-cyclodextrin (MSCD) and di-thiolated poly(ethylene glycol) (DSPEG). Using all-trans retinoic acid (RA) as a model hydrophobic drug, a sustained release from these cyclodextrin modified hydrogels was observed in vitro without an initial burst. This is because the cyclodextrin moiety in these hydrogels acts as a binding site for the RA. Furthermore, the nanosized hydrogel particles were injected into early stage zebrafish embryos in order to test in vivo release of RA and biocompatibility. We found the gel particles prepared from Dex-mal, MSCD and DSPEG were suitable for use in zebrafish embryos and it showed the release of RA in the embryos occurs in a controlled manner.

Molecular structure and expression patterns of flounder ( Paralichthys olivaceus) Myf-5, a myogenic regulatory factor

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MRF4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder ( Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine.

Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos

Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction.

Identification of the F1F0 mitochondrial ATPase as a target for modulating skin pigmentation by screening a tagged triazine library in zebrafish

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells.

Overexpression of neurogenin induces ectopic expression of HuC in zebrafish

Several basic helix-loop-helix (bHLH) transcription factors are known to be involved in vertebrate neurogenesis. To investigate their roles in zebrafish neurogenesis, we isolated cDNAs for homologues of neurogenin and Math-1/atonal. The transcription of neurogenin was first detectable in zebrafish nervous system at late gastrulation stage. The expression of zebrafish neurogenin precedes and overlaps that of HuC, one of the earliest neuronal precursor markers. Injection of neurogenin mRNA into early stage zebrafish embryos induced ectopic expression of HuC. These results suggest that neurogenin may participate in the generation of HuC-expressing cells, implying its role in neuronal determination in zebrafish.