Zebrafish Blastomeres Research Articles

Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors

The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.

An orthotopic glioblastoma animal model suitable for high-throughput screenings.

Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens. We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy. We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease. We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.

Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

Non-directional radial intercalation dominates deep cell behavior during zebrafish epiboly

SummaryEpiboly is the first coordinated cell movement in most vertebrates and marks the onset of gastrulation. During zebrafish epiboly, enveloping layer (EVL) and deep cells spread over the vegetal yolk mass with a concomitant thinning of the deep cell layer. A prevailing model suggests that deep cell radial intercalations directed towards the EVL would drive deep cell epiboly. To test this model, we have globally recorded 3D cell trajectories for zebrafish blastomeres between sphere and 50% epiboly stages, and developed an image analysis framework to determine intercalation events, intercalation directionality, and migration speed for cells at specific positions within the embryo. This framework uses Voronoi diagrams to compute cell-to-cell contact areas, defines a feature-based spatio-temporal model for intercalation events and fits an anatomical coordinate system to the recorded datasets. We further investigate whether epiboly defects in MZspg mutant embryos devoid of Pou5f1/Oct4 may be caused by changes in intercalation behavior. In wild-type and mutant embryos, intercalations orthogonal to the EVL occur with no directional bias towards or away from the EVL, suggesting that there are no directional cues that would direct intercalations towards the EVL. Further, we find that intercalation direction is independent of the previous intercalation history of individual deep cells, arguing against cues that would program specific intrinsic directed migration behaviors. Our data support a dynamic model in which deep cells during epiboly migrate into space opening between the EVL and the yolk syncytial layer. Genetic programs determining cell motility may control deep cell dynamic behavior and epiboly progress.

Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos

The mechanical properties of cells change as they proceed through the cell cycle, primarily owing to regulation of actin and myosin II. Most models for cell mechanics focus on actomyosin in the cortex and ignore possible roles in bulk cytoplasm. We explored cell cycle regulation of bulk cytoplasmic actomyosin in Xenopus egg extracts, which is almost undiluted cytoplasm from unfertilized eggs. We observed dramatic gelation-contraction of actomyosin in mitotic (M phase) extract where Cdk1 activity is high, but not in interphase (I-phase) extract. In spread droplets, M-phase extract exhibited regular, periodic pulses of gelation-contraction a few minutes apart that continued for many minutes. Comparing actin nucleation, disassembly and myosin II activity between M-phase and I-phase extracts, we conclude that regulation of nucleation is likely to be the most important for cell cycle regulation. We then imaged F-actin in early zebrafish blastomeres using a GFP–Utrophin probe. Polymerization in bulk cytoplasm around vesicles increased dramatically during mitosis, consistent with enhanced nucleation. We conclude that F-actin polymerization in bulk cytoplasm is cell cycle regulated in early vertebrate embryos and discuss possible biological functions of this regulation.

Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos

The mechanical properties of cells change as they proceed through the cell cycle, primarily owing to regulation of actin and myosin II. Most models for cell mechanics focus on actomyosin in the cortex and ignore possible roles in bulk cytoplasm. We explored cell cycle regulation of bulk cytoplasmic actomyosin in Xenopus egg extracts, which is almost undiluted cytoplasm from unfertilized eggs. We observed dramatic gelation-contraction of actomyosin in mitotic (M phase) extract where Cdk1 activity is high, but not in interphase (I-phase) extract. In spread droplets, M-phase extract exhibited regular, periodic pulses of gelation-contraction a few minutes apart that continued for many minutes. Comparing actin nucleation, disassembly and myosin II activity between M-phase and I-phase extracts, we conclude that regulation of nucleation is likely to be the most important for cell cycle regulation. We then imaged F-actin in early zebrafish blastomeres using a GFP-Utrophin probe. Polymerization in bulk cytoplasm around vesicles increased dramatically during mitosis, consistent with enhanced nucleation. We conclude that F-actin polymerization in bulk cytoplasm is cell cycle regulated in early vertebrate embryos and discuss possible biological functions of this regulation.

The formation of Ca2+ gradients at the cleavage furrows during cytokinesis of Zebrafish embryos

In dividing embryos, a localized elevation in intracellular Ca2+ ([Ca2+]i) at the cleavage furrow has been shown to be essential for cytokinesis. However, the underlying mechanisms for generating and maintaining these [Ca2+]i gradients throughout cytokinesis are not fully understood. In the present study, we analyzed the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) and endoplasmic reticulum (ER) distribution in determining the intracellular Ca2+ gradients in early zebrafish blastomeres. Application of the injected Ca2+ indicator, Indo-1, showed that during the first cell division a standing Ca2+ gradient was formed ∼35 min after fertilization, with the [Ca2+]i spatially decaying from 500–600 nmol/L at the cleavage furrow to 100–200 nmol/L around the nucleus. While the IP3R immunohistochemical fluorescence was relatively concentrated in the peri-furrow region, ER labeling was relatively enriched in both peri-furrow and peri-nuclear regions. Numeric simulation suggested that a divergence in the spatial distribution of IP3R and the locations of Ca2+ uptake within the ER was essential for the formation of a standing Ca2+ gradient, and the Ca2+ gradient could only be well-established under an optimal stoichiometry of Ca2+ uptake and release. Indeed, while inhibition of IP3R Ca2+ release blocked the generation of the Ca2+ gradient at a lower [Ca2+]i level, both Ca2+ release stimulation by inositol 1,4,5-trisphosphate (IP3) injection and ER Ca2+ pump inhibition by cyclopiazonic acid also eliminated the Ca2+ gradients at higher [Ca2+]i levels. Our results suggest a dynamic relationship between ER-mediated Ca2+ release and uptake that underlies the maintenance of the perifurrow Ca2+ gradient and is essential for cytokinesis of zebrafish embryos.

Live Analysis of Endodermal Layer Formation Identifies Random Walk as a Novel Gastrulation Movement

During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.

Single-Cell Detection of MicroRNAs in Developing Vertebrate Embryos After Acute Administration of a Dual-Fluorescence Reporter/Sensor Plasmid

The detection of microRNAs (miRNAs) at single-cell resolution is important for studying the role of these posttranscriptional regulators. Here, we use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid, injected into zebrafish blastomeres or electroporated into defined tissues of mouse embryos in utero or ex utero, to monitor the dynamics of specific miRNAs in individual live cells. This approach reveals, for example, that in the developing mouse central nervous system, miR-124a is expressed not only in postmitotic neurons but also in neuronal progenitor cells. Collectively, our results demonstrate that acute administration of DFRS plasmids offers an alternative to previous in situ hybridization and transgenic approaches and allows the monitoring of miRNA appearance and disappearance in defined cell lineages during vertebrate development.

Zebrafish fat-free is required for intestinal lipid absorption and Golgi apparatus structure

The zebrafish fat-free (ffr) mutation was identified in a physiological screen for genes that regulate lipid metabolism. ffr mutant larvae are morphologically indistinguishable from wild-type sibling larvae, but their absorption of fluorescent lipids is severely impaired. Through positional cloning, we have identified a causative mutation in a highly conserved and ubiquitously expressed gene within the ffr locus. The Ffr protein contains a Dor-1 like domain typical of oligomeric Golgi complex (COG) gene, cog8. Golgi complex ultrastructure is disrupted in the ffr digestive tract. Consistent with a possible role in COG-mediated Golgi function, wild-type Ffr-GFP and COG8-mRFP fusion proteins partially colocalize in zebrafish blastomeres. Enterocyte retention of an endosomal lipid marker in ffr larvae support the idea that altered vesicle trafficking contributes to the ffr mutant defect. These data indicate that ffr is required for both Golgi structure and vesicular trafficking, and ultimately lipid transport.

Furrow-Specific Endocytosis during Cytokinesis of Zebrafish Blastomeres

Mutations affecting endocytosis, such as those in clathrin and dynamin, unexpectedly cause defects in cytokinesis in a number of organisms. To explore the relationship between endocytosis and cytokinesis, we used the relatively large cells of the transparent zebrafish embryo. Using fluorescent markers for fluid-phase as well as plasma membrane uptake, we demonstrate that cytokinesis involves furrow-specific endocytosis. Clathrin-coated pits are visible near the furrow in ultrathin sections, while immunolabeling demonstrates that clathrin and caveolin are localized to the cleavage furrow. Hence, it is likely that both clathrin- and caveolae-mediated endocytosis occurs at the furrow during cytokinesis. Dynamin II is also localized to the furrow and may mediate furrow-specific endocytosis. Treatment of embryos with chlorpromazine or with methyl-β-cyclodextrin, both of which inhibit endocytosis, prevents the normal completion of cytokinesis. These data suggest that furrow-specific endocytosis is an integral part of cytokinesis.

Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis.

During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and beta-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and beta-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of alpha-tubulin labelled embryos. Transient incubation of embryos in 1 microg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of beta-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent. These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.

Conversion of zebrafish blastomeres to an endodermal fate by TGF-beta-related signaling.

The endoderm contributes cells to the gut, and participates in the induction and patterning of the vertebrate head and heart. The mechanisms controlling the formation of endoderm are poorly understood. Commitment of endoderm cells occurs at the onset of gastrulation and requires cell interactions; studies in vitro have implicated transforming growth factor Beta (TGF-beta)-related molecules in this process. TARAM-A is a zebrafish receptor kinase that is related to the type I subunit of the TGF-beta receptor, and is expressed in presumptive endomesodermal cells at gastrulation. We provide here evidence for its involvement in endoderm formation in vivo. Activation of TARAM-A was found to drive blastomeres towards an endodermal fate. The induced endoderm behaved ad endogenous endoderm during gastrulation: it migrated in contact with the yolk and expressed endoderm-specific markers. Loss-of-function mutations in the zebrafish one-eyed-pinhead (OEP) gene lead to defects in heart formation, defects of the ventral central nervous system (CNS) and cyclopia. Mutant embryos also lack endoderm and anterior mesoderm. Endoderm formation in oep mutant embryos was found to be restored by the activation of the TARAM-A signaling pathway. Cardiac and ocular defects, but not midline CNS structures, were rescued non-autonomously, demonstrating that endoderm may provide signals that can pattern the eye anlage, and which are distinct form those specifying the ventral midline of the CNS.

The DVR-1 (Vg1)Transcript of Zebrafish Is Maternally Supplied and Distributed throughout the Embryo

It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-β superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomate retains an equivalent developmental potential throughout the cleavage period.

Something's fishy here—rethinking cell movements and cell fate in the zebrafish embryo

A recent paper has challenged the prevailing view that zebrafish blastomeres undergo extensive cell mixing and are unrestricted in their cell fates during early development. Here, we offer a model that resolves apparent contradictions between studies that have examined the origins of cell fate in the zebrafish embryo. The model reconsiders cell movements during epiboly, and how cell mixing, or the lack of it, can affect the predictability of cell fate.

Cell lineage of zebrafish blastomeres: I. Cleavage pattern and cytoplasmic bridges between cells

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.