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In JoVE (1)
Other Publications (10)
- Journal of Molecular Biology
- Biochemical and Biophysical Research Communications
- The Biochemical Journal
- Cell
- Cell Stem Cell
- Development (Cambridge, England)
- Methods in Molecular Biology (Clifton, N.J.)
- Methods in Cell Biology
- Cell
- Cytometry. Part A : the Journal of the International Society for Analytical Cytology
Articles by Pulin Li in JoVE
JoVE General
Retro-orbital Injection in Adult Zebrafish
1Department of Hematology and Oncology, Children’s Hospital Boston, 2Harvard Stem Cell Institute, Harvard Medical School, 3Department of Medical Oncology, Dana Farber Cancer Institute
Here we show how to do retro-orbital injection in adult zebrafish.
Other articles by Pulin Li on PubMed
The Essential Role of the Flexible Termini in the Temperature-responsiveness of the Oligomeric State and Chaperone-like Activity for the Polydisperse Small Heat Shock Protein IbpB from Escherichia Coli
Small heat shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones that are believed to prevent irreversible aggregation of other cellular proteins under stress conditions. One of the most prominent features of sHSPs is that they exist as homo-oligomers. Examples of both monodisperse and polydisperse oligomers are found within this family. The small heat shock inclusion-body binding protein B (IbpB) of Escherichia coli, originally discovered as a component of inclusion bodies, exhibits a pronounced polydispersity in its oligomeric state. This research was performed to elucidate the temperature effect on the oligomeric state and chaperone-like activity of the polydisperse IbpB oligomers, as well as the structural basis for such a temperature effect. The data presented here demonstrate that the large oligomers of IbpB progressively dissociate into smaller ones at increasing heat-shock temperatures, accompanied by a notable enhancement of chaperone-like activities. The secondary structure, enriched mainly by beta-strands, is slightly changed with such temperature increases. The dimeric building blocks, which seem to be highly stable, act as the functional unit of IbpB. Limited proteolysis was used to identify the susceptible sites in IbpB that may compose the subunit interfaces, which indicated that the 11 residues at both the N and the C terminus are highly flexible and the removal of each will lead to the formation of dimers, as well as the disappearance of chaperone-like activities. Truncation of 11 residues from either end, using recombinant DNA technology, also led to the formation of dimeric mutant IbpB proteins lacking chaperone-like activities. Taken together, the flexible termini appear to be essential for small heat shock protein IbpB to generate various temperature-responsive oligomers, which exhibit various levels of chaperone-like activities, by interlinking or separating the dimer building blocks.
Small Heat-shock Proteins Function in the Insoluble Protein Complex
Small heat-shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones. The current model proposes that sHSPs function to prevent irreversible aggregation of non-native proteins by forming soluble complex. The chaperone activity of sHSPs is usually determined by the capacity to suppress thermally or chemically induced protein aggregation. However, sHSPs were frequently found in the insoluble complex particularly in vivo. In this report, it is clearly revealed that the insoluble sHSP/substrate complex is formed when sHSP is overloaded with non-native substrates, which is the very case under in vivo conditions. The proposal that sHSPs function to prevent the protein aggregation seems misleading. sHSPs appear to promote the elimination of protein aggregates by incorporating into the insoluble protein complex.
The Dramatically Increased Chaperone Activity of Small Heat-shock Protein IbpB is Retained for an Extended Period of Time After the Stress Condition is Removed
sHSP (small heat-shock protein) IbpB (inclusion-body-binding protein B) from Escherichia coli is known as an ATP-independent holding chaperone which prevents the insolubilization of aggregation-prone proteins by forming stable complexes with them. It was found that the chaperone function of IbpB is greatly modulated by the ambient temperature, i.e. when the temperature increases from normal to heat-shock, the chaperone activity of IbpB is dramatically elevated to a level that allows it to effectively bind the aggregation-prone client proteins. Although it is generally believed that the release and refolding of the client protein from the sHSPs depends on the aid of the ATP-dependent chaperones such as Hsp (heat-shock protein) 70 and Hsp100 when the ambient temperature recovers from heat-shock to normal, the behaviour of the sHSPs during this recovery stage has not yet been investigated. In the present study, we examined the behaviour and properties of IbpB upon temperature decrease from heat-shock to normal. We found that IbpB, which becomes functional only under heat-shock conditions, retains the chaperone activity for an extended period of time after the heat-shock stress condition is removed. A detail comparison demonstrates that such preconditioned IbpB is distinguished from the non-preconditioned IbpB by a remarkable conformational transformation, including a significant increase in the flexibility of the N- and C-terminal regions, as well as enhanced dynamic subunit dissociation/reassociation. Intriguingly, the preconditioned IbpB displayed a dramatic decrease in its surface hydrophobicity, suggesting that the exposure of hydrophobic sites might not be the sole determinant for IbpB to exhibit chaperone activity. We propose that the maintenance of the chaperone activity for such 'holdases' as sHSPs would be important for cells to recover from heat-shock stress.
Hematopoietic Stem Cell Development is Dependent on Blood Flow
During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and identifies NO as a conserved downstream regulator of HSC development.
Resolving the Controversy About N-cadherin and Hematopoietic Stem Cells
Discrepancies in published results about the role of N-cadherin in hematopoietic stem cells have led to confusion in the field. Attempting to settle the disagreements and reach a consensus, we undertook a collective discussion approach. This process clarified a number of issues but left some questions still unresolved.
Ubiquitous Transgene Expression and Cre-based Recombination Driven by the Ubiquitin Promoter in Zebrafish
Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.
Stem Cell Migration: a Zebrafish Model
Compared with other vertebrate animal models, zebrafish (Danio rerio) has its superior advantages for studying stem cell migration. Zebrafish have similar tissues and organs as mammals, where tissue-specific stem cells reside in. Zebrafish eggs are externally fertilized and remain transparent until most of the organs are fully developed. This allows imaging stem cells in vivo very easily. Recently, a zebrafish double pigmentation mutant, casper, became a new popular imaging model in the zebrafish field due to its completely transparent bodies in adulthood. It has been used as an excellent model to study adult hematopoietic stem cell (HSC) in the transplantation setting. The unparalleled imaging power of zebrafish provides great opportunities of tracing stem cells in vivo in the developmental and regenerative context. In this chapter, we use HSC as an example and combine the powerful imaging techniques in zebrafish, to provide protocols for in vivo imaging fluorescence-labeled stem cell migration, stem cell fate tracing in zebrafish embryos, HSC transplantation, and in vivo imaging in both zebrafish embryos and adults. These techniques can also be applied to other types of stem cells in zebrafish embryos and adults.
Transplantation in Zebrafish
Tissue or cell transplantation has been an extremely valuable technique for studying developmental potential of certain cell population, dissecting cell-environment interaction relationship, identifying stem cells, and many other applications. One key technical requirement for performing transplantation assay is the capability of distinguishing the transplanted donor cells from the endogenous host cells, and tracing the donor cells over time. Zebrafish has emerged as an excellent model organism for performing transplantation assay, thanks to the transparency of embryos during development and even certain adults. Using transgenic techniques and fast-evolving imaging technology, fluorescence-labeled donor cells can be easily identified and studied in vivo. In this chapter, we will first discuss the rationale of different types of zebrafish transplantation in both embryos and adults, and then focus on detailed methods of three types of transplantation: blastula/gastrula transplantation for mosaic analysis, stem cell transplantation, and tumor transplantation.
Lineage Regulators Direct BMP and Wnt Pathways to Cell-specific Programs During Differentiation and Regeneration
BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.
An Optical Platform for Cell Tracking in Adult Zebrafish
Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research. © 2011 International Society for Advancement of Cytometry.
