From fish tank to bedside in cancer therapy: an interview with Leonard Zon

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Portrait of a Stem Cell

AbstractThere is great enthusiasm for the potential use of stem cells in treating tissue degenerative disorders, but little is known about the intrinsic molecular programs defining self-renewal and differentiation. New data sets produced by transcriptional profiling of purified stem cell populations begin to establish the nature of “stemness.

Hematopoietic Stem Cells, Hematopoiesis and Disease: Lessons from the Zebrafish Model

The zebrafish model is rapidly gaining prominence in the study of development, hematopoiesis, and disease. The zebrafish provides distinct advantages over other vertebrate models during early embryonic development by producing transparent, externally fertilized embryos. Embryonic zebrafish are easily visualized and manipulated through microinjection, chemical treatment, and mutagenesis. These procedures have contributed to large-scale chemical, suppressor, and genetic screens to identify hematopoietic gene mutations. Genomic conservation and local synteny between the human and zebrafish genomes make genome-scale and epigenetic analysis of these mutations (by microarray, chromatin immunoprecipitation sequencing, and RNA sequencing procedures) powerful methods for translational research and medical discovery. In addition, large-scale screening techniques have resulted in the identification of several small molecules capable of rescuing hematopoietic defects and inhibiting disease. Here, we discuss the contributions of the zebrafish model to the understanding of hematopoiesis, hematopoietic stem cell development, and disease-related discovery. We also highlight the recent discovery of small molecules with clinical promise, such as dimethyl prostaglandin E2, 3F8, and thiazole-carboxamide 10A

Histocompatibility and Hematopoietic Transplantation in the Zebrafish

The zebrafish has proven to be an excellent model for human disease, particularly hematopoietic diseases, since these fish make similar types of blood cells as humans and other mammals. The genetic program that regulates the development and differentiation of hematopoietic cells is highly conserved. Hematopoietic stem cells (HSCs) are the source of all the blood cells needed by an organism during its lifetime. Identifying an HSC requires a functional assay, namely, a transplantation assay consisting of multilineage engraftment of a recipient and subsequent serial transplant recipients. In the past decade, several types of hematopoietic transplant assays have been developed in the zebrafish. An understanding of the major histocompatibility complex (MHC) genes in the zebrafish has lagged behind transplantation experiments, limiting the ability to perform unbiased competitive transplantation assays. This paper summarizes the different hematopoietic transplantation experiments performed in the zebrafish, both with and without immunologic matching, and discusses future directions for this powerful experimental model of human blood diseases

Taking the Stem Cell Debate to the Public

In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholder

A New System for the Rapid Collection of Large Numbers of Developmentally Staged Zebrafish Embryos

The zebrafish is an excellent genetic and developmental model system used to study biology and disease. While the zebrafish model is associated with high fecundity, its reproductive potential has not been completely realized by scientists. One major issue is that embryo collection is inefficient. Here, we have developed an innovative breeding vessel designed to stimulate the natural reproductive behavior of the fish. This novel apparatus allows us to collect large numbers of developmentally synchronized embryos in brief and defined windows of time, and with minimal investments in labor and space. To demonstrate the efficacy of this approach, we placed three separate groups (n = 180) of fish in the vessel and allowed them to spawn for 10-minute intervals. During these trials, which were repeated three times, the fish produced 86006917, 84006794, and 680061997 embryos, respectively. This level of embryo production is nearly twice what we were able to achieve when using conventional crossing equipment with some of the same fish, and it required significantly less room and time to set up and break down. This system overcomes major space and labor restrictions inherent in spawning equipment currently used in the field, and will greatly accelerate efforts to improve the scale and throughput of experiments.Stem Cell and Regenerative Biolog

Zebrafish scl functions independently in hematopoietic and endothelial development

AbstractThe SCL transcription factor is critically important for vertebrate hematopoiesis and angiogenesis, and has been postulated to induce hemangioblasts, bipotential precursors for blood and endothelial cells. To investigate the function of scl during zebrafish hematopoietic and endothelial development, we utilized site-directed, anti-sense morpholinos to inhibit scl mRNA. Knockdown of scl resulted in a loss of primitive and definitive hematopoietic cell lineages. However, the expression of early hematopoietic genes, gata2 and lmo2, was unaffected, suggesting that hematopoietic cells were present but unable to further differentiate. Using gene expression analysis and visualization of vessel formation in live animals harboring an lmo2 promoter-green fluorescent protein reporter transgene (Tg(lmo2:EGFP)), we show that angioblasts were specified normally in the absence of scl, but later defects in angiogenesis were evident. While scl was not required for angioblast specification, forced expression of exogenous scl caused an expansion of both hematopoietic and endothelial gene expression, and a loss of somitic tissue. In cloche and spadetail mutants, forced expression of scl resulted in an expansion of hematopoietic but not endothelial tissue. Surprisingly, in cloche, lmo2 was not induced in response to scl over-expression. Taken together, these findings support distinct roles for scl in hematopoietic and endothelial development, downstream of hemangioblast development

A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish

SummaryCRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish