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The precise assembly of an integrated network of blood vessels is essential for the survival of vertebrate embryos. However, the processes by which primitive endothelial cells form mature vessels capable of supplying oxygen and nutrients to developing tissues remain incompletely understood. Here, we propose a role for Radar, one of the zebrafish orthologues of gdf6, in establishing integrity of the trunk vasculature in zebrafish embryos. We show that radar expression is appropriately placed, both spatially and temporally, to perform such a role. Transcripts for radar are detected in the hypochord and the primitive gut endoderm. These tissues intimately flank developing axial vessels in the trunk and have been previously implicated in the regulation of vascular development. Morpholino-based targeted gene knock-down has generated a Radar-specific loss-of-function zebrafish model. These embryos display normal initiation of vascular patterning and commencement of circulation. However, by day 2 of development, the integrity of the axial vasculature is compromised with hemorrhages and circulation short-circuits throughout the developing trunk. We show that this aberrant vascular development is specific to a reduction of the radar gene product. These results suggest that Radar is involved in a signaling pathway required for establishing the integrity of the axial vessels during zebrafish development, Key Words: radar; GDF6a; BMP; TGF-[beta]; zebrafish; angiogenesis; hypochord; vasculogenesis; morpholino; microangiography.

Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens. Developmental Dynamics, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein-labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter-transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

Zebrafish are ideally suited for the live imaging of early immune cell compartments. Macrophages that initially appear on the yolk surface prior to the onset of circulation are the first functional immune cells within the embryo, predating the emergence of the first granulocytic cells-the heterophilic neutrophils. Both cell types have been shown in zebrafish to contribute to a robust early innate immune system, capable of clearing systemic infections and participating in wound healing. Early imaging of these cells within zebrafish relied on differential interference contrast (DIC) optics because of their superficial locations in the embryo and the optical transparency of embryonic tissues. Recently, the creation of a number of transgenic reporter lines possessing fluorescently marked myelomonocytic compartments provides the potential to live image these cells during the inflammatory response, in real-time, within a whole animal context. Live imaging during the different stages of inflammation using this expanding library of reporter lines, coupled with the ability to model aspects of human disease in the zebrafish system, have the potential to provide significant insights into inflammation and diseases associated with its dysregulation.

Studies in zebrafish have potential to contribute to understanding of the vertebrate hematopoietic and vasculogenic systems. Our research has examined the roles of several molecules in pathways that lead to the development of blood and vessels in zebrafish, and has provided insights into the regulation of these processes. Gdf6a/radar, a member of the bone morphogenetic protein (BMP) family, is expressed in the zebrafish hypochord and primitive gut endoderm; structures that flank the developing dorsal aorta and posterior cardinal vein. This pattern of expression positions Gdf6a/radar as a candidate regulator of vasculogenesis. Support for such a role has come from experiments where Gdf6a/radar function was depleted with antisense morpholino oligonucleotides. This resulted in vascular leakiness, suggesting that Gdf6a/radar is involved in maintenance of vascular integrity. The transcription factor Runx1 is known to play a critical role in mammalian definitive hematopoiesis. When Runx1 expression domains and function were analyzed in zebrafish, the importance of this gene in definitive hematopoiesis was confirmed. However there was also evidence for a wider role, including involvement in vascular development and neuropoiesis. This work has laid the foundation for an ethylnitrosourea (ENU) mutagenesis screen based on runx1 whole-mount in situ hybridzation, that aims to identify genes operative in the runx1 pathway. An additional member of the Runx family, Runx3, is also involved in developmental hematopoiesis, with a function distinct from that of Runx1. We hypothesize that Runx1 and Runx3 form a continuum of transcriptional control within the hematopoietic system. An added attraction of zebrafish is that models of human disease can be generated, and we have shown that this system has potential for the study of Runx1-mediated leukemogenesis.