Characterization of immune-matched hematopoietic transplantation in zebrafish.

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia.

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence.

Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonad-mesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plcgamma1) mutant, fail to specify HSCs, overexpression of notch or runx1 can rescue their hematopoietic defect. The most proximal HSC mutants, such as hdac1, were found to have no defect in vessel or artery formation. Further analysis demonstrated that hdac1 acts downstream of Notch signaling but upstream or in parallel to runx1 to promote AGM hematopoiesis. Together, our results establish a hierarchy of signaling programs required and sufficient for HSC emergence in the AGM.

Characterization of the zebrafish T cell receptor beta locus.

Zebrafish (Danio rerio) has become an increasingly important model for immunological study. Its immune system is remarkably similar to that of mammals and includes both the adaptive and innate branches. Zebrafish T cells express functional T cell receptors (TCR), and all four TCR loci are present within the genome. Using 5'-rapid amplification of cDNA ends, we cloned and sequenced zebrafish TCRbeta transcripts. TCRbeta VDJ coding joints demonstrate conservation of mechanisms used by other vertebrate species to increase junctional diversity. Using the sequences obtained, along with previously published data, we comprehensively annotated the zebrafish TCRbeta locus. Overall, organization of the locus resembles that seen in mammals. There are 51 V segments, a single D segment, 27 Jbeta1 segments, a single Jbeta2 segment, and two constant regions. This description of the zebrafish TCRbeta locus has the potential to enhance immunological research in zebrafish and further our understanding of mammalian TCR repertoire generation.

T2-TrpRS inhibits preretinal neovascularization and enhances physiological vascular regrowth in OIR as assessed by a new method of quantification.

PURPOSE: A carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS) has demonstrated potent angiostatic activity during retinal developmental neovascularization in vivo. The effects of T2-TrpRS on pathologic neovascularization were tested and compared with a potent VEGF antagonist using the mouse model of oxygen-induced retinopathy (OIR). METHODS: C57BL/6J mice were transiently exposed to hyperoxic conditions (75% O2) between postnatal day 7 (P7) and P12 and then returned to room air. Retinas were isolated, blood vessels stained with isolectin Griffonia simplicifolia, images of retinal whole-mounts acquired, and the area of vascular obliteration and extent of preretinal neovascularization quantified. This method was compared to the commonly used method of OIR quantification in which the number of pre-inner limiting membrane (ILM) nuclei is counted in serial sections of whole eyes. To assess the angiostatic activity of T2-TrpRS, mice were injected intravitreally at P12 with either T2-TrpRS, a VEGF aptamer, or vehicle (PBS) alone, and the effects on area of obliteration and on preretinal neovascular tuft formation were assessed. RESULTS: Using a modified method of quantification in the mouse OIR model based on images of isolectin-stained retinal wholemounts, we were able to assess reliably and consistently both vascular obliteration and preretinal neovascular tuft formation in the same specimen. T2-TrpRS demonstrated potent angiostatic activity, reducing the appearance of pathologic neovascular tufts by up to 90%. Surprisingly, T2-TrpRS also enhanced physiological revascularization of the obliterated retinal vasculature, reducing these areas by up to 60% compared with PBS-injected eyes. In contrast, the VEGF antagonist, while similarly reducing preretinal neovascular tuft formation, did not enhance revascularization of the obliterated areas. CONCLUSIONS: Use of a rapid, quantifiable method to assess the effect of T2-TrpRS on retinal angiogenesis in the OIR model demonstrates the importance of a quantification system that permits simultaneous analysis of a drug's effect on vascular obliteration as well as on preretinal neovascularization. The results obtained using this method suggest enhanced clinical value for compounds such as T2-TrpRS that not only inhibit pathologic neovascularization, but also facilitate physiological revascularization of ischemic tissue.

Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.

Quantification and localization of the IGF/insulin system expression in retinal blood vessels and neurons during oxygen-induced retinopathy in mice.

PURPOSE: Retinopathy is a result of pathologic angiogenesis influenced by insulinlike growth factor (IGF)-1. The authors examined the local expression of the IGF/insulin family. METHODS: In retinas with and without oxygen-induced retinopathy, the authors assessed with real-time RT-PCR mRNA expression of the IGF-1 receptor (IGF-1R), insulin receptor (IR), IGF-1, IGF-2, insulin (Ins2), and IGF-binding protein 1 (IGFBP1) to IGFBP6 in total retina from postnatal day (P) 7 to P33 to examine changes over time with the induction of retinopathy and at P17 on laser-captured retinal components to quantitatively localize mRNA expression in the ganglion cell layer, the outer nuclear layer, the inner nuclear layer, normal blood vessels, and neovascular tufts. RESULTS: IGF-1R and IR are expressed predominantly in photoreceptors and in vessels, with scant expression in the rest of the neural retina. IGF-1R expression is more than 100-fold greater than IR. The major local growth factor (expressed in photoreceptors and in blood vessels) is IGF-2 (approximately 1000-fold greater than IGF-1). IGF-1 (approximately 600 copies/10(6) cyclophilin) is expressed throughout the retina. IGFBP2, IGFBP4, and IGFBP5 expression is unchanged with increasing retinal development and with the induction of retinopathy. In contrast, IGFBP3 expression increased more than 5-fold with hypoxia, found in neovascular tufts. CONCLUSIONS: IGF-1R, IR, and the ligand IGF-2 are expressed almost exclusively in photoreceptors and blood vessels. IGFBP3 and IGFBP5 expression increases in neovascular tufts compared with normal vessels. IGF-1 is expressed throughout the retina at much lower levels. These results suggest cross-talk between vessels and photoreceptors in the development of retinopathy and retinal vasculature.

IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth.

Vessel loss precipitates many diseases. In particular, vessel loss resulting in hypoxia induces retinal neovascularization in diabetic retinopathy and in retinopathy of prematurity (ROP), major causes of blindness. Here we define insulin-like growth factor binding protein-3 (IGFBP3) as a new modulator of vascular survival and regrowth in oxygen-induced retinopathy. In IGFBP3-deficient mice, there was a dose-dependent increase in oxygen-induced retinal vessel loss. Subsequent to oxygen-induced retinal vessel loss, Igfbp3(-/-) mice had a 31% decrease in retinal vessel regrowth versus controls after returning to room air. No difference in serum insulin-like growth factor 1 (IGF1) levels was observed among groups. Wild-type mice treated with exogenous IGFBP3 had a significant increase in vessel regrowth. This correlated with a 30% increase in endothelial progenitor cells in the retina at postnatal day 15, indicating that IGFBP3 could be serving as a progenitor cell chemoattractant. In a prospective clinical study, we measured IGFBP3 (and IGF1) plasma levels weekly and examined retinas in all premature infants born at gestational ages <32 weeks at high risk for ROP. The mean level of IGFBP3 at 30-35 weeks postmenstrual age (PMA) for infants with proliferative ROP (ROP stages 3>, n = 13) was 802 microg/liter, and for infants with no ROP (ROP stage 0, n = 38) the mean level was 974 microg/liter (P < 0.03). These results suggest that IGFBP3, acting independently of IGF1, helps to prevent oxygen-induced vessel loss and to promote vascular regrowth after vascular destruction in vivo in a dose-dependent manner, resulting in less retinal neovascularization.