Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes.

During vertebrate embryogenesis, hematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM) region. A zebrafish chemical genetic screen identified compounds that regulate blood flow as modulators of HSC formation. silent heart (sih) embryos that lack a heartbeat and blood circulation exhibited severely reduced HSCs. Blood flow modifiers exerted their effects after the onset of heartbeat; however, nitric oxide (NO) donors affected HSC induction even when treatment occurred prior to the initiation of circulation, and rescued HSCs in sih mutants. NO synthase (Nos) inhibitors and morpholino-knockdown of nos1 (nnos/enos) blocked HSC development. Embryonic transplantation assays demonstrated a cell-autonomous requirement for nos1. Nos3 (eNos) was expressed in HSCs in the murine AGM. Intrauterine Nos inhibition or Nos3 deficiency in mice resulted in the absence of hematopoietic clusters and reduced transplantable progenitors and HSCs. This work links blood flow to AGM hematopoiesis, and identifies NO as a conserved downstream regulator of HSC development.

Iron is required to produce haem and iron-sulphur (Fe-S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe-S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of grx5 yeast Fe-S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe-S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5' iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe-S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe-S cluster assembly.

Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b) are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.

Cyclosporin A, a potent and clinically-important immunosuppressive drug (SandimmunR), is synthesized from its precursor amino acids by cyclosporin synthetase, a single multi-functional enzyme. In this study we report the cloning of the corresponding coding region of this synthetase. It contains an open reading frame of 45.8 kb which encodes a peptide with a calculated M(r) of 1,689,243. The predicted gene product contains 11 amino-acid-activating domains that are very similar to one another and to the domains of other peptide synthetases. Seven of these domains harbour N-methyltransferase functions. This is the largest genomic ORF described so far.

Septic cardiomyopathy is an important contributor to organ dysfunction in sepsis. Guided treatment of septic cardiomyopathy may affect patients' prognosis, especially when their cardiac index is substantially decreased. The implication of septic cardiomyopathy for both short- and long-term outcomes is an important area for future investigation.

A major goal of cancer research has been to identify genes that contribute to cancer formation. The similar pathology between zebrafish and human tumors, as well as the past success of large-scale genetic screens in uncovering human disease genes, makes zebrafish an ideal system in which to find such new genes. Here, we show that a zebrafish forward genetic screen uncovered multiple cell proliferation mutants including one mutant, crash&burn (crb), that represents a loss-of-function mutation in bmyb, a transcriptional regulator and member of a putative protooncogene family. crb mutant embryos have defects in mitotic progression and spindle formation, and exhibit genome instability. Regulation of cyclin B levels by bmyb appears to be the mechanism of mitotic accumulation in crb. Carcinogenesis studies reveal increased cancer susceptibility in adult crb heterozygotes. Geneexpression signatures associated with loss of bmyb in zebrafish are also correlated with conserved signatures in human tumor samples, and down-regulation of the B-myb signature genes is associated with retention of p53 function. Our findings show that zebrafish screens can uncover cancer pathways, and demonstrate that loss of function of bmyb is associated with cancer.cell cycle

Vascular endothelial growth factor A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of endothelial cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholinodisrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa 121 and VegfAa 165 isoforms, the VegfAb isoforms VegfAb 171 IntroductionDifferentiation of endothelial cells to form the vascular network of arteries and veins requires a critical mitogenic signal, vascular endothelial growth factor A (VEGFA), which is transduced to the nucleus through a number of functionally redundant receptors. These receptors include the type III receptor tyrosine kinases VEGFR1/FLT1, VEGFR2/FLK1 (human KDR), and type I nonkinase transmembrane receptors neuropilin 1 (NRP1) and NRP2, 1 which are critical mediators of both vasculogenesis (de novo formation of blood vessels) and angiogenesis (sprouting of vessels from preexisting vessels). 2 Mice hemizygous at their single VegfA locus show defective vasculogenesis and die between 11 and 12 days after conception (dpc). [3][4][5] Embryos homozygous for a targeted disruption of Flk1 (Vegfr2) die between 8.5 and 9.5 dpc from defects in both hematopoiesis and vasculogenesis, but disruption of Flt1 (Vegfr1) only leads to perturbed vascular patterning in full-term embryos. 6,7 The tight regulation of VEGFA levels and the requirement for its receptors in hematopoiesis and vasculogenesis point to its critical role in the formation of blood vessels during development.Differential splicing of the single VEGFA locus in humans gives rise to multiple protein isoforms, ranging in size from 121 to 206 amino acids (aa's) (VEGFA 121 , VEGFA 145 , VEGFA 165 , VEGFA 189 , and VEGF 206 ). 8,9 The VEGF isoforms differ mainly by the presence or absence of 2 heparin-binding domains encoded within exon 6 and 7 sequences. [10][11][12] Expression of a single VEGFA isoform rescues VegfA mutant mice through birth, although the vascular networks differ with expression of different isoforms. [13][14][15][16] VEGFA splicing is regulated during development and in disease, resulting in tissue-and stage-specific isoform ratios, which allow for distinct, context-dependent VEGFA signaling. 17 Zebrafish have proven useful for dissecting vascular developmental pathways. 18 A single zebrafish vegfA gene has been previously described, encoding the 121-and 165-aa isoforms. 19,20 Although VegfA 121 is the predominant isoform in early embryos and VegfA 165 in adults, other alternate transcripts exist in specific tissues. 21 The initial establishment of axial vasculature patterning does not require VegfA, but the sprouting of intersegmental vessels does. 22 Together, these results show that VegfA...