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Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis

¹ Division of Molecular Medicine, The University of Auckland, Auckland, New Zealand
² Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

View larger version (61K): [in a new window]   Fig. 1. (A) Sequence analysis (GenBank Accession Number, AF391125). The runt domain (RD) is in red. The region encompassed by two perpendicular bars indicates the longer template, and the region in green, the shorter template for in situ probes. The runx1-MO1 and runx1-MO2 binding sites are indicated by horizontal lines. (B) Percentage amino acid identity of zebrafish Runx1 when compared with the Xenopus, mouse and human orthologs. Overall identity is indicated in black, and identity within the RD in red. (C) Phylogenetic analysis of runx1. The GenBank Accession Numbers of the genes included in the analysis are: human RUNX1, L34598; mouse Runx1, D13802; Xenopus runx1, AF035446; human RUNX2, XM_004126; mouse Runx2, AF010284; human RUNX3, X79550; mouse Runx3, AF155880; and zebrafish runx3 (runxb transcript 1), AB043788. Bootstrap support values are given in the nodes. (D) The runx1 locus maps to the upper portion of zebrafish LG1 in a region, showing conserved syntenies with a portion of human chromosome 21. Distances on LG1 are in centiMorgans (cM) (the entire chromosome is 122 cM long), and distances on Hsa21 are in megabases (Mb). Abbreviations: gart (AF257743; D. B. Slavov and K. Gardiner, unpublished), ortholog of GART in human; app (AF257742; D. B. Slavov and K. Gardiner, unpublished), ortholog of APP in human; mx1 (AW202878; Washington University Zebrafish EST Project 1998, unpublished), apparent ortholog of MX1 in human (Woods et al., 2000); other markers of the form Z4593 are from Shimoda et al. (Shimoda et al., 1999) and http://zebrafish.mgh.harvard.edu/.

View larger version (73K): [in a new window]   Fig. 2. Expression of runx1 in zebrafish wild-type (A-N) and mutant (O,P) embryos. In situ was performed with the longer probe, except D,E, which were carried out with the shorter probe. (A-E,O,P) Whole embryos; dorsal views, anterior upwards (A-C), lateral views, anterior towards the left (D,E,O,P). (F-H) Posterior halves of embryos; dorsal view, posterior downwards (F), lateral views, anterior towards the left (G,H). (I,J) Transverse sections of the embryo corresponding to a line in H, with J a higher magnification of the area marked in I. (K-N) Dorsal views of head areas, except K, dorsolateral. Stages in hpf are indicated. (A) Diffuse pre-zygotic expression. (B-F) Expression within the LPM (arrowheads) and in Rohon-Beard cells (arrows). (G-J) Expression in the ICM (arrowheads), Rohon-Beard cells (arrow in G) and ventral wall of the dorsal aorta (arrows in H-J). Endothelial cells are indicated in J. Cells with runx1 expression show a different nuclear morphology. (K-N) Expression in the olfactory epithelium (arrowheads) and in putative cranial nerve VIII ganglia (arrows). (O,P) Arrows indicate markedly reduced runx1 expression in the ICM in spadetail and cloche respectively. NT, neural tube; NO, notochord; AV, axial vein; e, endothelial cell, ov, otic vesicle. Scale bars: 25 µm.

View larger version (72K): [in a new window]   Fig. 3. Characterization of runx1 expression. (A-E) Two-color in situ hybridization using runx1 (purple; NBT/BCIP) and scl (red; Fast Red) riboprobes (purple and red arrowheads, respectively). (C,E) Fluorescence of scl signal using rhodamine filter. (F-I) Co-labeling of embryos hybridized with runx1 riboprobe (purple) with anti-HNK-1 (brown) (purple and brown arrows respectively). (A) Dorsal view of whole embryo. (B,C) Higher magnifications of an area boxed in A. (D,E) Lateral view of posterior portion of embryo, anterior towards the left. (F-I) Dorsal views of mid-trunk embryo regions, anterior towards the left. (G,I) Higher magnifications of areas boxed in F,H, respectively. Stages in hpf are indicated. (A) runx1 and scl overlap in the LPM (arrowheads). (B,C) Overlap of runx1 and scl in individual cells (arrowheads). (D,E) runx1 expression is weaker than scl in the posterior ICM. (F,G) Rohon-Beard cells with no runx1 expression (brown arrowheads), and a cell with dual expression (purple arrowhead). (H,I) Putative cranial nerve VIII nuclei with overlapping runx1 and HNK-1 expression (purple arrowheads). Otic vesicle (ov) is indicated.

View larger version (76K): [in a new window]   Fig. 4. Effects of runx1 expression in cloche (A,B,D-F) and wild-type (C) zebrafish embryos at 48 hpf. Lateral views of anterior regions (A-C) and whole embryos (D-F), anterior towards the left. (A-C) Morphology and (D-F) expression of hbbe3 (globin). (A,B) Blood in the trunk of clo embryo injected with runx1 (arrowhead) compared with uninjected clo. Arrows indicate dilated heart. (C) Ectopic blood (arrowhead). (D-F) hbbe3 (globin) expression on the yolk (arrowhead) and in the trunk (arrow) in clo injected with runx1 compared with uninjected clo and wild type.

View larger version (80K): [in a new window]   Fig. 5. Phenotypic effects of runx1-MO injections. (A-H) Morphological changes at 24 hpf (A-C), 48 hpf (D-H) and circulation defect visualized by microangiography at 52 hpf (I,J). All are lateral views of whole embryos (A-E), posterior halves of embryos (F-H) and mid-trunk regions (I,J), anterior towards the left. Control embryos are indicated. (A,B) Blood cells accumulated in the anterior (arrows) and posterior (arrowheads) ICM in comparison with control in C. (D) Lack of normal circulation. Blood cells accumulated in the aorta (region encompassed by two black arrows). Empty heart and edematous vitelline vessels (red arrow). Underdeveloped head (red arrowhead). Otic vesicle containing three otoliths (black arrowhead). (E) Normal circulating blood cells (arrow). (F,G) Collections of blood cells in ventral tail ICM (arrowheads). (H) Normal tail circulation with caudal artery (arrowhead) and vein (arrow). (I) Interrupted aortic blood flow (red arrow) with lack of flow in cardinal vein and intersegmental vessels. (J) Normal circulation; dorsal aorta (red arrow), posterior cardinal vein (green arrow) and intersegmental vessel (red arrowhead).

View larger version (58K): [in a new window]   Fig. 6. Abnormal vasculature in the runx1-mo embryos demonstrated by molecular (A,B,E,F,I,J) and histological (C,D,G,H,K,L) analyses at 48 hpf. Expression of hbbe3 (globin) (A,B) and flk-1 (E,F,I,J). Lateral views of whole embryos (A,B) and posterior embryo region (E,F,I,J); anterior towards the left. Cross sections of the mid-trunk (C,D) and tail (G,H,K,L) regions. (K,L) Higher magnifications of G,H, respectively. Controls are indicated. (A) hbbe3 (globin) expression is limited to the posterior embryo (arrow) with lack of expression in the circulation (arrowhead). (B) Normal hbbe3 (globin) expression within vitelline vessels (arrowhead) and aorta (arrow). (E) ‘Wavy’ pattern of flk-1 expression in axial vessels (arrowhead). (F) Ectopic (arrowhead) and missing (arrow) flk-1 expression. (I) Interrupted expression in the axial vein (arrow), expansion of expression in the tail (black arrowhead) and a loss of expression in intersegmental vessels (blue arrowhead). (J) Normal flk-1 expression in the axial vessels (arrow) and intersegmental vessels (blue arrowhead). (C) Multiple and disorganized vascular channels replacing normal trunk vessels (arrow). (D) Normal dorsal aorta (arrow) and axial vein (arrowhead). (G,K) Expansion of tail region with multiple dilated capillaries (arrow). (H,L) Normal caudal vessels (arrows). Scale bars: 50 µm.

View larger version (71K): [in a new window]   Fig. 7. Abnormal hematopoiesis in runx1-mo embryos. Two-color in situ hybridization for flk-1 (purple) and scl (red) expression (A,B,E,F,I,J,M,N) and single hybridization with the myb riboprobe (O,P). Lateral views of the posterior (A,B,I,J,M,N) and anterior (E,F) embryo regions, and whole embryos (O,P); anterior towards the left. (M,N) Higher magnification of the ICM regions. Histological cross-sections of the tail (C) and trunk (G,K). Cytology of cells collected from ICM collections (D) and vitelline vessels (H,L). All are 48 hpf, except A,B, which are 24 hpf. Controls are indicated. (A,B) scl-positive cells (red arrow) accumulate in the ICM of runx1-mo embryo. (E,F) Lack of scl expression in circulation (red arrowhead) in runx1-mo embryo. (I) Cells that accumulate in the tail maintain scl expression (red arrowhead). In A,E,I, axial vessels (purple arrows) and intersegmental vessels (purple arrowheads) are poorly formed when compared with controls (B,F,J). (M,N) scl-positive cells (red arrow) predominate in runx1-mo embryo when compared with the predominantly flk-1-positive population in control (purple arrows). (C) Large immature cells (arrow) mixed with necrotic cells (arrowhead). (D) Blast-like morphology of accumulated cells with a mitotic figure (arrow). (G, arrow; H) Erythroid cells with delayed maturation compared with normal in K,L. (O,P) Reduction in myb expression in the aorta of runx1-mo embryo compared with control (arrowheads). Scale bars: 10 µm.

View larger version (88K): [in a new window]   Fig. 8. Neurological effects of Runx1 depletion. Morphological phenotype (A-D) and effect on HNK-1 expression (E,F) at 48 hpf. (A,B) Lateral and (E,F) dorsal views of head regions. (C,D) Lateral views of tail regions; anterior towards the left. Controls are indicated. (A,B) Enlarged ventricular space (arrowhead) and supernumerary otoliths (arrow) in runx1-mo embryo compared with normal. (C,D) Interrupted notochord (arrow indicates its anterior and arrowhead indicates posterior region in runx1-mo embryo). The red arrow in C indicates an accumulation of blood cells. In control, notochord (black arrow) and caudal vessels (red arrow) are shown. (E,F) Reduction in trigeminal descending (arrow) and Rohon-Beard ascending (arrowhead) central axons and abnormal localization of Rohon-Beard cells (brown arrow).

View larger version (68K): [in a new window]   Fig. 9. Effects of RUNX1-CBF2T1 expression in zebrafish embryos. (A) Western analysis of Jurkat cell lysate (lane 1) and extracts from 18 hpf embryos injected with pCS2cmv-RUNX1-CBF2T1 (lane 2), pCS2 vector alone (lane 3) and uninjected embryos (lane 4). (B) Tabulated summary of phenotypes generated by transient expression of RUNX1-CBF2T1 following injection of construct at two doses, compared with pCS2 vector alone and uninjected embryos. (C-N) Morphological changes at 24 hpf (C,D) and 48 hpf (E-N). (C-J) Whole embryos and (K-N) head regions; lateral views except L, which is ventral; anterior towards the left for all. Controls are indicated. (C,D) Blood accumulated in the ICM region compared with normal (arrowheads). (E,F) Embryos with no circulation and blood accumulated in the proximal aorta (arrow) and ICM (arrowhead). (G,H) Aberrant circulation with blood pooling in the ICM (arrows) and associated CNS bleed (arrowhead). (I) Normal circulation with CNS hemorrhage (arrowhead). (K-M) Arrowheads indicate intraventricular, intracerebral and pericardial hemorrhages respectively.

View larger version (83K): [in a new window]   Fig. 10. Histology of RUNX1-CBF2T1-related intracranial hemorrhages. Hematoxylin and Eosin staining of sagittal head sections at 48 hpf. Control is indicated. Arrows in A and B indicate intraventricular and intracerebral hemorrhages, respectively. (D,E) Higher magnifications of areas boxed in A,B respectively, with arrows indicating areas of bleeding. In D, an associated small intracerebral hemorrhage is shown (arrowhead). (F) At higher magnification, immature erythroid cells were present within hemorrhages (arrow). Scale bars: 50 µm.

View larger version (69K): [in a new window]   Fig. 11. Molecular and cellular effects of RUNX1-CBF2T1 expression at 48 hpf. Whole-mount in situ hybridization for hbbe3 (globin) (A,B), flk-1 (E,F) and myb (I,J) expression. Lateral views, anterior towards the left. Cells aspirated from the ICM region (C,D) and circulation (G,H,K,L). Controls are indicated. (A,B) hbbe3 (globin) expression is reduced in the circulation (arrowhead) and tail (arrow) compared with control. (E,F) flk-1 expression in the tail (arrows) is abnormal. (I,J) myb expression is markedly reduced in the dorsal aorta (arrows). (C) Immature, blast-like cells accumulated in the ICM. (D) Cluster of atypical early cells (arrow). (G) Nuclear bridge (arrowhead), doughnut-shape nucleus (arrow). (H) Binuclear cell (arrowhead). (K) Cleaved nuclei (black arrowheads), mitotic figure (arrow) and a megaloblast (purple arrowhead). Scale bars: 10 µm.

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