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K-ras/PI3K-Akt signaling is essential for zebrafish hematopoiesis and angiogenesis.

Liu L, Zhu S, Gong Z, Low BC - PLoS ONE (2008)

Bottom Line: Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels.Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor.Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Cell Signaling and Developmental Biology Laboratory, The National University of Singapore, Singapore, The Republic of Singapore.

ABSTRACT
The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.

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Related in: MedlinePlus

Sequence and expression analyses of zebrafish k-ras.(A) Zebrafish k-ras nucleotide and putative amino acid sequence. k-ras-MO1 and k-ras-MO2 binding sites are underlined. (B) Alignment of zebrafish K-ras with known Ras proteins of human, mouse and zebrafish. (C) Phylogenetic analysis of Ras proteins. (D) k-ras expression during embryonic development. k-ras transcripts were detectable from one cell stage (i) and then persist throughout the whole embryos (ii–vi). (i), one cell stage, side view; (ii), 3 hpf (hours-post fertilization), top view; (iii), 10 hpf, dorsal view, anterior to the top; (iv), 10 hpf, bottom view, dorsal to the top; (v), 20 hpf, lateral view, anterior to the left and dorsal to the top; (vi), 20 hpf, dorsal view, anterior to the left.
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pone-0002850-g001: Sequence and expression analyses of zebrafish k-ras.(A) Zebrafish k-ras nucleotide and putative amino acid sequence. k-ras-MO1 and k-ras-MO2 binding sites are underlined. (B) Alignment of zebrafish K-ras with known Ras proteins of human, mouse and zebrafish. (C) Phylogenetic analysis of Ras proteins. (D) k-ras expression during embryonic development. k-ras transcripts were detectable from one cell stage (i) and then persist throughout the whole embryos (ii–vi). (i), one cell stage, side view; (ii), 3 hpf (hours-post fertilization), top view; (iii), 10 hpf, dorsal view, anterior to the top; (iv), 10 hpf, bottom view, dorsal to the top; (v), 20 hpf, lateral view, anterior to the left and dorsal to the top; (vi), 20 hpf, dorsal view, anterior to the left.

Mentions: First, zebrafish k-ras cDNA (GenBank DQ486868) was isolated by reverse transcription-PCR. The encoded protein (Figure 1A) is highly homologous to human K-RAS2B and mouse K-RAS2, signified by a poly-lysine tract at its C-terminus (Figure 1A–1C). It is distinct from two other zebrafish Ras, N-ras [11] and BC048875 (Figure 1B and 1C), especially at their 5′UTR (supporting information Figure S1) that allows subsequent use of specific morpholino to knockdown K-ras. Zebrafish k-ras transcripts were detected from one-cell stage and continued to be detected throughout the whole embryos (Figure 1D). In adult, k-ras expression was detectable in most tissues (supporting information Figure S2).


K-ras/PI3K-Akt signaling is essential for zebrafish hematopoiesis and angiogenesis.

Liu L, Zhu S, Gong Z, Low BC - PLoS ONE (2008)

Sequence and expression analyses of zebrafish k-ras.(A) Zebrafish k-ras nucleotide and putative amino acid sequence. k-ras-MO1 and k-ras-MO2 binding sites are underlined. (B) Alignment of zebrafish K-ras with known Ras proteins of human, mouse and zebrafish. (C) Phylogenetic analysis of Ras proteins. (D) k-ras expression during embryonic development. k-ras transcripts were detectable from one cell stage (i) and then persist throughout the whole embryos (ii–vi). (i), one cell stage, side view; (ii), 3 hpf (hours-post fertilization), top view; (iii), 10 hpf, dorsal view, anterior to the top; (iv), 10 hpf, bottom view, dorsal to the top; (v), 20 hpf, lateral view, anterior to the left and dorsal to the top; (vi), 20 hpf, dorsal view, anterior to the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483249&req=5

pone-0002850-g001: Sequence and expression analyses of zebrafish k-ras.(A) Zebrafish k-ras nucleotide and putative amino acid sequence. k-ras-MO1 and k-ras-MO2 binding sites are underlined. (B) Alignment of zebrafish K-ras with known Ras proteins of human, mouse and zebrafish. (C) Phylogenetic analysis of Ras proteins. (D) k-ras expression during embryonic development. k-ras transcripts were detectable from one cell stage (i) and then persist throughout the whole embryos (ii–vi). (i), one cell stage, side view; (ii), 3 hpf (hours-post fertilization), top view; (iii), 10 hpf, dorsal view, anterior to the top; (iv), 10 hpf, bottom view, dorsal to the top; (v), 20 hpf, lateral view, anterior to the left and dorsal to the top; (vi), 20 hpf, dorsal view, anterior to the left.
Mentions: First, zebrafish k-ras cDNA (GenBank DQ486868) was isolated by reverse transcription-PCR. The encoded protein (Figure 1A) is highly homologous to human K-RAS2B and mouse K-RAS2, signified by a poly-lysine tract at its C-terminus (Figure 1A–1C). It is distinct from two other zebrafish Ras, N-ras [11] and BC048875 (Figure 1B and 1C), especially at their 5′UTR (supporting information Figure S1) that allows subsequent use of specific morpholino to knockdown K-ras. Zebrafish k-ras transcripts were detected from one-cell stage and continued to be detected throughout the whole embryos (Figure 1D). In adult, k-ras expression was detectable in most tissues (supporting information Figure S2).

Bottom Line: Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels.Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor.Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Cell Signaling and Developmental Biology Laboratory, The National University of Singapore, Singapore, The Republic of Singapore.

ABSTRACT
The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.

Show MeSH
Related in: MedlinePlus