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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

Disruption of zebrafish K-ras signaling resulted in the defective hematopoiesis.(A) k-ras-MO injected embryo showed empty heart, without or with few red blood cells inside (indicated by arrows in ii and iii), in comparison to wild type embryo, which showed plenty of red blood cells inside the heart (indicated by arrow in i). Embryos at 3 dpf (days-post fertilization). (B) Plenty of circulating red blood cells inside dorsal aorta (DA), posterior cardinal vein (PCV), inter-segmental vessels (Se), caudal artery (CA) and caudal vein (CV) in wild type embryos (i and iii), while no or less circulating red blood cells were found in k-ras-MO injected embryos inside DA, PCV, CA, CV and Se (ii and iv). Embryos at 3 dpf. (C) k-ras-MO injected embryos showed accumulated red blood cells in some sites that away from the circulation. Embryos at 3 dpf. (D) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside branchial arches (indicated by arrow in i) and heart chambers (indicated by arrow in ii ), while k-ras-MO injected embryo showed less/negative o-Dianisidine staining for branchial arches (indicated by arrow in iii) and heart (indicated by arrow in iv). Embryos at 6 dpf. (E) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside Se (indicated by arrows in i ), dorsal longitudinal anastomotic vessels (DLAV), CA and CV (indicated by arrows in ii), while k-ras-MO injected embryo failed to give the positive o-Dianisidine staining in these corresponding positions (indicated by arrows in iii and iv). Embryos at 6 dpf. All embryos shown are lateral view, with anterior to the left and dorsal to the top.
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pone-0002850-g002: Disruption of zebrafish K-ras signaling resulted in the defective hematopoiesis.(A) k-ras-MO injected embryo showed empty heart, without or with few red blood cells inside (indicated by arrows in ii and iii), in comparison to wild type embryo, which showed plenty of red blood cells inside the heart (indicated by arrow in i). Embryos at 3 dpf (days-post fertilization). (B) Plenty of circulating red blood cells inside dorsal aorta (DA), posterior cardinal vein (PCV), inter-segmental vessels (Se), caudal artery (CA) and caudal vein (CV) in wild type embryos (i and iii), while no or less circulating red blood cells were found in k-ras-MO injected embryos inside DA, PCV, CA, CV and Se (ii and iv). Embryos at 3 dpf. (C) k-ras-MO injected embryos showed accumulated red blood cells in some sites that away from the circulation. Embryos at 3 dpf. (D) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside branchial arches (indicated by arrow in i) and heart chambers (indicated by arrow in ii ), while k-ras-MO injected embryo showed less/negative o-Dianisidine staining for branchial arches (indicated by arrow in iii) and heart (indicated by arrow in iv). Embryos at 6 dpf. (E) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside Se (indicated by arrows in i ), dorsal longitudinal anastomotic vessels (DLAV), CA and CV (indicated by arrows in ii), while k-ras-MO injected embryo failed to give the positive o-Dianisidine staining in these corresponding positions (indicated by arrows in iii and iv). Embryos at 6 dpf. All embryos shown are lateral view, with anterior to the left and dorsal to the top.

Mentions: To identify the functional role of K-ras in vivo, translation of endogenous K-ras was suppressed by targeting k-ras mRNA with its specific antisense morpholino, k-ras-MO1 or k-ras-MO2 (Figure 1A). Optimal dose for microinjection was obtained that could result in specific defects but without gross lethality and global defects (materials and methods, Figure 2–Figure 4, and supporting information Table S1, Movie S1, S2, S3, S4, S5 and S6). From 24 hpf (hours-post fertilization) onwards, compared to control embryos, injected embryos showed reduced circulation of blood cells in the presence of a beating heart, albeit with lower beating rate (Figure 2A, Figure 2B, supporting information Figure S3 and Movie S1, S2, S3, S4, S5 and S6). Negligible or fewer circulating blood cells were seen inside the heart and blood vessels. This phenotype was observed in 76% of k-ras-MO1 injected embryos (75.8%±9.8, n>500, from 15 independent experiments). Moreover, accumulated red blood cells were often found at sites away from circulation (Figure 2C). As a negative control, one four-base mismatch morpholino k-ras-MO1-mis did not cause any of the above phenotypes as in k-ras-MO1 injected embryos (supporting information Figure S4). The specificity of K-ras knockdown was further confirmed by using a second morpholino, k-ras-MO2 that resulted in similar extents of defects in hematopoiesis (supporting information Table S1). Furthermore, the specificity and the efficiency of K-ras knockdown was confirmed by the reduced level of K-ras, rather than N-ras and H-ras, protein expression in K-ras MO injected embryos (supporting information Figure S5), and by the reduced level of the expression of a red fluorescent protein reporter fused downstream of k-ras 5′UTR in k-ras-MO injected embryos (supporting information Figure S6). Importantly, when k-ras mRNA was co-injected with k-ras-MO, such hematopoietic defects could be rescued effectively (Figure 5A and Figure 5B, supporting information Table S2), further supporting the specificity of K-ras knockdown. On the other hand, k-ras mRNA failed to rescue the gastrulation defects induced by the knock down of RhoA [12] (supporting information Figure S7), another small GTPase protein, demonstrating the specificity of the k-ras mRNA and the K-ras knock down.


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

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

Disruption of zebrafish K-ras signaling resulted in the defective hematopoiesis.(A) k-ras-MO injected embryo showed empty heart, without or with few red blood cells inside (indicated by arrows in ii and iii), in comparison to wild type embryo, which showed plenty of red blood cells inside the heart (indicated by arrow in i). Embryos at 3 dpf (days-post fertilization). (B) Plenty of circulating red blood cells inside dorsal aorta (DA), posterior cardinal vein (PCV), inter-segmental vessels (Se), caudal artery (CA) and caudal vein (CV) in wild type embryos (i and iii), while no or less circulating red blood cells were found in k-ras-MO injected embryos inside DA, PCV, CA, CV and Se (ii and iv). Embryos at 3 dpf. (C) k-ras-MO injected embryos showed accumulated red blood cells in some sites that away from the circulation. Embryos at 3 dpf. (D) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside branchial arches (indicated by arrow in i) and heart chambers (indicated by arrow in ii ), while k-ras-MO injected embryo showed less/negative o-Dianisidine staining for branchial arches (indicated by arrow in iii) and heart (indicated by arrow in iv). Embryos at 6 dpf. (E) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside Se (indicated by arrows in i ), dorsal longitudinal anastomotic vessels (DLAV), CA and CV (indicated by arrows in ii), while k-ras-MO injected embryo failed to give the positive o-Dianisidine staining in these corresponding positions (indicated by arrows in iii and iv). Embryos at 6 dpf. All embryos shown are lateral view, with anterior to the left and dorsal to the top.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002850-g002: Disruption of zebrafish K-ras signaling resulted in the defective hematopoiesis.(A) k-ras-MO injected embryo showed empty heart, without or with few red blood cells inside (indicated by arrows in ii and iii), in comparison to wild type embryo, which showed plenty of red blood cells inside the heart (indicated by arrow in i). Embryos at 3 dpf (days-post fertilization). (B) Plenty of circulating red blood cells inside dorsal aorta (DA), posterior cardinal vein (PCV), inter-segmental vessels (Se), caudal artery (CA) and caudal vein (CV) in wild type embryos (i and iii), while no or less circulating red blood cells were found in k-ras-MO injected embryos inside DA, PCV, CA, CV and Se (ii and iv). Embryos at 3 dpf. (C) k-ras-MO injected embryos showed accumulated red blood cells in some sites that away from the circulation. Embryos at 3 dpf. (D) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside branchial arches (indicated by arrow in i) and heart chambers (indicated by arrow in ii ), while k-ras-MO injected embryo showed less/negative o-Dianisidine staining for branchial arches (indicated by arrow in iii) and heart (indicated by arrow in iv). Embryos at 6 dpf. (E) o-Dianisidine staining for wild type embryo showed hemoglobin positive cells inside Se (indicated by arrows in i ), dorsal longitudinal anastomotic vessels (DLAV), CA and CV (indicated by arrows in ii), while k-ras-MO injected embryo failed to give the positive o-Dianisidine staining in these corresponding positions (indicated by arrows in iii and iv). Embryos at 6 dpf. All embryos shown are lateral view, with anterior to the left and dorsal to the top.
Mentions: To identify the functional role of K-ras in vivo, translation of endogenous K-ras was suppressed by targeting k-ras mRNA with its specific antisense morpholino, k-ras-MO1 or k-ras-MO2 (Figure 1A). Optimal dose for microinjection was obtained that could result in specific defects but without gross lethality and global defects (materials and methods, Figure 2–Figure 4, and supporting information Table S1, Movie S1, S2, S3, S4, S5 and S6). From 24 hpf (hours-post fertilization) onwards, compared to control embryos, injected embryos showed reduced circulation of blood cells in the presence of a beating heart, albeit with lower beating rate (Figure 2A, Figure 2B, supporting information Figure S3 and Movie S1, S2, S3, S4, S5 and S6). Negligible or fewer circulating blood cells were seen inside the heart and blood vessels. This phenotype was observed in 76% of k-ras-MO1 injected embryos (75.8%±9.8, n>500, from 15 independent experiments). Moreover, accumulated red blood cells were often found at sites away from circulation (Figure 2C). As a negative control, one four-base mismatch morpholino k-ras-MO1-mis did not cause any of the above phenotypes as in k-ras-MO1 injected embryos (supporting information Figure S4). The specificity of K-ras knockdown was further confirmed by using a second morpholino, k-ras-MO2 that resulted in similar extents of defects in hematopoiesis (supporting information Table S1). Furthermore, the specificity and the efficiency of K-ras knockdown was confirmed by the reduced level of K-ras, rather than N-ras and H-ras, protein expression in K-ras MO injected embryos (supporting information Figure S5), and by the reduced level of the expression of a red fluorescent protein reporter fused downstream of k-ras 5′UTR in k-ras-MO injected embryos (supporting information Figure S6). Importantly, when k-ras mRNA was co-injected with k-ras-MO, such hematopoietic defects could be rescued effectively (Figure 5A and Figure 5B, supporting information Table S2), further supporting the specificity of K-ras knockdown. On the other hand, k-ras mRNA failed to rescue the gastrulation defects induced by the knock down of RhoA [12] (supporting information Figure S7), another small GTPase protein, demonstrating the specificity of the k-ras mRNA and the K-ras knock down.

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