Snrk-1 is involved in multiple steps of angioblast development and acts via notch signaling pathway in artery-vein specification in vertebrates - PubMed (original) (raw)

Snrk-1 is involved in multiple steps of angioblast development and acts via notch signaling pathway in artery-vein specification in vertebrates

Chang Z Chun et al. Blood. 2009.

Abstract

In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase (snrk-1) family of serine/threonine kinases expressed specifically in the embryonic zebrafish vasculature and investigated its function in vivo. Using gain- and loss-of-function studies in vivo, we show that Snrk-1 plays an essential role in the migration, maintenance, and differentiation of angioblasts. The kinase function of Snrk-1 is critical for migration and maintenance, but not for the differentiation of angioblasts. In vitro, snrk-1 knockdown endothelial cells show only defects in migration. The snrk-1 gene acts downstream or parallel to notch and upstream of gridlock during artery-vein specification, and the human gene compensates for zebrafish snrk-1 knockdown, suggesting evolutionary conservation of function.

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Figures

Figure 1

Figure 1

Expression of snrk-1 during embryonic zebrafish development. (A-F) Whole-mount in situ hybridizations using digoxigenin-labeled snrk-1 antisense probes. At 24 hpf (A,B) and beyond (C-E) until 96 hpf (F), snrk-1 expression is restricted to head and trunk regions. In the trunk, section of 24 hpf snrk-1 in situ embryo (H) show expression in the dorsal aorta (DA), posterior cardinal vein (PCV), and the regions surrounding these 2 structures. No indicates notochord. Red arrow points to PCV and black arrow points to DA. The plane of the section in panel H is shown in panel G. (I) The orientation of the embryos.

Figure 2

Figure 2

Snrk-1 gene knockdown analysis in vivo. (A-D) Endothelial cells at 18 hpf and 28 hpf in wild-type (WT) and MO1-injected embryos are visualized by in situ hybridization using a fli antisense RNA probe. (C,D) High-magnification images of the trunk and head, respectively, of WT and MO1-injected embryos from panel B. Reduction of ECs is observed at (A) 18 hpf and (B) 28 hpf in MO1-injected embryos. Red bracket in panel C shows reduction of fli+ region composed of DA and PCV, and black asterisk shows that intersomitic vessels (ISVs) are missing at 28 hpf in MO1-injected embryos. (A-C) Lateral views. (D) Dorsal view. etsrp+ angioblasts in the head, trunk, and tail at (E-G) 6 som and (H-M) 17 hpf in WT, MO1-, and MO2-injected embryos. White asterisks in panels E through G are included for etsrp+ cell comparison across comparable regions in the 3 samples. At 17 hpf, in the head, compare 4 populations of etsrp+ angioblasts—of which the most anterior are enclosed in red box. In the trunk, 2 bilateral stripes of etsrp+ angioblasts are noticed, of which one stripe is missing in MO1-injected embryo (I, trunk). In the tail, 2 distinct etsrp+ angioblast populations merge at the tail tip indicated by the black box, and differences are noticed in their intensities and patterning in MO-injected embryos compared with WT. (K-M) Embryos overstained with etsrp probe. (L,M) Overstained embryos in which etsrp+ angioblasts are mispatterned. (N) The percentage of MO1- and MO2-injected embryos with mislocalized angioblasts at 17 hpf; *P < .001 between MO1- or MO2-injected groups compared with UI. (O) The fold change of etsrp transcript levels at 6 som in MO1- and MO2-injected embryos as analyzed by QPCR. Error bars represent SEM.

Figure 3

Figure 3

Gain-of-function analysis by overexpression of snrk-1 and snrk-1 kinase mutant (snrk-1 KM). (A-C) etsrp+ cells in the head (Hd), head to trunk (Hd-Tr), trunk (Tr), trunk to tail (Tr-Ta), tail (Ta), and lateral regions (La) of WT embryos (A), snrk-1 mRNA–injected (B), and snrk-1 KM mRNA–injected (C) embryos. (B) Ectopic induction of etsrp+ cells in snrk-1 mRNA–injected embryos. (A,B) Black asterisks in Tr-Ta and Ta panels indicate thicker etsrp-stained region at LPM in panel B compared with panel A. (C) snrk-1 KM mRNA–injected embryos with early migration of angioblasts from the LPM to the midline. (C, Tr-Ta) Inset is higher magnification of prematurely migrating angioblasts at 6 som. (D-F) etsrp+ cells in Hd, Tr, and Ta regions at 19 hpf in WT, snrk-1 mRNA–, and KM mRNA–injected embryos. (G) The fold change of etsrp transcript levels in snrk-1 mRNA– and KM mRNA–injected embryos at 6 som by QPCR. (H-K) An in vivo TUNEL apoptosis assay at 24 hpf in WT, MO1-, MO2-, and snrk-1 mRNA–injected embryos. (J,K) Arrows indicate apoptotic cells. (L) The mean number of apoptotic nuclei (x-axis) in the vessel region in each sample (y-axis: WT, n = 10; MO1, n = 14; MO2, n = 18; snrk-1 mRNA, n = 11), with the error bars representing SD. Paired t test: *P < .05 between WT and MO1; **P < .05 between WT and MO2; ***P < .005 between WT and snrk-1 mRNA samples.

Figure 4

Figure 4

Snrk-1 gene knockdown analysis in vitro. (A) RT-PCR for snrk-1 and actin genes in snrk-1 siRNA–transfected (500 ng) and control lacZ siRNA–transfected (500 ng) HUVECs. Lanes 7 through 9 show reduced snrk-1 transcripts compared with lanes 1 through 6. (B) In vitro KD studies show that there are fewer migratory ECs in snrk-1 siRNA–transfected HUVECs. The graph shows the effect of snrk-1 gene KD on migration of transfected HUVECs to serum-free medium (□) or 10% serum (■) for 5 hours at 37°C. All migration experiments have been conducted using HUVECs from passage numbers 2 through 4. The plotted data are pooled from 3 independent experiments and the error bars represent plus or minus SD. *P < .05 between snrk-1 siRNA and lacZ siRNA cells response to serum. (C) Graphic representation of the number of adherent ECs in snrk-1 siRNA–transfected, lacZ control siRNA–transfected, or untransfected HUVECs to an uncoated surface (□), laminin (formula image), or fibronectin (■). Error bars represent SD.

Figure 5

Figure 5

Snrk-1 functions in A/V specification. (A,B) grl in situ hybridization of 18 hpf WT and MO2-injected embryos, respectively. ephrin-B2a in situ hybridization of 26 hpf (C) WT, (D) _notch2-ICD_–injected, or (E) notch2-ICD + snrk-1 MO1–injected embryos. flt-4 in situ hybridization of 30 hpf (F) WT, (G) _notch2-ICD_–injected, and (H) notch2-ICD + snrk-1 MO1–injected embryos. (F-H) White asterisks show flt-4 expression in intersomitic arteries. (C-E) The black brackets indicate the space between the most dorsal stained regions of the embryo to the yolk extension. (D) Note the shrinkage in space in notch2-ICD_–injected embryo, indicating an expansion of ephrin-B2a_–stained cells. fli in situ hybridization of 26 to 28 hpf (I) WT, (J) MO1-injected, and (K) MO1 + h_snrk-1 RNA–injected embryos. (J,M) MO1-injected embryos missing ISVs compared with (I,L) WT or (K,N) MO1 + h_snrk-1_–coinjected embryos. (O) Graphic representation of the number of embryos displaying faint or absent ISVs in the trunk region of samples in panels I through K. *P < .001 across MO1 and MO1 + h_snrk-1 group. (P) A model placing Snrk-1 parallel to Notch in A/V specification. The dotted line indicates that definitive evidence is needed to show that snrk-1 is downstream of notch. Images in panels C through H were taken using an Observer Z1 inverted microscope (Carl Zeiss, Thornwood, NY) with 10× objective and 10× eyepiece. The image acquisition software used was AxioVision Rel4.6 (Carl Zeiss, Oberkochen, Germany); images were corrected for brightness and contrast in Photoshop 7.0.

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