Inui M et al. (2006), Xapelin and Xmsr are required for cardiovascula...

XB-ART-87

Dev Biol 2006 Oct 01;2981:188-200. doi: 10.1016/j.ydbio.2006.06.028.

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Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis.

Inui M , Fukui A , Ito Y , Asashima M .

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The cardiovascular development is the elaborate process, and despite the extensive studies, the mechanisms underlying endothelial, hematopoietic, and cardiac developments, as well as the interrelation between these processes, are not fully understood. In this study, we demonstrated that Xenopus apelin and Xmsr play pivotal roles in cardiovascular development. Apelin is a recently identified ligand for an orphan G-protein-coupled receptor APJ and is involved in fluid homeostasis in mammals. Xenopus preproapelin (Xpreproapelin) was isolated and its mRNA localized to the region around the presumptive blood vessels, which are overlapping or adjacent to those expressing Xmsr, the Xenopus homologue of APJ. Overexpression of Xpreproapelin disorganized the expression of the endothelial precursor cell marker XlFli and the hematopoietic precursor cell marker SCL at the neurula, whereas embryos injected with morpholino antisense oligonucleotides for Xapelin and Xmsr displayed attenuated expression of Tie2, alpha-globin, XPOX2, and cTnI, markers of endothelium, erythrocytes, myeloid cells, and cardiomyocytes, respectively. XlFli morpholino had similar effects to Xapelin and Xmsr morpholinos on cardiac differentiation, suggesting an unexpected potential relationship between the endothelium and cardiac differentiation. Forced expression of constitutive active G alpha i rescued the phenotypes of Xmsr morpholino-injected embryos, indicating that the i/o type of G protein alpha subunit acts downstream of Xmsr.

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Species referenced: Xenopus laevis
Genes referenced: apln aplnr cfd fli1 hba1 mpo nkx2-5 tal1 tek tnni3
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	Fig. 2. Expression patterns of Xpreproapelin and Xmsr mRNA. (A) RT-PCR analysis of Xpreproapelin and Xmsr mRNA during development. Ornithine decarboxylase (ODC) was used as an internal control. (B, C) Expression of Xpreproapelin mRNA at stage 10. Xpreproapelin mRNA was detected in the marginal zone, at the bilateral area of the dorsal lip (arrowheads). Inset in panel B shows a magnified view of the expression. (D, E) Expression of Xpreproapelin mRNA at stage 18, in the anterior ventral region of the embryo (arrowheads). (F, G) Expression of Xmsr mRNA at stage 10, in the marginal zone except for dorsal marginal zone (arrowheads). (H, I) Expression of Xmsr mRNA at stage 18, in the anterior ventral region of the embryo (arrowheads). (J–L) Expression of Xpreproapelin mRNA at stage 22, in regions lateral and posterior to the cement gland (CG), the dorsal posterior part of the embryo, and the dorsal midline. (M–O) Expression of Xmsr mRNA at stage 22, in areas lateral and posterior to the cement gland (CG), the dorsal posterior part of the embryo, and the dorsal midline. (P–R) Expression of Xpreproapelin mRNA at stage 30, around the anterior and posterior cardinal vein (ACV white arrowhead, PCV black arrowhead), aortic arcs (AA, arrowhead), dorsal longitudinal anastomosing vessel (DLAV, arrowhead), notochord (NC), neural tube (NT), and tailbud. (S–U) Expression of Xmsr mRNA at stage 30, in the anterior and posterior cardinal vein (ACV white arrowhead, PCV black arrowheads), aortic arcs (AA, arrowheads), endocardium (EC arrowhead), vascular vitelline network (VVN arrowhead), and tailbud. B, F, L, O: dorsal view;, C, G: vegetal view, dorsal upward, D H; anterior view, E, I, K, N, R, U: ventral view, J, M, P, Q, S, T: lateral view. Panels Q and T are magnified views of the dorsal part of the cleared embryos shown in panels P and S, respectively.
	Fig. 3. Comparison of expression patterns of Xpreproapelin, Xmsr, and Nkx2.5 mRNA. (A, D) Expression of Nkx2.5 mRNA at stage 22, in the mesodermal and endodermal layers of the anterior ventral region. (B, E) Expression of Xpreproapelin mRNA in stage 22, in the endodermal layer of anterior ventral region. (C, F) Expression of Xmsr mRNA in stage 22 embryo, in the mesodermal layer of the anterior ventral region. Cement glands are indicated by the dotted line in panels A–C. en: endodermal layer, mes: mesodermal layer, Scale bar: 100 μm.
	Fig. 4. Overexpression of Xpreproapelin. (A) Control embryo at stage 45. The position of heart is indicated by an arrowhead. (B) The section through the heart region of control embryo at stage 45, showing separate atria (a) and ventricle (v) and prominent trabeculae (arrowhead). (C) Xpreproapelin mRNA (1 ng)-injected embryo at stage 45, showing edema and abnormal heart morphology (arrowhead). (D) Section of heart region of the Xpreproapelin mRNA-injected embryo at stage 45. The heart had thin walls and poor chamber separation (arrowhead). (E) Graph showing the frequencies of edematous embryos with 125 pg, 250 pg, 500 pg, and 1000 pg of Xpreproapelin mRNA (Ap mRNA) or 1000 pg of beta-galactosidase mRNA (Beta-gal)-injected. Three independent experiments were performed and similar results obtained. The graph shows the representative result of one experiment. (F–H) Expression of XlFli, SCL, and Nkx2.5 at stage 17 in the control embryos. (I) Expression of cTnI in the control embryo at stage 30. (J) Section through the heart region of control embryo at stage 30 showing a single heart tube (arrowhead). (K–M) Expression of XlFli, SCL, and Nkx2.5 at stage 17 in the Xpreproapelin mRNA (1 ng)-injected embryo. (N) Expression of cTnI in the Xpreproapelin-injected embryo at stage 30. (O) Section through the heart region of the Xpreproapelin-injected embryo at stage 30, showing a separated heart tube (arrowheads). The black line in panels I and N indicates the level of the sections shown in panels J and O. Scale bar: 100 μm.
	Fig. 5. Xapelin and Xmsr knocked down embryos exhibited abnormal cardiovascular systems. (A, B) Western blotting assessing the specificities of XapelinMO and XmsrMO. XapelinMO (40 ng) and XmsrMO (20 ng) inhibited translation of mRNAs with 5′ UTR sequences (250 pg) (lane 2), but not from mRNAs that lack the 5′ UTR sequences (250 pg) (lane 4). (C, E, H) ControlMO (40 g)-, XapelinMO (40 ng)-, and XmsrMO (20 ng)-injected embryo at stage 45. (D, F, I) Section of the heart region of controlMO-, XapelinMO-, and XmsrMO-injected embryo at stage 45. Arrowheads indicate the position of the heart. (G, J) Embryos coinjected plasmid DNA (10 pg of pCS2-Xapelin 5m and 50 pg of pCS2-Xmsr 5m, respectively) with MOs (40 ng of XapelinMO and 20 ng of XmsrMO, respectively). (K, L, M) Graphs of the frequencies of edematous embryos in MO- and DNA-injected embryos. Three independent experiments were performed and showed similar results. The graphs show the representative results of one experiment. aMO: XapelinMO, mMO: XmsrMO, cMO: controlMO, Scale bar: 100 μm.
	Fig. 6. Xapelin and Xmsr are required for the expression of endothelial marker gene. (A, B) Expression of Ami in the controlMO (40 ng)-injected embryo at stage 35, in the posterior cardinal vein (PCV arrowhead), intersomitic veins (ISV arrowhead), and vascular vitelline network (VVN, arrowhead). (C, D) Expression of Ami in the XapelinMO (40 ng)-injected embryo. (E, F) Expression of Ami in the XmsrMO (20 ng)-injected embryo.
	Fig. 7. Xapelin and Xmsr are required for the expression of cardiovascular genes. (A) Expressions of XlFli, SCL, and Nkx2.5 in the controlMO (40 ng)-injected embryos at stage 17. (D) Expressions of XlFli, SCL, and Nkx2.5 in the XapelinMO (40 ng)-injected embryos at stage 17. (G) Expressions of XlFli, SCL, and Nkx2.5 in the XmsrMO (20 ng)-injected embryos at stage 17. (J) Expressions of Tie2, α-globin, XPOX2, cTnI, and Nkx2.5 in the controlMO-injected embryos. (O) Expressions of Tie2, α-globin, XPOX2, cTnI, and Nkx2.5 in the XapelinMO-injected embryos. (T) Expressions of Tie2, α-globin, XPOX2, cTnI, and Nkx2.5 in the XmsrMO-injected embryos. (a) Section through the heart region of controlMO-, XapelinMO-, and XmsrMO-injected embryos at stage 30. The black line in panels N, S, and X indicates the level of the sections shown in panels a. Arrow: endocardium. Arrowhead: myocardium, Scale bar: 100 μm.
	Fig. 8. XlFli-depleted embryos displayed phenotypes similar to Xapelin- and Xmsr-depleted embryos. (A) Western blotting assessing the target sequence specificity of XlFliMO. XlFliMO (20 ng) inhibited translation of injected mRNA with its target sequence (lane 4), but not of mRNA with 5 silent mutations around the first methionine (lane 2). (B) ControlMO (20 ng)-injected embryo at stage 45. (C) XlFliMO (20 ng)-injected embryo at stage 45. (D) Embryos coinjected pCS2-XlFli 5m (20 pg) with XlFliMO (20 ng). (E) Graphs showing the frequencies of edematous embryos in MO- and/or DNA-injected embryos. Three independent experiments were performed and showed similar results. The graph shows the representative result of one experiment. (G) Expression of SCL, Nkx2.5, Tie2, α-globin, and cTnI in the controlMO-injected embryos. (L) Expressions of SCL, Nkx2.5, Tie2, α-globin, and cTnI in the XlFliMO-injected embryos.
	Fig. 9. Gαi/o acts downstream of Xmsr. (A) ControlMO (20 ng)-injected embryo at stage 45. (B) XmsrMO (mMO) (20 ng)-injected embryo at stage 45. (C) Embryos coinjected pCS2-Xmsr 5m DNA (50 pg) (C), pCS2-EGFP DNA (50 pg) (D), pCS2-Gαi2QL (10 pg) (E), pCS2-Gαs short (10 pg) (F), pCS2-GαqQL (10 pg) (G), and pCS2-Gα12QL (10 pg) (H) with XmsrMO at stage 45. The embryos shown in the left of panels C and E are the embryos with normal phenotypes and the embryos shown in right of the panels are the embryos with edema phenotypes. The numbers shown in the bottom right of panels A indicate the percentage of edematous embryos, and the numbers shown in the bottom left of the panels indicate the percentage of normal embryos. (I) cTnI expression in controlMO (20 ng)- injected embryo. (J) cTnI expression in XmsrMO (mMO) (20 ng)-injected embryo. (K) cTnI expression in embryos coinjected with pCS2-Xmsr 5m DNA (50 pg) (K), pCS2-Gαi2QL (10 pg) (L), pCS2-Gαs short (10 pg) (M), pCS2-GαqQL (10 pg) (N), and pCS2-Gα12QL (10 pg) (O) with XmsrMO. The embryos shown in the left of panels J are the embryos with normal cTnI expression, and the embryos shown in the right of the panels are the embryos with reduced cTnI expression. The numbers shown at the bottom right of panels I indicate the percentage of the embryos with reduced cTnI expression, and the numbers shown at the bottom left of the panels indicate the percentage of the embryos with normal cTnI expression. (P, Q) Graphs summarizing the results above.