Identification of novel vascular endothelial-specific genes by the microarray analysis of the zebrafish cloche mutants

Abstract

The zebrafish cloche (clo) mutation affects the earliest known step in differentiation of blood and endothelial cells in vertebrates. We established clo/gata1-GFP transgenic line with erythroid-specific green fluorescent protein (GFP) expression, which allowed differentiation of clo and wild-type siblings at the midsomitogenesis stages before morphologically visible phenotypes appeared. To discover novel genes potentially involved in hematopoietic and vascular development, we performed microarray analysis of more than 15,000 zebrafish genes or expressed sequence tags (ESTs) in clo mutant embryos. We isolated the full-length sequences and determined the expression patterns for 8 novel cDNAs that were significantly down-regulated in clo-/- embryos. Dual specificity phosphatase 5 (dusp5), cadherin 5 (cdh5; VE-cadherin), aquaporin 8 (aqp8), adrenomedullin receptor (admr), complement receptor C1qR-like (crl), scavenger receptor class F, member 1 (scarf1), and ETS1-like protein (etsrp) were specifically expressed in the vascular endothelial cells, while retinol binding protein 4 (rbp4) was expressed in the yolk syncytial layer and the hypochord. Further functional studies of these novel genes should help to elucidate critical early steps leading to the formation of vertebrate blood vessels.

Figure 1.

Gata1-GFP homozygous transgenic fish were used to identify clo^–/– mutants at the 15-somite stage. (A) Wild-type embryo; (B) clo^–/– embryo. Note that GFP fluorescence in hematopoietic precursor cells (arrows) is absent in the clo^–/– embryo, while nonspecific GFP expression in the anterior region is not affected (arrowheads).

Figure 2.

Schematic view of the protein domains present in the novel vasculature-specific zebrafish proteins. RHOD denotes a Rhodanese homology domain; DSP, dual specificity phosphatase; CADH, cadherin repeat; CADH_C, cadherin cytoplasmic domain; 7-TM, 7 transmembrane domains; MIP, major intrinsic protein family domain; CLECT, C-type lectin; EGF-Ca, calcium binding EGF-like homology domain; EGF, EGF-like homology domain; TM, transmembrane domain; and ETS, ETS family DNA binding domain.

Figure 3.

Expression pattern of the dual specificity phosphatase 5 (dusp5) as analyzed by in situ hybridization. Anterior is to the left in all panels. (A)The 3-somite stage. A flat mount of the deyolked embryo. dusp5 is expressed in presumptive endothelial precursor cells, which form 2 bilateral stripes of cells that merge in the posterior. The 15-somite stage: (B) lateral view and (C) dorsal view. dusp5 is expressed in 2 bilateral stripes of cells within the lateral mesoderm in the anterior (arrow in B) and middle/posterior (arrowhead in B) parts of an embryo. In addition, a stripe of dusp5-expressing cells is apparent at the midline and extends through the middle and posterior parts of an embryo (arrowhead in C). (D) The 26-hpf stage. dusp5 expressed in all endothelial cells of the axial, head, and intersomitic vessels. The 52-hpf stage: (E) lateral view, (F) ventral view, and (inset) rostral view. dusp5 is expressed in a subset of nonvascular cells in the forebrain (arrowhead).

Figure 4.

Expression patterns of the cadherin 5 and aquaporin 8. Anterior is to the left in all panels. (A-D) cdh5 expression; (E-H) aqp8 expression. (A) The 15-somite stage. cdh5 mRNA is expressed in 2 distinct anterior (arrow) and middle/posterior (arrowhead) domains within lateral mesoderm. (B) The 26-hpf stage. cdh5 mRNA is expressed in all vascular endothelial cells of axial, intersegmental, and head vessels. (C) The 52-hpf stage, lateral view. cdh5 mRNA is localized to multiple head vessels, aortic arches (aa), pectoral fin bud (fb) vessel, heart (h) endocardium, the common cardinal vein (ccv), the cardinal vein plexus region at the tail (pl), posterior intersegmental vessels, and the dorsal longitudinal anastomotic vessel (dlav). (D) The 52-hpf stage, dorsal view. cdh5 mRNA is expressed in a group of cells within the diencephalon (arrowhead). Inset, ventral view of heart area. cdh5 is expressed in heart endocardium. (E) The 15-somite stage. aqp8 mRNA is expressed within lateral mesoderm in the posterior half on embryo (arrowhead). (F) The 26-hpf stage. aqp8 mRNA is localized to the dorsal aorta, cardinal vein plexus region, and intersegmental vessels. (G) The 36-hpf stage. aqp8 is expressed in the posterior part of the dorsal aorta, intersegmental vessels, the dorsal longitudinal anastomotic vessel (dlav), the cardinal vein plexus region (pl), and heart endocardium. (H) The 72-hpf stage. aqp8 is expressed in the dorsal longitudinal anastomotic vessel (dlav), the pectoral finbud vessel (fb), and faintly in the posterior intersegmental vessels.

Figure 5.

Expression patterns of the adrenomedullin receptor (admr), C1qR-like (crl), scavenger receptor class F (scarf1), and retinol binding protein 4 (rbp4) mRNAs. (A-B) admr expression, (C-D) crl expression, (E) scarf1 expression, and (F) rbp4 expression. (A) The 26-hpf stage. admr is localized to endothelial cells of axial, head, and intersegmental vessels. (B) The 52-hpf stage. admr expression is restricted to the main axial vessels, a subset of head vessels, and the common cardinal vein (ccv). (C) The 26-hpf stage. crl is expressed in endothelial cells of axial, head, and intersegmental vessels. (D) The 52-hpf stage. crl is expressed in the common cardinal vein (ccv), the pectoral fin bud vessel (fb), and a subset of head vessels. (E) The 22-somite stage. scarf1 is localized within lateral mesoderm to the presumptive endothelial cell precursors (arrowhead). (F) The 24-hpf stage. rbp4 mRNA is localized to the yolk syncytial layer and several hypochordal cells (arrowheads); higher magnification in the inset.

Figure 6.

Zebrafish C1qR-like protein is distantly related to the human complement receptor C1qR precursor protein (hC1qR1). Lectin homology domain is underlined; a line above a section indicates EGF-Ca domain; identical and similar amino acids are shaded. GeneWorks 2.5 software (Oxford Molecular Group, Campbell, CA) was used for the alignment.

Figure 7.

Expression of vascular-specific genes in cloche mutants at 22 to 24 hpf. (A-B) etsrp expression in the vasculature of wild-type siblings (A) and clo^–/– embryos (B). Note that only a few etsrp-expressing cells in the cardinal vein plexus region are remaining in clo mutant embryos. (C-D) dusp5 expression in wild-type siblings (C) and clo^–/– embryos (D). Note that only a few cells with faint dusp5 expression in the cardinal vein plexus region are remaining in clo mutant embryos. (E-F) cdh5 mRNA expression in wild-type siblings (E) and clo^–/– embryos (F). Note that only a few cdh5-expressing cells in the cardinal vein plexus region are remaining in clo mutant embryos. (G-H) aqp8 expression in wild-type siblings (G) and clo^–/– embryos (H). Note that only a few aqp8-expressing cells in the cardinal vein plexus region are remaining in clo mutant embryos. (I-J) admr expression in wild-type siblings (I) and clo^–/– embryos (J). Note that only a few cells with a very faint admr expression in the cardinal vein plexus region are remaining in clo mutant embryos. (K-L) crl expression in wild-type siblings (K) and clo^–/– embryos (L). Note that no crl-expressing cells are remaining in clo mutant embryos.