BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish

Abstract

For the proper development of vertebrate embryos as well as for survival of the adult organism, it is essential to form a functional vascular system. Molecules involved in this process are members of highly conserved families of proteins that exert conserved functions across species. Bone morphogenetic proteins (BMP) are extracellular factors that are regulated by extracellular modulators and bind to BMP receptors, which in turn activate intracellular signaling cascades. BMPs are necessary not only for induction of endothelial and hematopoietic lineages but also for further endothelial and hematopoietic cell differentiation. Previously, we identified BMPER (BMP endothelial cell precursor derived regulator) and demonstrated its spatiotemporal expression at sites of vasculogenesis and direct modulation of BMP activity. To directly investigate the role of BMPER in vascular development, we cloned the BMPER ortholog in zebrafish (zbmper). It is expressed at sites of high BMP activity, including vascular precursor cells located in the aortic arches and the intermediate cell mass during zebrafish embryonic development. Knockdown of zbmper results in a dorsalized phenotype, a reduced number of gata1 expressing hematopoietic precursor cells and of circulating blood cells as well as in a vascular phenotype. The generation of the caudal vein is compromised and the pattern guiding of the intersomitic vessels is disturbed, indicating that zbmper is required for early steps in vascular pattern formation and hematopoiesis in zebrafish.

Figure 1. Comparison of orthologous BMPER sequences

Boxshade plot of the amino acid sequences of BMPER orthologs in vertebrates (Mus musculus [12,42], Homo sapiens [14], Pan troglodytes, Canis familiaris, Gallus gallus [13], Danio rerio, Fugu rubripes, and in Drosophila melanogaster [15]. Black boxes indicate identical amino acids, grey boxes indicate similar amino acids. Solid lines identify vWC domains 1–5, the dotted line denotes the vWD domain and the dashed line indicates the trypsin inhibitor domain (TI). BMPER orthologs share a high degree of homology in vertebrates. The TI domain is absent in Drosophila.

Figure 2. Analysis of BMPER proteins

(A) A phylogenetic tree was calculated for the orthologs shown in Figure 1. The distance of branches from each node indicates increased divergence in sequence similarity among orthologs. The scale represents the fraction of non-identical amino acids along each branch. (B) C-Myc tagged zbmper was analyzed by western blotting on a 12% SDS-PAGE gel. A full length zbmper protein and a cleaved c-terminal degradation product can be detected.

Figure 3. RNA expression of zbmper in whole mount zebrafish embryos

(A–C and E–G) Embryos were analyzed by in situ hybridization using a zbmper specific probe [purple]. (A) Dorsal view of a 24 hpf zebrafish embryo. Zbmper is expressed in the lateral dorsal aorta (lda) associated with the aortic arches as well as within a cranial transverse connection (c), the eye primordia (e), and the dorsal midline (dm) of the head area. (B and C) Higher magnifications of the yolk sac extension. (B) Oblique dorsolateral view, anterior to the left. zbmper is expressed in the ICM (*) located at the distal end of the yolk sac extension. (C) Dorsal view, anterior to the left. Zbmper is expressed in the paraxial ICM more concentrated at the distal end of the yolk sac extension.(D) Dorsolateral view of a 24 hpf flk1:GFP transgenic fish embryo shows flk:GFP expression in the symmetric lateral dorsal aorta (lda) and anterior cardinal vein (acv) as well as in the transverse connection (c). Anterior to the left. (E) Dorsolateral view of a 48 hpf zebrafish embryo. Zbmper is strongly expressed in the lateral dorsal aorta and the aortic arches. Weaker expression is detectable in the pineal gland (pg) and in the eye lenses (el). (F) Transverse section at the level of the aortic arches at 48 hpf. As observed in whole mount preparations zbmper is strongly expressed in the lateral dorsal aorta associated with the aortic arches. Zbmper is also expressed in the lateral mesoderm (lm) that hosts early vascular precursor cells in vertebrate organisms. (G) Transverse section at the level of the dotted line in (E). Zbmper is expressed around the ventral wall of the dorsal aorta but not in the axial vein. (nc) notochord. (da) dorsal aorta. (av) axial vein. (y) yolk sac.

Figure 4. Loss of zbmper causes a dorsalized phenotype

(A) Western blot of zebrafish embryo lysates at 36hpf. Zbmper is effectively knocked down in embryos displaying the characteristic phenotype. (B–D) Lateral views of zebrafish embryos at different developmental stages. (B) Tail of a control zebrafish embryo at 48 hpf. (C) Tail of a representative zbmper morphant embryo at 36 hpf. The shape of the tail indicates a dorsalized phenotype. Somitic segments distal of the dorsal tail kick are smaller than in controls and are dysmorphic. Blood cells are pooled in the dorsal aorta proximal to the tail deformation (arrowhead). (D) Tail of a zbmper morphant embryo at 72 hpf. The tail has adopted a pigtail shape indicating a severely dorsalized phenotype.

Figure 5. Loss of zbmper results in disturbed blood circulation and loss of blood cells

(A) High magnification of a lateral view of the ventral part of the trunk of a control zebrafish. Anterior to the left. (nc) notochord, (ca) caudal aorta. Note the normal number of circulating blood cells in the dorsal aorta. (B) Lateral view of the trunk of a mildly dorsalized mutant embryo (48 hpf). Note the packed mass of circulating cells within the dorsal aorta. In live fish, this cell mass moves slightly back and forth in the rhythm of the beating heart, suggesting an obstructed outflow from the dorsal aorta. (C) In a more severe phenotype (36 hpf) only a few circulating blood cells (bc) can be observed in the same area of the dorsal aorta. (D) Cranial view of a control embryo at 72 hpf. Note the blood cells in the sinus venosus. (E) Cranial view of the same embryo as in Figure 4D (72 hpf) displaying a strong loss of blood cells in zbmper morphants. Note only few blood cells in the sinus venosus.

Figure 6. Loss of zbmper reduces gata1 expression in ICM

(A–C) Embryos in identical position. (*) ICM. (A) Tail view (phase) of a control embryo at 20 hpf. The ICM extends as indicated by the arrows. (B) Fluorescent image the tail region (as seen in A) of a gata1:GFP transgenic embryo. Note fluorescent signal in the ICM as a reporter for gata1 expression. (C) Tail view of a gata1:GFP transgenic zbmper morphant embryo. Note the reduced expression of gata1 in the ICM.

Figure 7. Loss of zbmper results in a vascular phenotype

(A–E) Lateral views of flk1:GFP transgenic zebrafish embryos at different developmental stages. Anterior to the left. (ye) yolk sac extension. (A) Tail of a control zebrafish embryo at 48 hpf. Note the regular, organized patterning of intersegmental blood vessels and the size of the caudal vein plexus. (B–E) zbmper morpholino injected embryos. (B) Tail of zbmper morphant at 48 hpf. Note the dysmorphic caudal vein with a localized reduction of vessel size (arrow) and aberrant ISV formation (arrowhead). (C) Tail of a zbmper morphant at 72 hpf displaying a severe tail phenotype. Intersegmental vessels cross segmental boundaries (arrowheads). The caudal vein is absent. At the tip of the tail the normal vascular organization is completely lost (*). In this area endothelial cells are detectable but do not form patent blood vessels. (D) Tail of a zbmper morphant at 72 hpf displaying a weak tail phenotype. The indicated intersegmental vessel crosses the respective vessel of the adjacent somite before it connects dorsally. The caudal vein is severely dysmorphic (arrow). (E) Tail of zbmper morphant at 7 dpf. Note the absence of the dorsal ends of ISVs and aberrant sprouting. ye = yolk extension, CA = caudal artery, CV = caudal vein, DLAV = dorsal longitudinal anastomotic vessel, ISV = intersegmental vessel.