Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP) y1 zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.Calponin 2 is a member of the calponin family, which includes two other distinct members, basic calponin 1 (h1 calponin, CNN1) (1) and acidic calponin 3 (h3 calponin, CNN3) (2). These calponins share high sequence homology at the amino-terminal two-thirds of the molecule, which contains the calponin homology (CH) 3 domain followed by three calponin repeats (3, 4). The carboxyl-terminal sequences beyond Cys-273 are unique for the three variants, accounting for differences in their isoelectric points. Calponin 1 is specific to smooth muscle cells and serves as a marker for smooth muscle cell differentiation. It inhibits the actin-activated ATPase activity of myosin (5) and may play a role in regulating smooth muscle contraction. As an actin-, tropomyosin-and calmodulin-binding protein, it interacts in vitro with cytoskeletal components, such as myosin (6, 7), caldesmon (8, 9), desmin (10), and tubulin (11). Calponin 1 has been shown to play an important role in agonist-induced signal transduction in smooth muscle cells (12). It binds to extracellular regulated Ser/Thr kinases (ERK1 and ERK2) (13, 14) through the aminoterminal CH domain and protein kinase C (PKC) (15).Less is known about the function of calponin 2. Although the sequence conservation between calponin 2 and calponin 1 points to a similar function, they have diverged during vertebrate evolution, and the se...

In this report, we use zebrafish as a model system to understand the importance of CD146 in vascular development. Endothelial-specific expression of CD146 was verified by whole-mount in situ hybridization. Suppression of CD146 protein expression by antisense morpholino oligonucleotides (MO) resulted in poorly developed intersomitic vessels (ISVs). In CD146 morphants, we observed a lack of blood flow through the ISV region, despite that fluorescence microangiography showed that the ISVs were present. This finding suggests that the lumens of the developing ISVs may be too narrow for proper circulation. Additionally, remodeling of the caudal vein plexus into functional vascular tubes appeared to be affected. Suppression of CD146 protein expression resulted in a circulation shunt that caused the circulation to by-pass part of the caudal artery/vein system. The same vascular defects were recapitulated by using a second morpholino oligonucleotide. This morphant expressed a truncated CD146 protein with amino acids V32 to T57 at the N terminus deleted in an in-frame manner. This region, therefore, is likely to contain elements critical for CD146 function. This study provides the first in vivo functional assessment of CD146 in embryonic development by showing that knockdown of CD146 protein expression severely hinders vascular development in zebrafish. Developmental Dynamics 232:232-244, 2005.