The Role Of Connexin43 In The Cardiovascular System Development In Zebrafish Embryos

Gap junctions are membrane channels that serve as major conduits for cell - cell communication by mediating the intercellular passage of ions and small molecules (<1200 Daltons). Connexins are the subunits of gap junctions. Many different connexin genes have been identified in humans, with subsets of connexin genes expressed in different tissues. Mutations in different connexin genes cause distinct developmental defects in humans including deafness, skin disorders, peripheral neuropathies, cataracts etc.The connexin gene known as connexin43 or al connexin (referred to as Cx43a1; Gja1 - Mouse Genome Informatics) plays a crucial role in cardiac development, as Cx43al knockout mice die shortly after birth due to pulmonary outflow obstruction. Typically, the Cx43al knockout mouse heart exhibits two prominent pouches at the base of the pulmonary outflow tract, a region known as the infundibulum of the heart. Studies using transgenic mouse models to manipulate Cx43al function indicate that the outflow obstruction probably involves the perturbation of cardiac neural crest cell migration.Despite the fact that gap junctions are usually not present in circulating blood cells, connexin expression is known to occur in leukocytes during inflammatory reactions in blood vessels, and gap junctions have been described in the hematopoietic bone marrow. Cx43 has been identified in human and mouse bone marrow preparations. In vitro studies of cultured bone marrow or thymic stromal cells revealed cx43 as the principal gap junction protein expressed by these primary cell types.In recent years, zebrafish has emerged as an exciting animal model system for studying vertebrate organ development, in particular, the development of hematopoietic system, as zebrafish embryos are optically transparent, easily manipulated. Zebrafish lacks yolk sac blood islands; instead, embryonic hematopoiesis occurs in a relatively dorsal location above the yolk tube called intermediate cell mass (ICM). ICM is formed in two paraxial stripes of mesoderm that arise during gastrulation. The site is similar to the extraembryonic location of the earliest hematopoietic progenitors on the yolk sac of higher vertebrates. In the present study, we turn to the zebrafish embryos to pursue the role of cx43 GJ during hematopoiesis.To study the effects of connexin43 down regulation on the development of the embryonic heart and vasculature in zebrafish, two types of well designed morpholino oligonucleotide antisenses were injected into zebrafish embryos to block the translation of cx43 at one or two cells stage. After injection, the phenotypes of heart and vasculature were monitored by whole mount in situ hybridization, whole-mount immunofluorescence and microangiography. Whole-mount in situ hybridization with vmhc and amhc RNA probes showed that the vmhc expression cell domain was reduced; meanwhile, amhc expression cell domain was increased in cx43 down regulation group. Whole-mount immunofluorescence provided the evidence that down regulation of cx43 resulted in enlarged atrium and retrenched ventricle. Both in situ hybridization and microangiography indicated that vasculature pattern of cx43 morphants are almost normal compared with wildtype. Besides, the function of heart was affected obviously. Down regulation of cx43 caused the development defects of zebrafish embryonic heart, which may be involved in the wrong destination of two migratory cell populations, but it did not nearly affect vascular development.In vitro studies suggested that cx43 expression was of particular importance during establishment or regeneration of the mammalian hematopoietic system. But little is known about its in vivo functions during hematopoiesis because of embryonic lethality of mammalian knockout models. In the present study, we noticed for the first time that cx43 was expressed in zebrafish intermediate cell mass (ICM). We also found that cx43 knockdown by Morpholino-modified antisense oligonucleotides resulted in striking decrease of erythrocytes, and hydropsia of ICM during embryogenesis. Further investigation showed that hamatopoietic stem cells in cx43 morphant were decreased. Besides, we found the accumulation of blood cells in cx43 morphants which drops a hint that cx43 are involved in appropriate migration of blood cells. Based on these findings we put forward an assumption that down regulation of cx43 expression leads to advanced onset of erythroid differentiation, which affects not only the proliferation of hematopoietic stem cells but also the destination of cells from ICM.