Calcitonin (CT) is one of the hormones involved in vertebrate calcium regulation. It has been proposed to act as a hypocalcemic factor, but the regulatory pathways remain to be clarified. We investigated the CT/calcitonin gene-related peptide (CGRP) family in zebrafish and its potential involvement in calcium homeostasis. We identified the presence of four receptors: CTR, CRLR1, CRLR2, and CRLR3. From the phylogenetic analysis, together with the effect observed after CT and CGRP overexpression, we concluded that CTR appears to be a CT receptor and CRLR1 a CGRP receptor. The distribution of these two receptors shows a major presence in the central nervous system and in tissues involved in ionoregulation. Zebrafish embryos kept in high-Ca 2þ -concentration medium showed upregulation of CT and CTR expression and downregulation of the epithelial calcium channel (ECaC). Embryos injected with CT morpholino (CALC MO) incubated in high-Ca 2þ medium, showed downregulation of CTR together with upregulation on ECaC mRNA expression. In contrast, overexpression of CT cRNA induced the downregulation of ECaC mRNA synthesis, concomitant with the downregulation in the calcium content after 30 hours postfertilization. At 4 days postfertilization, CT cRNA injection induced upregulation of hypercalcemic factors, with subsequent increase in the calcium content. These results suggest that CT acts as a hypocalcemic factor in calcium regulation, probably through inhibition of ECaC synthesis. ß
In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as aA1-and bA1-globin, but not the embryonic be1-globin, was affected.
Human synuclein family consists of ␣-, -, and ␥-synucleins. Here, we cloned three genes, sncb, sncga and sncgb from zebrafish. They encode -, ␥1-, and ␥2-synucleins, respectively. The zSyn-, zSyn-␥1, and zSyn-␥2 proteins display 69%, 47%, and 50% identity to human -synuclein and ␥-synuclein, respectively. By reverse transcriptase-polymerase chain reaction, we demonstrated that sncb and sncga mRNA were abundant in brain and eye, while sncgb expression was moderate in brain, kidney, ovary and testis. The 1.8-kb 5-upstream/promoter region of the sncga gene was sufficient to direct green fluorescent protein (GFP) expression in the central nervous system and cranial ganglions. A transgenic line, Tg(sncga:GFP), was generated and its GFP expression is similar to that of endogenous sncga mRNA. Moreover, this line also labels the habenular complex and the domain of GFP expression is larger in the left than in the right habenula. Thus, this line can be used to study sncga gene regulation and for left-right asymmetry study in zebrafish brain. Developmental Dynamics 238:746 -754, 2009.
BackgroundMammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos.Methodology/Principal FindingsWe demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth.Conclusions/SignificanceOur results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.
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