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The spatiotemporally controlled expression of G-CaMP fluorescent calcium indicator proteins can facilitate reliable imaging of brain circuit activity. Here, we generated a transgenic mouse line that expresses G-CaMP7 under a tetracycline response element. When crossed with a forebrain-specific tetracycline-controlled transactivator driver line, the mice expressed G-CaMP7 in defined cell populations in a tetracycline-controlled manner, notably in pyramidal neurons in layer 2/3 of the cortex and in the CA1 area of the hippocampus; this expression allowed for imaging of the in vivo activity of these circuits. This mouse line thus provides a useful genetic tool for controlled G-CaMP expression in vivo.

Presynaptic assembly requires the packaging of requisite proteins into vesicular cargoes in the cell soma, their long-distance microtubule-dependent transport down the axon, and, finally, their reconstitution into functional complexes at prespecified sites. Despite the identification of several molecules that contribute to these events, the regulatory mechanisms defining such discrete states remain elusive. We report the characterization of an Arf-like small G protein, ARL-8, required during this process. arl-8 mutants prematurely accumulate presynaptic cargoes within the proximal axon of several neuronal classes, with a corresponding failure to assemble presynapses distally. This proximal accumulation requires the activity of several molecules known to catalyze presynaptic assembly. Dynamic imaging studies reveal that arl-8 mutant vesicles exhibit an increased tendency to form immotile aggregates during transport. Together, these results suggest that arl-8 promotes a trafficking identity for presynaptic cargoes, facilitating their efficient transport by repressing premature self-association., (Copyright 2010 Elsevier Inc. All rights reserved.)

Semaphorins are extracellular proteins that regulate axon guidance and morphogenesis by interacting with a variety of cell surface receptors. Most semaphorins interact with plexin-containing receptor complexes, although some interact with non-plexin receptors. Class 2 semaphorins are secreted molecules that control axon guidance and epidermal morphogenesis in Drosophila and Caenorhabditis elegans. We show that the C. elegans class 2 semaphorin MAB-20 binds the plexin PLX-2. plx-2 mutations enhance the phenotypes of hypomorphic mab-20 alleles but not those of mab-20 null alleles, indicating that plx-2 and mab-20 act in a common pathway. Both mab-20 and plx-2 mutations affect epidermal morphogenesis during embryonic and in postembryonic development. In both contexts, plx-2 null mutant phenotypes are much less severe than mab-20 null phenotypes, indicating that PLX-2 is not essential for MAB-20 signaling. Mutations in the ephrin efn-4 do not synergize with mab-20, indicating that EFN-4 may act in MAB-20 signaling. EFN-4 and PLX-2 are coexpressed in the late embryonic epidermis where they play redundant roles in MAB-20-dependent cell sorting.

Chondroitin polymerization was first demonstrated in vitro when human chondroitin synthase (ChSy) was coexpressed with human chondroitin polymerizing factor (ChPF), which is homologous to ChSy but has little glycosyltransferase activity. To analyze the biological function of chondroitin, the Caenorhabditis elegans ortholog of human ChSy (sqv-5) was recently cloned, and the expression of its product was depleted by RNA-mediated interference (RNAi) and deletion mutagenesis. Blocking of chondroitin synthesis resulted in defects of cytokinesis in early embryogenesis, and eventually, cell division stopped. Here, we cloned the ortholog of human ChPF in C. elegans, PAR2.4. Despite little glycosyltransferase activity of the gene product, chondroitin polymerization was demonstrated as in the case of mammals when PAR2.4 was coexpressed with cChSy in vitro. The worm phenotypes including the reversion of cytokinesis, observed after the depletion of PAR2.4 by RNAi, were very similar to the cChSy (sqv-5)-RNAi phenotypes. Thus, PAR2.4 in addition to cChSy is indispensable for the biosynthesis of chondroitin in C. elegans, and the two cooperate to synthesize chondroitin in vivo. The expression of the PAR2.4 protein was observed in seam cells, which can act as neural stem cells in early embryonic lineages. The expression was also detected in vulva and distal tip cells of the growing gonad arms from L3 through to the young adult stage. These findings are consistent with the notion that chondroitin is involved in the organogenesis of the vulva and maturation of the gonad and also indicative of an involvement in distal tip cell migration and neural development.

During apoptosis, phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and has been suggested to act as an "eat-me" signal to trigger phagocytosis. It is unclear how phagocytes recognize phosphatidylserine. Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate recognition of phosphatidylserine and phagocytosis. We report that psr-1, the Caenorhabditis elegans homolog of PSR, is important for cell corpse engulfment. In vitro PSR-1 binds preferentially phosphatidylserine or cells with exposed phosphatidylserine. In C. elegans, PSR-1 acts in the same cell corpse engulfment pathway mediated by intracellular signaling molecules CED-2 (homologous to the human CrkII protein), CED-5 (DOCK180), CED-10 (Rac GTPase), and CED-12 (ELMO), possibly through direct interaction with CED-5 and CED-12. Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10, and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis.