Summary In animals, many cells reach their destinations by migrating towards higher concentrations of an attractant. However, the nature, generation and interpretation of attractant gradients are poorly understood. Using a GFP fusion and a signaling sensor, we analyzed the distribution of the attractant chemokine Sdf1 during migration of the zebrafish posterior lateral line primordium, a cohort of about 200 cells that migrates over a stripe of cells uniformly expressing sdf1. We find that a small fraction of the total Sdf1 pool is available to signal and induces a linear Sdf1-signaling gradient across the primordium. This signaling gradient is initiated at the rear of the primordium, equilibrates across the primordium within 200 minutes, and operates near steady-state. The rear of the primordium generates this gradient through continuous sequestration of Sdf1 protein by the alternate Sdf1-receptor Cxcr7. Modeling shows that this is a physically plausible scenario.

Neuronal Ca 2؉ signals can affect excitability and neural circuit formation. Ca 2؉ signals are modified by Ca 2؉ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca 2؉ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca 2؉ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP3R) can partially compensate for their loss of flight. Ca 2؉ measurements show that Orai gain-of-function contributes to the quanta of Ca 2؉ -release through mutant InsP3Rs and elevates store-operated Ca 2؉ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca 2؉ in neurons is required for Drosophila flight.calcium homeostasis ͉ flight patterns ͉ inositol 1,4,5-trisphosphate receptor ͉ sarco-endoplasmic reticulum-associated Ca 2ϩ ATPase ͉ STIM S everal aspects of neuronal function are regulated by ionic calcium (Ca 2ϩ ). Specific attributes of a Ca 2ϩ ''signature'' such as amplitude, duration, and frequency of the signal can determine the morphology of a neural circuit by affecting the outcome of cell migration, the direction taken by a growth-cone, dendritic development, and synaptogenesis (1). Ca 2ϩ signals also determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors (2). Most of these Ca 2ϩ signals have been attributed to the entry of extracellular Ca 2ϩ through voltage-operated channels or ionotropic receptors. However, other components of the ''Ca 2ϩ tool-kit'' coupled to Ca 2ϩ release from intracellular Ca 2ϩ stores are also present in neurons. These molecules include the store-operated Ca 2ϩ (SOC) channel, encoded by the Orai gene, identified recently in siRNA screens for molecules that reduce or abolish Ca 2ϩ influx from the extracellular milieu after intracellular Ca 2ϩ store depletion (3-5). Several reports have confirmed its identity as the pore forming subunit of the Ca 2ϩ -release activated Ca 2ϩ (CRAC) channel (6-8). Activation of this CRAC channel is mediated by the endoplasmic reticulum (ER) resident protein STIM (stromal interaction molecule), also identified in an RNAi screen for molecules that regulate SOC influx (9, 10). STIM senses the drop in ER Ca 2ϩ levels, and interacts with Orai by a mechanism which is only just being understood (11). Orai and STIM function in conjunction with the sarco-endoplasmic reticular Ca 2ϩ -ATPase pump (SERCA) to maintain ER store Ca 2ϩ and basal Ca 2ϩ . The importance of intracellular Ca 2ϩ homeostasis and SOC entry (E) in neural circuit formation and in neuronal function and physiology remains to be elucidated.Here, we report how Orai and STIM me...

Calcium (Ca

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Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

In neurons a well-defined source of signaling Ca2+ is the extracellular medium. However, as in all metazoan cells, Ca2+ is also stored in endoplasmic reticular compartments inside neurons. The relevance of these stores in neuronal function has been debatable. The Orai gene encodes a channel that helps refill these stores from the extracellular medium in non-excitable cells through a process called store-operated Ca2+ entry or SOCE. Recent findings have shown that raising the level of Orai or its activator STIM, and consequently SOCE in neurons, can restore flight to varying extents to Drosophila mutants for an intracellular Ca2+-release channel – the inositol 1,4,5-trisphosphate receptor (InsP3R). Both intracellular Ca2+-release and SOCE appear to function in neuro-modulatory domains of the flight circuit during development and acute flight. These findings raise exciting new possibilities for the role of SOCE in vertebrate motor circuit function and the treatment of neurodegenerative disorders where intracellular Ca2+ signaling has been implicated as causative.