A negative-feedback loop maintains optimal chemokine concentrations for directional cell migration.

Chemoattractant gradients frequently guide migrating cells. To achieve the most directional signal, such gradients should be maintained with concentrations around the dissociation constant (Kd)1-6 of the chemoreceptor. Whether this actually occurs in animals is unknown. Here we investigate whether a moving tissue, the zebrafish posterior lateral line primordium, buffers its attractant in this concentration range to achieve robust migration. We find that the Cxcl12 (also known as Sdf1) attractant gradient ranges from 0 to 12 nM, values similar to the 3.4 nM Kd of its receptor Cxcr4. When we increase the Kd of Cxcl12 for Cxcr4, primordium migration is less directional. Furthermore, a negative-feedback loop between Cxcl12 and its clearance receptor Ackr3 (also known as Cxcr7) regulates the Cxcl12 concentrations. Breaking this negative feedback by blocking the phosphorylation of the cytoplasmic tail of Ackr3 also results in less directional primordium migration. Thus, directed migration of the primordium is dependent on a close match between the Cxcl12 concentration and the Kd of Cxcl12 for Cxcr4, which is maintained by buffering of the chemokine levels. Quantitative modelling confirms the plausibility of this mechanism. We anticipate that buffering of attractant concentration is a general mechanism for ensuring robust cell migration.

Cadherin-Mediated Cell Coupling Coordinates Chemokine Sensing across Collectively Migrating Cells.

The directed migration of cells sculpts the embryo, contributes to homeostasis in the adult, and, when dysregulated, underlies many diseases [1, 2]. During these processes, cells move singly or as a collective. In both cases, they follow guidance cues, which direct them to their destination [3-6]. In contrast to single cells, collectively migrating cells need to coordinate with their neighbors to move together in the same direction. Recent studies suggest that leader cells in the front sense the guidance cue, relay the directional information to the follower cells in the back, and can pull the follower cells along [7-19]. In this manner, leader cells steer the collective and set the collective's overall speed. However, whether follower cells also participate in steering and speed setting of the collective is largely unclear. Using chimeras, we analyzed the role of leader and follower cells in the collectively migrating zebrafish posterior lateral line primordium. This tissue expresses the chemokine receptor Cxcr4 and is guided by the chemokine Cxcl12a [20-23]. We find that leader and follower cells need to sense the attractant Cxcl12a for efficient migration, are coupled to each other through cadherins, and require coupling to pull Cxcl12a-insensitive cells along. Analysis of cell dynamics in chimeric and protein-depleted primordia shows that Cxcl12a-sensing and cadherin-mediated adhesion contribute jointly to direct migration at both single-cell and tissue levels. These results suggest that all cells in the primordium need to sense the attractant and adhere to each other to coordinate their movements and migrate with robust directionality.

A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish.

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.

Generation and dynamics of an endogenous, self-generated signaling gradient across a migrating tissue.

In animals, many cells reach their destinations by migrating toward 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 min, 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.

The enigma of store-operated ca-entry in neurons: answers from the Drosophila flight circuit.

In neurons a well-defined source of signaling Ca(2+) is the extracellular medium. However, as in all metazoan cells, Ca(2+) 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 Ca(2+) 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 Ca(2+)-release channel - the inositol 1,4,5-trisphosphate receptor (InsP(3)R). Both intracellular Ca(2+)-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 Ca(2+) signaling has been implicated as causative.

Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight.

Calcium (Ca(2+)) signaling is known to regulate the development, maintenance and modulation of activity in neuronal circuits that underlie organismal behavior. In Drosophila, intracellular Ca(2+) signaling by the inositol 1,4,5-trisphosphate receptor and the store-operated channel (dOrai) regulates the formation and function of neuronal circuits that control flight. Here, we show that restoring InsP(3)R activity in insulin-producing neurons of flightless InsP(3)R mutants (itpr) during pupal development can rescue systemic flight ability. Expression of the store operated Ca(2+) entry (SOCE) regulator dSTIM in insulin-producing neurons also suppresses compromised flight ability of InsP(3)R mutants suggesting that SOCE can compensate for impaired InsP(3)R function. Despite restricted expression of wild-type InsP(3)R and dSTIM in insulin-producing neurons, a global restoration of SOCE and store Ca(2+) is observed in primary neuronal cultures from the itpr mutant. These results suggest that restoring InsP(3)R-mediated Ca(2+) release and SOCE in a limited subset of neuromodulatory cells can influence systemic behaviors such as flight by regulating intracellular Ca(2+) homeostasis in a large population of neurons through a non-cell-autonomous mechanism.

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight.

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 (InsP(3)R) 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 InsP(3)Rs 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.

Compensation of inositol 1,4,5-trisphosphate receptor function by altering sarco-endoplasmic reticulum calcium ATPase activity in the Drosophila flight circuit.

Ionic Ca2+ functions as a second messenger to control several intracellular processes. It also influences intercellular communication. The release of Ca2+ from intracellular stores through the inositol 1,4,5-trisphosphate receptor (InsP3R) occurs in both excitable and nonexcitable cells. In Drosophila, InsP3R activity is required in aminergic interneurons during pupal development for normal flight behavior. By altering intracellular Ca2+ and InsP3 levels through genetic means, we now show that signaling through the InsP3R is required at multiple steps for generating the neural circuit required in air puff-stimulated Drosophila flight. Decreased Ca2+ release in aminergic neurons during development of the flight circuit can be compensated by reducing Ca2+ uptake from the cytosol to intracellular stores. However, this mode of increasing intracellular Ca2+ is insufficient for maintenance of flight patterns over time periods necessary for normal flight. Our study suggests that processes such as maintenance of wing posture and formation of the flight circuit require InsP3 receptor function at a slow timescale and can thus be modulated by altering levels of cytosolic Ca2+ and InsP3. In contrast, maintenance of flight patterns probably requires fast modulation of Ca2+ levels, in which the intrinsic properties of the InsP3R play a pivotal role.