Piezo buffers mechanical stress via modulation of intracellular Ca 2+ handling in the Drosophila heart

Throughout its lifetime the heart is buffeted continuously by dynamic mechanical forces resulting from contraction of the heart muscle itself and fluctuations in haemodynamic load and pressure. These forces are in flux on a beat-by-beat basis, resulting from changes in posture, physical activity or emotional state, and over longer timescales due to altered physiology (e.g. pregnancy) or as a consequence of ageing or disease (e.g. hypertension). It has been known for over a century of the heart’s ability to sense differences in haemodynamic load and adjust contractile force accordingly[1-4]. These adaptive behaviours are important for cardiovascular homeostasis, but the mechanism(s) underpinning them are incompletely understood. Here we present evidence that the mechanically-activated ion channel, Piezo, is an important component of the Drosophila heart’s ability to adapt to mechanical force. We find Piezo is a sarcoplasmic reticulum (SR)-resident channel and is part of a mechanism that regulates Ca2+ handling in cardiomyocytes in response to mechanical stress. Our data support a simple model in which Drosophila Piezo transduces mechanical force such as stretch into a Ca2+ signal, originating from the SR, that modulates cardiomyocyte contraction. We show that Piezo mutant hearts fail to buffer mechanical stress, have altered Ca2+ handling, become prone to arrhythmias and undergo pathological remodelling

NHA1 is a cation/proton antiporter essential for the water-conserving functions of the rectal complex in Tribolium castaneum

More than half of all extant metazoan species on earth are insects. The evolutionary success of insects is linked with their ability to osmoregulate, suggesting that they have evolved unique physiological mechanisms to maintain water balance. In beetles (Coleoptera)—the largest group of insects—a specialized rectal (“cryptonephridial”) complex has evolved that recovers water from the rectum destined for excretion and recycles it back to the body. However, the molecular mechanisms underpinning the remarkable water-conserving functions of this system are unknown. Here, we introduce a transcriptomic resource, BeetleAtlas.org, for the exceptionally desiccation-tolerant red flour beetle Tribolium castaneum, and demonstrate its utility by identifying a cation/H+ antiporter (NHA1) that is enriched and functionally significant in the Tribolium rectal complex. NHA1 localizes exclusively to a specialized cell type, the leptophragmata, in the distal region of the Malpighian tubules associated with the rectal complex. Computational modeling and electrophysiological characterization in Xenopus oocytes show that NHA1 acts as an electroneutral K+/H+ antiporter. Furthermore, genetic silencing of Nha1 dramatically increases excretory water loss and reduces organismal survival during desiccation stress, implying that NHA1 activity is essential for maintaining systemic water balance. Finally, we show that Tiptop, a conserved transcription factor, regulates NHA1 expression in leptophragmata and controls leptophragmata maturation, illuminating the developmental mechanism that establishes the functions of this cell. Together, our work provides insights into the molecular architecture underpinning the function of one of the most powerful water-conserving mechanisms in nature, the beetle rectal complex

Epidermal growth factor signalling controls Myosin II planar polarity to orchestrate convergent extension movements during Drosophila tubulogenesis

Aditya Saxena was funded by the Cambridge Commonwealth Trust. Barry Denholm was funded by Kidney Research UK PDF1/2010. Stephanie Bunt was funded by The Rae and Edith Bennett Travelling Scholarship and John Stanley Gardiner Trust Fund. Marcus Bischoff was supported by WG086986 and WT096645MA to Peter Lawrence. Krishnaswamy VijayRaghavan was funded by The Wellcome Trust: 079221/B/06/Z. Helen Skaer was funded by The Wellcome Trust: 079221/B/06/Z and 094879/A/10/Z.Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis we show that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, we demonstrate that the vector of cell movement is regulated by localised epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality.Publisher PDFPeer reviewe

The role of enhancer of split and groucho during neurogenesis in Drosophila melanogaster and Musca Domestica

This study has investigated the role of the E(spl) and groucho genes during neural fate commitment in the fly. It is known that the carboxy-terminal tryptophan-arginine-proline-tryptophan (WRPW) motif of E(spl) binds Groucho to form a complex, which represses the transcription of target genes. The importance of specific residues within WRPW has been investigated by generating a number of mutant derivatives containing single amino acid substitutions within the motif. It has been found that changes in WRPW abolish the in vivo function of the protein, and attenuate interaction with the Groucho protein. To determine the mode of E(spl)-mediated regulation, a series of co-expression assays were performed. E(spl) has been ectopically co-expressed with proneural genes scute or daughterless during allocation of imaginal SOP cells. It was found that E(spl) did repress the neural fate in the context of the co-expression assay, suggesting that, in addition to transcriptional repression of the proneural genes, post-transcriptional modes of regulation also occur. The requirement for an intact WRPW motif further suggest that this mode of repression may involve Groucho. Finally, a region of the groucho gene from the housefly (Musca domestica) has been cloned which encodes the C-terminal WD40 repeats and part of the variable region and displays a high degree of identity with Drosophila Groucho in these regions. In the Musca blastoderm embryo groucho mRNA is ubiquitously expressed, but later becomes confined to the developing CNS. A preliminary functional analysis using the technique of RNA interference suggests that groucho plays a role during neurogenesis in the Musca embryo

The tiptop/teashirt genes regulate cell differentiation and renal physiology in Drosophila.

International audienceThe physiological activities of organs are underpinned by an interplay between the distinct cell types they contain. However, little is known about the genetic control of patterned cell differentiation during organ development. We show that the conserved Teashirt transcription factors are decisive for the differentiation of a subset of secretory cells, stellate cells, in Drosophila melanogaster renal tubules. Teashirt controls the expression of the water channel Drip, the chloride conductance channel CLC-a and the Leukokinin receptor (LKR), all of which characterise differentiated stellate cells and are required for primary urine production and responsiveness to diuretic stimuli. Teashirt also controls a dramatic transformation in cell morphology, from cuboidal to the eponymous stellate shape, during metamorphosis. teashirt interacts with cut, which encodes a transcription factor that underlies the differentiation of the primary, principal secretory cells, establishing a reciprocal negative-feedback loop that ensures the full differentiation of both cell types. Loss of teashirt leads to ineffective urine production, failure of homeostasis and premature lethality. Stellate cell-specific expression of the teashirt paralogue tiptop, which is not normally expressed in larval or adult stellate cells, almost completely rescues teashirt loss of expression from stellate cells. We demonstrate conservation in the expression of the family of tiptop/teashirt genes in lower insects and establish conservation in the targets of Teashirt transcription factors in mouse embryonic kidney

Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis

During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination.During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination

A model for MpT cell intercalation.

<p>(A) Schematic showing a stage 13 MpT with distal to proximal (D–P) and circumferential (C) coordinates indicated. An asymmetric source of EGF ligand from the distally placed TC (TC) establishes a gradient of EGF pathway activity (red shading) in the distal tubule extending to the kink (k). A small cluster of tubule cells is highlighted. (B) Basal view of this cluster of cells during elongation. Individual cells read differential EGF activity across their D-P axis (higher distal relative to proximal; dashed lines in i), to produce an asymmetric accumulation of Myosin II (green, ii) at the basal, proximal cortex (buff cell). This leads to contraction along the circumferential axis (dashed arrows, ii). The resulting change in cell shape facilitates progressive, small movements (red arrows) between circumferential neighbours (pink and orange cells, iii). Multiple cycles of Myosin II pulses lead to cell intercalation (iv). Asynchronous pulses in a neighbouring cell (cyan), contracts its circumferential axis (black arrows, iii) facilitating intercalation of the buff cell, and producing a change in cell shape to initiate another cell intercalation event between the orange and grey cells (red arrows).</p