The insect cryptonephridial complex.

Beaven et al. introduce the insect cryptonephridial complex, a multi-organ system that is one of the most powerful water-extraction systems in nature.

The take-off of Drosophila research in 1930-1950s Edinburgh.

The fruit fly, Drosophila melanogaster, is a simple and powerful model organism. It has played a critical role over more than a century, for example in establishing the field of genetics, and in foundational insights into the molecular basis of development. From the 1930s until today, researchers at the University of Edinburgh have used Drosophila to tackle questions in basic and biomedical science. Here the history of the initial decades of this research is explored, beginning with the introduction of Drosophila research to Edinburgh by Francis Albert Eley Crew, in the newly established Institute of Animal Genetics. This period of research includes the discovery that chemicals can cause genetic mutation. This was demonstrated by research of the effects of mustard gas on flies by Charlotte Auerbach and colleagues, guided by the future Nobel laureate Hermann Muller. Drosophila research was also formative in Conrad Hal Waddington's conceptual thinking about developmental biology, including in his vision of the epigenetic landscape. This holistic, systems-level view of the control of development was far before its time and has continued to be influential to this day in our conceptualisation of developmental biology and in the increasingly important field of systems biology. Waddington's experiments with Drosophila in Edinburgh also gave rise to the evolutionary concept of genetic assimilation, in which an environmentally induced phenotype subsequently becomes genetically encoded.

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.

Piezo buffers mechanical stress via modulation of intracellular Ca2+ 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 (Frank, Z. biology, 1895, 32, 370-447; Anrep, J. Physiol., 1912, 45 (5), 307-317; Patterson and Starling, J. Physiol., 1914, 48 (5), 357-79; Starling, The law of the heart (Linacre Lecture, given at Cambridge, 1915), 1918). 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.

Early patterning followed by tissue growth establishes distal identity in Drosophila Malpighian tubules.

Specification and elaboration of proximo-distal (P-D) axes for structures or tissues within a body occurs secondarily from that of the main axes of the body. Our understanding of the mechanism(s) that pattern P-D axes is limited to a few examples such as vertebrate and invertebrate limbs. Drosophila Malpighian/renal tubules (MpTs) are simple epithelial tubules, with a defined P-D axis. How this axis is patterned is not known, and provides an ideal context to understand patterning mechanisms of a secondary axis. Furthermore, epithelial tubules are widespread, and their patterning is not well understood. Here, we describe the mechanism that establishes distal tubule and show this is a radically different mechanism to that patterning the proximal MpT. The distal domain is patterned in two steps: distal identity is specified in a small group of cells very early in MpT development through Wingless/Wnt signalling. Subsequently, this population is expanded by proliferation to generate the distal MpT domain. This mechanism enables distal identity to be established in the tubule in a domain of cells much greater than the effective range of Wingless.

Release and spread of Wingless is required to pattern the proximo-distal axis of Drosophila renal tubules.

Wingless/Wnts are signalling molecules, traditionally considered to pattern tissues as long-range morphogens. However, more recently the spread of Wingless was shown to be dispensable in diverse developmental contexts in Drosophila and vertebrates. Here we demonstrate that release and spread of Wingless is required to pattern the proximo-distal (P-D) axis of Drosophila Malpighian tubules. Wingless signalling, emanating from the midgut, directly activates odd skipped expression several cells distant in the proximal tubule. Replacing Wingless with a membrane-tethered version that is unable to diffuse from the Wingless producing cells results in aberrant patterning of the Malpighian tubule P-D axis and development of short, deformed ureters. This work directly demonstrates a patterning role for a released Wingless signal. As well as extending our understanding about the functional modes by which Wnts shape animal development, we anticipate this mechanism to be relevant to patterning epithelial tubes in other organs, such as the vertebrate kidney.

14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes.

The meiotic spindle is formed without centrosomes in a large volume of oocytes. Local activation of crucial spindle proteins around chromosomes is important for formation and maintenance of a bipolar spindle in oocytes. We found that phosphodocking 14-3-3 proteins stabilize spindle bipolarity in Drosophila melanogaster oocytes. A critical 14-3-3 target is the minus end-directed motor Ncd (human HSET; kinesin-14), which has well-documented roles in stabilizing a bipolar spindle in oocytes. Phospho docking by 14-3-3 inhibits the microtubule binding activity of the nonmotor Ncd tail. Further phosphorylation by Aurora B kinase can release Ncd from this inhibitory effect of 14-3-3. As Aurora B localizes to chromosomes and spindles, 14-3-3 facilitates specific association of Ncd with spindle microtubules by preventing Ncd from binding to nonspindle microtubules in oocytes. Therefore, 14-3-3 translates a spatial cue provided by Aurora B to target Ncd selectively to the spindle within the large volume of oocytes.

Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system.

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end-tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain-containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin-dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.

Using fly genetics to dissect the cytoskeletal machinery of neurons during axonal growth and maintenance.

The extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. For this, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream of these signals is implemented by their actin and microtubule cytoskeleton. In the first part of this Commentary, we discuss the fundamental roles of the cytoskeleton during axon growth. We present the various classes of actin- and microtubule-binding proteins that regulate the cytoskeleton, and highlight the important gaps in our understanding of how these proteins functionally integrate into the complex machinery that implements growth cone behaviour. Deciphering such machinery requires multidisciplinary approaches, including genetics and the use of simple model organisms. In the second part of this Commentary, we discuss how the application of combinatorial genetics in the versatile genetic model organism Drosophila melanogaster has started to contribute to the understanding of actin and microtubule regulation during axon growth. Using the example of dystonin-linked neuron degeneration, we explain how knowledge acquired by studying axonal growth in flies can also deliver new understanding in other aspects of neuron biology, such as axon maintenance in higher animals and humans.

Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

Drosophila growth cones: a genetically tractable platform for the analysis of axonal growth dynamics.

The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F-actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross-talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F-actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin-MT linking factor Short stop, thus identifying an essential molecular player in this context.