Mechanisms of Systemic Osmoregulation in Insects.

Water is essential to life. Terrestrial insects lose water by evaporation from the body surface and respiratory surfaces, as well as in the excretory products, posing a challenge made more acute by their high surface-to-volume ratio. These losses must be kept to a minimum and be offset by water gained from other sources. By contrast, insects such as the blood-sucking bug Rhodnius prolixus consume up to 10 times their body weight in a single blood meal, necessitating rapid expulsion of excess water and ions. How do insects manage their ion and water budgets? A century of study has revealed a great deal about the organ systems that insects use to maintain their ion and water balance and their regulation. Traditionally, a taxonomically wide range of species were studied, whereas more recent research has focused on model organisms to leverage the power of the molecular genetic approach. Key advances in new technologies have become available for a wider range of species in the past decade. We document how these approaches have already begun to inform our understanding of the diversity and conservation of insect systemic osmoregulation. We advocate that these technologies be combined with traditional approaches to study a broader range of nonmodel species to gain a comprehensive overview of the mechanism underpinning systemic osmoregulation in the most species-rich group of animals on earth, the insects.

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.

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.

A protective role for <i>Drosophila</i> Filamin in nephrocytes via Yorkie mediated hypertrophy.

Podocytes are specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Because of their position, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury. It is likely podocytes have adaptative responses to help buffer against deleterious mechanical force and thus reduce injury. However, these responses remain largely unknown. Here, using the <i>Drosophila</i> model, we show the mechanosensor Cheerio (dFilamin) provides a key protective role in nephrocytes. We found expression of an activated mechanosensitive variant of Cheerio rescued filtration function and induced compensatory and hypertrophic growth in nephrocytes depleted of the nephrocyte diaphragm proteins Sns or Duf. Delineating the protective pathway downstream of Cheerio we found repression of the Hippo pathway induces nephrocyte hypertrophy, whereas Hippo activation reversed the Cheerio-mediated hypertrophy. Furthermore, we find Yorkie was activated upon expression of active Cheerio. Taken together, our data suggest that Cheerio acts via the Hippo pathway to induce hypertrophic growth, as a protective response in abnormal nephrocytes.

Scaffold polarity proteins Par3A and Par3B share redundant functions while Par3B acts independent of atypical protein kinase C/Par6 in podocytes to maintain the kidney filtration barrier.

Glomerular diseases are a major cause for chronic kidney disorders. In most cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and loss of podocytes. Here, we establish a link between the Par3 polarity complex and actin regulators necessary to establish and maintain podocyte architecture by utilizing mouse and Drosophila models to characterize the functional role of Par3A and Par3B and its fly homologue Bazooka in vivo. Only simultaneous inactivation of both Par3 proteins caused a severe disease phenotype. Rescue experiments in Drosophila nephrocytes revealed atypical protein kinase C (aPKC)-Par6 dependent and independent effects. While Par3A primarily acts via aPKC-Par6, Par3B function was independent of Par6. Actin-associated synaptopodin protein levels were found to be significantly upregulated upon loss of Par3A/B in mouse podocytes. Tropomyosin2, which shares functional similarities with synaptopodin, was also elevated in Bazooka depleted nephrocytes. The simultaneous depletion of Bazooka and Tropomyosin2 resulted in a partial rescue of the Bazooka knockdown phenotype and prevented increased Rho1-GTP, a member of a GTPase protein family regulating the cytoskeleton. The latter contribute to the nephrocyte phenotype observed upon loss of Bazooka. Thus, we demonstrate that Par3 proteins share a high functional redundancy but also have specific functions. Par3A acts in an aPKC-Par6 dependent way and regulates RhoA-GTP levels, while Par3B exploits Par6 independent functions influencing synaptopodin localization. Hence, Par3A and Par3B link elements of polarity signaling and actin regulators to maintain podocyte architecture.

A unique Malpighian tubule architecture in Tribolium castaneum informs the evolutionary origins of systemic osmoregulation in beetles.

Maintaining internal salt and water balance in response to fluctuating external conditions is essential for animal survival. This is particularly true for insects as their high surface-to-volume ratio makes them highly susceptible to osmotic stress. However, the cellular and hormonal mechanisms that mediate the systemic control of osmotic homeostasis in beetles (Coleoptera), the largest group of insects, remain largely unidentified. Here, we demonstrate that eight neurons in the brain of the red flour beetle Tribolium castaneum respond to internal changes in osmolality by releasing diuretic hormone (DH) 37 and DH47-homologs of vertebrate corticotropin-releasing factor (CRF) hormones-to control systemic water balance. Knockdown of the gene encoding the two hormones (Urinate, Urn8) reduces Malpighian tubule secretion and restricts organismal fluid loss, whereas injection of DH37 or DH47 reverses these phenotypes. We further identify a CRF-like receptor, Urinate receptor (Urn8R), which is exclusively expressed in a functionally unique secondary cell in the beetle tubules, as underlying this response. Activation of Urn8R increases K+ secretion, creating a lumen-positive transepithelial potential that drives fluid secretion. Together, these data show that beetle Malpighian tubules operate by a fundamentally different mechanism than those of other insects. Finally, we adopt a fluorescent labeling strategy to identify the evolutionary origin of this unusual tubule architecture, revealing that it evolved in the last common ancestor of the higher beetle families. Our work thus uncovers an important homeostatic program that is key to maintaining osmotic control in beetles, which evolved parallel to the radiation of the "advanced" beetle lineages.

Transgenic Tarantula Toxin: A novel tool to study mechanosensitive ion channels in Drosophila.

The tarantula venom toxin GsMTx4 is the only known specific inhibitor of cation-selective mechanosensitive ion channels (MSCs). Its specificity, potency, and ease of use on isolated tissues and cells have made it a powerful pharmacological tool to identify and probe the physiological function of MSCs. In some contexts, however, it would be desirable to deliver the toxin in a controlled way in vivo. Here we describe a novel tool to allow spatial and temporal control of GsMTx4 delivery in vivo in Drosophila. To test the tool, we targeted MSCs required for mechanical nociception in a specific subset of sensory neurons in intact larvae. Expression of GsMTx4 in these neurons results in robust inhibition of mechanical nociception, demonstrating the toxin is active when expressed in vivo. The tool will be particularly useful to manipulate MSC activity in a spatially and temporally-controlled manner to study their role in development, physiology and behaviour in intact, free moving animals.

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.

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

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.

Malpighian tubule development in the red flour beetle (Tribolium castaneum).

Malpighian tubules (MpTs) are the major organ for excretion and osmoregulation in most insects. MpT development is characterised for Drosophila melanogaster, but not other species. We therefore do not know the extent to which the MpT developmental programme is conserved across insects. To redress this we provide a comprehensive description of MpT development in the beetle Tribolium castaneum (Coleoptera), a species separated from Drosophila by >315 million years. We identify similarities with Drosophila MpT development including: 1) the onset of morphological development, beginning when tubules bud from the gut and proliferate to increase organ size. 2) the tubule is shaped by convergent-extension movements and oriented cell divisions. 3) differentiated tip cells activate EGF-signalling in distal MpT cells through the ligand Spitz. 4) MpTs contain two main cell types - principal and stellate cells, differing in morphology and gene expression. We also describe development of the beetle cryptonephridial system, an adaptation for water conservation, which represents a major modification of the MpT ground plan characterised by intimate association between MpTs and rectum. This work establishes a new model to compare MpT development across insects, and provides a framework to help understand how an evolutionary novelty - the cryptonephridial system - arose during organ evolution.

Shaping up for action: the path to physiological maturation in the renal tubules of Drosophila.

The Malpighian tubule is the main organ for excretion and osmoregulation in most insects. During a short period of embryonic development the tubules of Drosophila are shaped, undergo differentiation and become precisely positioned in the body cavity, so they become fully functional at the time of larval hatching a few hours later. In this review I explore three developmental events on the path to physiological maturation. First, I examine the molecular and cellular mechanisms that generate organ shape, focusing on the process of cell intercalation that drives tubule elongation, the roles of the cytoskeleton, the extracellular matrix and how intercalation is coordinated at the tissue level. Second, I look at the genetic networks that control the physiological differentiation of tubule cells and consider how distinctive physiological domains in the tubule are patterned. Finally, I explore how the organ is positioned within the body cavity and consider the relationship between organ position and function.

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

The 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.

Why flies? Inexpensive public engagement exercises to explain the value of basic biomedical research on Drosophila melanogaster.

Invertebrate model organisms are powerful systems for uncovering conserved principles of animal biology. Despite widespread use in scientific communities, invertebrate research is often severely undervalued by laypeople. Here, we present a set of simple, inexpensive public outreach exercises aimed at explaining to the public why basic research on one particular invertebrate, the insect Drosophila melanogaster, is valuable. First, we designed seven teaching modules that highlight cutting-edge research in Drosophila genetics, metabolism, physiology, and behavior. We then implemented these exercises in a public outreach event that included both children and adults. Quantitative evaluation of participant feedback suggests that these exercises 1) teach principles of animal biology, 2) help laypeople better understand why researchers study fruit flies, and 3) are effective over a wide range of age groups. Overall, this work provides a blueprint for how to use Drosophila as a vehicle for increasing public awareness and appreciation of basic research on genetically tractable insects in particular and invertebrates in general.

Characterisation of the Drosophila procollagen lysyl hydroxylase, dPlod.

The lysyl hydroxylase (LH) family of enzymes has important roles in the biosynthesis of collagen. In this paper we present the first description of Drosophila LH3 (dPlod), the only lysyl hydroxylase encoded in the fly genome. We have characterised in detail the developmental expression patterns of dPlod RNA and protein during embryogenesis. Consistent with its predicted function as a collagen-modifying enzyme, we find that dPlod is highly expressed in type-IV collagen-producing cells, particularly the haemocytes and fat body. Examination of dPlod subcellular localisation reveals that it is an endoplasmic reticulum resident protein, that partially overlaps with intracellular type-IV collagen. Furthermore, we show that dPlod is required for type-IV collagen secretion from haemocytes and fat body, and thus establish that LH3 enzyme function is conserved across widely separated animal phyla. Our findings, and the new tools we describe, establish the fly as an attractive model in which to study this important collagen biosynthesis enzyme.

Bringing together components of the fly renal system.

The function of all animal excretory systems is to rid the body of toxins and to maintain homeostatic balance. Although excretory organs in diverse animal species appear superficially different they are often built on two common principals: filtration and tubular secretion/reabsorbtion. The Drosophila excretory system is composed of filtration nephrocytes and Malpighian (renal) tubules. Here we review recent molecular genetic data on the development and differentiation of nephrocytes and renal tubules. We focus in particular on the molecular mechanisms that underpin key cell and tissue behaviours during morphogenesis, drawing parallels with other species where they exist. Finally we assess the implications of patterned tissue differentiation for the subsequent regulation of renal function. These studies highlight the continuing usefulness of the fly to provide fundamental insights into the complexities of organ formation.

The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm.

The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation, 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1-a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.

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.

crossveinless-c is a RhoGAP required for actin reorganisation during morphogenesis.

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In our search for genes that regulate Drosophila morphogenesis, we have isolated several lethal alleles of crossveinless-c (cv-c). Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the reorganisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and we propose that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions.