Biology subjects

Denholm, B.

Publications and source records attributed to Denholm, B..

9 recordsLinked to original sources

bioRxiv2024

Something old, something new: the origins of an unusual renal cell underpinning a beetle water-conserving mechanism

Tenebrionid beetles have been highly successful in colonising environments where water is scarce, underpinned by their unique osmoregulatory adaptations. These include a cryptonephridial arrangement of their organs, in which part of their renal/Malpighian tubules are bound to the surface of the rectum. This allows them to generate a steep osmotic gradient to draw water from within the rectum and return it to the body. Within the cryptonephridial tubules a seemingly novel cell type, the leptophragmata, is considered to play a key role in transporting potassium chloride to generate this osmotic gradient. Nothing was known about the developmental mechanisms or evolution of these unusual renal cells. Here we investigate the mechanisms underpinning development of the leptophragmata in the red flour beetle, Tribolium castaneum. We find that leptophragmata express and require a teashirt/tiptop transcription factor gene, as do the secondary renal cells of Drosophila melanogaster which lack a cryptonephridial arrangement. We also find an additional transcription factor, Dachshund, is required to establish leptophragmata identity and to distinguish them from the secondary cells in Triboliums non-cryptonephridial region of renal tubule. Dachshund is also expressed in a sub-population of secondary cells in Drosophila. So leptophragmata, which are unique to the beetle lineage, appear to have originated from a specific renal cell type present ancestrally, and specified by a conserved repertoire of transcription factors. SignificanceBeetles are a highly successful insect group and represent a quarter of all known animal species. Their digestive/renal systems have undergone major evolutionary change compared to other insects, likely contributing to their success. A dramatic example is the cryptonephridial complex, an evolutionary innovation of the gut and renal system which integrate as a powerful water-conservation system; an adaptation for survival in arid conditions. An unusual renal cell type--the leptophragmata--underpin the functions of the complex, but their developmental and evolutionary origins are unknown. Here we reveal the developmental mechanism that establish leptophragmata identity and, by studying a species lacking a cryptonephridial complex, shed light on their evolutionary origin. More broadly, the work illuminates the evolution of novel cell types.

developmental biology↗

bioRxiv2024

Evidence for the independent evolution of a rectal complex within the beetle superfamily Scarabaeoidea

Rectal or cryptonephridial complexes have evolved repeatedly in arthropods, including in beetles where they occur in [~]190,000 species of Cucujiformia and Bostrichoidea, and Lepidoptera where they occur in [~]160,000 species. Sections of the Malpighian/renal tubules coat the outer surface of the rectum, acting as powerful recycling systems of the gut contents, recovering water and specific solutes. There are hints that a rectal complex evolved independently within another beetle group, Scarabaeoidea. Here we report our observations of rectal complexes in Scarabaeoidea, which support this view. We did not find a rectal complex in the related group, Staphylinoidea, or in Lucanidae, a basal group of Scarabaeoidea. We did observe rectal complexes in Melolontha melolontha (Melolonthini), Pachnoda marginata and Cetonia aurata (Cetoniinae), consistent with previous reports from these groups. Intriguingly we found that rectal complexes occur in adult, but not M. melolontha larvae, and larvae but not adults within Cetoniinae, indicating dramatic pupal remodelling of these organ systems. Insights into the structure of the rectal complexes of Scarabaeoidea are compared with the well-studied rectal complexes of Cucujiformia. Finally we discuss possible functions of the rectal complexes of beetles within Scarabaeoidea, and future approaches to address this question.

evolutionary biology↗

bioRxiv2022

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 intrinsically 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 waterconserving functions of this system are unknown. Here, we introduce a transcriptomic resource, BeetleAtlas.org, for 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 modelling 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 novel functions of this cell. Together, our work provides the first insights into the molecular architecture underpinning the function of one most powerful water-conserving mechanisms in nature, the beetle rectal complex. Significance StatementBeetles are the largest group of insects, inhabiting a wide range of habitats on earth. Unique adaptations in overcoming water stress is critical to their success, yet the mechanisms underpinning this ability are unknown. Using genetics, electrophysiology, imaging and behavioral studies we show that a cation/H+ (NHA1) transporter is exclusively localized to specialized cell type, the leptophragmata, in the Malpighian tubules associated with the rectal complex. Ion transport functions of NHA1 in leptophragmata underpin the movement of water from the rectum, from where it would be destined for excretion, to the Malpighian tubule and then recycled back to the body. This water recovery capability of rectal complex is essential for maintaining systemic water balance in beetles. This work provides the first insight into to the molecular architecture of one of most powerful water-conservation mechanisms in biology, and provides an important clue to the ecological and evolutionary success of the beetles.

systems biology↗

bioRxiv2022

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 hearts ability to sense differences in haemodynamic load and adjust contractile force accordingly1-4. 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 hearts 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.

physiology↗

bioRxiv2022

Early patterning followed by tissue growth establishes proximo-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 (MipTs) 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 at the earliest stage of 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 wider than the effective range of Wingless. Summary statementHow does proximo-distal patterning occur in the epithelial Malpighian/renal tubules? Cells are patterned early by a mechanism involving Wingless/Wnt, and expand by cell proliferation to generate a distal domain.

developmental biology↗

bioRxiv2021

The mechano-sensitive ion channel Piezo mediates Rho activation and actin stress fibre formation in Drosophila nephrocytes

Due to their position on glomerular capillaries, podocytes are continuously counteracting biomechanical filtration forces. Most therapeutic interventions known to generally slow or prevent the progression of chronic kidney disease appear to lower these biomechanical forces on podocytes, highlighting the critical need to better understand podocyte mechano-signalling pathways. Here we investigated whether the mechanotransducer Piezo is involved in a mechanosensation pathway in Drosophila nephrocytes, the podocyte homologue in the fly. Loss of function analysis in Piezo depleted nephrocytes reveal a severe morphological and functional phenotype. Further, pharmacological activation of endogenous Piezo with Yoda1 causes a significant increase of intracellular Ca++ upon exposure to a mechanical stimulus in nephrocytes, as well as filtration disturbances. Elevated Piezo expression levels also result in a severe nephrocyte phenotype. Interestingly, expression of Piezo which lacks mechanosensitive channel activity, does not result in a severe nephrocyte phenotype, suggesting the observed changes in Piezo wildtype overexpressing cells are caused by the mechanosensitive channel activity. Moreover, blocking Piezo activity using the tarantula toxin GsMTx4 reverses the phenotypes observed in nephrocytes overexpressing Piezo. Taken together, here we provide evidence that Piezo activity levels need to be tightly regulated to maintain normal pericardial nephrocyte morphology and function.

cell biology↗

bioRxiv2021

Drosophila Filamin exhibits a mechano-protective role during nephrocyte injury via induction of hypertrophic growth

Podocytes are highly specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Due to their position and function in the kidney, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury and loss. The mechanism by which biomechanical forces are sensed and transduced to elicit an adaptive and protective response remains largely unknown. Here we show, using the Drosophila nephrocyte model, that the mechanosensor Cheerio (dFilamin) is central to this mechano-protective mechanism. We found expression of an activated mechanosensitive variant of Cheerio induced hypertrophy and rescued filtration function in injured nephrocytes. Additional analysis with human Filamin B confirmed this mechano-protective role. We delineated the mechano-protective pathway downstream of Cheerio and found activation of TOR and Yorkie induce nephrocyte hypertrophy, whereas their repression reversed the Cheerio-mediated hypertrophy. Although Cheerio/Filamin B pathway mediates a mechano-protective role in the face of injury, we found excessive activity resulted in a pathological phenotype, indicating activity levels must be tightly controlled. Taken together, our data suggest that Cheerio acts via the TOR and YAP pathway to induce hypertrophic growth, as a mechano-protective response to nephrocyte injury.

cell biology↗

bioRxiv2020

A Unique Renal 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 - homologues of vertebrate corticotropinreleasing factor (CRF) hormones - to control systemic water balance. Knockdown of the gene encoding the two hormones (Urinate, Urn8) reduces renal secretion and restricts organismal fluid loss, whereas injection of DH37 or DH47 reverses these phenotypes. We further identify a novel CRF-like receptor, Urinate Receptor (Urn8R), which is exclusively expressed in a unique secondary cell (SC) in the beetle renal organs, as underlying this response. Activation of Urn8R increases K+ secretion specifically through SCs, creating a lumen-positive transepithelial potential that drives fluid secretion. Together, these data show that beetle renal organs operate by fundamentally different mechanism than those of other insects. Finally, we adopt a fluorescent labelling strategy to identify the evolutionary origin of this unusual renal architecture within the large Order of Coleoptera. Our work thus uncovers an important homeostatic program that is key to maintaining osmotic control in beetles, which evolved in parallel to the radiation of the higher beetle families. Significance StatementBeetles are the most diverse animal group on the planet. Their evolutionary success suggests unique physiological adaptations in overcoming water stress, yet the mechanisms underlying this ability are unknown. Here we use molecular genetic, electrophysiology and behavioral studies to show that a group of brain neurons responds to osmotic disturbances by releasing diuretic hormones that regulate salt and water balance. These hormones bind to their receptor exclusively localized to a unique secondary cell in the renal organs to modulate fluid secretion and organismal water loss. This renal architecture, common to all higher beetle families, is novel within the insects, and provides an important clue to the evolutionary success of the beetles in colonizing an astounding range of habitats on Earth.

physiology↗

bioRxiv2020

Par3A and Par3B orchestrate podocyte architecture by regulating RhoA levels

Glomerular diseases are a major cause for chronic kidney disorders. In the majority of cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and subsequent loss of podocytes. Here, we establish a link between components of the Par3 polarity complex and actin regulators, which are necessary to establish and maintain the podocytes architecture utilizing both, mouse and Drosophila models. We demonstrate that the two mammalian Par3 proteins, Par3A and Par3B, share redundant functions despite differing in their ability to interact with other components of the Par complex. Only simultaneous inactivation of both Par3 proteins causes a severe disease phenotype in mouse podocytes by regulating Rho-GTP levels involving the actin regulators Synaptopodin and CD2AP in an aPKC independent manner.

cell biology↗