Adult Tubules Research Articles

Hippo effector, Yorkie, is a tumor suppressor in select Drosophila squamous epithelia

Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.

Compromised junctional integrity phenocopies age-dependent renal dysfunction in Drosophila Snakeskin mutants.

Transporting epithelia provide a protective barrier against pathogenic insults while allowing the controlled exchange of ions, solutes and water with the external environment. In invertebrates, these functions depend on formation and maintenance of 'tight' septate junctions (SJs). However, the mechanism by which SJs affect transport competence and tissue homeostasis, and how these are modulated by ageing, remain incompletely understood. Here, we demonstrate that the Drosophila renal (Malpighian) tubules undergo an age-dependent decline in secretory capacity, which correlates with mislocalisation of SJ proteins and progressive degeneration in cellular morphology and tissue homeostasis. Acute loss of the SJ protein Snakeskin in adult tubules induced progressive changes in cellular and tissue architecture, including altered expression and localisation of junctional proteins with concomitant loss of cell polarity and barrier integrity, demonstrating that compromised junctional integrity is sufficient to replicate these ageing-related phenotypes. Taken together, our work demonstrates a crucial link between epithelial barrier integrity, tubule transport competence, renal homeostasis and organismal viability, as well as providing novel insights into the mechanisms underpinning ageing and renal disease.

Testis-on-chip platform to study ex vivo primate spermatogenesis and endocrine dynamics

Here, we report a testis-on-chip platform for the ex vivo culture of seminiferous tubules isolated from human and non-human primate testis. Tissues are cultured in a dedicated chamber with continuous perfusion via a vascular-like channel. The platform is fabricated from PDMS using a 3D printed mold, after design has been optimized, e.g., for the barrier between the culture chamber and the perfusion channel. COMSOL modeling revealed no direct negative impact of the flow on the tissues for the applied flowrate in the device, shear rate remaining in the physiological range. Culture experiments were performed using adult human seminiferous tubules from gender dysphoria patients and prepubertal seminiferous tubules from a 6-month-old marmoset for up to 11 and 9 days, respectively. First, microscopic, and live imaging revealed the presence of viable cell populations in both types of samples. Next, marmoset tissues were exposed to stimulatory conditions through perfusion of gonadotropins at different doses. The tissue response was characterized by histological analysis after their recovery from the device and testosterone and estradiol analysis in the effluent. Histological observations suggested improved maintenance of marmoset testicular tissues under stimulatory conditions, which similarly resulted in an increase in both testosterone and estradiol production, with yet different patterns for the low-dose and high-dose stimulation. The herein reported testis-on-chip platform shows great promise to evaluate endocrine and toxic effects on the testis.

Molecular Cloning and Expression Profiling of CncC in Bactrocera dorsalis Hendel.

Simple SummaryWe cloned the CncC gene of Bactrocera dorsalis and performed a domain analysis of the protein to clarify the expression levels of the gene in different developmental stages and tissue sites of B. dorsalis and to provide a theoretical basis for further investigating the function of CncC in regulating pesticide resistance in B. dorsalis.The cap ‘n’ collar isoform C (CncC) transcription factor is thought to be a regulator associated with antioxidant and detoxification genes that can enhance pest resistance by regulating the expression of detoxification enzyme genes. However, this transcription factor has not been well studied in the important agricultural pest Bactrocera dorsalis. In this study, the cDNA sequence of CncC in B. dorsalis was cloned, and the complete ORF sequence was obtained; it had a sequence length of 3378 bp, encoding a total of 1125 amino acids. Phylogenetic tree analysis showed that B. dorsalis CncC belonged to the CNC family and that its amino acid sequence showed the closest relationship with B. tryoni. The conserved structural region of BdCncC was analyzed and was found to include a conserved bZIP superfamily structural domain. Spatiotemporal expression analysis revealed that BdCncC was most highly expressed in the adult Malpighian tubules, followed by the antennae, foregut, and midgut, and then the brain, hemolymph, hindgut, and fat body. BdCncC was expressed at every developmental stage, and the highest expression was found in mature males. This study provides a theoretical basis for an in-depth investigation of the function of BdCncC in regulating pesticide resistance in B. dorsalis.

An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine.

In recent years, live-imaging techniques have been developed for the adult midgut of Drosophila melanogaster that allow temporal characterization of key processes involved in stem cell and tissue homeostasis. However, these organ culture techniques have been limited to imaging sessions of <16 hours, an interval too short to track dynamic processes such as damage responses and regeneration, which can unfold over several days. Therefore, we developed an organ explant culture protocol capable of sustaining midguts ex vivo for up to 3 days. This was made possible by the formulation of a culture medium specifically designed for adult Drosophila tissues with an increased Na+/K+ ratio and trehalose concentration, and by placing midguts at an air-liquid interface for enhanced oxygenation. We show that midgut progenitor cells can respond to gut epithelial damage ex vivo, proliferating and differentiating to replace lost cells, but are quiescent in healthy intestines. Using ex vivo gene induction to promote stem cell proliferation using RasG12V or string and Cyclin E overexpression, we demonstrate that progenitor cell lineages can be traced through multiple cell divisions using live imaging. We show that the same culture set-up is useful for imaging adult renal tubules and ovaries for up to 3 days and hearts for up to 10 days. By enabling both long-term imaging and real-time ex vivo gene manipulation, our simple culture protocol provides a powerful tool for studies of epithelial biology and cell lineage behavior.

Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids

The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on organic anion transporter 1 (OAT1), one of the most important proteins involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters, resulting in improved OAT1 transport capacity. Next, a more in-depth proteomic data analysis showed that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation. Moreover, we demonstrated that the combination of EVs and tubuloid-derived cells can be used as part of a bioartificial kidney to generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from kidney tubular epithelial cells can phenotypically improve in vitro tubuloid maturation, thereby enhancing their potential as functional units in regenerative or renal replacement therapies.Graphical

Dent Disease in Drosophila: An Unexpected Model to Study the Relationship Between a Cl‐ Transporter and Ca2+

Mutations in the 2Cl‐/H+ transporter CLC‐5 cause Dent Disease Type 1 (DD1) and lead to progressive renal failure by age 20‐40. A major characteristic of DD1 is the renal mishandling of Ca2+that increases urinary Ca2+ excretion, calcium oxalate (CaOx) kidney stones, and kidney calcification. Interestingly, none of the DD1 symptoms include or relate to Cl‐ homeostasis despite impaired transport function. Thus, how does a Cl‐ transporter cause Ca2+ dysregulation?Our laboratory has identified Drosophila Clc‐c to be the homolog of CLC‐5 with conserved amino acids at DD1 mutation sites. We hypothesize that Clc‐c shares functional similarities in Cl‐transport and Ca2+ homeostasis. To test our hypothesis, we used electrophysiology to measure ion transport, and then assessed CaOx crystal formation in adult fly renal tubules (Malpighian tubules, MTs) of Drosophila.Voltage clamp experiments with Xenopus oocytes show that Clc‐c is electrogenic with the same outward‐rectifying current in Cl‐ solutions as observed with human CLC‐5. Next, knockdown flies (Clc‐c‐KD) were generated by crossing Uro‐Gal4 (MT driver) with UAS‐Clc‐c‐RNAi flies, resulting in 50% Clc‐c mRNA expression in the MTs. Crystals in adult MT (7 days after eclosure) were present in 100% of Clc‐c‐KD compared to 71% of control flies. The average number of crystals in adult tubule pairs was significantly greater in Clc‐c‐KD flies (15±4 crystals per pair) than control flies (5±2 crystals, P= 0.02). When fed a diet with 2mM NaOx ClcC‐KD for 4 days, Clc‐c‐KD flies had a significantly greater number of crystals in each tubule (24±5 crystals) than controls (4±2 crystals, P=0.005). However, the KD flies did not produce more crystals than control flies when fed a supraphysiological concentration of sodium oxalate (20 mM NaOx diet) for 1 or 4 days and in ex vivo assays of dissected MTs submerged in solution containing 10 mM NaOx for 1 hour.In conclusion, Drosophila Clc‐c has both identical sequences at DD1 mutation sites and similar biophysical functions to human CLC‐5. Furthermore, Clc‐c‐KD increases incidence of CaOx crystals which corresponds to the increased risks for kidney stones in DD1 patients. With this fly‐DD1 model, we will be able to assess the relationship of Clc‐c Cl‐ transport to renal Ca deposits. Future experiments will evaluate renal pathophysiology of DD1 mutations and their relationship to Ca2+ transport in Clc‐c transgenic flies.

The role of mineralocorticoid receptor in the potassium switch: effect on NCC regulation+

Thiazide sensitive sodium‐chloride cotransporter (NCC) is a major salt transport pathway in the apical membrane of the nephron distal convoluted tubule. While we know the importance of its function in maintaining sodium‐potassium homeostasis, the regulation of NCC is complicated and the mechanisms are not well understood. The aim of this study is to evaluate the role of mineralocorticoid receptors (MRs) in the regulation of NCC. Here, we generated DCT‐specific MRs Knockout (KO) mice, in which MRs were deleted in cells expressing NCC, taking advantage of a cre recombinase under the control of the NCC gene. Under standard and Low‐Na+ diets, these DCT‐specific MRs‐KO mice display normal Na+/K+ balance but exhibit problems with Cl‐ regulation. These KO mice presented hypochloremia under normal diet but unexpectedly, hyperchloremia after 6d on low Na+ diet. The expression of NCC and phosphorylated NCC were both decreased in KO mice compared to that in control mice, under both standard and low‐Na+ diet. This decrease in NCC protein levels is related to a decrease in transcription levels. Under standard diet, decreased NCC expression is compensated by an increase in αENaC and pendrin expression, which is not the case under low‐salt diet. Under a low‐K+ diet, KO mice exhibit hypokalemia and volume depletion with increased blood CO2 levels and Renin mRNA levels. NCC expression and its phosphorylation are both increased in control and KO groups under low‐K+ diet compared to standard diet but, the levels of NCC protein and mRNA are still decreased in the KO group compared to the control group mice. Taken together, these results demonstrate that MRs is not necessary for the complete regulation of NCC under low‐Na+ and low‐K+ diet. On the other hand, MRs appears to be indispensable to assure proper NCC expression in adult DCT renal tubules.

Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation

The Pax family of developmental control genes are frequently deregulated in human disease. In the kidney, Pax2 is expressed in developing nephrons but not in adult proximal and distal tubules, whereas polycystic kidney epithelia or renal cell carcinoma continues to express high levels. Pax2 reduction in mice or cell culture can slow proliferation of cystic epithelial cells or renal cancer cells. Thus, inhibition of Pax activity may be a viable, cell-type-specific therapy. We designed an unbiased, cell-based, high-throughput screen that identified triazolo pyrimidine derivatives that attenuate Pax transactivation ability. We show that BG-1 inhibits Pax2-positive cancer cell growth and target gene expression but has little effect on Pax2-negative cells. Chromatin immunoprecipitation suggests that these inhibitors prevent Pax protein interactions with the histone H3K4 methylation complex at Pax target genes in renal cells. Thus, these compounds may provide structural scaffolds for kidney-specific inhibitors with therapeutic potential.

Studies on Postnatal Development of Testis of Guinea Pig (Cavia porcellus)

The histology and histochemistry of the testis of guinea pig of various postnatal age groups was conducted. A total of 24 guinea pigs of four different postnatal ages with six male animals each were collected from the Department of Laboratory Animal Medicine, Madhavaram Milk Colony, Chennai as per the Ethical committee approval. After collection, animals were euthanized as per CPCSEA norms and testis was dissected out and was cut into small pieces, fixed and processed for paraffin embedding. Sections of 4–5 µm thickness were cut and used for the routine and special histological and histochemical staining techniques. Testes wereencapsulated by tunica vaginalis and tunica albuginea. Septa from the capsule divided the testicular parenchyma into lobules. Each lobule consisted of seminiferous tubules which consisted of spermatogenic cells in stratified layers and sertoli cells. Pre-weaning and weaning group of guinea pigs seminiferous tubules showed wide lumen with only type 1 and type 2 spermatogonia and sertoli cells. Young and adult animals seminiferous tubules showed narrow lumen with type 1 and type 2 spermatogonia, primary spermatocytes in various stages of differentiation, secondary spermatocytes, spermatids (early and late) and sertoli cells. Sertoli cells were large oval shaped cells with lightly stained irregular shaped nucleus. Interstitial tissue contained leydig cells in all ages.Leydig cells appeared as varied in shape. In all the age groups studied, PAS activity was noticed in the capsule and basement membrane. The micrometrical parameters increased as age advanced in both right and left testis.

Differentiation of fetal sertoli cells in the adult testis.

Sertoli cells proliferate and construct seminiferous tubules during fetal life, then undergo differentiation and maturation in the prepubertal testes. In the adult testes, mature Sertoli cells maintain spermatogonia and support spermatogenesis during the entire lifetime. Although Sertoli-like cells have been derived from iPS cells, they tend to remain immature. To investigate whether Sertoli cells can spontaneously acquire the ability to support spermatogenesis when transferred into the adult testis, we transplanted mouse fetal testicular cells into a Sertoli-depleted adult testis. We found that donor E12.5, E14.5 and E16.5 Sertoli cells colonized adult seminiferous tubules and supported host spermatogenesis 2 months after transplantation, demonstrating that immature fetal Sertoli cells can undergo sufficient maturation in the adult testis to become functional. This technique will be useful to analyze the developmental process of Sertoli cell maturation and to investigate the potential of iPS-derived Sertoli cells to colonize, undergo maturation, and support spermatogenesis within the testis environment.

Molecular basis for epithelial morphogenesis and ion transport in the Malpighian tubule.

During development, the insect Malpighian tubule undergoes several programmed morphogenetic events that give rise to the tubule's ability to transport ions and water at unparalleled speed. Studies in Diptera, in particular, have greatly increased our understanding of the molecular pathways underlying embryonic tubule development. In this review, we discuss recent work that has revealed new insights into the molecular players required for the development and maintenance of structurally and functionally intact adult Malpighian tubules. We highlight the contribution of the smooth septate junction (sSJ) proteins to the morphogenesis and transport function of the epithelial cells of the Drosophila melanogaster Malpighian tubule and also discuss new findings on the role of the GATAe transcription factor. We also consider the roles of sSJ proteins in the fly midgut, as compared to the Malpighian tubule, and the importance of cellular context for the functions of these proteins.

Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men.

Sperm develop from puberty in the seminiferous tubules, inside the blood‐testis barrier to prevent their recognition as “non‐self” by the immune system, and it is widely assumed that human sperm‐specific proteins cannot access the circulatory or immune systems. Sperm‐specific proteins aberrantly expressed in cancer, known as cancer‐testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell‐derived and sperm‐specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is “outside” the blood‐testis barrier. From TIF, the proteins can access the circulatory‐ and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm‐specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood‐based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex‐specific and male‐fertility‐related responses.

Abstract IA25: Adult deletion of Lats1/2 leads to sarcomatoid renal cell carcinoma

Abstract Alterations of the Hippo signaling pathway have been found in patients with sarcomatoid renal cell carcinoma, a highly aggressive kidney disease with rapid progression and poor prognosis. It is unclear whether deregulated Hippo pathway is a primary cause of sarcomatoid cancers. To investigate whether conditional (kidney-specific) Hippo knockout leads to renal cancer development and/or sarcomatoid dedifferentiation, we conditionally inactivated both Lats1 and Lats2 from the adult renal proximal tubule epithelial cells. Mosaic deletion of Lats1 and Lats2 in the adult proximal tubules results in cell-autonomous sarcomatoid renal cell carcinoma ((54%) as early as 8 weeks after gene deletion) and lung metastasis. RNA sequencing of tumors compared to control renal cortices revealed massive transcriptional changes, with 2,292 genes significantly upregulated (FC&amp;gt;2) and 2261 genes significantly downregulated (FCË‚0.5). To visualize the cellular onset and progression of renal defects, we conducted histologic examination at different time points after induction of Lats1/2 deletion. Mutant kidneys displayed tubular atrophy with glomerular cysts. Loss of proximal tubule epithelial markers, and de novo expression of the mesenchymal marker vimentin, indicated that kidney epithelial cells had undergone an epithelial-to-mesenchymal transition (EMT). These studies demonstrate that loss of Lats1/2 rapidly leads to aggressive, metastatic sarcomatoid renal cell carcinoma. Citation Format: Antoine Reginensi, Sanjay Jain, Benjamin Humphreys, Jess Shen, Didier Hodzic, Jeff Wrana, Helen McNeill. Adult deletion of Lats1/2 leads to sarcomatoid renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA25.

Kidney Organoids and Tubuloids.

In the past five years, pluripotent stem cell (PSC)-derived kidney organoids and adult stem or progenitor cell (ASC)-based kidney tubuloids have emerged as advanced in vitro models of kidney development, physiology, and disease. PSC-derived organoids mimic nephrogenesis. After differentiation towards the kidney precursor tissues ureteric bud and metanephric mesenchyme, their reciprocal interaction causes self-organization and patterning in vitro to generate nephron structures that resemble the fetal kidney. ASC tubuloids on the other hand recapitulate renewal and repair in the adult kidney tubule and give rise to long-term expandable and genetically stable cultures that consist of adult proximal tubule, loop of Henle, distal tubule, and collecting duct epithelium. Both organoid types hold great potential for: (1) studies of kidney physiology, (2) disease modeling, (3) high-throughput screening for drug efficacy and toxicity, and (4) regenerative medicine. Currently, organoids and tubuloids are successfully used to model hereditary, infectious, toxic, metabolic, and malignant kidney diseases and to screen for effective therapies. Furthermore, a tumor tubuloid biobank was established, which allows studies of pathogenic mutations and novel drug targets in a large group of patients. In this review, we discuss the nature of kidney organoids and tubuloids and their current and future applications in science and medicine.

Regeneration after acute kidney injury requires PTIP-mediated epigenetic modifications.

A terminally differentiated cellular phenotype is thought to be maintained, at least in part, by both active and repressive histone marks. However, it is unclear whether regenerating cells after injury need to replicate such epigenetic marks to recover. To test whether renal epithelial cell regeneration is dependent on histone H3K4 methylation, we generated a mouse model that deleted the Paxip1 gene in mature renal proximal tubules. Paxip1 encodes PTIP, an essential protein in the Mll3/4 histone H3K4 methyltransferase complex. Mice with PTIP deletions in the adult kidney proximal tubules were viable and fertile. Upon acute kidney injury, such mice failed to regenerate damaged tubules, leading to scarring and interstitial fibrosis. The inability to repair damage was likely due to a failure to reenter mitosis and reactivate regulatory genes such as Sox9. PTIP deletion reduced histone H3K4 methylation in uninjured adult kidneys but did not significantly affect function or the expression of epithelial specific markers. Strikingly, cell lineage tracing revealed that surviving PTIP mutant cells could alter their phenotype and lose epithelial markers. These data demonstrate that PTIP and associated MLL3/4-mediated histone methylation are needed for regenerating proximal tubules and to maintain or reestablish the cellular epithelial phenotype.

The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule.

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.

Rab11 plays a key role in stellate cell differentiation via non-canonical Notch pathway in Malpighian tubules of Drosophila melanogaster

Rab11, a member of Rab-GTPase family, and a marker of recycling endosomes has been reported to be involved in the differentiation of various tissues in Drosophila. Here we report a novel role of Rab11 in the differentiation of stellate cells via the non-canonical Notch pathway in Malpighian tubules. During Malpighian tubule development caudal visceral mesodermal cells intercalate into the epithelial tubule of ectodermal origin consisting of principal cells, undergo mesenchymal to epithelial transition and differentiate into star shaped stellate cells in adult Malpighian tubule. Two transcription factors, Teashirt and Cut (antagonistic to each other) are known to be expressed in stellate cells and principal cells, respectively, from early stages of development and serve as markers for these cells. Inhibition of Rab11 function or over-expression of activated Notch in stellate cells resulted in the expression of Cut that leads to down-regulation of Teashirt or vice-versa that leads to hampered differentiation of stellate cells. The stellate cells do not transform to star/bar shaped and remain in mesenchymal state in adult Malpighian tubule. Over-expression of Deltex, which plays important role in non-canonical Notch signaling pathway, shows similar phenotype of stellate cells as seen in individuals with down-regulated Rab11, while down-regulation of Deltex in genetic background of Rab11RNAi rescues Teashirt expression and shape of stellate cells. Our experiments suggest that an inhibition or reduction of Rab11 function in stellate cells results in the faulty recycling of Notch receptors to plasma membrane as they accumulate in early and late endosomes, leading to Deltex mediated non-canonical Notch activation.

TmDorX2 positively regulates antimicrobial peptides in Tenebrio molitor gut, fat body, and hemocytes in response to bacterial and fungal infection

Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Insect glutathione S-transferases (GSTs) are important in insecticide detoxification and Insect-specific GSTs, Epsilon and Delta, have largely expanded in insects. In this study, we functionally expressed and characterized an epsilon class GST gene (BdGSTe8), predominant in the adult Malpighian tubules of Bactrocera dorsalis. This gene may be associated with malathion resistance based on transcriptional studies of resistant and susceptible strains. RNA interference-mediated knockdown of this gene significantly recovered malathion susceptibility in the adults of a malathion-resistant strain, and overexpression of BdGSTe8 enhanced resistance in transgenic Drosophila. Analysis of BdGSTe8 polymorphism showed that several point mutations may be associated with metabolic resistance to malathion. A cytotoxicity assay in Escherichia coli indicated that both of the recombinant BdGSTe8 proteins may play a functional role in protecting cells from toxicity. The allele of BdGSTe8-B conferred higher levels of malathion detoxification capability. Liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that the BdGSTe8-A allele did not metabolize malathion directly. However, the BdGSTe8-B allele was involved in the direct metabolism of malathion, which was caused by a mutation in V128A. Further analysis of the sequence suggests that BdGSTe8 evolved rapidly. It maybe play the role of a backup gene and could become a new gene in the future in order to retain the ability of detoxification of malathion, which was driven by positive selection. These results suggest that divergent molecular evolution in BdGSTe8 has played a role in metabolic resistance to malathion in B.dorsalis.