Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm.

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.

An acytokinetic cell division creates PIP2-enriched membrane asymmetries leading to slit diaphragm assembly in Drosophila nephrocytes.

Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly we have studied their formation in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. We find that their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms we describe allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.

Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes.

The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of Amnionless and two cubilin paralogues, Cubilin and Cubilin2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, which is strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Of note, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes suggest a possible unexpected conserved role for this endocytic receptor in vertebrate SD remodelling.

A specific isoform of Pyd/ZO-1 mediates junctional remodeling and formation of slit diaphragms.

The podocyte slit diaphragm (SD), responsible for blood filtration in vertebrates, is a major target of injury in chronic kidney disease. The damage includes severe morphological changes with destabilization of SDs and their replacement by junctional complexes between abnormally broadened foot processes. In Drosophila melanogaster, SDs are present in nephrocytes, which filter the fly's hemolymph. Here, we show that a specific isoform of Polychaetoid/ZO-1, Pyd-P, is essential for Drosophila SDs, since, in pyd mutants devoid of Pyd-P, SDs do not form and the SD component Dumbfounded accumulates at ectopic septate-like junctions between abnormally aggregated nephrocytes. Reintroduction of Pyd-P leads to junctional remodeling and their progressive normalization toward SDs. This transition requires the coiled-coil domain of Pyd-P and implies formation of nonclathrin vesicles containing SD components and their trafficking to the nephrocyte external membrane, where SDs assemble. Analyses in zebrafish suggest a conserved role for Tjp1a/ZO-1 in promoting junctional remodeling in podocytes.

Fear-of-intimacy-mediated zinc transport controls the function of zinc-finger transcription factors involved in myogenesis.

Zinc is a component of one-tenth of all human proteins. Its cellular concentration is tightly regulated because its dyshomeostasis has catastrophic health consequences. Two families of zinc transporters control zinc homeostasis in organisms, but there is little information about their specific developmental roles. We show that the ZIP transporter Fear-of-intimacy (Foi) is necessary for the formation of Drosophila muscles. In foi mutants, myoblasts segregate normally, but their specification is affected, leading to the formation of a misshapen muscle pattern and distorted midgut. The observed phenotypes could be ascribed to the inactivation of specific zinc-finger transcription factors (ZFTFs), supporting the hypothesis that they are a consequence of intracellular depletion of zinc. Accordingly, foi phenotypes can be rescued by mesodermal expression of other ZIP members with similar subcellular localization. We propose that Foi acts mostly as a transporter to regulate zinc intracellular homeostasis, thereby impacting on the activity of ZFTFs that control specific developmental processes. Our results additionally suggest a possible explanation for the presence of large numbers of zinc transporters in organisms based on differences in ion transport specificity and/or degrees of activity among transporters.

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is an important serine/threonine-kinase regulating different membrane receptors and intracellular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant (K220R) caused precocious differentiation of cells into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.

Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies.

Drosophila nephrocytes are functionally homologous to vertebrate kidney podocytes. Both share the presence of slit diaphragms that function as molecular filters during the process of blood and haemolymph ultrafiltration. The protein components of the slit diaphragm are likewise conserved between flies and humans, but the mechanisms that regulate slit diaphragm dynamics in response to injury or nutritional changes are still poorly characterised. Here, we show that Dumbfounded/Neph1, a key diaphragm constituent, is a target of the Src kinase Src64B. Loss of Src64B activity leads to a reduction in the number of diaphragms, and this effect is in part mediated by loss of Dumbfounded/Neph1 tyrosine phosphorylation. The phosphorylation of Duf by Src64B, in turn, regulates Duf association with the actin regulator Dock. We also find that diaphragm damage induced by administration of the drug puromycin aminonucleoside (PAN model) directly associates with Src64B hyperactivation, suggesting that diaphragm stability is controlled by Src-dependent phosphorylation of diaphragm components. Our findings indicate that the balance between diaphragm damage and repair is controlled by Src-dependent phosphorylation of diaphragm components, and point to Src family kinases as novel targets for the development of pharmacological therapies for the treatment of kidney diseases that affect the function of the glomerular filtration barrier.

Drosophila araucan and caupolican integrate intrinsic and signalling inputs for the acquisition by muscle progenitors of the lateral transverse fate.

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. Here we identify, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, we show that Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity.

The complex spatio-temporal regulation of the Drosophila myoblast attractant gene duf/kirre.

A key early player in the regulation of myoblast fusion is the gene dumbfounded (duf, also known as kirre). Duf must be expressed, and function, in founder cells (FCs). A fixed number of FCs are chosen from a pool of equivalent myoblasts and serve to attract fusion-competent myoblasts (FCMs) to fuse with them to form a multinucleate muscle-fibre. The spatial and temporal regulation of duf expression and function are important and play a deciding role in choice of fibre number, location and perhaps size. We have used a combination of bioinformatics and functional enhancer deletion approaches to understand the regulation of duf. By transgenic enhancer-reporter deletion analysis of the duf regulatory region, we found that several distinct enhancer modules regulate duf expression in specific muscle founders of the embryo and the adult. In addition to existing bioinformatics tools, we used a new program for analysis of regulatory sequence, PhyloGibbs-MP, whose development was largely motivated by the requirements of this work. The results complement our deletion analysis by identifying transcription factors whose predicted binding regions match with our deletion constructs. Experimental evidence for the relevance of some of these TF binding sites comes from available ChIP-on-chip from the literature, and from our analysis of localization of myogenic transcription factors with duf enhancer reporter gene expression. Our results demonstrate the complex regulation in each founder cell of a gene that is expressed in all founder cells. They provide evidence for transcriptional control--both activation and repression--as an important player in the regulation of myoblast fusion. The set of enhancer constructs generated will be valuable in identifying novel trans-acting factor-binding sites and chromatin regulation during myoblast fusion in Drosophila. Our results and the bioinformatics tools developed provide a basis for the study of the transcriptional regulation of other complex genes.

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.

Mind bomb 2, a founder myoblast-specific protein, regulates myoblast fusion and muscle stability.

A fundamental step during Drosophila myogenesis is the specification of founder myoblasts (FMs). Founders possess the information required for the acquisition of muscle identity and for the execution of the myogenic programme, whereas fusion-competent myoblasts (FCMs) acquire this information after fusing to founders. Very little is known about genes that implement the execution of the myogenic programme. Here we characterise Mind bomb 2 (Mib2), a protein with putative E3 ubiquitin ligase activity that is exclusive of FMs and necessary for at least two distinct steps of the founder/myotube differentiation programme. Thus, in mib2 mutants, the early process of myoblast fusion is compromised, as FMs undergo a reduced number of rounds of fusion with FCMs. At later stages, with the onset of muscle contraction, many muscles degenerate, display aberrant sarcomeric structure and detach from tendons. The fusion process requires intact E3-RING-finger domains of Mib2 (the putative catalytic sites), probably to eliminate the FCM-specific activator Lmd from nascent myotubes. However, these sites appear dispensable for muscle integrity. This, and the subcellular accumulation of Mib2 in Z and M bands of sarcomeres, plus its physical interaction with nonmuscle myosin (a Z-band-localised protein necessary for the formation of myofibrils), suggest a structural role for Mib2 in maintaining sarcomeric stability. We suggest that Mib2 acts sequentially in myoblast fusion and sarcomeric stability by two separable processes involving distinct functions of Mib2.

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.

Conserved cross-interactions in Drosophila and Xenopus between Ras/MAPK signaling and the dual-specificity phosphatase MKP3.

The extracellular signal-regulated kinase (ERK) is a key transducer of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) signaling pathways, and its function is required in multiple processes during animal development. The activity of ERK depends on the phosphorylation state of conserved threonine and tyrosine residues, and this state is regulated by different kinases and phosphatases. A family of phosphatases with specificity toward both threonine and tyrosine residues in ERK (dual-specificity phosphatases) play a conserved role in its dephosphorylation and consequent inactivation. Here, we characterize the function of the dual-specificity phosphatase MKP3 in Drosophila EGFR and Xenopus FGFR signaling. The function of MKP3 is required during Drosophila wing vein formation and Xenopus anteroposterior neural patterning. We find that the expression of the MKP3 gene is localized in places of high EGFR and FGFR signaling. Furthermore, this restricted expression depends on ERK function both in Drosophila and Xenopus, suggesting that MKP3 constitutes a conserved negative feedback loop on the activity of the Ras/ERK signaling pathway.

Founder myoblasts and fibre number during adult myogenesis in Drosophila.

We have examined the mechanisms underlying the setting of myotubes and choice of myotube number in adult Drosophila. We find that the pattern of adult myotubes is prefigured by a pattern of duf-lacZ-expressing myoblasts at appropriate locations. Selective expression of duf-lacZ in single myoblasts emerges from generalized, low-level expression in all adult myoblasts during the third larval instar. The number of founders, thus chosen, corresponds to the number of fibres in a muscle. In contrast to the embryo, the selection of individual adult founder cells during myogenesis does not depend on Notch-mediated lateral inhibition. Our results suggest a general mechanism by which multi-fibre muscles can be patterned.

Tufted is a gain-of-function allele that promotes ectopic expression of the proneural gene amos in Drosophila.

The Tufted(1) (Tft(1)) dominant mutation promotes the generation of ectopic bristles (macrochaetae) in the dorsal mesothorax of Drosophila. Here we show that Tft(1) corresponds to a gain-of-function allele of the proneural gene amos that is associated with a chromosomal aberration at 36F-37A. This causes ectopic expression of amos in large domains of the lateral-dorsal embryonic ectoderm, which results in supernumerary neurons of the PNS, and in the notum region of the third instar imaginal wing, which gives rise to the mesothoracic extra bristles. Revertants of Tft(1), which lack ectopic neurons and bristles, do not show ectopic expression of amos. One revertant is a loss-of-function allele of amos and has a recessive phenotype in the embryonic PNS. Our results suggest that both normal and ectopic Tft(1) bristles are generated following similar rules, and both are subjected to Notch-mediated lateral inhibition. The ability of Tft(1) bristles to appear close together may be due to amos having a stronger proneural capacity than that of other proneural genes like asense and scute. This ability might be related to the wild-type function of amos in promoting development of large clusters of closely spaced olfactory sensilla.

myoblasts incompetent encodes a zinc finger transcription factor required to specify fusion-competent myoblasts in Drosophila.

We report a new gene, myoblasts incompetent, essential for normal myogenesis and myoblast fusion in Drosophila. myoblasts incompetent encodes a putative zinc finger transcription factor related to vertebrate Gli proteins and to Drosophila Cubitus interruptus. myoblasts incompetent is expressed in immature somatic and visceral myoblasts. Expression is predominantly in fusion-competent myoblasts and a loss-of-function mutation in myoblasts incompetent leads to a failure in the normal differentiation of these cells and a complete lack of myoblast fusion. In the mutant embryos, founder myoblasts differentiate normally and form mononucleate muscles, but genes that are specifically expressed in fusion-competent cells are not activated and the normal downregulation of twist expression in these cells fails to occur. In addition, fusion-competent myoblasts fail to express proteins characteristic of the general pathway of myogenesis such as myosin and Dmef2. Thus myoblasts incompetent appears to function specifically in the general pathway of myogenesis to control the differentiation of fusion-competent myoblasts.