RESUMEN
Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional macromolecular assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the elements that maintain nephrocyte architecture and the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Rac1 and Cdc42 activity as well as proper microtubule organization and endoplasmic reticulum structure, are required to position the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/citología , Drosophila/metabolismo , Riñón/citología , Riñón/metabolismo , Podocitos/metabolismo , Animales , Citoesqueleto/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Riñón/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Nocodazol/farmacología , Podocitos/efectos de los fármacosRESUMEN
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.
Asunto(s)
Movimiento Celular , Polaridad Celular , Drosophila melanogaster/citología , Factor de Crecimiento Epidérmico/metabolismo , Túbulos de Malpighi/embriología , Morfogénesis , Miosina Tipo II/metabolismo , Transducción de Señal , Animales , Linaje de la Célula , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Epitelio/embriología , Epitelio/metabolismo , Receptores ErbB/metabolismo , Genes de Insecto , Homeostasis , Túbulos de Malpighi/citología , Modelos BiológicosRESUMEN
The normal development of an organ depends on the coordinated regulation of multiple cell activities. Focusing on tubulogenesis, we review the role of specialised cells or groups of cells that are selected from within tissue primordia and differentiate at the outgrowing tips or leading edge of developing tubules. Tip or leading cells develop distinctive patterns of gene expression that enable them to act both as sensors and transmitters of intercellular signalling. This enables them to explore the environment, respond to both tissue intrinsic signals and extrinsic cues from surrounding tissues and to regulate the behaviour of their neighbours, including the setting of cell fate, patterning cell division, inducing polarity and promoting cell movement and cell rearrangements by neighbour exchange. Tip cells are also able to transmit mechanical tension to promote tissue remodelling and, by interacting with the extracellular matrix, they can dictate migratory pathways and organ shape. Where separate tubular structures fuse to form networks, as in the airways of insects or the vascular system of vertebrates, specialised fusion tip cells act to interconnect disparate elements of the developing network. Finally, we consider their importance in the maturation of mature physiological function and in the development of disease.
Asunto(s)
Células/metabolismo , Morfogénesis , Animales , Proliferación Celular , Células/citología , HumanosRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster , Riñón/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Túbulos Renales/embriología , Túbulos Renales/crecimiento & desarrollo , Túbulos Renales/metabolismo , Ratones , Modelos Biológicos , Organogénesis/genética , Organogénesis/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Equilibrio Hidroelectrolítico/genéticaRESUMEN
Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2α kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2α phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. We report the characterisation of the first non-mammalian eIF2α phosphatase and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5'UTR, enabling induction in response to eIF2α phosphorylation. Moreover, we show that its expression is necessary for embryonic and larval development and that this is to oppose the inhibitory effects of GCN2 on anabolic growth.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 1/metabolismo , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Células COS , Chlorocebus aethiops , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/genética , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosforilación/genética , Proteínas Quinasas/genética , Proteína Fosfatasa 1/genética , Procesamiento Postranscripcional del ARN/genética , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Podocitos/citología , Podocitos/fisiología , Animales , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/fisiología , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Podocitos/metabolismoRESUMEN
During embryonic development, Drosophila macrophages (haemocytes) undergo a series of stereotypical migrations to disperse throughout the embryo. One major migratory route is along the ventral nerve cord (VNC), where haemocytes are required for the correct development of this tissue. We show, for the first time, that a reciprocal relationship exists between haemocytes and the VNC and that defects in nerve cord development prevent haemocyte migration along this structure. Using live imaging, we demonstrate that the axonal guidance cue Slit and its receptor Robo are both required for haemocyte migration, but signalling is not autonomously required in haemocytes. We show that the failure of haemocyte migration along the VNC in slit mutants is not due to a lack of chemotactic signals within this structure, but rather to a failure in its detachment from the overlying epithelium, creating a physical barrier to haemocyte migration. This block of haemocyte migration in turn disrupts the formation of the dorsoventral channels within the VNC, further highlighting the importance of haemocyte migration for correct neural development. This study illustrates the important role played by the three-dimensional environment in directing cell migration in vivo and reveals an intriguing interplay between the developing nervous system and the blood cells within the fly, demonstrating that their development is both closely coupled and interdependent.
Asunto(s)
Movimiento Celular/fisiología , Drosophila/embriología , Macrófagos/fisiología , Sistema Nervioso/embriología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Movimiento Celular/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Embrión no Mamífero , Desarrollo Embrionario/genética , Hemocitos/metabolismo , Hemocitos/fisiología , Macrófagos/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas RoundaboutRESUMEN
Molecular biology is reaching new depths in our understanding of the development and physiology of Malpighian tubules. In Diptera, Malpighian tubules derive from ectodermal cells that evaginate from the primitive hindgut and subsequently undergo a sequence of orderly events that culminates in an active excretory organ by the time the larva takes its first meal. Thereafter, the tubules enlarge by cell growth. Just as modern experimental strategies have illuminated the development of tubules, genomic, transcriptomic, and proteomic studies have uncovered new tubule functions that serve immune defenses and the breakdown and renal clearance of toxic substances. Moreover, genes associated with specific diseases in humans are also found in flies, some of which, astonishingly, express similar pathophenotypes. However, classical experimental approaches continue to show their worth by distinguishing between -omic possibilities and physiological reality while providing further detail about the rapid regulation of the transport pathway through septate junctions and the reversible assembly of proton pumps.
Asunto(s)
Dípteros/embriología , Túbulos de Malpighi/embriología , Animales , Dípteros/genética , Dípteros/metabolismo , Genómica , Túbulos de Malpighi/metabolismoRESUMEN
Organs are made up of cells from separate origins, whose development and differentiation must be integrated to produce a physiologically coherent structure. For example, during the development of the kidney, a series of interactions between the epithelial mesonephric duct and the surrounding metanephric mesenchyme leads to the formation of renal tubules. Cells of the metanephric mesenchyme first induce branching of the mesonephric duct to form the ureteric buds, and they then respond to signals derived from them. As a result, mesenchymal cells are recruited to the buds, where they undergo a mesenchymal-to-epithelial transition as they condense to form nephrons. In contrast, the simple renal tubules of invertebrates, such as insect Malpighian tubules (MpTs), have always been thought to arise from single tissue primordia, epithelial buds that grow by cell division and enlargement and from which a range of specialized subtypes differentiate. Here, we reveal unexpected parallels between the development of Drosophila MpTs and vertebrate nephrogenesis by showing that the MpTs also derive from two cell populations: ectodermal epithelial buds and the surrounding mesenchymal mesoderm. The mesenchymal cells are recruited to the growing tubules, where they undergo a mesenchymal-to-epithelial transition as they integrate and subsequently differentiate as a physiologically distinctive subset of tubule cells, the stellate cells. Strikingly, the normal incorporation of stellate cells and the later physiological activity of the mature tubules depend on the activity of hibris, an ortholog of mammalian NEPHRIN.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Túbulos de Malpighi/embriología , Mesodermo/fisiología , Organogénesis/fisiología , Animales , Linaje de la Célula/fisiología , AMP Cíclico/metabolismo , Epitelio/embriología , Inmunohistoquímica , Túbulos de Malpighi/metabolismo , Proteínas de la Membrana/fisiologíaRESUMEN
Tissue morphogenesis involves both the sculpting of tissue shape and the positioning of tissues relative to one another in the body. Using the renal tubules of Drosophila, we show that a specific distal tubule cell regulates both tissue architecture and position in the body cavity. Focusing on the anterior tubules, we demonstrate that tip cells make transient contacts with alary muscles at abdominal segment boundaries, moving progressively forward as convergent extension movements lengthen the tubule. Tip cell anchorage antagonizes forward-directed, TGF-ß-guided tubule elongation, thereby ensuring the looped morphology characteristic of renal tubules from worms to humans. Distinctive tip cell exploratory behavior, adhesion, and basement membrane clearing underlie target recognition and dynamic interactions. Defects in these features obliterate tip cell anchorage, producing misshapen and misplaced tubules with impaired physiological function.
Asunto(s)
Adhesión Celular/fisiología , Membrana Celular/metabolismo , Drosophila/crecimiento & desarrollo , Túbulos Renales/citología , Morfogénesis/fisiología , Animales , Drosophila/metabolismo , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Hibridación in Situ , Túbulos Renales/metabolismo , Rayos Láser , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
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.
Asunto(s)
Drosophila melanogaster/enzimología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Secuencia de Aminoácidos , Animales , Colágeno/biosíntesis , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Datos de Secuencia Molecular , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/química , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Alineación de SecuenciaRESUMEN
The intercalation of mesenchymal cells into epithelia, through mesenchymal-to-epithelial transition (MET), underlies organogenesis, for example, in nephrogenesis, and tissue regeneration, during cell renewal and wound repair. Despite its importance, surprisingly little is known about the mechanisms that bring about MET in comparison with the related and much-studied, reverse process, epithelial-to-mesenchymal transition (EMT). We analyse the molecular events that regulate MET as stellate cells integrate into the established epithelium of the developing renal tubules in Drosophila. We show that stellate cells polarise as they integrate between epithelial principal cells and that the normal, localised expression of cell polarity proteins in principal cells is required for stellate cells to become epithelial. While the basolateral and apical membranes act as cues for stellate cell polarity, adherens junction integrity is required to regulate their movement through the epithelium; in the absence of these junctions stellate cells continue migrating into the tubule lumen. We also show that expression of basolateral proteins in stellate cells is a prerequisite for their ingression between principal cells. We present a model in which the contacts with successive principal cell membrane domains made by stellate cells as they integrate between them act as a cue for the elaboration of stellate cell polarity. We suggest that the formation of zonula adherens junctions between new cell neighbours establishes their apico-basal positions and stabilises them in the epithelium.
Asunto(s)
Polaridad Celular , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Células Epiteliales/citología , Epitelio/embriología , Túbulos Renales/citología , Mesodermo/citología , Uniones Adherentes/metabolismo , Animales , Movimiento Celular , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Epitelio/metabolismo , Túbulos Renales/embriología , Mesodermo/metabolismoRESUMEN
Details of the mechanisms that determine the shape and positioning of organs in the body cavity remain largely obscure. We show that stereotypic positioning of outgrowing Drosophila renal tubules depends on signaling in a subset of tubule cells and results from enhanced sensitivity to guidance signals by targeted matrix deposition. VEGF/PDGF ligands from the tubules attract hemocytes, which secrete components of the basement membrane to ensheath them. Collagen IV sensitizes tubule cells to localized BMP guidance cues. Signaling results in pathway activation in a subset of tubule cells that lead outgrowth through the body cavity. Failure of hemocyte migration, loss of collagen IV, or abrogation of BMP signaling results in tubule misrouting and defective organ shape and positioning. Such regulated interplay between cell-cell and cell-matrix interactions is likely to have wide relevance in organogenesis and congenital disease.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Colágeno Tipo IV/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Hemocitos/metabolismo , Animales , Proteínas Morfogenéticas Óseas/genética , Colágeno Tipo IV/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/metabolismo , Hemocitos/citología , Túbulos Renales/embriología , Morfogénesis , Transducción de SeñalRESUMEN
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.
Asunto(s)
Drosophila/embriología , Riñón/embriología , Sistema Urinario/embriología , Animales , Embrión no Mamífero , Riñón/fisiología , Modelos Biológicos , Fenómenos Fisiológicos del Sistema Urinario , Micción/fisiologíaRESUMEN
The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? We analyse this question in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. Here we show that, whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. We also identify a requirement for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery.
Asunto(s)
Polaridad Celular/fisiología , Proteínas de Drosophila/fisiología , Embrión no Mamífero/fisiología , Células Epiteliales/fisiología , Regulación del Desarrollo de la Expresión Génica , Túbulos Renales/embriología , Proteínas de la Membrana/fisiología , Uniones Adherentes/metabolismo , Animales , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Técnicas para Inmunoenzimas , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Morfogénesis/fisiologíaRESUMEN
Ureteric contractions propel foetal urine from the kidney to the urinary bladder. Here, we show that mouse ureteric smooth muscle cell (SMC) precursors express the transcription factor teashirt 3 (TSHZ3), and that Tshz3-null mutant mice have congenital hydronephrosis without anatomical obstruction. Ex vivo, the spontaneous contractions that occurred in proximal segments of wild-type embryonic ureter explants were absent in Tshz3 mutant ureters. In vivo, prior to the onset of hydronephrosis, mutant proximal ureters failed to express contractile SMC markers, whereas these molecules were detected in controls. Mutant embryonic ureters expressed Shh and Bmp4 transcripts as normal, with appropriate expression of Ptch1 and pSMAD1/5/8 in target SM precursors, whereas myocardin, a key regulator for SMC differentiation, was not expressed in Tshz3-null ureters. In wild-type embryonic renal tract explants, exogenous BMP4 upregulated Tshz3 and myocardin expression. More interestingly, in Tshz3 mutant renal tract explants, exogenous BMP4 did not improve the Tshz3 phenotype. Thus, Tshz3 is required for proximal ureteric SMC differentiation downstream of SHH and BMP4. Furthermore, the Tshz3 mutant mouse model of ;functional' urinary obstruction resembles congenital pelvi-ureteric junction obstruction, a common human malformation, suggesting that TSHZ, or related, gene variants may contribute to this disorder.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Proteínas Hedgehog/metabolismo , Factores de Transcripción/metabolismo , Uréter/embriología , Uréter/metabolismo , Animales , Secuencia de Bases , Tipificación del Cuerpo , Proteína Morfogenética Ósea 4/genética , Diferenciación Celular , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Humanos , Hidronefrosis/congénito , Hidronefrosis/embriología , Hidronefrosis/genética , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Fenotipo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Uréter/citologíaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Células Epiteliales/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Morfogénesis , Proteínas de Unión al GTP rho/metabolismo , Animales , Polaridad Celular , Forma de la Célula , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Activación Enzimática , Células Epiteliales/citología , Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Hibridación in Situ , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Proteínas de Unión al GTP rho/genéticaRESUMEN
The function of excretion in insects is performed by the Malpighian tubules, a functional equivalent of the vertebrate kidney. Malpighian tubules are long, thin tubes connected to the hindgut. Upon the determination of the Malpighian tubule major cell type early in embryogenesis, the tubular architecture is achieved by extensive cell division and cell rearrangements. During the tube elongation process, cells exchange their neighbors, allowing the short and fat Malpighian tubule primordia to grow and become a thin tube. Cell rearrangement and intercalation underlie the morphogenesis of other epithelial tissues in Drosophila melanogaster, such as the embryonic epidermis. Recent work has provided insights in the cellular and molecular basis of cell intercalation. These advances are reviewed and discussed with regard to what is known about Malpighian tubule morphogenesis. Mature Malpighian tubules are composed of two cell types, each having a specific function in excretion: The principal cells and the stellate cells. Drosophila and mammalian kidney development show striking similarities, as the recruitment of the stellate cells to the Malpighian tubules, like the cells of the metanephric mesenchyme, requires that cells undergo a mesenchymal-to-epithelial transition. The molecular similarities between these two cases is reviewed here.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/embriología , Túbulos de Malpighi/embriología , Morfogénesis/genética , Animales , Embrión no Mamífero , Desarrollo Embrionario/fisiología , Túbulos Renales/embriología , Modelos Animales , Biología Molecular , Morfogénesis/fisiología , Organogénesis , Sensibilidad y EspecificidadRESUMEN
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.