RESUMEN
In the mid-1960s, bone morphogenetic proteins (BMPs) were first identified in the extracts of bone to have the remarkable ability to induce heterotopic bone. When the Drosophila gene decapentaplegic (dpp) was first identified to share sequence similarity with mammalian BMP2/BMP4 in the late-1980s, it became clear that secreted BMP ligands can mediate processes other than bone formation. Following this discovery, collaborative efforts between Drosophila geneticists and mammalian biochemists made use of the strengths of their respective model systems to identify BMP signaling components and delineate the pathway. The ability to conduct genetic modifier screens in Drosophila with relative ease was critical in identifying the intracellular signal transducers for BMP signaling and the related transforming growth factor-beta/activin signaling pathway. Such screens also revealed a host of genes that encode other core signaling components and regulators of the pathway. In this review, we provide a historical account of this exciting time of gene discovery and discuss how the field has advanced over the past 30 years. We have learned that while the core BMP pathway is quite simple, composed of 3 components (ligand, receptor, and signal transducer), behind the versatility of this pathway lies multiple layers of regulation that ensures precise tissue-specific signaling output. We provide a sampling of these discoveries and highlight many questions that remain to be answered to fully understand the complexity of BMP signaling.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genética , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
During Drosophila oogenesis, somatic follicle cells (FCs) differentiate to secrete components of the eggshell. Before secretion, the epithelium reorganizes to shape eggshell specializations, including border FC collective cell migration and later dorsal formation. These FC movements provide valuable insights into collective cell migration. However, little is known about centripetal migration, which encloses the oocyte after secretion has begun. Centripetal migration begins with apical extension of a few FCs that move away from the basement membrane to invade between germ cells. We define a timeline of reproducible milestones, using time-lapse imaging of egg chamber explants. Inward migration occurs in two phases. First, leading centripetal FCs ingress, extending apically over the anterior oocyte, and constricting basally. Second, following FCs move collectively toward the anterior, then around the corner to move inward with minimal change in aspect ratio. E-cadherin was required in leading centripetal FCs for their normal ingression, assessed with homozygous shotgun mutant or RNAi knockdown clones; ingression was influenced non-autonomously by mutant following FCs. This work establishes centripetal migration as an accessible model for biphasic E-cadherin-adhesion-mediated collective migration.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Femenino , Drosophila melanogaster/metabolismo , Folículo Ovárico/metabolismo , Drosophila/metabolismo , Oogénesis , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Movimiento Celular , Cadherinas/metabolismoRESUMEN
A long-standing problem in biology is how to dissect traits for which no tractable model exists. Here, we screen for genes like the nude locus (Foxn1)-genes central to mammalian hair and thymus development-using animals that never evolved hair, thymi, or Foxn1. Fruit flies are morphologically disrupted by the FOXN1 transcription factor and rescued by weak reductions in fly gene function, revealing molecules that potently synergize with FOXN1 to effect dramatic, chaotic change. Strong synergy/effectivity in flies is expected to reflect strong selection/functionality (purpose) in mammals; the more disruptive a molecular interaction is in alien contexts (flies), the more beneficial it will be in its natural, formative contexts (mammals). The approach identifies Aff4 as the first nude-like locus, as murine AFF4 and FOXN1 cooperatively induce similar cutaneous/thymic phenotypes, similar gene expression programs, and the same step of transcription, pre-initiation complex formation. These AFF4 functions are unexpected, as AFF4 also serves as a scaffold in common transcriptional-elongation complexes. Most likely, the approach works because an interaction's power to disrupt is the inevitable consequence of its selected-for power to benefit.
Asunto(s)
Factores de Transcripción Forkhead , Piel , Animales , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Desnudos , Fenotipo , Piel/metabolismo , Timo/metabolismoRESUMEN
Studies in yeast first delineated the function of Mob proteins in kinase pathways that regulate cell division and shape; in multicellular eukaryotes Mobs regulate tissue growth and morphogenesis. In animals, Mobs are adaptors in Hippo signaling, an intracellular signal-transduction pathway that restricts growth, impacting the development and homeostasis of animal organs. Central to Hippo signaling are the Nuclear Dbf2-Related (NDR) kinases, Warts and LATS1 and LATS2, in flies and mammals, respectively. A second Hippo-like signaling pathway has been uncovered in animals, which regulates cell and tissue morphogenesis. Central to this emergent pathway are the NDR kinases, Tricornered, STK38, and STK38L. In Hippo signaling, NDR kinase activation is controlled by three activating interactions with a conserved set of proteins. This review focuses on one co-activator family, the highly conserved, non-catalytic Mps1-binder-related (Mob) proteins. In this context, Mobs are allosteric activators of NDR kinases and adaptors that contribute to assembly of multiprotein NDR kinase activation complexes. In multicellular eukaryotes, the Mob family has expanded relative to model unicellular yeasts; accumulating evidence points to Mob functional diversification. A striking example comes from the most sequence-divergent class of Mobs, which are components of the highly conserved Striatin Interacting Phosphatase and Kinase (STRIPAK) complex, that antagonizes Hippo signaling. Mobs stand out for their potential to modulate the output from Hippo and Hippo-like kinases, through their roles both in activating NDR kinases and in antagonizing upstream Hippo or Hippo-like kinase activity. These opposing Mob functions suggest that they coordinate the relative activities of the Tricornered/STK38/STK38L and Warts/LATS kinases, and thus have potential to assemble nodes for pathway signaling output. We survey the different facets of Mob-dependent regulation of Hippo and Hippo-like signaling and highlight open questions that hinge on unresolved aspects of Mob functions.
RESUMEN
Epithelial structures are foundational for tissue organization in all metazoans. Sheets of epithelial cells form lateral adhesive junctions and acquire apico-basal polarity perpendicular to the surface of the sheet. Genetic analyses in the insect model, Drosophila melanogaster, have greatly advanced our understanding of how epithelial organization is established, and how it is modulated during tissue morphogenesis. Major insights into collective cell migrations have come from analyses of morphogenetic movements within the adult follicular epithelium that cooperates with female germ cells to build a mature egg. Epithelial follicle cells progress through tightly choreographed phases of proliferation, patterning, reorganization and migrations, before they differentiate to form the elaborate structures of the eggshell. Distinct structural domains are organized by differential adhesion, within which lateral junctions are remodeled to further shape the organized epithelia. During collective cell migrations, adhesive interactions mediate supracellular organization of planar polarized macromolecules, and facilitate crawling over the basement membrane or traction against adjacent cell surfaces. Comparative studies with other insects are revealing the diversification of morphogenetic movements for elaboration of epithelial structures. This review surveys the repertoire of follicle cell morphogenesis, to highlight the coordination of epithelial plasticity with progressive differentiation of a secretory epithelium. Technological advances will keep this tissue at the leading edge for interrogating the precise spatiotemporal regulation of normal epithelial reorganization events, and provide a framework for understanding pathological tissue dysplasia.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Epitelio/crecimiento & desarrollo , Morfogénesis/genética , Óvulo/crecimiento & desarrollo , Animales , Diferenciación Celular/genética , Movimiento Celular/genética , Drosophila melanogaster/genética , Femenino , Oogénesis/genética , Folículo Ovárico/crecimiento & desarrolloRESUMEN
The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. We show here that the leucine zipper protein Bunched and the adaptor protein Madm are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, our data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells.
Asunto(s)
Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Células Madre/citología , Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genética , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/metabolismo , Intestinos/citología , Proteínas de Unión al GTP Monoméricas/biosíntesis , Neuropéptidos/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro , Transducción de Señal , Células Madre/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Bone Morphogenetic Proteins (BMPs) are critical for pattern formation in many animals. In numerous tissues, BMPs become distributed in spatially non-uniform profiles. The gradients of signaling activity can be detected by a number of biological assays involving fluorescence microscopy. Quantitative analyses of BMP gradients are powerful tools to investigate the regulation of BMP signaling pathways during development. These approaches rely heavily on images as spatial representations of BMP activity levels, using them to infer signaling distributions that inform on regulatory mechanisms. In this perspective, we discuss current imaging assays and normalization methods used to quantify BMP activity profiles with a focus on the Drosophila wing primordium. We find that normalization tends to lower the number of samples required to establish statistical significance between profiles in controls and experiments, but the increased resolvability comes with a cost. Each normalization strategy makes implicit assumptions about the biology that impacts our interpretation of the data. We examine the tradeoffs for normalizing versus not normalizing, and discuss their impacts on experimental design and the interpretation of resultant data.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/fisiología , Alas de Animales/metabolismo , Animales , Simulación por Computador , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Colorantes Fluorescentes/farmacología , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , Modelos Biológicos , Modelos Estadísticos , Modelos Teóricos , Transducción de Señal , Programas Informáticos , Alas de Animales/crecimiento & desarrolloRESUMEN
Animals use TGF-ß superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-ß/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, "BMP-specific" Smads can be phosphorylated in response to TGF-ß/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-ß/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-ß/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-ß signaling output.
Asunto(s)
Receptores de Activinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteína Smad2/metabolismo , Factores de Transcripción/metabolismo , Receptores de Activinas/genética , Animales , Western Blotting , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Cross-Talk , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad Reguladas por Receptores , Proteína Smad2/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Bone morphogenetic protein (BMP) signaling controls development and maintenance of many tissues. Genetic and quantitative approaches in Drosophila reveal that ligand isoforms show distinct function in wing development. Spatiotemporal control of BMP patterning depends on a network of extracellular proteins Pent, Ltl and Dally that regulate BMP signaling strength and morphogen range. BMP-mediated feedback regulation of Pent, Ltl, and Dally expression provides a system where cells actively respond to, and modify, the extracellular morphogen landscape to form a gradient that exhibits remarkable properties, including proportional scaling of BMP patterning with tissue size and the modulation of uniform tissue growth. This system provides valuable insights into mechanisms that mitigate the influence of variability to regulate cell-cell interactions and maintain organ function.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Transducción de Señal , Alas de Animales/crecimiento & desarrollo , Animales , Comunicación Celular , Drosophila/citología , Drosophila/metabolismo , Alas de Animales/metabolismoRESUMEN
Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. We demonstrate that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. We identify Med as the primary SUMOylation target in the Dpp pathway, and show that failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and we provide evidence that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, our results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/embriología , Transducción de Señal , Proteína Smad4/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Mutación , Procesamiento Proteico-PostraduccionalRESUMEN
We identify Balpha (PPP2R2A) and Bdelta (PPP2R2D), two highly related members of the B family of regulatory subunits of the protein phosphatase PP2A, as important modulators of TGF-beta/Activin/Nodal signalling that affect the pathway in opposite ways. Knockdown of Balpha in Xenopus embryos or mammalian tissue culture cells suppresses TGF-beta/Activin/Nodal-dependent responses, whereas knockdown of Bdelta enhances these responses. Moreover, in Drosophila, overexpression of Smad2 rescues a severe wing phenotype caused by overexpression of the single Drosophila PP2A B subunit Twins. We show that, in vertebrates, Balpha enhances TGF-beta/Activin/Nodal signalling by stabilising the basal levels of type I receptor, whereas Bdelta negatively modulates these pathways by restricting receptor activity. Thus, these highly related members of the same subfamily of PP2A regulatory subunits differentially regulate TGF-beta/Activin/Nodal signalling to elicit opposing biological outcomes.
Asunto(s)
Activinas/metabolismo , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Xenopus/embriología , Receptores de Activinas/metabolismo , Receptores de Activinas Tipo I , Animales , Línea Celular , Drosophila , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Proteína Nodal , Fenotipo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Alas de Animales/citología , Proteínas de Xenopus/metabolismoRESUMEN
TSC22D1, which encodes transforming growth factor beta-stimulated clone 22 (TSC-22), is thought to be a tumor suppressor because its expression is lost in many glioblastoma, salivary gland, and prostate cancers. TSC-22 is the founding member of the TSC-22/DIP/Bun family of leucine zipper transcription factors; its functions have not been investigated in a multicellular environment. Genetic studies in the model organism Drosophila melanogaster often provide fundamental insights into mechanisms disrupted in carcinogenesis, because of the strong evolutionary conservation of molecular mechanisms between flies and humans. Whereas humans and mice have four TSC-22 domain genes with numerous isoforms, Drosophila has only one TSC-22 domain gene, bunched (bun), which encodes both large and small protein isoforms. Surprisingly, Drosophila Bun proteins promote cellular growth and proliferation in ovarian follicle cells. Loss of both large isoforms has the strongest phenotypes, including increased apoptosis. Cultured S2 cells depleted for large Bun isoforms show increased apoptosis and less frequent cell division, with decreased cell size. Altogether, these data indicate that Drosophila TSC-22/DIP/Bun proteins are necessary for cellular growth, proliferation, and survival both in culture and in an epithelial context. Previous work demonstrated that bun prevents recruitment of epithelial cells to a migratory fate and, thus, maintains epithelial organization. We speculate that reduced TSC22D1 expression generally reduces cellular fitness and only contributes to carcinogenesis in specific tissue environments.
Asunto(s)
Proliferación Celular , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/química , Proteínas Supresoras de Tumor/fisiología , Animales , Aumento de la Célula , Supervivencia Celular , Células Epiteliales/citologíaRESUMEN
Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. This epithelium of follicle cells encases germline cells to create an egg. In this review, we summarize progress toward understanding mechanisms that maintain the epithelium or permit migrations essential for oogenesis. Cell-cell communication is important, but the same signals are used repeatedly to control distinct events. Understanding intrinsic mechanisms that alter responses to developmental signals will be important to understand regulation of cell shape and organization.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Morfogénesis , Folículo Ovárico/citología , Óvulo/citología , Animales , División Celular , Femenino , Óvulo/ultraestructura , Células Madre/citologíaRESUMEN
BACKGROUND: Transforming Growth Factor-beta1 stimulated clone-22 (TSC-22) is assumed to act as a negative growth regulator and tumor suppressor. TSC-22 belongs to a family of putative transcription factors encoded by four distinct loci in mammals. Possible redundancy among the members of the TSC-22/Dip/Bun protein family complicates a genetic analysis. In Drosophila, all proteins homologous to the TSC-22/Dip/Bun family members are derived from a single locus called bunched (bun). RESULTS: We have identified bun in an unbiased genetic screen for growth regulators in Drosophila. Rather unexpectedly, bun mutations result in a growth deficit. Under standard conditions, only the long protein isoform BunA - but not the short isoforms BunB and BunC - is essential and affects growth. Whereas reducing bunA function diminishes cell number and cell size, overexpression of the short isoforms BunB and BunC antagonizes bunA function. CONCLUSION: Our findings establish a growth-promoting function of Drosophila BunA. Since the published studies on mammalian systems have largely neglected the long TSC-22 protein version, we hypothesize that the long TSC-22 protein is a functional homolog of BunA in growth regulation, and that it is antagonized by the short TSC-22 protein.
Asunto(s)
División Celular/genética , Drosophila melanogaster/genética , Genes de Insecto , Factor de Crecimiento Transformador beta1/genética , Alelos , Animales , Recuento de Células , Tamaño de la Célula , Drosophila melanogaster/crecimiento & desarrollo , Genes Reguladores , Genotipo , MutaciónRESUMEN
Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched (bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. Here, we show that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Folículo Ovárico/fisiología , Receptores Notch/metabolismo , Factores de Transcripción/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Movimiento Celular , Forma de la Célula , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oogénesis , Folículo Ovárico/metabolismo , Receptores Notch/genética , Proteínas Serrate-Jagged , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Bone morphogenetic proteins (BMP) direct dorsal-ventral patterning in both invertebrate and vertebrate embryos, with strong evolutionary conservation of molecular components of the pathway. Dorsal-ventral patterning of the early Drosophila embryo is a powerful experimental system to probe mechanisms of BMP gradient formation and interpretation. Recent studies have found that spatial patterns of activated BMP signal transducers in Drosophila go through an unexpected transition: a shallow gradient of weak responses at mid-cellularization changes to a step gradient of stronger responses in cellularized embryos. The transition between two gradients of different shape yields new insights into the progression of Drosophila dorsal-ventral patterning and raises new issues about the mechanisms of gradient formation.
Asunto(s)
Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Modelos Biológicos , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inducción Embrionaria , MorfogénesisRESUMEN
Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Here we compare patterns of two related cellular responses, both signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Our data demonstrate that nuclear accumulation of the co-SMAD Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (SOG) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Transducción de Señal/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Anticuerpos/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/fisiología , Dosificación de Gen , Ligandos , Morfogénesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Smad4 , Transactivadores/genética , Transactivadores/inmunología , Factores de Transcripción/genéticaRESUMEN
Peroxisome proliferator-activated receptor gamma (PPARgamma) and transforming growth factor-beta (TGF-beta) are key regulators of epithelial cell biology. However, the molecular mechanisms by which either pathway induces growth inhibition and differentiation are incompletely understood. We have identified transforming growth factor-simulated clone-22 (TSC-22) as a target gene of both pathways in intestinal epithelial cells. TSC-22 is member of a family of leucine zipper containing transcription factors with repressor activity. Although little is known regarding its function in mammals, the Drosophila homolog of TSC-22, bunched, plays an essential role in fly development. The ability of PPARgamma to induce TSC-22 was not dependent on an intact TGF-beta1 signaling pathway and was specific for the gamma isoform. Localization studies revealed that TSC-22 mRNA is enriched in the postmitotic epithelial compartment of the normal human colon. Cells transfected with wild-type TSC-22 exhibited reduced growth rates and increased levels of p21 compared with vector-transfected cells. Furthermore, transfection with a dominant negative TSC-22 in which both repressor domains were deleted was able to reverse the p21 induction and growth inhibition caused by activation of either the PPARgamma or TGF-beta pathways. These results place TSC-22 as an important downstream component of PPARgamma and TGF-beta signaling during intestinal epithelial cell differentiation.
Asunto(s)
Células Epiteliales/citología , Intestinos/citología , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/biosíntesis , Tiazolidinedionas , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Northern Blotting , Western Blotting , Células COS , Diferenciación Celular , División Celular , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , ADN/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Genes Dominantes , Vectores Genéticos , Humanos , Hibridación in Situ , Proteínas de Filamentos Intermediarios/metabolismo , Queratina-20 , Ligandos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Precipitina , Estructura Terciaria de Proteína , Rosiglitazona , Transducción de Señal , Tiazoles/farmacología , Factores de Tiempo , Transcripción Genética , TransfecciónRESUMEN
Transforming growth factor-beta (TGF-beta) appears to promote the regression phase of the mammalian hair cycle, in vivo in mice and in organ culture of human hair follicles. To assess the relationship between TGF-beta activity and apoptosis of epithelial cells during the murine hair cycle, we identified active TGF-beta responses using phospho-Smad2/3-specific antibodies (PS2). Strong, nuclear PS2 staining was observed in the outer root sheath throughout the anagen growth phase. Some bulb matrix cells were positive for PS2 during late anagen. Extensive, but weak, staining was observed in this region at the anagen-catagen transition. We also examined expression of TGF-beta-stimulated clone-22 (TSC-22), which is associated with TGF-beta-induced apoptosis of some cell lines. Recombinant rat TSC-22 was used to generate a rabbit anti-TSC-22 antibody useful for immunohistochemistry. TSC-22 RNA accumulation and immunoreactivity were observed in follicles throughout the murine hair cycle, including the dermal papilla and lower epithelial strand of late-catagen hair follicles. Neither the expression pattern nor the presence of nuclear TSC-22 correlated with the sites of apoptosis, suggesting that TSC-22 is not an effector of apoptosis in mouse catagen hair follicles. These studies support a complex role for TGF-beta in regulating the regression phase of the cycle, with potential for indirect promotion of apoptosis during the anagen-catagen transition.
Asunto(s)
Folículo Piloso/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/fisiología , Animales , Apoptosis , Núcleo Celular/química , Proteínas de Unión al ADN/análisis , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Conejos , Ratas , Proteínas Represoras/análisis , Proteínas Represoras/fisiología , Proteína Smad2 , Transactivadores/análisisRESUMEN
TSC-22 (transforming growth factor-beta-stimulated clone 22) belongs to a family of leucine zipper transcription factors that includes sequences from invertebrates and vertebrates. The single Drosophila family member, encoded by the bunched gene, serves to integrate opposing bone morphogenic protein (BMP) and epidermal growth factor (EGF) signals during oogenesis. Similarly, mammalian TSC-22 expression is regulated by several families of secreted signaling molecules in cultured cells. Here, we show that chick TSC-22 is dynamically expressed in the condensing feather bud, as well as in many tissues of the chick embryo. BMP-2/4, previously shown to inhibit bud development, repress TSC-22 expression during feather bud formation in vivo. Noggin, a BMP antagonist, promotes TSC-22 expression. EGF, TGF-alpha, and fibroblast growth factor all promote both feather bud development and TSC-22 expression; each can promote ectopic feather buds that are regularly spaced between existing feather buds. Thus, TSC-22 is a candidate to integrate small imbalances in receptor tyrosine kinase and BMP signaling during feather tract development to generate stable and reproducible morphogenetic responses.