RESUMO
Migration of the Drosophila tracheal cells relies on cues provided by nearby cells; however, little is known about how these signals specify a migratory path. Here we investigate the role of cell surface proteins in the definition of such a pathway. We have found that the PS1 integrin is required in the tracheal cells of the visceral branch, whereas the PS2 integrin is required in the visceral mesoderm; both integrins are necessary for the spreading of the visceral branch over its substratum. This is the first identification of a cell surface molecule with expression restricted to a subset of tracheal cells that all migrate in a given direction. We have also found that expression of PS1 in the visceral branch is regulated by the genes that direct tracheal cell migration, showing that integrin expression is part of the cell-fate program that they specify. These results support a model in which signal transduction determines the tracheal migratory pathways by regulating the expression of cell surface proteins, which in turn interact with surface molecules on the surrounding cell population.
Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila , Integrinas/fisiologia , Traqueia/citologia , Animais , Drosophila , Cadeias alfa de Integrinas , Integrinas/metabolismo , MorfogêneseRESUMO
The assembly of an organism requires the interaction between different layers of cells, in many cases via an extracellular matrix. In the developing Drosophila larva, muscles attach in an integrin-dependent manner to the epidermis, via a specialized extracellular matrix called tendon matrix. Tiggrin, a tendon matrix integrin ligand, is primarily synthesized by cells distant to the muscle attachment sites, yet it accumulates specifically at these sites. Previous work has shown that the PS integrins are not required for tiggrin localization, suggesting that there is redundancy among tiggrin receptors. We have examined this by testing whether the PS2 integrin can recruit tiggrin to ectopic locations within the Drosophila embryo. We found that neither the wild type nor modified forms of the PS2 integrin, which have higher affinity for tiggrin, can recruit tiggrin to new cellular contexts. Next, we genetically manipulated the fate of the muscles and the epidermal muscle attachment cells, which demonstrated that muscles have the primary role in recruiting tiggrin to the tendon matrix and that cell-cell contact is necessary for this recruitment. Thus we propose that the inherent polarity of the muscle cells leads to a molecular specialization of their ends, and interactions between the ends produces an integrin-independent tiggrin receptor. Thus, interaction between cells generates an extracellular environment capable of nucleating extracellular matrix assembly.
Assuntos
Adesão Celular , Comunicação Celular , Proteínas da Matriz Extracelular/metabolismo , Integrinas/metabolismo , Animais , Drosophila/embriologia , Proteínas de Drosophila , Epiderme/metabolismo , Ligantes , Músculos/metabolismoRESUMO
Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Receptores ErbB/fisiologia , Integrinas/fisiologia , Neurregulinas , Tendões/embriologia , Animais , Adesão Celular , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Matriz Extracelular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Morfogênese , Fatores de Transcrição/genética , Tubulina (Proteína)/genética , Dedos de ZincoRESUMO
Cell migration during embryogenesis involves two populations of cells: the migrating cells and the underlying cells that provide the substratum for migration. The formation of the Drosophila larval midgut involves the migration of the primordial midgut cells along a visceral mesoderm substratum. We show that integrin adhesion receptors are required in both populations of cells for normal rates of migration. In the absence of the PS integrins, the visceral mesoderm is disorganised, the primordial midgut cells do not display their normal motile appearance and their migration is delayed by 2 hours. Removing PS integrin function from the visceral mesoderm alone results in visceral mesoderm disorganization, but only causes a modest delay in migration and does not affect the appearance of the migrating cells. Removing PS integrin function from the migrating cells causes as severe a delay in migration as the complete loss of PS integrin function. The functions of PS1 and PS2 are specific in the two tissues, endoderm and mesoderm, since they cannot substitute for each other. In addition there is a partial redundancy in the function of the two PS integrins expressed in the endoderm, PS1 (alphaPS1betaPS) and PS3 (alphaPS3betaPS), since loss of just one alpha subunit in the midgut results in either a modest delay (alphaPS1) or no effect (alphaPS3). We have also examined the roles of small GTPases in promoting migration of the primordial midgut cells. We find that dominant negative (N17) versions of Rac and Cdc42 cause a very similar defect in migration as loss of integrins, while those of Rho and Ras have no effect. Thus integrins are involved in mediating migration by creating an optimal substratum for adhesion, adhering to that substratum and possibly by activating Rac and Cdc42.
Assuntos
Proteínas de Drosophila , Integrinas/fisiologia , Animais , Movimento Celular , Sistema Digestório/embriologia , Drosophila/embriologia , Endoderma/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Cadeias alfa de Integrinas , Proteínas de Membrana/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Transdução de SinaisRESUMO
Integrin cell surface receptors are ideally suited to coordinate cellular differentiation and tissue assembly during embryogenesis, as they can mediate both signaling and adhesion. We show that integrins regulate gene expression in the intact developing embryo by identifying two genes that require integrin function for their normal expression in Drosophila midgut endodermal cells. We determined the relative roles of integrin adhesion versus signaling in the regulation of these integrin target genes. We find that integrin-mediated adhesion is not required between the endodermal cells and the surrounding visceral mesoderm for integrin target gene expression. In addition, a chimeric protein that lacks integrin-adhesive function, but maintains the ability to signal, can substitute for the endogenous integrin and regulate integrin target genes. This chimera consists of an oligomeric extracellular domain fused to the integrin betaPS subunit cytoplasmic domain; a control monomeric extracellular domain fusion does not alter integrin target gene expression. Therefore, oligomerization of the 47-amino-acid betaPS intracellular domain is sufficient to initiate a signaling pathway that regulates gene expression in the developing embryo.
Assuntos
Adesão Celular/fisiologia , Proteínas de Drosophila , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Integrinas/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Citoplasma/metabolismo , Primers do DNA , Endoderma/citologia , Cadeias alfa de Integrinas , Integrinas/genética , Integrinas/metabolismo , Mesoderma/citologia , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the alpha subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (alphaPS2betaPS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type alphaPS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the alphaPS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the alphaPS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin alphaIIbbeta3.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Embrião não Mamífero/fisiologia , Integrinas/biossíntese , Integrinas/fisiologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Citoplasma/fisiologia , Primers do DNA , Drosophila/genética , Éxons , Genes Letais , Cadeias alfa de Integrinas , Integrinas/química , Substâncias Macromoleculares , Dados de Sequência Molecular , Morfogênese , Mutagênese Sítio-Dirigida , Fenótipo , Alinhamento de SequênciaRESUMO
We have examined the role of integrins in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). To this end we have analyzed muscle attachments and neuromuscular junctions ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions (HAJs) to extracellular matrix, but not for their intracellular link to the cytoskeleton. The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. It is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component laminin A, while adhesion mediated by PS2 integrin does not. At neuromuscular junctions the formation of functional synapses occurs normally in embryos lacking PS integrins and/or laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly. We suggest that neuromuscular contact in part requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of laminin A.
Assuntos
Proteínas de Drosophila , Integrinas/deficiência , Junções Intercelulares , Laminina/deficiência , Junção Neuromuscular/embriologia , Animais , Membrana Basal , Adesão Celular , Drosophila/embriologia , Drosophila/genética , Epiderme/embriologia , Cadeias alfa de Integrinas , Integrinas/genética , Junções Intercelulares/ultraestrutura , Laminina/genética , Modelos Estruturais , Músculos/embriologia , Músculos/ultraestrutura , Sistema Nervoso/embriologia , Junção Neuromuscular/ultraestrutura , Tendões/embriologiaRESUMO
We have isolated and characterized mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development. Analysis of both loss and gain of gene function conditions during embryonic and postembryonic development revealed specific requirements for neurotactin during axon outgrowth, fasciculation, and guidance. Furthermore, embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, displayed phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes.
Assuntos
Moléculas de Adesão Celular/genética , Proteínas de Drosophila , Drosophila/genética , Glicoproteínas de Membrana/genética , Neuritos/fisiologia , Proteínas Tirosina Fosfatases , Alelos , Animais , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Sistema Nervoso Central/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/química , Neurônios Aferentes/enzimologia , Neurônios Aferentes/ultraestrutura , Fenótipo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
We tested the ability of different integrin alpha subunits to substitute for each other during embryonic development. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers where they are required for normal development. Using the GAL4 system, we are able to rescue fully the embryonic lethality of an alphaPS2 null mutation with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, due to partial rescue of both muscle and midgut phenotypes. Similarly we are able to rescue the embryonic/first instar larval lethality of an alphaPS1 null mutation gene with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. We conclude that the two alpha subunits are not equivalent and have distinct functions which reside in the extracellular domains.
Assuntos
Proteínas de Drosophila , Embrião não Mamífero/metabolismo , Integrinas/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Dimerização , Drosophila/embriologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Cadeias alfa de Integrinas , Integrinas/química , Integrinas/genética , Larva/citologia , Larva/genética , Dados de Sequência Molecular , Morfogênese/genética , Mutação/genética , Fenótipo , Filogenia , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transgenes/genéticaRESUMO
In the Drosophila embryo, the alphaPS2betaPS integrin heterodimer is localized tightly at the termini of the multinucleate muscles where they attach to the alphaPS1betaPS-containing epidermal tendon cells. Here we examine the basis for alphaPS2betaPS integrin subcellular localization. We show that the betaPS cytoplasmic tail is sufficient to direct the localization of a heterologous transmembrane protein, CD2, to the muscle termini in vivo. This localization does not occur via an association with structures set up by the endogenous betaPS integrins, since it can occur even in the absence of the betaPS protein. Furthermore, the subcellular localization of the alphaPS2betaPS integrin is not dependent on any other interactions between the muscles and the tendon cells. In embryos that lack the segmental tendon cells, due to a mutation removing the related segment polarity genes engrailed and invected, alphaPS2betaPS is still localized to the muscle termini even though the ventral longitudinal muscles are not attached to the epidermis, but instead are attached end to end. Thus the alphaPS2betaPS integrin can be localized by an intracellular mechanism within the muscles. Our results challenge the view that the transmission of signals from the extracellular environment via integrins is required for the organization of the cytoskeleton and the resultant cellular polarity.
Assuntos
Adesão Celular , Drosophila melanogaster/embriologia , Integrinas/metabolismo , Junções Intercelulares/fisiologia , Músculos/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Compartimento Celular , Citoplasma/metabolismo , Células Epidérmicas , Proteínas da Matriz Extracelular/fisiologia , Ligantes , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Proteínas Recombinantes de Fusão , Relação Estrutura-AtividadeRESUMO
The development of the central nervous system in the Drosophila embryo is initiated by the acquisition of neural potential by clusters of ectodermal cells, promoted by the activity of proneural genes. Proneural gene function is antagonized by neurogenic genes, resulting in the realization of the neural potential in a single cell per cluster. To analyse the relationship between proneural and neurogenic genes, we have studied, in specific proneural clusters and neuroblasts of wild-type and neurogenic mutants embryos, the expression at the RNA and protein levels of lethal of scute, the most important known proneural gene in central neurogenesis. We find that the restriction of lethal of scute expression that accompanies the restriction of the neural potential to the delaminating neuroblast is regulated at the transcriptional level by neurogenic genes. These genes, however, do not control the size of proneural clusters. Moreover, available antibodies do not provide evidence for an hypothetical posttranscriptional regulation of proneural proteins by neurogenic genes. We also find that neurogenic genes are required for the specification of the mesectoderm. This has been shown for neuralized and Notch, and could also be the case for Delta and for the Enhancer of split gene complex. Neurogenic genes would control at the transcriptional level the repression of proneural genes and the activation of single-minded in the anlage of the mesectoderm.
Assuntos
Sistema Nervoso Central/embriologia , Drosophila/genética , Ectoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Animais , Sistema Nervoso Central/fisiologia , Drosophila/embriologia , Imuno-Histoquímica , Hibridização In Situ , MutaçãoRESUMO
The lethal of scute (l'sc) genetic function, which plays an essential role in the early development of the central nervous system of the Drosophila embryo, is localized within the achaete-scute complex (AS-C). Several lines of evidence have suggested that the AS-C T3 transcription unit corresponds to the l'sc function. We demonstrate that short fragments of DNA, containing the T3 transcribed region and a few kilobases of flanking sequences, rescue, albeit partially, the lethality and neural phenotype of l'sc deletions. Still, the complex wild-type pattern of expression of T3 is not reproduced by the transduced genes. This depends on cis-control elements scattered within the entire AS-C DNA and intermingled with regulatory elements specific for other AS-C transcription units. These elements are necessary for the initial activation of T3 in the neuroectoderm, probably mediated by axis-patterning genes. The presence of a cluster of E-boxes, upstream of the T3 transcribed region, suggests another level of control of T3 expression by basic-helix-loop-helix proteins, among them its own gene product.
Assuntos
Sistema Nervoso Central/embriologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genes de Insetos , Genes Letais , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/embriologia , Genes Reguladores , Sequências Hélice-Alça-Hélice/genética , Dados de Sequência Molecular , Morfogênese/genética , Fenótipo , Regiões Promotoras Genéticas , Deleção de Sequência , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The Drosophila position specific (PS) integrins consist of two cell surface heterodimers, PS1 (alpha PS1 beta PS) and PS2 (alpha OS2 beta PS), which are expressed on complementary sides of attachments between cell layers and are essential for these attachments. Current evidence suggests that the PS integrins bind to components of the extracellular matrix, similar to the majority of vertebrate integrins, but specific Drosophila ligands have not yet been identified. In the embryo PS1 is found on the surface of the epidermis and endoderm, while PS2 is restricted to the mesoderm. The integrins are concentrated at the sites where the somatic muscles attach to the epidermis and at the interface between the visceral mesoderm and the endoderm. In myospheroid mutant embryos, which lack the beta PS subunit, the adhesion between the mesoderm and the other cell layers fails. The PS integrins are also required for the adhesion of the dorsal to the ventral surface of the wing during metamorphosis. PS1 is expressed on the basal surface of the dorsal cells and PS2 is expressed on the ventral cells. Loss of PS integrin function in the wing results in balloon shaped wings because of the failure of the two surfaces of the wing blade to adhere to each other. These and other aspects of the phenotypes of mutations in the genes encoding the PS integrins indicate that integrins play an important role in the adhesion of different cell layers to each other and thus an essential role in the morphogenesis of the organism. The use of extracellular matrix receptors in this role may aid in keeping the different cell layers distinct.
Assuntos
Drosophila/embriologia , Matriz Extracelular/metabolismo , Integrinas/fisiologia , Animais , Comunicação Celular/fisiologia , Integrinas/genética , Morfogênese/fisiologia , Mutação/genética , FenótipoRESUMO
Genes of the achaete-scute complex (ASC) participate in the formation of the central nervous system in the Drosophila embryo. Previous genetic analyses have indicated that lethal of scute (l'sc) is the most important gene of the complex in that process. We have obtained antibodies against the l'sc protein to study the expression of the gene during early neurogenesis. The protein is found in groups of embryonic neuroectodermal cells, analogous to the proneural clusters that precede the appearance of precursors of peripheral sensory organs in imaginal epithelia. The groups appear in different regions of the neuroectoderm, accompanying the three successive waves of neuroblast segregation. Most neuroblasts delaminate from these clusters and express position-specific levels of l'sc protein. No significant differences have been found between the distribution of l'sc RNA and protein. Phenotypic analysis of a l'sc deficiency has shown that the gene is required for neuroblast commitment, although this requirement is less widespread than the domain of l'sc expression, suggesting a high degree of redundancy in the function of genes that participate in the process of neuroblast segregation. The ASC genes have been postulated to play a role in the control of NB identity, revealed by the generation of a defined lineage of identifiable neurons. However, our study in l'sc mutants of the expression of fushi tarazu, engrailed, and even-skipped, used as markers of neuronal identity, has not provided evidence to support this hypothesis.