RESUMEN
The basic helix-loop-helix transcription factor Twist regulates a series of distinct cell fate decisions within the Drosophila mesodermal lineage. These twist functions are reflected in its dynamic pattern of expression, which is characterized by initial uniform expression during mesoderm induction, followed by modulated expression at high and low levels in each mesodermal segment, and finally restricted expression in adult muscle progenitors. We show two distinct partner-dependent functions for Twist that are crucial for cell fate choice. We find that Twist can form homodimers and heterodimers with the Drosophila E protein homologue, Daughterless, in vitro. Using tethered dimers to assess directly the function of these two particular dimers in vivo, we show that Twist homodimers specify mesoderm and the subsequent allocation of mesodermal cells to the somatic muscle fate. Misexpression of Twist-tethered homodimers in the ectoderm or mesoderm leads to ectopic somatic muscle formation overriding other developmental cell fates. In addition, expression of tethered Twist homodimers in embryos null for twist can rescue mesoderm induction as well as somatic muscle development. Loss of function analyses, misexpression and dosage experiments, and biochemical studies indicate that heterodimers of Twist and Daughterless repress genes required for somatic myogenesis. We propose that these two opposing roles explain how modulated Twist levels promote the allocation of cells to the somatic muscle fate during the subdivision of the mesoderm. Moreover, this work provides a paradigm for understanding how the same protein controls a sequence of events within a single lineage.
Asunto(s)
Proteínas de Unión al ADN/química , Dimerización , Proteínas de Drosophila , Drosophila/embriología , Mesodermo/metabolismo , Proteínas Nucleares/química , Factores de Transcripción/química , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Linaje de la Célula , Células Cultivadas , ADN/metabolismo , ADN Complementario/metabolismo , Proteínas de Unión al ADN/metabolismo , Inmunohistoquímica , Modelos Genéticos , Músculos/embriología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismo , Transfección , Proteína 1 Relacionada con TwistRESUMEN
Hibris (Hbs) is a transmembrane immunoglobulin-like protein that shows extensive homology to Drosophila Sticks and stones (Sns) and human kidney protein Nephrin. Hbs is expressed in embryonic visceral, somatic and pharyngeal mesoderm among other tissues. In the somatic mesoderm, Hbs is restricted to fusion competent myoblasts and is regulated by Notch and Ras signaling pathways. Embryos that lack or overexpress hbs show a partial block of myoblast fusion, followed by abnormal muscle morphogenesis. Abnormalities in visceral mesoderm are also observed. In vivo mapping of functional domains suggests that the intracellular domain mediates Hbs activity. Hbs and its paralog, Sns, co-localize at the cell membrane of fusion-competent myoblasts. The two proteins act antagonistically: loss of sns dominantly suppresses the hbs myoblast fusion and visceral mesoderm phenotypes, and enhances Hbs overexpression phenotypes. Data from a P-homed enhancer reporter into hbs and co-localization studies with Sns suggest that hbs is not continuously expressed in all fusion-competent myoblasts during the fusion process. We propose that the temporal pattern of hbs expression within fusion-competent myoblasts may reflect previously undescribed functional differences within this myoblast population.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Músculo Esquelético/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/genética , Fusión Celular , Clonación Molecular , Drosophila/embriología , Embrión no Mamífero , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Mesodermo , Datos de Secuencia Molecular , Músculo Esquelético/citología , Mutación , Receptores Notch , Homología de Secuencia de Aminoácido , Transducción de Señal , Vísceras/anomalías , Vísceras/embriología , Proteínas ras/metabolismoRESUMEN
Recent studies in invertebrates have provided important mechanistic insights into several general aspects of muscle development. Two new genes have been identified that are involved in muscle fusion in Drosophila and a novel maternal component was shown to be responsible for myogenic determination in an ascidian. In addition, genetic analyses of nematode and Drosophila homologues of factors known to be myogenic regulators in other species yielded surprising findings about both the evolutionary conservation and divergence of these functions. Drosophila myogenesis has become a highly informative model for understanding the interplay between the signaling and transcriptional networks that underlie cell-fate specification during embryonic development.
Asunto(s)
Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Músculos/embriología , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Proteínas Portadoras/genética , Fusión Celular , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas del Huevo/genética , Humanos , Invertebrados , Factores de Transcripción MEF2 , Fibras Musculares Esqueléticas , Proteína MioD/genética , Factores Reguladores Miogénicos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist , Urocordados/genéticaRESUMEN
Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed-functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.
Asunto(s)
Proteínas Bacterianas , Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Proteínas de Drosophila , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas ras/genética , Proteínas ras/metabolismo , Animales , Sitios de Unión/genética , Proteínas de Unión al ADN , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Corazón/embriología , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Mesodermo/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Miocardio/citología , Miocardio/metabolismo , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteína Wnt1RESUMEN
We report the embryonic phenotype of muscleblind (mbl), a recently described Drosophila gene involved in terminal differentiation of adult ommatidia. mbl is a nuclear protein expressed late in the embryo in pharyngeal, visceral, and somatic muscles, the ventral nerve cord, and the larval photoreceptor system. All three mbl alleles studied exhibit a lethal phenotype and die as stage 17 embryos or first instar larvae. These larvae are partially paralyzed, show a characteristically contracted abdomen, and lack striation of muscles. Our analysis of the somatic musculature shows that the pattern of muscles is established correctly, and they form morphologically normal synapses. Ultrastructural analysis, however, reveals two defects in the terminal differentiation of the muscles: inability to differentiate Z-bands in the sarcomeric apparatus and reduction of extracellular tendon matrix at attachment sites to the epidermis. Failure to differentiate both structures could explain the partial paralysis and contracted abdomen phenotype. Analysis of mbl expression in embryos that are either mutant for Dmef2 or ectopically express Dmef2 places mbl downstream of Dmef2 function in the myogenic differentiation program. mbl, therefore, may act as a critical element in the execution of two Dmef2-dependent processes in the terminal differentiation of muscles.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila , Drosophila/embriología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Núcleo Celular/química , Sistema Nervioso Central/química , Conectina , Proteínas de Unión al ADN/genética , Drosophila/genética , Ectodermo/química , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto/fisiología , Proteínas de Insectos/análisis , Factores de Transcripción MEF2 , Mesodermo/química , Fibras Musculares Esqueléticas/citología , Proteínas Musculares/análisis , Músculo Esquelético/química , Músculo Esquelético/citología , Músculo Esquelético/embriología , Mutación , Factores Reguladores Miogénicos , Unión Neuromuscular/embriología , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Células Fotorreceptoras de Invertebrados/química , ARN Mensajero/análisis , Tendones/embriología , Factores de Transcripción/genéticaRESUMEN
The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer-promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions.
Asunto(s)
Proteínas de Drosophila , Drosophila/fisiología , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cruzamientos Genéticos , Cartilla de ADN , Elementos Transponibles de ADN , Proteínas de Unión al ADN/biosíntesis , Drosophila/genética , Femenino , Rayos gamma , Biblioteca de Genes , Genes de Insecto , Proteínas de Insectos/química , Larva , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Insercional , Proteínas Nucleares/química , Reacción en Cadena de la Polimerasa , Proteínas Represoras/biosíntesis , Retroviridae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Alas de AnimalesAsunto(s)
Tipificación del Cuerpo , Drosophila/embriología , Embrión no Mamífero/fisiología , Factores de Transcripción , Animales , Regulación del Desarrollo de la Expresión Génica , Mesodermo/citología , Mesodermo/fisiología , Músculos/embriología , Proteínas Nucleares/biosíntesis , Proteína 1 Relacionada con TwistRESUMEN
Somatic muscle is derived from a subset of embryonic mesoderm. In Drosophila, Twist (Twi), a basic helix-loop-helix transcription factor, is a candidate regulator of mesodermal differentiation and myogenesis. Altering amounts of Twist after gastrulation revealed that high levels of Twist are required for somatic myogenesis and block the formation of other mesodermal derivatives. Expression of twist in the ectoderm drives these cells into myogenesis. Thus, after an initial role in gastrulation, twist regulates mesodermal differentiation and propels a specific subset of mesodermal cells into somatic myogenesis. Vertebrate homologs of twist may also participate in the subdivision of mesoderm.
Asunto(s)
Drosophila/embriología , Secuencias Hélice-Asa-Hélice/fisiología , Mesodermo/citología , Músculos/citología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , División Celular , Drosophila/metabolismo , Proteínas de Drosophila , Ectodermo/citología , Ectodermo/metabolismo , Gástrula/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Músculos/embriología , Proteínas Nucleares/genética , Células Madre/citología , Proteína 1 Relacionada con TwistRESUMEN
The final pattern of the Drosophila larval body wall muscles depends critically on the prior segregation of muscle founder cells. We would like to understand the underlying molecular mechanisms which ensure the precise allocation and placement of these muscle founder cells. We have begun our analysis by examining the role of the segment polarity genes, known to be involved in the patterning of the ectoderm. Mutations in only one member of this class, wingless (wg), lead to the complete loss of a subset of muscle founder cells characterised by the expression of S59. Using the GAL4-targetted expression system, we find that Wingless, a secreted glycoprotein and well characterized signalling molecule, acts directly on the mesoderm to ensure the formation of S59-expressing founder cells. Moreover, we present evidence that Wg can signal across germ layers and that, in the wild-type embryo, Wg from the ectoderm could constitute an inductive signal for the initiation of the development of a subset of somatic muscles.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Drosophila/genética , Genes de Insecto , Mesodermo/fisiología , Músculos/embriología , Proteínas Proto-Oncogénicas/genética , Células Madre/fisiología , Animales , Ectodermo/fisiología , Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Morfogénesis/genética , Músculos/citología , Proteína Wnt1RESUMEN
We describe a group of Drosophila cDNAs that encode MADs box proteins and which are members of the MEF2 (myocyte enhancer-binding factor 2) family of transcription factors. Drosophila has a single MEF2 gene, DMEF2, that is alternatively spliced to produce different transcripts and which is expressed in the mesodermal primordium before gastrulation. The mechanisms responsible for the subsequent subdivision of the mesoderm are unknown. However, DMEF2 may play a role in this important event because our experiments show that it is a downstream target for twist and that its early expression pattern modulates as the mesoderm is organising into cell groupings with distinct fates. DMEF2 is also expressed in both the segregating primordia and the differentiated cells of the somatic, visceral and heart musculature. It is the only known gene expressed in these three main types of muscle throughout differentiation.
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes de Insecto , Músculos/embriología , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Diferenciación Celular/fisiología , Clonación Molecular , Proteínas de Drosophila , Corazón/embriología , Factores de Transcripción MEF2 , Mesodermo/metabolismo , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Miocardio/citología , Miocardio/metabolismo , Factores Reguladores Miogénicos , Vísceras/embriología , Vísceras/metabolismoRESUMEN
Inductive interactions between germ layers are an essential feature of the development of many organisms. In several species these interactions are mediated by members of the transforming growth factor-beta (TGF beta) family. In amphibians, different concentrations of activin can induce different types of mesoderm in the animal cap assay. In Drosophila, a member of the TGF beta family, decapentaplegic (dpp), acts as an inductive signal. Midway through embryogenesis, dpp is expressed in the visceral mesoderm, and enhances the expression of the homeotic gene labial in the underlying midgut endoderm. Earlier in development, however, dpp expression is limited to the dorsal ectoderm. At this stage in development, thickveins, a dpp receptor, is expressed in the mesoderm, and this suggests that ectodermal dpp might not only be required for development of dorsal ectoderm, but could also act inductively to mediate pattern formation in the underlying mesoderm. Here we show, by expressing dpp ectopically in the ectoderm and mesoderm and by examining dpp null mutant embryos, that dpp regulates expression of mesodermal genes.
Asunto(s)
Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Hormonas de Insectos/genética , Mesodermo/fisiología , Animales , Drosophila , Ectodermo/fisiología , Inducción Embrionaria/genética , Transducción de SeñalRESUMEN
Earlier work has indicated that the period length of Drosophila circadian behavioral rhythms is dependent on the abundance of the period (per) gene product. Increased expression of this gene has been associated with period shortening for both the circadian eclosion (pupal hatching) rhythm and circadian locomotor activity rhythms of adult Drosophila. In this study it is shown that a wide variety of missense mutations, affecting a region of the per protein consisting of approximately 20 aa, predominantly generate short period phenotypes. The prevalence of such mutations suggests that short period phenotypes may result from loss or depression of function in this domain of the per protein. Possibly mutations in the region eliminate a regulatory function provided by this segment, or substantially increase stability of the mutant protein.
Asunto(s)
Relojes Biológicos/fisiología , Drosophila/genética , Genes , Mutación , Proteínas Nucleares , Proteínas/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Mapeo Cromosómico , Proteínas de Drosophila , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Proteínas Circadianas Period , Proteínas/química , Proteínas/metabolismoRESUMEN
Antibodies to Notch show that it is a stable, high-molecular-weight transmembrane glycoprotein, with epidermal growth factor (EGF)-like elements exposed on the cell surface. The protein is phosphorylated variably on serines of the cytoplasmic domain. Individual Notch polypeptide chains appear to be associated with one another by disulfide bonds, suggesting that homotypic interaction of these proteins is required for function. Immunocytochemistry has revealed striking features of Notch expression that might clarify its function: Cells of the ventral neurogenic ectoderm become conspicuously labeled with the protein prior to embryonic neurogenesis, and Notch appears to be associated with cells destined for both neural and epidermal lineages. High levels of Notch become restricted to neuroblasts as they delaminate from the embryonic ectoderm and are apposed to mesoderm. Mesodermal cells express Notch also, suggesting a possible involvement in neurogenesis, or an unknown role in mesoderm differentiation. In larvae and pupae, a correlation of expression and neuroblast mitotic activity is seen for many cells. Notch produced by a dividing neuroblast may persist on derivative cells, including terminally differentiated neurons and nerve processes. In the larval eye imaginal disk, strong Notch expression appears in the morphogenetic furrow, uniformly on cell surfaces as they cluster to form ommatidia. Expression persists on ommatidia after release from the furrow. These patterns suggest a role for Notch in position-dependent development in both initiation and maintenance of cell-surface interactions. In the eye and embryonic ectoderm, uniform expression on cells interacting to produce different developmental lineages from a single primordium suggests that Notch alone may not be sufficient to elaborate cell fates.
Asunto(s)
Drosophila/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Diferenciación Celular , Membrana Celular/metabolismo , Drosophila/genética , Drosophila/crecimiento & desarrollo , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/genética , Peso Molecular , Mutación , Fosforilación , Distribución TisularRESUMEN
The per locus of Drosophila has been implicated in the control of behavioural rhythms. In fruitfly embryos and larvae per is expressed in salivary glands. Per mutations have striking effects on intercellular communication in salivary glands: gap junction channels are modulated so that their conductance varies inversely with the period of behavioural rhythms in the mutants. A similar effect on junctional communication in the nervous system may explain how per influences behavioural rhythms.
Asunto(s)
Conducta Animal , Relojes Biológicos , Comunicación Celular , Drosophila/genética , Uniones Intercelulares/fisiología , ARN/análisis , Animales , Electrofisiología , Histocitoquímica , Mutación , Permeabilidad , Glándulas Salivales/químicaRESUMEN
The period (per) locus, which controls biological rhythms in Drosophila, was originally defined by three chemically induced mutations. Flies carrying the pero mutation were arrhythmic, whereas pers and perl mutants had circadian behavioural rhythms with 19-hour and 29-hour periodicities, respectively. Wild-type flies have 24-hour rhythms. Here we compare the per locus DNA sequences of the three mutants with the parental wild-type. The pers and perl mutations lead to amino-acid substitutions, whereas pero introduces an early translation stop (amber). The results indicate that the protein product of per controls biological rhythms. We also report that the abundance of this protein may set the pace of the Drosophila clock. Although circadian rhythms are restored when arrhythmic (per-) Drosophila are transformed with per locus DNA, flies receiving identical transforming DNA segments can produce rhythms with periods that differ by more than 12 hours. Transcription studies reveal a tenfold variation in the level of per RNA among transformed lines. Levels of per RNA are inversely correlated with period length, so that flies with lowest levels of the per product have slow-running biological clocks. On the basis of the combined studies we suggest that perl and pers mutants produce hypoactive and hyperactive per proteins, respectively.