RESUMEN

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/metabolismo

RESUMEN

Using DNA probes labeled with digoxygenin-11-dUTP, a simplified method of electron microscopic (EM) in situ hybridization was developed for standard squashes of Drosophila melanogaster polytene chromosomes. The developed method is efficient and reproducible: its high resolution and specificity was shown for the transformed strain 148, in which the insertion was localized by EM as a new thin band. The method was applied for fine mapping of the developmentally regulated complex gene, muscleblind (mbl), which was shown to cover the 54B1-2 large band and the adjacent interbands in 2R polytene chromosome.

Asunto(s)

Cromosomas , Nucleótidos de Desoxiuracil/química , Digoxigenina/química , Drosophila melanogaster/genética , Animales , Sondas de ADN , Hibridación in Situ , Microscopía Electrónica , Reproducibilidad de los Resultados

RESUMEN

saliva (slv) transcription begins at the salivary gland placodes and continues on throughout development as salivary glands invaginate and reach their final location and morphology. saliva is located cytogenetically in 76A/B, and encodes a 226-amino-acid protein with four hydrophobic domains. A Northern blot detects a 1.6-kb transcript throughout development. Database similarity searches reveal homology to proteins from Caenorhabditis, Lilium, Medicago and mouse.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto/genética , Genes de Plantas/genética , Glándulas Salivales/embriología , Proteínas y Péptidos Salivales/genética , Vertebrados/genética , Secuencia de Aminoácidos , Animales , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Drosophila melanogaster/química , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Plantas/genética , Glándulas Salivales/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

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/genética

RESUMEN

We report a simplified method of electron microscopic (EM) in situ hybridization for standard squashes of Drosophila melanogaster polytene chromosomes using digoxigenin-11-dUTP labelled DNA probes. The method is efficient and reproducible: its high resolution and specificity were demonstrated for the transformed strain 148, in which the insertion was localized precisely as a new thin band both by conventional EM and according to our method. In addition, the method was applied to the fine mapping of the developmentally regulated gene muscle-blind (mbl). On the one hand, mbl was shown to cover the 54B1-2 large band and the adjacent interbands in the 2R polytene chromosome. On the other hand, the use of distantly located DNA probes in the mbl gene allowed us to orientate the transcription unit in the chromosome.

Asunto(s)

Cromosomas , Nucleótidos de Desoxiuracil , Digoxigenina/análogos & derivados , Proteínas de Drosophila , Drosophila melanogaster/genética , Hibridación in Situ/métodos , Animales , Mapeo Cromosómico , Sondas de ADN , Microscopía Electrónica/métodos , Proteínas Nucleares/genética

RESUMEN

We have isolated the embryonic lethal gene muscleblind (mbl) as a suppressor of the sev-svp2 eye phenotype. Analysis of clones mutant for mbl during eye development shows that it is autonomously required for photoreceptor differentiation. Mutant cells are recruited into developing ommatidia and initiate neural differentiation, but they fail to properly differentiate as photoreceptors. Molecular analysis reveals that the mbl locus is large and complex, giving rise to multiple different proteins with common 5' sequences but different carboxy termini. Mbl proteins are nuclear and share a Cys3His zinc-finger motif which is also found in the TIS11/NUP475/TTP family of proteins and is highly conserved in vertebrates and invertebrates. Functional analysis of mbl, the observation that it also dominantly suppresses the sE-Jun(Asp) gain-of-function phenotype and the phenotypic similarity to mutants in the photoreceptor-specific glass gene suggest that mbl is a general factor required for photoreceptor differentiation.

Asunto(s)

Proteínas de Drosophila , Drosophila/genética , Proteínas de Insectos/genética , Proteínas Nucleares/genética , Células Fotorreceptoras de Invertebrados/embriología , Proteínas Tirosina Quinasas Receptoras , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Diferenciación Celular/genética , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Drosophila/embriología , Drosophila/crecimiento & desarrollo , Proteínas del Ojo/genética , Genes Letales , Genes Supresores , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Proteínas Proto-Oncogénicas c-jun/genética , Receptores de Esteroides/genética , Homología de Secuencia de Aminoácido , Transcripción Genética , Vertebrados/genética

RESUMEN

The homeotic gene Ultrabithorax encodes a family of six homeoproteins translated from alternatively spliced mRNAs. The structures of these UBX isoforms have been conserved among anciently diverged Drosoph-ila species and functional distinctions between some isoforms have been reported that suggest subtle but important roles in Ubx action. We present a detailed analysis of the expression patterns of Ubx mRNAs and proteins during embryogenesis, using isoform-specific monoclonal antibodies and synthetic oligonucleotide probes. These patterns are remarkably complex, each mRNA and corresponding protein isoform being expressed in a partially overlapping but distinct stage and tissue-specific pattern. The complexity is greatest in the central nervous system, where different isoforms predominate during successive developmental stages and where their relative proportions differ from one metamere to another and even among individual neurons within a given metamere. The distributions of UBX isoforms are consistent with those functional distinctions that have been described; they also suggest that different isoforms may be specialized or optimized to control different aspects of central nervous system development. The close correspondence between the mRNA and protein patterns indicates that the mRNAs do not differ strongly in translatability, despite the abundance of rare codons in the optional exons. There is a delay between the detection of particular splicing events in the nucleus and the detection of the 3' end of the message or the appearance of the corresponding mRNAs and proteins in the cytoplasm. This delay is consistent with the size of the Ubx introns and indicates a cotranscriptional mechanism of splicing.

Asunto(s)

Clonación Molecular/métodos , Proteínas de Drosophila , Drosophila/genética , Hormonas de Insectos/genética , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Poli C/genética , Animales , Artefactos , Composición de Base , Secuencia de Bases , Cartilla de ADN , ADN Complementario/genética , Bases de Datos Factuales , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa

RESUMEN

We describe a method for the in situ detection of specific splicing variants. The method is based on the use of antisense oligonucleotides designed to span splice junctions labelled with digoxigenin by terminal transferase tailing. We find that the spatial patterns of Ubx splicing variants Ia and IIa are similar in early embryos, but differ in late embryos. Variant IVa is only detected in the CNS (ps6) at stages 16 and 17. We also present evidence indicating that the first splicing event is cotranscriptional.

Asunto(s)

Empalme Alternativo , Hibridación in Situ/métodos , Sondas de Oligonucleótidos , Animales , Secuencia de Bases , Digoxigenina , Drosophila , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Transcripción Genética