Ubiquitous overexpression of a transgene encoding the extracellular portion of the Drosophila Roughest-Irregular Chiasm C protein induces early embryonic lethality
Abstract:The cell adhesion molecule Rst-irreC is a transmembrane glycoprotein of the immunoglobulin superfamily involved in several important developmental processes in Drosophila, including axonal pathfinding in the optic lobe and programmed cell death and pigment cell differentiation in the pupal retina. As an initial step towards the "in vivo" functional analysis of this protein we have generated transgenic fly stocks carrying a truncated cDNA construct encoding only the extracellular domain of Rst-IrreC under the t… Show more
“…We had previously shown that ubiquitous expression of a heat shock-inducible transgenic cDNA construct encoding the Rst protein ectodomain (pCa18D3.1) (Moda et al, 2000) at different embryonic stages can severely disrupt myogenesis by preventing myoblast fusion (Strünkelnberg et al, 2001) as well as block blastoderm cellularization and interfere with embryonic central nervous system midline formation (Moda and Ramos, unpublished). Induction of this construct during larval stages had no obvious developmental effects, but when white prepupae carrying pCa18D3.1 were heat shocked for a 22-hour period at 338C a small but consistent reduction in viability was observed in comparison with similarly treated nontransgenic siblings (see Fig.…”
Section: Misexpression Of the Rst Ectodomain Interferes With Salivarymentioning
confidence: 99%
“…Unless otherwise noted, all stocks used in this work were previously described (Araújo et al, 2003;Boschert et al, 1990;Lindsley and Zimm, 1992;Moda et al, 2000;Ramos et al, 1993).…”
Section: Fly Stocks and Genetic Manipulationsmentioning
confidence: 99%
“…Flies were normally kept at 258C on standard Drosophila food and crosses were performed according to standard procedures. Unless otherwise noted, all stocks used in this work were previously described (Araújo et al, 2003;Boschert et al, 1990;Lindsley and Zimm, 1992;Moda et al, 2000;Ramos et al, 1993).…”
Section: Fly Stocks and Genetic Manipulationsmentioning
The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst(D) mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time.
“…We had previously shown that ubiquitous expression of a heat shock-inducible transgenic cDNA construct encoding the Rst protein ectodomain (pCa18D3.1) (Moda et al, 2000) at different embryonic stages can severely disrupt myogenesis by preventing myoblast fusion (Strünkelnberg et al, 2001) as well as block blastoderm cellularization and interfere with embryonic central nervous system midline formation (Moda and Ramos, unpublished). Induction of this construct during larval stages had no obvious developmental effects, but when white prepupae carrying pCa18D3.1 were heat shocked for a 22-hour period at 338C a small but consistent reduction in viability was observed in comparison with similarly treated nontransgenic siblings (see Fig.…”
Section: Misexpression Of the Rst Ectodomain Interferes With Salivarymentioning
confidence: 99%
“…Unless otherwise noted, all stocks used in this work were previously described (Araújo et al, 2003;Boschert et al, 1990;Lindsley and Zimm, 1992;Moda et al, 2000;Ramos et al, 1993).…”
Section: Fly Stocks and Genetic Manipulationsmentioning
confidence: 99%
“…Flies were normally kept at 258C on standard Drosophila food and crosses were performed according to standard procedures. Unless otherwise noted, all stocks used in this work were previously described (Araújo et al, 2003;Boschert et al, 1990;Lindsley and Zimm, 1992;Moda et al, 2000;Ramos et al, 1993).…”
Section: Fly Stocks and Genetic Manipulationsmentioning
The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst(D) mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time.
“…We are interested in developmental processes mediated by cell adhesion molecules. Studies of Roughest (Rst) function have shown that its precise temporal expression is important for proper development (Araujo et al 2003) and ectopic expression of Rst leads to interference with its normal function (Schneider et al 1995;Reiter et al 1996;Moda et al 2000). Rst is a cell adhesion molecule of the immunoglobulin superfamily (Ramos et al 1993) functioning in several developmental processes.…”
Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily with pleiotropic functions during the development of Drosophila melanogaster. It has been shown to be involved in cell sorting before apoptosis in the developing compound eye, in fusion processes of embryonic muscle development and in axonal pathfinding. In accordance with its multiple functions, the rst gene shows a dynamic expression pattern throughout the development of Drosophila. In order to understand the transcriptional regulation of rst expression we have identified rst cis regulatory sequences in an enhancer detection screen. By dissection of the identified rst cis regulatory sequences we identified several distinct rst regulatory modules. Among others these include elements for expression in interommatidial cells of the pupal eye disc at a time when apoptotic decisions are made in these cells and elements for expression in the embryonic mesoderm. The expression of rst in the embryonic mesoderm is regulated by at least two separate modules.
“…A proteína Rst está envolvida em vários eventos no desenvolvimento de D. melanogaster, tais como a fusão de mioblastos durante a diferenciação da musculatura somática embrionária (STRÜNKELNBERG et al, 2001); o direcionamento axonal do lobo óptico (BOSCHERT et al, 1990; SCHNEIDER, 1995); a morfogênese de células precursoras interomatidiais (REITER et al 1996;ARAÚJO et al, 2003;BAO;CAGAN, 2005), a padronização dos órgãos sensoriais da antena (REDDY et al, 1999); diferenciação das células pigmentares do olho composto (ARAÚJO et al, 2003) e a morte celular programada no mesmo (WOLF; READY, 1991;RAMOS et al, 1993;REITER et al, 1996;ARAÚJO et al 2003); além da autofagia da glândula salivar larval (SIMON et al, 2009); foi também demonstrado que quando a porção extracelular desta proteína é superexpressa, ocorre uma interferência na celularização do blastoderma (MODA et al, 2000). Simon et al (2009) observaram que pupas portadoras de uma mutação dominante na região regulatória do gene roughest (rst D ) apresentaram suas glândulas larvais em períodos superiores a 16 horas após a formação do pupário, chegando até mesmo 24 horas após a formação do mesmo.…”
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