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Deciphering the Alk signaling pathway in Drosophila
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Ruth H. Palmer)ORCID iD: 0000-0002-8332-7011
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In Drosophila melanogaster the visceral mesoderm (VM) develops during embryogenesis in a process where myoblasts become specified to generate two distinct cell types, the founder cells (FCs) and the fusion competent myoblasts (FCMs) that consequently fuses. The cell specification is dependent on cell signaling mediated by the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb), how this further sets up different identity programs that drive myoblasts to differentiate into FCs and FCMs is still not well understood.

We have analysed whether the Midkine (MDK)/Pleiotrophin (PTN) homologues in Drosophila, Miple1 and Miple2 activate the Alk RTK in vivo. Earlier results from cell culture experiments suggested that vertebrate MDK/PTN is capable of activating ALK, findings that have become controversial with other studies showing contradictory results. We wanted to use Drosophila that have conserved homologues of both MDK/PTN and ALK, to address the question in vivo. We analysed the contribution of Miple in Alk dependent developmental processes such as visceral mesoderm (VM) specification during embryogenesis and in body size regulation of adult flies. Specification of VM as well as body size are not effected by loss of Miple proteins, and over expression of Miple proteins do not effect VM specification or body size. All together we conclude that there is no evidence that Miple1 or Miple2 can activate Alk in vivo. We found that loss of Miple protein effect the median lifespan of the fly which is reduced, interestingly the over expression of Miple proteins can promote an increased median life span in Drosophila.

We have also analysed how Alk RTK signaling regulates the Gli-like transcription factor Lame duck (Lmd) in vivo on a post-translational level. It has already been reported that Lmd plays an essential role in specification of FCMs in the somatic mesoderm during embryogenesis. We detect Lmd protein exclusively in FCMs of VM in control embryos, but in Alk mutants Lmd protein is present in all cells of VM and opposite to this when Alk is activated in all cells in VM by over expression of Jeb this results in total loss of Lmd protein. This suggests that Alk signaling is regulating Lmd, and we additionally show that Lmd persist in FCMs in mutants where VM is specified but where myoblast fusion do not occur, supporting that Alk activity in FCs is regulating the downregulation of Lmd in FCMs upon fusion.

Finally we have characterised the Rap1GEF C3G in vivo in Drosophila. In cell culture systems, the GTPase Rap1 has been identified to mediate Alk signaling and that this is regulated by the GEF C3G and interestingly the Drosophila C3G is expressed in the FCs of VM. We generated deletion mutants of C3G which exhibit semi-lethality and reduced life span, but no defects in visceral mesoderm development during embryogenesis. Instead we detected distinct phenotypes in somatic muscles of 3rd instar mutant larvae, with detachment and mistargeting of muscles, which effect localisation of integrins. We suggest that Drosophila C3G regulates Rap1 via inside out signaling of integrins which in turn effects cell adhesion in vivo in Drosophila larval muscles.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2015. , 73 p.
Keyword [en]
Drosophila, Alk, RTK, visceral mesoderm, signal transduction, growth factor, transcription factor
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-101009ISBN: 978-91-7601-247-5 (print)OAI: oai:DiVA.org:umu-101009DiVA: diva2:800986
Public defence
2015-05-05, Hörsal E04, Byggnad 6E, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-04-14 Created: 2015-03-16 Last updated: 2015-05-05Bibliographically approved
List of papers
1. The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation
Open this publication in new window or tab >>The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 11, e112250- p.Article in journal (Refereed) Published
Abstract [en]

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-97588 (URN)10.1371/journal.pone.0112250 (DOI)000344863100069 ()
Available from: 2015-01-04 Created: 2014-12-23 Last updated: 2017-12-05Bibliographically approved
2. Jeb/Alk signalling regulates the Lame duck GLI family transcription factor in the Drosophila visceral mesoderm
Open this publication in new window or tab >>Jeb/Alk signalling regulates the Lame duck GLI family transcription factor in the Drosophila visceral mesoderm
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2013 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 140, no 15, 3156-3166 p.Article in journal (Refereed) Published
Abstract [en]

The Jelly belly (Jeb)/Anaplastic Lymphoma Kinase (Alk) signalling pathway regulates myoblast fusion in the circular visceral mesoderm (VM) of Drosophila embryos via specification of founder cells. However, only a limited number of target molecules for this pathway are described. We have investigated the role of the Lame Duck (Lmd) transcription factor in VM development in relationship to Jeb/Alk signal transduction. We show that Alk signalling negatively regulates Lmd activity post-transcriptionally through the MEK/MAPK (ERK) cascade resulting in a relocalisation of Lmd protein from the nucleus to cytoplasm. It has previously been shown that downregulation of Lmd protein is necessary for the correct specification of founder cells. In the visceral mesoderm of lmd mutant embryos, fusion-competent myoblasts seem to be converted to 'founder-like' cells that are still able to build a gut musculature even in the absence of fusion. The ability of Alk signalling to downregulate Lmd protein requires the N-terminal 140 amino acids, as a Lmd(141-866) mutant remains nuclear in the presence of active ALK and is able to drive robust expression of the Lmd downstream target Vrp1 in the developing VM. Our results suggest that Lmd is a target of Jeb/Alk signalling in the VM of Drosophila embryos.

Keyword
Lmd, Alk, Jeb, Drosophila, Visceral muscle, Founder cell, Fusion competent myoblast
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-79229 (URN)10.1242/dev.094466 (DOI)000321864900009 ()
Available from: 2013-09-16 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
3. The Rap1 guanine nucleotide exchange factor C3G is required for preservation of larval muscle integrity in Drosophila melanogaster
Open this publication in new window or tab >>The Rap1 guanine nucleotide exchange factor C3G is required for preservation of larval muscle integrity in Drosophila melanogaster
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2010 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 3, e9403Article in journal (Refereed) Published
Abstract [en]

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize betaPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages.

National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-33098 (URN)10.1371/journal.pone.0009403 (DOI)000275063400001 ()20209136 (PubMedID)
Available from: 2010-04-12 Created: 2010-04-12 Last updated: 2017-12-12Bibliographically approved

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