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The study of sensory cilia development in caenorhabditis elegans
Södertörn University, School of Life Sciences. Karolinska Institutet.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cilia and flagella are widespread eukaryotic subcellular components that are conserved from green algae to mammals. In different organisms they function in cell motility, movement of extracellular fluids and sensory reception. While the function and structural description of cilia and flagella are well established, very little is known about the developmental mechanisms by which cilia are generated and shaped and how their components are assembled into functional machineries. To answer these questions, we used sensory cilia development in the nematode Caenorhabditis elegansas a model system.

The work described here developed from the initial discovery of the ciliogenic properties of the gene daf-19, which encodes the sole C. elegans member of the RFX-type transcription factors. All members of the RFX transcription factor family are characterized by the presence of a conserved DNA binding domain, which recognizes special motifs (X-boxes) in promoters of its target genes. By using a genome search approach for X-box promoter motif-containing genes (xbx genes) we identified a list of about 750 xbx genes (candidates). This list comprises some already known ciliary genes as well as new genes, many of which we hypothesize to be important for cilia development and functioning.

A computational search for X-box motifs in the C. briggsae genome has demonstrated strong conservation of this motif between closely related nematode species. To find out whether RFX-type transcription factors can also regulate ciliogenic pathways in other organisms, we applied a similar search strategy to distant species such as the fruit fly Drosophila. Using X-box consensus sequences with varying degrees of refinement and subsequent gene expression analysis, we were able to identify a set of Drosophila xbx genes. Intriguingly, the majority of fly xbx genes that have homologs in C. elegans were down regulated in dRfx fly mutants, suggesting an evolutionary conserved role for RFX-type transcription factors in the regulation of ciliary genes.

Using X-box matches as a prediction tool we were able to identify novel ciliary genes, dyf-2 and dyf-11, in the C. elegans genome. We cloned these genes by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 and DYF-11 functions selectively affects the assembly and motility of different intraflagellar transport (IFT) components, resulting in compromised protein transport within cilia, and subsequently in defective cilia structures and sensory functions. Importantly, the mouse orthologs of DYF-2 and DYF-11 also localize to cilia, pointing to evolutionarily conserved roles for these proteins in cilia biogenesis.

In conclusion, our studies of the regulation of sensory cilia formation demonstrated how contributions of multiple factors are integrated into a developmental module that leads to the formation of the primary sensory organs, cilia. In addition, data obtained during the course of this study provide a useful resource for researchers interested in further identification and study of new genes implicated in cilia biogenesis and will have a significant impact on the understanding and treatment of cilia-based pathologies in humans.

Place, publisher, year, edition, pages
Stockholm: Karolinska Institutet , 2008. , 44 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:sh:diva-31234ISBN: 978-91-7140-992-8 (print)OAI: oai:DiVA.org:sh-31234DiVA: diva2:1050318
Public defence
2008-01-25, MA648, Alfred Nobels allé 7, Huddinge, 09:00 (English)
Opponent
Supervisors
Available from: 2016-11-28 Created: 2016-11-28 Last updated: 2016-11-28Bibliographically approved
List of papers
1. Analysis of xbx genes in C-elegans
Open this publication in new window or tab >>Analysis of xbx genes in C-elegans
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2005 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 132, no 8, 1923-1934 p.Article in journal (Refereed) Published
Abstract [en]

Cilia and flagella are widespread eukaryotic subcellular components that are conserved from green algae to mammals. In different organisms they function in cell motility, movement of extracellular fluids and sensory reception. While the function and structural description of cilia and flagella are well established, there are many questions that remain unanswered. In particular, very little is known about the developmental mechanisms by which cilia are generated and shaped and how their components are assembled into functional machineries. To find genes involved in cilia development we used as a search tool a promoter motif, the X-box, which participates in the regulation of certain ciliary genes in the nematode Caenorhabditis elegans. By using a genome search approach for X-box promoter motif-containing genes (xbx genes) we identified a list of about 750 xbx genes (candidates). This list comprises some already known ciliary genes as well as new genes, many of which we hypothesize to be important for cilium structure and function. We derived a C elegans X-box consensus sequence by in vivo expression analysis. We found that xbx gene expression patterns were dependent on particular X-box nucleotide compositions and the distance from the respective gene start. We propose a model where DAF-19, the RFX-type transcription factor binding to the X-box, is responsible for the development of a ciliary module in C elegans, which includes genes for cilium structure, transport machinery, receptors and other factors.

National Category
Developmental Biology
Identifiers
urn:nbn:se:sh:diva-14466 (URN)10.1242/dev.01775 (DOI)000229103300016 ()15790967 (PubMedID)2-s2.0-18844446127 (ScopusID)
Available from: 2011-12-23 Created: 2011-12-23 Last updated: 2016-11-28Bibliographically approved
2. Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species
Open this publication in new window or tab >>Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species
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2007 (English)In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 8, no 9, R195- p.Article in journal (Refereed) Published
Abstract [en]

Background: Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. Results: We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. Conclusion: Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-BiedI syndrome.

National Category
Microbiology Genetics
Identifiers
urn:nbn:se:sh:diva-14245 (URN)10.1186/gb-2007-8-9-r195 (DOI)000252100800021 ()17875208 (PubMedID)2-s2.0-44049101446 (ScopusID)
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2016-11-28Bibliographically approved
3. Caenorhabditis elegans DYF-2, an orthologue of human WDR19, is a component of the intraflagellar transport machinery in sensory Cilia
Open this publication in new window or tab >>Caenorhabditis elegans DYF-2, an orthologue of human WDR19, is a component of the intraflagellar transport machinery in sensory Cilia
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2006 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 17, no 11, 4801-4811 p.Article in journal (Refereed) Published
Abstract [en]

The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet-Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-14274 (URN)10.1091/mbc.E06-04-0260 (DOI)000241993500019 ()16957054 (PubMedID)2-s2.0-33750514794 (ScopusID)
Available from: 2011-12-21 Created: 2011-12-20 Last updated: 2016-11-28Bibliographically approved
4. Central role for DYF-11/MIP-T3 in assembling kinesin motor-intraflagellar transport complexes
Open this publication in new window or tab >>Central role for DYF-11/MIP-T3 in assembling kinesin motor-intraflagellar transport complexes
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31235 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-11-28 Created: 2016-11-28 Last updated: 2016-11-28Bibliographically approved

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