Forkhead transcription factor FKH-8 cooperates with RFX in the direct regulation of sensory cilia in Caenorhabditis elegans
- Developmental Neurobiology Unit, Instituto de Biomedicina de Valencia IBV-CSIC, Spain
- Molecular Basis of Neurodegeneration Unit, Instituto de Biomedicina de Valencia IBV-CSIC, Spain
- Department of Biosciences and Nutrition. Karolinska Institute. Campus Flemingsberg, Sweden
- Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de Investigaciones Científicas/Universidad Pablo de Olavide, Spain
Figures
Figure 1 with 3 supplements
FKH-8 is a candidate for direct regulation of ciliome gene expression in C. elegans.
(A) Schema for a sensory cilium. Cilia components (ciliome) can be divided into core and subtype-specific categories. Core genes whose reporters are analyzed in panel C and Figure 1—figure …
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Figure 1—source data 1
Raw quantification data of ciliome gene reporters in daf-19(m86); daf-12(sa204) included in Figure 1 and Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig1-data1-v2.xlsx
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Figure 1—source data 2
Gene lists and raw data for bioinformatics analysis in Figure 1 and Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Ciliome reporters used in this work.
Schematic representation of reporter constructs used in the manuscript. Selected core cilia components contain at least one experimentally validated X-box motif in their sequences (marked as a blue …
Figure 1—figure supplement 2
Reporter expression for some core ciliome genes is abolished in daf-19(m86) mutant.
Expression of short reporters for the core cilia components tmem-107, mks-1 and osm-5 is completely abolished in daf-12(sa204); daf-19(m86) double mutants. The same extrachromosomal line was …
Figure 1—figure supplement 3
Available -omics data identifies FKH-8 as a candidate transcriptional regulator of ciliome genes in C.elegans.
(A) DAF-19/RFX motifs (X-box) are enriched in regulatory sequences of core and broadly expressed ciliome genes more prominently than in subtype-specific features. Motif logo for the palindromic …
Figure 2 with 2 supplements
FKH-8 is expressed in sensory ciliated neurons, binds ciliome genes near DAF-19 X-boxes and physically interacts with DAF-19.
(A) fkh-8 locus (top) and fosmid based fkh-8 reporter (bottom). Grey boxes represent exons and orange boxes correspond to exons coding for the FKH DNA binding domain (DBD). Putative daf-19/RFX …
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Figure 2—source data 1
Raw quantification data of FKH-8 and ift-20 reporter co-expression represented in Figure 2B.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig2-data1-v2.xlsx
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Figure 2—source data 2
Raw data for bioinformatics analysis in Figure 2 and Figure 2—figure supplements 1 and 2.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig2-data2-v2.xlsx
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Figure 2—source data 3
Co-IP original files of unedited gels in Figure 2.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig2-data3-v2.zip
Figure 2—figure supplement 1
fkh-8 expression along ciliated system development.
(A) Representative Z projections of developmental embryonic milestones until hatching (L1) shows FKH-8::GFP fosmid reporter expression matches in time and space panciliary reporter ift-20::gfp …
Figure 2—figure supplement 2
FKH-8 binds near X-BOX motifs.
(A) Analysis of DAF-19/RFX binding motifs (PWM M1534_1.02) in peaks for ChIP-seq datasets of other C. elegans FKH TFs present in ENCODE database. There is no significant enrichment for DAF-19 motif …
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Figure 2—figure supplement 2—source data 1
Co-IP original files of unedited gels for the chromatin fraction.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig2-figsupp2-data1-v2.zip
Figure 3 with 2 supplements
FKH-8 TF and FKH-binding sites are required for correct core ciliome gene reporter expression.
(A) Dorso-ventral images from young adult heads expressing different core ciliome multicopy array gene reporters in wild type and fkh-8(vlc43) null mutant animals. All reporters are extrachromosomal …
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Figure 3—source data 1
Raw quantification data of ciliome gene reporter expression defects in fkh-8 mutants and cis-regulatory point mutation analysis corresponding to Figure 3 and Figure 3—figure supplements 1 and 2.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig3-data1-v2.xlsx
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Figure 3—source data 2
Motif enrichment analysis of xbx-1 and ift-20 reporters.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
f. fkh-8(tm292) is a hypomorphic recessive allele.
(A) Quantification of the number of gfp-positive cells in five distinct anatomical regions for each reporter in wild type, fkh-8(tm292) hypomorphic allele and fkh-8(vlc43) null mutant. To facilitate …
Figure 3—figure supplement 2
f. Functional characterization of putative FKH sites in cis-regulatory modules of two core ciliome components.
(A) Schematics for the ift-20 and xbx-1 loci and reporters. Dark grey boxes represent exons whereas light grey boxes correspond to UTRs. FKH-8 Chip-seq peaks are depicted with an orange horizontal …
Figure 4 with 2 supplements
FKH-8 and DAF-19 exhibit crosstalk and synergistic effects in the transcriptional regulation of ciliome genes.
(A) daf-19 locus codes for five different daf-19 isoforms. Grey boxes represent exons whereas blue boxes correspond to exons coding for the RFX DNA binding domain (DBD). FKH-8 binding events are …
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Figure 4—source data 1
Raw quantification data of synergistic actions of fkh-8 and daf-19 depicted in Figure 4, Figure 4—figure supplements 1 and 2.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Lack of FKH-8 has no major effect on DAF-19 expression.
(A) Representative lateral views from heads of young adult hermaphrodites co-expressing a fosmid-based DAF-19::GFP reporter and dat-1::mcherry reporter labeling the dopaminergic neurons. Lack of …
Figure 4—figure supplement 2
FKH-8 and DAF-19 show synergistic effects in the transcriptional regulation of the ciliome.
(A) Representative micrographs of kap-1 reporter expression in different genetic backgrounds. kap-1::gfp is unaffected in fkh-8(tm292) and ectopically expressed both in daf-19(m86); daf-12(sa204) …
Figure 5 with 1 supplement
fkh-8(vlc43) null mutants display morphological defects in cilia.
Integrated reporters unaffected in fkh-8 mutant are used to label the cilia of several distinct subpopulations of ciliated neurons. CEP: otIs259(dat-1::gfp); ADF: zdIs13(tph-1::gfp); AWB: kyIs104(str…
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Figure 5—source data 1
Raw quantification data of cilia morphology analysis depicted in Figure 5 and Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
fkh-8(vlc43) null mutants display morphological defects in cilia using physical immobilization with polystyrene beads.
Integrated reporters unaffected in fkh-8 mutant are used to label the cilia of several distinct subpopulations of ciliated neurons. CEP: otIs259(dat-1::gfp); ADF: zdIs13(tph-1::gfp); AWB: kyIs104(str…
Figure 6 with 1 supplement
FKH-8 is required for the correct display of several sensory mediated behaviors.
(A) Mutations in fkh-8 significantly impair appropriate backward response to nose touch, revealing functionality defects for the ASH, FLP and/or OLQ ciliated neurons. This phenotype is stronger in fk…
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Figure 6—source data 1
Raw quantification data for behavioral analysis in Figure 6 and Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
FKH-8 is not required for correct display of mechanosensory behaviors mediated by non-ciliated neurons.
(A to C) fkh-8 mutants show normal avoidance behaviors elicited by mechanical stimuli known as gentle touch and harsh touch paradigms, suggesting FKH-8 is not required for the correct functionality …
Figure 7
Mammalian FKH TFs with known motile cilia regulatory functions can rescue fkh-8 mutant phenotype.
(A) Rescue strategy: dat-1 promoter, unaffected in fkh-8 mutants, is used to drive FKH TF cDNA and eGFP expression specifically in the dopaminergic ciliated system. (B) Representative images of …
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Figure 7—source data 1
Raw quantification data for rescuing experiments in Figure 7.
- https://cdn.elifesciences.org/articles/89702/elife-89702-fig7-data1-v2.xlsx
Additional files
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Supplementary file 1
List of reagents: Strains, plasmids and primers.
- https://cdn.elifesciences.org/articles/89702/elife-89702-supp1-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/89702/elife-89702-mdarchecklist1-v2.docx
