Loss-of-function of β-catenin bar-1 slows development and
activates the Wnt pathway in Caenorhabditis elegans
M. Leontien van der Bent1*, Mark G. Sterken1*, Rita J.M. Volkers1*, Joost A.G.
Riksen1, Tobias Schmid2, Alex Hajnal2, Jan E. Kammenga1# & L. Basten Snoek1#.
1Laboratory
of Nematology, Wageningen University, Droevendaalsesteeg 1, 6708 PB
Wageningen, The Netherlands
2Institute
of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland
Supplementary information
Supplemental figure A. Developmental delay between N2 and bar-1(ga80).
Age estimation of the samples used in this study (triangles), based on gene
expression profiles obtained from Snoek et al. 2014 (light blue circles) 23. N2 samples
are shown in blue, bar-1(ga80) samples in yellow.
Supplementary figure B. Egg hatching in N2 and bar-1(ga80).
Here we show the hatching of eggs after bleaching three N2 (blue) and three bar1(ga80) (yellow) populations. The dots indicate the individual observations of the six
populations and the line shows the average over these observations per time-point.
The total number of hatched eggs counted was 2,330 for N2 and 943 for bar-1(ga80).
There was no significant difference in the hatching of the eggs between N2 and bar1(ga80) (ANOVA over the average per time-point, p = 0.159).
Supplementary Figure C. Significances of genes affected by bar-1 taking
development in account. This figure shows the outcome when a model is used
explaining the differences between bar-1(ga80) and N2 by both genotype and
developmental age (44 hours for bar-1(ga80) versus 48 hours for N2 in this
experiment and 44 to 58 hours for N2 in the developmental series (Snoek et al.
2014). Nine different groups of genes can be found. First three groups are genes
higher expressed in bar-1(ga80) compared to N2. Within this group there are genes
that show: increasing expression levels during L4 development (8: 1,494 genes),
decreasing expression levels during L4 development (6: 666 genes) or are not
affected during L4 development (7: 2,055 genes). Second three groups are genes
lower expressed in bar-1(ga80) compared to N2. Within this group there are genes
that show: decreasing expression levels during L4 development (2: 1,094 genes),
decreasing expression during L4 development (4: 1,631 genes) or are not affected
during L4 development (3: 1,018 genes). Last three groups are not affected by bar1(ga80). Genes with increasing expression levels during L4 development (1: 3,145
genes), genes with decreasing expression levels (5: 3,634 genes) and genes not
affected during L4 development (9: 9,871 genes). Sums of groups may be different
than numbers mentioned in the text as gene represented by multiple different
probes/spots can be present in more than one category. For example: there are 3920
genes showing higher expression levels in bar-1(ga80) compared to N2 yet the sum
of groups 6, 7 and 8 is 4215. There are 3637 genes showing a decreased expression
in bar-1(ga80) compared to N2, yet the sum of groups 2,3 and 4 is 3743.
Supplementary Figure D. Visualization of the transcriptional network of up41
regulated genes in bar-1(ga80).
This figure shows the relation between the up-regulated genes in bar-1(ga80) and
the four transcription factors that were enriched for binding sites (from 30) in this set.
The colors of the edges indicate the transcription factor that makes the connection:
red for PQM-1, blue for ELT-3, orange for MDL-1, and grey for PHA-4. The color of
the nodes indicates whether it is a transcription factor (dark red) or how strongly a
gene is up-regulated (darker green, is stronger up-regulated). PQM-1 and MDL-1
both have binding sites near PHA-4 and ELT-3, thus potentially regulating expression
of these transcription facors.
Supplementary Figure E. The expression of class I and class II genes in bar-1
relative to N2.
The class I (DAF-16 up-regulated genes) and class II (DAF-16 down-regulated
genes) expression in EW15 relative to N2. The boxplots show the distribution of the
expression of all the genes belonging to either category. As can be seen, there is
quite some variation in the expression differences between N2 and EW15 for these
genes. Taken as a whole, the class I genes are significantly higher expressed than
the class II genes (two-sided t-test, p < 1*10-13). The class I genes are also
significantly up-regulated in bar-1(ga80) (two-sided t-test, p < 1*10-8) and the class II
genes are significantly down-regulated (two-sided t-test, p < 1*10-5). In both cases
the effect sizes are small, 10% higher and 5% lower than N2 respectively.
Supplementary Table 1. Genes significantly up and down regulated in bar-1lof
mutant. These are the genes which results from the model including age.
Supplementary Table 2. Enrichment results
The outcome of the enrichment analysis for the up and down regulated genes in bar1(ga80) compared to N2, both for the sets including and excluding genes with a
developmental effect. Enrichment was tested for: gene ontology terms, gene classes,
anatomy terms, KEGG pathway component, protein domains, and Wormbook
chapter occurrence.
Supplementary Table 3.
Raw data underlying supplementary figure E. The relative expression of genes
belonging to Class I (PQM-1 and/or DAF-16 regulated) and Class II (PQM-1
regulated) identified by Tepper et al, 2013.
Supplementary Text 1. Polymorphisms in Wnt-pathway genes and loci with
possible Wnt-pathway modifiers.
Supplementary text.
Title: Loss-of-function of β-catenin bar-1 slows development and activates the
Wnt pathway in Caenorhabditis elegans
Merged Supplementary figures and text. M. Leontien van der Bent, Mark G. Sterken,
Rita J.M. Volkers, Joost A.G. Riksen, Tobias Schmid, Alex Hajnal, Jan E. Kammenga
& L. Basten Snoek
Suppl. Figure A
Title: Loss-of-function of β-catenin bar-1 slows development and activates the
Wnt pathway in Caenorhabditis elegans
Merged Supplementary figures and text. M. Leontien van der Bent, Mark G. Sterken,
Rita J.M. Volkers, Joost A.G. Riksen, Tobias Schmid, Alex Hajnal, Jan E. Kammenga
& L. Basten Snoek
Suppl. Figure B
Title: Loss-of-function of β-catenin bar-1 slows development and activates the
Wnt pathway in Caenorhabditis elegans
Merged Supplementary figures and text. M. Leontien van der Bent, Mark G. Sterken,
Rita J.M. Volkers, Joost A.G. Riksen, Tobias Schmid, Alex Hajnal, Jan E. Kammenga
& L. Basten Snoek
Suppl. Figure C
Title: Loss-of-function of β-catenin bar-1 slows development and activates the
Wnt pathway in Caenorhabditis elegans
Merged Supplementary figures and text. M. Leontien van der Bent, Mark G. Sterken,
Rita J.M. Volkers, Joost A.G. Riksen, Tobias Schmid, Alex Hajnal, Jan E. Kammenga
& L. Basten Snoek
Suppl. Figure D
Title: Loss-of-function of β-catenin bar-1 slows development and activates the
Wnt pathway in Caenorhabditis elegans
Merged Supplementary figures and text. M. Leontien van der Bent, Mark G. Sterken,
Rita J.M. Volkers, Joost A.G. Riksen, Tobias Schmid, Alex Hajnal, Jan E. Kammenga
& L. Basten Snoek
Suppl. Figure E
Merged Supplementary figures and text. M. Leontien van der Bent, Mark G. Sterken,
Rita J.M. Volkers, Joost A.G. Riksen, Tobias Schmid, Alex Hajnal, Jan E. Kammenga
& L. Basten Snoek
Suppl. Text 1
Genetic polymorphisms in Wnt-pathway genes
Table: SNPs and AA changes obtained from Wormbase (www.wormbase.org) in
Wnt-pathway components (genes shown in figure 3)
Gene
Has SNP
Has
SNP
between
CB4856 and
N2
AA change
between N2
and CB4856
Gene
Has SNP
Has
SNP
between
CB4856 and
N2
AA change
between N2
and CB4856
Wrm-1
Hmp-1
Sys-1
Bar-1
Mom-2
Egl-20
Cwn-2
Cwn-1
Lin-44
Apr-1
Gsk-3
Kin-19
Pry-1
Axl-1
Dsh-2
Mig-5
Dsh-1
Mom-5
Cfz-2
Mig-1
Lin-17
Lin-18
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
N
?
Y
Y
N
N
N
Y
?
Y
N
Y
?
Y
N
N
N
Vps-35
Vps-29
Vps-26
Ceh-13
Psa-3
Ceh-22
Lin-39
Mab-5
Egl-5
Lit-1
Mom-4
Par-5
Crm-1
Had-1
Pop-1
Mig-14
Lin-23
Mom-1
Unc-37
Cam-1
Sfrp-1
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
?
Y
N
Y
Y
Y
N
N
N
Y
?
Y
?
N
Y
N
Y
Y
N
Y
Downstream targets of bar-1 show signs of natural variation
Using data from expression quantitative trait loci (eQTL) studies, it is possible to
identify loci harbouring regulators acting downstream or parallel to bar-1. An eQTL
shows a locus harbouring allelic variants causal for gene expression levels
differences between genotypes of the gene for which the eQTL was found.
Overlapping eQTLs of multiple genes indicate a regulator of gene expression as the
expression of all these genes seems to be affected by one locus. The genes
differently expressed between bar-1(ga80) and N2 can be compared to genes with
eQTLs at a specific marker in several experiments (Vinuela et al. 2010, 2012,
Rockman et al. 2010). Enrichment would indicate a gene polymorphic between N2
and CB4856 involved in processes affected by Wnt-signalling downstream of bar-1.
Table: eQTL loci enriched with genes affected by bar-1(ga80) including genes with a
development effect
bar-1(ga80) effect
Down
Locus (markes)
65-74 (peak at 6869)
UP
6-8
16-19
96-101 (peak at 96)
Locus(bp)
IV 3.9Mb-11.6Mb
(peak between
6.5Mb-7.4Mb)
I 4.3Mb-5.9Mb
I 11.7Mb-14.2Mb
V 15.2Mb-20.7Mb
Paper
Vinuela et al. 2010
Vinuela et al. 2010
Vinuela et al. 2010
Vinuela et al. 2010
Table: eQTL loci enriched with genes affected by bar-1(ga80) excluding genes with a
development effect
bar-1(ga80) effect
Down
UP
Locus (markes)
25
96-101 (peak at 96)
1129-1139
Locus(bp)
II 3.4Mb
V 15.2Mb-20.7Mb
V 16.25Mb16.39Mb
Paper
Vinuela et al. 2010
Vinuela et al. 2010
Rockman et al. 2010
By eQTL enrichment we identified 2 possible polymorphic loci between
CB4856 and N2 for genes down regulated in bar-1(ga80) as well as 3 different loci
for genes up regulated in bar-1(ga80). The enriched eQTL locus on chromosome V
was found in two independent experiments (Rockman et al. 2010 and Vinuela et al.
2010). This shows that multiple polymorphic loci between N2 and CB4856 might
affect genes specifically downstream of or modulated by bar-1 and wnt-signalling.