Bieder et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00483-4(2023) 24:20

RESEARCH ARTICLE

BMC Molecular and Cell
BiologyOpen Access

Genetic and protein interaction studies
between the ciliary dyslexia candidate genes
DYX1C1 and DCDC2
Andrea Bieder1, Gayathri Chandrasekar1, Arpit Wason2, Steffen Erkelenz3, Jay Gopalakrishnan3, Juha Kere1,4 andIsabel Tapia-Páez5*
Abstract
Background DYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic
studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both arecytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact
molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and
DCDC2 interact on the genetic and the protein level.
Results Here, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective
interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models
including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b inzebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among
DYX1C1 and DCDC2 in a cellular model.
Conclusions In summary, we describe the physical and functional interaction between the two genes DYX1C1 and
DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set
the stage for future functional studies.
Keywords Cilia, Centrosome, Dyslexia, Genetic interaction, Zebrafish*Correspondence:
Isabel Tapia-Páez
isabel.tapia@ki.se
1
Department of Biosciences and Nutrition, Karolinska Institutet,
Huddinge, Sweden
2
Center for Molecular Medicine, Institute for Biochemistry I of the
University of Cologne, Cologne, Germany
3Institute of Human Genetics, Universitätsklinikum, Heinrich Heine
University, Düsseldorf, Germany
4
Molecular Neurology Research Program, University of Helsinki,
Folkhälsan Institute of Genetics, Helsinki, Finland5
Department of Medicine, Solna, Karolinska Institutet, Solnavägen 30,
SE-171 76 Solna, Sweden

Background
Developmental dyslexia (DD) is the most common neurodevelopmental learning disability. Its heredity has been
established and the underlying genetic causes are beginning to emerge with the identification of dyslexia susceptibility genes [1]. Two of the most replicated dyslexia
candidate genes are DYX1C1 (DNAAF4) and DCDC2[2–4]. Dyx1c1 and Dcdc2 have been shown to have roles
in neuronal migration in rodent knockdown models andin in vitro models, reminiscent of early studies showing
neuronal migration abnormalities in postmortem dyslexic brains [3, 5–8].

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

In addition to their shared association with DD, bothDYX1C1 and DCDC2 have established roles in cilia andin ciliopathies. Cilia are microtubule-based organelles
protruding from the cell surface of most eukaryotic cells
and are anchored to the cell via the basal body, which
grows from the mother centriole of the centrosome. Ciliopathies are a class of genetic disorders linked to malfunctioning of the cilium and in some cases involving
neurodevelopmental phenotypes [9, 10]. Many ciliopathies display a brain phenotype, and a growing body ofevidence is emerging for a role of cilia in neuropsychiatric disorders such as schizophrenia and autism [11–15].
Mutations in DYX1C1 cause primary ciliary dyskinesia(PCD) and loss-of function mutations in DCDC2 cancause a wide spectrum of cilia-related disorders suchas nephronophthisis-related ciliopathies (NPHP-RC),
hereditary hearing loss, and neonatal sclerosing cholangitis [16–21]. Several high-throughput transcriptomics
studies show that DYX1C1 and DCDC2 are upregulated
in highly ciliated tissues [22–24]. Furthermore, knockdown of dyx1c1 in zebrafish embryos produces a broad
ciliary phenotype and dyx1c1 mutant zebrafish display
idiopathic scoliosis due to motile ciliary defects [16, 25,26]. Similarly, zebrafish morphants of dcdc2b display aciliopathy phenotype including curved body, pronephriccysts and left-right asymmetry defects [18]. DYX1C1 has
been proposed as a cytoplasmic axonemal dynein assembly factor [16, 27–30]. Recent studies have strengthened
a role of DCDC2 and DYX1C1 also in primary cilia andhuman neurons [31, 32].
We have shown previously by proteomic studies that
DYX1C1 interacts with CPAP (Centrosomal-P4.1-associated protein, Centromere protein J (CENPJ)) in the neuroblastoma cell line SH-SY5Y [7]. CPAP was identified asa regulator of brain size and its mutation causes autosomal recessive primary microcephaly (MCPH) and Seckel
syndrome [33, 34]. It is required for cilia formation inneuronal cells in vitro and is crucial for cilia length control, cilia disassembly, cell cycle reentry, neural stem cellmaintenance and microtubule regulation [35–40]. In
addition to this well-studied centrosomal role, a role inneuronal migration has been described, downstream of
the neurogenic transcription factor Ascl1/Mash1 [41].
Indeed, knockdown of Cpap in mouse produces a neuronal migration phenotype reminiscent of Dyx1c1 andDcdc2 knockdown phenotypes in rats [3, 6, 41].
It is important to understand the relationship among
dyslexia candidate genes and their interacting partners and their relation to ciliary processes. Based on the
results that, 1) DYX1C1 and DCDC2 are both dyslexia
candidate genes, 2) they are both involved in neuronalmigration and 3) they are both involved in ciliary processes, we asked whether there is a functional relationship between DYX1C1 and DCDC2. Here, we study aPage 2 of 11

possible functional interaction between DYX1C1 and
DCDC2 by using protein-protein interaction, subcellular
localization and perturbation approaches in vitro and a
genetic interaction model in vivo. We show on exogenousand endogenous levels that the CPAP protein interacts
with both DCDC2 and DYX1C1 in human cell models,
supporting further an interacting role of DYX1C1 and
DCDC2. Building on the previous results with loss-offunction experiments in zebrafish [16, 18, 25], we showthat a combined knockdown of dcdc2b and dyx1c1 produces an exacerbated phenotype suggesting a synergistic effect between these two genes. In addition, weshow mutual transcriptional regulation of DYX1C1 and
DCDC2 in the hTERT-RPE1 cell line.
In summary, we demonstrate an interaction between
DYX1C1, DCDC2 and CPAP on the gene and protein
levels.

Results
CPAP interacts with both DCDC2 and DYX1C1 proteins

We have previously observed a protein-protein interaction between DCDC2 and DYX1C1 by pulldown
approaches in SH-SY5Y cells. In addition, we observed aninteraction between CPAP and DYX1C1 by LC/MS-MS
in SH-SY5Y cells [7]. Here, we sought to confirm theinteraction between DYX1C1 and CPAP by co-immunoprecipitation and asked whether DCDC2 also interacts
with CPAP. First, we investigated whether these proteinprotein interactions can be observed endogenously. We
performed immunoprecipitations using HeLa whole cell
extracts. α-tubulin, γ-tubulin and CEP152 were included
as positive controls for interaction with CPAP and
CEP350 as a negative control for interaction with CPAP[35]. Indeed, we observed a protein-protein interaction
between DYX1C1 and CPAP and between DCDC2 and
CPAP on endogenous protein levels (Fig. 1A). As bothDYX1C1 and CPAP have roles in brain development, wenext tested if there is an interaction in 15 day old hiPSCderived brain organoids. Indeed, we observed successful
immunoprecipitation of CPAP using a DYX1C1 antibody
and, reciprocally, pulldown of DYX1C1 using a CPAP
antibody (Fig. 1B).
Next, we asked which protein domains in DYX1C1
are mediating these interactions. We used the stable
doxycycline-inducible cell line hTERT-RPE1-DOXCPAP-GFP, where CPAP is epitope-tagged to GFP. We
transfected doxycycline-induced cells growing in normal
serum conditions with the epitope-tagged expression
constructs DYX1C1-V5 and DCDC2-V5 and used wholecell extracts to perform immunoprecipitations. We foundthat both DYX1C1-V5 and DCDC2-V5 were successfully
pulled down by the GFP-trap (Fig. 1D). The p23 and TPR(tetratricopeptide repeat) domains have previously beenreported to be crucial in protein-protein interactions,Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

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Fig. 1 CPAP interacts with DCDC2 and DYX1C1. (A) CPAP endogenously interacts with DCDC2 and DYX1C1. Endogenous immunoprecipitations using
HeLa cell extracts. CPAP was used as a bait to pull down interactors. CEP350 was used as a negative control and CEP152 was used as a positive control
for interaction with CPAP. Beads alone were used as a negative control for the IP´s. (B) CPAP and DYX1C1 interaction in brain organoids. CPAP was immunoprecipitated by anti- DYX1C1 in 15 day old brain organoids. Reciprocally, DYX1C1 was pulled down by anti-CPAP. (C) Schematic representation of
domain structures of DYX1C1, CPAP and the deletion constructs DYX1C1ΔTPR, DYX1C1Δp23, DYX1C1ΔDYX. p23 = p23 domain, TPR = tetratricopeptide
repeat domain, DYX = DYX domain. (D) CPAP interacts with DYX1C1 via the p23 domain. hTERT-RPE1 cells stably expressing DOX-CPAP-GFP and growing
in normal serum conditions were induced with doxycycline and transiently transfected with the indicated constructs. GFP-Trap was used to pull down
CPAP-GFP. anti-V5 antibody was used for immunodetection of interactors.

while the DYX domain was identified as a novel, highly
conserved domain specific for DYX1C1 [7, 42–45]. We
explored which of the domains in DYX1C1 mediates theinteraction with CPAP by using epitope-tagged expression constructs with deletions in defined domains fortransfection: DYX1C1ΔDYX-V5, DYX1C1ΔTPR-V5,
DYX1C1Δp23-V5 (Fig. 1C). We found that DYX1C1
interaction with CPAP was abrogated upon deletion of
the p23 domain, but not the TPR or the DYX domains.
We concluded that DYX1C1 interacts with CPAP via the
p23 domain (Fig. 1D).
In summary, we confirm the previously reported protein-protein interaction between DYX1C1 and CPAP,
characterize their interacting domain and report a novel
interaction between DCDC2 and CPAP.
Co-occurrence of DYX1C1 and CPAP at the centrosome

Several studies have reported a localization of DYX1C1 at
or around the centrosome [6, 7, 24, 31, 46]. We therefore
hypothesized that DYX1C1 and CPAP might co-occur atthe centrosome. We performed immunofluorescence on
hTERT-RPE1 cells grown in normal serum conditions.

Indeed, we observed a co-occurrence of DYX1C1 and
CPAP at the centrosome (Fig. 2A). DCDC2 has beenreported to dynamically localize to the ciliary axoneme,
the abscission structure (midbody) and the mitotic spindle fibers [18]. DCDC2 does not co-occur with CPAP
during cytokinesis (Fig. 2B). During metaphase, DCDC2
is at the centrosomes, where it co-occurs with CPAP, andat the mitotic spindle (Fig. 2C).
In summary, we observed that DYX1C1 co-occurs
with CPAP at the centrosome, while DCDC2 localization depends on cell cycle stages and may co-occur with
CPAP during certain stages.
Exacerbated ciliary phenotype by double morpholino
knockdown of dyx1c1 and dcdc2b in zebrafish

Our previous in vitro analyses support the interaction
between the two proteins DYX1C1 and DCDC2 [7], (thisstudy). In addition, strong ciliary phenotypes including ventrally curved body axis, hydrocephalus, situs
inversus and kidney cysts have been observed in zebrafish following morpholinobased knockdown of dyx1c1
or dcdc2b [18, 25]. Moreover, a growing body of genetic

Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

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Fig. 2 Co-occurrence of DYX1C1 and CPAP at the subcellular level. A) DYX1C1 and CPAP co-occur around the centrosome. Confocal immunofluorescence images of endogenous protein in hTERT-RPE1 cells grown in normal serum conditions. B)-C) DCDC2 co-occurs with CPAP depending on cell cycle
stage. Nuclei are stained with DRAQ5. Scale bar = 10 μm

and functional evidence suggests digenic interactions
among ciliopathy genes, which can be addressed by using
combined morpholino knockdowns in the zebrafish[47–51]. To investigate whether there is a digenic interaction between dyx1c1 and dcdc2b in vivo, we performed
morpholino coinjection studies in zebrafish embryos
and examined their phenotypes. A subphenotypic doseof 20 µM for dyx1c1 and 100 µM for dcdc2b was chosen
for coinjection experiments as they did not produce any
ciliary phenotype when injected either alone or in combination with 100 µM or 20 µM of stdctlMO, respectively,

into 1-cell stage embryos. Interestingly, when a combined dose of 20 µM of dyx1c1 and 100 µM of dcdc2b was
injected, a dramatic exacerbation of the phenotype wasobserved in 60% of the embryos injected with the morpholinos (Fig. 3). These results suggest a genetic interaction between the two genes in vivo. In summary, weobserved that the two genes dyx1c1 and dcdc2b synergize
to produce an exacerbated ciliary phenotype in zebrafish.

Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

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Fig. 3 Genetic interaction between dyx1c1 and dcdc2b in zebrafish. A,B,C) Injection of 20 µM of dyx1c1MO or 100 µM of dcdc2bMO individually in zebrafish embryos resulted in a maximum number of embryos with straight body axis similar to wildtype embryos at 2dpf. D) Co-injection of the subphenotypic doses of dyx1c1MO and dcdc2bMO (20 µM dyx1c1 + 100µM dcdc2b) resulted in a strong curved body axis in a greater number of embryos at 2dpf.
StdctlMO was used in order to maintain a uniform concentration of the morpholinos. E) Graphical representation of percentage of embryos with strong
curly phenotype following MO injection. Coinjection of dyx1c1 and dcdc2b resulted in a dramatic increase in number of embryos with strong curly body
axis as compared to individual knockdown of the two genes. The number of embryos examined in control and knockdown groups were 100 (n = 100).

Transcriptional regulation between dyx1c1/DYX1C1 anddcdc2 / DCDC2

Next, we asked whether there is transcriptional regulation between dyx1c1/DYX1C1 and dcdc2b/DCDC2. We
knocked down DYX1C1 and DCDC2 in the human ciliated hTERT-RPE1 cell line using siRNA, which efficiently
reduced expression of DYX1C1 and DCDC2 (Fig. 4A,
C). We observed that DCDC2 expression was significantly downregulated upon knockdown of DYX1C1(Fig. 4B). However, overexpression of DYX1C1 did notalter DCDC2 expression in hTERT-RPE1 cells (data notshown). Similarly, knockdown of DCDC2 led to a downregulation of DYX1C1 (Fig. 4D).
We also collected RNA from zebrafish at 1 dpf and 2
dpf after dyx1c1 -morpholino injection into 1-cell stageembryos and performed qRT-PCR analysis to measure
the expression of dcdc2b. dcdc2b expression was notaltered in dyx1c1 morphant embryos compared to control embryos at 1 dpf and at 2 dpf (Fig. 4E). In summary,we observed that dcdc2b expression is unchanged in the
dyx1c1 morpholino injected zebrafish, but that expression of DCDC2 and DYX1C1 are changed upon knockdown of DYX1C1 and DCDC2, respectively, in a humanciliated cell line. Taken together, these results suggestthat there is transcriptional control among DYX1C1 and
DCDC2 in certain cell types indicating that DYX1C1 and
DCDC2 may act in common transcriptional pathways ina tissue-specific manner.

Discussion
In recent years, the detailed molecular functions of
DYX1C1 and DCDC2 have been emerging. Both genes
have been assigned roles in developmental dyslexia, neuronal migration and cilia. Given these overlapping functions, the question arises whether there is a functional
relationship between these two genes.
Here, we show that DYX1C1 and DCDC2 proteins bothinteract with CPAP. We validate the previously suggested

Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

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Fig. 4 Transcriptional control between DYX1C1/dyx1c1 and DCDC2/dcdc2b.A-D). hTERT-RPE1 cells were transfected with siDYX1C1, siDCDC2 or siCtrl, respectively, RNA was extracted and qRT-PCR was run (n = 3 experiments with 3 replicates, mean ± sem). A, C) Knockdown of DYX1C1 and DCDC2 efficiently
reduced the respective transcripts (p-value < 0.0001, t-test). B, D) Expression of DCDC2 and DYX1C1 is reduced after knockdown of siDYX1C1 and siDCDC2,
respectively (p-value < 0.05, t-test). E) dcdc2b expression was unchanged in dyx1c1 morphants: RNA of dyx1c1 morphant zebrafish was collected at 1dpf
and 2dpf after morpholino injection at 1-cell stage. RT-qPCR analysis shows that dcdc2b expression is unchanged in dyx1c1 morphant embryos compared
to control embryos (n = 3, mean ± sem, p-value (1dpf ) = 0.2597, p-value (2dpf ) = 0.4521), t-test). dpf = days post fertilization

protein-protein interaction between CPAP and DYX1C1
by co-immunoprecipitation and identify the p23 domain
as the interaction-mediating domain. This result is consistent with a previous study showing protein-protein
interactions of DYX1C1 mainly via p23 [7]. P23 domains
are known to be important for protein-protein interactions, for example with the chaperone Hsp90 [43–45].
In addition, it is known that DYX1C1 is interacting with
Hsp70 and Hsp90 and has a chaperone function [16, 52,53]. TPR domain containing proteins (TTC proteins)
have been reported to have important roles in cilia functioning in IFT and dynein assembly [54]. A ciliary function of the CCT/TRiC chaperone complex has previously
been described affecting the BBSome [55]. Interestingly,
it has been shown that Dyx1c1 interacts with the chaperones Cct3, Cct4, Cct5 and Cct8 while CCT4, CCT5 and
CCT8 are reportedly localizing to the centrosome [16,55]. It remains to be determined whether the chaperonefunctions and cilia-related functions of DYX1C1 are disparate or functionally related.
Both Dyx1c1 and Dcdc2 have been shown to produce
neuronal migration defects when knocked down in rats
[3, 6]. Similarly, CPAP has been shown to regulate neuronal migration independently of its function at the centrosome, via its microtubule-destabilizing domain PN2-3[41]. CPAP controls, via its PN2-3 domain, ciliary lengthand centriolar and ciliary tubulin assembly and disassembly [39, 56–58]. CPAP promotes cilia disassembly thereby
keeping a neural stem cell pool [35]. Ascl1— a proneuraltranscription factor known for its role in neurogenesis— is a transcriptional regulator of Cpap, while DYX1C1
perturbation affects ASCL1 expression [7, 41, 59]. This
indicates a complex regulatory mechanism of CPAP by
DYX1C1 and ASCL1. FOXP2, a gene often associatedwith speech and language disorders, affects DCDC2
expression in SH-SY5Y cells providing a further link
between language-related gene expression and DCDC2[60]. Interestingly, DYX1C1 is highly upregulated in differentiating human neurons [32]. Further studies inhuman stem cell derived neurons might shed more lighton the role of these genes in neuronal migration.
Both DYX1C1 and DCDC2 have been reported as
cytoskeletal interactors [7, 61]. DYX1C1 associates withmicrotubule proteins and DCDC2 has a role in microtubule stabilization [7, 61]. DCDC2 localizes to primary cilia and is involved in ciliary signaling [18, 62].
Some studies report a physical localization of DYX1C1
at or around the basal body [6, 7, 31, 46]. It might be a
dynamic process, shuttling between the cytoplasm andtemporal accumulation around the basal body. DCDC2

Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

and DYX1C1 do not co-occur at the cilium and ourimmunoprecipitations were carried out on non-starved,
cycling cells both suggesting that their combined action
may take place in the cytoplasm, consistent with in situ
proximity ligation assay (PLA) data [7]. Future studies
using PLA might pinpoint the subcellular localization of
their interaction with CPAP.
Genetic interaction studies have been successfully used
to study cellular pathways in model systems, for example
zebrafish [63]. They can provide useful information aboutprotein-protein complexes, downstream pathways andparallel pathways. This approach has been used to dissectinteractions of ciliary genes – for example, the genetic
interactions of BBS and PCD genes have been modelledin zebrafish [47–50]. Exacerbation of a ciliary phenotype
can take place among physically interacting and among
non-interacting proteins. For example, concomitant lossof bbs7 and bbs1 – members of the BBsome protein complex – and of dnah6 with dnai1 and dnah5 - where no
physical interaction takes place – produces an aggravatedphenotype [48, 49]. Previous studies have reported a ciliary phenotype in dyx1c1 and dcdc2 morphant zebrafish[18, 25]. Here, we show an exacerbated phenotype indouble dyx1c1 and dcdc2b morpholino injected zebrafish. The tissue expression specificity as well as the subcellular localization might overlap only partially raising thequestion whether dyx1c1 and dcdc2 might act in parallel pathways thereby aggravating the ciliary phenotype.
Future studies should address whether dyx1c1 morphants
can be rescued by dcdc2b overexpression and vice-versa.
We show a mutual transcriptional regulation between
DYX1C1 and DCDC2 in the human ciliated RPE1 cellline, but not in the morphant zebrafish. This might bedue to dilution of the effect in the whole organism ascompared to an isolated cell type. Transcriptional control
likely is indirect as there is no evidence that either proteins would act as transcription factors. The regulation of
DCDC2 by DYX1C1 has not been reported previously [7,62, 64]. This might point to a cell-specific or cilia-specificeffect, which could be further studied using starved andnon-starved cells as well as different non-ciliated and ciliated cell lines.
Our study stands out in applying many different model
systems, from different cell lines to brain organoids tozebrafish. We are the first to address a possible functional
link between DYX1C1, DCDC2 and CPAP by combining
different experimental readouts. However, some limitations need to be considered: the DCDC2-CPAP interaction needs further dissection to determine the interacting
domain of DCDC2. In the zebrafish, a rescue experiment would be needed in order to strengthen the case.
In the cell models, cellular localization upon perturbation should be studied. Transcriptional regulation of
DYX1C1 and DCDC2 will have to be confirmed by rescue

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experiments via overexpression of knockdown-resistant
DYX1C1. These are some experiments that may help tofurther strengthen the notion that DYX1C1, DCDC2 and
CPAP act via a common pathway.

Conclusions
In conclusion, we report genetic- and protein interactions among DYX1C1, DCDC2 and CPAP. We thereby
contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2. Recently, studies combining patient cohorts, cellular models, zebrafish andmouse behavior assays have elegantly demonstrated thelink between ciliary and centrosomal defects and neuropsychiatric disorders such as schizophrenia [14, 15].
Future studies should make use of a combination ofgenetic, behavioral and molecular approaches in order toprovide a more integrative picture of the roles of ciliary
dyslexia candidate genes in homeostasis and disease.
Methods
Zebrafish maintenanceWild-type zebrafish were obtained from the zebrafish
core facility at Karolinska Institutet and were reared andmaintained as described previously [65]. Briefly, zebrafish were maintained at a temperature of 28oC and were
kept under a 14:10 h light:dark cycle. They were fed
daily with fish flakes and live brine shrimp. For breeding,
one female and one male AB strain fish were placed ina breeding tank and were separated by a divider. Whenthe lights turned on the following morning, the divider
was removed to allow the fish to breed. Embryos thatwere obtained were collected and transferred to a petri
dish containing E3 medium (5 mM NaCl, 0.17 mM KCl,
0.33 mM CaCl2, and 0.33 mM MgSO4). Methylene blue
(0.1%) was added to the medium to prevent fungal contamination. Embryos were maintained at 28 °C until theyreached the desired developmental stage. Developmental
staging is shown as days post fertilization (dpf ).
Zebrafish embryos and microinjections of morpholinos
and mRNA

Morpholinos against zebrafish dyx1c1 and dcdc2b were
designed as described previously [18, 25]. The following
morpholinos (MO) were purchased from Gene Tools,
LLC. dyx1c1 AUGMO: 5’-GTGATCTCTCACTATCAGCGGCATC-3’, dyx1c1 spliceMO: 5’-TGACAGTCAACATGTCTTACCGATG-3’,
dcdc2b
AUGMO:
5’-CCGGTGGATGCCATGACTTTTCAGT-3’
andstandard control morpholino (stdctlMO): 5’-CCTCTTACCTCAGTTACAATTTATA-3’. Individual knockdown of dyx1c1 and dcdc2b genes have been reported
previously [18, 25]. Wildtype AB embryos of 1-cell stage
were placed in furrows on a 2% agarose plate and a subphenotypic dose of 20 µM of dyx1c1 together with 100

Bieder et al. BMC Molecular and Cell Biology(2023) 24:20µM of dcdc2b was injected into them to study the synergistic effect of the two genes. As controls, a combineddose of 20 µM of dyx1c1 plus 100 µM of stdctl or 100µM of dcdc2b plus 20 µM of stdctl was injected into
1-cell stage embryos in order to keep the final concentration at 120 µM. After injection, the embryos were transferred to petri dishes with E3 medium and raised in an
incubator maintained at 28oC until they were ready to be
imaged. Dead and uninjected embryos were discardedand E3 medium was changed daily. Two days post fertilized embryos were examined for phenotype scoring
and later anesthetized with tricaine (MS-222, 40 µg/ml)
for imaging. Wild-type embryos were imaged and used
as uninjected controls. After imaging all embryos wereeuthanized using an overdose of MS-222.
Cell culture

The human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1, ATCC, CRL-4000™)
was cultured in DMEM/F12, 10% fetal bovine serum(FBS), 100 U/ml penicillin and 100 µg/ml streptomycin,
0.01 mg/ml hygromycin B at 5% CO2. To induce ciliogenesis, cells were starved using OptiMEM reduced serum
medium for 24 h unless indicated otherwise. The doxycycline-inducible CPAP-GFP-hTERT-RPE1 cell line wasdescribed previously and cultured as described above[39]. CPAP expression was induced using 2 µg/ml of
doxycycline for 2–12 h. CPAP-GFP expression was verified on an inverted fluorescence microscope (Olympus).
Transient transfections were done using lipofectamine
2000 (Thermo Fisher Scientific). HeLa cells were cultured
in a DMEM medium containing 10% FBS, 0.1 mM MEM
non-essential amino acids (NAA), 100 µg/ml streptomycin, 100U/ml penicillin (Life Technologies GmbH, Darmstadt, Germany). hiPSC-derived brain organoids were
cultured as described previously [35] and were differentiated for 15 days.
Expression constructs

Cloning and validation of the expression constructs
DYX1C1-V5,
DCDC2-V5,
DYX1C1ΔDYX-V5,
DYX1C1ΔTPR-V5, DYX1C1Δp23-V5 in pcDNA3.1/
V5-His-TOPO vector were described previously [7, 62,
66, 67].
siRNA knockdown

hTERT-RPE1 cells were seeded in six-well plates andgrown to 80% confluency. Cells were transfected at a final
concentration of 25 nM of each siGENOME SMARTpool
targeting DYX1C1 (Cat. Nr. M-015300-02) or DCDC2(Cat. Nr. M-020868-01) and non-targeting control (Cat.
Nr. D-001206-14-20) (all Thermo Scientific, Dharmacon) using Lipofectamine 2000 (Thermo Fisher Scientific). After 24 h, hTERT-RPE1 cells were serum-starved

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for 24 h and harvested. RNA was prepared using the
NucleoSpin RNA kit (Macherey-Nagel) according to the
manufacturer’s instructions. All the experiments were
performed in triplicates and were repeated independently three times.
Quantitative real-time PCR (qRT-PCR)

cDNA was synthesized with Maxima Kit (Thermo scientific) using 500 ng of RNA. qRT-PCR was analyzed with
cDNA diluted 1:5 using Taqman expression assays andTaqMan fast Universal PCR Master Mix (Life Technologies, 4,352,042) (DYX1C1: Hs00370049_m1; DCDC2:
Hs00393203_m1; HPRT1: Hs02800695_m1). HPRT1
was used as an internal control to normalize expression
levels. For zebrafish, SYBR Green primers were used
(dcdc2b: F:ATG ACA CGT CAG CTC CAC AG; R: TGG
AAT GGT GTG ACT CGC TC; β-actin, F-5′-TGC CCC
TCG TGC TGT TTT-3′, R- 5′-TCT GTC CCA TGCCAA CCA T-3′) and FastStart Universal SYBR Green
Master Mix (04913850001, Roche). The reactions were
amplified using the 7500 Fast Real-Time PCR system(Applied Biosystems). The ΔΔCt method was used to calculate relative expression levels and shown as fold change(2−∆∆Ct)[68]. Student’s t-test was used for statistical analysis between conditions using GraphPad Prism 7 software.
Immunoprecipitations

Figure 1 A: The endogenous immunoprecipitations were
performed with the mouse monoclonal anti-CPAP antibody [39], coated beads or IgG control beads. Protein G
beads were coated with anti-CPAP antibodies overnightat 4 °C, mixed with HeLa cell extracts, and incubated at
4 °C for 4 h. Cell extracts were prepared in a buffer containing 80 mM BRB, 100 mM KCl, 1 mM MgCl2, 1 mM
EGTA, protease inhibitor cocktails, and 1 mM PMSF. The
extracts were centrifuged at 100,000 g, and the supernatant was collected. These high-speed extracts were thenused for immunopurification. The protein-bound beads
were washed with BRB buffer containing 0.1% Triton
X-100 and 100 mM NaCl and washed twice with highsalt
buffer containing 500 mM salt. After a final wash withbuffer containing 100 mM NaCl, the samples were eluted
using Laemmli buffer for Western blot analyses. Figure 1B: Brain organoid samples were immediately lysed in
50 mM Tris–HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA
(pH 8.0), 1% Triton X-100, 0.01% Igepal and proteaseinhibitors (Roche Applied Science Cat #11 873 580 001).
Subsequently, samples were cleared of debris and DNA
by centrifugation (12 000 × g at 4 °C for 10 min). Protein
concentrations were determined by Bradford assay (RothGmbH Cat# K015.1) according to manufacturer’s instructions. For immunoprecipitation experiments, 2500 µg of
total protein were incubated overnight at 4 °C with protein G Dynabeads (Invitrogen Cat# 10004D) coupled toBieder et al. BMC Molecular and Cell Biology(2023) 24:20

CPAP (Hybridoma C44) or DYX1C1 (Proteintech, Cat#
14522-1-AP) antibody. After five washes in lysis buffer
containing 150 mM NaCl, bound proteins were eluted
with 0.1 M glycine–HCl (pH 3.0) for 5 min followed byneutralization with 0.5 M Tris–HCl (pH 7.8) and 1.5 M
NaCl. Figure 1D: Immunoprecipitations were performed
using GFP-trap. Cells were first lysed using 200 µl icecold Lysis buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl;
0.5 mM EDTA; 0.5% NP-40) containing protease inhibitors (Roche). Beads were equilibrated by adding 25 µl ofbead slurry into 500 µl ice-cold buffer (10 mM Tris/Cl pH
7.5; 150 mM NaCl; 0.5 mM EDTA). Proteins were boundby adding the diluted lysate to the equilibrated GFP-trap
beads, and washed 3 times with 500 µl of ice-cold dilution buffer. The experiments were repeated at least threetimes; a representative example is shown.
SDS-PAGE and Western blotting

Figure 1 A: Protein samples were resolved in 8% or
12% acrylamide gels and transferred to nitrocellulose
membranes. After incubating with primary antibodies
overnight at 4 °C, the blots were treated with secondary antibodies at RT for 1 h. Super Signal West Pico or
Femto Chemiluminescent substrates (Pierce) were used
for detection. Antibodies for Western blots: monoclonal mouse anti-CPAP (1:50), mouse anti-γ-tubulin(1:3,000; Sigma-Aldrich), mouse anti-α-tubulin (1:3,000;
Sigma), rabbit anti-Cep152 (E. Nigg), anti-Cep350, antiDYX1C1, anti-DCDC2, and peroxidase-conjugatedsecondary antibodies (1:3,000; Life Technologies). Figure 1B: Samples were resolved by 10% SDS-PAGE. Target proteins were detected by immunoblotting withprimary antibodies: mouse anti-CPAP (1:50, Hybridoma
C44), rabbit anti-DYX1C1 (1:1.000, Proteintech Cat#
14522-1-AP) and mouse anti-GAPDH (1:10.000, Proteintech Cat# 60,004-I-Ig) followed by incubation withthe corresponding goat-anti-mouse (1:5000, Thermo
Fisher Scientific Cat#626,520) and donkey anti-rabbit(1:5000, Thermo Fisher Scientific Cat#A16023) HRPcoupled secondary antibodies. Figure 1D: Cells werelysed in NP40 cell lysis buffer (Thermo Fisher Scientific)
containing protease inhibitors cocktail (Complete Mini,
Roche, #11,836,153,001). The samples were then passed
several times through a 26-gauge needle, placed on ice
for 10 min and centrifuged in a pre-cooled microcentrifuge. Protein concentrations were determined using
the Quant-iT Protein Assay Kit and Qubit fluorometer(Thermo Fisher Scientific). Gel electrophoresis was performed using a 4–12% gradient gel (Invitrogen Bis-Tris
NuPAGE gels) and proteins were transferred to a Amersham Hybond-P nylon membrane (GE healthcare). The
transfer was done with a semi-dry transfer apparatus(Biorad) at 20 V for 1 h using transfer buffer (25mM Tris,
190 mM glycine, 20% MeOH, 0,05% SDS). The blot wasPage 9 of 11

then incubated overnight at 4 °C with blocking buffer(5% non-fat dry milk, 10 mM Tris pH 7.5, 100 mM NaCl,
0.1% Tween 20 PBS). Probing was done using anti-V5HRP antibody (Invitrogen, 1:8000) overnight at 4oC and asecondary anti-HRP antibody (Invitrogen, 1:106). Detection was performed with Luminol Enhancer Solution andPeroxide Solution (Thermo Fisher Scientific) for 5 min.
ImmunofluorescencehTERT-RPE1 cells were seeded on coverslips, grown
to 80% confluency and fixed. (For ciliary stainings,
cells additionally were serum-starved for 24 h using
OptiMEM reduced serum medium before fixation). Cells
were incubated on ice for 45 min, then fixed in a 50:50
solution of 4% formaldehyde (Thermo Fisher Scientific,
Prod. Nr. 28,906) and methanol or in pure methanol for
15 min at -20 °C. The fixed cells were blocked and permeabilized in 5% horse serum (Life Technologies), 0.05%
PBS-Tween for 1 h at room temperature and incubatedo/n at 4 °C with a primary antibody (rabbit anti-DYX1C1,
1:500, Sigma, SAB4200128[16]; rabbit anti-DCDC2,
1:200, Abcam, ab157186[18]; mouse anti-CPAP (Gopalakrishnan lab), 1:20; mouse anti-acetylated tubulin,
1:5000, Sigma T7451; mouse anti-gamma-tubulin, 1:500,
Abcam, ab11316). Antibodies have been previously validated for specificity [16, 18, 35]. Cells were incubated for
1 h at room temperature with secondary antibodies (donkey anti-rabbit 568, Invitrogen, A10042; donkey antimouse 568, Invitrogen, A10037). Nuclei were stainedwith DRAQ5 (Cell Signaling Technology, Cambridge,UK) at 1:1000 for 10 min at room temperature and coverslips were mounted in Prolong Gold antifade reagent(Invitrogen). Images were acquired on a A1R confocal(Nikon Instruments, Inc.) using NIS elements software.
Brightness and contrast were adjusted using the NIS elements software and the far-red channel was pseudo colored in blue.
List of abbreviations
DCDC2	Doublecortin domain containing 2 geneDD	Developmental dyslexia
DYX1C1/DNAAF4	Dyslexia 1 candidate 1 gene/ Dynein axonemal
assembly factor 4
hTERT-RPE1	human retinal pigmented epithelial cell line
immortalized with hTERTCENPJ/CPAP	Centrosomal protein J/ Centrosomal-P4.1-associatedprotein
PCD	primary ciliary dyskinesia
PLA	in situ proximity ligation assay
NPHP-RC	nephronophthisis-related ciliopathies
BBS	Bardet-Biedl-Syndrome
TPR	tetratricopeptide repeat domain

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00483-4.

Bieder et al. BMC Molecular and Cell Biology(2023) 24:20

Supplementary Material 1
Acknowledgements
We thank Daniel Peñaranda Garcia for help with laboratory work. We thankthe zebrafish core facility at Karolinska Institutet for their help in providing
embryos.
Authors’ contributions
ITP, JK and GC conceived the study. AB, GC, AW, SE and ITP designed andperformed experiments and analysed the data. AB wrote the manuscript. JG
provided expertise to the study. All authors edited and approved the final
manuscript.
Funding
This study was supported by the Swedish Research Council (VR) and theSwedish Brain Foundation (Hjärnfonden). JK is a recipient of The Royal Society
Wolfson Research Merit Award. This study was in part performed at the Live
Cell Imaging unit/Nikon Center of Excellence, Department of Biosciences andNutrition, Karolinska Institutet, Huddinge, Sweden, supported by grants fromthe Knut and Alice Wallenberg Foundation, the Swedish Research Council, theCentre for Innovative Medicine and the Jonasson donation to the School ofTechnology and Health, Kungliga Tekniska Högskolan, Huddinge, Sweden.
Open access funding provided by Karolinska Institute.
Data Availability
Not applicable.

Page 10 of 11

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Declarations
Ethics approval and consent to participate
All zebrafish experiments were carried out in accordance with ethical permits
approved by the relevant ethical committee (Stockholm North Experimental
Animal Committee Dnr N29-12, Dnr N122-15).
Consent for publication
Not applicable.
Competing interests
The authors declare no conflict of interest.

19.

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Received: 17 November 2022 / Accepted: 9 May 2023
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