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Int J Dev Biol
2017 Jan 01;613-4-5:267-276. doi: 10.1387/ijdb.160442yc.
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Roles of the cilium-associated gene CCDC11 in left-right patterning and in laterality disorders in humans.
Gur M
,
Cohen EB
,
Genin O
,
Fainsod A
,
Perles Z
,
Cinnamon Y
.
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Axial determination occurs during early stages of embryogenesis. Flaws in laterality patterning result in abnormal positioning of visceral organs, as manifested in heterotaxy syndrome, or complete left-right inversion as in situs inversus totalis. These malformations are often associated with ciliopathies, as seen in primary ciliary dyskinesia. We have recently described a novel mutation in the Coiled-Coil Domain-Containing 11 (CCDC11) gene associated with laterality disorders in a consanguineous family of Arab-Muslim origin with two affected siblings presenting with diverse phenotypes, one with heterotaxy syndrome and the other with non-primary ciliary dyskinesia situs inversus totalis. This study further characterizes the roles of CCDC11 and the implications of the identified mutation on left-right axial patterning in patient-derived cells and in the frog embryo as a model organism. We analyzed patient-derived cells and manipulated Ccdc11 levels in Xenopus laevis frog embryos. Cilia length in patient cells was longer than in controls, and CCDC11 was localized to the centriole and the actin cytoskeleton. Mutated truncated protein accumulated and was also localized to the centriole and actin cytoskeleton. In frog embryos, Ccdc11 was regulated downstream of FoxJ1, and overexpression of the full-length or truncated protein, or downregulation of the gene resulted in severe disruption of embryonic left-right axial patterning. Taken together, our initial description of the deleterious mutation in CCDC11 in patients, the current results and more recent supportive studies highlight the important role of CCDC11 in axial patterning.
Fig. 1. Ciliogenesis analysis of patient-derived monociliated skinfibroblast cells and subcellular localization of CCDC11. (A,B) Immunostaining
with anti-ARL13B antibody (red) and DAPI nuclear staining (blue). Representative images of cilia showing longer cilia in patient’s fibroblasts. (C) Cilium
length was measured and compared between groups. Average cilium length in patient’s cells was significantly longer (by 0.3 mm; P < 0.01). (D,F)
Control and (E,G) patient cells were stained with rhodamine phalloidin (red), anti-CCDC11 antibody (green) and DAPI (blue). While in control cells, only
a minute amount of CCDC11 was localized to the actin cytoskeleton (D), in the patient cells, where truncated protein accumulates, the protein was
widely localized to the stress fibers (E,G). Both WT and mutated proteins were also localized to the centriole (arrowheads in F and G).
Fig. 2. Spatiotemporal expression
of Ccdc11 in Xenopus laevis and its
regulation by FoxJ1. To determine the
temporal expression pattern of xCcdc11,
embryos were collected at different
developmental stages from blastula
to early tailbud and level of expression
was determined by qPCR (A). Temporal
expression shows that xCcdc11 starts to
be expressed at gastrulation (St. 10.5), increases
during neurulation and continues
to accumulate through development. (B)
Spatial expression of xCcdc11 by wholemount
in-situ hybridization shows that at
late gastrulation, xCcdc11 is expressed
in the dorsal lip of the blastopore (St.
12). (C) At neurulation (St. 18) and (D)
at the tailbud stage (St. 28), embryo
staining shows a scattered pattern of
ectodermal layer, stereotypic to ciliated
cells. (E) Section of neurula embryo
shows expression at the GRP (St. 17).
IHC staining with anti-CCDC11 antibody
shows similar expression patterns in the
ectodermal layer in (F) late neurula (St.
20) and (G) tailbud (St. 30). Breakage of
bilateral symmetry in frogs depends on
the development of ciliated epithelium
at the GRP, emerging from the dorsal lip
of the blastopore. The qPCR and in-situ
hybridization assays gave a spatiotemporal
pattern of xCcdc11 expression
that suggests a role in ciliogenesis
and in axial patterning. (H,I) xCcdc11
expression is regulated by xFoxj1. The
Forkhead transcription factor Foxj1 is
a master regulator gene of motile cilia.
To test its regulatory effect on xCcdc11
expression, embryos were injected
at 2- to 4-cell stage with xFoxj1 RNA.
For in-situ hybridization analysis, embryos were injected unilaterally with xFoxj1 RNA together with FITC, as lineage tracer, and collected at St. 12.5.
In-situ hybridization staining shows that on the injected side (right, marked by blue staining), xCcdc11 levels are dramatically upregulated compare to
the uninjected side, which serves as an internal control (H). For quantitative analysis, embryos were injected radially and total RNA was extracted at
St. 9, 10 and 13. qPCR shows that similar to other genes expressed in the GRP and critical for cilia motility (xTekt2 and xDnah9), xCcdc11 expression
increases with xFoxj1 overexpression, but is not sufficient to induce ectopic expression prior to gastrulation (I). Both qPCR and in-situ hybridization
analyses indicate that the master regulator of motile cilia, FOXJ1, positively regulates Ccdc11 expression, suggesting a role for CCDC11 in ciliogenesis.
Fig. 3. Misexpression of xCcdc11 impairs L–R patterning. To study the role of CCDC11 in
frogs, we knocked down its expression using xCcdc11–MO. Embryos were injected at the 2- to
4-cell stage and analyzed for xCoco expression by whole-mount in-situ hybridization on St. 20
GRPs. Embryos injected with control–MO or uninjected embryos served as controls. In normal
development, xCoco expression on the left is inhibited (blue arrowhead in A), while it continues
to be expressed on the right (A). When leftward flow is defective, xCoco expression may be either
uniform, reflecting no inhibition at all (B), or reduced on the right side (blue arrowhead in
C). Analysis of embryos injected with xCcdc11–MO revealed disruption in the proper inhibition of
xCoco expression. While only 25% of the embryos injected with control–MO exhibited deviation from
normal expression, 49% of xCcdc11–MO-injected embryos showed abnormal expression (P < 0.05). To verify that the laterality disorders induced by xCcdc11 knockdown persist with time, we manipulated xCcdc11 levels and evaluated L–R patterning by in-situ hybridization staining for xPitx2 during the tailbud stages (blue arrowheads in E–H). xPitx2 is normally expressed unilaterally on the left, thus it serves as a reliable marker for laterality (E). Laterality defects may result in either bilateral (F), completely absent (G) or right expression (H) of xPitx2. Analysis of xCcdc11 downregulation by xCcdc11–MO injections resulted in laterality defects, as exhibited by abnormal expression of xPitx2 ( I, P < 0.001). The ambiguous phenotype obtained in xCcdc11 knockdown mimics the diverse phenotype between the two siblings carrying the mutation, and is often observed when GRP flow is disrupted. Overexpression of xCcdc11 also resulted in abnormal expression of xPitx2 (J, P < 0.01), indicating the need for tight regulation of CCDC11 levels for proper L–R patterning. The efficiency xCcdc11–MO in blocking translation of xCcdc11 mRNA was assessed by western blot analysis of total proteins extracted from embryos injected with xCcdc11–MO, uninjected embryos, and embryos injected with control–MO. Analysis with anti-CCDC11 antibody revealed that xCcdc11–MO inhibits xCcdc11 expression by 84% compared to control–MO-injected embryos. Ponceau S total protein staining of the same membrane was used to validate equal loading in all lanes (K).
Fig. 4. Ccdc11 overexpression impairs L–R patterning.
The accumulation of truncated CCDC11 in
skin fibroblast and nasal epithelial cells from the ISIT
sibling implies tight regulation and suggests that
high levels of CCDC11 might interfere with normal
ciliary function. To test this hypothesis, embryos
were injected with mRNA encoding either the WT
or the truncated form of Ccdc11 of both human
and frog orthologs (hCcdc11, thCcdc11, xCcdc11
and txCcdc11 respectively). Embryos injected with
mRNA for GFP and xFoxj1 served as controls.
To verify dorsal injection, mRNA encoding GFP
was added and embryos were screened at early
neurula stages. Embryos were collected at St. 20
and laterality was assessed by in-situ hybridization
for xCoco expression (as shown in Fig. 3). All four
forms of Ccdc11 overexpression resulted in abnormal xCoco expression, as with xFoxj1 overexpression. The disruption of proper axial patterning following
CCDC11 misexpression indicates an essential role for CCDC11 in L–R patterning, and the prominence of controlled levels of expression.
cfap53 (cilia and flagella associated protein 53) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, vegetal view, dorsal up.
