Prostaglandin signalling regulates ciliogenesis by modulating intraflagellar transport

ABSTRACT Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation

remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish _leakytail_ (_lkt_)

mutants show ciliogenesis defects, and the _lkt_ locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2

(PGE2), a function that is abrogated by the Lkt/ABCC4T804M mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the

cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes

ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP

synthesis and increase anterograde IFT, thereby promoting ciliogenesis. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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* Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS CILIARY TRANSITION ZONE PROTEINS COORDINATE CILIARY PROTEIN

COMPOSITION AND ECTOSOME SHEDDING Article Open access 09 July 2022 A NOVEL ROLE FOR THE CHLORIDE INTRACELLULAR CHANNEL PROTEIN CLIC5 IN CILIARY FUNCTION Article Open access 17 October 2023

MUCOCILIARY WNT SIGNALING PROMOTES CILIA BIOGENESIS AND BEATING Article Open access 06 March 2023 ACCESSION CODES ACCESSIONS GENBANK/EMBL/DDBJ * EU586042 * EU586042.1 REFERENCES * Smith, W.

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Download references ACKNOWLEDGEMENTS We acknowledge P. Yuanyuan and J. Guan for assistance in fish care, X. Zhu for hRPE1 cells, J. Shah for IFT88–EYFP cells, C. Yi for cAMP assays, Q. Li

for diagram drawing and L. Cai and G. Zhu for spin-disk confocal microscopy analysis. We are grateful to H. Ma, B. Appel, Z. Sun, J. Gamse and members of our laboratories for comments on the

manuscript and helpful discussions. This research was supported in part by grants from the National Basic Research Program of China (CMST2013CB945301, CMST2012CB944501; T.P.Z.), National

Natural Science Foundation of China (NSFC31172173; T.P.Z.) and Shanghai Pujiang Program (11PJ1401600; T.P.Z.) as well as the National Institute of Health of America (T.P.Z., J.D.S., J.M.,

I.A.D.) and Canadian Institutes of Health Research (MOP106513, S.P.C.C.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Genetics, State Key Laboratory of Genetic Engineering,

Fudan University School of Life Sciences, Shanghai 200433, China Daqing Jin, Jianjian Sun, Guangju Yu, Wenyan Li & Tao P. Zhong * Department of Medicine, Vanderbilt University School of

Medicine, Tennessee 37232, USA Terri T. Ni, Haiyan Wan & Tao P. Zhong * Department of Cell & Developmental Biology, State University of New York Upstate Medical University, New York

13210, USA Jeffrey D. Amack * Department of Cell & Developmental Biology, Vanderbilt University School of Medicine, Tennessee 37232, USA Jonathan Fleming & Chin Chiang * MRC Centre

for Developmental and Biomedical Genetics, The University of Sheffield, Sheffield S10 2TN, UK Anna Papierniak & Jarema Malicki * Department of Pharmaceutical Science, St Jude Children’s

Research Hospital, Tennessee 38163, USA Satish Cheepala & John D. Schuetz * Division of Cancer Biology and Genetics, Queen’s University, Kingston, Ontario K7L 3N6, Canada Gwenaëlle

Conseil & Susan P. C. Cole * Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai 200031, China Bin Zhou * Department of Medicine, Massachusetts General Hospital,

Harvard Medical School, Massachusetts 02148, USA Iain A. Drummond Authors * Daqing Jin View author publications You can also search for this author inPubMed Google Scholar * Terri T. Ni View

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Google Scholar CONTRIBUTIONS T.P.Z. conceived and directed the project. T.T.N. and H.W. initiated the project and discovered the _lkt_ gene product as ABCC4. D.J. carried out most

experiments and discovered the roles of COX, ABCC4 and EP4 in ciliogenesis. J.S. and G.Y. conducted cell culture experiments. J.D.A. carried out KV flow experiments. S.C. and J.D.S.

conducted PGE2 efflux experiments. J.F., C.C. and B.Z. carried out some of the cell culture experiments. G.C. and S.P.C.C. carried out vesicular transport assays. W.L. conducted double _in

situ_ hybridization. A.P. and J.M. tested roles of PGE2 in _ift_ mutants and were involved in early mutant analyses. I.A.D. carried out histology and provided reagents. T.P.Z., D.J. and J.M.

prepared figures and wrote the paper. CORRESPONDING AUTHOR Correspondence to Tao P. Zhong. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 _LKT/ABCC4_-DEFICIENT EMBRYOS DISPLAY RANDOMIZATION OF ORGAN LATERALITY. (A) _cmlc2_ expression showing the laterality

(left-jagging versus right-jagging) of heart tube in wild-type (wt) embryos and _lkt_ mutants. (B) _foxA3_ expression depicting the sidedness of liver (black arrow) and pancreas (white

arrow) in wt embryos and _lkt_ mutants. (C) _insulin_ expression displaying the pancreas laterality in wt embryos and _lkt_ mutants. (D) _lft1_ expression showing brain laterality in wt

embryos and _lkt_ mutants. (E,F) Acetylated tubulin immunostaining reveals normal pronepheric cilia development in _lkt_ mutants compared with wild-type embryos. Scale bar: 100 μm (A–F); 10

μm (E,F). (G,H) Analysis of sidedness of asymmetric gene expression in wt embryos, _lkt_ mutants and _lkt/abcc4_ morphant embryos. The total numbers of embryos analysed pooled from n = 3

independent experiments are indicated on the top of each bar. The number of embryos with sided expression of each marker divided by total number of embryos. The percentages of embryos with

LR defects were significantly higher in _lkt_ mutants or _abcc4_ morphants as compared with wt embryos, but not statistically different among _lkt_ mutants or _abcc4_ morphant embryos (Yates

corrected Chi-square test, ∗∗∗_P_ < 0.0001, one degree of freedom). Statistics source data for Supplementary Fig. 1g, h can be found in Supplementary Table 2. SUPPLEMENTARY FIGURE 2

ANNOTATED ALIGNMENT OF DEDUCED FULL-LENGTH ZEBRAFISH LKT/ABCC4 PROTEIN WITH ORTHOLOGS IN OTHER SPECIES. Black: identical across four species. Grey: identical in 2 or more species or

conservative amino acid substitution. IDs: human (NP005836), mouse (NP001028508), chicken (NP001025990) and zebrafish (EU586042). TM: transmembrane domain. NBD: nucleotide binding domain.

Red star: _leakytail_ zebrafish mutation site (T804M). Walker A/B (ATP-binding sites) and signature C are conserved motifs found within the NBD of ABC transporters. SUPPLEMENTARY FIGURE 3

INJECTION OF _LKT/ABCC4_ SPLICING MORPHOLINOS PHENOCOPIES _LKT_ MUTANTS. (A) Schematic diagram depicting zebrafish _lkt/abcc4_ gene structure, including exon 9, 10, 11, and intron 9, 10. Red

bar: _abcc4_ morpholinos target site. Black arrows: primer sites for RT-PCR. (B) RT-PCR analyses showed that wild-type _lkt/abcc4_ transcripts contained fused exons 9, 10 and 11, whereas

mis-spliced transcripts contained only fused exons 9 and 11 in embryos injected with different doses of _lkt/abcc4_-MO. Total RNAs were isolated from morphants and controls, and subjected to

RT-PCR using primers located in exon 9 and 11. (C–E) Acetylated tubulin immunostaining showing a reduction of KV cilia (C), a loss of OV short cilia (D) and lack of some ear kinocilia in

_lkt/abcc4_ morphants (E), similar to those of _lkt_ mutants. (F–H) _lkt/abcc4_ morphants showing a curved body axis (F; 81%; n = 123), hydrocephalus (F; 78%, n = 123; red arrow), three

otoliths (G; 80%; n = 123; red arrows) and reversed (left)-looped heart (H; 41%; n = 93). (I,J) In situ hybridization showing expression of _abcc4_ (E; arrowhead) and _insulin_, a pancreas

marker (F; arrowhead). (K) Double in situ hybridization with _abcc4_ and _insulin_ probes showing expression of both genes in the pancreas (arrowhead). Inset displays a high-magnification

image of _abcc4_ and _insulin_ expression patterns, which largely overlap. (L,M) In situ hybridization showing expression of _abcc4_ (L; arrowhead) and _wt1b_, a glomerulus marker (M;

arrows). (N) Double in situ with _abcc4_ (arrowhead) and _wt1b_ (arrows) probes showing expression in the pancreas and glomerulus, respectively. Inset shows an enlarged view of _abcc4_ and

_wt1b_ expression. Double in situ hybridization analyses were conducted using digoxingenin-labeled _lkt_ antisense probe (I,K,L,N). _insulin_ (J,K) and _wt1b_ antisense probes (M,N) were

fluorescein-labeled. Scale bar: 10 μm (C–E); 100 μm (F–H; I–N). SUPPLEMENTARY FIGURE 4 ECTOPIC EXPRESSION OF _ABCC4_ MRNA RESCUES _LKT_ MUTANT PHENOTYPES. (A–D) _lkt_ mutants displaying a

ventrally curved axis (A), three otoliths (B; red arrows), hydrocephalus (C; red arrow) and reversed (left)-looped heart (D). (E–H) _abcc4_ mRNA injection rescued mutant phenotypes,

including a straight body axis (E), two otoliths (F), normal brain (G) and normal (right)-looped heart (H). Scale bar: 100 μm (A–H). (I) Percentages of rescued _lkt_ mutants grouped in heart

looping, otolith biogenesis, axis formation or brain development. Approximately 160 pg of _abcc4_ mRNA was injected into embryos derived from crosses of _lkt_ heterozygouts. Injected

embryos were genotyped using SSLP markers (Z17212 and Z6907). Embryos linking with the _lkt_ mutant genotype but showing wild-type phenotypes were scored as rescued mutants. %: The number of

rescued _lkt_ mutants divided by number of all _lkt_ mutants. The total numbers of embryos pooled from n = 3 independent experiments are indicated. Graph shows mean ± s.d.; Student’s t

test: ∗∗∗_P_ < 0.001. (J) Bar graph depicting percentages of normal (rescued) brain phenotypes in embryos co-injected with control morpholinos, antisense morpholinos and antisense

morpholinos with specific mRNAs. Wild-type embryos were co-injected with _cox1_-MO (8 ng) with _cox1_ mRNA (130 pg), _cox2_-MO (8 ng) with _cox2_ mRNA (125 pg), _ep4_-MO (1 ng) with _ep4_

mRNA (120 pg) or _abcc4_-MO (12 ng) with _abcc4_ mRNA (160 ng). Ctrl: Ctrl-MO (8 ng). The total numbers of embryos pooled from n = 3 independent experiments are indicated. Graph shows mean ±

s.d.; Student’s t test: ∗∗∗_P_ < 0.001. Statistics source data for Supplementary Fig. 4i, j can be found in Supplementary Table 2. SUPPLEMENTARY FIGURE 5 PGE2 SIGNALING AFFECTS

CILIA-DEPENDENT PROCESSES. (A) Bar chart depicting percentages of phenotypically wild-type embryos in several experimental groups as indicated. _lkt_ mutants were treated using PGE2 (50μm)

or PGF2〈 (50μm) to rescue reversed heart looping (3–24 hpf), otolith defects (10–48 hpf) and hydrocephalus (10–72 hpf). The total number of embryos pooled from n = 3 independent experiments

are indicated. Error bars represent mean ± s.d.; Student’s t test: ∗∗_P_ < 0.001. NS: not significant. (B,C) _cox2-MO_ injection (8 ng) caused hydrocephalus and curved axis (31%; n =

110), and three otoliths (21%; n = 110; arrows). (D–G) Acetylated tubulin immunostaining showing a reduction of ependymal cell cilia in the spinal canal in embryos injected with _cox1-_MO

(E; 8 ng, n = 24), _cox2_-MO (F; 8ng, n = 27) and _ep4_-MO (G; 1 ng, n = 25), compared with control embryos at 72 hpf (D). Scale bar: 100 μm (B,C); 10 μm (D–G). (H,I) Stacked bar graphs

depicting percentages of embryos with phenotypes grouped in otolith defect (H) and curved axis (I). _cox1/2_ morphants were incubated in PGE2 (50μM) to rescue otolith defect (10 to 48 hpf)

and curved axis (10 to 72 hpf). Treatment of _ep4_ morphants with FSK (50 μM) but not PGE2 rescued otolith defect (10 to 48 hpf) and curved axis (10 to 72 hpf) in _ep4_ morphants. Ctrl:

embryos injected with Ctrl-MO. The total numbers of embryos pooled from n = 3 independent experiments are indicated on the top of each bar. Yates corrected Chi-square test, ∗∗∗_P_ <

0.001, degree of freedom = 1. NS: not significant. Statistics source data for Supplementary Fig. 5a, d, e can be found in Supplementary Table 2. SUPPLEMENTARY FIGURE 6 EXPRESSION OF _FOXJ1A_

AND _FOXJ1B_ ARE NOT ALTERED IN EMBRYOS DEFICIENT IN _LKT/ABCC4_, _COX1/COX2_ OR _EP4_ ACTIVITIES. (A,B) _lkt_+/− heterozygous and wild-type homozygous embryos injected with different doses

of _cox1_-MO and _ep4_-MO display comparable percentages of LR randomization and hydrocephalus. Embryos derived from crosses between _lkt_+/− heterozygotes and wild-type zebrafish were

injected with _cox1_-MO, _ep4_-MO or control-MO (Ctrl). The injected embryos were genotyped using SSLP markers (Z17212) to distinguish _lkt_+/− heterozygous embryos (∼50%) from wild-type

homozygotes. Percentages reflect the frequency of phenotypic abnormalities. The total numbers of injected embryos pooled from n = 3 independent experiments are indicated above each bar.

Yates corrected Chi-square test, ∗∗∗_P_ < 0.0001, one degree of freedom. NS: Not significant. Statistics source data for Supplementary Fig. 6a, b can be found in Supplementary Table 2.

(C–F) In situ hybridization analyses revealing the comparable expression of _foxj1a_ in DFC cells in wild-type embryos, _lkt_ mutants, _cox1/cox2_ morphants or _ep4_ morphants at 9 hpf.

(G–J) The expression of _foxj1a_ in the spinal cord precursors (arrowhead) and rudiments of the pronephric ducts (arrows) was not altered in _lkt_ mutants, _cox1/cox2_ morphants or _ep4_

morphants, compared with wild-type embryos at 13 hpf. (K–N) The _foxj1b_ expression in developing otic placodes was not altered in _lkt_ mutants, _cox1/cox2_ morphants or _ep4_ morphants

compared with wild-type embryos at 13 hpf. Scale bar: 100 μm (A–L). SUPPLEMENTARY FIGURE 7 ANTEROGRADE IFT VELOCITY IS INCREASED IN PGE2-TREATED IMCD3 CELLS. Histograms showing IFT88-EYFP

particle velocity distribution in PGE2-treated IMCD3 cells and control cells (n > 50). Blue: Anterograde velocity; Red: Retrograde velocity. Arrows represent mean velocity in untreated

cells. PGE2 treatment increased the anterograde velocity but not the retrograde velocity. SUPPLEMENTARY FIGURE 8 ABCC4- OR EP4-DEPLETION CAUSES CILIOGENESIS DEFICIENCY IN IMCD3 CELLS. (A–D)

Immunostaining analyses showing individual cilia in IMCD3 cells treated with control siRNA, ABCC4-siRNA, EP4-siRNA or PGE2. Insets reveal high-magnification images of cilia (red arrows).

Green: ARL13B for cilium; Red: γ-tubulin for centrosome; Blue: DAPI; Arrow: cilium. PGE2 treatment: 10 μM; 12 h. Scale bar: 10 μm (A–D). (E,F) Immunoblot analysis showing ABCC4 or EP4

protein levels in IMCD3 cells transfected with control siRNA, ABCC4-siRNA or EP4-siRNA. (G,H) Statistical analyses of percentages of ciliated cells and average length of cilia in IMCD3 cells

transfected with control siRNA (470), ABCC4 siRNA (654), EP4 siRNA (594) and control siRNA plus PGE2 (371). Student’s t-test: ∗∗_P_ < 0.01,∗∗∗_P_ < 0.001; error bar represents mean ±

s.d.; n = 3 independent experiments with total number of cells provided in parentheses. (I,J) Bar graphs showing steady-state levels of intracellular cAMP (I) and intracellular Ca2+ (J) in

IMCD3 cells treated with DMSO (314), PGE2 (303) and FSK (305). PGE2 treatment causes an increase of intracellular cAMP but not Ca2+ in IMCD3 cells. Forskolin (FSK) is used as a positive

control. cAMP concentrations were measured using cAMP complete ELISA kit (ENZO, Life Science). Intracellular Ca2+ intensity levels (340/380nm) were measured using Fura-2 probe. DMSO: 0.5%;

PGE2: 10 uM; Forskolin: 100 uM. Error bar represents mean ± s.d.; Student’s t-test: ∗_P_ < 0.05,∗∗_P_ < 0.01,∗∗∗_P_ < 0.001; n = 3 independent experiments with total number of cells

analysed provided in parentheses. Statistics source data for Supplementary Fig. 8g–j can be found in Supplementary Table 2. Uncropped images of the immunoblots are shown in Supplementary

Fig. 9. SUPPLEMENTARY FIGURE 9 SCANS OF FULL-SIZE IMMUNOBLOTS. The red rectangles indicate the immunoblot fragments presented in the main body of the paper. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Information (PDF 2912 kb) SUPPLEMENTARY TABLE 1 Supplementary Information (XLS 34 kb) SUPPLEMENTARY TABLE 2 Supplementary Information (XLSX 110 kb) KV

FLUID FLOW IN WILD-TYPE EMBRYOS. This is a time-lapse of fluorescent bead movement within the KV of an 8-somite wild-type embryo. Imaged were taken at 25 frames per second using a Zeiss

Axio Imager M1 microscope with a 63 × Plan Apochromat objective. (MOV 9776 kb) ABSENCE OF KV FLUID FLOW IN _LKT_ MUTANTS. This is a time-lapse of fluorescent bead movement within the mutant

KV at the 8-somite stage. KV fluid flow was imaged (25 frames per second) under a 63 × Plan Apochromat objective using a Zeiss Axio Imager M1 microscope. (MOV 13409 kb) RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Jin, D., Ni, T., Sun, J. _et al._ Prostaglandin signalling regulates ciliogenesis by modulating intraflagellar

transport. _Nat Cell Biol_ 16, 841–851 (2014). https://doi.org/10.1038/ncb3029 Download citation * Received: 16 December 2013 * Accepted: 16 July 2014 * Published: 31 August 2014 * Issue

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