INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse

Monique Jacoby, James J. Cox, Stéphanie Gayral, Daniel J. Hampshire, Mohammed Ayub, Marianne Blockmans, Eileen Pernot, Marina V. Kisseleva, Philippe Compère, Serge N. Schiffmann, Fanni

Gergely, John H. Riley, David Pérez-Morga, C. Geoffrey Woods, & Stéphane Schurmans

Nature Genetics: doi:10.1038/ng.427

Supplementary Figure 1: Targeted disruption of Inpp5e in mouse. (a). Wild-type (+), floxed and ∆ Inpp5e alleles. Filled boxes, exons. Filled circles, loxP sites. Primers used in PCR on genomic DNA (arrows) and in RT-PCR on brain RNA (arrow heads) are shown. (b) PCR analysis of genomic DNA extracted from Inpp5e mice. The + and ∆ alleles are respectively 1422 and 683 bp long.

b

+/+

+∆

∆/∆

∆/+

5 6 7 8 9 10

5 6 7 8 9 10

5 6 9 10

1 kba

+ allele

flox allele

∆ allele

Nature Genetics: doi:10.1038/ng.427

AQ

P2

+ D

AP

I

+/+

Normal tubule

∆/∆

Cyst

b

AQ

P1

+ D

AP

I

a

Supplementary Figure 2: Immunofluorescence studies on kidney section from Inpp5e+/+ and Inpp5e∆/∆ E18.5 embryos with (a) anti-AQP1 and (b) anti-AQP2 antibodies (red). Kidney cysts positives for AQP1 and AQP2 markers are shown. Nuclei are stained with DAPI (blue). Scale bars = 5 µm.

Nature Genetics: doi:10.1038/ng.427

Eye

EyeThyroid

Spinalchord

Olfactoryepithelium

Kidney

a

c

d e

b

Thymus

Eye BrainSpinalchord

LiverLung

Limb

ThymusBrain

Supplementary Figure 3: Tissue expression of Inpp5e mRNA during mouse embryogenesis. (a) Serial transversal sections through an E10.5 Inpp5e+/+

head incubated with the radiolabeled antisense oligonucleotide probe encoded in exon 8 of the murine Inpp5e gene. The arrow indicates the eye in development. The same radiolabeled antisense oligonucleotide Inpp5e probe was used on sagital and parasagital sections through (b) an E14.5 and (c) an E18.5 Inpp5e+/+ embryo. (d) No signal is detected when the same radiolabeled antisense oligonucleotide Inpp5e probe is used on a Inpp5e∆/∆

embryo section, demonstrating the specificity of the antisense probe. (e) A Inpp5e+/+ embryo is shown as positive control. Scale bars = 5 mm.

Nature Genetics: doi:10.1038/ng.427

+/+

∆/∆

b

c

∆/∆a

Supplementary Figure 4: SEM observations of primary cilia (arrows) emerging from cells (a) of a kidney cystic tubule from Inpp5e∆/∆ mice, of the Bowman’s capsule from (b) a Inpp5e+/+ glomerulus and (c) a cystic Inpp5e∆/∆ glomerulus. Scale bars = 2 µm.

Nature Genetics: doi:10.1038/ng.427

b

c d

a

Supplementary Figure 5: Detection of apoptotic cells in the kidney cysts of E18.8 Inpp5e∆/∆ embryos. (a) SEM, (b) TEM, and (c, d) TUNEL assay on kidney from Inpp5e∆/∆ embryos. In SEM observations, apoptotic bodies in the lumen of a cyst are shown (arrow, scale bar = 10 µm). In TEM observations, apoptotic cells have condensed and marginalized chromatin (arrows; scale bars = 10 µm). In the TUNEL assay, a TUNEL positive cell (arrow) is present among the cells lining a cyst in mutant kidney. (d) No apoptotic cell is detected in Inpp5e+/+ kidney by TUNEL. Nuclei are stained with DAPI (blue). Scale bars = 5 µm.

Nature Genetics: doi:10.1038/ng.427

a Inpp5e +secondary Ab +DAPI

Secondary Ab +DAPI

c Inpp5e +secondary Ab +DAPI

b d

Inpp5e

Actin

NC C

Inpp5e+/+ MEF

Supplementary Figure 6: Inpp5e protein expression in ciliated and non-ciliated Inpp5e+/+ MEF. Immunofluorescence studies on (a, b) non-ciliated and (c) ciliated Inpp5e+/+ MEF using an anti-mouse Inpp5e antibody and a secondary antibody (red) (a, c) or only the secondary antibody (red) (b). Arrows indicate the primary cilia. Scale bars = 5 µm. (d) Western blot analysis of protein extracts from non-ciliated (NC) and ciliated (C) Inpp5e+/+

MEF with an anti-mouse Inpp5e antibody. Actin served as loading control.

Nature Genetics: doi:10.1038/ng.427

∆/∆+/+

Supplementary Figure 7: SEM views of primary cilia protruding from Inpp5e+/+ (left panel) and Inpp5e∆/∆ (right panel) MEF after 4h following addition of 10% serum. Scale bars = 2 µm

Nature Genetics: doi:10.1038/ng.427

+/∆ Cre flox/∆Crea

b

Supplementary Figure 8: Phenotypic characterization of Inpp5eflox/∆

CAGG-Cre-ERTM mice. Four week-old mice were treated with Tamoxifen to activate the Cre recombinase and analysed 5 months later. (a) Retina and (b) kidney sections of Tamoxifen-treated Inpp5e+/∆ CAGG-Cre-ERTM

and Inpp5eflox/∆ CAGG-Cre-ERTM mice showed the absence of the photoreceptor layer and cystic glomeruli in mutant adult mice. The photoreceptor cell layer is marked with a bracket. Scale bars = 10 µm.

Nature Genetics: doi:10.1038/ng.427

rerio

Supplementary Figure 9: Sequence comparison of known INPP5E proteins. The C-terminal 33 amino acids of INPP5E are shown for all available mammals and bird. The conserved CaaX box is shown in grey. The effect of the MORM mutation is shown first, followed by wild-type human INPP5E, then the other species. The length of the protein is indicated as is the amino acid identities compared to human INPP5E.

Nature Genetics: doi:10.1038/ng.427

-250

0

250

500

750

1000

1250

NT GFP WT MORM

PtdIns(3,4,5)P3

0

100

200

300

400

NT GFP WT MORM

PtdIns(4,5)P2aR

elea

sed

phos

phat

e (p

mol

)

Rel

ease

d ph

osph

ate

(pm

ol)

b

MO

RM

C-

MO

RM

WT

C-

WT

GFP-INPP5E WT

GFP-INPP5E MORM

Supplementary Figure 10: Phosphatase activity of GFP-tagged MORM and WT Inpp5e proteins. (a) Anti-GFP immunoprecipitates were incubated with PtdIns(3,4,5)P3 or PtdIns(4,5)P2 as substrates. The amount of released phosphate is presented as the mean ± standard deviation. NT: not transfected. (b) Western blot analysis showing the amount of GFP-tagged WT and MORM Inpp5e proteins after immunoprecipitation (WT, MORM)with the anti-GFP antibody and engaged in the phosphatase reaction. C-WT and C-MORM are controls which were treated in the absence of anti-GFP antibody during immunoprecipitation.

Nature Genetics: doi:10.1038/ng.427

Inpp5e

- peptide + peptide

NT T NT T

Actin

Inpp5e

+/+ ∆/∆

- peptide + peptide

Actin

a bInpp5e-

pept

ide

+ p

eptid

e

c

+/+ ∆/∆

Supplementary Figure 11: Characterization of the anti-mouse Inpp5e antibody. (a) Immunofluorescence study on ciliated Inpp5e+/+ MEF with an anti-mouse Inpp5e antibody (red) pre-incubated or not with the immunization peptide. Arrows indicate cilia. Nuclei are stained with DAPI (blue). Scale bars = 5 µm. (b) Western blot analysis of brain protein extracts from Inpp5e+/+ and Inpp5e∆/∆ mice with an anti-mouse Inpp5e antibody pre-incubated or not with the immunization peptide. Actin served as loading control. (c) Western blot analysis of protein extracts from COS-7 cells transfected (T) or not (NT) with a mouse Inpp5e expression vector. The anti-mouse Inpp5e antibody was pre-incubated or not with the immunization peptide. Actin served as loading control. A marked reduction in the Inpp5e signal is observed.

Nature Genetics: doi:10.1038/ng.427

Supplementary Table 1: Oligonucleotides sequences.

GFP-WT INPP5E F1: 5’-CCGCTCGAGCCGG CATGCCGTCCAAGGCGGAG-3’

R1: 5’-CGGGGTACCCCGTCAAGAAACGG AGCAGAT-3’

GFP-MORM INPP5E F1: 5’-CCGCTCGAGCCGG CATGCCGTCCAAGGCGGAG-3’

R2: 5’-CGGGGTACCCCGCTAAATCTCCTTCGAAATCCG-3’

HA-WT INPP5E F2: 5’-GGGGTACCCCCGCCACCATGGTGTACCCCTACGA

CGTGCCCGACTACGCCATGCCGTCCAAGGCGGAGA-3’

R3: 5’-TCGCGGATCCAATCAAGAAACGGAGCAGATG-3’

HA-MORM INPP5E F2: 5’-GGGGTACCCCCGCCACCATGGTGTACCCCTACGA

CGTGCCCGACTACGCCATGCCGTCCAAGGCGGAGA-3’

R4: 5’-TCGCGGATCCAACTAAATCTC CTTCGAAATC-3’

In situ hybridization probes Sense: 5’TTGGGAAGGACACCTACGACAGCACCTCCAAGCAAAGGACACCCT-3’

Antisense:5’AGGGTGTCCTTTGCTTGGAGGTGCTGTCGTAGGTGTCCTTCCCAA-3’

Nature Genetics: doi:10.1038/ng.427