The phosphatase Dullard negatively regulates BMP signalling and is essential for nephron maintenance after birth 

Supplementary Information

Masaji Sakaguchi1,2, Sazia Sharmin1, Atsuhiro Taguchi1, Tomoko Ohmori1,

Sayoko Fujimura3, Takaya Abe4, Hiroshi Kiyonari4, Yoshihiro Komatsu5,

Yuji Mishina5, Makoto Asashima6, Eiichi Araki2, and Ryuichi Nishinakamura1,*

Supplementary Figures S1-S6"Supplementary Table S1

1Department of Kidney Development, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan 2Department of Metabolic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan

3Liaison Laboratory Promotion Facility, Institute of Molecular Embryology and

Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan 4Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology (CDB), RIKEN Kobe, 2-2-3 Minatojima Minami, Chuo-ku, Kobe 650-0047, Japan

5Department of Biologic and Materials Sciences, School of Dentistry, University of

Michigan, 1011 North University Avenue. Ann Arbor, MI 48109-1078, USA

6Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan

*Corresponding author: Ryuichi Nishinakamura M.D., Ph.D., Department of

Kidney Development, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan

E-mail: ryuichi@gpo.kumamoto-u.ac.jp

Fax: +81-96-373-6618; Tel: +81-96-373-6615

Flox"WT"16.5 kb"

14.5 kb" 13.5 kb"11.4 kb"

5´probe" 3´probe"

c d

b

(a) In situ hybridization of Dullard in the kidneys at E14.5, P0 (newborn), and P14 (14 days after birth).  Scale bar: 100 μm. (b) Targeting strategy of the floxed Dullard allele. (c) Targeted ES clones were confirmed by Southern blot analysis. Genomic DNA was digested with AflIII and the 5' probe detected 14.5-kb wild-type and 16.5-kb floxed bands. Upon KpnI digestion, the 3' probe detected 11.4-kb wild-type and 13.5-kb floxed bands. (c) PCR-based genotyping was performed using DNA extracted from mouse tails for genotyping of Dullard+/+, Dullardflox/+, and Dullardflox/flox mice. The primer positions used for the genomic PCR genotyping are indicated by the arrows in (b)."

Supplementary Figure S1 | Generation of a floxed Dullard allele. !

5’ Probe 3’ Probe

5’ Probe

loxP loxP frt frt R2

R1 F1

AflⅡ

AflⅡ AflⅡ

KpnⅠ

KpnⅠ

13.5 kb

11.4 kb

14.5 kb

16.5 kb

Pr DT-A pA Pr Neo pA

Pr Neo pA

WT allele

Floxed allele

Targeting Vector

AflⅡ 3’ Probe

KpnⅠ

AflⅡ KpnⅠ

exon 2 3 4

F2

aInner medulla "Outer medulla "Cortex "

Dul

lard

E14.5

P14

P０

a! b!

Cortex "

Outer medulla "

Inner medulla "

Dullardflox/flox 　"

Hoxb7Cre; Dullardflox/flox "

Dullardflox/flox 　"

Hoxb7Cre; Dullardflox/flox"

c! d! Dullardflox/flox　 　　

Hoxb7 Cre; Dullardflox/flox"

MM UB MM

UB Flox

Deleted

Cre

1.0

0.1

0.01

0.001 MM UB MM UB

Six2 Cre; Dullardflox/flox"

Dullard

UB

Rel

ativ

e ex

pres

sion

　(A

U)"

Six2 Cre; Dullardflox/+　 　　

Supplementary Figure S2 | Ureteric bud-specific Dullard deletion shows no defects.

(a) Macroscopic evaluation of 8-week-old Dullardflox/flox and Hoxb7Cre; Dullardflox/flox mutant kidneys. No obvious abnormalities are observed. Scale bar: 1 mm. (b) Haematoxylin and eosin staining of Dullardflox/flox and Hoxb7Cre; Dullardflox/flox mutant kidneys at 8 weeks after birth. The Hoxb7Cre; Dullardflox/flox mutant kidney shows normal cortical and medullary structures. Scale bar, 100 μm. (c) Deletion of Dullard genome in the ureteric bud-derived lineages of the Hoxb7Cre; Dullardflox/flox adult mice. The kidney cortex (mainly mesenchyme-derived) and a block containing calices and the pelvis (ureteric bud-derived) were anatomically dissected, followed by genomic PCR. (d) Reduced Dullard expression in the metanephric mesenchyme of the Six2Cre; Dullardflox/flox kidney at E18.5. For sorting of the metanephric mesenchyme, the GFP in Six2Cre mice was used, since the GFP-Cre fusion was driven by the Six2 promoter. DBA staining was used for sorting of the ureteric bud-derived cells. Six2Cre; Dullardflox/+ and six Six2Cre; Dullardflox/flox embryos were analysed  (n=3 and 6, respectively). Mean ± s.d. MM: metanephric mesenchyme-derived cells, UB: ureteric bud-derived cells.

YFP"DBA"Caspase-3"

Cortex "

Inner"medulla"

Outer "medulla"

""Capase-3"

ba

c d

CD31

F4/80 DAPI

HE SMA

Dullardflox/flox 　 　　

Six2Cre; Dullardflox/flox"

e

Six2Cre; Dullardflox/flox"

Dullardflox/flox 　 　　

Supplementary Figure S3 | No urinary tract obstruction in Six2Cre; Dullardflox/flox

kidneys.

(a) HE staining of a Six2Cre; Dullardflox/flox kidney at P14 showing the severe phenotype. Note that the cavity in the outer medulla is connected to the pelvic space. Scale bar: 100 μm. (b) Immunostaining for smooth muscle actin at P21 shows unaffected ureteric muscle layer formation in the mutant kidney. Note that the ureter is not dilated despite the expanded cavity in the kidney. Scale bar: 0.5 mm. (c) Apoptotic cells are not detected in Six2Cre; R26RYFP kidneys at P7. Few cleaved caspase-3-positive cells (red) are seen in control Six2Cre; R26RYFP kidneys throughout the cortex, outer medulla, and inner medulla. YFP: green, DBA: purple. Scale bar: 25μm. (d) No enhanced infiltration of macrophages to the neighbouring region of the cavity in the Dullard mutant kidney, as revealed by F4/80 staining at P14. Scale bar: 100 μm. (e) Immunostaining of CD31 at P14 shows no enhanced vascularization in the mutant kidney. Scale bar: 100 μm.

a!

Rel

ativ

e ex

pres

sion

(AU

)"b!

Rel

ativ

e ex

pres

sion

(AU

)"

BMP2"

0.5"

1.0"

WT Mt

Rel

ativ

e ex

pres

sion

(AU

)"

BMP4"

0.5"

1.0"

WT Mt

BMP7"

0.5"

1.0"

WT Mt

BMP2"

0.5"

1.0"

P0 P7 P14 P21

BMP4"

0.5"

1.0"

P0 P7 P14 P21

BMP7"

0.5"

1.0"

P0 P7 P14 P21

c!

Rel

ativ

e ex

pres

sion

(AU

)" Id2"

WT Mt

Rel

ativ

e ex

pres

sion

(AU

)" Id1"

WT Mt

Rel

ativ

e ex

pres

sion

(AU

)" Sox9"

2.0"

WT Mt

*

1.0" 1.0"

1.0"0.5" 0.5"

(a) The expression levels of three BMP ligand genes, BMP2, BMP4, and BMP7, were analysed by qRT-PCR. The mean values of three individual samples at each stage are shown. The relative expression (in arbitrary units [AU]) was calculated by setting the stage P0 expression of each gene at 1. Mean ± s.d. (b) qRT-PCR of BMP ligand genes at P7 in control (WT) and Six2Cre; Dullardflox/flox mutant (Mt) kidneys (n=3 in each group). (c) qRT-PCR of Id1, Id2, and Sox9 at P7 in control (WT) and Six2Cre; Dullardflox/flox mutant (Mt) kidneys (n=3 in each group). Mean ± s.d., *P<0.001.

Rel

ativ

e ex

pres

sion

　(A

U)"

Rel

ativ

e ex

pres

sion

　(A

U)"

Rel

ativ

e ex

pres

sion

　(A

U)"

Supplementary Figure S4 | Expression of BMP-related genes in neonatal kidneys. !

c! d

Sox9"

Cor

tex"

Out

er

med

ulla"

Inne

r

med

ulla"

Sox9"

Dullardflox/flox　　　

Rel

ativ

e ex

pres

sion

　(A

U)" NS

1.0"

0.5"

"Dullardflox/flox"

Six2Cre; "Dullardflox/+"

Six2Cre; Dullardflox/flox 　 　　

Sox9"

GAPDH"

b!

Six2Cre;"Dullardflox/flox"

Six2Cre; "Dullardflox/+"

"Smad1/5/8"

"p-Smad1/5/8"

"Dullardflox/flox"

GAPDH"

a

(a) Western blot analysis using kidney lysates from newborn mice shows that p-Smad1/5/8 is increased in the Six2Cre; Dullardflox/flox mutant mice. (b,c) Sox9 is not upregulated in the Dullard mutant mice at P0, as shown by western blot and qRT-PCR analyses (n=3 in each group). Mean ± s.d. (d) Immunostaining of Sox9 in the cortex, outer medulla, and inner medulla of newborn kidneys. Sox9 is detected at the ureteric bud tips in both the control and mutant kidneys, but is not increased in the mutant mesenchyme. Scale bar: 100 μm.

Six2 Cre; Dullardflox/flox"

Dullardflox/flox　 　　

Six2 Cre; "Dullardflox/flox"

kDa

50

50

37

75

37

kDa

Supplementary Figure S5 | p-Smad 1/5/8 is upregulated in Dullard mutants at P0.

GAPDH"

b!

Six2Cre;"Dullardflox/flox"

"Smad1/5/8"

"p-Smad1/5/8"

"Dullardflox/flox"

GAPDH"

a

(a) Western blot analyses of kidney lysates at P7 showing an inhibitory effect of LDN-198189 against the BMP activity. LDN-198189 was injected daily from P1 to mothers nursing pups, including Six2Cre; Dullardflox/flox mutant mice. (b) Western blot analyses of BMP receptors in the untreated Six2Cre; Dullardflox/flox kidney at P7.

BMPR2"

BMPR1A"

Six2Cre;"Dullardflox/flox"

"Dullardflox/flox"

kDa

50

50

37

kDa

150

37

Supplementary Figure S6 | Suppression of the p-Smad1/5/8 increase by LDN-193189.

75

Primer Sequence (5’ to 3’)

BMP2 forward TGACTGGATCGTGGCACCTC

BMP2 reverse CAGAGTCTGCACTATGGCATGGTTA

BMP4 forward AGCCGAGCCAACACTGTGAG

BMP4 reverse TCACTGGTCCCTGGGATGTTC

BMP7 forward CAGCTGAAGGCTATGCTGCTCTACTA

BMP7 reverse AGCAGGGCTTGGGTACTGTGTC

Id1 forward TGGACGAGCAGCAGCAGGTGAAC

Id1 reverse GGATCTCCACCTTGCTCACTTTG

Id2 forward GGACATCAGCATCCTGTCCTTG

Id2 reverse CTCCTGGTGAAATGGCTGATAAC

Sox9 forward GGAGATGAAATCTGTTCTGGGAATG

Sox9 reverse TGAAGGTTAACTGCTGGTGTTCTGA

β-actin forward CATCCGTAAAGACCTCTATGCCAAC

β-actin reverse ATTGGAGCCACCGATCCACA

Dullard forward GGTGCTGAGGCCTACAGTGAGA

Dullard reverse CCACAAGCTCATACCACTGGCTTA

Supplementary Table S1. List of primers for qRT-PCR