Mouse models recapitulate human FRIZZLED2 autosomal

dominant omodysplasia and identify causal mechanisms

Sanika Vaidya1, R.P. Liegel1, C.A. Menke1, Rolf W. Stottmann1,2
1 Division of Human Genetics, 2 Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

Background Phenotypes of Frizzled2 mouse models Planar cell polarity is disrupted

• Fzd2D4/D4 and
• Fzd2D4/D4 embryos exhibit cleft palate,

wild-type (wt)
• A nonsense mutation in FRIZZLED2 was previously identified Fzd2D3/D3 cleft • The planar cell polarity (PCP) pathway is one branch of non-
in a mother and daughter with autosomal dominant micrognathia, and shortened limbs mutants have canonical Wnt signaling
Body Weight Humerus Radius Ulna wide faces
omodysplasia, presenting with short humeri, radial dislocation, • WNT binds FZD, which triggers a cascade that activates small

Humerus Length/Body Wt (um/g)

Radius Length/Body Wt (um/g)

Ulna Length/Body Wt (um/g)

1.05 2600 2200 2800

1.00 2400 2600

round faces, frontal bossing, small noses with long tips, long GTPases that phosphorylate c-Jun N-terminal kinase (JNK),
2000

Body Wt (g)
0.95 2200
2400
2000 1800
0.90
2200

philtrums, cleft lip and palate, and genitourinary anomalies (1) which regulates transcription to direct cell polarization and
1800
0.85
1600
1600 2000
0.80
1400 1400

• FRIZZLED2 (FZD2) is a transmembrane WINGLESS-

0.75 1800

cytoskeletal organization
RELATED (WNT) receptor that recruits DISHEVELLED (DVL) Mandible Femur Tibia • Irregular chondrocyte

D4/+

Length to width ratio (LWR)

Fibula 4

Mandible Length/Body Wt (um/g)

Femur Length/Body Wt (um/g)

Fibula Length/Body Wt (um/g)

orientation is an

Tibia Length/Body Wt (um/g)

in canonical and non-canonical WNT signal transduction 6500

6000
2000

1800
2400

2200
2500

2000
W

• The patient variant results in a premature stop codon that 5500

5000
1600
2000

1800
1500

1000
indicator of disrupted L

causes the loss of 17 amino acids, disrupting the DVL binding 4500 1400
1600 500 +/+ PCP mechanisms
4000 1200 1400 0
3
sequence. We hypothesize that the deleterious phenotypes • Wild-type limb

D4/D4
are caused by disrupted WNT signaling sections at E12.5
• Fzd2D3/D3 embryos exhibit cleft palate p<0.0001
• To construct an animal model with CRISPR- (50% penetrance) and shortened limbs
show flattened
2
Cas9 transgenesis, we obtained an allelic Body Weight Humerus Radius Ulna
chondrocytes
Frizzled

Humerus Length/Body Wt (um/g)

stacked along the

Radius Length/Body Wt (um/g)

series of two small deletions that can

Ulna Length/Body Wt (um/g)

1.0 2500 2200
2600

Body Wt (g)
2000

replicate the human disease in mice proximal-distal axis,

0.9
2000
2400
1800
D3/+ 0.8
2200
D3/D3 1
1500
1600
as expected

Palate width/interorbital distance

0.7 2000

* * • Fzd2D4/D4 limbs show

wt 0.65

irregular patterning
Mandible Femur Tibia Fibula
0.60

of chondrocytes 0
D3

Mandible Length/Body Wt (um/g)

Femur Length/Body Wt (um/g)

Femur Length/Body Wt (um/g)

wt D4/D4

Tibia Length/Body Wt (um/g)

7000 2000 2500
2500
1800
D3/D3

6000 0.55
2000
2000

Distal
1600

D4 5000
1400
1500
1500

0.50
4000
1200 1000 1000
Dvl binding consensus: KTxxxW

PDZ-interacting: S/T-X-V

cleft, c.; no cleft, n.c.; measured at E17.5

Canonical WNT signaling is DKK inhibitor IIIc3a can rescue mutant phenotypes
downregulated

Proximal
wt D4/+ D4/D4

• In the absence of WNT, Mandible Tibia wt D4/D4

Mandible Length/Body Wt (um/g)
a destruction complex

Tibia Length/Body Wt (um/g)

100x; DAPI highlight nuclei (blue), wheat germ agglutinin stains cell membranes (red)
Control

6500 3000
made of AXIN, APC,
and GSK3 6000
phosphorylates β- * 2500 * Conclusions & Future Directions
catenin, targeting it for 5500
IIIc3a Treatment

degradation. Once
WNT binds FZD, DVL is 5000
2000 • Mutations in FZD2 cause deleterious phenotypes by disrupting
recruited and activated. both canonical and non-canonical WNT signaling
4500
This induces AXIN to
bind LRP, resulting in 4000 • Shortened limb and micrognathia phenotypes can be rescued
1500
degradation of the • IIIc3a is a small molecule competitive inhibitor of DIKKOPF with IIIc3a, a DKK inhibitor which activates canonical WNT
3a

3a

3a
ol

ol

ol

3a

3a

3a
ol

ol

ol
destruction complex; β-
tr

tr

tr

tr

tr

tr
c

c
(DKK), a canonical WNT signaling inhibitor signaling
on

on

on
III

III

III

on

on

on
III

III

III
catenin can translocate
C

C
• Daily 25 mg/kg i.p. injections of IIIc3a at E10.5-14.5 partially
into the nucleus and rescue micrognathia and shortened limb phenotype of • Lithium chloride injections may downregulate canonical WNT
target gene expression Fzd2D4/D4 embryos measured at E17.5; controls are uninjected signaling
Similar results were seen in the radius, ulna, femur, and fibula
ß-catenin/tubulin

0.15
• Western blot • Further biochemical analysis can elucidate the nature of non-
p=0.0003
analysis of face canonical WNT signaling disruption
0.10 and limb tissue at Lithium chloride fails to augment canonical WNT signaling
E13.5 shows
90
• Co-immunoprecipitation studies can determine interaction
0.05
decreased active β- Radius Femur between mutant FZD2 and DVL
• Lithium chloride (LiCl) has been Limbs Faces
catenin in Fzd2D4/D4
Radius Length/Body Wt (um/g)

NaCl
Femur Length/Body Wt (um/g)

0.00 shown to inhibit GSK, LiCl NaCl LiCl

wt D4/D4 embryos
preventing β-catenin destruction
2200
* * * 2000
* * 160

2000 90
• qRT-PCR analysis of embryo face tissue at complex function Β-catenin 95 kDa

E13.5 shows Fzd2D4/D4 mutants display • 10 µL/g i.p. LiCl injections at 1800
1500 50
Acknowledgements & References
decreased expression of AXIN2 and DKK1, E8.5, 9.5, and 10.5 fail to rescue 15

Fzd2D4/D4 phenotypes measured 1600

two direct transcriptional targets of β-catenin 8 30
GAPDH 37 kDa
at E17.5; some injected embryo 1400 Funding for this project was provided by the Division of Human Genetics.
AXIN2 Relative Quantity DKK1 Relative Quantity E10.5 Limbs E10.5 Faces
3 p=0.0001
1.5
p<0.0001 limbs have shorter lengths than 1000 p= 0.0308 p= 0.0614
1.6 1.5
1200 (1) Saal et al. (2015). A mutation in FRIZZLED2 impairs Wnt signaling
β catenin/GAPDH

β catenin/GAPDH

uninjected controls
AXIN2/GAPDH

DKK1/GAPDH

• LiCl injections decrease active and causes autosomal dominant omodysplasia. Human Molecular
l

l
ol

ol

ol

l
ol

ol

ol
C

2 1.0 1.4 1.0

tr

tr

tr

tr

tr

tr
Li

Li

Li

Li

Li

Li
on

on

on

on

on

on

Genetics, 24(12), 3399-3409. doi:10.1093/hmg/ddv088

β-catenin level in limb and face
C

1 0.5 1.2 0.5

tissue dissected 6 hours after

0
wt D4/D4
0.0
wt D4/D4 final injection Similar results were seen in the humerus, ulna, tibia, and fibula
1.0
NaCl LiCL
0.0
NaCl LiCl

10 ul/g injection at E8.5, 9.5, 10.5 10 ul/g injection at E8.5, 9.5, 10.5 */ : p<0.05, one-way ANOVA, Tukey’s multiple comparisons test