Download edn1 - University of Oregon (SPUR)

More than just a sucker
“Double mutant analysis of bmp4 and edn1 in zebrafish
craniofacial development”
Hypothesis: bmp4 and edn1 genetically interact to
pattern the developing zebrafish skeleton
Braden Larson
PI: Prof. Charles Kimmel
Mentor: Dr. Jamie Nichols
SPUR Program 2010
8/11/2010
Zebrafish
•Amenable to genetics:
-Easy to take care of
-lay in large numbers
•Amenable to embryology:
-Clear embryos
-Develop rapidly
•And they’re vertebrates!
-Can study skeletal development
-Relevant to human development/disease
Double Mutant Analysis
Additive: aa + bb = aabb
+
WT
=
Green + wrinkled = Grinkled
If phenotypes are additive, genes act in separate pathways
and do not genetically interact!
Double Mutant Analysis
•Enhancement of phenotype: aabb > aa + bb
-Suggests that the genes interact positively
WT
+
=
Green + Blue = White
•Supression of phenotype: aabb < aa + bb
-Suggests that one gene suppresses the other or has an
opposite effect
+
WT
=
Green + Blue = Yellow
In both cases, these genes act in the same pathway
and genetically interact with each other!
Zebrafish larval head skeleton
Kimmel et al., 2001
Endothelin-1
(sucker)
-Key regulator of ventral
craniofacial development
-Mutation causes severely
reduced ventral cartilages
WT
Miller, Schilling, Lee, Parker, Kimmel, 2000
edn1 -/-
Bone morphagenetic
protein-4
•Incompletely penetrant tail
phenotype in zebrafish (Stickney et al)
•Has no skeletal phenotype
Albertson R C et al. PNAS 2005;102:16287-16292
WT
edn1 and bmp4 both regulate bapx1
In zebrafish, edn1 regulates bapx1
36hpf
Miller et al. 2003
edn1  bapx1
In chicks Bmp4 regulates bapx1
bapx1
bmp4 –l bapx1
Wilson, Tucker et al.
Both bmp4 and edn1 regulate bapx1,
suggesting that they act in the same pathway!
Collecting Data
•Embryos were stained with alcian
blue and alizarin red at 6dpf
•Genotyped by PCR
WT
Jack
•Scored
Kinked PQ
Tapered or flared
meckels
“Jack”
Clutch A
Phenotype
Jack
edn1-/- bmp4 +/+ edn1-/-bmp4+/80% (4/5)
76.9% (10/13)
edn1-/-bmp4-/80% (4/5)
Clutch B
Jack
100% (7/7)
25% (1/4)
100% (18/18)
Kinked PQ phenotype
Clutch A
Phenotype
Kink
Clutch B
Kink
edn1-/- bmp4 +/+ edn1-/-bmp4+/0% (0/10)
15.4% (4/26)
edn1-/-bmp4-/60% (6/10)
7.1% (1/14)
0% (0/8)
27.8% (5/18)
Meckels phenotype
Tapered vs flared
Clutch A
Phenotype
Flared M
Tapered M
Clutch B
Flared M
Tapered M
edn1-/- bmp4 +/+ edn1-/-bmp4-/+
80% (8/10)
53.8% (14/26)
20% (2/10)
46.2% (12/26)
edn1-/-bmp4-/40% (4/10)
60% (6/10)
50% (7/14)
50% (7/14)
12.5% (1/8)
87.5% (7/8)
38.9% (14/36)
61.1% (22/36)
Hypothesis: bmp4 and edn1 genetically interact to
pattern the developing zebrafish skeleton
Conclusions
•The Jack phenotype appears to have no correlation with
genotypes
•The kinked PQ phenotype also saw no correlation
between genotypes
•The tapered meckels phenotype saw an increase in
frequency from edn1 mutants to edn1/bmp4 double
mutants.
Phenotype is enhanced!?
Preliminary data says yes, supporting my hypothesis!
Future Directions
-Increase sample size for statistical analysis
-In Situ Hybridization to examine expression of
shared downstream targets (eg. bapx1)
-Pair bmp4 with less severe endothilin
pathway member (eg. furinA/bmp4 double
mutants)
Thanks to…!
-Charles Kimmel
-Jamie Nichols
The Kimmel lab
-Peter O’Day
-Blakely Strand
The SPUR students
Questions?