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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?