Genetic tools

The tranparency of the zebrafish also enables identification of abnormal embryonic phenotypes. This makes it a possible to use the zebrafish in forward genetic screens to identify and dissect the genetic pathways important for development. The high fecundity of the zebrafish, coupled with the availability of numerous genomic tools and resources make it relatively straightforward to perform candidate or positional cloning of the genes responsible for observed mutant phenotypes. We have successfully applied this approach to identify mutants that affect blood vessel development.

Zebrafish are also amenable to reverse genetic approaches.  We were among the first labs to apply such approaches using programmable nucleases to introduce targeted deletions in the zebrafish genome.  Our initial efforts relied on engineered zinc finger nucleases and we now take advantage of CRISPR-mediated gene editing to generate mutant zebrafish.

Examples from our work using forward or reverse genetic approaches

Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D., and Wolfe, S. A. (2008) Targeted gene inactivation in zebrafish using engineered zinc finger nucleases. Nature Biotechnology, 26:695-701. - co-corresponding authors.  PMID: 18500337; PMCID: PMC2502069.

Covassin, L.D.; Siekmann, A. F., Kacergis, M. C., Laver, E., Moore, J.C.,  Villefranc, J. A., Weinstein, B.M., and Lawson, N. D. (2009) A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development. Dev. Biol. 329(2):212-26. PMID: 19269286; PMCID: PMC2791107.

Kok, F. O., Shin, M., Ni, C-W., Gupta, A., Grosse, A. S., van Impel, A., Kirchmaier, B. C., Peterson-Maduro, J., Kourkoulis, G., Male, I., DeSantis, D.F., Sheppard-Tindell, S., Ebarasi, L., Betsholtz, C., Schulte-Merker, S., Wolfe, S. A., and Lawson, N. D. (2015) Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish. Developmental Cell, 32:97-108.  PMID: 25533206 PMCID: PMC4487878

Shin, M., Male, I., Beane, T. J., Villefranc, J. A., Kok, F. O., Zhu, L. J., and Lawson, N. D. (2016) Vegfc acts through ERK to induce sprouting and differentiation of lymphatic progenitors.  Development, 143:3785-3795. PMID: 27621059 PMCID: PMC5087638