TY - JOUR
T1 - Examination of endogenous rotund expression and function in developing Drosophila olfactory system using CRISPR-Cas9-mediated protein tagging
AU - Li, Qingyun
AU - Barish, Scott
AU - Okuwa, Sumie
AU - Volkan, Pelin C.
N1 - Publisher Copyright:
© 2015 Li et al.
PY - 2015
Y1 - 2015
N2 - The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates. We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.
AB - The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates. We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.
KW - CRISPR
KW - Cas9
KW - Genome editing
KW - Homologous recombination
KW - Olfactory system development
KW - Rotund
KW - Tagging
UR - http://www.scopus.com/inward/record.url?scp=84949958028&partnerID=8YFLogxK
U2 - 10.1534/g3.115.021857
DO - 10.1534/g3.115.021857
M3 - Article
C2 - 26497147
AN - SCOPUS:84949958028
SN - 2160-1836
VL - 5
SP - 2809
EP - 2816
JO - G3: Genes, Genomes, Genetics
JF - G3: Genes, Genomes, Genetics
IS - 12
ER -