Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.
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.
Department of Biology, Duke University, Durham, North Carolina 27708.
Qingyun
Q
Li
Department of Biology, Duke University, Durham, North Carolina 27708.
Scott
S
Barish
Department of Biology, Duke University, Durham, North Carolina 27708.
Sumie
S
Okuwa
Department of Biology, Duke University, Durham, North Carolina 27708 Duke Institute for Brain Sciences, Duke University, Durham, North Carolina 27708 pc72@duke.edu.
Pelin C
PC
Volkan
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004330:Drosophila / Q000196:embryology* / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D059765:Homologous Recombination; D009830:Olfactory Bulb / Q000196:embryology* / Q000378:metabolism*; D021381:Protein Transport; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D029721:Drosophila Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D014157:Transcription Factors; C450829:rn protein, Drosophila
CRISPR; Cas9; genome editing; homologous recombination; olfactory system development; rotund; tagging