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\subsection*{DmCa\textsubscript{v}3 is broadly expressed in the adult brain}  To determine After several failed attempts to generate an antibody that works well for immunohistochemistry, we decided to tag  the endogenous DmCa\textsubscript{v}3 with GFP and then visualize its  expression patternof DmCa\textsubscript{v}3  in the adult brain, we generated GFP-tagged DmCa\textsubscript{v}3 allele. brain.  First, we generated a  founder line (\emph{DmCa\textsubscript{v}3\textsuperscript{Founder}}) by line, (\emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w+}}}}), using  end-out homologous recombination for to facilitate the  versatile allele generation\cite{Huang:2009ei} generation of a variety of different alleles\cite{Huang:2009ei}  (Fig. \ref{fig:2}a). In \emph{DmCa\textsubscript{v}3\textsuperscript{Founder}}, about 2 kb of genomic region including first coding exon was replaced by \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w+}}}} flies, an  \emph{attP} landing  site for $\phi$C31-mediated DNA integration and a  floxed \emph{white\textsuperscript{+}} marker.   After removing marker replace \sim2 kb of genomic DNA surrounding the first coding exon of DmCa\textsubscript{v}3.   Next, we removed the  \emph{white\textsuperscript{+}} marker in from  the \emph{DmCa\textsubscript{v}3\textsuperscript{Founder}} \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w+}}} line  by Cre-mediated recombination, \emph{attB} vector recombination to generate \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w-}}}.  We then used the $\phi$C31 integrase to insert into the \emph{attP} landing site of \emph{DmCa\textsubscript{v}3\textsuperscript{Founder, \emph{w-}}} an \emph{attB}vector  (\emph{pGE-attB\textsuperscript{GMR}}) containing the  deleted genomic region with plus an additional  GFP coding sequence  and linker sequences inserted sequence in-frame  before the start codonin frame was integrated into \emph{attP} site  of founder line by $\phi$C31 integrase.   Integration resulted in DmCa\textsubscript{v}3 tagged with GFP at its N-terminal end (\emph{GFP::DmCa\textsubscript{v}3}).   In \emph{GFP::DmCa\textsubscript{v}3} flies, GFP signal appears broadly across DmCa\textsubscript{v}3.   This produced  thebrain (Fig. \ref{fig:2}b).  Expressed region includes well-structured neuropils like antennal lobe, mushroom body, central complex in the central brain (Fig. \ref{fig:2}c-h) and optic lobe as well as less-structured neuropil regions.  The strongest expression was detected in the central complex consisting four substructures of fan-shaped body, ellipsoid body, noduli and protocerebral bridge (Fig. \ref{fig:2}e and g) and the expression was predominent in ventral part of fan-shaped body and ventral noduli (Fig. \ref{fig:2}e).   In mushroom body,  \emph{GFP::DmCa\textsubscript{v}3} was localized in postsynapse-rich calyx in dorso-posterior brain (Fig. \ref{fig:2}h) rather than axonal lobes in anterior brain (Fig. \ref{fig:2}d).  We observed \emph{GFP::DmCa\textsubscript{v}3} expression only in the posterior part of peduncle, a fiber track of mushroom body joining the posterior calyx and anterior lobes (Fig. \ref{fig:2}f).  These results suggest strict regulation of subcellular localization of line, which expresses an N-terminally GFP-tagged  DmCa\textsubscript{v}3 channels in under  the brain. control of its own endogenous promoter.  In these \emph{GFP::DmCa\textsubscript{v}3} flies, GFP fluorescence appears broadly across the brain (Fig. \ref{fig:2}b).  GFP::DmCa\textsubscript{v}3 is expressed in well-structured neuropils like the antennal lobes, the mushroom bodies, the central complex (Fig. \ref{fig:2}c-h), the optic lobes, as well as in some of the less-structured neuropils.  The central complex---comprising the fan-shaped body, ellipsoid body, noduli, and protocerebral bridge---shows the strongest expression with the ventral fan-shaped body and ventral noduli particularly prominent (Fig. \ref{fig:2}e and g).  In mushroom body neurons, there is far more GFP::DmCa\textsubscript{v}3 in the dendrite-rich calyx of the dorso-posterior brain (Fig. \ref{fig:2}h) than the axon-rich lobes of the anterior brain (Fig. \ref{fig:2}d).  GFP::DmCa\textsubscript{v}3 is also limited to the posterior mushroom body peduncles, which are the fiber tracks that join the posterior calyces with the anterior mushroom body lobes (Fig. \ref{fig:2}f).  These results suggest strict regulation of the subcellular localization of DmCa\textsubscript{v}3 channels in the brain.  We also next  visualized the projections of DmCa\textsubscript{v}3-expressing neurons using another knock-in allele, \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}. In \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}, theGal4 coding sequence replaces the  first coding exonof DmCa\textsubscript{v}3  andits  flanking introns to drive of DmCa\textsubscript{v}3 are replaced by the Gal4 coding sequence.  This puts  GAL4 expression under the control of the endogenous DmCa\textsubscript{v}3 promoter (Fig. \ref{fig:3}a). We visualized Consistent with our results using GFP::DmCa\textsubscript{v}3, \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} drives  the projections expression  ofDmCa\textsubscript{v}3-positive neurons by using DmCa\textsubscript{v}3\textsuperscript{Gal4} to drive  a membrane-tethered mCherry (UAS-mChRFP).  Consistent with \emph{GFP::DmCa\textsubscript{v}3}, \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} showed broad expression pattern in (UAS-mChRFP) broadly across  the brain (Fig. \ref{fig:S1}a). We observed strong co-localization of the \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}-driven mCherry signal The DmCa\textsubscript{v}3\textsuperscript{Gal4}}>mCherry  and \emph{GFP::DmCa\textsubscript{v}3} GFP::DmCa\textsubscript{v}3 signals are strongly co-localized, including  in the neuropils central complex and mushroom bodies  (\ref{fig:S1}b and c}), suggesting c}).  This suggests  both expressions are driven by reagents reflect proper expression from  the same endogenous DmCa\textsubscript{v}3 promoters. promoter.