Identification of the Rpd3 mutation
MARCM-based mosaic screen and SNP mapping leading to the identification of Rpd312-37
were analogous to procedures previously described (Chihara et al., 2007
). After narrowing the region to ~67 kb, all open reading frames and splice acceptor and donor sites were sequenced.
Identification of the prospero mutation
pros38 was isolated from a MARCM-based P-element forward genetic screen. pros38 possesses a p[mCD8-GFP, y+] transposon inserted early in the open reading frame of prospero, at amino acid 188. The insertion site was confirmed by inverse PCR.
MARCM was performed as described (Wu and Luo, 2006a
). Fly brains of both genders were dissected, fixed, and stained as described (Wu and Luo, 2006b
). Antibody conditions: rabbit anti-Rpd3 1:1000 (gift from J.T. Kadonaga), rabbit anti-acetylated lysine 1:1000 (Cell Signaling Technology #9441), mouse anti-Prospero 1:4 (Developmental Studies Hybridoma Bank #MR1A, C.Q. Doe), rat anti-mCD8α 1:100 (Invitrogen Caltag #RM2200), and mouse anti-nc82 1:40 (Developmental Studies Hybridoma Bank #nc82, E. Buchner).
Quantification of DL1 PN dendrite targeting
DL targeting (Figures , , ) includes full innervation of the DL1 glomerulus, as well as minor dendrite spillover into nearby glomeruli in the dorsolateral quadrant of the antennal lobe.
Prospero acts in postmitotic neurons to regulate PN dendrite targeting and axon terminal arborization
Postmitotic expression of Prospero suppresses Rpd3−/− PN phenotypes
Axon rank order
Confocal stacks of axons were blindly rank ordered by two independent observers with consistent results. Axons were ranked by degree of axon overbranching, with the lowest score denoting the least branching (closest to wild-type) (Figures , , ).
Rpd3 limits PN axon terminal arborization
Calculation of normalized fluorescence for Pros protein level
Confocal images were taken with all pixel values in the linear range. Background fluorescence was subtracted from the average pixel intensities in the DL1 PN nucleus and the highest Pros expressing nucleus in the section. Ratio of DL1 to highest intensity was calculated as (DL1-background)/(highest Pros-background) ().
Construction of UAS-Rpd3-V5
To generate UAS-Rpd3-V5
, we obtained Rpd3-V5
cDNA from K.T. Min (NIH, USA) (Cho et al., 2005
). The Rpd3-V5
fragment was amplified using the following primers (5′-3′): GGGGTACCCCAAAATGCAGTCTCACAGCAAAAAGCGCG and GACTAGTCTACGTAGAATCGAGACCGAGGAGAGGGTTAGG. The first primer adds a KpnI site and a Kozak sequence, and the second primer adds a stop codon and an SpeI site to the amplified Rpd3-V5
fragment. The PCR product was subcloned into the pUAST
vector (KpnI and XbaI). Germline transformation was performed using standard P-element transformation; an insertion on the 2nd
chromosome was used in all experiments.
Construction of UAS-HDAC3-3xFLAG
To generate UAS-HDAC3-3xFLAG, a full length cDNA (LD23745, Berkeley Drosophila Genome Project Gold cDNA, Drosophila Genomics Resource Center, Bloomington, IN) was amplified using the following primers (5′-3′): CACCCAAAATGACGGACCGTAGGGTGTC and CTACTTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGG TCTTTGTAGTCACTTTCTGCCGAATCGGGCTTGTCTTG. The first primer amplifies from the 5′ end and adds a CACC overhang for the TOPO reaction and a Kozak sequence. The second primer adds a C-terminal 3xFLAG tag. The PCR product was subcloned into pENTR-D/TOPO (Invitrogen) and recombined into the destination vector pUAST-Gateway-attB (described below) using LR Clonase II (Invitrogen).
Another UAS-HDAC3 was constructed similarly with a C-terminal V5 tag an alternative second primer (5′-3′): CTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCACTTTCTGCCGAATCGGGCTTGTCTTG. This construct yielded similar results to the 3xFLAG tagged construct (data not shown).
constructs were integrated into the P24 landing site (Markstein et al., 2008
) on the 2nd
Construction of pUAST-Gateway-attB
destination vector was created by PCR amplifying the Gateway cassette from pBPGUw
(Pfeiffer et al., 2008
) using the following primers (5′-3′): CACCTCGAGGTATCACGAGGCCCTTTC and CACTCTAGACTCGGCCGGCCGTTTATCAC. The first primer amplifies from the 5′ end and adds an XhoI site. The second primer adds an XbaI site to the 3′ end. The PCR product was cut and ligated into pUAST-attB
(XhoI and XbaI) (Bischof et al., 2007
Construction of UAS-3xFLAG-pros-L
UAS-1xFLAG-pros-L DNA was obtained from F. Matsuzaki. We amplified approximately 4.5kb of the 5′ side of pros-L using the following primers (5′-3′): GAAGATCTTCATGAGTAGCGATTACAAGGATGATG and CTCCCGCAGAGTCGATTCGACCACG. The first primer adds a BglII site to the 5′ end. The amplified fragment was digested with BglII and KpnI. The fragment was then subcloned into a pUAST vector (BglII and KpnI), which already contained the N-terminal 3xFLAG fragment between EcoRI and BglII sites. Then we amplified approximately 2.5kb of the 3′ side of pros-L, digested the fragment with KpnI and XbaI, and ligated with the plasmid above to obtain UAS-3xFLAG-pros-L.
UAS-3xFLAG-pros-L was used for transfection of S2 cell culture (see below).
For all genetic experiments, flies containing an insertion of UAS-pros-L on the X chromosome were kindly provided by C.Q. Doe.
Pros immunoprecipitation from wild-type embryos
Fly embryos were collected on grape plates for 14 hours, washed, and homogenized using a dounce homogenizer in RIPA buffer (150mM NaCl, 0.5% deoxycholic acid, 0.1% SDS, 50mM Tris pH 8.0, protease inhibitor cocktail (1:100, Sigma-Aldrich #P8340), 150nM trichostatin A (Sigma-Aldrich #T1952), 5mM sodium butyrate (Sigma-Aldrich #B5887), 10mM niacinamide (Sigma-Aldrich #N0636), and with or without 1% Triton-X). Immunoprecipitation was performed using anti-Pros antibody bound to Protein G beads.
The final precipitate was denatured in SDS sample buffer and protein was run on a standard SDS-PAGE gel, transferred, and blotted using anti-Rpd3 or anti-acetylated lysine antibody.
Pros immunoprecipitation from S2 cell culture
S2 cells were raised in Shields and Sang M3 Insect Medium (Sigma-Aldrich #S8398) supplemented with potassium bicarbonate, penicillin, streptomycin, and 10% fetal bovine serum. Cells were split at 24 hours prior and immediately before co-transfection of tubP-GAL4 and UAS-3xFLAG-pros-L using Effectene Transfection Reagent (Qiagen #301425). S2 cells endogenously express Rpd3.
48 hours after transfection, HDAC inhibitors were added for 4 hours at the following final concentrations: trichostatin A 150nM and sodium butyrate 5mM. Cells were lysed in the following immunoprecipitation buffer: 150mM NaCl, 1% Triton-X, 2mM EDTA, 50mM Tris pH 8.0, 10% glycerol, protease inhibitor cocktail 1:100, 150nM trichostatin A, and 5mM sodium butyrate. Immunoprecipitation was performed using anti-Pros antibody bound to Protein G beads.
The final precipitate was denatured in SDS sample buffer, run on a standard SDS-PAGE gel, transferred, and blotted using anti-Pros, anti-Rpd3, or anti-acetylated lysine antibody. Alternatively, the final precipitate was denatured in SDS sample buffer, run on a standard SDS-PAGE gel, stained using GelCode Blue Stain Reagent (Thermo Scientific #24590), and the Pros band cut from the gel for mass spectrometry. Mass spectrometry was performed by NextGen Sciences (Ann Arbor, MI) to determine the acetylation state of Pros.