Strains and genetics
Worms were raised at room temperature using OP50 Escherichia coli seeded on NGM plates. Strains with a pdk-1(sa680) or daf-2(e1370) mutation were raised at a permissive temperature of 16°C and analyzed at 22°C or 25°C, respectively. To control for maternal rescue in the first generation, age-1(mg44) and daf-18(mg198); age-1(mg44) mutants were analyzed as second-generation age-1(mg44) homozygotes. N2 Bristol was utilized as the wild-type reference strain. Strains obtained through the Caenorhabditis Genetics Center include: GR1032 age-1(mg44) II/mnC1 dpy-10(e128) unc-52(e444) II, VC204 akt-2(ok393) X, GR1308 daf-16(mg54) I; daf-2(e1370) III, JT9609 pdk-1(sa680) X, KR344 let-363(h98) dpy-5(e61) unc-13(e450) I; sDp2(I;f), HT1881 daf-16(mgDF50) I; daf-2(e1370) unc-119(ed3) III; lpIS12, HT1882 daf-16(mgDF50) I; daf-2(e1370) unc-119(ed3) III; lpIS13, HT1883 daf-16(mgDF50) I; daf-2(e1370) unc-119(ed3) III; lpIS14, KQ1366 rict-1(ft7) II, CF1038 daf-16(mu86) I, VC1027 daf-15(ok1412)/nT1 IV; +/nT1 V, CB1370 daf-2(e1370) III. SO26 daf-18(mg198) IV was provided by the Solari laboratory, Centre Leon Berard, Leon, France. GR1309 daf-16(mgDF47) I; daf-2(e1370) III was provided by the Ruvkun laboratory, Boston, MA, USA. OH99 mgIS18 IV and LE311 lqIS4 X were provided by the Hobert laboratory, New York, NY, USA. FX00399 akt-1(tm399) V was provided by the Japanese Knockout Consortium, Tokyo, Japan.
Molecular biology and transgenic lines
Expression clones were made in the pSM vector, a derivative of pPD49.26 (A. Fire, Stanford University School of Medicine, Stanford, CA, USA) with extra cloning sites (S. McCarroll and C. I. Bargmann, unpublished data). The plasmids and transgenic strains (0.5-30 ng/μl) were generated using standard techniques and co-injected with markers Punc-122::gfp or Punc122::dsRed (15-30 ng/μl): wyIs45 [Pttx3::gfp::rab3], wyIs92 [Pmig-13::snb-1::yfp+odr-1::rfp], olaEx20 [Pttx3::mch, Pglr3::mch, Pdaf-18::daf-18 cDNA, Punc-122::GFP], olaEx25 [Pttx3::mch, Pglr3::mch, Pdaf-18::daf-18 cDNA, Punc-122::GFP], olaEx72 [Pttx-3b::daf-18 cDNA, punc-122::GFP], olaEx73 [Pttx-3b::daf-18 cDNA, Punc-122::GFP], olaEx528 [Pttx-3b::GFP, Punc-122::GFP], olaEx529 [Pttx-3b::GFP, Punc-122::GFP], olaEx531 [Pttx-3b::GFP, Punc-122::GFP], olaEx532 [Pttx-3b::GFP, Punc-122::GFP], olaEx533 [Pttx-3b::GFP, Punc-122::GFP], olaEx534 [Pttx-3g::HRP::CD2::GFP, Punc-122::GFP], olaEx760 [Pttx-3g::GFP, Punc-122::GFP], olaEx761 [Pttx-3g::GFP, Punc-122::GFP], olaEx762 [Pttx-3g::GFP, Punc-122::GFP], olaEx763 [Pttx-3g::mCH, Pdaf-16b::GFP], olaEx764 [Pttx3::mch, Pglr3::mch, cosmid R13H8, Punc-122::GFP].
Fluorescence microscopy and confocal imaging
Images of fluorescently tagged fusion proteins were captured in live C. elegans using a 60× CFI Plan Apo VC, NA 1.4, oil objective on an UltraView VoX spinning disc confocal microscope (PerkinElmer). Worms were immobilized using 50 nM levamisole (Sigma), oriented anterior to the left and dorsal up.
Mosaic analysis was conducted on daf-18(mg198)
animals as described previously by expressing unstable transgenes with the rescuing pdaf-18::daf-18
cDNA (Solari et al., 2005
) or cosmid R13H8 (for daf-16
mosaics), and cytoplasmic cell-specific markers in RIA and AIY (Colon-Ramos et al., 2007
; Yochem and Herman, 2003
). Animals were inspected for retention of the transgene and rescue using a Leica DM5000 B microscope.
Quantification of AIY outgrowth in wild-type and mutant animals was carried out on a Leica DM5000 B microscope. Neurite truncations were scored as a failure of the two AIY neurites to meet at the dorsal midline. Neurite outgrowth in embryos was quantified by measuring the length of the whole neurite and Zone 3 (dorsal portion of the neurite) regions in confocal micrographs using Volocity 5 software (Improvision). Zone 3 length was averaged using images of several embryos (three to six) taken at each developmental time point, with individual Zone 3 lengths determined as described above. Embryos were assigned a stage based on morphological characteristics and developmental time points, such as the beginning of twitching.
Quantification of AIY neurite length in wild-type, daf-18(mg198), daf-16(mgDF47); and daf-16(mgDF47); daf-18(mg198) L4 animals was carried out by imaging the length of the dorsal portion of both AIY axons (Zone 2 and Zone 3) using a 60× CFI Plan Apo VC, NA 1.4, oil objective on an UltraView VoX spinning disc confocal microscope (PerkinElmer). Zone 2 and Zone 3 were defined as the portion of the AIY neurite that turned and extended dorsally, respectively. These regions were measured in 3D by using Volocity software (Improvision).
Statistical significance was calculated using Student’s t-test or Fisher’s Exact Test.
Transfection and immunocytochemistry
Primary cerebellar granule neurons were prepared from P6 Long Evans rat pups as described (Konishi et al., 2002
). One day after culture preparation, neurons were treated with cytosine arabinofuranoside (AraC) at a final concentration of 10 μM to prevent glial proliferation. Granule neurons were transfected using a modified calcium phosphate method as described (de la Torre-Ubieta et al., 2010
). Cells were fixed at the indicated time points and subjected to immunocytochemistry with the GFP (Molecular Probes) antibody together with the MAP2 (Sigma) or Tau1 (Chemicon) antibodies, and stained with the DNA-binding dye bisbenzimide (Hoechst 33258).
Morphological analysis of cerebellar granule neurons
To characterize the morphology of cerebellar granule neurons, individual images were captured randomly and in a blinded manner on a Nikon eclipse TE2000 epifluorescence microscope using a digital CCD camera (Diagnostic Instruments). Images were imported into Spot Imaging Software (Diagnostic Instruments) and the length of neuronal processes was analyzed by tracing. Total length is the length of processes including all its branches added together for a given neuron. To analyze neuron polarization, neurons were scored in a blinded manner as polarized or non-polarized as previously described (de la Torre-Ubieta et al., 2010
; Shi et al., 2003
). A neuron in which the longest neurite was at least twice as long as the other neurites was considered to be polarized. Data were collected from three independent experiments with 50-100 neurons scored per condition per experiment.
RNAi and rescue constructs
A DNA template-based method of RNAi was used to express short hairpin RNAs (shRNAs) targeting the sequence GAGCGTGCCCTACTTCAAGG in FOXO1, FOXO3 and FOXO6 (de la Torre-Ubieta et al., 2010
). Sequences for the scrambled shRNAs are TACGCGCATAAGATTAGGGTG (U6/scr1) and AAGTGCCAATTTCGATGATAT (U6/scr2). The rescue construct for FOXO6 (FOXO6-Res) was generated by engineering silent mutations (indicated by bold font) on FOXO6 as follows: CGTC
TTCAAGG (de la Torre-Ubieta et al., 2010
Statistical analyses were performed using GraphPad software. In experiments in which only two groups were analyzed, comparison of the two groups was carried out using Student’s t-test. Pairwise comparison within multiple groups was carried out by analysis of variance (ANOVA) followed by the Bonferroni post-hoc test. All histogram data were obtained from three or more independent experiments and are presented as mean ± s.e.m. unless otherwise specified. Statistical information and the total number of cells analyzed per experiment are provided in the figure legends.