Supplemental Materials and Methods s1

Sokol et al., Supplemental Material

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Supplemental Material

Supplemental Materials and Methods

RACE

The full-length pri-let-7-C transcript was determined by RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE) using the GeneRacer Kit (Invitrogen). Two independently isolated samples of total RNA, one from mixed stages of pupae and the other from Ecdysone-treated S2 cells, were used to generate cDNAs according to the manufacturer's instructions. The 5' and 3' ends of the pri-let-7-C transcript were amplified by nested PCR using pairs of oligos located between the miR-100 and let-7 coding regions and oligos supplied by the manufacturer. PCR products were subcloned and sequenced by standard methods. The pri-let-7-C sequence was deposited in GenBank under the accession number EU624487.

Plasmid Construction

let-7-CKO1 knockout vector. A portion of the let-7-C locus was subcloned from Bac clone BACR13N02 (BACPAC Resources, Oakland, CA) into the pP{Target70} vector (a gift from J. Sekelsky, University of North Carolina, Chapel Hill, NC) as a 11,545 bp SalI-SalI fragment creating pP{Target70, let-7-CWT}. A 4,472 bp NsiI-NsiI fragment containing the let-7-C miRNA coding sequences was subsequently subcloned from pP{Target70, let-7-CWT} into pLitmus29 (New England Biolabs). Two changes were made to this plasmid using the Transformer Site-Directed Mutagenesis Kit (BD Biosciences). The AgeI site located 1,589 bp upstream of miR-100 was converted to an I-SceI site and a 1,071 bp fragment containing the three mature let-7-C miRNA sequences was deleted. Both of these changes were confirmed by PCR. This modified NsiI-NsiI fragment was subsequently cloned back into pP{Target70, let-7-CWT} generating the knockout vector, pP{Target70, let-7-CKO1}.

let-7-CGKI knockout vector. A modified ends-out targeting vector, pKIKO (Yang Hong, personal communication), containing 5' cloning polylinker sites, 3' cloning polylinker sites and the mini-white marker gene, was used in making the construct. The 2.7 kb right arm of the targeting vector was amplified by PCR from BACR04D05 clone and cloned into the BstB I and Xho I sites of the pKIKO vector. The Gal4-containing fragment was amplified from the pGATN vector by PCR and cloned into pBluescript SK(+) vector (Stratagene) using EcoR I and Kpn I sites. The 4.1 kb left arm of the targeting vector was amplified by PCR from BACR04D05 clone and fused with the Gal4 in pBluescript vector using Not I and EcoR I sites. The left arm and Gal4 fusion fragment was then subcloned into the pKIKO targeting vector, which already contained the 2.7kb right arm, using Not I and Sac II sites. Sequences of oligos used in PCR are available upon request.

let-7-C rescuing transgenes. The let-7-C locus was subcloned from Bac clone BACR13N02 into the pP{W8} vector as a 13,288 bp BamHI-BamHI fragment and a 4,695 bp BamHI-XbaI fragment creating pP{W8, let-7-C}. A 3,231 bp AgeI-XbaI fragment containing the let-7-C miRNA coding sequences was then subcloned from pP{W8, let-7-C} into pBluescript SK(+) (Stratagene). The following sequences were subsequently deleted from this plasmid using the Transformer Site-Directed Mutagenesis Kit (BD Biosciences): AACCCGTAAATCCGA (miR-100 seed sequence), GAGGTAGTAGGTT (let-7 seed sequence), and TCCCTGAGACCCTAA (miR-125 seed sequence). The deletion of these sequences was confirmed by PCR and the modified fragments were cloned back into pP{W8, let-7-C}, generating pP{W8, let-7-CDmiR-100}, pP{W8, let-7-CDlet-7}, and pP{W8, let-7-CDmiR-125} respectively. In addition to the pP{W8, let-7-C}, pP{W8, let-7-CDmiR-100}, pP{W8, let-7-CDlet-7}, and pP{W8, let-7-CDmiR-125}, a fifth let-7-C derivative transgene was generated, pP{W8, let-7-CDlet-7-C}, that removed the 3kb encoding miR-100, let-7 and miR-125 and contained the remaining 15 kb of let-7-C genomic. This control transgene was used when analyzing transheterozygous let-7-CKO1/let-7-CGKI mutants in behavioral assays (see below).

Generation of let-7-C knockouts

let-7-CKO1 knockout. Generation of the let-7-CKO1 mutant flies was initiated using ends-in homologous recombination (Rong and Golic 2000). Briefly, an X chromosome insertion of P{Target70, let-7-CKO} was used as a donor. y1w*; P{ry+,70 FLP}23, P{v+, 70 I-SceI}4A/TM6 virgins were crossed to P{Target70, let-7-CKO1} males and their progeny were heat shocked for 1 hour at 38oC on the 3rd and 4th days of development. Putative targeting events were identified by the loss of red/white eye mosaicism induced by FLP. Stocks of flies were generated from independent flies carrying putative targeting events. Subsequent Southern blotting and PCR analyses identified those stocks that contained targeting events at the let-7-C locus, which resulted in a cis duplication of the locus. However, for all stocks in which the P{Target70, let-7-CKO} had been correctly targeted to the let-7-C locus, I-CreI-mediated reduction to a single allele proved unsuccessful. Therefore, we employed an alternative, FRT-based strategy to delete the let-7-C locus using an FRT site targeted to the let-7-C locus in the P{Target70, let-7-CKO1} transgene and an FRT site in trans located in a piggyBac transposon insertion in the neighboring CG10283 locus (Figure S2). Briefly, hsFLP; al, P{Target70, let-7-CKO1}, sp virgins were crossed to PBac{WH}CG10283F03185 males and their progeny were heat shocked for 1 hour at 38oC on the 2nd and 3rd days of development. Upon eclosion, flies displayed variegated eye color, suggesting that recombination events between the two FRT sites had resulted in the deletion of the white gene contained within the PBac{WH}CG10283F03185 insertion. To select such recombination events, males with variegated eye color were individually crossed to al sp virgins and al- sp+ progeny were selected and backcrossed to generate a balanced stock. Deletion of a portion of the let-7-C locus in this al- sp+ stock was confirmed by PCR and therefore the stock was renamed and referred to as the let-7-CKO strain. let-7-CKO1 stocks were maintained over either CyO, P{Gal4-Kr.C}DC3, P{UAS-GFP.S65T}DC7 (hereafter referred to as CyO-GFP) or T(2;3)B3, CyO: TM6B, Tb1 balancer chromosomes.

let-7-CGKI knockout. Ends-out homologous recombination was used to generate the let-7-CGKI mutants. Standard P-element transformation of the let-7-CGKI targeting construct was used to generate transgenic donor lines. Female homozygous donor flies were crossed to yw/Y, hs-hid; hs-FLP, hs-SceI/CyO, hs-hid males (Yang Hong, unpublished). The resulting progeny larvae were heat-shocked at 38°C for 75 minutes twice during their development, once at 48 hrs after egg laying (AEL) and then again at 72 hrs AEL. Mosaic-eyed females were crossed to w; CyO/Sco balancer stock and targeting candidates were screened in the next generation as nonvariegated red-eye flies. Male candidates were crossed to w; CyO/Sco balancer stock again for chromosome mapping and balancing. The candidates with mine-white marker mapped to 2nd chromosome were screened and confirmed by genomic PCR.

Mutant Strain Construction

Balanced let-7-CKO1 stocks containing a P{W8, let-7-C} or P{W8, let-7-C} derivative transgene were separately crossed to a common, independently maintained, balanced let-7-CGKI stock. Transheterozygous let-7-CKO1/let-7-CGKI mutants carrying a single copy of the P{W8, let-7-C} or P{W8, let-7-C} derivative transgene were selected for further analysis. Because the P{W8, let-7-C} or P{W8, let-7-C} derivative transgenes were generated from the same parental stock, we assumed that the progeny resulting from these independent crosses differed from one another only by their let-7-C miRNA genotype.

Developmental Viability Analysis

Terminal phase and timing of pupariation were examined as follows. let-7-CGKI/ CyO-GFP flies were crossed to let-7-CKO1/CyO-GFP flies. Resulting embryos were dechorionated and scored for GFP expression between 15 and 18 hours after egg laying using a Zeiss M2 fluorescent stereo microscope. Transheterozygous let-7-CKO1/let-7-CGKI embryos, identified based on the absence of GFP expression, were selected. Wildtype control embryos were dechorionated and aged in parallel. Hatched first instar wildtype, and transheterozygous let-7-CKO1/let-7-CGKI larvae were collected, placed into new vials in batches of 20-25 larvae per vial, and grown at 25oC. Viability was calculated as the percentage of hatched first instar larvae that survived to adulthood. Once adults had eclosed, pre-adult terminal phase was determined by analyzing the stage of arrested pupae as defined in Bainbridge and Bownes (1981). Developmental viability of P{W8, let-7-C} or P{W8, let-7-C} derivative transgenic strains was conducted by crossing let-7-CGKI/ CyO-GFP flies to a let-7-CKO1/CyO-GFP strain that contained one of the transgenes and performing the same analysis as decribed here. To analyze the developmental progression of metamorphosis (Fig. S3), white prepupae were collected within an hour of pupariation, placed in a petri dish on a wetted Kimwipe, and grown at 25oC.

Adult Behavioral Assays

As described above, all flies tested in behavioral assays carried the P{W8} vector which restores wildtype eye color to w1118 derivative strains. Behavioral assays analyzing motility were all performed at a fixed 2 hr interval, 6 to 8 hrs after lights-on in a fixed light/dark cycle.

Spontaneous Locomotion Assay. Newly eclosed males were transferred in batches of 15 to fresh vials and aged for 3-4 days. They were then individually transferred, without anesthesia, to an approximately 1 cm3 chamber. Locomotion was quantified as the number of times that the fly walked across the midline of the chamber over a 4-minute period (Stockinger et al. 2005). Results from eight flies of each genotype are presented in Figure 2.

Climbing Assay. Newly eclosed males were transferred in batches of 10-15 to fresh vials and aged for 3-4 days. They were then transferred, without anesthesia, to a 15-ml conical tube, tapped to the bottom of the tube, and their subsequent climbing activity was videorecorded for 20 seconds. To quantify climbing activity, the location of each fly relative to the bottom of the tube was noted after 10 seconds of climbing. The assay was performed three times for each group of 15 flies. For the data presented in Figure 2, the following number of flies were analyzed: wildtype, 60; let-7-CK01/GKI, 51; rescued let-7-CK01/GKI, 59; mir-100D, 64; let-7D , 70; mir-125D, 60.

Flight Assay. Newly eclosed males were transferred in batches of 15 to fresh vials and aged for 3-4 days. They were then dumped into the top of a cylinder of 450 mm height and 80 mm diameter with a plate of mineral oil at the bottom and the number of flies trapped in the oil was counted immediately. For each genotype, flight was quantified as the percentage of total flies tested that were not trapped in oil. For the data presented in Figure 2, the following number of flies were analyzed: wildtype, 50; let-7-CK01/GKI, 96; rescued let-7-CK01/GKI, 132; mir-100D, 87; let-7D , 105; mir-125D, 161.

Fertility Assay. Mating tests were used to assay fertility. Individual virgin males or females were mated to three wildtype females or males, respectively, in single vials for ten days. Vials were then scored for the progeny, and sterility was defined as either the complete absence of progeny, or between one and ten progeny. Vials in which the tested fly has died were not scored. For the data presented in Figure 2, the following numbers of males and females were analyzed: wildtype, 15 males, 32 females; let-7-CK01/GKI, 22 males, 34 females; rescued let-7-CK01/GKI, 37 males, 39 females; mir-100D, 37 males, 35 females; let-7D , 38 males, 125 females; mir-125D, 22 males, 38 females.

Oviposition Assay. Individual virgin females were mated to three wildtype males in single vials for one day. They were then transferred to a second fresh vial, and the number of eggs that the female laid over the subsequent 24-hour period was counted. For the data presented in Figure 2, the following number of flies were analyzed: wildtype, 15; let-7-CK01/GKI, 10; rescued let-7-CK01/GKI, 20; mir-100D, 20; let-7D , 19; mir-125D, 21.

Quantification of DM Neuromuscular Features

After abdomen dissection and rhodamine phalloidin and HRP double staining, dorsal DM muscles in one A4 hemisegment per animal were imaged with a confocal laser-scanning microscope. This set of ~ 6-8 DM muscles are readily identifiable, situated between two landmarks (the dorsal vessel and a more ventral DM that displays a unique HRP pattern). The dorsalmost ~ 3 DM muscles were not scored because pericardial cells displaying intense HRP staining usually occluded them. A 15-25 mm deep Z-series was collected that contained the entire DM set as well as there innervating neuromuscular junctions. Z-series were collected from at least five sets of DM muscles per genotype. Using this collection of images, the width of the DMs and the length of the neuromuscular junctions were measured using a ruler, and the measurements were scaled appropriately. DM nuclei number was quantified by visually identifying DM muscle nuclei based on triple labeling of dorsal abdomens with DAPI, rhodamine phalloidin, and HRP.

Supplemental Figures and Figure Legends

Figure S1: Phylogenetic conservation of the genomic organization of let-7-C miRNAs. The tree is based on the current view of metazoan phylogeny (Adoutte et al. 2000). The nucleotide distances (in kb) between the sequences encoding the mature forms of miR-100, let-7, and miR-125 are indicated. The absence of triangles indicates the absence of the mature miRNA sequence in the corresponding genome. Numbers in parentheses indicate the chromosomal locations of the three let-7-C loci in Homo sapiens and the unlinked let-7 and miR-125 (lin-4) loci in C. elegans. Phylogenetic conservation indicates that miR-100 was present in the last common ancestor of cnidarians and bilaterians (shaded circle), let-7 and miR-125 as well as the clustering of miR-100, let-7 and miR-125 was present in the last common ancestor of protosotomes and deuterostomes (white circle), and the relative ordering of miR-100 g let-7 g miR-125 has been widely maintained in the genomes of bilaterians. Portions of this data have been previously reported in Sempere et al. 2003 and Prochnik et al. 2007.

Notes: a. The human genome contains three miR-100 and mir-125 paralogues and, as presented in the diagram, each is located near a let-7 encoding sequence. In addition to the three let-7 encoding genes depicted, the human genome contains nine other let-7 genes.

b. The identification of miR-100 and let-7 sequences in the mate pair sequence traces of opposing ends of a single fragment (BGOB98823) of the Lottia gigantea genome indicates that miR-100 and let-7 are located in tandem, but by an unknown distance on the partially-assembled Lottia gigantea genome sequence.

c. The nonoverlap of contigs assembled around let-7 and miR-125 sequence from Capitella sp I sequence traces indicates that these two miRNA are not located within 8 kb of each other in the Capitella genome.

Figure S2: Generation of let-7-CKO1 mutant. (A) Schematic representation of the wildtype let-7-C locus. (B) Schematic representation of the pP{Target70, let-7-CKO1} targeting vector. Parentheses indicate location of the ~1.1 kb fragment containing miR-100, let-7 and miR-125 deleted in the targeting vector. (C) Schematic representation of the duplication of the let-7-C locus resulting from ends-in targeting of the P{Target70, let-7-CKO1} transgene. (D) Schematic representation of the PBac{WH}CG10283F03185 insertion located in the CG10283 gene neighboring let-7-C. (E) Schematic representation of the let-7-CKO1 mutation. The let-7-CKO1 mutation was generated by FRT-mediated recombination between two FRT sites, shown in (C) and (D) respectively, that resulted in the deletion of the intervening region. (F) PCR analysis confirming that a ~1.1kb fragment, containing miR-100, let-7 and miR-125, is deleted in let-7-CKO1 mutant animals. PCR analysis was performed on genomic DNA extracted from wildtype (+/+), let-7-CKO1 heterozygote (+/-) and let-7-CKO1 homozygous (-/-) adults using primers indicated as black arrows in (E).