1. Volume 8, Issue 3, Pages 321-330 (March 2005)
The Temporal Patterning MicroRNA let-7 Regulates Several Transcription Factors at the Larval to Adult Transition in C. elegans 
Helge Großhans, Ted Johnson, Kristy L. Reinert, Mark Gerstein, Frank J. Slack 
Developmental Cell 
Volume 8, Issue 3, Pages (March 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
RNAi of Both Weak and Strong let-7(n2853) Suppressors Causes a Significant Reduction of the Levels of the Respective Target mRNAs
Aliquots of undiluted or 1:5 diluted templates were used for RT-PCR as indicated to determine whether target mRNA levels were reduced compared to the control eft-2 mRNA; “-” indicates the control reaction without reverse transcriptase.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2
daf-12 Interacts with let-7 and Affects the Timing of Seam Cell Fusion
(A) let-7(n2853) mutants die at the L4 molt by bursting through the vulva (arrowhead), while (B) daf-12(m20) let-7(n2853) double mutant adult animals survive into adulthood, as indicated by the presence of oocytes (asterisk) and a functional vulva (arrowhead) (B). (C) These animals produce viable embryos. (D) daf-12(m20) let-7(mn112) adults are viable and fertile (arrows, embryos). (E and F) Alae are absent from (E) let-7(n2853) single mutant worms but present (arrows) in (F) let-7(n2853) daf-12(m20) double mutant worms grown at 20°C. (G) Wild-type N2; wIs79 animals have unfused seam cells at the early L4 stage as indicated by AJM-1/GFP signal at the cell junctions (arrowheads). (H) daf-12(RNAi) animals occasionally display precocious seam cell fusion as indicated by lack of GFP signal at cell junctions. Arrows in lower DIC panels in (G) and (H) point to distal gonad tips at a different part of the animals, confirming appropriate developmental stage. (I) Precocious seam cell fusion is strongly enhanced in lin-41(ma104); daf-12(RNAi) double mutant animals at the L3 molt. Magnification in (A)–(D) is 400× except for the left panel in (C), which was observed at 100×; in (E)–(H) it is 630×. Anterior of each animal is left, ventral down.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
The daf-12 3′UTR Mediates Posttranscriptional, let-7-Dependent Control of Gene Expression
(A) Examples of LCS:let-7 duplexes predicted for daf-12; let-7 are shown in gray. Arrows indicate where the sequence of C. briggsae daf-12 (uppercase) differs from C. elegans daf-12. (B) Diagram of the col-10::lacZ::daf-12 reporter gene construct. (C) The reporter gene is efficiently expressed in early larval (L1–L3 stage) animals but downregulated in L4 and adult animals. Deletion of the first four (ΔLCS1–4) or the last four (ΔLCS5–8) LCSs impairs downregulation (*p < 0.05, **p < 0.01, paired Student’s t test). (D) Repression is relieved in the let-7(n2853) mutant background (four lines) (*p < 0.05). Error bars indicate standard deviations.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
The pha-4 3′UTR Regulates let-7-Dependent Downregulation of Intestinal pha-4::gfp Expression
(A) Examples of LCS:let-7 duplexes predicted for pha-4. (B and D) A pha-4::gfp::pha-43′UTR transgene is highly expressed in the intestinal cells of early stage (L3) larvae but is only weakly visible in L4 and adult animals. (C) Strong intestinal expression persists in adult animals when the pha-4 3′UTR of the transgene is replaced by the unc-54 3′UTR. (E) let-7(n2853) mutant animals continue to express the pha-4::gfp::pha3′UTR transgene as adults. Images in (D) and (E) were taken for the same exposure time and processed identically.
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5
The 3′UTRs of Additional Predicted Transcription Factor Targets Confer let-7-Dependent Regulation
(A and B) Examples of LCS:let-7 duplexes predicted for the indicated transcription factors; the let-7 sequence is shown in gray. (C) The expression of the col-10::lacZ reporter construct is downregulated in wild-type (N2) adult animals when the reporter contains the lss-4 or die-1 3′UTRs (*p < 0.05, **p < 0.01, paired Student’s t test; error bars are standard deviations between independent lines). (D and E) In contrast, expression of the reporter genes persists in adult let-7(n2853) mutant animals (right columns). Four let-7(n2853); col-10::lacZ::lss-4 and seven let-7(n2853); col-10::lacZ::die-1 lines, respectively, were assayed along with their respective isogenic parent (*p < 0.05, **p < 0.01, paired Student’s t test).
Developmental Cell 2005 8, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions