A. Sequence alignment of the lin-4 family designated in the 5′ to 3′ orientation. B. Northern blot analysis of lin-4 and miR-237 shows distinct expression patterns at early stages of nematode development. U6 RNA is shown as a loading control. C. Sequence alignment of the let-7 family. mir-84 shares the closest sequence homology with let-7 compared to the other family members. D. Northern blot analysis of let-7, miR-84, miR-48 and miR-241 reveals distinct temporal expression patterns for these closely related miRNAs. U6 is shown as a loading control. Abbreviations: eL1, early larval stage 1; mL1, mid-L1; eL2, early L2; mL2, mid-L2; eL3, early L3; mL3, mid-L3; eL4, early L4, mL4, mid-L4; eAd, early adult stage; mAd, mid-Ad; Ad/egg, late stage adults carrying eggs.

Transgenic animals carrying mir∷gfp fusion constructs for lin-4, mir-237, let-7, mir-84, and mir-48 were analyzed throughout C. elegans development for seam cell expression. The larval stages shown in each panel denotes the earliest stage at which seam cell expression was first detected for the specified mir∷gfp construct. White lines indicate positive expressing seam cells. The schematic diagram to the left depicts the characteristic division pattern of the hypodermal seam cells during larval development, in which after each seam cell division, one daughter cell fuses with the hypodermis (hyp7) and the other daughter cell divides again, terminally differentiating at the adult stage (indicated by the horizontal black bars) (Rougvie, 2001).

Animals carrying mir∷gfp fusion constructs for lin-4, the lin-4 homologue, mir-237, let-7, and the let-7 family members, mir-84 and mir-48 were analyzed for expression in the vulval precursor cells (VPCs), P3.p-P8.p, and in the anchor cell of the somatic gonad (white arrows) at third larval (L3) stage. A schematic diagram of the spatial expression patterns for the lin-4 and let-7 family members during vulva development at the L3 stages is shown to the right. The morphogenic signal, LIN-3, secreted by the anchor cell (AC) induces the closest vulva precursor cell, P6.p, to adopt the 1° cell fate and the two adjacent VPCs, P5.p and P7.p, to adopt the 2° cell fates. 1° and 2° cells will later differentiate into the mature vulva (V). VPCs that are not induced by the anchor cell (P3.p, P4.p, and P8.p) will take on non-vulval 3° cell fates, fuse with the hyp 7 cell, and become part of the outlaying epidermis (E). Our mir∷gfp fusion studies for lin-4 (blue), let-7 (blue), mir-237 (green), mir-84 (green), and mir-48 (red) showed unique and partially overlapping expression patterns in both the anchor cell of the somatic gonad and the VPCs, indicating that these miRNAs may play an important role during cell fate specification and vulva formation. The (*) indicates that mir-84 shows dynamic expression in the VPCs and by the very late L3 stage, mir-84 expression was noted exclusively in the P5.p, P6.p, and P7.p cells (Johnson et al., 2005). Note that Pn.p cells depicted in panels for lin-4, let-7, and mir-48 have undergone one cell division.

Northern blot analysis of miR-237, miR-84, miR-48, and miR-241 expression during the third larval (L3) stage 3, the forth larval (L4) stage, and in the adult (Ad) with total RNA taken from wild-type worms (N2), lin-4 (e912) loss-of-function mutants, and daf-12 (rh61) ligand binding domain mutants. All the miRNAs analyzed showed some degree of regulation by both heterochronic genes. U6 is shown as a loading control. The numbers shown in red are the normalized ratios of miRNA expression in heterochronic mutants vs. wild-type N2 animals.

Animals carrying mir∷gfp fusion constructs for lin-4 (A), the lin-4 homologue, mir-237 (B), and the let-7 homologue, mir-84 (C), were analyzed for expression in the developing gonad at the first larval (L1) stage and the third larval (L3) stage 3. Note that lin-4 was not detected in the gonad at the L1 stage (white arrow). Abbreviations: AC, anchor cell; dtc, distal tip cell; u, uterine cells including the anchor cell; Z1 and Z4, somatic gonad progenitor cells.