The miR-51 family is redundantly required for embryogenesis, growth, male mating, and pharyngeal attachment. (A) Summary of abnormal phenotypes observed in mir-51 family mutants. The nDf58 allele is a deletion covering mir-54, mir-55, and mir-56. The nDf67 allele is a deletion covering mir-51 and mir-53. mjEx123 is an extrachromosomal transgene that includes a mir-52 genomic fragment. Single mutants of the mir-51 family show no obvious abnormal defects, but animals multiply mutant for mir-51 family genes show synthetic abnormalities. Let, lethal. “Yes” in the “Let” column indicates that a strain was not viable under normal laboratory conditions. Pun, pharynx unattached. Number of animals scored as Pun and total number of animals scored are given (percentage is in parentheses). Gro, slow growth. The asterisk in A indicates that only ∼5% of progeny show a slow growth phenotype. Mating defective column: “Yes” indicates that males fail to mate successfully under standard conditions. For nonviable genotypes, phenotypes were assessed in offspring of rescued homozygotes. (B–G) In embryos lacking all members of the miR-51 family, the pharynx detaches from the anterior hypodermis. mir-51 family mutant animals carrying (B, D, and F) or not carrying a (C, E, and G) a miR-52 expressing extrachromosomal array (mjEx123) were observed. Six different animals are shown. Red arrow: anterior pharynx. White arrow: anterior hypodermis. Scale bar: 20 μm.

The miR-51 family is sufficient in the pharynx to maintain attachment of the pharynx to the mouth. (A) Diagrammatic representation of morphogenesis of the buccal cavity (mouth). Arcade cells and arcade (green), pharyngeal epithelium and pharynx (orange), and the hypodermal cell Hyp1 (beige) are shown. (B–G) The anterior pharynx is polarized in wild-type and mir-51 family mutant embryos. Embryos were fixed and stained using an antibody against mCHERRY to identify those carrying a mir-52 rescuing transgene marked by DLG-1∷mCHERRY (mjEx123) (data not shown) and with α-MH27 antibody (B and E), which is specific for AJM-1, to visualize adherens junctions. (C and F) DAPI stain. (D and G) Merge. (H–O) Arcade cells are often incorrectly positioned in mir-51 family mutant embryos. Live imaging. (H and J) plasma membrane localized GFP in arcade and pharynx. Insets show the region bounded by the dotted lines in more detail. (L–O) Arcade cells become detached from the developing pharynx. Live imaging as in H–K. (L and N) White brackets: arcade cell bodies. (P) Tissue-specific rescue of the Pun phenotype. Animals carrying pha-4∷mir-52∷unc-54 transgenic arrays in the mir-51 family mutant background. Five independent transgenic lines showed an increased number of animals with an attached pharynx as compared to a control pha-4∷gfp∷unc-54 transgenic array.

The miR-51 family acts in part through CDH-3 to regulate pharyngeal attachment. (A) Venn diagram showing the number of unique genes predicted to be targeted by all six members of the miR-51 family by three different target prediction algorithms. Those predicted by two or more programs are outlined in black. (B–M) miR-52 can regulate the cdh-3 3′ UTR in the hypodermis. Transgenes: dlg-1∷mcherry, col-10∷gfp∷cdh-3 (WT, MUT), and myo-2∷mcherry∷unc-54. (N) cdh-3 3′ UTR regulation through miR-52 was assessed in the arcade cells using bath-15∷gfp∷cdh-3 transgenes. Wild-type (WT) and two different mutant cdh-3 3′ UTRs were used (DEL, MUT; see Figure S3). Larvae were imaged at the L4 stage. The mean pixel intensity for arcade cells was quantified. Error bars indicate the standard error of the mean (n > 30, P > 0.0001, Student's t-test). (O) Overexpression of CDH-3 (mjEx288 or mjEx289) enhances the unattached pharynx phenotype of mir-51 mir-52 mir-54 mir-55 mir-56 mutants (n4473 n4114; nDf58). The Pun phenotype was quantified in these mutants and in mir-52 rescued embryos (mjEx123) in the presence or absence of a transgene carrying cdh-3 genomic sequence (P < 0.0001, χ² test). (P) Hypothetical model of miR-51 family activity during pharyngeal adhesion. CDH-3 is in blue; CDH-4 is in red.

The conserved miR-51 family is widely expressed throughout development in C. elegans. (A) The miR-51 is highly conserved. Its origin predates the origin of Bilateria. Bases 2–8 (“seed region”) are highlighted. (B) Genomic location of miR-51 family genes in the C. elegans genome. Mutant alleles shown in boldface type. mir-54, mir-55, and mir-56 are likely expressed from the same transcript. Not drawn to scale. (C–J) GFP fluorescence and DIC/Nomarski imaging of animals carrying promoter∷gfp fusion transgenes. mir-51∷gfp is expressed in anterior ventral cells (C and D, Z-projection; 10 μm), and mir-52∷gfp is ubiquitously expressed in the soma (E and F). mir-53∷gfp is also ubiquitously expressed, but expression is weak in the gut and in anterior pharyngeal cells (G and H), and mir-54–56∷gfp is very weakly expressed in anterior ventral cells (I and J, gain adjusted, Z-projection; 10 μm). Germline expression cannot be assessed using these reporters due to possible transgene silencing. Coelomocyte expression may represent uptake of GFP exported from other cells. Developmental stage: embryos at 1.5-fold stage. Scale bar: 20 μm. (K) Summary of miR-51 family expression patterns as assessed by GFP expression in transgenes in embryos and/or larvae. (L) Northern blotting confirms the absence of miR-51 family miRNAs in mir-51 family mutants. A total of 20 μg total RNA extracted from mixed-stage animals was used for each lane. Two blots were probed and stripped three times in the following order: (i) miR-51, miR-52, miR-54; (ii) miR-53, miR-56, miR-55. The probe for miR-54 was not specific and is not shown, but probes for miR-55 and miR-56 confirmed the absence of the miR-54–56 cluster in nDf58 mutants. miR-52 is detected by the miR-53 probe (compare lanes 4 and 7) because these two miRNAs differ by only one nucleotide. However, miR-53 is not detected by the miR-52 probe at the same exposure, likely because miR-53 is expressed at a lower level than miR-52.