Makorin ortholog LEP-2 regulates LIN-28 stability to promote the juvenile-to-adult transition in Caenorhabditis elegans

Abstract

The heterochronic genes lin-28, let-7 and lin-41 regulate fundamental developmental transitions in animals, such as stemness versus differentiation and juvenile versus adult states. We identify a new heterochronic gene, lep-2, in Caenorhabditis elegans. Mutations in lep-2 cause a delay in the juvenile-to-adult transition, with adult males retaining pointed, juvenile tail tips, and displaying defective sexual behaviors. In both sexes, lep-2 mutants fail to cease molting or produce an adult cuticle. We find that LEP-2 post-translationally regulates LIN-28 by promoting LIN-28 protein degradation. lep-2 encodes the sole C. elegans ortholog of the Makorin (Mkrn) family of proteins. Like lin-28 and other heterochronic pathway members, vertebrate Mkrns are involved in developmental switches, including the timing of pubertal onset in humans. Based on shared roles, conservation and the interaction between lep-2 and lin-28 shown here, we propose that Mkrns, together with other heterochronic genes, constitute an evolutionarily ancient conserved module regulating switches in development.

Keywords: Developmental timing; Heterochronic pathway; LIN-28/let-7 axis; Mkrn.

Fig. 1.

Tail tip morphogenesis in wild-type and lep-2(lf) mutant C. elegans. (A,B) Developmental profiles of tail tip morphogenesis (TTM) in wild-type (A) and lep-2(lf) mutant (B) males. Left panels show DIC images; right panels shows adherens junctions at the cell boundaries visualized by AJM-1::GFP. The tail tip is conical in early L4. In wild type, tail tip retraction (arrow) and cell fusion (visible as breakdown of the adherens junctions, arrowhead and inset) begins in mid L4. The adult tail tip is fully retracted. lep-2(bx73) male tail tips do not undergo morphogenesis during L4. The adult tail tips are long, unretracted (arrows) and the tail tip cells are unfused. Punctate AJM-1::GFP staining indicates tail tip cell fusion in adult males (arrowhead and inset). (C,D) Expression of a dmd-3 transcriptional reporter (right) during TTM in wild-type males (C) and lep-2(lf) mutants (D). In wild type, dmd-3>YFP is expressed in the tail tip from early to mid-L4. Expression diminishes in late L4. In lep-2(bx73) mutants, the reporter is not expressed in early or mid-L4, but turns on in the tail tips of some late L4 and adult males. (E) DIC images of tail tips of an adult lep-2(bx73) and lep-2(ok900) mutant, showing adult tail tip retraction.

Fig. 2.

Male mating behavior is impaired in lep-2(lf). Mating behavior of individual wild-type (WT) and lep-2 mutant males was recorded over a period of 60 min. Wild-type males showed the known suite of mating behaviors (Barr and Garcia, 2006), which include the initial contact of the male tail with the hermaphrodite, scanning the hermaphrodite for the vulva, precise ventral turns when the male reaches the end of its mate, and a successful copulation. lep-2(lf) males were unable to perform normal mating behavior. Shown are data for (A) the time it took a male to make first contact with a hermaphrodite and (B) the time it took for the male to copulate. Wild-type males contacted a hermaphrodite and copulated in less than 20 min. Most mutant males took much longer to contact a mate and all but two males never copulated. The defect is rescued in males that had developed as dauer larvae (PD).

Fig. 3.

Location and mapping of lep-2. (A) Array comparative genomic hybridization (array CGH) graph of the C. elegans whole-genome tiling array, color-coded by chromosome. The dots represent the log₂ ratio of wild-type to mutant [lep-2(ny4)] signal for each probe. Chromosome IV shows a ∼8 kb long region with a higher DNA content in the wild-type sample relative to the mutant, indicating a deletion. (B) lep-2 gene structure, depicting two isoforms. The lesions affecting this locus are labeled: ny4 and ok900 are deletions; bx73, bx147 and sy68 harbor point mutations within the coding sequence of lep-2. (C) Schematic of the predicted LEP-2 protein: two predicted isoforms (LEP-2A and LEP-2B), both of which include the full complement of Mkrn motifs (zinc fingers in blue, RING domain in green) with the lesions in lep-2 alleles indicated by a bracket (deletion in ok900), red bars (amino acid substitutions in bx147 and sy68) and an asterisk (premature stop in bx73). Human MKRN1 is shown for comparison. (D) An alignment of the first Mkrn zinc finger from LEP-2, two mutant forms, and three human Mkrns. Blue letters indicate conserved amino acids; red letters indicate amino acid substitutions in lep-2 mutants.

Fig. 4.

Genetic interaction of lep-2 with other heterochronic genes. (A) Penetrance and expressivity of the adult tail tip phenotypes in lep-2(lf) (left) and wild-type (right) males alone and in combination with RNAi knockdown, gain-of-function or loss-of-function of other heterochronic genes. Data are for lep-2(ok900), except in lin-28(−) and lin-14(−) double mutants, where lep-2(ny4) was used. The penetrance of the Ore phenotype in RNAi-treated wild-type males indicates that lin-41(RNAi) had an efficiency of ∼75%, which is comparable to the findings of Del Rio-Albrechtsen et al. (2006). lin-28(RNAi) was 50% efficient, since loss of lin-28 by mutation led to 100% Ore tails. OP50 indicates that the animals were raised under standard culture conditions. HT115 is the bacterial strain used as control for RNAi experiments. let-7(+++) represents the transgene zaIs3, which leads to overexpression of let-7. (B) Genetic interactions between heterochronic genes evaluated in the epistasis experiments. Red letters indicate retarded phenotypes, blue letters precocious phenotypes in loss-of-function mutants. (C,D) DIC micrographs of male tail tips in single-mutant and double-mutant or RNAi-treated animals. (C) Wild-type and lep-2(lf) mutant males missing LIN-28 either by mutation or RNAi show precocious TTM as early as L3, have rounded L4 tails and display the Ore phenotype in adults. The lep-2(lf) phenotype is shown for comparison on the left. (D) TTM occurs precociously in lin-14(n179) males, but not in lin-14(n179); lep-2(bx73) males.

Fig. 5.

LIN-28 is regulated by LEP-2. (A) Western blot analysis of LIN-28 levels in wild-type (wt) and lep-2(ny4) L1 and L3 larvae. (B) lin-28 mRNA levels at L1 and L3 stages in wild-type and lep-2(bx73) mutants normalized to wild-type L1 levels. Error bars indicate s.d. (C,D) Expression of LIN-28::Dendra2 in the pharynx region of wild-type and lep-2(ny4) animals. (C) Images of the same focal plane in the pharynx region showing signal in (from top to bottom), bright field (BF), green channel before photoconversion, green channel after photoconversion, red channel after photoconversion. The dashed line indicates the position of the cross-section view through the z-plane shown in D. (D) Optical cross-section through the pharynx in one wild-type (top row) and one lep-2(ny4) (bottom row) animal at the L2 and L3 stage showing LIN-28::Dendra2 expression after photoconversion. The absence of green fluorescence in L2 animals shows that photoconversion was near complete. In the wild-type L3 animal, LIN-28::Dendra2 could not be detected in either channel, showing that LIN-28::Dendra2 is degraded normally. In the lep-2(ny4) animal, fluorescence was detected in the red channel only, indicating that LIN-28::Dendra2 persisted and no new protein was produced. 22 wild-type and 23 lep-2 mutant animals were examined, with results similar to the examples shown in D.