The Makorin lep-2 and the lncRNA lep-5 regulate lin-28 to schedule sexual maturation of the C. elegans nervous system.

Sexual maturation must occur on a controlled developmental schedule. In mammals, Makorin3 (MKRN3) and the miRNA regulators LIN28A/B are key regulators of this process, but how they act is unclear. In C. elegans, sexual maturation of the nervous system includes the functional remodeling of postmitotic neurons and the onset of adult-specific behaviors. Here, we find that the lin-28-let-7 axis (the 'heterochronic pathway') determines the timing of these events. Upstream of lin-28, the Makorin lep-2 and the lncRNA lep-5 regulate maturation cell-autonomously, indicating that distributed clocks, not a central timer, coordinate sexual differentiation of the C. elegans nervous system. Overexpression of human MKRN3 delays aspects of C. elegans sexual maturation, suggesting the conservation of Makorin function. These studies reveal roles for a Makorin and a lncRNA in timing of sexual differentiation; moreover, they demonstrate deep conservation of the lin-28-let-7 system in controlling the functional maturation of the nervous system.

Introduction

The timing of sexual maturation is subject to complex regulation that reflects competing demands on biological systems (Bogin et al., 2011; Gluckman and Hanson, 2006; Parent et al., 2003). Over evolutionary time, the pressure on a species to reproduce rapidly is countered by the need to allow sufficient time for robust juvenile development. Optimum balance of these pressures is encoded by internal, genetic mechanisms that guide the timing of the juvenile-to-adult transition (Zhu et al., 2018). At the level of an individual, internal and external stimuli—for example the presence of mates and food, as well as health and nutritional status—interact with these timers to determine the onset of sexual maturation (Abreu and Kaiser, 2016; Avendaño et al., 2017; Livadas and Chrousos, 2016; Plant, 2015).

In general, the nature of these genetic timers and their interaction with other signals is not well understood. Furthermore, while the hallmark of the juvenile-to-adult transition is the functional maturation of the germline and genitalia, reproductive maturation is typically accompanied by a much broader suite of changes in morphology, physiology, and behavior. These behavioral changes entail not only the activation of copulatory programs themselves, but also include modifications to decision-making circuits, allowing animals to incorporate reproductive drives when prioritizing behavioral programs. Whether genetic timers function at a single hub to coordinate behavioral transitions, or whether instead they may have distributed functions across different cell types, is unclear.

The timing of sexual maturation has been extensively studied in mammals, where it is coordinated by the temporally controlled production of gonadal steroids at puberty (Abreu and Kaiser, 2016; Avendaño et al., 2017; Livadas and Chrousos, 2016; Plant, 2015). This process is activated by the hypothalamic-pituitary-gonadal (HPG) axis, in which the pulsatile release of gonadotropin-releasing hormone (GnRH) by neurons in the hypothalamus activates gonadotropin production in the pituitary gland. This, in turn, triggers gonadal maturation and subsequent steroid hormone production. Upstream of HPG axis activation, a complex set of regulatory inputs converges on the release of the neuropeptide kisspeptin in the arcuate nucleus of the hypothalamus to stimulate GnRH production. Multiple studies suggest that kisspeptin release is influenced by environmental signals as well as internal timing cues (Harter et al., 2018). Recent studies in mice and humans have identified a number of genes important for this process, including the miRNA regulators LIN28A/B, their target LET7, and the Makorin MKRN3, whose loss causes Central Precocious Puberty in humans (Abreu et al., 2013; Corre et al., 2016; Gajdos et al., 2010; Ong et al., 2009; Park et al., 2012; Yi et al., 2018; Zhu et al., 2010; Zhu et al., 2018). However, where and how these genes act to regulate the onset of reproductive maturation remains unclear. Moreover, some aspects of this model are likely to be specific to mammals, highlighting the importance of studying these questions in diverse animals.

In the nematode C. elegans, juveniles pass through four larval stages before becoming sexually mature adults, offering an ideal opportunity to understand the mechanisms that regulate the juvenile-to-adult transition. Most studies addressing this question have focused on stage-specific events during larval development, particularly in the hypodermal seam cells, epidermal-like cells that lie along the sides of the body. In seam cells, stage-specific patterns of proliferation and differentiation are controlled by a complex mechanism known as the ‘heterochronic pathway’ (Rougvie and Moss, 2013). In this mechanism, temporally controlled waves of miRNA expression (including the first two miRNAs discovered in any system, lin-4 and let-7) act on a variety of targets to specify stage-specific events and regulate the transition from one stage to the next. The conserved miRNA regulator lin-28 is also a key component of this mechanism. The heterochronic pathway also controls some events that occur during the larval-to-adult transition, including the cessation of molting, the fusion of the seam cells, and male-specific tail tip morphogenesis (TTM), a remodeling of four postmitotic posterior hypodermal cells (Del Rio-Albrechtsen et al., 2006; Rougvie and Moss, 2013). In some heterochronic mutants (‘precocious’ mutants), particular stage-specific events are skipped or occur too early; in others (‘retarded’ or ‘delayed’ mutants), events are delayed or absent.

Recent studies of TTM have identified two additional heterochronic genes, lep-2/Makorin and the lncRNA lep-5, that influence developmental timing by regulating LIN-28 stability (Herrera et al., 2016; Kiontke et al., 2019). Mutations in these genes cause a delayed phenotype, the retention of a larval-like, pointed (‘Lep’) tail tip in males. These mutants also have developmental defects in the body hypodermis, but they lack the seam cell lineage disruptions of other heterochronic mutants. Interestingly, lep-2 mutant males have defects in mating behavior, suggesting a role for lep-2 in the sexual maturation of the nervous system (Herrera et al., 2016).

While the maturation of the hypodermis has been well studied, the mechanisms that control the timing of adult-specific sexual behavior in C. elegans are poorly understood. In both sexes, males and hermaphrodites (somatic females that produce both eggs and sperm), sexual behavior requires larval neurogenesis that produces sex-specific circuits for copulation and egg-laying behavior (Emmons, 2018; García and Portman, 2016). The development of these circuits is independent of the gonad, relying instead on intrinsic lineage programs in which chromosomal sex acts via the sex-determination hierarchy to regulate neurogenesis and cell fate specification (Barr et al., 2018). Furthermore, multiple aspects of adult male behavior also require changes in synaptic connectivity and the functional modulation of pre-existing, post-mitotic neurons and circuits at the juvenile-to-adult transition (Portman, 2017). For example, the AWA olfactory neurons express the food-associated chemoreceptor odr-10 in larvae of both sexes, but in males, odr-10 expression is repressed at the larval-to-adult transition. This repression desensitizes males to food stimuli and promotes mate-searching behavior (Ryan et al., 2014). In other cases, adult-specific activation of gene expression in males, such as the expression of daf-7 in the ASJ neurons (Hilbert and Kim, 2017) facilitates adult behavior. The extent to which sex-shared, postmitotic neurons are functionally modulated at the juvenile-to-adult transition remains unclear, and the mechanisms that control the timing of this modulation are unknown.

The striking involvement in mammalian puberty of orthologs of three C. elegans heterochronic genes (lep-2, lin-28, and let-7), together with the emerging appreciation that C. elegans sexual differentiation involves functional transitions in post-mitotic neurons, led us to explore the role of the heterochronic pathway in the timing of these changes. We find that the lep-2/lep-5 branch of the heterochronic pathway plays a key role in timing the sexual differentiation of the C. elegans nervous system, demonstrating conservation of mechanism across species and raising the possibility that an unidentified lep-5-like lncRNA may play a key role in mammalian sexual maturation. Moreover, we find that this pathway acts cell-autonomously in multiple neurons, suggesting that genetic timing information is widely distributed across the nervous system. Multiple targets of lin-41, including mab-3 and lin-29a, integrate the heterochronic and sex-determination pathways to regulate downstream effector genes and the activation of multiple adult behaviors. Interestingly, although the mammalian Makorin MKRN3 functions in the opposite direction as its C. elegans ortholog lep-2, we find that MRKN3 expression in the C. elegans nervous system is sufficient to delay sexual maturation, indicating that the pathway in which it interacts is functionally conserved across species. These findings identify the heterochronic pathway as an ancient regulator of sexual differentiation of the nervous system, provide specific mechanistic hypotheses for the functions of these genes in the mammalian brain, and suggest an important role for a yet-unidentified mammalian lncRNA in the timing of puberty.

Results

Multiple C. elegans neurons undergo male-specific functional maturation during the juvenile-to-adult transition

To visualize the sexual maturation of the C. elegans nervous system, we studied the developmental dynamics of the expression of five genes known to display sexually dimorphic expression in adults (Figure 1). Four of these reflect sexual dimorphism in shared (i.e., non-sex-specific) neurons: srj-54, a GPCR of unknown function expressed male-specifically in the AIM interneurons (Lee and Portman, 2007; Portman, 2007); odr-10, a chemoreceptor whose male-specific downregulation in the AWA olfactory neurons is associated with decreased diacetyl and food detection (Lee and Portman, 2007; Ryan et al., 2014; Sengupta et al., 1996); mab-3, a doublesex-family transcription factor that is male-specifically expressed in the ADF chemosensory neurons, where it promotes the detection of hermaphrodite sex pheromone (Fagan et al., 2018; Yi et al., 2000); and daf-7, a TGFβ-superfamily ligand that is male-specifically expressed in the ASJ chemosensory neurons and promotes the food-leaving behavior of adult males (Hilbert and Kim, 2017; Ren et al., 1996). We also studied pkd-2, a marker for the male-specific CEM pheromone-detection neurons, which exist in an undifferentiated state until late larval development (Barr and Sternberg, 1999; Narayan et al., 2016; Srinivasan et al., 2008; Sulston et al., 1980; Sulston et al., 1983). Importantly, all of these neurons are generated during embryogenesis and, with the exception of the CEMs, are thought to be functional components of larval circuits.

Figure 1.

Five classes of neurons undergo male-specific functional maturation during the larval-to-adult transition.

(A–E) Rows show expression of reporter genes for srj-54, odr-10, mab-3, daf-7, and pkd-2, respectively. At the left, the name of each marker, a brief description of its gene product, and the cell type whose maturation it marks are indicated. For all rows, representative epifluorescence (above; dashed line indicates outline of body) and DIC (below) images are shown for the indicated sex and stage. The graph at the right of each row shows expression levels in L3, L4, and adult males. Asterisks indicate the statistical significance of comparisons between L3 and L4 and between L4 and adult. Numbers above each bar indicate numbers of animals scored.

For all of the markers we examined, we found that sex-specific expression patterns were absent in young larvae, emerging only late in larval development (Figure 1). In L3 males, we detected virtually no expression of srj-54 in AIM, mab-3 in ADF, or, consistent with a previous report (Hilbert and Kim, 2017), daf-7 in ASJ. Moderate expression of these markers was observed in L4 males, but the full extent of sex differences in expression was not fully apparent until adulthood (Figure 1A,C,D). With regard to odr-10 expression in AWA, we observed the opposite pattern, confirming our earlier findings (Ryan et al., 2014): reporter expression was readily detectable in L3 males, as it is in adult hermaphrodites, and diminished in males through L4 into adulthood (Figure 1B). Thus, at least with respect to these markers, the AWA, AIM, ADF, and ASJ neurons in larval males exist in a hermaphrodite-like ‘ground state’ before they acquire adult-specific characteristics during the juvenile-to-adult transition. The CEMs, which are absent in hermaphrodites, underwent maturation with similar timing. As expected (Barr and Sternberg, 1999), we detected no expression of pkd-2 in L3 males; intermediate expression was observed in L4 animals and was further strengthened in day-one adults (Figure 1E). This timing is consistent with that previously reported for other CEM-specific genes (Barr et al., 2001; Portman and Emmons, 2004; Wang et al., 2015) as well as the cholinergic maturation of these neurons (Pereira et al., 2015). Together, these findings reinforce the idea that the larval-to-adult transition involves not only the incorporation of new male-specific neurons into the nervous system (Barr et al., 2018), but also the molecular maturation of multiple classes of pre-existing neurons.

The lin-28–let-7 regulatory module controls the timing of sexual maturation of the C. elegans nervous system

To ask whether the heterochronic pathway has a role in the timing of these molecular maturation events, we examined animals carrying mutations in lin-28 and let-7, as these genes are at the core of the late heterochronic timer (Rougvie and Moss, 2013) (Figure 2A,B). In the hypodermal seam and the male tail tip hypodermis, lin-28 mutants precociously execute late-larval patterns of proliferation, differentiation, and morphogenesis (Ambros, 1989; Ambros and Horvitz, 1984; Herrera et al., 2016; Moss et al., 1997); conversely, let-7 mutants retain larval-specific fates and male tail morphology as adults (Reinhart et al., 2000; Slack et al., 2000; Vadla et al., 2012).

Figure 2.

Heterochronic genes regulate the sexual maturation of multiple C. elegans neurons.

(A) A subset of the heterochronic pathway, indicating genes and regulatory events important for the timing of the juvenile-to-adult transition in hypodermal tissues. (B) The temporal dynamics of lep-2, lep-5, lin-28, and let-7 expression during larval development. (C–G) Expression of markers for the sexual maturation of five neuron classes in wild-type and heterochronic mutants. For each marker, expression is shown in the males of the indicated stage (L3, L4, and Adult) and genotype. ‘–” indicates that a null allele was used, while ‘lf’ and ‘gf’ indicate hypomorphic and hypermorphic alleles, respectively. For lin-28(–) and lin-41(lf), only L3 and L4 males were scored. All strains were grown at 20°C except for let-7(lf) and lin-41(bx42) and their paired wild-type controls, which were grown at 25°C. Asterisks indicate the statistical significance of comparisons to paired wild-type controls of the same stage. Paired wild-type control data was pooled to create the WT graph at the left of each row. Numbers above each bar indicate numbers of animals scored. Graphs are shaded to indicate temporal phenotypes as shown in the legend.

(A) A predicted model for lep-5 secondary structure; see Kiontke et al., 2019 for details. Colored shading indicates regions disrupted by the mutants tested. (B) Expression of Psrj-54::YFP in adult males of the indicated genotype. Numbers above each bar indicate numbers of animals scored. (C) The fs21 allele mutates six nucleotides (shown in red) important for the predicted zipper ‘a’ region. The fs21 fs25 allele mutates an additional seven positions ~ 325 nt in the 3´ direction in a manner predicted to allow zipper ‘a’ to re-form, even in the presence of significant changes in its primary sequence (Kiontke et al., 2019).

We found that gene expression dynamics in the nervous system were similarly altered in these mutants (Figure 2C–G). lin-28 mutant males exhibited precocious activation of srj-54 in AIM, with clear reporter expression in over half of L3 males and over 80% of L4 males. In contrast, the sexual maturation of AIM was delayed in let-7 mutant males, with very little srj-54 expression detectable in L4 and only moderate expression in one-day adults (Figure 2C). (Since let-7 null mutants are inviable, these experiments used the hypomorphic allele n2853; null phenotypes would likely be stronger (Reinhart et al., 2000).) We observed similar effects of lin-28 and let-7 mutations on pkd-2 reporter expression in the CEMs (Figure 2G): loss of lin-28 resulted in precocious expression in L3 males, while loss of let-7 slightly reduced pkd-2 expression in young adults. let-7 mutant adults also retained larval-like expression of odr-10 in AWA. Unexpectedly, however, odr-10 downregulation did not occur precociously in lin-28 mutant males; instead, levels increased in L4 (Figure 2D). Because odr-10 downregulation does depend on genes downstream of lin-28 (see below), some adult-specific characteristic(s) that are independent of lin-28 are likely to be required for odr-10 downregulation (e.g., signals from male-specific components of the adult nervous system). Finally, we also observed precocious ADF expression of mab-3 in lin-28 mutant males (Figure 2E), with strong expression initiating in L3. Together, these results demonstrate that the lin-28–let-7 regulatory axis governs the timing of key events in the sexual maturation of the C. elegans nervous system.

In the hypodermis, a key target of let-7 is lin-41, which encodes an RBCC protein of the NHL family (Slack et al., 2000). We found that loss of lin-41 function resulted in precocious activation of srj-54 in AIM and pkd-2 in CEM, and precocious loss of odr-10 in AWA (Figure 2C,D,G). Reciprocally, a lin-41(gf) allele (Del Rio-Albrechtsen et al., 2006) caused somewhat delayed maturation of the expression of srj-54, though it had no statistically significant effects on odr-10 or pkd-2 expression (Figure 2C,D,G). This discrepancy could reflect cell-type-specific thresholds for the degree of lin-41 function necessary to elicit a gain-of-function phenotype. Together, these results indicate that lin-41 is an important effector of let-7 function in the nervous system, as it is in the hypodermis.

The makorin lep-2 and the lncRNA lep-5 act upstream of lin-28 to promote sexual differentiation

In the timing of male tail tip morphogenesis, the Makorin lep-2 and the lncRNA lep-5 act upstream of lin-28 (Herrera et al., 2016; Kiontke et al., 2019). Because loss-of-function mutations in the lep-2 ortholog MKRN3 cause Central Precocious Puberty in humans (Abreu et al., 2013) and because of the sexual behavior defects in C. elegans lep-2 mutant males (Herrera et al., 2016), we considered the possibility that the lep-2/lep-5 branch of the heterochronic pathway might function in sexual differentiation of the C. elegans nervous system. Consistent with this idea, we found that these animals exhibited striking defects in neuronal maturation: for srj-54, odr-10, and mab-3, day one adult lep-5 males retained the expression pattern typically seen in wild-type L3 males (Figure 2C,D,E). We observed weaker phenotypes for daf-7 and pkd-2: in both cases, expression increased during larval development, but failed to reach the level typically seen in young adults (Figure 2F,G). Thus, lep-2 is required for the sexual differentiation of AIM, AWA, and ADF, and also contributes to this process in ASJ and CEM. Intriguingly, the absence of sexually mature characteristics in young lep-2 adult males stands in contrast to the Central Precocious Puberty that results from loss of human MKRN3 function, a distinction to which we return below.

As is the case for tail tip morphogenesis (Kiontke et al., 2019), the lep-5 mutant phenotype almost completely phenocopied that of lep-2. Mutant males failed to adopt adult-specific gene expression patterns in AIM, AWA, and ADF, but had only modest defects in marker expression in ASJ and CEMs (Figure 2C–G). lep-5 encodes a lncRNA that is predicted to adopt a compact secondary structure with prominent stem-loop structures near its 5´ and 3´ ends, as well as a two-part central double-stranded ‘zipper’ region (Figure 2—figure supplement 1A) (Kiontke et al., 2019). lep-5 has been proposed to function as an RNA scaffold, forming a tripartite complex with LEP-2 and LIN-28 to promote LIN-28 degradation (Kiontke et al., 2019). We found that a Crispr-mediated deletion of the predicted 3´ hairpin, lep-5(fs20), phenocopied the lep-5 null allele, showing a nearly complete lack of srj-54 expression in AIM in adult males (Figure 2—figure supplement 1B). lep-5(fs8), a point mutation that disrupts the 5´ hairpin, as well as lep-5(fs21), a Crispr-engineered allele bearing a cluster of five point mutations predicted to disrupt the central zipper, caused a complete loss of srj-54 expression in adults (Figure 2—figure supplement 1B, C). Furthermore, lep-5(fs21fs25), a double mutant that carries a set of compensatory changes predicted to restore the central zipper, completely restored adult expression of srj-54 (Figure 2—figure supplement 1B, C). The phenotypes of these mutants are consistent with those seen in male tail tip morphogenesis (Kiontke et al., 2019), indicating that lep-5 likely acts through a similar mechanism in both tail tip morphogenesis and neuronal maturation.

Figure 2—figure supplement 1.

Structural features of the lep-5 lncRNA required for its function in the nervous system.

(A) A predicted model for lep-5 secondary structure; see Kiontke et al., 2019 for details. Colored shading indicates regions disrupted by the mutants tested. (B) Expression of Psrj-54::YFP in adult males of the indicated genotype. Numbers above each bar indicate numbers of animals scored. (C) The fs21 allele mutates six nucleotides (shown in red) important for the predicted zipper ‘a’ region. The fs21 fs25 allele mutates an additional seven positions ~ 325 nt in the 3´ direction in a manner predicted to allow zipper ‘a’ to re-form, even in the presence of significant changes in its primary sequence (Kiontke et al., 2019).

The lep-2–lin-28–let-7 axis acts cell-autonomously to time the onset of sexual maturation in the nervous system

We considered several scenarios by which heterochronic genes could control the timing of neuronal maturation (Figure 3A). In one, heterochronic genes might act in the gonad to control the secretion of hormonal signals. However, this seems unlikely, as the development of the C. elegans gonad is largely independent of the heterochronic pathway (Rougvie and Moss, 2013) and, with the exception of vulva and hindgut development, (Kimble, 1981; Sulston et al., 1980), the gonad does not play a significant role in the sexual differentiation of the C. elegans soma (Barr et al., 2018). Alternatively, the heterochronic pathway might act as a central somatic timer (e.g., in seam cells or a master ‘clock’ neuron) from which it broadcasts temporal information to the nervous system. Finally, individual neurons might possess their own timers, such that temporal control is distributed throughout the nervous system.

Figure 3.

The heterochronic pathway is a cell-autonomous, distributed timer of sexual maturation.

(A) Models for non-autonomous and autonomous functions of the heterochronic pathway, as described in the text. (B) Expression of LIN-28::GFP and the pan-neural nuclear marker Prab-3::nRFP. Strong GFP expression is detected throughout the nervous system of L1 animals; this decreases during L2 and is virtually undetectable in L3. (C) Plep-5::GFP expression in L1, L2, and L3 animals. GFP is essentially undetectable in L1 but is seen in many head neurons in L2 and L3. (D) Plep-2::GFP::LEP-2 expression in L3, L4, and Adult males. Broad GFP expression is seen in the nervous system at all stages examined. (E–H) Cell-type specific rescue. Numbers above each bar indicate numbers of animals scored. Asterisks indicate the significance of the comparison between transgenic animals and non-transgenic control siblings. (E) Expression of Psrj-54::YFP in wild-type, lep-2 (non-transgenic siblings), and lep-2; AIM::lep-2 males of the indicated stage. (F) Expression of ODR-10::GFP in wild-type, lep-2 (non-transgenic siblings), and lep-2; AIM::lep-2 males of the indicated stage. (G) Expression of Psrj-54::YFP in wild-type, lin-28 (non-transgenic siblings), and lin-28; AIM::lin-28 males of the indicated stage. (H) Expression of ODR-10::GFP in wild-type, lep-2 (non-transgenic siblings), and lep-2; AWA::lep-2 adult males.

Images show expression of the Plep-5::GFP reporter (left), the pan-neural nuclear marker Prab-3::nlsRFP (right) and the merged image (center) in the indicated stages of (A) wild-type and (B) lin-4(e912) mutants. Dashed lines indicate the body outline. The images in (A) are of the same individual as in Figure 3C, shown here in wider-view versions.

To address this, we first confirmed earlier reports suggesting that lin-28, lep-2, and lep-5 are broadly expressed in the nervous system (Herrera et al., 2016; Kiontke et al., 2019; Moss et al., 1997). Using a translational LIN-28::GFP reporter, we observed widespread expression in L1 and L2 animals, including extensive expression in the nervous system (Figure 3B). LIN-28::GFP abundance decreased during L2 in the nervous system and elsewhere, and by L3, expression was virtually undetectable, consistent with previous reports (Moss et al., 1997). We observed an inverse pattern of Plep-5::GFP expression in the nervous system, consistent with its previously described dynamics in the hypodermis (Kiontke et al., 2019). Plep-5::GFP was detectable in very few neurons in L1 larvae, but broad neuronal expression was apparent by L2 and L3 (Figure 3C). Interestingly, lep-5 reporter expression appears to be restricted to a subset of the nervous system (Figure 3C), an important issue for future work. With a Plep-2::lep-2::GFP translational reporter (Herrera et al., 2016), we observed expression in many head neurons in early larvae (not shown) and in L3, L4, and adult males (Figure 3C). Because lep-2 expression levels remain essentially constant during larval development (Herrera et al., 2016), lep-2 likely does not itself provide timing information but rather seems to provide a permissive cue for the transition into adulthood. Instead, lep-5 likely provides an instructive signal important for determining the onset of the sexual differentiation of the nervous system, as it does in the hypodermis (Kiontke et al., 2019).

To ask whether the heterochronic pathway functions in the nervous system itself, we restored expression of wild-type lep-2 or lin-28 in the nervous system of lep-2 or lin-28 mutants. Expression of lep-2 under the control of the AIM-specific promoter Peat-4prom11 (Serrano-Saiz et al., 2017) restored srj-54 expression to lep-2 mutant adults, but had no effect on odr-10 expression in AWA (Figure 3E,F), indicating that lep-2 acts cell-autonomously to control timing in AIM. Furthermore, Peat-4prom11::lin-28 rescued the precocious maturation of AIM in lin-28 mutant larvae (Figure 3G). Thus, timing information intrinsic to AIM itself is critical for the timely control of AIM’s maturation. This property is not limited to AIM, as AWA-specific expression of lep-2 (Pgpa-4∆6::lep-2(+)) rescued the lack of odr-10 downregulation in AWA in lep-2 mutants (Figure 3H). Together, these findings indicate that the heterochronic pathway does not control an organism-wide temporal signal that is broadcast from a central timer; rather, timing information is distributed, such that individual neurons maintain their own internal clocks.

lep-2 and lep-5 function upstream of lin-28 and its miRNA target let-7 in the sexual differentiation of the nervous system

Multiple studies have shown that lin-28 negatively regulates the biogenesis and activity of multiple miRNA targets, including let-7, in C. elegans, Drosophila, and mammals (Tsialikas and Romer-Seibert, 2015). The decay of lin-28 activity is a key regulatory point in the progression into adulthood, allowing the activation of let-7 and the subsequent downregulation of its targets. In the hypodermal seam, lin-28 decay is controlled at both the mRNA and protein levels (Huang et al., 2011; Morita and Han, 2006; Seggerson et al., 2002; Weaver et al., 2017; Weaver et al., 2014). In the male tail tip, lep-2 and lep-5 function together to promote the degradation of LIN-28 protein during L3 (Herrera et al., 2016; Kiontke et al., 2019). We carried out a series of genetic and molecular epistasis experiments to ask which aspects of this regulatory logic extend to the nervous system.

Unlike wild-type males, in which neuronal LIN-28::GFP decays during L2 and is virtually absent by L3, lep-2 mutant males exhibited strong neuronal LIN-28::GFP that persisted into L3 and L4 (Figure 4A and data not shown). Thus, consistent with its role in the tail tip and other hypodermal cells (Herrera et al., 2016), lep-2 promotes the timely decay of LIN-28 in the nervous system. We also examined expression of the AIM maturation marker srj-54 in lin-28; lep-2 double mutants. Like lin-28 single mutants, these animals exhibited precocious AIM maturation (Figure 4B), confirming that lep-2 acts genetically upstream of lin-28. We observed a similar phenotype in lin-28; lep-5 double mutants (Figure 4C), indicating that lep-5 also acts upstream of lin-28 with respect to nervous system maturation. Finally, we confirmed that lin-28 acts upstream of let-7 in the nervous system by examining srj-54 expression in lin-28; let-7 mutants. As expected, AIM maturation was delayed compared to wild-type (Figure 4D). Thus, lep-2 and lep-5 regulate the lin-28–let-7 regulatory module to control the onset of sexual differentiation of the C. elegans nervous system.

Figure 4.

Regulatory relationships and conservation of heterochronic function.

(A) LIN-28::GFP expression persists into L3 in lep-2 mutants (right), unlike wild-type (left). The images on the left are the same as those in Figure 3C, but the GFP signal intensity is doubled to highlight the difference in expression in L3. Images for wild-type and mutant were taken with identical settings and images were processed identically. (B–D) Psrj-54::YFP expression in the indicated single and double mutant adult males. Numbers above each bar indicate numbers of animals scored. (E–G) MKRN3 overexpression in the nervous system. Non-transgenic siblings were used as WT controls. Numbers above each bar indicate numbers of animals scored. Asterisks indicate significance of the comparison of transgenic animals to control animals of the same stage. (E) Psrj-54::YFP expression in animals of the indicated genotype and stage. (F) ODR-10::GFP expression in animals of the indicated genotype and stage. (G) Ppkd-2::GFP expression in animals of the indicated genotype and stage.

(A) MKRN3 overexpression in the nervous system of lep-2 mutants. Bars indicate distributions of the intensity of Psrj-54::YFP expression. Non-transgenic siblings were used as WT controls. Numbers above each bar indicate numbers of animals scored. (B–D) Expression of Psrj-54::YFP (B) and ODR-10::GFP (C,D) in mab-3 (B–C) and lin-29a (D) mutants. Numbers indicate number of animals scored. Asterisks indicate statistical comparisons between WT and mutant animals. (E) Attraction to ascaroside pheromones using a quadrant-style assay. Each point represents the chemotaxis index calculated from one assay (ten animals). Asterisks indicate the significance of the indicated comparisons.

Together with previous work (Kiontke et al., 2019), our results support the idea that the onset of lep-5 expression is a critical step in determining the timing of LIN-28 decay and the subsequent progression into adulthood. To investigate mechanisms controlling the timing of lep-5 activation, we considered a role for genes involved in the timing of early larval development. The miRNA lin-4 and its key target, the transcription factor lin-14, regulate stage-specific development in L1 and L2 (Rougvie and Moss, 2013). We found that neuronal expression of Plep-5::GFP was significantly disrupted in lin-4 mutants: very little reporter expression was detectable in the nervous system of larvae of any stage (Figure 3—figure supplement 1). This suggests that lin-4 is necessary to trigger the onset of lep-5 expression in the nervous system, consistent with its role in promoting the L1 to L2 transition (Rougvie and Moss, 2013).

Figure 3—figure supplement 1.

lin-4 is necessary for proper lep-5::GFP expression in the nervous system.

Images show expression of the Plep-5::GFP reporter (left), the pan-neural nuclear marker Prab-3::nlsRFP (right) and the merged image (center) in the indicated stages of (A) wild-type and (B) lin-4(e912) mutants. Dashed lines indicate the body outline. The images in (A) are of the same individual as in Figure 3C, shown here in wider-view versions.

Human MKRN3 can inhibit sexual differentiation of the C. elegans nervous system

Because the lep-2 and lin-28 orthologs MKRN3 and LIN28A/B are both implicated in the timing of human puberty, our results and those of Herrera et al. (2016) raise the possibility that MKRN3 might act upstream of LIN28A/B in the mammalian nervous system. However, while loss of the C. elegans Makorin lep-2 causes a delay in sexual differentiation, loss-of-function mutations in MKRN3 are associated with the opposite phenotype, Central Precocious Puberty (Abreu et al., 2013). Several models can explain this apparent discrepancy. For example, it could be the case that both MKRN3 and LEP-2 inhibit LIN28A/B and LIN-28, but LIN28A/B might have multiple functions in mammalian puberty, acting both to promote and inhibit its onset (Corre et al., 2016). Alternatively, MKRN3 might act to stabilize LIN28A/B, possibly by inhibiting another mammalian Makorin that might function more like C. elegans lep-2. Finally, MKRN3 might regulate other substrates that function downstream of, or in parallel with, mammalian LIN28A/B in regulating puberty.

The first model predicts that MKRN3 might be able to functionally substitute for lep-2. However, we found that pan-neural expression of MKRN3 in lep-2 mutants had no apparent effect on srj-54 expression (Figure 4—figure supplement 1A). Though we cannot rule out the possibility that technical issues hindered rescue, this result suggests that LEP-2 and MKRN3 do not have equivalent biochemical functions.

Figure 4—figure supplement 1.

Genetic control of male sexual differentiation and behavior.

(A) MKRN3 overexpression in the nervous system of lep-2 mutants. Bars indicate distributions of the intensity of Psrj-54::YFP expression. Non-transgenic siblings were used as WT controls. Numbers above each bar indicate numbers of animals scored. (B–D) Expression of Psrj-54::YFP (B) and ODR-10::GFP (C,D) in mab-3 (B–C) and lin-29a (D) mutants. Numbers indicate number of animals scored. Asterisks indicate statistical comparisons between WT and mutant animals. (E) Attraction to ascaroside pheromones using a quadrant-style assay. Each point represents the chemotaxis index calculated from one assay (ten animals). Asterisks indicate the significance of the indicated comparisons.

Because wild-type MKRN3 inhibits sexual differentiation in humans, we next asked whether it might have the same effect in C. elegans. Remarkably, expression of MKRN3 from a pan-neuronal promoter in a wild-type background caused a modest but statistically significant delay in the activation of srj-54 expression in AIM and the downregulation of odr-10 expression in AWA (Figure 4E,F). However, we observed no effect of MKRN3 overexpression on pkd-2 expression (Figure 4G). The observation that MKRN3 retains its ability to inhibit some aspects of sexual maturation when expressed in the C. elegans nervous system raises the intriguing possibility that MKRN3 exerts an inhibitory effect on the conserved lin-28–let-7-dependent heterochronic timer and, moreover, that there is deep functional conservation of the mechanism in which it participates. The lack of an apparent effect of MKRN3 overexpression on CEM maturation is consistent with our finding that these neurons rely less on lep-2 and lep-5 function (Figure 2G), indicating that their maturation occurs through mechanisms that are at least partially distinct from those that operate in AWA and AIM.

Multiple effectors connect lin-41 to the sexual maturation of the male nervous system

Our survey of heterochronic mutants (Figure 2) indicated that the lin-28–let-7 pathway controls sexual maturity via the RNA-binding protein LIN-41, a direct target of let-7 (Slack et al., 2000). LIN-41 regulates gene expression by binding to the 5´ or 3´ UTR of multiple target genes, including lin-29a, mab-3, dmd-3, and mab-10 (Aeschimann et al., 2017). In the case of ADF, whose maturation is marked by the activation of mab-3, this provides a continuous molecular pathway connecting the lep-2–lep-5–lin-28 timer to the sexual differentiation of ADF. With regard to the other neurons we studied, mab-3 and lin-29a appear to have overlapping but distinct roles in sexual maturation. Studies carried out in parallel with this work found that lin-29a regulates expression of srj-54 in AIM and daf-7 in ASJ (Pereira et al., 2019). We found that the full extent of srj-54 expression in AIM also depended on mab-3 (Figure 4—figure supplement 1B). Thus, both lin-29a and mab-3 promote AIM maturation, though the role of mab-3 may be secondary and/or indirect. Furthermore, we found that the adult-specific repression of odr-10 in AWA was lost in mab-3 mutants but was unaffected by loss of lin-29a (Figure 4—figure supplement 1C,D). Thus, with respect to the molecular maturation events we have investigated here, both mab-3 and lin-29a link the heterochronic pathway to downstream effectors of sexual differentiation. Because both mab-3 and lin-29a are also targets of the master sexual regulator tra-1 (Fagan et al., 2018; Pereira et al., 2019; Yi et al., 2000), these genes integrate sexual and temporal information to specify male-specific aspects of the sexual maturation of the C. elegans nervous system.

The heterochronic neuronal timer is necessary for the onset of sex differences in adult C. elegans behavior

With the exception of srj-54, whose function is unknown, each of the molecular markers used in this work is associated with adult-specific male behavior. Indeed, previous research has found that lep-2 mutants have defects in male mating and food-leaving behaviors (Herrera et al., 2016), although the underlying mechanisms were unclear. We therefore surveyed these and other aspects of adult male behavior in both lep-2 and lep-5 mutants. While many heterochronic mutants have drastic, pleiotropic effects that interfere with behavioral assays, lep-2 and lep-5 males are, with the exception of a moderate defect in tail tip retraction, morphologically wild type. The retention of a pointed tail tip into adulthood is in itself thought not to significantly compromise male sexual behavior (Del Rio-Albrechtsen et al., 2006).

Food-leaving behavior, the propensity of well-fed males to leave a food source in search of mates, is manifested only in adulthood (Lipton et al., 2004). Multiple mechanisms promote male food-leaving, including signals from the male-specific ray sensory neurons, the neuropeptide receptor PDFR-1, the TGFβ-superfamily ligand DAF-7, and downregulation of the food-associated chemoreceptor odr-10 (Barrios et al., 2012; Barrios et al., 2008; Hilbert and Kim, 2017; Lipton et al., 2004; Ryan et al., 2014). We found that lep-2 and lep-5 mutant males exhibited markedly decreased food-leaving behavior as adults (Figure 5A), indicating that both of these genes are important for the acquisition of male sexual drive.

Figure 5.

Adult-specific male behavior requires the function of the heterochronic pathway.

(A) Food-leaving behavior. The maximum distance that a single animal had travelled from a small food spot was determined at 3, 6, and 24 hr after the beginning of the assay. Numbers indicate number of animals assayed. Asterisks indicate significance of comparisons to wild-type adults. (B) Attraction to a blend of ascaroside pheromones (ascr#2/#3/#8) using a quadrant-style assay. Each point represents the chemotaxis index calculated from one assay (ten animals). (C, D) Contact-response (Rsp) and Vulva-location (Lov) behavior. Numbers indicate number of animals tested. pkd-2 mutant males, known to be defective in both behaviors (Barr et al., 2001), are used as controls.

We also examined attraction to ascaroside sex pheromones, a male-specific behavior that depends partially on mab-3 function in ADF (Fagan et al., 2018; Srinivasan et al., 2008). We found that ascaroside attraction was absent in male larvae: L3 males were slightly repelled by an ascr#2/#3/#8 blend, reminiscent of the behavior of adult hermaphrodites (Fagan et al., 2018) (Figure 5B). The transition to the adult behavioral state requires lep-2 and lep-5, as pheromone attraction was abolished in these mutants (Figure 5B). In contrast, lin-29a mutant males exhibited no apparent defects in ascaroside attraction (Figure 3—figure supplement 1D), suggesting that mab-3 is the primary effector of the sexual maturation of ADF.

Finally, we examined two sub-steps of male mating, response behavior (‘Rsp’, the typical backward-locomotion response of males to physical contact with hermaphrodites) and the subsequent vulva-location behavior (‘Lov’, the ability of the male to locate the hermaphrodite vulva) (Liu and Sternberg, 1995). While these behaviors depend primarily on male-specific sensorimotor circuits in the tail, sex-specific characteristics of shared neurons are also likely to be important for their execution (Barr and Sternberg, 1999; Koo et al., 2011; Liu and Sternberg, 1995; Liu et al., 2011; Sherlekar et al., 2013) (R.M.M. and D.S.P., unpublished results). We found that response behavior was reduced in both lep-2 and lep-5 mutant males, while vulva-location behavior was almost completely absent (Figure 5C,D). Together, these findings demonstrate that the Makorin lep-2 and the lncRNA lep-5, and by extension, the heterochronic pathway, are important for implementing adult-specific sexual behavior in C. elegans males.

Discussion

Although it has long been appreciated that animal behavior undergoes functional transitions commensurate with sexual maturation (Spear, 2000), the mechanisms that determine the timing of these changes are poorly understood. Indeed, in humans, adolescence is a period of heightened susceptibility to a variety of neuropsychiatric disorders (Jones, 2013; Walker et al., 2017), highlighting the importance of understanding the regulatory mechanisms that control these transitions. Here, we unite disparate findings from studies of mammalian puberty and C. elegans developmental biology to establish that a conserved developmental timer functions cell-autonomously to coordinate the sexual maturation of the C. elegans nervous system (Figure 6). Central to this mechanism is the temporal control provided by the lncRNA lep-5, which, together with the Makorin lep-2, promotes the timely decay of the conserved miRNA inhibitor LIN-28 (Kiontke et al., 2019). This decay permits the biosynthesis of mature let-7 miRNA and, through the pathway shown in Figure 6, promotes the juvenile-to-adult transition.

Figure 6.

The heterochronic and sex-determination pathways intersect to control adult-specific male gene expression and behavior.

Genes are color-coded to indicate function as indicated. Grey arrows from MKRN3 represent hypothetical interactions that could explain the ability of MKRN3 to inhibit C. elegans sexual differentiation. See text for details.

The maturation events we have studied here entail not the incorporation of new neurons and circuits into the nervous system, but rather gene expression and functional changes in pre-existing, postmitotic neurons. As such, these events parallel similar changes in the mammalian hypothalamus, where both gene expression and physiological changes determine the timing of HPG axis maturation and the onset of puberty (Abreu and Kaiser, 2016; Avendaño et al., 2017; Livadas and Chrousos, 2016; Plant, 2015). Because MKRN3, LIN28A/B, and LET7 are expressed in the hypothalamus and elsewhere in the mammalian brain, our results indicate that the neuronal function of the heterochronic pathway is an ancient regulator of the juvenile-to-adult transition. In this view, the activational effects of gonadal steroids at puberty (McCarthy et al., 2012) are likely to be a recent evolutionary adaptation that reinforces sexual differentiation of the mammalian nervous system via an endocrine feedback loop. Moreover, it suggests that puberty-independent events outside the hypothalamus that contribute to behavioral maturation during mammalian adolescence, such as the transition from play to aggression in Siberian hamsters (Paul et al., 2018), could also be timed by the heterochronic pathway.

In addition to identifying neuronal functions for lep-2, lin-28, and let-7, we also show that the recently identified lncRNA lep-5 functions in this pathway. lep-5 was identified in forward genetic screens for male tail morphogenesis defects; in lep-5 mutants, LIN-28 protein persists longer than in wild-type, delaying the onset of expression of dmd-3, the master regulator of tail tip morphogenesis (Kiontke et al., 2019). Because lep-5 is expressed in a temporal wave whose rise corresponds to the time at which lin-28 expression decays, lep-5 is thought to provide an instructive temporal cue for the destabilization of LIN-28 and, in turn, allow production of the let-7 miRNA and the progression through late-larval development. Because lep-2 mutants have a phenotype essentially identical to that of lep-5, Kiontke et al., 2019 have proposed that lep-5 serves as an RNA scaffold that recruits both LEP-2 and LIN-28; such a model is supported by in vivo crosslinking experiments demonstrating that lep-5 binds to both LEP-2 and LIN-28. As a member of the Makorin family, LEP-2 may possess both RNA-binding and putative E3 ubiquitin ligase activities (Herrera et al., 2016), suggesting that a tripartite LEP-2–lep-5–LIN-28 complex could destabilize LIN-28 through ubiquitination by LEP-2 (Kiontke et al., 2019). Sequence homologs of lep-5 have not been found outside of nematodes; however, lncRNAs are known to evolve rapidly, usually making it impossible to identify orthologs based on primary sequence alone (Diederichs, 2014). It is tempting to speculate that structural and/or functional orthologs of lep-5 are present in mammals and could have an important role in the regulation of LIN28 and its control of puberty.

Intriguingly, mammalian MKRN3 has the opposite effect on developmental timing as its C. elegans ortholog lep-2. While lep-2 mutant adults exhibit juvenile characteristics in the male tail tip (Herrera et al., 2016) and nervous system (this work), null or hypomorphic mutations in human MKRN3 lead to Central Precocious Puberty (Abreu et al., 2013), in which children younger than eight (girls) or nine (boys) years of age develop secondary sexual characteristics. This may reflect a generally more complex role for a putative MKRN3–LIN28–LET7 regulatory module in mammalian puberty compared to C. elegans sexual maturation. For example, overexpression of Lin28a delays puberty in male and female mice (Corre et al., 2016; Zhu et al., 2010), consistent with the maturation-inhibiting function of C. elegans lin-28. However, Lin28b–/– mice, as well as mice overexpressing let-7, also exhibit delayed sexual maturation, in contrast to the roles of lin-28 and let-7 in C. elegans; moreover, these effects were seen only in males (Corre et al., 2016). In mammals, these genes can be easily imagined to function at multiple steps in sexual development, such that apparently antagonistic activities could arise from roles for the mammalian heterochronic pathway in the production of both maturation-promoting and maturation-inhibiting signals. Clarification of these issues will require regionally and temporally controlled manipulations of mammalian gene activity. Here, we found that human MKRN3 was not able to rescue the defects of C. elegans lep-2 mutants. Remarkably, however, MKRN3 overexpression caused a slight delay in the sexual maturation of two neuron types, AIM and AWA, in the C. elegans nervous system. This strongly suggests that the biochemical functions of LEP-2 and MKRN3 are not equivalent, and, moreover, that the mechanism by which MKRN3 regulates pubertal timing is conserved in C. elegans. Several potential models are consistent with these observations (Figure 6). MKRN3 might inhibit LEP-2, perhaps through a dominant-negative mechanism in which MKRN3 binds to lep-5 but is unable to recognize C. elegans LIN-28. Alternatively, MKRN3 might somehow stabilize LIN-28, rather than promoting its degradation, or, less likely, MKRN3 could function in parallel with the lep-2—lin-28 module. Further, we cannot rule out the possibility that MKRN3 overexpression artifactually inhibits lep-2 function.

Ours are not the first studies to examine roles for the heterochronic pathway in the C. elegans nervous system. Early in larval development, the timing of synaptic remodeling of a class of GABAergic motorneurons is controlled by the heterochronic gene lin-14, which specifies multiple aspects of the L1 stage (Hallam and Jin, 1998). Later, a hermaphrodite-specific aspect of nervous system maturation, the outgrowth of the HSN axon, is controlled by lin-14 and lin-28 (Olsson-Carter and Slack, 2010). However, in neither case have the relevant targets of heterochronic function been identified. Interestingly, the heterochronic pathway, particularly let-7 and lin-41, also has a role in regulating damaged-induced regeneration of the AVM axon in adults, an ability that declines with age (Zou et al., 2013). Moreover, studies in Drosophila also support a role for the heterochronic pathway in the timing of major transitions in the nervous system (Faunes and Larraín, 2016). let-7 and related miRNAs control temporal specification of neuronal fate at the larval-to-pupal transition (Chawla et al., 2016; Marchetti and Tavosanis, 2017; Wu et al., 2012); they also regulate remodeling of neuromuscular anatomy and function at the pupal-to-adult transition (Caygill and Johnston, 2008; Sokol et al., 2008). Recently, Drosophila lin-28 has been found to be widely expressed in the nervous system in late third instar larvae, and loss of lin-28 function accelerates the larval-to-pupal transition (González-Itier et al., 2018). Together, these findings indicate an ancient role for this regulatory module in orchestrating developmental transitions in the nervous system.

Work carried out in parallel with the studies reported here (Pereira et al., 2019) found that the heterochronic pathway acts cell-autonomously to control several other aspects of male-specific sexual differentiation, including a neurotransmitter fate switch and changes in synaptic connectivity. Interestingly, Pereira et al. identified lin-29a, a male-specific isoform of lin-29, as a key mediator of many of these transitions, acting to integrate temporal and sexual signals. Here, our studies consider other aspects of male-specific functional maturation and focus on upstream components of the heterochronic pathway, particularly lep-2 and lep-5, best known for their roles in male tail development (Herrera et al., 2016; Kiontke et al., 2019). The strong nervous system phenotypes of these mutants indicate that this specialized branch of the heterochronic pathway is critical for the sexual maturation of the nervous system. Moreover, these studies strongly suggest that the function of MKRN3 in regulating human puberty is related to the LIN28–LET7 axis and predict the central involvement of a yet-unidentified lncRNA in this process.

Though all of the maturation events we have studied here are male-specific, the heterochronic pathway almost certainly has a role in hermaphrodite-specific and non-sex-specific maturation of the C. elegans nervous system as well. Indeed, as mentioned above, differentiation of the hermaphrodite-specific HSN neuron requires heterochronic function (Olsson-Carter and Slack, 2010); however, little is known about non-male-specific changes in neuronal function that accompany sexual maturation. Interestingly, two recent studies have demonstrated functional transitions in chemosensory coding and behavior that occur at the juvenile-to-adult transition in hermaphrodites (Fujiwara et al., 2016; Hale et al., 2016). In one of these cases (Fujiwara et al., 2016), signaling from the gonad plays an important role, raising the more general possibility that signals from the gonad could intersect with cell-autonomous timers to provide more flexible control of behavior.

Although the phenomena we have studied here derive their sex-specificity by the integration of temporal and sexual signaling at the level of mab-3 and lin-29a, other interactions between sex-determination and heterochronic timing are likely. By ChIP-seq studies, lin-28 has been identified as a direct target of tra-1 (Berkseth et al., 2013); both tra-1 and tra-2 are potential targets of the miRNA let-7 (targetscan.org); the abundance of TRA-1 protein has been shown to be developmentally regulated (Weinberg et al., 2018) (H.L. and D.S.P., in prep.); and some phenotypes of heterochronic mutants can be considered sexual transformations (Ambros and Horvitz, 1984). Interestingly, many aspects of the timing of mammalian puberty also appear to be sex-specific (Abreu and Kaiser, 2016; Cousminer et al., 2016). Together, these observations blur the distinctions between sexual differentiation mechanisms and developmental timing pathways, helping provide a mechanistic understanding of the observation that sexual dimorphism often arises through heterochrony (McNamara, 2012). Because the deep crosstalk between sexual and temporal information is unlikely to be limited to C. elegans, reproductive maturation disorders and sex-biased disorders of the nervous system in humans will both be informed by a deeper appreciation of these intersections.

Materials and methods

C. elegans culture and staging

In general, C. elegans were cultured using standard methods (Stiernagle, 2006). All strains used here carried the mutation him-5(e1490) to increase the numbers of males, such that we refer to him-5(e1490) as the wild-type. All strains used in this work are listed in the Key resources table..

To isolate populations at specific larval stages, adult hermaphrodites were allowed to lay eggs for two hours, roughly synchronizing their progeny. Because male tail morphogenesis cannot be used to reliably stage some heterochronic mutant males under low magnification, sibling hermaphrodites were used as proxies to roughly stage animals. For all individuals scored, larval stage was definitively determined by the progression of gonadal development using DIC microscopy (Zeiss Axioplan 2, 63x Plan-Apo objective). Males in which the linker cell had migrated dorsally but not yet ventrally were staged as L3 larvae. Males in which the linker cell had migrated ventrally and extended posteriorly were staged as L4 larvae. One-day old adults were obtained by picking L3-L4 larvae and allowing them to mature overnight. Because lin-28 hermaphrodites have severe egg-laying defects, we passed all strains containing lin-28 mutations through the dauer stage to suppresses this phenotype (Liu and Ambros, 1991), allowing us to use timed egg-laying for rough synchronization.

Imaging and quantitation of fsEx295, fsIs5, fsEx527, ksIs2, and myIs4

Animals were mounted on 4% agarose pads in M9 buffer supplemented with levamisole. GFP and DIC brightfield images were obtained using a 63x PlanApo objective on a Zeiss Axioplan 2. GFP intensity was qualitatively determined to be bright (3), moderate (2), faint (1), or absent (0). For rescue and overexpression experiments, the experimenter was blind to the presence or absence of the expression array when scoring reporter fluorescence. Animals not bearing the array were used as non-transgenic sibling controls. Representative images were taken using the same illumination intensity and exposure time. Images of ksIs2 were captured using an Apotome. Fluorescence intensity data were compared using Mann-Whitney tests (for pairwise analyses) or Kruskal-Wallis tests with Dunn’s correction (for multiple comparisons). For these and all other statistical analyses reported in this work, statistical significance is indicated in the figures as follows: *0.01 < p < 0.05; **0.001 < p < 0.01; ***p<0.001.

Imaging of strains containing maIs108 and nyEx53

Animals were mounted as described above and images were obtained using a Leica SP5 Confocal Microscope with a HyD hybrid detector and 63x objective. Z-series were obtained starting at the most superficial neuron and ending at the deepest neuron as determined by expression of the pan-neural nuclear marker otIs355[Prab-3::tagRFP::2xNLS]. Images were taken at 0.5 µm steps, 200 Hz, and 2x zoom. FIJI (Schindelin et al., 2012) was used to create max-intensity z-projections of the entire z-stack. Images were pseudocolored and merged using Adobe Photoshop. Quantitation and statistical analysis was carried out as described in the preceding paragraph.

Imaging of strains containing nyEx62

Animals were mounted as above but were anesthetized with 20 mM NaN3. Images were obtained using a Zeiss AxioImager equipped with an Apotome using the 40x objective. Ten to fifteen slices, taken with a spacing of 0.5 µm, were merged into a Z projection using Image J.

Transgenic strains

cDNAs were amplified from RNA extracted from mixed-stage him-5 cultures. lep-2 and lin-28 cDNAs were amplified using primers shown in Table S1. Human MKRN3 cDNA was amplified from the plasmid SC319872 (Origene) using primers shown in Table S1. The resulting cDNAs were cloned into pDONR221 and recombined via Multisite Gateway Cloning (Invitrogen) to make following expression clones:

Peat-4prom11::lep-2::SL2::mCherry::unc-54 3´UTR

Peat-4prom11::lin-28::SL2::mCherry::unc-54 3´UTR

Pgpa-4del6::lep-2::SL2::mCherry::unc-54 3´UTR

Prab-3::MKRN3::SL2::mCherry::unc-54 3´UTR

Transgenes were injected into young adult hermaphrodites using standard methods (Evans, 2006) to generate fsEx transgenic lines.

The Plep-5::GFP reporter was constructed by overlap extension PCR (Nelson and Fitch, 2011) using primers KKOlp-5_1 and plep-5_GFP_B to amplify C. elegans genomic DNA (for fragment A) and primers pPD95.75.C and pPD95.75D to amplify from plasmid pPD95.75 (for fragment B). Final extension used primers KKlep5_expr-9 and pPD95.75.D*. Transgenes were injected with standard methods using the co-injection marker pRF4[rol-6(d)].

Behavioral assays

Food-Leaving assay

Males were isolated to a newly seeded plate approximately 16 hr prior to the start of the assay, at the L4 stage (to assay adults) or L1 (to assay L3s). Single males were placed on a 7 µL spot of E. coli OP50 in the center of a 10 cm plate containing 10 mL of standard agar lacking cholesterol based on the method of Lipton et al. (2004). At designated intervals, tracks were examined to determine the farthest distance traveled by the animal: zero (never left food),<1 cm (a ‘minor excursion’ off the food),>1 cm but <3.5 cm (a ‘major excursion’ off the food), or >3.5 cm (left the plate). Animals that died on the plate were censored. The fraction in each category for each genotype or age was compared with controls using a chi-square test (Prism 7.0).

Ascaroside attraction

Pheromone attraction assays were performed and data were analyzed as previously described (Fagan et al., 2018).

Response and location of vulva

Adult males were placed on a mating assay plate with 15 to 20 unc-31; him-5 hermaphrodites and allowed four minutes to display mating behavior as previously described (Barr and Sternberg, 1999; Liu and Sternberg, 1995), Males were assayed in pairs and watched in real time to determine whether they could (1) respond to hermaphrodite contact and (2) locate the vulva. Data is reported as follows: Response = # of males responding/total # of males assayed. Location = # of males that locate the vulva/total # of males that responded. For each step, successful individuals were scored as one and unsuccessful individuals as 0. Kruskal-Wallis tests (with Dunn’s correction) were used to analyze differences between groups.

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The Makorin lep-2 and the lncRNA lep-5 regulate lin-28 to schedule sexual maturation of the C. elegans nervous system" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Catherine Dulac as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: L René García (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript, "The Makorin lep-2 and the IncRNA lep-5 regulate lin-28 to schedule sexual maturation of the C. elegans nervous system", demonstrates that the lin-28/let-7/lin-41 heterochronic gene pathway is used to direct the developmentally programmed sexual dimorphism of C. elegans male neurons. In previous work, the authors have beautifully worked out the genetics that describe the molecular regulation of sexual dimorphism in neurons common to male and hermaphrodite C. elegans. In this manuscript, the research builds on this foundation to identify the pathway that determines when sexual dimorphism is expressed. The work uses genetic analysis to place lep-2, a homolog of mammalian Makorin, and lep-5, a long noncoding RNA, upstream of lin-28. Assayed neurons in lep-2 and lep-5 mutants show delayed transition from larval to adult gene expression changes. Consistent with the retardation in developmental sexual dimorphism, the adult lep mutants show abnormal copulatory drive characteristics. The researchers also show that LEP-2 and LIN-28 regulate the timing of neural sexual remodeling cell-autonomously, which is an important observation. Finally, the researchers make an argument that the mammalian Makorin's function, as a regulator that delays reproductive maturity, can be capitulated experimentally in C. elegans.

General assessment:

Both reviewers find the work interesting and important, especially praising the finding that LEP-2 and LIN-28 act cell-autonomously. However, both of them point out the need for clarification in several points.

Essential revisions:

1) Both reviewers raise concern about MKRN3, because in the MKRN3 overexpression experiments the authors looked at only changes in srj-54 expression in AIM and the effect seen here was rather modest. The reviewers request looking at other maturation marker genes in other neurons. These experiments need to be performed upon revision. Specifically,

- Regarding the experiment showing that expression of human MKRN3 in worms delays sexual maturation, it would be nice to see that this is a general effect that occurs with more than one marker in more than one neuron (the authors only show delay in srj-54 expression in AIM).

- In the last paragraph of the subsection “Human MKRN3 can inhibit sexual differentiation of the C. elegans nervous system”, the authors state that transgenic expression of MKRN3, has a statistical effect of delaying adult male gene expression in AIM during L4 to adult stage. For AIM, the effect is modest, which is ok, but I do not feel by itself is strong enough to support the claim that Makorin's function in mammals can be recapitulated in the C. elegans model. If one can show data for modest (statistical) retardation in developmental gene expression for the other neurons studied in the paper: AWA(odr-10), ADF(mab-3), ASJ (daf-7), CEM (pkd-2), then I would feel more confident that MKRN3 generally affects the worm's heterochronic pathway in many neurons. One might need to use stronger promoters than the rab-3 promoter for each neuron tested, but this would provide more robust evidence for placing Makorin in the model presented in Figure 6.

2) One of the reviewers points out the need for quantification of the results in Figure 4—figure supplement 1. Indeed, while quantitative data are shown for Figure 4—figure supplement 1D, it is not the case for Figure 4—figure supplement 1A and B. Quantification of expression levels and qualitative description of the expression pattern need to be added.

- Supplementary Figure 2A needs quantification or more details about the phenotype: is it faint in 100% males (as the image shows) or what kind of srj-54 phenotype do mab-3 mutants display?

Essential revisions:

1) Both reviewers raise concern about MKRN3, because in the MKRN3 overexpression experiments the authors looked at only changes in srj-54 expression in AIM and the effect seen here was rather modest. The reviewers request looking at other maturation marker genes in other neurons. These experiments need to be performed upon revision. Specifically,

- Regarding the experiment showing that expression of human MKRN3 in worms delays sexual maturation, it would be nice to see that this is a general effect that occurs with more than one marker in more than one neuron (the authors only show delay in srj-54 expression in AIM).

- In the last paragraph of the subsection “Human MKRN3 can inhibit sexual differentiation of the C. elegans nervous system”, the authors state that transgenic expression of MKRN3, has a statistical effect of delaying adult male gene expression in AIM during L4 to adult stage. For AIM, the effect is modest, which is ok, but I do not feel by itself is strong enough to support the claim that Makorin's function in mammals can be recapitulated in the C. elegans model. If one can show data for modest (statistical) retardation in developmental gene expression for the other neurons studied in the paper: AWA(odr-10), ADF(mab-3), ASJ (daf-7), CEM (pkd-2), then I would feel more confident that MKRN3 generally affects the worm's heterochronic pathway in many neurons. One might need to use stronger promoters than the rab-3 promoter for each neuron tested, but this would provide more robust evidence for placing Makorin in the model presented in Figure 6.

We agree that our analysis of the MKRN3 overexpression phenotype was somewhat preliminary. In response to these concerns, the revised paper now includes data for two additional markers, odr-10 (AWA) and pkd-2 (CEM). We find that MKRN3 overexpression causes a significant delay in the temporally controlled downregulation of odr-10 expression, strengthening our initial conclusion that MKRN3 can inhibit C. elegans nervous system maturation. In contrast, we observed no detectable effect of MKRN3 on pkd-2 expression, raising the possibility that it may not regulate CEM maturation. This is consistent with our earlier observation that CEM maturation seems to rely less on lep-2 and lep-5 than does AWA maturation (Figure 2) and that the CEMs therefore likely employ other mechanisms to time their differentiation.

For technical reasons, we were unable to score expression of mab-3 or daf-7 in our MKRN3 overexpression strains. The transmission of our mab-3::GFP transgene was too low for us to be able to reliably assess expression in the simultaneous presence of the MKRN3 arrays. Furthermore, we were unable to obtain stable lines carrying both the daf-7 reporter transgene (ksIs2) and a MKRN3 overexpression array. While the reasons for this are unknown, we feel that our scoring of odr-10 and pkd-2 substantially strengthens our analysis of the MKRN3 phenotype. Our paper now demonstrates that human MKRN3 can inhibit two distinct aspects of the maturation of the C. elegans nervous system (srj-54/AIM and odr-10/AWA), but that other aspects (pkd-2/CEM) may not be influenced by this mechanism.

2) One of the reviewers points out the need for quantification of the results in Figure 4—figure supplement 1. Indeed, while quantitative data are shown for Figure 4—figure supplement 1D, it is not the case for Figure 4—figure supplement 1A and B. Quantification of expression levels and qualitative description of the expression pattern need to be added.

- Supplementary Figure 2A needs quantification or more details about the phenotype: is it faint in 100% males (as the image shows) or what kind of srj-54 phenotype do mab-3 mutants display?

We agree with these points. The revised manuscript now reports careful quantitative scoring of both srj-54 and odr-10 expression in mab-3 mutants. In agreement with our previous qualitative findings, this reveals that there are significant defects in the adult expression of both markers.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (C. elegans)	him-5	NA	WormBase ID: WBGene00001864
Gene (C. elegans)	lep-2	NA	WormBase ID: WBGene00002278
Gene (C. elegans)	lep-5	NA	WormBase ID: WBGene00010424
Gene (C. elegans)	let-7	NA	WormBase ID: WBGene00002285
Gene (C. elegans)	lin-28	NA	WormBase ID: WBGene00003014
Gene (C. elegans)	lin-29	NA	WormBase ID: WBGene00003015
Gene (C. elegans)	lin-4	NA	WormBase ID: WBGene00002993
Gene (C. elegans)	lin-41	NA	WormBase ID: WBGene00003026
Gene (C. elegans)	mab-3	NA	WormBase ID: WBGene00003100
Gene (C. elegans)	pha-1	NA	WormBase ID: WBGene00004010
Gene (C. elegans)	unc-31	NA	WormBase ID: WBGene00006767
Gene (human)	MKRN3	NA	HGNC:7114
Strain, strain background (C. elegans)	otIs355[rab-3p(prom1):: 2xNLS::TagRFP] IV; him-5(e1490) V; nyEx62[Plep-5::GFP + pRF4]	this paper	DF305	otIs355: OH10689; e1490: CB4088; nyEx62 see below
Strain, strain background (C. elegans)	lin-4(e912) II; otIs355[rab-3p(prom1):: 2xNLS::TagRFP] IV ; him-5(e1490) V; nyEx62[Plep-5::GFP + pRF4]	this paper	DF306	e912: CB2627; otIs355: OH10689; e1490: CB4088; nyEx62 see below
Strain, strain background (C. elegans)	pkd-2(sy606) IV; him-5(e1490) V	Caenorhabditis Genetics Center	PT8	RRID:WB-STRAIN:PT8
Strain, strain background (C. elegans)	fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V	Lee and Portman, 2007	UR219
Strain, strain background (C. elegans)	him-5(e1490) V; kyIs53[Podr-10::odr-10::GFP] X	Ryan et al., 2014	UR460
Strain, strain background (C. elegans)	unc-31(e169) IV; him-5(e1490) V	PMID: 22023935	UR624
Strain, strain background (C. elegans)	mab-3(e1240) II; him-5(e1490) V; kyIs53[ODR-10::GFP] X	this paper	UR626	e1240: UR278; e1490: UR926; kyIs53: CX3344
Strain, strain background (C. elegans)	pha-1(e2123) III; him-5(e1490) V; fsEx295[ODR-10::GFP fosmid + pBx1]	Ryan et al., 2014	UR773
Strain, strain background (C. elegans)	him-5(e1490) V; lin-41(bx42) X; fsEx295[ODR-10::GFP fosmid + pBx1]	this paper	UR786	e1490: UR926; bx42: EM106; fsEx295: UR773
Strain, strain background (C. elegans)	lin-41(bx42) I; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V	this paper	UR869	bx42: EM106; fsIs5: UR218; e1490: UR926
Strain, strain background (C. elegans)	lin-41(ma104) I; myIs4[Ppkd-2::pkd-2:: GFP + cc::GFP] him-5 (e1490) V	this paper	UR871	ma104: CT8; myIs4: UR1258; e1490: UR1258
Strain, strain background (C. elegans)	lin-28(n719) I; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V	this paper	UR872	n719: MT1524; fsIs5: UR218; e1490: UR218
Strain, strain background (C. elegans)	lin-28(n719) I; myIs4[Ppkd-2::pkd-2:: GFP + cc::GFP] him-5 (e1490) V	this paper	UR873	n719: MT1524; myIs4: UR1258; e1490: UR1258
Strain, strain background (C. elegans)	fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; lep-5(fs8) X	this paper	UR874	fsIs5: UR218; e1490: UR218; fs8: UR290
Strain, strain background (C. elegans)	myIs4[Ppkd-2::pkd-2::GFP  + cc::GFP] him-5(e1490) V; lep-5(fs8) X	this paper	UR876	myIs4: UR1258; e1490: UR1258; fs8: UR290
Strain, strain background (C. elegans)	myIs4[Ppkd-2::pkd-2::GFP  + cc::GFP] him-5(e1490) V; let-7(n2853) X	this paper	UR878	myIs4: UR1258; e1490: UR1258; n2853: MT7626
Strain, strain background (C. elegans)	him-5(e1490) V	this paper	UR926	e1490: CB4088
Strain, strain background (C. elegans)	fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; lep-5(fs20) X	this paper	UR1248	fsIs5: UR218; e1490: UR218; fs20: UR1142
Strain, strain background (C. elegans)	lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V	this paper	UR1249	ok900: RB986; fsIs5: UR218; e1490: UR218
Strain, strain background (C. elegans)	fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; let-7(n2853) X	this paper	UR1250	fsIs5: UR218; e1490: UR218; n2853: MT7626
Strain, strain background (C. elegans)	pha-1(e2123) III; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx295[ODR-10:: GFP fosmid + pBx1]	this paper	UR1251	e2123: GE24; e1490: UR926; fsEx295: UR773
Strain, strain background (C. elegans)	pha-1(e2123) III; lep-2(ok900) IV; him-5(e1490) V; fsEx295[ODR-10::GFP fosmid + pBx1]	this paper	UR1252	e2123: GE24; ok900: RB986; e1490: UR926; fsEx295: UR773
Strain, strain background (C. elegans)	lin-28(n719) I; pha-1(e2123) III; him-5(e1490) V; fsEx295[ODR-10::GFP fosmid + pBx1]	this paper	UR1253	n719: MT1524; e2123: GE24; e1490: UR926; fsEx295: UR773
Strain, strain background (C. elegans)	him-5(e1490) V; let-7(n2853) X; fsEx295[ODR-10::GFP fosmid + pBx1]	this paper	UR1254	e1490: UR926; n2853: MT7626; fsEx295: UR773
Strain, strain background (C. elegans)	pha-1(e2123) III; him-5(e1490) V; let-41(ma104) X; fsEx295[ODR-10::GFP fosmid + pBx1]	this paper	UR1255	e2123: GE24; e1490: UR926; ma104: CT8; fsEx294: UR773
Strain, strain background (C. elegans)	lin-29a(xe38) II; him-5(e1490) V; kyIs53[Podr-10::odr- 10::GFP] X	this paper	UR1256	xe38: HW1693; e1490: UR926; kyIs53: CX3344
Strain, strain background (C. elegans)	lin-29a(xe40) II; him-5(e1490) V; kyIs53[Podr-10::odr -10::GFP] X	this paper	UR1257	xe40: HW1695; e1490: UR926; kyIs53: CX3344
Strain, strain background (C. elegans)	myIs4[Ppkd-2::pkd-2:: GFP + cc::GFP] him-5 (e1490) V	this paper	UR1258	myIs4: PT621; e1490: PT621
Strain, strain background (C. elegans)	myIs4[Ppkd-2::pkd-2:: GFP + cc::GFP] him-5(e1490) V; lep-5(ny28) X	this paper	UR1259	myIs4: PT621; e1490: PT621; ny28: UR1022
Strain, strain background (C. elegans)	lep-2(ok900) IV; myIs4[Ppkd-2::pkd-2:: GFP + cc::GFP] him-5 (e1490) V	this paper	UR1260	ok900: RB986; myIs4: UR1258; e1490: UR1258
Strain, strain background (C. elegans)	lin-41(bx42) I; myIs4[Ppkd-2::pkd-2:: GFP + cc::GFP] him-5 (e1490) V	this paper	UR1261	bx42: EM106; myIs4: UR1258; e1490: UR1258
Strain, strain background (C. elegans)	him-5(e1490) V; lep-5(ny28) X; fsEx527[1.5 kb MAB-3::GFP (pDZ162) +  cc::GFP]	this paper	UR1262	e1490: UR926; ny28: UR1022; fsEx527: UR1131
strain, strain background (C. elegans)	lep-2(ok900) IV; him-5(e1490) V; fsEx527[1.5 kb MAB-3::GFP (pDZ162) + cc::GFP]	this paper	UR1263	ok900: RB986; e1490: UR926; fsEx527: UR1131
Strain, strain background (C. elegans)	lin-28(n719) I; him-5(e1490) V; fsEx527[1.5 kb MAB-3::GFP (pDZ162)  + cc::GFP]	this paper	UR1264	n719: MT1524; e1490: UR926; fsEx527: UR1131
Strain, strain background (C. elegans)	him-5(e1490) V; ksIs2[Pdaf-7::GFP +  rol-6(su1006)]	this paper	UR1265	e1490: UR926; ksIs2: FK181
Strain, strain background (C. elegans)	him-5(e1490) V; lep-5(ny28) X; ksIs2[Pdaf-7:: GFP + rol-6(su1006)]	this paper	UR1266	e1490: UR926; ny28: UR1022; ksIs2: FK181
Strain, strain background (C. elegans)	lep-2(ok900) IV; him-5(e1490) V; ksIs2[Pdaf-7::GFP + rol-6(su1006)]	this paper	UR1267	ok900: RB986; e1490: UR926; ksIs2: FK181
Strain, strain background (C. elegans)	fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx559[Prab-3::MKRN3::SL2:: mCherry::unc-54 3' UTR  + Pmyo-3::mCherry Line 1]	this paper	UR1268	fsIs5: UR218; e1490: UR926; fsEx559: see below
Strain, strain background (C. elegans)	fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx560[Prab-3::MKRN3::SL2:: mCherry::unc-54 3' UTR  + Pmyo-3::mCherry Line 2]	this paper	UR1269	fsIs5: UR218; e1490: UR926; fsEx560: see below
Strain, strain background (C. elegans)	lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx561[Peat-4prom11::lep- 2::Sl2::mCherry::unc-54 3' UTR + Pmyo-3::mCherry Line 1]	this paper	UR1270	ok900: RB986; fsIs5: UR218; e1490: UR218; fsEx561: see below
Strain, strain background (C. elegans)	lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx562[Peat-4prom11::lep- 2::Sl2::mCherry::unc-54 3' UTR + Pmyo-3::mCherry Line 2]	this paper	UR1271	ok900: RB986; fsIs5: UR218; e1490: UR218; fsEx562: see below
Strain, strain background (C. elegans)	lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx563[Peat- 4prom11::lep-2::Sl2::mCherry::unc-54 3' UTR + Pmyo-3::mCherry Line 3]	this paper	UR1272	ok900: RB986; fsIs5: UR218; e1490: UR218; fsEx563: see below
Strain, strain background (C. elegans)	lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx564[Prab-3::MKRN3::SL2:: mCherry::unc-54 3' UTR + Pmyo-3::mCherry Line 1]	this paper	UR1273	ok900: RB986; fsIs5: UR218; e1490: UR218; fsEx564: see below
Strain, strain background (C. elegans)	lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx565[Prab-3::MKRN3::SL2:: mCherry::unc-54 3' UTR +  Pmyo-3::mCherry Line 2]	this paper	UR1274	ok900: RB986; fsIs5: UR218; e1490: UR218; fsEx565: see below
Strain, strain background (C. elegans)	lin-28(n719) I; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx566[Peat-4prom11::lin- 28::Sl2::mCherry::unc-54 3' UTR + Pmyo-3::mCherry Line 1]	this paper	UR1275	n719: MT1524; fsIs5: UR218; e1490: UR218; fsEx566: see below
Strain, strain background (C. elegans)	lin-28(n719) I; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; fsEx567[Peat-4prom11::lin-28:: Sl2::mCherry::unc-54 3' UTR + Pmyo-3::mCherry Line 2]	this paper	UR1276	n719: MT1524; fsIs5: UR218; e1490: UR218; fsEx567: see below
Strain, strain background (C. elegans)	pha-1(e2123) III; lep-2(ok900) IV; him-5(e1490) V; fsEx295[ODR-10::GFP fosmid + pBx1]; fsEx568[Pgpa-4del6::lep- 2::Sl2::mCherry::unc-54 3' UTR + Pelt-2::GFP Line 1]	this paper	UR1277	pha-1: GE24; ok900: RB986; e1490: UR926; fsEx295: UR773; fsEx568: see below
Strain, strain background (C. elegans)	pha-1(e2123) III; lep-2(ok900) IV; him-5(e1490) V; fsEx295[ODR-10::GFP fosmid + pBx1]; fsEx569[Pgpa-4del6 ::lep-2::Sl2::mCherry::unc-54 3' UTR + Pelt-2::GFP Line 2]	this paper	UR1278	e2123: GE24; ok900: RB986; e1490: UR926; fsEx295: UR773; fsEx569: see below
Strain, strain background (C. elegans)	lin-28(n719) I; lep-2(ok900) IV; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V	this paper	UR1279	n719: MT1524; ok900: RB986; fsIs5: UR218; e1490: UR218
Strain, strain background (C. elegans)	lin-28(n719) I; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; lep-5(ny28) X	this paper	UR1280	n719: MT1524; fsIs5: UR218; e1490: UR218; ny28: UR1022
Strain, strain background (C. elegans)	lin-28(n719) I; fsIs5[Psrj-54::YFP + cc::GFP] him-5(e1490) V; let-7(n2853) X	this paper	UR1281	n719: MT1524; fsIs5: UR218; e1490: UR218; n2853: MT7626
Recombinant DNA reagent	MKRN3 (NM_005664) Human Untagged Clone	Origene	Cat# SC319872
Recombinant DNA reagent	pDONR221	Invitrogen		https://www.addgene.org/vector-database/2394/
Recombinant DNA reagent	Peat-4prom11::lep- 2::SL2::mCherry::unc-54 3´UTR	this paper		Construction described below
Recombinant DNA reagent	Peat-4prom11::lin- 28::SL2::mCherry::unc-54 3´UTR	this paper		Construction described below
Recombinant DNA reagent	Pgpa-4del6::lep-2::SL2: :mCherry::unc-54 3´UTR	this paper		Construction described below
Recombinant DNA reagent	Prab-3::MKRN3::SL2:: mCherry::unc-54 3´UTR	this paper		Construction described below
Software, algorithm	Prism 8		GraphPad Software	RRID:SCR_002798
Software, algorithm	ApE, A Plasmid Editor		M Wayne Davis	RRID:SCR_014266 http://jorgensen.biology.utah.edu/wayned/ape/