Timing mechanism of sexually dimorphic nervous system differentiation
The nervous systems of males and females of many animal species display anatomical and functional differences ( Jazin and Cahill , 2010; McCarthy and Arnold , 2011; Portman , 2017; Yang and Shah , 2014 ) . Sex-specific maturation events in the brain are typically triggered at a specific window of postnatal life . Sexual maturation in mammals , called puberty , represents a key developmental transition and is accompanied by substantial sexually dimorphic changes in the brain ( Avendaño et al . , 2017; Juraska and Willing , 2017; Sisk and Foster , 2004 ) . The onset of puberty in mammals is characterized by the activation of neurons in the hypothalamus that produce hormonal signals ( Avendaño et al . , 2017 ) . However , the mechanisms that control the activation of these neurons and hence the timing of pubertal onset are poorly understood . Like in other animals , most of the sex-specific features of the nervous system of the nematode Caenorhabditis elegans are also established during postembryonic development ( Barr et al . , 2018; Sulston et al . , 1980; Sulston and Horvitz , 1977 ) . Sexual dimorphisms in the C . elegans nervous system fall into two broad types: ( 1 ) Some neurons are present only in one sex but not the opposite sex ( Sulston et al . , 1980; Sulston et al . , 1983 ) . These sex-specific neurons are directly involved in distinct aspects of sex-specific mating and reproductive behavior ( Emmons , 2018 ) . ( 2 ) Some embryonically born , postmitotic neurons that are present in both sexes ( ‘sex-shared’ neurons ) can adopt sex-specific features during sexual maturation ( Portman , 2017 ) . Sexually dimorphic features of sex-shared neurons include sex-specific synaptic wiring patterns ( Jarrell et al . , 2012 ) and sex-specific expression of signaling molecules , such as neurotransmitters , neuropeptides and chemoreceptors ( Hilbert and Kim , 2017; Pereira et al . , 2015; Ryan et al . , 2014; Serrano-Saiz et al . , 2017a; Serrano-Saiz et al . , 2017b ) . For example , the AIM interneuron class is glutamatergic in juvenile , pre-sexual maturation stages of both sexes , but during sexual maturation will turn off glutamatergic identity and turn on cholinergic neurotransmitter identity specifically in males ( Pereira et al . , 2015 ) . In other cases , sexually dimorphic features emerge from a ‘mixed’ juvenile state . For example , juvenile phasmid sensory neurons innervate several distinct interneuron targets in both sexes before sexual maturation , but during sexual maturation specific subsets of these connections get pruned in a sex-specific manner ( Oren-Suissa et al . , 2016 ) . Most if not all of these sexually dimorphic features of the C . elegans nervous system terminally differentiate during the fourth larval stage ( Desai et al . , 1988; Oren-Suissa et al . , 2016; Pereira et al . , 2015; Serrano-Saiz et al . , 2017a; Sulston and Horvitz , 1977 ) . The molecular mechanisms that control the timing of sexual maturation in postmitotic neurons have largely remained obscure . Candidate regulators of sex-specific developmental timing events are so-called heterochronic genes , first identified in C . elegans ( Ambros and Horvitz , 1984 ) . Heterochronies refer to offsets of the relative timing of somatic developmental events versus gonadal developmental events ( Slack and Ruvkun , 1997 ) . In heterochronic mutants , the timing of cellular cleavage patterns in postembryonic skin cell lineages is disrupted in a highly stereotyped manner; in some mutants , lineage patterning events normally occurring late will occur earlier ( ‘precocious mutants’ ) while in other mutants specific cell lineage patterning events will be delayed or never occur ( Ambros , 1989; Ambros and Horvitz , 1984; Slack and Ruvkun , 1997 ) . Molecular analysis of these heterochronic mutants revealed a gene regulatory pathway , which contains several deeply conserved post-transcriptional regulatory factors at its core , including the miRNA let-7 , the LIN-28 protein , a negative regulator of let-7 processing , and the RNA-binding , posttranscriptional regulator LIN-41 , a key target of the let-7 miRNA ( Figure 1A ) ( Ambros , 1989; Ecsedi et al . , 2015; Moss et al . , 1997; Reinhart et al . , 2000; Slack et al . , 2000 ) . While these regulatory factors are well known to control cell division patterns in the ectoderm , their role in postmitotic cell types is much less well defined ( Del Rio-Albrechtsen et al . , 2006; Hallam and Jin , 1998; Howell et al . , 2015; Olsson-Carter and Slack , 2010; Zou et al . , 2013 ) . Within the nervous system , the impact of heterochronic genes on sex-specific differentiation events during sexual maturation at the juvenile to adult transition has been poorly defined . Such a role is not an obvious matter because heterochronic genes do not control timing in all tissues . For example , heterochronic genes are not involved in controlling the timing of gonadal maturation ( Ambros and Horvitz , 1984; Euling and Ambros , 1996 ) . We show here that the phylogenetically conserved core heterochronic pathway genes lin-28 , let-7 and lin-41 control the timing of maturation of sexual dimorphisms in different neuron classes . We show that these broadly expressed timing genes act through distinct cell-specific effector modules , among them a specific isoform of the lin-29 Zn finger transcription factor and the two DM domain ( DMD ) containing transcription factors mab-3 and dmd-3 . These temporally regulated effector genes are expressed in a sexually dimorphic manner in distinct sets of sex-shared neurons to control specific aspects of sexually dimorphic features and functions . Remarkably , genome-wide association studies have linked one component of the heterochronic pathway to aberrant onset of puberty in humans ( Faunes and Larraín , 2016 ) . Our findings therefore suggest the emergence of a phylogenetically conserved mechanism to control the timing of sexual maturation .
As an entry point to identify the molecular mechanisms required for the correct timing of nervous system sexual maturation , we used the previously described neurotransmitter switch of the AIM interneurons ( Figure 1B–D ) , a sex-shared neuron class that is required for mate searching behavior ( Barrios et al . , 2012; Pereira et al . , 2015 ) . A fosmid-based reporter of the eat-4/VGLUT vesicular glutamate transporter is expressed during juvenile stages in the AIM interneuron of both sexes . At the onset of sexual maturation , during the fourth larval stage , AIM neurons of the male turn off the glutamatergic gene reporter and turn on expression of fosmid-based reporters for the cholinergic genes unc-17/VACHT and cho-1/CHT ( Pereira et al . , 2015 ) . The acquisition of AIM cholinergic identity during sexual maturation only in males was confirmed by analyzing the expression of the cholinergic unc-17/VACHT locus , tagged with mKate2 using CRISPR/Cas9 genome engineering ( Figure 1—figure supplement 1 ) . Sexual maturation in many animal species is controlled by gonadal hormones ( Avendaño et al . , 2017 ) . In C . elegans , the gonad has been shown to be required for the establishment of sex-specific adult behaviors ( Arantes-Oliveira et al . , 2002; Fujiwara et al . , 2016; Kleemann et al . , 2008; Lipton et al . , 2004 ) . Therefore , we first examined whether signals from the germline or gonad may affect the AIM neurotransmitter switch . We found that neither genetic removal of the germ cells nor laser ablation of gonadal precursor cells ( which results in the loss of both gonad and germline ) affects the execution of the AIM neurotransmitter switch ( Figure 1—figure supplement 2 ) . Temporal control of cell lineage decisions during early larval stages in C . elegans has been shown to depend on the activity of the evolutionary conserved heterochronic pathway ( Ambros and Horvitz , 1984; Moss and Romer-Seibert , 2014; Slack and Ruvkun , 1997 ) . To test if members of the heterochronic pathway were required for the correct timing of nervous system differentiation during sexual maturation , we examined the impact of heterochronic mutants on the male-specific AIM neurotransmitter switch . We observed no defects in the timing of cholinergic gene battery induction in L4 in lin-4 null mutant animals which controls the timing of L2-specific aspects of cellular patterning ( Ambros and Horvitz , 1984 ) ( data not shown ) . However , we observed a premature onset of both unc-17/VACHT and cho-1/CHT expressions in AIM neurons of lin-28 mutant males ( Figure 1B , D ) , mirroring the precocious cellular cleavage defects of lin-28 in epidermal tissues ( Ambros and Horvitz , 1984 ) . Matching the precocious induction of cholinergic identity , lin-28 mutant males also showed a precocious downregulation of the glutamatergic marker eat-4/VGLUT in the AIM interneurons ( Figure 1B , D ) . We also found that in males carrying a loss-of-function mutation in the miRNA let-7 , whose processing is negatively controlled by lin-28 ( Lehrbach et al . , 2009 ) , the neurotransmitter switch of the AIM interneurons is eliminated , that is , male AIM neurons remain glutamatergic ( Figure 1C–D ) . Since let-7 is a repressor of gene expression , the loss of the neurotransmitter switch suggests that let-7 may normally act to repress a factor that prevents the neurotransmitter switch . We found that lin-41 , the first of many let-7 targets to be identified ( Slack et al . , 2000 ) encodes this repressor factor . In lin-41 loss-of-function mutants ( bch28 ) , the neurotransmitter switch was prematurely executed , while in lin-41 gain-of-function mutants ( xe8 ) , in which let-7 is unable to downregulate lin-41 activity due to a deletion of the let-7-binding sites in the lin-41 3’UTR ( Ecsedi et al . , 2015 ) , the neurotransmitter switch of AIM was abrogated and AIM neurons remained glutamatergic ( Figure 1B , D ) . The heterochronic pathway genes also controlled the onset of differentiation of the male-specific CEM sensory neurons . Even though these neurons are already born in the embryo ( and removed by apoptosis in hermaphrodites [Sulston et al . , 1983] ) , the CEM neurons only fully differentiate at the fourth larval stage ( Pereira et al . , 2015 ) . By analyzing the expression of cholinergic reporters , we found that this L4-specific maturation event is also precisely timed by the heterochronic pathway genes . In lin-28 mutants , CEM neurons prematurely expressed cholinergic genes while in let-7 mutants CEM neurons’ terminal differentiation was blocked ( Figure 1E , F ) . As we had seen for the AIM neurotransmitter switch , lin-41 mutants showed precocious cholinergic gene expression in CEM neurons and lin-41 gain-of-function mutants ( in which lin-41 expression fails to be downregulated ) also fail to show CEM cholinergic gene expression during sexual maturation suggesting that let-7 miRNA acts through regulating lin-41 for the correct timing of this process ( Figure 1E , F ) . However , as in the case of the AIM neurotransmitter switch , lin-4 mutants show no defect in the timing of CEM differentiation ( data not shown ) . In conclusion , mutant analysis of the heterochronic pathway genes showed that the lin-28/let-7/lin-41 regulatory pathway is required for the correct timing and for the acquisition of male-specific features of two distinct neuron classes; however , this regulatory cassette may couple to upstream regulatory inputs other than lin-4 . lin-41 encodes a versatile regulatory protein with RING and NHL domains , involved in both post-transcriptional RNA regulation ( including translation ) and protein ubiquitination ( Ecsedi and Großhans , 2013 ) . To ask which of the many previously described effectors of LIN-41 ( Aeschimann et al . , 2017; Ecsedi and Großhans , 2013 ) are relevant in the context of the AIM neurotransmitter switch , we turned to the lin-29 Zn finger transcription factor , whose expression is translationally inhibited by lin-41 in the context of timing of skin cell proliferation ( Aeschimann et al . , 2017; Ambros and Horvitz , 1984; Rougvie and Ambros , 1995; Slack et al . , 2000 ) ( Figure 1A ) . Orthology analysis with several tools ( Kim et al . , 2018; Wang et al . , 2017 ) as well as a detailed analysis of predicted DNA binding features suggests that LIN-29 is an ortholog of the uncharacterized vertebrate ZNF362 and ZNF384 proteins ( Figure 1A ) . During the larva-to-adult transition , let-7 inhibition of lin-41 derepresses lin-29 expression and induces adult gene expression programs in the epidermis . Consistent with lin-29 being a functional effector of lin-41 in the AIM interneurons , we find that the AIM neurotransmitter switch is entirely lost in lin-29 null mutants ( Figure 1C , D ) . In contrast , we found that the onset of differentiation of the male-specific CEM neurons in the L4 stage , as measured by the induction of cholinergic identity , is unaffected in lin-29 null mutants ( Figure 1E , F ) . These findings indicate that lin-41 controls distinct effector genes in different neuron types . The transcriptional co-factor mab-10/NAB has been shown to act with lin-29 to control epidermal cell differentiation during the larval to adult transition ( Harris and Horvitz , 2011 ) . Moreover , biochemical studies revealed that the mab-10 transcript , like lin-29 , is a direct target of the LIN-41 protein ( Aeschimann et al . , 2017 ) . We confirmed this notion by CRISPR/Cas9-mediated tagging of the mab-10 locus with mCherry , finding that ubiquitous mab-10 expression initiates at the L4 stage ( Figure 2A ) . However , analyzing the effects of a newly generated mab-10 null allele , we found that in the absence of mab-10 , the AIM neurotransmitter switch and CEM cholinergic gene expression occurred normally ( Figure 1D , F ) . Hence , while mab-10 functions with lin-29 in seam cell differentiation , it is not required for lin-29 function in AIM interneurons in males . To address in which cells the heterochronic pathway acts to control the AIM neurotransmitter switch , we first examined the expression pattern of lin-28 , let-7 , lin-41 and lin-29 in the nervous system . While expression of these genes in other cells , specifically , the epidermis , has previously been examined in detail ( Moss et al . , 1997; Rougvie , 2001; Slack et al . , 2000 ) , much less is known about their neuronal expression . We analyzed heterochronic gene expression relative to either a panneuronally expressed RFP marker or a cholinergic RFP marker ( cholinergic labeling of the AIM neuron allowed us to correlate the timing of heterochronic gene expression relative to the AIM neurotransmitter switch ) . Consistent with previous reporter gene analysis ( Moss et al . , 1997 ) , we found that a fosmid-based reporter construct of the lin-28 gene is expressed very broadly in the nervous system in a sexually non-dimorphic manner and downregulated by the end of the L2 stage ( Figure 2B ) . To analyze lin-41 expression , we used an endogenously gfp tagged lin-41 allele ( Spike et al . , 2014 ) . This lin-41 reporter allele showed broad expression in the nervous system from the L1 until the L4 stage in both sexes , when it becomes downregulated ( Figure 2C ) . Previous work showed that lin-41 downregulation in hypodermal tissue depends on let-7 ( Ecsedi et al . , 2015; Slack et al . , 2000 ) and to corroborate this notion in the context of the nervous system , we analyzed let-7 activity using an ‘activity sensor’ construct in which the lin-41 promoter is fused to gfp and to the let-7 responsive 3’UTR of the lin-41 locus . This reporter shows ubiquitous expression , including in the nervous system , from the L1 stage to L4 , when GFP levels start to drop and disappear from most tissues , including neurons , by the adult stage ( Figure 2D ) . A control construct carrying the unc-54 3’ UTR is not downregulated at the L4 and young adult stages ( Figure 2D ) . We conclude that let-7 activity is , as expected , temporally controlled in the nervous system . We did not observe any sexual dimorphisms of lin-41 or let-7 sensor expression in the nervous system . To determine the expression pattern of the lin-41 effector lin-29 in the nervous system , we used strains in which the lin-29 locus was tagged with gfp , using CRISPR/Cas9-mediated genome engineering . lin-29 produces two distinct transcripts , lin-29a and lin-29b that differ at their 5’end; both contain five DNA-binding Zn finger domains ( Rougvie and Ambros , 1995 ) ( Figure 3A ) . We used a strain in which both isoforms were simultaneously tagged by introducing gfp at the 3’ end of the locus ( Aeschimann et al . , 2017 ) ( Figure 3A ) . In addition , we examined the expression pattern exclusively of the b-isoform , by introducing the same 3’ terminal gfp tag in the a-isoform-specific mutant allele ( xe40 ) via CRISPR/Cas9-mediate genome engineering ( Figure 3A ) . To examine the expression pattern of the a-isoform , we inserted gfp at the 5’ end of the a-isoform ( Figure 3A ) . Generally , the expression pattern of the gfp allele that tags both isoforms matches the expression pattern reported using an antibody directed against both LIN-29 isoforms ( Figure 3B ) ( Bettinger et al . , 1996; Euling et al . , 1999 ) . As previously reported , we observed expression in head neurons and the ventral nerve cord . However , we found that this neuronal expression is almost completely restricted to the male and this male-specific expression can be entirely ascribed to the a-isoform ( Figure 3B–D ) . The male-specific neuronal expression of lin-29a is not observed in male-specific neurons but is , interestingly , entirely restricted to sex-shared neurons ( i . e . neurons that are generated in both sexes ) . Intriguingly , most of these neurons had not been previously reported to show sexually dimorphic gene expression patterns or functions . Specifically , 21 out of 116 sex-shared neuron classes express lin-29a , including seven sensory neuron classes in the head and tail ( AWA , ASG , ASJ , ASK , ADF , PHB , PLM ) , seven interneuron classes ( AVA , RIA , AIM , AVG , RIF , PVC , PVN ) , a few head and tail motor neurons ( SAB , PDA , PDB ) and most of the sex-shared ventral nerve cord motor neurons ( dorsal and ventral A , B and D-type and AS neurons ) . Many of these LIN-29A expressing neurons are synaptically connected to one another ( White et al . , 1986 ) and some of the synaptic connections are known to be sexually dimorphic ( Jarrell et al . , 2012 ) . Expression of lin-29a in all these neurons in the male nervous system is precisely temporally controlled; it is first observed in the early L4 stage and persists throughout adulthood ( Figure 3D ) . In addition to the male-specific neuronal expression of the lin-29a isoform , we also found that the lin-29b isoform is expressed in three postembryonically generated touch neurons ( AVM , PVM , PDE ) , where its expression is not temporally regulated , that is , its expression is induced right after the neurons are born in the first two larval stages and expression is observed in both sexes ( data not shown ) . The late larval onset of expression of lin-29 in non-neuronal cells has been reported to be controlled by the heterochronic pathway ( Bettinger et al . , 1996; Moss et al . , 1997 ) . To assess whether onset of male-specific , neuronal expression of the lin-29a isoform is controlled by lin-41 and let-7 , we analyzed lin-29a neuronal expression in these mutant backgrounds . Indeed , we found that in lin-41 ( lf ) mutants lin-29a::gfp expression is observed prematurely including in the sex-shared neurons of the male ( Figure 4A ) . As expected , in let-7 ( lf ) and lin-41 ( gf ) mutants ( both of which maintain expression of LIN-41 ) , lin-29a::gfp expression levels are significantly reduced compared to wild-type adult males ( Figure 4B ) . This decrease in lin-29a expression is consistent with the similarity of loss-of-function phenotypes of let-7 and lin-29 null mutants and lin-41 gain-of-function mutants in which the AIM neurotransmitter switch does not occur ( Figure 1 ) . Together with our previous biochemical analysis ( Aeschimann et al . , 2017 ) , we conclude that let-7-controlled LIN-41 directly represses lin-29a translation to control the precise timing of LIN-29A protein accumulation in the nervous system . To assess the functional relevance of the lin-29a isoform in controlling the AIM neurotransmitter switch , we generated two lin-29a-isoform specific alleles ( xe38 and xe40 ) using CRISPR/Cas9 genome engineering ( Figure 3A ) ( Aeschimann et al . , 2018 ) . We indeed find that in both isoform-specific alleles the male-specific AIM neurotransmitter switch failed to occur ( Figure 5A ) . A newly generated lin-29 null allele that removes both isoforms ( Aeschimann et al . , 2017 ) also confirmed the results previously reported with the canonical null alleles tested ( Figure 5A ) . To assess whether lin-29a acts cell-autonomously in AIM to control its neurotransmitter switch , we expressed lin-29a under an AIM-specific promoter ( Serrano-Saiz et al . , 2017a ) . This driver is not only active in both sexes , but it is already active in embryos and early larval stages , hence allowing us to assess three key issues: ( 1 ) whether LIN-29A acts cell-autonomously , ( 2 ) whether LIN-29A can induce the neurotransmitter switch prematurely in males and ( 3 ) whether LIN-29A can ectopically induce the AIM neurotransmitter switch in hermaphrodites . We generated two independent transgenic lines that cell type-specifically express lin-29a in a lin-29a ( xe38 and xe40 ) mutant background . We found that in adult male animals , the loss of the AIM neurotransmitter switch ( as assayed by cho-1 and unc-17 induction and eat-4 downregulation in AIM ) is rescued by the transgene , thereby demonstrating cell autonomy of LIN-29A function ( Figure 5B ) . Examining juvenile males , before the onset of overt sexual maturation , we observed that the neurotransmitter switch is executed prematurely already by the first larval stage in these transgenic animals ectopically expressing LIN-29A in AIM neurons ( Figure 5B ) . This sufficiency experiment indicates that the temporal control of lin-29a expression , and specifically the repression of lin-29a by the heterochronic pathway is required to prevent lin-29a from triggering the neurotransmitter switch precociously . Lastly , examining adult hermaphrodite animals expressing this construct , we found that lin-29a is sufficient to induce the neurotransmitter switch in the opposite sex ( Figure 5B ) . We conclude that lin-29a is required and sufficient to control the AIM neurotransmitter switch . To assess whether lin-29a function in AIM goes beyond controlling AIM’s neurotransmitter switch , we examined another molecular marker of sexually dimorphic AIM differentiation , the orphan GPCR , srj-54 , which is induced in the AIM neurons upon male sexual differentiation ( Portman , 2007 ) . We found that srj-54 fails to be expressed in lin-29a mutants ( Figure 6A ) adding another AIM male-specific molecular feature that fails to be normally induced in the absence of lin-29 . As we will describe further below , lin-29a mutants are also defective in an AIM-mediated sexual behavioral paradigm . The expression of lin-29a in a number of distinct neuron types indicated that lin-29a may play additional roles in controlling sexually dimorphic , that is , male-specific nervous system features . To this end , we assessed two other molecular markers that were previously found to be expressed in a sexually dimorphic manner in two distinct , sex-shared neuron classes , both of which express lin-29a . First , the dmd-5 transcription factor was reported to be expressed in AVG neurons in adult males but not in hermaphrodites ( Oren-Suissa et al . , 2016 ) . By analyzing the expression of a dmd-5 reporter in wild type versus lin-29a mutants , we found that lin-29a is required for dmd-5 expression in AVG in males ( Figure 6B ) . Second , the daf-7 gene , encoding a neuroendocrine TGFβ-like signal , is turned on during sexual maturation in the sex-shared ASJ neurons only in males ( Hilbert and Kim , 2017 ) . This induction is also abrogated in lin-29a mutants ( Figure 6B ) . Non-sexually dimorphic expression of dmd-5 and daf-7 in other neuron types was not affected by lin-29a . The electron micrographical reconstruction of the nervous system of C . elegans male revealed sexually dimorphic neuronal morphology and connectivity of several sex-shared neurons ( Jarrell et al . , 2012; White et al . , 1986 ) . Specifically , the lin-29a-expressing PDB motorneuron located in the pre-anal ganglion grows elaborate branches off from its cell body and initial segment of its posteriorly directed main process . This was only observed in the male but not in the hermaphrodite ( Jarrell et al . , 2012; White et al . , 1986 ) . These male-specific branches make extensive chemical and electrical synaptic contacts with both male-specific neurons , as well as sex-shared neurons ( Jarrell et al . , 2012 ) . We corroborated the existence and reproducibility of male-specific branches by visualizing PDB with a reporter transgene ( Figure 6C ) . We found that these branches grow specifically during sexual maturation in the L4 stage ( Figure 6C ) , co-incident with the onset of lin-29a expression . Growth of these branches is disrupted in lin-29a mutants ( Figure 6C ) . These findings demonstrate a role for lin-29a in regulating the temporal and sexually dimorphic acquisition of male-specific neuronal morphologies and predict that PDB fails to make its branch-specific synaptic contacts in the absence of lin-29a . We conclude that lin-29a affects multiple distinct types of sexual maturation events , from gene expression to anatomy , in distinct neuron classes . We next sought to broaden our understanding of lin-29a function in the nervous system by testing if lin-29a was required for the establishment of adult-specific sexually dimorphic behaviors . Critical for such an analysis is the fact that lin-29a mutant males do not display the obvious morphological and patterning defects displayed by the lin-29 null allele . Specifically , the male tail , whose overall morphogenesis is disrupted in lin-29 null mutants ( Euling et al . , 1999 ) appears morphologically normal in lin-29a mutants ( Figure 7A ) . We precisely quantified other morphological differences between hermaphrodites and males using an automated visualization/measurement tool ( Yemini et al . , 2013 ) and found no overt morphological differences between wild-type and lin-29a mutant males ( Figure 7B ) . The normal overall appearance of lin-29a mutant males allowed us to first ask whether lin-29a mutant males displayed mating-related behavioral defects . One aspect of mate attraction relates to the mating process itself , in which the male detects various sensory cues from the hermaphrodites to engage in direct contact ( Liu and Sternberg , 1995 ) . Previous work with the lin-29 null mutant revealed multiple defects in these direct interactions and these defects were ascribed to spicule patterning defects ( Euling et al . , 1999 ) . In spite of spicule patterning appearing normal in lin-29a isoform-specific mutants , we observed that these mutants were unable to mate ( Figure 7C ) . We found that one aspect of mating behavior defective in these mutants is vulva location behavior ( Figure 7C ) . The AIM and ASJ neurons , both of which express lin-29a specifically in males , were previously implicated in mate-searching , which constitutes another aspect of mating behavior ( Barrios et al . , 2012; Hilbert and Kim , 2017 ) . In this behavioral paradigm , well-fed single adult males leave a source of food to explore their environment in search for mating partners . We indeed found that lin-29a mutants are defective in mate searching behavior ( Figure 7D ) . These defects are similar to the defects caused by loss of daf-7/TGFβ ( Hilbert and Kim , 2017 ) and , consistent with this phenotypic similarity , we had described above that daf-7/TGFβ expression is lost in the ASJ neurons of lin-29a mutants . Nevertheless , we could partially rescue the mate searching defects of the lin-29a mutant males by restoring LIN-29A expression exclusively in the AIM neurons ( Figure 7D ) , indicating that lin-29a may function in several distinct neurons to control mate searching behavior . The male-specific expression of lin-29a in ventral nerve cord and head motor neurons and in a command interneuron pair made us consider whether lin-29a may be involved in controlling aspects of sexually dimorphic locomotor behavior . It has previously been shown that adult male and hermaphrodites display distinct locomotor behavior when crawling on a lawn of bacteria ( Mowrey et al . , 2014 ) . We extended the analysis of sexually dimorphic adult-specific behavior using an automated worm tracker system which quantifies several hundred distinct postural and locomotory features of an animal ( Yemini et al . , 2013 ) . We found a large number of sexually dimorphic features . While many of them were not affected in lin-29a mutant animals ( Figure 8 and Supplementary file 1 ) , we found that lin-29a mutants displayed a striking feminization of a number of body posture and locomotory features . Postural differences included head bend , neck bend , midbody bend , hips bend , amplitude ratio and primary wavelength . ( Figure 8 ) . Other lin-29a-dependent locomotory features included midbody crawling amplitude and tail crawling amplitude ( Figure 8 ) . The lack of morphological differences between wild-type and lin-29a mutant males indicates that these locomotory phenotypes are the result of neuronal defects rather than being a secondary consequence of overall body morphology . Taken together , these results show that lin-29a is required for the acquisition of male-specific postural and locomotory features and that the absence of lin-29a results in a feminization of these behavioral parameters . We sought to determine in which of the neurons that express lin-29a in a sexually dimorphic manner lin-29a acts to control aspects of the locomotor behaviors described above . We particularly considered the so-called ‘motor circuit’ , composed of a set of command interneurons and ventral nerve cord motor neurons ( Von Stetina et al . , 2006 ) . Expression of lin-29a in the motor circuit , using a cis-regulatory element from the unc-17/VAChT locus rescued a defined subset of the feminized behavioral features ( wavelength , crawling amplitude and midbody bend ) ( Figure 8 ) . Other lin-29a-expressing neurons may be involved in controlling locomotory and postural features not rescued by this transgene . Having established the function of lin-29a in sexually dimorphic development and function of the C . elegans nervous system , we sought to better understand the mode of lin-29a regulation . lin-29a shows a striking specificity of expression in three different dimensions . Within the nervous system , lin-29a is expressed in a temporally controlled manner ( onset in embryonically born neurons only in the L4 stage ) , in a sex-specific manner ( only in males ) and it is expressed in a highly neuron type-specific manner . In as far as the first dimension , time , is concerned , we have described above that lin-29a expression in the nervous system is temporally controlled by the lin-28/let-7/lin-41 heterochronic pathway . To assess the control of sex-specificity of lin-29a expression , we genetically removed tra-1 , the master regulatory transcription factor of sex determination which is normally expressed in all hermaphroditic cells ( XX sex chromosome genotype ) where it suppresses male identities ( Schvarzstein and Spence , 2006 ) . We found that in tra-1 ( e1488 ) mutants , lin-29a expression becomes derepressed in XX animals ( Figure 9A ) . Moreover , we induced the degradation of tra-1 exclusively in the nervous system by force-expressing fem-3 , which promotes TRA-1 protein destruction ( Starostina et al . , 2007 ) , under the control of a panneuronal driver . We found that lin-29a expression is derepressed in the nervous system of the hermaphrodite , with the same pattern of cellular specificity seen normally in males ( Figure 9A ) . To address the cellular specificity of lin-29a expression , we considered the possibility that neuron class-specific terminal selector-type transcription factors that control the expression of neuron-class specific identity programs ( Hobert , 2016 ) may also control the neuronal specificity of lin-29a expression . For example , the terminal differentiation program of the lin-29a expressing ASK neurons is controlled by the LIM homeobox gene ttx-3/LHX2 and the identity of the AIM neuron is defined by the unc-86/BRN3 POU homeobox gene ( Serrano-Saiz et al . , 2013 ) . We found that lin-29a expression is eliminated in the ASK neurons of ttx-3 mutants and in the AIM neurons of unc-86 mutants ( Figure 9B ) . The unc-3/COE-type transcription factor controls the identity of multiple neurons of the motor circuit , including the lin-29a expressing ventral nerve cord motorneurons , the SAB head motor neurons , the PDA and PDB tail motor neurons and the command interneurons AVA and PVC ( Kratsios et al . , 2011; Pereira et al . , 2015 ) . We found that in unc-3 mutants lin-29a expression is lost in all these neurons ( Figure 9B ) . We conclude that the complex lin-29a expression pattern can be explained by an intersectional mechanism . Temporal control is exerted via the heterochronic pathway , sexual specificity by the sex determination pathway and cellular specificity by terminal selector-type neuronal identity factors . The sexually dimorphic expression pattern of lin-29a in the male nervous system does not extend to all sex-shared neurons that are known to display sexually dimorphic features . For example , the sex-shared PHC sensory neuron functionally and anatomically remodels during sexual maturation ( Serrano-Saiz et al . , 2017a ) , but does not express lin-29a . This suggests that lin-29a is possibly just one of several targets of LIN-41 in the context of male-specific nervous system differentiation . Previous whole animal RNA profiling of let-7 and lin-41 mutants , as well as RNA-binding assays demonstrated that LIN-41 also represses mab-3 and dmd-3 mRNA ( Aeschimann et al . , 2017 ) . Both genes are members of the DMD family of sex-specifically expressed transcription factors ( Mason et al . , 2008; Yi and Zarkower , 1999 ) and were previously shown to be expressed in male-specific neurons and in a male-specific manner in distinct , but sex-shared neurons ( Mason et al . , 2008; Serrano-Saiz et al . , 2017a; Yi et al . , 2000 ) . Their timing of expression had not been previously examined . We gfp-tagged the endogenous mab-3 and dmd-3 loci using CRISPR/Cas9 genome engineering . Within the sex-shared nervous system , we found that mab-3 is not only male-specifically expressed in the ADF neuron , as previously reported ( Yi et al . , 2000 ) , but also in additional head neurons , including the embryonically generated SMD head motor neurons ( Figure 9C ) . We found that like the male-specific , neuronal expression of lin-29a , male-specific neuronal expression of mab-3 is precisely timed to commence at the L4 stage ( Figure 9C ) . Similarly , the gfp-tagged dmd-3 locus is expressed in a sex-shared neuron class , the PHC neurons ( Figure 9D ) , where dmd-3 acts to control the sexually dimorphic remodeling of PHC ( Mason et al . , 2008; Serrano-Saiz et al . , 2017a ) . dmd-3::gfp expression in PHC also commences at the L4 stage ( Figure 9D ) , at the time when PHC neurons remodel in a male-specific manner ( Serrano-Saiz et al . , 2017a ) . We corroborated our previous data of LIN-41 protein affecting mab-3 and dmd-3 expression ( Aeschimann et al . , 2017 ) by showing that the temporal specificity of nervous system expression of mab-3 and dmd-3 is indeed lost in let-7 mutants ( Figure 9C–D ) . Like lin-29a , the male-specificity of mab-3 and dmd-3 expression in the nervous system is controlled by the tra-1 transcription factor ( Figure 9E , G ) Moreover , the neuronal specificity of mab-3 and dmd-3 expression is , like lin-29a , also controlled by neuron-type specific terminal selectors . unc-42 is a terminal selector transcription factor that controls terminal differentiation of the RMD and SMD neurons ( Pereira et al . , 2015 ) , and we found that mab-3 expression in these neurons fails to be induced in an unc-42 mutant background ( Figure 9C ) . Similarly , ceh-14 is a terminal selector of PHC neuron identity ( Serrano-Saiz et al . , 2017a ) and is required for dmd-3 expression in PHC ( Figure 9F ) . We conclude that lin-41 operates through multiple distinct , male-specific effector genes and that the temporal , sexual and spatial specificity of expression of these distinct effectors is controlled by a similar , intersectional regulatory logic ( Figure 10A , B ) .
We have described here that the timing of multiple distinct , neuron-type specific sexually dimorphic maturation events in the nervous system of C . elegans are controlled by a phylogenetically conserved regulatory cassette composed of ubiquitously expressed and temporally controlled genes , lin-28 ( a regulator of let-7 miRNA processing ) , let-7 ( a miRNA ) and its target lin-41 ( a translational regulator ) . The conservation of the deployment of this pathway in the timing of sexual maturation is remarkable . Genetic manipulations in mice , as well as genome-wide association studies of humans with abnormally timed puberty demonstrate that lin-28 and let-7 also control timing of sexual maturation ( puberty ) onset in mammals ( Chen et al . , 2017; Corre et al . , 2016; He et al . , 2009; Ong et al . , 2009; Perry et al . , 2009; Sulem et al . , 2009; Zhu et al . , 2010 ) . This conclusion was further corroborated by the finding that mutations in human makorin , the orthologue of lep-2 , a regulator of lin-28 activity in C . elegans ( Herrera et al . , 2016 ) , also affect the timing of puberty in humans ( Abreu et al . , 2013; Bulcao Macedo et al . , 2014 ) . Similarly , in the fly nervous system , lin-28 and let-7 are involved in controlling the timing of pupal to adult transitions ( González-Itier et al . , 2018; Sempere et al . , 2003; Wu et al . , 2012 ) that are accompanied by sexual maturation in the nervous system , as assessed by expression of the male-specific isoform of Fruitless , a master regulator of male-specific nervous system differentiation ( Lee et al . , 2000 ) . However , a regulatory linkage between the heterochronic pathway and transcriptional regulators of sexual differentiation ( hormone receptors in vertebrates or Fruitless in flies ) has not yet been established in these systems . Apart from suggesting a remarkable conservation of timing mechanisms of sexual maturation , we have provided insights into the mechanisms by which the lin-28/let-7 axis affects the timing of sexual maturation . The let-7 miRNA is known to have a plethora of distinct mRNA targets ( Büssing et al . , 2008 ) , but the one that we define here as being relevant in the context of sexual maturation is the translational regulator lin-41 ( Slack et al . , 2000 ) . C . elegans lin-41 had previously been implicated as an output of let-7 in the timing of skin cell maturation events ( Slack et al . , 2000 ) as well as vulval patterning ( Ecsedi et al . , 2015 ) . lin-41 is also highly conserved in vertebrates where its expression appears to be temporally regulated during embryogenesis with its expression being complementary to let-7 , as observed in C . elegans ( Schulman et al . , 2005 ) . The dynamics of lin-28 , let-7 and lin-41 expression has not yet been comprehensively examined in the context of sexual maturation in the vertebrate brain , but notably , the expression of Lin28 and Let7 in the hypothalamus , a site of release of peptides that control pubertal onset , appears to be temporally controlled ( Sangiao-Alvarellos et al . , 2013 ) . In Drosophila , let-7 expression has also been found to be temporally regulated; its expression increases during late pupal stages ( Faunes and Larraín , 2016 ) . This is the time at which at least some parts of the Drosophila nervous system sexually mature ( Lee et al . , 2000 ) , but a role of Drosophila let-7 in the timing of sexual maturation has not yet been assessed . One key remaining question , raised by our studies in C . elegans , is how lin-28/Lin28 function is precisely timed in the nervous system . Surprisingly , we found that a previously described upstream regulator of lin-28 , the lin-4 miRNA ( Moss et al . , 1997 ) , does not affect male-specific sexual maturation in the nervous system , suggesting the existence of alternative pathways that determine when lin-28’s function in controlling let-7 processing is downregulated . Recent work in C . elegans has discovered LIN-28 protein degradation , mediated by the conserved LEP-2/Makorin protein , as another means to control lin-28 function ( Herrera et al . , 2016 ) . It remains to be explored how LEP-2/Makorin-mediated LIN-28 degradation is temporally controlled . We showed here that the key target of let-7 , the lin-41 translational regulator , relays temporal information in a modular manner to distinct , neuron class-specific outputs ( Figure 10A ) . Among them is the phylogenetically conserved LIN-29 Zn finger transcription factor ( whose vertebrate orthologs ZNF362 and ZNF384 are completely unstudied ) , which we identified as being sexually dimorphically expressed in one fifth of all sex-shared neuron classes . Two other targets of lin-41 that are relevant for sexual maturation are two male-specifically expressed DM domain transcription factors ( Aeschimann et al . , 2017 ) ( this paper ) . Neurons that express these sexually dimorphic output regulators ( lin-29 , mab-3 , dmd-3 ) act at distinct levels of information processing , from sensory , to inter- to motorneurons . Intriguingly , many of the sex-shared , LIN-29 ( + ) interneurons are synaptically connected and some of these connections are sexually dimorphic . Some of the neurons that express LIN-29 or MAB-3 in a male-specific manner were not previously shown to display any sexual dimorphisms , demonstrating that the C . elegans nervous system may be even more sexually dimorphic than previously appreciated . lin-29 , mab-3 and dmd-3 expression mark many , but not quite all sex-shared neurons with sexually dimorphic features . We indeed found evidence for two other dmd genes whose expression is both sexually and temporally controlled in yet distinct neuron classes ( Oren-Suissa et al . , 2016 ) ( unpubl . data ) . Altogether , these findings suggest a scenario in which a ubiquitously expressed temporal coordinate system ( heterochronic pathway ) , intersects with a ubiquitously expressed sexual coordinate system ( sex determination pathway ) to control the sexual and temporal specificity of a number of modular , neuron-type specific output systems that control sexual dimorphisms in distinct cellular circuit modules ( Figure 10A , B ) . This modular organization displays striking conceptual similarities with the modular organization of sexual dimorphic control mechanisms in vertebrates ( Yang and Shah , 2014 ) . How is the neuron type-specificity of the modular outputs ( lin-29 , mab-3 , dmd-3 etc . ) of the temporal and sex-determination system controlled ? We provided evidence here that the neuron-type specificity of the sex-specific regulator is controlled by neuron-type specific terminal selector transcription factors which define the overall differentiated state of individual neuron classes ( Hobert , 2016 ) ( Figure 10B ) . For example , unc-3 is required for many ( non-sexually dimorphic ) aspects of motor neuron differentiation in the ventral nerve cord and we find that unc-3 also is required to target lin-29 expression specifically in ventral nerve cord neurons . Like the regulation of lin-29 by the temporal cue lin-41 , the neuron-type specific regulation of lin-29 could potentially be direct since we noted several phylogenetically conserved binding sites for UNC-3 protein in the lin-29 locus . The regulation of sex-specific output regulators , like lin-29 , by terminal selectors reveals the potential existence of a specific gene regulatory network architecture . For example , in the male AIM neurons , unc-86 and ceh-14 are required for the induction of unc-17 and cho-1 expression ( Pereira et al . , 2015 ) . Both are apparently not sufficient to induce unc-17 and cho-1 expression in hermaphrodites , but via the unc-86-mediated induction of lin-29 expression in the AIM neurons in males , unc-86 and ceh-14 are now apparently able to induce , with the help of lin-29 , the expression of unc-17 and cho-1 . Further biochemical studies will be required to corroborate the existence of such feedforward architecture ( Figure 10B ) . In conclusion , we have identified an intersectional regulatory mechanism in which temporal cues , sexual cues and neuron-type specific cues define the onset of expression of sex-specific , neuron-type specific ‘output regulators’ which define sexual dimorphic features of the C . elegans nervous system ( Figure 10 ) .
Worm strains used in this study are listed in Table 1 . The wild-type strain was Bristol N2 . Worms were grown on nematode growth media ( NGM ) seeded with bacteria ( OP50 ) as a food source . To synchronize worms 10 adult gravid hermaphrodites were transferred to a new plate with OP50 . Worms were allowed to lay eggs for approximately two hours and adult gravid hermaphrodites were removed afterwards . srj-54::yfp is a PCR fragment that was constructed using standard PCR fusion methods by fusing fragment A , comprising 664 bp upstream of the predicted ATG of srj-54 and the first four codons of srj-54 coding sequence , to fragment B , comprising yfp fused to the unc-54 3´ UTR . srj-54::yfp was injected with unc-122::gfp into him-5 ( e1490 ) to generate a stable extrachromosomal array . This array was integrated into the genome using standard UV irradiation methods followed by several rounds of backcrossing to him-5 ( e1490 ) to generate fsIs5 . For neuron-specific expression of lin-29a , promoter constructs driving lin-29a cDNA were generated by Gibson assembly or RF cloning . cDNA was amplified from pDH06 , kindly provided by Dr . Horvitz ( Harris and Horvitz , 2011 ) . A 1 kb of the unc-17/VACHT promoter that drives expression in AVA , SAB , DA , DB , VA , VB , PDA and PDB neurons ( our unpublished data ) was amplified by PCR from the fosmid construct ( FW AATGAAATAAGCTTGCATGGTATACACCAATCATTTCTCC and REV TCCTCTAGAGTCGACCTGCAGATAATTTAATTAAAATTGAGTTCCAAC ) . The PCR fragments were assembled into pPD95 . 75 by Gibson . For AIM-specific expression a fragment of the eat-4 locus was used to drive LIN-29A expression . The lin-29a cDNA was inserted to replace the fem-3 cDNA in this construct ( Pereira et al . , 2015 ) . For nervous system masculinization , a rab-3 pan-neuronal promoter driving fem-3::mCherry was used ( White et al . , 2007 ) . Transgenic lines carrying extrachromosomal arrays were generated by germline injection with 20 ng/ul of plasmid DNA and unc-122::gfp as a co-injection marker , also at 20 ng/ul . Isoform-specific gfp::3xflag tagging of endogenous lin-29 using CRISPR/Cas9 . In order to obtain the lin-29 ( xe65 ) allele , the following mix was injected into lin-29a ( xe40 ) mutant worms: 50 ng/µl pIK155 , 100 ng/µl of pIK198 with a cloned sgRNA ( atattatttatcagtgattg ) , 2 . 5 ng/µl pCFJ90 , 5 ng/µl pCFJ104 and 10 ng/µl pFA26 ( pDD282 with cloned homology arms ) . Recombinants were isolated according to the protocol by Dickinson et al . ( Dickinson et al . , 2015 ) , verified by DNA sequencing and outcrossed three times . The plasmid for homologous recombination ( pFA26 ) was previously described ( Aeschimann et al . , 2017 ) . The lin-29 ( xe63 ) allele specifically tags lin-29a with GFP::3xFLAG at the N-terminus ( Aeschimann et al . , 2018 ) . Tagging of endogenous mab-10 with flag::mCherry using CRISPR/Cas9 . In order to tag mab-10 with flag::mCherry at the C-terminus , the following mix was injected into unc-119 ( ed3 ) worms ( Dickinson et al . , 2015; Katic et al . , 2015 ) : 25 ng/ul pIK155 , 65 ng/ul of pIK198 with a cloned sgRNA ( gctcccggaatcttgaagct ) , 10 ng/ul pIK127 ( Peft-3::gfp::h2b ) , 5 ng/ul Pmyo-3::gfp and 65 ng/ul pIK284 . The plasmid serving as a template for homologous recombination ( pIK284 ) was obtained using the SapTrap protocol ( Schwartz et al . , 2016 ) , by combining SapTrap component vectors pMLS257 , pMLS291 and pMLS382 with two pairs of annealed oligos ( oIK1052/oIK1053 , oIK1054/oIK1055 ) . Wild-type moving recombinants were isolated , verified by DNA sequencing and outcrossed three times . The mab-10::mCherry allele is not fully functional since mab-10 ( xe75 ) and mab-10 ( xe44 ) both enhance the bursting phenotype of let-7 ( n2853ts ) ( data not shown ) . lin-29 , lin-29a and mab-10 null mutant alleles . In order to specifically mutate lin-29a without affecting expression of lin-29b , we introduced deletions into the coding exons specific to lin-29a , at the same time introducing a frame-shift in the downstream lin-29a reading frame . The lin-29a ( xe38 ) allele is a 10 bp deletion in exon 2 , resulting in a frame-shift and a stop codon in exon 3 . It was obtained by injecting a single sgRNA ( gctggaaccaccactggctc ) and has the following flanking sequences: 5’ gaatagctggaaccaccact – xe38 deletion – ctaccacccattttggtgtt 3’ . The lin-29a ( xe40 ) allele is a 1102 bp deletion covering exons 2–4 , introducing a frame-shift in the lin-29a reading frame with a predicted stop codon in exon 6 ( Aeschimann et al . , 2018 ) . We used deletions of almost the entire coding regions as null alleles for lin-29 and mab-10: The lin-29 ( xe37 ) allele is a 14 . 8 kbp deletion spanning lin-29 exons 2–11 and the mab-10 ( xe44 ) allele is a 2 . 9 kbp deletion spanning mab-10 exons 3–9 ( Aeschimann et al . , 2018 ) . To obtain mutant worm lines , wild-type worms were injected with a mix containing 50 ng/µl pIK155 , 100 ng/µl of each pIK198 with a cloned sgRNA , 5 ng/µl pCFJ90 and 5 ng/µl pCFJ104 , as previously described ( Katic et al . , 2015 ) . Single F1 progeny of injected wild-type worms were picked to individual plates and the F2 progeny screened for deletions using PCR assays . After analysis by DNA sequencing , the alleles were outcrossed three times to the wild-type strain . Tagging of endogenous unc-17 with 3xflag::mKate2 using CRISPR/Cas9 . The unc-17 locus was tagged at the C-terminus as previously described ( Dickinson et al . , 2015; Katic et al . , 2015 ) . The plasmid serving as a template for homologous recombination was cloned using Gibson and included 700 bp-long homology arms . Wild-type moving recombinants were isolated , verified by DNA sequencing and outcrossed three times . Tagging of endogenous mab-3 and dmd-3 with 3xflag::gfp using CRISPR/Cas9 . The mab-3 and dmd-3 loci were tagged at the C-terminus as previously described ( Dickinson et al . , 2015; Katic et al . , 2015 ) . The plasmid serving as a template for homologous recombination was cloned using Gibson and included 700 bp-long homology arms . Wild-type moving recombinants were isolated , verified by DNA sequencing and outcrossed three times . Expression analysis of the lin-29 endogenously tagged alleles with neuron-specific resolution was done by assessing nuclear position and size using Nomarski optics and crossing these strains with neuronal landmark reporter strains eat-4 ( otIs518 ) ( Serrano-Saiz et al . , 2017a ) and cho-1 ( otIs544 ) ( Pereira et al . , 2015 ) . Laser ablation of Z1/Z4 was performed at the L1 stage as previously described ( Hsin and Kenyon , 1999 ) using a MicroPoint Laser System Basic Unit ( N2 pulsed laser ( dye pump ) , ANDOR Technology ) attached to a Zeiss Axio Imager Z2 microscope ( Objective EC Plan-Neofluar 63X/1 . 30 Oil ) . Worms were recovered by washing with M9 buffer and imaged at the young adult stage . Control animals were treated in the same manner , but not subjected to laser exposure . Gonad and germline removal were confirmed by DIC microscopy . Worms were anesthetized using 100 mM of sodium azide and mounted on 5% agarose on glass slides . All images were acquired using a Zeiss confocal microscope ( LSM880; Zeiss [Carl Zeiss] , Thornwood , NY ) . Image reconstruction was performed using the ZEN software tool . Maximum intensity projections of representative images are shown . For neurite tracing , confocal Z-stacks were opened using FIJI , and loaded into the Simple Neurite Tracer plugin . All neurites emerging from the soma or posterior process of PDB were traced . The simple neurite tracer plugin was used to analyze the skeletons for neurite length , which were summed to calculate total neurite . Animals were synchronized at the L1 stage and grown to the early L4 stage group housed . The day before the mating assay experiment , males at the early L4 stage were separated to a new plate . Male mating behavior was assayed by direct observation of the mating process . A single young adult male was placed with 10–15 L4 unc-3 ( e151 ) hermaphrodites on a mating plate . The male mating behavior was recorded as previously described ( Euling et al . , 1999 ) . Males were tested for their ability to locate vulva in a mating assay , calculated as location efficiency . The number of passes or hesitations at the vulva until the male firs stops were counted: location efficiency = 1/number of encounters to stop , expressed as percentage . Animals were synchronized at the L1 stage and grown to the early L4 stage . The day before the mating assay experiment , hermaphrodites and males at the early L4 stage were separated to a new plate . The mate-searching assay was performed as previously described ( Lipton et al . , 2004 ) . A single animal was placed in the center of a 15 µ﻿l patch of food per 10 cm plate . Each animal that had left the food was scored blindly at 7 time points for a period of 24 hr . A worm was considered a leaver if it was 3 cm away from the food source at the scoring time . Automated single worm tracking was performed using the Wormtracker 2 . 0 system at room temperature ( Yemini et al . , 2013 ) . Animals were synchronized at the L1 stage and grown to the early L4 stage . The day before the tracking experiments , hermaphrodites and males were separated to same-sex group housed plates . Animals were recorded at the young adult stage for 5 min to ensure sufficient sampling of locomotion related behavioral features . All animals were tracked on NGM plates uniformly covered with food ( OP50 ) . To avoid potential variability arising due to room conditions , all strains that were compared in a single experiment were recorded simultaneously in identical room condition , along with the controls . Analysis of the tracking videos was performed as previously described ( Yemini et al . , 2013 ) . After correction for multiple testing ( Bonferroni correction ) , features shown in Figure 8 and Supplementary file 1 emerged as the ones with most significantly different q-value among the test groups . For the rescue experiment , we only measured these features , permitting us to use the p-value .
The molecular mechanisms that control the timing of sexual differentiation in the brain are poorly understood . We found that the timing of sexually dimorphic differentiation of postmitotic , sex-shared neurons in the nervous system of the Caenorhabditis elegans male is controlled by the temporally regulated miRNA let-7 and its target lin-41 , a translational regulator . lin-41 acts through lin-29a , an isoform of a conserved Zn finger transcription factor , expressed in a subset of sex-shared neurons only in the male . Ectopic lin-29a is sufficient to impose male-specific features at earlier stages of development and in the opposite sex . The temporal , sexual and spatial specificity of lin-29a expression is controlled intersectionally through the lin-28/let-7/lin-41 heterochronic pathway , sex chromosome configuration and neuron-type-specific terminal selector transcription factors . Two Doublesex-like transcription factors represent additional sex- and neuron-type specific targets of LIN-41 and are regulated in a similar intersectional manner .
In most adult animals , male and female brains are slightly different . For example , in the nematode worm Caenorhabditis elegans , certain neurons exist in one sex but not the other . Nerve cells that are shared in both sexes may also activate different genes , or form different connections in males and females . Most of these differences – which ultimately give rise to sex-specific behaviors – emerge during a period of development called sexual maturation . Yet , the mechanisms that control when sexual differentiation takes place in the brain are largely unknown . To investigate this , Pereira et al . set out to determine how sex differences arise in the nervous system of C . elegans , a small animal with two sexes , male and hermaphrodite . In particular , Pereira et al . wanted to know which genes cause certain neurons that are present in both sexes to switch to the male-specific form when the worm gets old enough . The experiments revealed that a genetic pathway formed of three genes , let-7 , lin-28 and lin-41 , controls when sexual maturation takes place throughout the worm nervous system . When the worm is young , lin-41 is active and represses a gene called lin-29A . As the animal reaches maturity , let-7 ‘switches off’ lin-41 , and lin-29A gets activated in a subset of neurons . These brain cells then turn on male-specific genes and acquire a shape only found in males . The anatomy of male mutant worms that lack lin-29A is normal , but the animals show features found in hermaphrodites , for example in the way they crawl across a dish . This shows that activating lin-29A may also trigger male-specific behaviors . Switching on sex-specific neuronal circuits at the correct time is essential for animals to develop correctly . The lin-7 and let-28 genes also control when sexual maturation takes place in mammals , so studying these genes in C . elegans could help to understand how male and female brains are shaped during development in other species , and why some diseases affect the sexes differently .