HLH-3 Is Required for the Maturation of Neurons Necessary for Male Specific Exploration Behavior

HLH-3 Is Required for the Maturation of Neurons Necessary for Male Specific Exploration Behavior | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results HLH-3 Is Required for the Maturation of Neurons Necessary for Male Specific Exploration Behavior View ORCID Profile Kimberly Goodwin , View ORCID Profile Aixa Alfonso doi: https://doi.org/10.1101/2025.03.20.644401 Kimberly Goodwin * Biological Sciences, University of Illinois at Chicago , Chicago Illinois 60607, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kimberly Goodwin Aixa Alfonso * Biological Sciences, University of Illinois at Chicago , Chicago Illinois 60607, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aixa Alfonso For correspondence: aalfonso{at}uic.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Sexually dimorphic gene expression within the nervous system of many organisms gives rise to features that underline sexually dimorphic behaviors. In C. elegans , terminal differentiation of neurons with sexually dimorphic features often occur not at the time of neuronal birth, but rather during the juvenile to adult stage transition, when sexual maturation occurs. Previous works have shown that the bHLH transcription factor, HLH-3, is necessary for the differentiation of sex-specific neurons in hermaphrodites. Expression of the bHLH transcription factor, hlh-3 , is dynamic and widespread throughout the embryo. Post-embryonic expression in the hermaphrodite is restricted to the HSNs, and the P cells and their descendants, including the VCs. In this work, we characterize the post-embryonic expression pattern of hlh-3 in the male. Our findings show that the pattern of expression observed in males varies dramatically from that observed in hermaphrodites. Males show expanded expression in the head at the L1 and late L3/early L4 stage, as well as broader expression in the tail at the early L3/Late L4 stage. This leads us to the conclusion that the post-embryonic expression pattern of hlh-3 is sexually dimorphic. We also describe the role of hlh-3 in the maturation of a subset of neurons required for male specific exploration behavior. Our findings show that in the absence of hlh-3 function, the AWA, ASJ, and AIM neurons fail to acquire terminally differentiated features and therefore contribute to the failure of males to perform behaviors mediated by these cells. INTRODUCTION In multicellular organisms, neuronal tissue comprises a variety of specialized cell types which take part in regulatory circuitries that govern behavior. These different cell types acquire their morphology, neurotransmitter phenotype, and connectivity as a function of time and based on the transcriptomic profile that promotes their differentiation ( Hobert, 2016 ; Tekieli et al., 2021 ). Sexually dimorphic neuronal maturation is controlled by two major processes, acquisition of sexual identity and developmental timing (H. Lawson et al., 2019 ). A complete cell lineage map, connectome, transcriptome, and a variety of transgenic reporters make the nematode Caenorhabditis elegans an ideal model organism to elucidate the mechanisms through which neurons obtain their identity ( Cook et al., 2019 ; Sulston & Horvitz, 1977 ; The C. elegans Sequencing Consortium, 1998; Tintori et al., 2016 ). Many sex shared neurons in C. elegans males have sexually dimorphic functions which control behaviors that support the drive to locate a mate and reproduce ( Portman, 2017 ). One such behavior is sexually dimorphic exploration. This behavior is displayed by well fed, sexually mature, adult males which prioritize leaving a food source, increasing their likelihood of encountering a potential mate ( Lipton et al., 2004 ; Ryan et al., 2014 ). This behavior is controlled by a network of neurons which sense internal signals that report reproductive drive and nutritional status ( Barrios, 2014 ; Barrios et al., 2012 ; Hilbert & Kim, 2017 , 2018 ; Ryan et al 2014 ; Lawson et. al., 2014; Sanzo-Machuca et al., 2019 ). The maturation of the sex shared neurons involved in this regulatory circuit takes place during the larval to adult transition ( Hilbert & Kim, 2018 ; Ryan et al., 2014 ; Serrano-Saiz et al., 2017 ). Both developmental timing and sex identity have been implicated in the acquisition of terminal identity features like the downregulation of the diacetyl GPCR, ODR-10, in the AWAs, up-regulation of the TGF-ꞵ-like DAF-7 in the ASJs, and the neurotransmitter identity switch in the AIMs ( Hilbert & Kim, 2018 ; H. Lawson et al., 2019 ; H. N. Lawson et al., 2020 ; Pereira et al., 2019 ; Ryan et al., 2014 ). Asymmetric expression of proneural genes in the descendants of ectodermal cells generate neuronal progenitor cells ( Duggan & Chalfie, 1995 ; Ghysen & Dambly-Chaudiere, 1989 ). Most proneural genes encode proteins in the bHLH family (Bertrand et. al. 2002, Grove et. al. 2009). While proneural genes have been traditionally thought to have an early role promoting neuronal lineages, recent evidence suggests that some bHLH transcription factors have a direct influence on terminal fate acquisition too ( Hobert, 2016 ; Masoudi et al., 2018 ; Poole et al., 2011 ). Previous findings implicate the bHLH (basic helix-loop-helix) transcription factor HLH-3 in the terminal differentiation of sex-specific neurons in C. elegans hermaphrodites ( Doonan et al., 2008 ; Lloret-Fernández et al., 2018 ; Perez & Alfonso, 2020 ; Raut, 2017 ). HLH-3 is a Class II bHLH transcription factor that heterodimerizes with the Class I bHLH transcription factor, HLH-2, to promote transcription of its targets ( Grove et al., 2009 ; Krause et al., 1997 ). The gene hlh-3 encodes a protein with 59% identity in the bHLH domain to the Asense proneural protein in Drosophila and 62% identity to MASH-1 in mice (Krause et. al. 1997, Doonan et. al. 2008). We have demonstrated that expression of hlh-3 is required for the differentiation of ventral cord type C motor neurons (VCs) and hermaphrodite specific neurons (HSNs) ( Doonan et al., 2008 ; Perez & Alfonso, 2020 ; Raut, 2017 ). Expression of the LIM homeobox transcription factor and terminal differentiation marker TTX-3 in the sex shared AIY neurons also requires HLH-3, however its function is redundant with two other bHLH proteins, HLH-16 and REF-2 ( Filippopoulou et al., 2021 ; Murgan et al., 2015 ; F. Zhang et al., 2014 ). The role of HLH-3 extends beyond traditional proneural functions. For example, the sisters of a pair of neurosecretory cells, the NSMs, die because HLH-3 promotes expression of the apoptosis signal EGL-1 in them ( Thellmann et al., 2003 ). Likewise, specification of the linker cell (LC), a migrating cell which leads the formation of the male somatic gonad, requires hlh-3 function ( Kimble & Hirsh, 1979 ; Sallee et al., 2017 ). Extensive work has been done to characterize the expression of hlh-3 in hermaphrodites ( Doonan et al., 2008 ; Lloret-Fernández et al., 2018 ; Perez & Alfonso, 2020 ). Expression data was first gathered using a full-length translational fusion as well as a fosmid reporter, which showed similar patterns of expression ( Doonan et al., 2008 ; Krause et al., 1997 ; Lloret-Fernández et al., 2018 ). This work showed that expression of hlh-3 is broad and dynamic at the embryonic stage but becomes restricted to the HSNs and P cells upon hatching ( Doonan et al., 2008 ; Krause et al., 1997 ; Lloret-Fernández et al., 2018 ). Further analysis showed widespread expression in the ring ganglia and ventral nerve cord (VNC). Expression of hlh-3 in Pn.a (neural precursor) descendants in the L1 stage became restricted to VCs (Pn.aap) and HSNs by the L3 stage ( Doonan et al., 2008 ). Generation and analysis of a CRISPR-Cas9 engineered allele, hlh-3(ic271[ic271[hlh-3::gfp]]) , also displayed hlh-3 expression restricted to HSNs and VCs after the L1 stage and throughout the lifetime of the hermaphrodite ( Perez & Alfonso, 2020 ). Previous efforts characterized the expression of hlh-3 in hermaphrodite development. In this work we characterized the endogenous expression pattern of hlh-3 in males throughout post-embryonic development utilizing the previously generated CRISPR-Cas9 allele, hlh-3(ic271[hlh-3::gfp]) . We also investigated the role that HLH-3 plays in the maturation of sex shared neurons that regulate male specific exploration behavior. RESULTS Post-embryonic expression of hlh-3 is sexually dimorphic To characterize expression in the first larval stage (L1) of males, individual males were selected by identifying the large nuclei of Y and B cells in their tail ( Sulston & Horvitz, 1977 ). In contrast to the pattern of expression observed in hermaphrodites, we find that hlh-3 is expressed in several cells in the head of males at the L1 stage, these are not detectable in hermaphrodites ( Figure 1 A and E). Like hermaphrodites, early L1 males show expression of hlh-3 in 12 P cells. However, expression of hlh-3 is seen in two additional cells in the mid body of hermaphrodites, the HSNs, which are absent in males ( Figure 1 B and F). Expression in the HSNs was observed throughout the L1 stage of hermaphrodites. In the mid to late stage of both sexes, hlh-3 expression remains present along the ventral cord ( Figure 1 C, D, G and H). Download figure Open in new tab Figure 1: Post-embryonic expression of ic271[hlh-3::gfp] in males is sexually dimorphic (A ) Representative images of ic271[hlh-3::gfp] expression in the head of mid stage L1 male. (B-D) Representative images of midbody ic271[hlh-3::gfp] expression in early (∼16hrs post egg lay), mid (∼18hrs post egg lay), and late (∼20hrs post egg lay) stage L1 male ventral cord. (E) Representative images of ic271[hlh-3::gfp] expression in the head of mid stage L1 hermaphrodite (F-H) Representative images of midbody ic271[hlh-3::gfp] expression in early (∼16hrs post egg lay), mid (∼18hrs post egg lay), and late (∼20hrs post egg lay) stage L1 hermaphrodite ventral cord and midbody. Arrowheads point to positive nuclei, anterior is to the left, ventral is down. (I-K) Representative images of ic271[hlh-3::gfp] expression in head, midbody and tail of late L3/early L4 males, respectively. (L-N) Representative images of ic271[hlh-3::gfp] expression in head, midbody and ventral cord, and tail of late L3/early L4 hermaphrodites, respectively. Arrowheads point to positive nuclei, anterior is to the left, ventral is down. (O-Q) Representative images of ic271[hlh-3::gfp] expression in head, midbody and tail of adult males, respectively. Arrowheads point to positive nuclei. (R-T) Representative images of ic271[hlh-3::gfp] expression in head, ventral cord and tail of adult hermaphrodites, respectively. Dashed lines outline embryos. Arrowheads point to positive nuclei. Arrowheads point to positive nuclei, anterior is to the left, ventral is down. Expression of hlh-3 in males is broader than that in hermaphrodites. At the late L3/early L4 stages, positive cells in males included two cells in the head, several cells along the ventral cord, and a cluster at the tail ganglia ( Figure 1 I-K ). In contrast, positive cells in hermaphrodites are restricted to six cells along the ventral cord corresponding to the location of the VCs, and two cells in the midbody located between VC4 and VC5 corresponding to the location of the HSNs ( Figure 1 L-N ). Expression of hlh-3 in males is completely extinguished by the adult stage ( Figure 1 O-Q). However, in adult hermaphrodites, expression was still observed in the VCs, as previously reported, but not in the head or tail ( Figure 1 H, I, and J) ( Perez & Alfonso, 2020 ). hlh-3 null males do not exhibit exploration behavior As published, loss of hlh-3 function results in the hermaphrodite specific egg-laying defective (Egl) behavior ( Doonan et al., 2008 ). Thus, we set out to determine if hlh-3(tm1688) adult males had any observable mating-related behavioral defects. A well characterized behavior specific to adult males is exploration ( Lipton et al., 2004 ; Ryan et al., 2014 ). Well-fed, sexually mature adult males will leave a food source to explore, thus increasing their probability of encountering a potential mate ( Lipton et al., 2004 ; Ryan et al., 2014 ). To address whether loss of hlh-3 function affects this behavior, we performed a food leaving assay ( Lipton et al., 2004 ). For these assays, we created a strain with the null hlh-3(tm1688 ) allele and a him-5(e1490) mutation to increase the production of males ( Doonan et al., 2008 ; Hodgkin et al., 1979). We find that hlh-3(tm1688);him-5(e1490) adult males had a significantly lower probability to leave their food source when compared to strains containing only him-5(e1490) ( Figure 2 A and B). This phenotype is not observed in hlh-3(tm1688);him-5(e1490) adult hermaphrodites. To address whether the lack of exploration resulted from a locomotion defect, we measured the ability of worms to reach the same distance as the leaving assay, when the entire area within the scorable distance is a lawn of bacteria ( Barrios et al., 2012 ). We find that hlh-3(tm1688);him-5(e1490) mutant males can reach the edge of the food source as well as him-5(e1490) males do, thus a locomotion defect is not the cause of the observed lack of exploration (Figure S1 A and B). These results suggest that HLH-3 has a role in the differentiation of one or more neurons regulating the male mate searching drive ( Barrios et al., 2012 ; Hilbert & Kim, 2017 ; Lipton et al., 2004 ; Ryan et al., 2014 ). Download figure Open in new tab Figure 2: HLH-3 is required for male specific exploration behavior (A) Representative graph depicting quantification of average male specific exploration behavior of him-5(e1490) adult males and hlh-3(tm1688);him-5(e1490) adult males (n = 45 for each genotype). Each point represents the percentage of worms remaining in the non-leaver area at the indicated time point. (B) Probability of leaving a food source for him-5(e1490) males and hlh-3(tm1688);him-5(e1490) adult males was calculated by the single exponential decay function N(t)/N(0)=exp(-λt), where N(t)/N(0) refers to the ratio of non-leavers to leavers at a given time (t), and the hazard value (λ) is the constant rate demonstrating the probability of leaving. Values plotted as averages with SEM for 3 independent experiments. n.s. p = 0.961256 as determined by chi-squared analysis. Adult mutant male AIMs fail to fully switch their neurotransmitter identity AIM interneurons are sex-shared neurons responsible for male mating drive and leaving behavior ( Barrios et al., 2012 ). In males, adult AIM cells display a cholinergic identity. The neurotransmitter identity change from glutamatergic to cholinergic happens in the L4 to adult stage transition, a sexually dimorphic change ( Pereira et al., 2015 ). Given that previous work has shown that hlh-3(tm1688) hermaphrodites show a lack of terminal differentiation in sex specific neurons, we wanted to determine if hlh-3(tm1688) adult male AIMs showed normal AIM sexual differentiation. To address whether adult mutant AIMs undergo maturation in the absence of hlh-3 function, we asked whether hlh-3(tm1688);him-5(e1490) adult male AIMs attain their mature cholinergic neurotransmitter identity. We assessed this by characterizing eat-4 /VGLUT expression, a marker for glutamatergic identity, and cho-1 /ChT expression, a marker for cholinergic identity (Lee et al., 1999; Matthies et al., 2006). We find that about 60% of adult hlh-3(tm1688);him-5(e1490) males retain expression of eat-4 /VGLUT in AIM cells ( Figure 3 A and C). To ascertain cho-1 /ChT expression levels in AIMs, we measured intensity of expression relative to that of neighboring cholinergic AIY cells ( Pereira et al., 2015 ). We find that AIMs in hlh-3(tm1688); him-5(e1490) adult males have a modest decrease in cho-1 /ChT intensity when compared to the AIY cells in him-5(e1490) adult males, but there is no significant difference between that of him-5(e1490) and hlh-3(tm1688);him-5(e1490) L4 males ( Figure 3 D-H ). To determine whether this dimmer cho-1 /ChT expression pattern in mutant adult males resembles that of him-5(e1490) L4 males we compared intensity of hlh-3(tm1688);him-5(e1490) adult male AIMs to that of him-5(e1490) L4 males. We find that while expression of cho-1 /ChT decreases in hlh-3(tm1688);him-5(e1490) adult males, it is still significantly brighter than him-5(e1490) L4 males ( Figure 3 H). Thus, while the AIMs in hlh-3(tm1688);him-5(e1490) adult males do not resemble those of him-5(e1490) L4 males, it is apparent they are not fully undergoing a neurotransmitter identity switch. Download figure Open in new tab Figure 3: hlh-3 is required for AIM terminal differentiation in adult males (A-B) Representative images of eat-4(prom11)::GFP and ttx-3p::RFP expression in a him -5(e1490) and a hlh-3(tm1688);him-5(e1490) adult male, respectively. Open arrowheads indicate AIM cells, dashed arrowhead indicates AIY cells. (C) Percent of eat-4(prom11)::GFP positive AIM cells present in him-5(e1490) adult males (n = 42), and hlh-3(tm1688);him-5(e1490) adult males (n = 46). (D-E) Representative images of cho-1p::RFP and ttx-3p::GFP expression in a him-5(e1490) and a hlh-3(tm1688);him-5(e1490) adult male. Open arrowheads indicate AIM cells, dashed arrowhead indicates AIY cells. (F-G) Representative images of cho-1p::RFP and ttx-3p::GFP expression in a wild type and a hlh-3(tm1688) L4 male. Open arrowheads indicate AIM cells, dashed arrowhead indicates AIY cells. (H) Quantification of the relative intensity of cho-1p::RFP in AIM cells. Relative AIM intensity was determined by comparing its intensity to that of the standard cho-1p::RFP intensity in AIY cells of him-5(e1490) adult males (n = 36), hlh-3(tm1688);him-5(e1490) adult males (n = 38), him-5(e1490) L4 males (n = 33), and hlh-3(tm1688);him-5(e1490) L4 males (n =15). * p = 0.031623, ** p = 0.001101 as determined by Student’s T-test Mutant male ASJ sensory neurons do not express the TGF-β like daf-7 Exploration behavior in adult males is promoted by PDF-1 secretion via AIMs ( Barrios et al., 2012 ). In males, reception of PDF-1 by the ASJ cells has been shown to increase daf-7 expression and promote exploration ( Hilbert & Kim, 2018 ). Due to the proximity of the two cells, it is possible that the secreted neuropeptide PDF-1 from AIM results in increased DAF-7/TGFß levels in ASJs of adult males ( Cook et al., 2019 ). Since in hlh-3(tm1688) mutants, male AIMs are not terminally differentiated, then we expect to see a decrease in the levels of daf-7 in males, but no measurable change in hermaphrodites. To address whether expression of daf-7 was altered in hlh-3(tm1688);him-5(e1490) adult males, we examined daf-7 expression using a daf-7p::gfp reporter (Hilbert ,& Kim, 2017). daf-7 is expressed in one set of cells, the more anteriorly positioned ASIs, in juvenile males and hermaphrodites of all stages, and two sets of cells in adult males, the more anterior ASIs and the more posterior ASJs ( Hilbert & Kim, 2017 , Cook et al., 2019 ). Utilizing this information, we were able to identify the pattern of expression in him-5(e1490) and hlh-3(tm1688);him-5(e1490) strains. We find that expression of daf-7 was absent in one or more ASJ cells in hlh-3(tm1688); him-5(e1490) adult males, whereas expression in another cell, the ASIs remain unaltered ( Figure 4 A, B, E, and F) ( Hilbert & Kim, 2017 ). Examination of daf-7 in hlh-3(tm1688);him-5(e1490) adult hermaphrodites showed no change in expression pattern ( Figure 4 C, D, E, and F). Download figure Open in new tab Figure 4: Upregulation of daf-7 in adult male ASJs requires hlh-3 (A-B) Representative images of daf-7p::gfp expression in him-5(e1490) and hlh-3(tm1688);him-5(e1490) adult males. (C-D) Representative images of daf-7p::gfp expression in him-5(e1490) and hlh-3(tm1688) him-5(e1490) adult hermaphrodites. Dashed arrowheads indicate ASI cells, Solid arrowheads indicate ASJ cells. (E) Quantification of individuals of the indicated genotype and sex containing < 2 or 2 daf-7p::gfp positive ASJ and ASI cells for him-5(e1490) adult males (n = 26), hlh-3(tm1688);him-5(e1490) adult males (n = 30), him-5(e1490) hermaphrodites (n = 20), and hlh-3(tm1688);him-5(e1490) adult hermaphrodites (n = 20). To address whether the lack of daf-7 expression meant that the ASJ cells in hlh-3(tm1688);him-5(e1490) adults were not present we examined an ASJ specific trx-1p::trx-1::gfp reporter in both genotypes ( Sanzo-Machuca et al., 2019 ). We find that both ASJ cells are detectable in the him-5(e1490) and hlh-3(tm1688);him-5(e1490) adult males (Figure S2 A-C). Mutant male AWA sensory neurons fail to downregulate expression of ODR-10 and show greater attraction to diacetyl DAF-7/TGF-ß mediated signaling in well fed, adult males has been shown to decrease expression of ODR-10 in adult male AWAs ( Wexler et al., 2020 ). A decrease in ODR-10 has been linked to increased exploration behavior ( Ryan et al., 2014 ). To determine whether ODR-10 expression is altered in hlh-3(tm1688);him-5(e1490) adult male AWAs, we measured ODR-10::GFP intensity levels relative to that of a 0.3% InSpeck standard fluorescent intensity bead (Modular Probes). We find that hlh-3(tm1688);him-5(e1490) adult male ODR-10::GFP expression was significantly brighter than that in adult him-5(e1490) males, but not significantly different than that in him-5(e1490) L4 males ( Figure 5 A, B, E, and F). hlh-3(tm1688);him-5(e1490) adult hermaphrodites ODR-10::GFP levels remain unaffected ( Figure 5 C, D, and F) Download figure Open in new tab Figure 5: HLH-3 is required for adult male odr-10 repression and subsequent chemosensation of diacetyl (A-E) Representative images of ODR-10::GFP expression in him-5(e1490) adult males, him-5(e1490) adult hermaphrodites, him-5(e1490) L4 males, and hlh-3(tm1688);him-5(e1490) adult males. (F) Quantification of ODR-10::GFP expression in relation to 0.3% standard intensity beads for him-5(e1490) adult males (n = 39), hlh-3(tm1688);him-5(e1490) adult males (n = 30), hlh-3(tm1688);him-5(e1490) adult hermaphrodites (n = 20), him-5(e1490) L4 males (n = 28), and him-5(e1490) adult hermaphrodites (n = 39). *** p = 0.000229 , ** p < 0.00001 , n.s. p = 0.403301, n.s. p value = 0.091906 as determined by Student’s T-test. Expression of ODR-10::GFP is significantly dimmer in hlh-3(tm1688);him-5(e1490) adult males than in him-5(e1490) adult males. (G) Diacetyl (1:1000 dilution) chemotaxis assay where hlh-3(tm1688);him-5(e1490) adult males have a mild attraction to diacetyl, while him-5(e1490) adult males do not. Values plotted as averages with SEM for 3 independent experiments (n = 40-50 per genotype). (H) Pyrazine (0.1mg/1ml) chemotaxis assay where both him-5(e1490) and hlh-3(tm1688);him-5(e1490) adult males have a strong attraction to pyrazine (n = 40-50 per genotype). Values plotted as averages with SEM for 3 independent experiments (n = 40-50 per genotype). Others have shown that increased levels of ODR-10 in L4 male AWAs lead to decreased levels of exploration and increased attraction to diacetyl (Colbert & Bargmann, 1995; Ryan et al., 2014 ). To determine if the observed decrease in ODR-10 expression translates to changes in chemoattraction to diacetyl, we performed a chemotaxis assay ( Bargmann et al., 1993 ; Margie et al., 2013 ). Consistent with increased ODR-10 expression levels, we found that hlh-3(tm1688);him-5(e1490) adult males have a significant increase in attraction to the odorant diacetyl ( Figure 5 G). To further determine the functionality of hlh-3(tm1688);him-5(e1490) AWA odorant detection, we then assayed attraction to another odorant detected by AWA, pyrazine ( Bargmann et al., 1993 ). Although the receptor to pyrazine is not currently identified, it is known that it is not a ligand of ODR-10 (Sengupta et. al. 1996). Our findings show that hlh-3(tm1688);him-5(e1490) adult males and hermaphrodites show chemoattraction of pyrazine similar to that of their him-5(e1490) counterparts ( Figure 5 H). This indicates that while some chemosensory functions of the AWAs of adult males are abnormal, others remain unaffected. Post-embryonic expression of hlh-3 is detectable in male AIMs, but not AWAs or ASJs AIMs act non-autonomously to influence the cellular circuitry regulating exploration behavior in adult males ( Barrios et al., 2012 ). AIMs are sex-shared head interneurons and at a position reminiscent of where we find ic271[hlh-3::gfp] positive cells ( Figure 1 B) in the L1 male. To determine if the identity of the cells present in the head of L1 worms were AIM cells, we introduced ttx-3p::rfp , a cell specific reporter for the landmark cell, AIY (Hobert et al., 1997). Given that one of the ic271[hlh-3::gfp] positive cells in the head is located immediately anterior to AIY, the landmark cell, we conclude one of the hlh-3 positive cells in the head are AIMs (Figure 7 A, C). It is noteworthy there is no adjacent hlh-3 positive cell in L1 hermaphrodites (Figure 7 B, C). ic271[hlh-3::gfp] positive AIM cells were not observed in early L3/late L4 stage males or hermaphrodites. We then determined whether hlh-3 expression was also detectable in the ASJs and AWAs as well. DiI staining was performed on ic271[hlh-3::gfp] L1 and late L3/early L4 staged males to co-label ASJ cells and determine if hlh-3 was expressed in these cells. There was no evidence that hlh-3 was present in ASJs at either of these stages (Figure 7 D and E). To determine if hlh-3 was expressed in AWA cells, we examined both L1 and late L3/early L4 males co-expressing podr-10::rfp and ic271[hlh-3::gfp] (Wan et al., 2019). We do not detect hlh-3 expression in male AWAs at either stage (Figure 7 F and G). Expression of hlh-3 in male AIMs is not sufficient to down-regulate expression of ODR-10 in AWAs Since we learned that hat hlh-3 is expressed post-embryonically in AIM cells, but not AWA or ASJ cells, we hypothesized that a loss of HLH-3 in the AIMs may be non-autonomously causing the phenotypes observed in both other sets of cells. To address whether the function of HLH-3 in the AIM cells is necessary to regulate ODR-10 levels in the AWA cell, we generated a construct in which we utilized a promoter fragment of eat-4 expressed only in AIMs and PHCs of both males and hermaphrodites to drive expression of a GFP tagged copy of hlh-3 (Ex[eat-4(prom11):: hlh-3::gfp + rol-6(su1006)]) ( Serrano-Saiz et al., 2017 ). We then measured ODR-10::GFP intensity of this strain in comparison to a control. We find that rescuing expression of HLH-3 in the AIMs of males is not sufficient to rescue ODR-10 levels in the AWAs (Figure 8 A, C, and E). DISCUSSION Expression of hlh-3 is sexually dimorphic The pattern of expression of hlh-3 in hermaphrodites has been described in the past ( Doonan et al., 2008 ; Krause et al., 1997 ; Lloret-Fernández et al., 2018 ; Perez & Alfonso, 2020 ). However, a description of hlh-3 expression in males has not been reported before this work. To determine the male expression pattern during post embryonic development, and to ascertain if there were any differences from that in hermaphrodites, we documented the expression patterns of ic271[hlh-3::gfp] in both sexes, at all post-embryonic stages, and compared them side by side. As shown before, in hermaphrodites, expression of hlh-3 was observed in P cells and Pn.a descendants at the L1 stage ( Doonan et al, 2008 ; Perez & Alfonso, 2020 ). In contrast, the expression pattern in males is expanded to several more cells along the VNC that we suspect to be precursors to the male specific descendants of the P cells, CAs and CPs. It is also interesting to note that hlh-3 expression is observed in a few other cells in the head of L1 males, but not hermaphrodites. The location of these cells suggests these positive cells are either the CEMs, or a subset of sex-shared neurons that are necessary for sex specific functions ( Shaham & Bargmann, 2002 ). This work corroborates previous findings that hlh-3 is expressed in regions of the adult and juvenile staged hermaphrodites. Unfortunately, characterization of the L2 expression pattern in either males or hermaphrodites was not possible because sex discrimination at this stage, using our equipment, is not feasible. Expression of hlh-3 at the L3 stage and beyond differs in males when compared to hermaphrodites as it is observed in a much higher number of cells in the tail. Given that HLH-3 is required for the terminal differentiation of both VCs and HSNs, it is reasonable to assume that a subset of the hlh-3 positive cells observed in male tails are also sex-specific ( Doonan et al., 2008 ; Krause et al., 1997 ; Perez & Alfonso, 2020 ). Because of its location, the cluster of expression observed in the tail ganglia is likely to include neurons that play major roles in male mating and mate sensing activities, such as ray A-type or B-type neurons (RnA and RnB), hook sensillum neurons (HOA and HOB), or phasmid neurons (PHA, PHB, and PHC) ( Barr & Sternberg, 1999 ; Garcia et al., 2001 ; K. S. Liu & Sternberg, 1995 ; Loer & Kenyon, 1993 ). Efforts to utilize the whole nervous system NeuroPal reporter to positively identify each of the cells that express hlh- 3 were not fruitful because nuclear expression of the CRISPR-Cas9 ic271[hlh-3::gfp] allele was dim ( Tekieli et al., 2021 ). Further investigation with an alternative hlh-3 reporter is required to confirm the identity of the hlh-3 positive cells in the male. Post-embryonic expression of hlh-3 in males is most prominent in the L1 stage as well as the late L3/early L4 stages in a sexually dimorphic pattern. Previous reports show sexually dimorphic neurons display ‘just-in-time’ development, in which neurons that acquire sexually dimorphic terminal identity features form these features in the L4 stage just before adulthood ( Hobert, 2016 ; Tekieli et al., 2021 ). The expression pattern of hlh-3 at the L3/L4 stages and in places such as the male tail support the idea that hlh-3 function is necessary for many male specific neurons to mature to adulthood; this is consistent with the hypothesis that it is necessary for the terminal differentiation of hermaphrodite specific neurons (Doonan et. al 2008; Perez & Alfonso 2020 ). However, the expression of hlh-3 in some cells in the male L1 stage, but not later stages, suggests that hlh-3 may be necessary to set into motion a cascade of events that contribute to terminal identity acquisition at a later stage. These contrasting patterns of expression suggest that the function of hlh-3 is necessary at different developmental stages to promote neuronal identity cascades in motion. Many neurons necessary for male exploration fail to obtain terminal differentiation features Our results have shown that the sex-shared AWAs, ASJs, and AIMs of adult mutant males fail to acquire terminal identity features, and subsequently do not result in a defect in male exploration. Mutant adult male AIM neurons fail to fully acquire the mature cholinergic identity ( Figure 3 ). In turn, male ASJ neurons are present but fail to upregulate expression of daf-7 upon adulthood ( Figure 4 ). As a result, expression of ODR-10 is not downregulated in adult male AWA neurons, resulting in increased attraction to diacetyl ( Figure 5 ). Since hlh-3 is not expressed post-embryonically in either the AWAs or ASJs ( Figure 6 ), we asked whether restoration of hlh-3 function in AIM, the only cell in the circuit where we see post-embryonic expression of hlh-3 , could restore function to the circuit in a non-cell autonomous way. We find it does not (Figure 7). Expression of hlh-3 in the L1 to L4 stages does not rescue the mutant phenotypes in the AWA cell and by inference the ASJ cell. This is surprising given that the current body of evidence suggest that the emergence of male sexual dimorphism results from “just-in-time” development ( Tekieli et al., 2021 ); a process in which dimorphic neurons are born in any given stage, but all begin the terminal differentiation program in the L4 stage ( Tekieli et al., 2021 ). Rescue of ODR-10 expression levels in the AWAs through rescue of hlh-3 expression in AIMs may be due to one of a few reasons. Download figure Open in new tab Figure 6: hlh-3 is expressed post-embryonically in male L1 AIMs (A-B) Representative images of ic271[hlh-3::gfp] and ttx-3p::rfp expression in the head of an L1 male and hermaphrodite, respectively. (C) Quantification of ic271[hlh-3::gfp] expression in the AIM cell of L1 males (n = 25) and hermaphrodites (n = 23). Open arrowheads indicate AIM cells, dashed arrowheads indicate AIY cells. (D-E) Representative images of ic271[hlh-3::gfp] and ttx-3p::rfp expression in the head of a late L3/earlyL4 male and hermaphrodite, respectively. (F-G) Representative images of ic271[hlh-3::gfp] and DiI stain in the head of an L1 and late L3/earlyL4 male, respectively. Dashed arrowhead represents the ASJ cells. (H-I) Representative images of ic271[hlh-3::gfp] and odr-10p::rfp expression in the head of an L1 and late L3/earlyL4 male, respectively. Dashed arrowhead indicates AWA cells. First, the eat-4(prom11)::gfp construct used to drive expression of hlh-3 in AIMs was not integrated. While expression of the reporter is present and detectable in hermaphrodites of all stages, in males, expression is only observed in the L1 through L4 stages. Assessment of L4 stage males shows the construct present in 46% of L4 worms, when the construct is still detectable. Thus, it is possible that in some assayed males the construct was lost in precursors to the AIM. Since the rescue construct is not expressed in adult males, choosing adult males in which it is certain that the construct is present was impossible. Thus, the lack of rescue could be explained by a skewed proportion of males in which the extra chromosomal array is lost in the AIM cells. Secondly, it could be suggested that the construct is unable to generate functional hlh-3 products. However, lack of function of the HLH-3 protein produced by the rescue construct is unlikely. The eat-4 construct was generated from a punc-86::hlh-3::gfp construct that has been shown to rescue HSN maturation defects in hlh-3(tm1688) adult hermaphrodites ( Raut, 2017 ). Moreover, the fact that we detect expression of the eat-4(prom11)::gfp construct in hermaphrodites suggests it has been transcribed and translated. One other possibility is that some other neuron influencing this circuitry is required for the down-regulation of ODR-10 in the AWAs. The network of neurons characterized in this study does not extend to all neurons that regulate male exploration behavior (Barrios, 2012). It is possible that other neurons in the head (URY) or tail (PHA and PQR), that have been shown to affect male exploration, may require expression of hlh-3 in order to repress ODR-10 in adult males. This would require further work to characterize their differentiation and functionality within hlh-3(lof) males. Lastly, it is possible that to rescue the mutant phenotype, it is necessary to restore hlh-3 function in embryonic stages. It is known that hlh-3 is expressed transiently in embryonic AIM, AWA, and ASJ cells of hermaphrodites (Ma et. al., 2021). Thus, it is possible that hlh-3 function is necessary embryonically in males to set in motion a regulatory cascade for one or all of these cell types. If so, our observations are, in the context of the AIM, inconsistent with the hypothesis of “just-in-time” maturation of sex-shared neurons with sexually dimorphic functions. To further explore the mechanism of hlh-3 function it would be necessary to determine whether hlh-3 expression is also detectable in male AIM, AWA, and ASJ neurons in the embryonic stage and a rescue construct driving embryonic expression would need to be generated. From this data we can conclude that hlh-3 expression is necessary for this subset of neurons to acquire terminal identity features, though further work is necessary to determine the mechanism by which that is accomplished. hlh-3 is a non-traditional bHLH protein with a late acting role in neuronal terminal identity acquisition of sex-specific and sex-shared neurons with sex-specific functions In this work we have characterized the phenotypes displayed by hlh-3(lof) adult male neurons that are sex-shared but have sex-specific roles in adulthood. Our findings regarding defects in sex shared neurons with sex-specific functions in hlh-3 mutant males adds support to the hypothesis that hlh-3 is necessary for the terminal differentiation of both sex-specific and sex-shared neurons involved in sex-specific behaviors, a novel finding. How HLH-3 regulates these processes is not yet clear. However, we can say that the role of hlh-3 is not that of a traditional proneural bHLH transcription factor but rather one of the many bHLH proteins in C. elegans that has non-traditional roles in development. Unlike what has been observed in the hermaphrodite specific HSNs and VCs, in males hlh-3 expression may not necessary throughout the remainder of developmental stages. This indicates that hlh-3 may be acting much earlier in development to set up a signaling cascade that leads to the differentiation of these neurons at a much later stage than previously hypothesized. Alternatively, it may be possible that our reporters are not able to detect minute quantities of expressed hlh-3 that may be required for the acquisition of these terminal differentiation features. In this scenario, if transcripts were detectable in the late L3/early L4 stage then this would much better fit the current model that indicates that many factors that contribute to the arising of sexually dimorphic features are expressed just before these changes are observed to occur. If embryonic expression of hlh-3 does truly set up a signaling cascade that results in attainment of sexually dimorphic features emerging in the L4 to adult transition, then it is possible that the process of sex-shared neurons gaining these sexually dimorphic features may start much earlier than previously thought. Early acting factors, such as hlh-3 and/or other bHLH proteins, may be necessary to specify sex-shared neurons with the potential to acquire these sexually dimorphic features earlier in development, but then factors such as mab-3 and lin-29a may be functioning in parallel, late acting pathways to more directly carry out acquisition of these features. Re-emergence of hlh-3 expression in a subset of these neurons may be indicative of re-enforcement of the identity of these cells. Further work deciphering the early acting, embryonic roles of hlh-3 versus the late acting, post-embryonic role of hlh-3 in sex-shared neurons through use of condition specific knockout strains would answer questions concerning this topic. MATERIALS AND METHODS Strain maintenance All strains were maintained at 22°C under standard conditions on nematode growth media seeded with OP50-1 ( Brenner, 1974 ; Johnson et al., 1988 ). The National Bioresource of Japan generated the hlh-3(tm1688) allele used in this work. Several strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). VZ797 was graciously provided by Dr. Antonio Miranda-Vizuete. podr-10::rfp constructs were graciously provided by Dr. King L Chow. Construction of transgenic strains The genomic region of chromosome III contained between -2609bp and -2155bp from the ATG of the eat-4 locus was amplified via PCR ( Serrano-Saiz et al., 2017 ). The DNA fragment was cloned into the pRD6 construct after excising the unc-86 promoter with SalI and HindIII enzymes to generate pKA1 ( Raut, 2017 ). pKA1 (50ng/µl) was injected into hlh-3(tm1688) II; kyIs37[ODR-10::GFP + lin15(+)] II; him-5(e1490) V (AL190) using rol-6(su1006) (50ng/µl) as a co-injection marker to generate [eat-4(prom11)::hlh-3::gfp + rol-6(su1006)]; hlh-3(tm1688) II; kyIs37[ODR-10::GFP + lin15(+)] II; him-5(e1490) V (Peixoto et al., 1998). This produced strains in which hlh-3 function was only rescued in AIMs and PHCs of both males and hermaphrodites. podr-10::rfp (50ng/ul) was injected into ic271[hlh-3::gfp]; him-5(e1490) using rol-6(su1006) (50ng/µl) as a co-injection marker (Peixoto et al., 1998; Perez & Alfonso, 2020 ; Wan et al., 2019). This generated [podr-10::rfp + rol-6(su1006)]; ic271[hlh-3::gfp]; him-5(e1490) V . This produced a strain which co-labels ic271[hlh-3::gfp] and podr10::rfp expressing cells. Microscopy Fluorescent Imaging Animals were segregated by sex at the L4 stage on uncrowded plated seeded with a lawn of OP50-1. After 26 hours they were mounted on 2.5% agarose pads and anesthetized with 10mM levamisole ( Johnson et al., 1988 ). Images were taken at either 63x or 100x with a Carl Zeiss Axioskop 2 fluorescence microscope utilizing Axiovision 2.002 software. Images were further visualized with Fiji version 1.54f software ( Schindelin et al 2012 ). Original grayscale images were converted with red and green Lookup tables and merged using Merge Channels. Figures were created using Adobe Illustrator version 28.6. Fluorescence Quantification GFP fluorescence was quantified by measuring the intensity of the reporter using Fiji software and comparing it to an InSpeck™ Green (505/515) microscope image of a 0.3% standard intensity bead mounted on the same agarose pad (Invitrogen, I7219, Schindelin et al., 2012 ). Statistical significance was determined using the Student’s T-test. About 30-40 cells were imaged per genotype. Quantification of cho-1p::rfp intensity in AIM cells was performed by comparing its intensity relative to that of the cho-1p::rfp intensity in the neighboring AIY cells (Serrano-Saiz, Pereira, et al., 2017). Statistical significance was determined using the Student’s T-test. About 25-35 cells were imaged per genotype. Leaving assay L4 males or hermaphrodites were picked 26 hours prior to the assay and isolated on an uncrowded plate seeded with OP50-1 bacteria. Exploration assays were conducted as previously described ( Lipton et al., 2004 ). A single animal was placed on a 10cm plate seeded with 20µl of OP50-1 bacteria. Animals were allowed to roam for 1 hour before their tracks were observed. If the animal reaches an area 1cm from the edge of the plate (the scoring distance), they are considered a “leaver”. The number of leavers is counted at the end of each hour time point for a total of 5 hours. The probability of leaving was determined by obtaining the hazard value (λ), using the single exponential decay function N(t)/N(0)=exp(-λt). These values were calculated using the exponential parametric survival using maximum likelihood (R Project for Statistical Computing with R survival package). Statistical significance was calculated using one-way ANOVA with Tukey HSD. For the locomotion control, 10cm plates were seeded with OP50-1 bacteria spread into a lawn covering the area to the 1cm scoring distance used in the exploration assay ( Barrios et al., 2012 ). Single animals were placed on these plates and their tracks were analyzed using the same methodology as the exploration assay. Statistical significance was calculated using chi-squared analysis. Chemotaxis assays About 50-60 L4 males were picked 26 hours prior to the assay and isolated on an uncrowded plate seeded with OP50-1 bacteria. To minimize transfer of bacteria, adult males were transferred to an unseeded plate before being washed off with M9 (Stiernagle, 2006).and collected in a 1.5mL tube. Worms were then spun down and transferred to the assay plate. The rest of the assay was conducted as previously described ( Margie et al., 2013 ). 2µl of diacetyl (1:1000µl dilution in ethanol) was used in diacetyl assays (Arcos Organics, 431-03-8). 2µl of a 1:100µl dilution of 10mg/ml pyrazine in ethanol was used in pyrazine assays (Oakwood Chemical, 290-37-9). For each chemotaxis assay, averages are shown pooled across 3 replicates (n = 40-50 per genotype). Significance was determined using the Student’s T-test. Data Availability Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Download figure Open in new tab Figure S1: HLH-3 null adult males can reach scoring distances at similar rates as wild type males (A) Representative graph depicting the quantification of average male specific exploration behavior of him-5(e1490) adult males and hlh-3(tm1688);him-5(e1490) adult males (n = 45 for each genotype). Each point represents the percentage of worms remaining in the non-leaver area at the indicated time point. (B) Probability of leaving a food source for him-5(e1490) males and hlh-3(tm1688);him-5(e1490) adult males was calculated by the single exponential decay function N(t)/N(0)=exp(-λt), where N(t)/N(0) refers to the ratio of non-leavers to leavers at a given time (t), and the hazard value (λ) is the constant rate demonstrating the probability of leaving. Values plotted as averages with SEM for 3 independent experiments. n.s. p = 0.961256 as determined by chi-squared analysis. Download figure Open in new tab Figure S2: Adult male ASJs are detectable in wild type and mutant adult males; they still express trx-1 (A-B) Representative images of trx-1p::trx-1::gfp expression in adult male him-5(e1490) and him-5(e1490);hlh-3(tm1688) , respectively. (C) Quantification of indicated genotype containing either 2 or <2 ASJ cells for him-5(e1490) adult males (n = 21) and him-5(e1490);hlh-3(tm1688) adult males (n = 29). ACKNOWLEDGEMENTS We would like to acknowledge Maryam Sabir for help with data acquisition; Dr. Suzanne McCutcheon and Dr. Janet Richmond for imaging equipment; and the Department of Biological Sciences for support with this publication. We thank Dr. Antonio Miranda-Vizuete and Dr. King L Chow for graciously providing strains and constructs. REFERENCES 1. ↵ Bargmann , C. I. , Hartwieg , E. , & Robert Horvitz , H . ( 1993 ). Odorant-Selective Genes and Neurons Mediate Olfaction in C. elegans . Cell , 74 , 515 – 527 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Barr , M. M. , & Sternberg , P. W . ( 1999 ). A polycystic kidney-disease gene homologue required for male mating behavior in C. elegans . 386 – 389 . 3. ↵ Barrios , A . ( 2014 ). Exploratory decisions of the Caenorhabditis elegans male: A conflict of two drives . Seminars in Cell and Developmental Biology , 33 , 10 – 17 . doi: 10.1016/j.semcdb.2014.06.003 OpenUrl CrossRef PubMed 4. ↵ Barrios , A. , Ghosh , R. , Fang , C. , Emmons , S. W. , & Barr , M. M . ( 2012 ). PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans . Nature Neuroscience , 15 ( 12 ), 1675 – 1682 . doi: 10.1038/nn.3253 OpenUrl CrossRef PubMed 5. Bertrand , N. , Castro , D. & Guillemot , F . Proneural genes and the specification of neural cell types . Nat Rev Neurosci 3 , 517 – 530 ( 2002 ). doi: 10.1038/nrn874 OpenUrl CrossRef PubMed Web of Science 6. ↵ Brenner , S . ( 1974 ). The Genetics of Caenorhabditis elegans . Genetics , 77 ( 1 ), 71 – 94 . doi: 10.1093/GENETICS/77.1.71 OpenUrl Abstract / FREE Full Text 7. ↵ Cook , S. J. , Jarrell, travis A., Brittin, Christopher A. , Wang , Y. , Bloniarz , A. , Yakovlev , M. A. , Nguyen , K. Q. , t-H tang , Leo , Bayer , Emily A. , Duerr , J. S. , Bülow , H. , Hobert , O. , Hall , D. H. , Emmons , S. W. , Cook , S. J. , & Jarrell , T. A. ( 2019 ). Whole-animal connectomes of both Caenorhabditis elegans sexes . Nature , 571 , 63 – 71 . doi: 10.1038/s41586-019-1352-7 OpenUrl CrossRef PubMed 8. ↵ Doonan , R. , Hatzold , J. , Raut , S. , Conradt , B. , & Alfonso , A . ( 2008 ). HLH-3 is a C. elegans Achaete/Scute protein required for differentiation of the hermaphrodite-specific motor neurons . Mechanisms of Development , 125 ( 9–10 ), 883 – 893 . doi: 10.1016/j.mod.2008.06.002 OpenUrl CrossRef PubMed 9. ↵ Duggan , A. , & Chalfie , M . ( 1995 ). Control of neuronal development in Caenorhabditis elegans . Current Opinion in Neurobiology , 5 ( 1 ), 6 – 9 . doi: 10.1016/0959-4388(95)80080-8 OpenUrl CrossRef PubMed 10. ↵ Filippopoulou , K. , Couillault , C. , & Bertrand , V . ( 2021 ). Multiple neural bHLHs ensure the precision of a neuronal specification event in Caenorhabditis elegans . Biology Open , 10 ( 12 ). doi: 10.1242/BIO.058976/273578/AM/MULTIPLE-NEURAL-BHLHS-ENSURE-THE-PRECISION-OF-A OpenUrl CrossRef 11. ↵ Garcia , L. R. , Mehta , P. , & Sternberg , P. W . ( 2001 ). Regulation of distinct muscle behaviors controls the C. elegans male’s copulatory spicules during mating . Cell , 107 ( 6 ), 777 – 788 . doi: 10.1016/S0092-8674(01)00600-6 OpenUrl CrossRef PubMed Web of Science 12. ↵ Ghysen , A. , & Dambly-Chaudiere , C . ( 1989 ). Genesis of the Drosophila peripheral nervous system . Trends in Genetics , 5 ( 8 ), 251 – 255 . OpenUrl CrossRef PubMed Web of Science 13. ↵ Grove , C. A. , De Masi , F. , Barrasa , M. I. , Newburger , D. E. , Alkema , M. J. , Bulyk , M. L. , & Walhout , A. J. M. ( 2009 ). A Multiparameter Network Reveals Extensive Divergence between C. elegans bHLH Transcription Factors . Cell , 138 ( 2 ), 314 – 327 . doi: 10.1016/J.CELL.2009.04.058 OpenUrl CrossRef PubMed Web of Science 14. ↵ Hilbert , Z. A. , & Kim , D. H . ( 2017 ). Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans . ELife , 6 . doi: 10.7554/ELIFE.21166 OpenUrl CrossRef 15. ↵ Hilbert , Z. A. , & Kim , D. H . ( 2018 ). PDF-1 neuropeptide signaling regulates sexually dimorphic gene expression in shared sensory neurons of C. elegans . ELife , 7 . doi: 10.7554/ELIFE.36547 OpenUrl CrossRef 16. ↵ Hobert , O . ( 2016 ). Terminal Selectors of Neuronal Identity . Current Topics in Developmental Biology , 116 , 455 – 475 . doi: 10.1016/BS.CTDB.2015.12.007 OpenUrl CrossRef PubMed 17. Hodgkin , J . ( 1983 ). Two types of sex determination in a nematode . Nature , 304 , 267 – 268 . OpenUrl CrossRef PubMed 18. ↵ Johnson , C. D. , Herman , R. K. , & Stern , B. D . ( 1988 ). The Acetylcholinesterase Genes of C. elegans: Identification of a Third Gene (ace-3) and Mosaic Mapping of a Synthetic Lethal Phenotype . Neuron , 1 , 165 – 173 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Kimble , J. , & Hirsh , D . ( 1979 ). The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans . Developmental Biology , 70 ( 2 ), 396 – 417 . doi: 10.1016/0012-1606(79)90035-6 OpenUrl CrossRef PubMed Web of Science 20. ↵ Krause , M. , Park , M. , Zhang , J. M. , Yuan , J. , Harfe , B. , Xu , S. Q. , Greenwald , I. , Cole , M. , Paterson , B. , & Fire , A . ( 1997 ). A C. elegans E/Daughterless bHLH protein marks neuronal but not striated muscle development . Development , 124 ( 11 ), 2179 – 2189 . doi: 10.1242/DEV.124.11.2179 OpenUrl Abstract 21. ↵ Lawson , H. N. , Wexler , L. R. , Wnuk , H. K. , & Portman , D. S . ( 2020 ). Dynamic, Non-binary Specification of Sexual State in the C. elegans Nervous System . Current Biology , 30 ( 18 ), 3617 – 3623.e3 . doi: 10.1016/j.cub.2020.07.007 OpenUrl CrossRef PubMed 22. ↵ Lawson , H. , Vuong , E. , Miller , R. M. , Kiontke , K. , Fitch , D. H. , & Portman , D. S . ( 2019 ). The makorin lep-2 and the lncrna lep-5 regulate lin-28 to schedule sexual maturation of the C. elegans nervous system . ELife , 8 . doi: 10.7554/ELIFE.43660 OpenUrl CrossRef 23. ↵ Lipton , J. , Kleemann , G. , Ghosh , R. , Lints , R. , & Emmons , S. W . ( 2004 ). Mate searching in Caenorhabditis elegans : A genetic model for sex drive in a simple invertebrate . Journal of Neuroscience , 24 ( 34 ), 7427 – 7434 . doi: 10.1523/JNEUROSCI.1746-04.2004 OpenUrl Abstract / FREE Full Text 24. ↵ Liu , K. S. , & Sternberg , P. W . ( 1995 ). Sensory Regulation of Male Mating Behavior in Caenorhabditis elegans . Neuron , 14 , 79 – 89 . OpenUrl CrossRef PubMed Web of Science 25. ↵ Lloret-Fernández , C. , Maicas , M. , Mora-Martínez , C. , Artacho , A. , Jimeno-Martín , Á. , Chirivella , L. , Weinberg , P. , & Flames , N . ( 2018 ). A transcription factor collective defines the HSN serotonergic neuron regulatory landscape . ELife , 7 . doi: 10.7554/ELIFE.32785 OpenUrl CrossRef 26. ↵ Loer , C. M. , & Kenyon , C. J . ( 1993 ). Serotonin-deficient mutants and male mating behavior in the nematode Caenorhabditis elegans . The Journal of Neuroscience : The Official Journal of the Society for Neuroscience , 13 ( 12 ), 5407 – 5417 . doi: 10.1523/JNEUROSCI.13-12-05407.1993 OpenUrl Abstract / FREE Full Text 27. Ma , X. , Zhao , Z. , Xiao , L. et al. A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis . Nat Methods 18 , 893 – 902 ( 2021 ). doi: 10.1038/s41592-021-01216-1 OpenUrl CrossRef PubMed 28. ↵ Margie , O. , Palmer , C. , & Chin-Sang , I . ( 2013 ). C. elegans Chemotaxis Assay . JoVE (Journal of Visualized Experiments ), 74 , e50069 . doi: 10.3791/50069 OpenUrl CrossRef 29. ↵ Masoudi , N. , Tavazoie , S. , Glenwinkel , L. , Ryu , L. , Kim , K. , & Hobert , O . ( 2018 ). Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity . PLOS Biology , 16 ( 4 ), e2004979 . doi: 10.1371/JOURNAL.PBIO.2004979 OpenUrl CrossRef PubMed 30. ↵ Murgan , S. , Kari , W. , Rothbächer , U. , Iché-Torres , M. , Mélénec , P. , Hobert , O. , & Bertrand , V . ( 2015 ). Atypical Transcriptional Activation by TCF via a Zic Transcription Factor in C. elegans Neuronal Precursors . Developmental Cell , 33 ( 6 ), 737 – 745 . doi: 10.1016/J.DEVCEL.2015.04.018 OpenUrl CrossRef PubMed 31. ↵ Pereira , L. , Aeschimann , F. , Wang , C. , Lawson , H. , Serrano-Saiz , E. , Portman , D. S. , Großhans , H. , & Hobert , O . ( 2019 ). Timing mechanism of sexually dimorphic nervous system differentiation . ELife , 8 . doi: 10.7554/ELIFE.42078 OpenUrl CrossRef 32. ↵ Pereira , L. , Kratsios , P. , Serrano-Saiz , E. , Sheftel , H. , Mayo , A. E. , Hall , D. H. , White , J. G. , LeBoeuf , B. , Garcia , L. R. , Alon , U. , & Hobert , O . ( 2015 ). A cellular and regulatory map of the cholinergic nervous system of C. elegans . ELife , 4 December 2015 . doi: 10.7554/ELIFE.12432 OpenUrl CrossRef 33. ↵ Perez , L. M. , & Alfonso , A . ( 2020 ). The Conserved ASCL1/MASH-1 Ortholog HLH-3 Specifies Sex-Specific Ventral Cord Motor Neuron Fate in Caenorhabditis elegans . G3: Genes|Genomes|Genetics , 10 ( 11 ), 4201 – 4213 . doi: 10.1534/g3.120.401458 OpenUrl Abstract / FREE Full Text 34. ↵ Poole , R. J. , Bashllari , E. , Cochella , L. , Flowers , E. B. , & Hobert , O . ( 2011 ). A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans . PLoS Genetics , 7 ( 6 ), 1002109 . doi: 10.1371/journal.pgen.1002109 OpenUrl CrossRef 35. ↵ Portman , D. S . ( 2017 ). Sexual modulation of sex-shared neurons and circuits in Caenorhabditis elegans . Journal of Neuroscience Research , 95 ( 1–2 ), 527 – 538 . doi: 10.1002/JNR.23912 OpenUrl CrossRef PubMed 36. ↵ Raut , S. ( 2017 ). Functional Analysis of HLH-3, a bHLH Achaete/Scute Protein, in HSN Maturation . https://indigo.uic.edu/articles/thesis/Functional_Analysis_of_HLH-3_a_bHLH_Achaete_Scute_Protein_in_HSN_Maturation/10824206 37. ↵ Ryan , D. A. , Miller , R. M. , Lee , K. , Neal , S. J. , Fagan , K. A. , Sengupta , P. , & Portman , D. S . ( 2014 ). Sex, Age, and Hunger Regulate Behavioral Prioritization through Dynamic Modulation of Chemoreceptor Expression . Current Biology , 24 ( 21 ), 2509 – 2517 . doi: 10.1016/J.CUB.2014.09.032 OpenUrl CrossRef PubMed 38. ↵ Sallee , M. D. , Littleford , H. E. , & Greenwald , I . ( 2017 ). A bHLH Code for Sexually Dimorphic Form and Function of the C. elegans Somatic Gonad . Current Biology , 27 ( 12 ), 1853 – 1860 .e5. doi: 10.1016/J.CUB.2017.05.059 OpenUrl CrossRef PubMed 39. ↵ Sanzo-Machuca , Á. , Monje Moreno , J. M. , Casado-Navarro , R. , Karakuzu , O. , Guerrero-Gómez , D. , Fierro-González , J. C. , Swoboda , P. , Muñoz , M. J. , Garsin , D. A. , Pedrajas , J. R. , Barrios , A. , & Miranda-Vizuete , A . ( 2019 ). Redox-dependent and redox-independent functions of Caenorhabditis elegans thioredoxin 1 . Redox Biology , 24 , 101178 . doi: 10.1016/J.REDOX.2019.101178 OpenUrl CrossRef PubMed 40. ↵ Schindelin , J. , Arganda-Carreras , I. , Frise , E. , et al. Fiji: an open-source platform for biological-image analysis . Nat Methods 9 , 676 – 682 ( 2012 ). doi: 10.1038/nmeth.2019 OpenUrl CrossRef PubMed Web of Science 41. Sengupta , P. , Chou , J. H. , & Bargmann , C. I . ( 1996 ). odr-10 Encodes a Seven Transmembrane Domain Olfactory Receptor Required for Responses to the Odorant Diacetyl . Cell , 84 ( 6 ), 899 – 909 . doi: 10.1016/S0092-8674(00)81068-5 OpenUrl CrossRef PubMed Web of Science 42. ↵ Serrano-Saiz , E. , Oren-Suissa , M. , Bayer , E. A. , & Hobert , O . ( 2017 ). Sexually Dimorphic Differentiation of a C. elegans Hub Neuron Is Cell Autonomously Controlled by a Conserved Transcription Factor . Current Biology : CB , 27 ( 2 ), 199 – 209 . doi: 10.1016/J.CUB.2016.11.045 OpenUrl CrossRef PubMed 43. ↵ Shaham , S. , & Bargmann , C. I . ( 2002 ). Control of neuronal subtype identity by the C. elegans ARID protein CFI-1 . Genes and Development , 16 ( 8 ), 972 – 983 . doi: 10.1101/gad.976002 OpenUrl Abstract / FREE Full Text 44. ↵ Sulston , J. E. , & Horvitz , H. R . ( 1977 ). Post-Embryonic Cell Lineages of the Nematode, Caenorhabditis elegans . Developmental Biology , 110 – 156 . 45. Sulston , J. E. , Schierenberg, ’ E, White , J. G. , & Thomson , J. N. ( 1983 ). The Embryonic Cell Lineage of the Nematode Caenorhabditis elegans . In Developmental Biology (Vol. 100 ). 46. ↵ Tekieli , T. , Yemini , E. , Nejatbakhsh , A. , Wang , C. , Varol , E. , Fernandez , R. W. , Masoudi , N. , Paninski , L. , & Hobert , O . ( 2021 ). Visualizing the organization and differentiation of the male-specific nervous system of C. elegans . doi: 10.1242/dev.199687 OpenUrl CrossRef PubMed 47. The C. elegans Sequencing Consortium. ( 1998 ). Genome Sequence of the Nematode C. elegans : A Platform for Investigating Biology . 48. ↵ Thellmann , M. , Hatzold , J. , & Conradt , B . ( 2003 ). The Snail-like CES-1 protein of C. elegans can block the expression of the BH3-only cell-death activator gene egl-1 by antagonizing the function of bHLH proteins . Development , 130 ( 17 ), 4057 – 4071 . doi: 10.1242/DEV.00597 OpenUrl Abstract / FREE Full Text 49. ↵ Tintori , S. C. , Osborne Nishimura , E. , Golden , P. , Lieb , J. D. , & Goldstein , B . ( 2016 ). A Transcriptional Lineage of the Early C. elegans Embryo . Developmental Cell , 38 ( 4 ), 430 – 444 . doi: 10.1016/J.DEVCEL.2016.07.025 OpenUrl CrossRef PubMed 50. ↵ Wexler , L. R. , Miller , R. M. , & Portman , D. S . ( 2020 ). C. elegans Males Integrate Food Signals and Biological Sex to Modulate State-Dependent Chemosensation and Behavioral Prioritization . Current Biology , 30 ( 14 ), 2695 – 2706 .e4. doi: 10.1016/J.CUB.2020.05.006 OpenUrl CrossRef PubMed 51. ↵ Zhang , F. , Bhattacharya , A. , Nelson , J. C. , Abe , N. , Gordon , P. , Lloret-Fernandez , C. , Maicas , M. , Flames , N. , Mann , R. S. , Colón-Ramos , D. A. , & Hobert , O . ( 2014 ). The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types . Development , 141 ( 2 ), 422 – 435 . doi: 10.1242/DEV.099721 OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted March 20, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following HLH-3 Is Required for the Maturation of Neurons Necessary for Male Specific Exploration Behavior Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share HLH-3 Is Required for the Maturation of Neurons Necessary for Male Specific Exploration Behavior Kimberly Goodwin , Aixa Alfonso bioRxiv 2025.03.20.644401; doi: https://doi.org/10.1101/2025.03.20.644401 Share This Article: Copy Citation Tools HLH-3 Is Required for the Maturation of Neurons Necessary for Male Specific Exploration Behavior Kimberly Goodwin , Aixa Alfonso bioRxiv 2025.03.20.644401; doi: https://doi.org/10.1101/2025.03.20.644401 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)