Patterning of sexually dimorphic neurogenesis in the Caenorhabditis elegans ventral cord by Hox and TALE homeodomain transcription factors

Authors


Abstract

Background: Reproduction in animals requires development of distinct neurons in each sex. In C. elegans, most ventral cord neurons (VCNs) are present in both sexes, with the exception of six hermaphrodite-specific neurons (VCs) and nine pairs of male-specific neurons (CAs and CPs) that arise from analogous precursor cells. How are the activities of sexual regulators and mediators of neuronal survival, division, and fate coordinated to generate sex-specificity in VCNs? Results: To address this, we have developed a toolkit of VCN markers that allows us to examine sex-specific neurogenesis, asymmetric fates of daughters of a neuroblast division, and regional specification on the anteroposterior axis. Here, we describe the roles of the Hox transcription factors LIN-39 and MAB-5 in promoting survival, differentiation, and regionalization of VCNs. We also find that the TALE class homeodomain proteins CEH-20 and UNC-62 contribute to specification of neurotransmitter fate in males. Furthermore, we identify that VCN sex is determined during the L1 larval stage. Conclusions: These findings, combined with future analyses made possible by the suite of VCN markers described here, will elucidate how Hox-mediated cell fate decisions and sex determination intersect to influence development of neuronal sex differences. Developmental Dynamics 243:159–171, 2014. © 2013 Wiley Periodicals, Inc.

INTRODUCTION

The development of sex-specific neurons is fundamental to reproduction in animals. The nematode Caenorhabditis elegans exemplifies this: the worm's nervous system is extensively specialized to promote the distinct reproductive behaviors of each sex, and when sex-specific neuronal development fails, reproduction is compromised (reviewed in Barr and Garcia, 2006; Portman, 2007; Wolff and Zarkower, 2008). In C. elegans, the male plays a more active role in mating; consistent with this, 89 of the male's 383 neurons are male-specific (Hodgkin, 1988). For example, male-specific head sensory neurons are used for mate searching, and an elaborate network of tail sensory, motor, and interneurons mediate copulation (Portman, 2007; Wolff and Zarkower, 2008). Hermaphrodites, in contrast, have eight sex-specific neurons, all of which regulate egg laying (reviewed in Schafer, 2005). Furthermore, some neurons common to both sexes are functionally specialized to mediate sex-specific behaviors such as response to pheromones (Jang et al., 2012; White and Jorgensen, 2012).

To better understand the genesis of nervous system sexual dimorphism, we have undertaken an analysis of C. elegans ventral cord neurons (VCNs). While most VCNs are present in both sexes, the ventral cord also includes lineally related, but sexually distinct neurons, the hermaphrodite VCs and the male CAs and CPs. These neurons arise from a group of analogous precursor cells, the Pn.aap cells (Fig. 1A) (Sulston and Horvitz, 1977). In hermaphrodites, six Pn.aap cells (P3.aap-P8.aap) differentiate into VC neurons (VC1-VC6), which are cholinergic and peptidergic and innervate vulval muscles to modulate egg laying (White et al., 1986; Schinkmann and Li, 1992; Bany et al., 2003; Duerr et al., 2008). In males, nine Pn.aap cells (P3.aap-P11.aap) divide to produce pairs of CA and CP neurons (CA1-CA9 and CP1-CP9). P2.aap does not divide, but instead differentiates into a single neuron designated CP0. Six of the CP neurons (CPs 1-6) are serotonergic and are required for effective ventral flexure of the tail during mating (Loer and Kenyon, 1993). Accordingly, the male connectome reveals synapses between CPs and musculature used for mating (Jarrell et al., 2012). CPs also synapse extensively with the gonad, and both CAs and CPs connect with copulatory spicule-associated neurons, indicating a possible role in insemination (Jarrell et al., 2012). Consistent with this, a subset of CAs has been shown to be required for the initiation of sperm transfer (Schindelman et al., 2006).

Figure 1.

The ventral nerve cord in C. elegans is sexually dimorphic. A: The Pn.aap precursor cell is born in late L1 (blue dot). In hermaphrodites 6 Pn.aap cells become VC neurons and in males 9 Pn.aap cells divide in late L3 to become CA and CP neurons. VA, VB, VD, and AS are non–sex-specific postembryonic motor neurons. B–I: Hermaphrodites (left); males (right); insets (C–I, bottom) represent expression pattern of reporters. All strains express tph-1::mCherry. B,C: Male-specific expression of tph-1::mCherry in CPs and sexually dimorphic expression of ida-1::gfp in hermaphrodite VCs and male CAs. D,E: flp-22::gfp expression in CPs 1-4. F,G: flp-21::gfp expression in CPs 7-9. H,I: Sexually dimorphic expression of lin-11::gfp(wgIs62) in hermaphrodite VCs and a subset of male CAs and CPs. Scale bars = 100 μm.

Sexual dimorphism in Pn.aap-derived neurons is characterized by sex-specific cell death, cell division, and neurotransmitter expression. These processes are influenced by two Hox transcription factors, with LIN-39 active in the midbody and MAB-5 active in more posterior cells (Costa et al., 1988; Clark et al., 1993; Salser et al., 1993; Wang et al., 1993; Kenyon et al., 1997). It is not yet known how LIN-39 and MAB-5 interact with sex-determining genes to promote sex-specificity in VCNs. To address this, we have assembled a toolkit of markers that allows us to dissect sex-, region-, and lineage-specific differences among VCNs. We have used these markers to reveal regulation of neuronal fate by LIN-39 and MAB-5 and the temporal window for sex determination in VCNs. To identify additional regulators, we have used screens and candidate gene testing. We find that the TALE class Hox cofactors CEH-20/Pbx and UNC-62/Meis play important, but distinct, roles in males and hermaphrodites and that the O/E transcription factor UNC-3 prevents inappropriate serotonergic gene expression in both sexes.

RESULTS

Ventral Cord Neurons are Sexually Distinct and Regionally Specialized

To examine the extent of sexually dimorphic differentiation in the ventral nerve cord, we analyzed expression of reporter transgenes in wild-type hermaphrodite and male Pn.aap descendants (Fig. 1; Table 1). Based on this analysis, these reporters can be organized into two classes. The first class is expressed male-specifically in the ventral cord. tph-1::mCherry, cat-1::gfp, cat-4::gfp, and bas-1::gfp, markers of serotonergic fate, are expressed in CPs 1-6, but not in most hermaphrodite VCs (cat-1::gfp is expressed in VCs 4-5). This expression pattern is consistent with previously described tph-1::gfp reporters and anti-serotonin immunostaining (Loer and Kenyon, 1993; Sze et al., 2000; Clark and Chiu, 2003). Of interest, expression of tph-1::mCherry (and bas-1::gfp) consistently appears more intense in CPs 5-6 than in CPs 1-4. The FMRF-amide-like neuropeptide reporters flp-22::gfp and flp-21::gfp are also specific to subsets of male CPs, with flp-22::gfp expressed in CPs 1-4 (and usually CP 0) and flp-21::gfp in CPs 7-9. The second class of reporters is expressed in both sexes, but in sexually dimorphic patterns. ida-1::gfp is expressed in VC neurons in hermaphrodites and in CAs 1-9 and CP 0 in males (Zahn et al., 2004). Expression of ida-1::gfp appears more intense in CAs 7-9 than in CAs 1-6. A LIM homeodomain translational reporter lin-11fosmid::gfp(wgIs62) is expressed in hermaphrodite VCs and in male CAs 1-4, 7 and CPs 0-9. These analyses establish a toolkit of markers that can be used to differentiate between male and hermaphrodite ventral cord neurons and between CA and CP in males. Additionally, they provide evidence for distinct regulatory modules along the anteroposterior (A-P) axis in males: CA/CPs 1-4, CA/CPs 5-6, and CA/CPs 7-9 each display a characteristic reporter expression profile.

Table 1. Reporter Expression in Sexually Dimorphic VCNs
ReporterGenotypeVCanCA1-9aCP0aCP1-9anComments
  1. a

    Average number of cells ± SEM; N.D. = not determined.

tph-1::mCherryWild-type0109006.0 ± 0.01*103*CP1-6
lin-39(n1760)N.D. 00081 
mab-5(e1239)N.D. 006.0 ± 0.0289 
ced-3(n1286)N.D. 006.0 ± 0.0375 
ced-3(n1286); lin-39(n1760)N.D. 00071 
ida-1::gfpWild-type6 ± 0.021099 ± 0.011 ± 0.020103 
lin-39(n1760)N.D. 5.6 ± 0.10*0.7 ± 0.06081*CA5-9
mab-5(e1239)N.D. 6.6 ± 0.09*1.0 ± 0.02089*CA1-6
ced-3(n1286)N.D. 8.9 ± 0.041 ± 0.00075 
ced-3(n1286); lin-39(n1760)N.D. 9.5 ± 0.24*071*CA1-9, CP0
flp-21::gfpWild-type068002.9 ± 0.03*82*CP7-9
lin-39(n1760)N.D. 005.1 ± 0.08*73*CP5-9
mab-5(e1239)N.D. 000.0 ± 0.0177 
flp-22::gfpWild-type05700.3 ± 0.054 ± 0.01*81*CP1-4
lin-39(n1760)N.D. 000.2 ± 0.0588 
mab-5(e1239)N.D. 00.5 ± 0.056 ± 0.03*94*CP1-6
ced-3(n1286)N.D. 00.6 ± 0.104 ± 0.00*28*CP1-4
ced-3(n1286); lin-39(n1760)N.D. 02.4 ± 0.16*88*CP0-4
lin-11::gfpWild-type5.8 ± 0.06884.8 ± 0.10*0.9 ± 0.038.7 ± 0.08109*CA1-4,7
cat-1::gfpWild-type2.0 ± 0.02*62005.9 ± 0.05111*VC4-5
lin-39(n1760)N.D. 000.0 ± 0.0282 
cat-4::gfpWild-type042005.5 ± 0.0972 
lin-39(n1760)N.D. 000.0 ± 0.01125 
bas-1::gfpWild-type046004.7 ± 0.13164 
lin-39(n1760)N.D. 00067 

Ventral Cord Sexual Dimorphism is Established During the L1 Larval Stage

Somatic sex difference in C. elegans is accomplished by a sex determination cascade in which the X chromosome to autosome (X:A) ratio ultimately controls the activity of the transcription factor TRA-1 (Zarkower, 2006). TRA-1 is active in XX animals, causing them to develop as hermaphrodites, whereas TRA-1 is inactive in XO animals, causing them to develop as males. In both sexes, the Pn.aap cells are generated during the late L1 larval stage, in a series of cell divisions that gives rise to most of the worm's postembryonic motor neurons (Fig. 1A) (Sulston and Horvitz, 1977). Hermaphrodite VCs, arising from differentiation of Pn.aap, are thus born during L1. In contrast, the male-specific division that generates CAs and CPs does not occur until much later, during the L3 larval stage. The temporally distinct “birthdays” of VCs and CA/CPs led us to investigate when the global sex-determining pathway is required for distinct sexual fates in the Pn.aap lineage. It is possible that Pn.aap sex is determined during L1, at the time that Pn.aap is generated. Alternatively, Pn.aap sex could remain plastic until L3, when the CA/CP division occurs in males. To explore these possibilities, we used an allele of tra-2(ar221ts) that causes the activity of the global sex determination pathway to be temperature dependent (Hodgkin, 2002). In tra-2(ar221ts) mutants, XX individuals raised at 25°C are phenotypically male in somatic cells, while those raised at 16°C are phenotypically hermaphrodite.

To determine the temperature sensitive period for Pn.aap sex determination, we first performed a series of “upshift” experiments (Fig. 2). We shifted tph-1::gfp (zdIs13); tra-2(ar221ts) XX worms from the hermaphrodite-promoting temperature (16°C) to the male-promoting temperature (25°C) at hatching and at each larval molt, then scored VCN expression of tph-1::gfp in young adults. Most worms grown at 16°C during embryogenesis, but shifted to 25°C at hatching express tph-1::gfp in five or six VCNs (Fig. 2B). However, those shifted after L1 rarely express tph-1::gfp in more than four VCNs (Fig. 2C). This result suggests that the global sex determination pathway can influence Pn.aap-derived neurons to assume a male-like fate (tph-1::gfp expression) during L1, but not later. Downshift experiments yielded essentially symmetrical results: worms grown at 25°C during embryogenesis, but shifted to 16°C at hatching exhibit hermaphrodite-like fates (no tph-1::gfp expression) (Fig. 2D). Those downshifted after L1 develop male-like VCNs (Fig. 2E). Taken together, these results indicate a critical period for VCN sex determination during L1.

Figure 2.

The temperature sensitive period for VCN sex determination is during L1. A: Percentage of male-like ventral nerve cords (five or greater CPs) for upshifted (16°C → 25°C, blue) or downshifted (25°C → 16°C, red) populations at each larval stage. B–E: Expression of tph-1::gfp(zdIs13) in tra-2(ar221ts) shifted animals. B: Male-like ventral nerve cords in animals upshifted at hatching, as represented by 6 CPs. C: Hermaphrodite-like ventral nerve cords in animals upshifted at L1, as represented by fewer than 5 CPs. D: Hermaphrodite-like ventral nerve cords in animals downshifted at hatching. E: Male-like ventral nerve cords in animals downshifted at L1. Arrowheads: CPs. Scale bars = 100 μm.

lin-39 and mab-5 are Expressed in Overlapping VCN Domains in Both Sexes

The regional specificity of marker expression in the VCNs we examined suggests regulation by Hox transcription factors. Consistent with this, both LIN-39 and MAB-5 have previously been shown to affect survival, division, and fate in VCNs (Clark et al., 1993; Salser et al., 1993). It is possible that sex-specific aspects of VCN neurogenesis could arise from sex-specific expression of lin-39 or mab-5 during the critical window for VCN sex determination. Published analyses of MAB-5 expression, however, do not support this idea: MAB-5 appears to be expressed in all posterior ventral cord neurons in both males and hermaphrodites in early larvae (Salser et al., 1993). Our analyses of a mab-5::gfp(wgIs27) fosmid reporter are consistent with this finding (data not shown). To examine whether lin-39 is sex-specifically expressed in VCNs during this developmental window, we examined expression of a translational lin-39fosmid::gfp reporter (Celniker et al., 2009; Zhong et al., 2010; Sarov et al., 2012). This functional fosmid construct includes 37.5 kb of lin-39 coding and surrounding sequence; this type of construct has been shown to faithfully represent endogenous gene expression. We find that, during late L1, lin-39fosmid::gfp(wgIs18) is expressed in nearly all midbody VCNs in both sexes in a region that includes the precursors to VCs 1-6 (P3-8.aap) and CA/CP pairs 1–6 and CP 0 (P2-8.aap) (Fig. 3). This expression pattern persists through later larval stages and into early adulthood in both sexes. Thus, lin-39, like mab-5, is expressed in VCNs in both sexes. The expression domains of LIN-39 and MAB-5 are consistent with distinct, but overlapping functional domains for LIN-39 and MAB-5 in the ventral cord at key points in VCN neurogenesis (Fig. 3E). Sex-specificity of Hox gene activity likely arises from interactions with additional regulators. In contrast to expression in early larvae, in L4 and adult males, lin-39::gfp expression appears to be up-regulated in male CAs and CPs relative to other VCNs (Fig. 3D), suggesting a later role for lin-39 in these neurons.

Figure 3.

lin-39::gfp is expressed in both sexes. A,C: Hermaphrodites. B,D: Males. A,B: lin-39::gfp expression at L1/L2, showing no obvious difference between sexes. White dotted line: outline of the gonad. Blue brackets: extent of lin-39::gfp expression. Arrowheads: rectum. C,D: Expression of lin-39::gfp at L4, showing prominent expression in CA/CP 1–6 pairs in males. Brackets: CA/CP pairs. E: Diagram of overlapping lin-39 and mab-5 expression in the ventral nerve cord. Scale bars = 50 μm.

LIN-39 and MAB-5 Regionally Influence VCN Fates

We next asked whether Hox gene function is required for regionalized expression of our panel of sexually dimorphic VCN markers (Figs. 1, 4; Table 1). The lin-39(n1760) null mutation affects cell fates in lin-39's expression domain in both sexes. Expression of ida-1::gfp is absent in lin-39(n1760) hermaphrodite VCs (data not shown, see Fig. 5). In males, neither tph-1::mCherry nor other markers of serotonergic fate (cat-1::gfp, cat-4::gfp, and bas-1::gfp) are expressed in the lin-39(n1760) ventral cord. Similarly flp-22::gfp expression is absent in lin-39(n1760) CPs. In contrast, ida-1::gfp expression is lost in CAs 1-4 but remains in CAs 5-9 with CAs 5 and 6 now resembling CAs 7-9 in their higher-intensity ida-1::gfp expression. Consistent with an apparent transformation of the fates of CAs 5 and 6 to that of CAs 7-9, flp-21::gfp expression expands to include CPs 5-6 in lin-39(n1760) mutants. Similarly, the mab-5(e1239) mutation affects cell fates in mab-5's expression domain. flp-21::gfp, normally expressed in CPs 7-9, is not expressed in the mab-5(e1239) ventral cord. ida-1::gfp expression is also lost in CAs 7-9, but remains in CAs 1-6 and CP 0. The normal tph-1::mCherry expression domain is retained, but with a reduction in intensity of expression in CPs 5-6. Consistent with an apparent transformation of CPs 5-6 fates to that of the more anterior CPs 1-4, the domain of flp-22::gfp expression is expanded to include CPs 5-6. Thus, we find that in domains unique to each Hox gene (P3-P6.aap for lin-39 and P9-P11.aap for mab-5), expression of characteristic differentiation markers is lost. The situation is more complex in the region of lin-39/mab-5 overlap (P7-P8.aap), with apparent fate transformations seeming to indicate reciprocal interactions between these two genes. Together, these results are consistent with the possibility of a requirement for Hox genes in cell survival and/or differentiation in male Pn.aap lineages.

Figure 4.

LIN-39 and MAB-5 define regions of sex-specific VCN identity. A,C,E: lin-39(n1760) mutant males, with (A) lack of tph-1::mCherry and anterior ida-1::gfp expression, (C) lack of flp-22::gfp expression, and (E) anterior expansion of flp-21::gfp expression. B,D,F: mab-5(e1239) mutant males, with (B) lack of ida-1::gfp expression in posterior CAs, (D) posterior expansion of flp-22::gfp, and (F) lack of flp-21::gfp expression. Insets represent expression pattern of reporters. G: Wild-type expression of ida-1::gfp and tph-1::mCherry in ced-3(n1286). H: VCN reporter expression in ced-3(n1286) lin-39(n1760), showing relatively wild-type ida-1::gfp and lack of tph-1::mCherry expression. For wild-type comparisons, see Figure 1. Scale bars = 100 μm.

Figure 5.

TALE class homeodomain transcription factors CEH-20 and UNC-62 are required for serotonergic fate in males. A–H: Expression of ida-1::gfp and tph-1::mCherry in RNAi-treated hermaphrodites (A,C,E,G) and males (B,D,F,H). Insets represent expression pattern of reporters. A,B: RNAi negative control (L4440 empty vector), showing wild-type expression patterns. C,D: Expression in lin-39(RNAi) are similar to those in lin-39(n1760) (see Fig. 4A). E: ceh-20(RNAi), showing a reduced number of ida-1::gfp(+) cells. F: ceh-20(RNAi), showing a reduced the number of tph-1::mCherry(+) cells but not ida-1::gfp(+) cells. G,H: unc-62(RNAi) produces similar results to ceh-20(RNAi). I: Summary of effects of TALE-HD RNAi on number of ida-1::gfp(+) and tph-1::mCherry(+), and flp-22::gfp(+) cells in the ventral cord. Error bars represent SEM; number of ventral cords scored is indicated above error bars. Scale bars = 100 μm.

lin-39 Influences VCN Survival and Gene Expression

lin-39 is required for survival of a subset of Pn.aap cells in both sexes (Clark et al., 1993; Salser et al., 1993). In lin-39(n1760) hermaphrodites, P3-P8.aap undergo programmed cell death, never differentiating as VCs. In lin-39(n1760) males, most P3-P6.aap die, but P7-P8.aap survive. These surviving Pn.aap cells divide normally into CA/CPs 5-6, but the CP neurons fail to express serotonin (Clark et al., 1993; Salser et al., 1993). Therefore, wild-type lin-39 is necessary to block an inappropriate cell fate, programmed cell death, and promote expression of serotonin. Expression of reporters normally seen in CA/CPs 1-4 is absent, of course, when P3-P6.aap die in lin-39 mutants because CA/CPs 1-4 are never generated. To see whether lin-39 function is necessary for expression of reporters in CA/CPs 1-4, we blocked programmed cell death with a ced-3 mutation: in lin-39(n1760); ced-3(n1286) double mutants, all P3-P8.aap cells survive. In these mutants, all CP neurons (CP1-CP6) lack serotonin immunoreactivity (data not shown) and fail to express tph-1::mCherry (Fig. 4; Table 1). This is consistent with previous findings demonstrating a requirement for lin-39 in serotonin production (Clark et al., 1993; Loer and Kenyon, 1993; Hunter and Kenyon, 1995). Further evidence that loss of serotonergic reporter expression cannot be explained by cell death alone comes from loss of expression of multiple serotonergic reporters (tph-1::mCherry, cat-1::gfp, cat-4::gfp, and bas-1::gfp) in CPs 5-6, which survive in lin-39(n1760) mutants (Table 1). In contrast, lin-39(n1760); ced-3(n1286) males retain some expression of the peptidergic reporter flp-22::gfp in CPs 1-4 (Table 1), suggesting that lin-39 is not absolutely required for flp-22::gfp expression. lin-39 is not required for expression of ida-1::gfp in either sex, as ida-1::gfp is expressed at nearly wild-type levels in VCs and CAs in lin-39(n1760); ced-3(n1286) double mutants (Fig. 4G,H; Table 1, and data not shown).

Hox Cofactors are Required for Sex-specific VCN Development

lin-39 acts in both sexes to promote survival of Pn.aap and its descendants, but acts male-specifically to promote the CA/CP cell divisions and expression of serotonin in CPs 1-6 (Clark et al., 1993; Salser et al., 1993). To identify additional regulators that act with lin-39 to promote expression of serotonergic genes in males, we performed a targeted RNAi screen, assaying post-embryonic loss of function of a library of 387 C. elegans genes known or predicted to encode transcription factors. We screened a neuronally RNAi-sensitive strain, tph-1::gfp (mgIs42); nre-1(hd20) lin15b(hd126) for changes in tph-1::gfp expression in VCNs. This screen identified lin-39 as well as two other significant regulators of tph-1::gfp expression, ceh-20 and unc-62. CEH-20 and UNC-62 belong to the TALE class of homeodomain (TALE-HD) proteins, a family of Hox cofactors (Liu and Fire, 2000; Van Auken et al., 2002; reviewed in Mann et al., 2009). While both ceh-20 and unc-62 are known to act with lin-39 to promote survival of P3-P8.aap in hermaphrodites (Liu et al., 2006; Potts et al., 2009), their function in male Pn.aap has not been described.

We, therefore, assessed the roles of unc-62 and ceh-20, as well as the three other C. elegans TALE-HD encoding genes, ceh-40, ceh-60, and psa-3, in differentiation of sex-specific VCNs. To do so, we targeted these genes post-embryonically by RNAi in neuronally sensitive strains expressing VCN reporters. Treatment with unc-62(RNAi) or ceh-20(RNAi) results in reduction of ida-1::gfp(+) VCNs in hermaphrodites and tph-1::mCherry(+) and flp-22::gfp(+) VCNs in males (Fig. 5). These phenotypes are similar, but less penetrant than those produced by lin-39(RNAi). The effect on ida-1::gfp in males is different: whereas lin-39(RNAi) phenocopies the lin-39(n1760) mutant, leading to loss of ida-1::gfp in CAs 1-4 (presumably due to cell death in P3-P6.aap), unc-62(RNAi) and ceh-20(RNAi) retain bright expression of ida-1::gfp in four anterior VCNs (likely CAs1-4). Moreover, a small number of ida-1::gfp(+) VCNs were observed in pairs, suggesting that, in the absence of unc-62 or ceh-20 function, normally asymmetric Pn.aap divisions were disrupted, transforming some posterior daughters into “CA-like” neurons.

To address possible redundancy of unc-62 and ceh-20 with respect to ventral cord phenotypes, we targeted both genes simultaneously by RNAi. While this double-RNAi enhanced larval arrest (data not shown), those males that reached adulthood expressed tph-1::gfp and ida-1::gfp at levels similar to those seen in experiments targeting either ceh-20(RNAi) or unc-62(RNAi) alone. One interpretation of these data may be that, in males, unc-62 and ceh-20 are required with lin-39 to promote CP-specific fate, but are not required for Pn.aap survival, ida-1::gfp expression, or cell division. RNAi targeting other TALE-HD genes (ceh-40, ceh-60, or psa-3) has no effect on ventral cord neuron reporter expression in our assays.

Males with a ceh-20(ay9) hypomorphic mutation (null alleles are embryonic lethal) also show altered expression of a tph-1 reporter, with 52% of males showing a reduction in tph-1::cfp reporter expression (n = 147). Surprisingly, these experiments also yielded 29% of males with additional tph-1::cfp(+) VCNs, perhaps suggesting complex dosage-dependent effects of ceh-20 on VCN fate. However, these data are hard to interpret given that the level of CEH-20 activity in these mutants is not known.

unc-3 Prevents Inappropriate Serotonergic Specification in VCNs

Genetic screens for mutations that alter tph-1::gfp expression in VCNs in our lab identified mutations that mapped to a region of the X chromosome that contains unc-3, which encodes a homolog of mouse Olf-1/Early B cell factor (O/E) genes (Prasad et al., 1998). Previous studies of unc-3(e151) hermaphrodites have identified ectopic VCs resulting from inappropriate transformation of the Pn.aaa cell to the fate of its posterior sister, Pn.aap (Prasad et al., 2008). We find that expression of a tph-1::gfp reporter in unc-3(e151) mutants is increased in the ventral cord in both sexes (Fig. 6). In unc-3(e151) mutants, 28% of males (n = 56) have 7-9 tph-1::gfp(+) VCNs. Other markers of serotonergic fate are also misregulated in unc-3(e151) males, showing an increase in the number of VCNs that express bas-1::gfp, cat-1::gfp, cat-4::gfp, and serotonin immunoreactivity (data not shown). Surprisingly, the inappropriate serotonergic specification appears to extend across sex boundaries: 40% (n = 154) of hermaphrodites have 1-3 tph-1::gfp(+) VCNs that express the normally male-specific tph-1::gfp.

Figure 6.

unc-3 prevents ectopic serotonergic VCNs in both sexes. A,C,E: Hermaphrodites. B,D,F: Males. A,B: Wild-type expression of tph-1::gfp. C: Ectopic tph-1::gfp(+) cells in VCNs in unc-3 hermaphrodites. D: Supernumerary tph-1::gfp(+) VCNs in unc-3 males. E,F: Ectopic tph-1::gfp(+) cells in both sexes are dependent on lin-39. Arrowheads: serotonergic VCNs. Asterisks: hermaphrodite-specific neurons (HSNs). Scale bars = 100 μm.

unc-3 is thus required to prevent Pn.aap fate outside the Pn.aap lineage. Consistent with this, an unc-3::mCherry reporter is not expressed in hermaphrodite VC neurons or in male CAs or CPs (Kratsios et al., 2012, and our unpublished observations). We wondered if lin-39 is required for serotonergic marker expression outside of Pn.aap, and find that to be the case: lin-39(n1760) fully suppresses the ectopic tph-1::gfp expression in the ventral cord in unc-3(e151) mutants of both sexes (hermaphrodites, n = 102, males n = 127). This requirement for lin-39 to promote tph-1::gfp expression in cells other than Pn.aap is consistent with broad expression of lin-39::gfp in midbody ventral cord neurons (Fig. 3).

DISCUSSION

Sexual differentiation in C. elegans ventral cord neurons requires the interplay of sex-determining genes and genes that regulate cell fate decisions such as survival, proliferation, and neurotransmitter profile. It has been understood for some time that the Hox genes mab-5 and lin-39 and the sex determination cascade culminating in TRA-1 must somehow interact to distinguish VCs in hermaphrodites from CAs and CPs in males. This study begins to fill in additional details of sex-specific VCN development. Specifically, our analysis has used a toolkit of VCN reporters that allows us to assess sex-specific neurogenesis, asymmetric fates of the daughters of a neuroblast division, and regional specification on the A-P axis. We further identify new roles for the Hox cofactors ceh-20 and unc-62 and the O/E transcription factor unc-3 in development of sexually dimorphic VCNs.

Three events, each influenced by Hox transcription factors, contribute to sexually dimorphic fates in VCNs (Fig. 7): 1) A different subset of Pn.aap cells survive in each sex. 2) In males, Pn.aap divide to produce two daughter cells. 3) These daughters of Pn.aap differentiate asymmetrically with distinct male-specific neuronal fates.

Figure 7.

Model for sex-specific development of ventral cord neurons. A: Sex-specific VCN development is regulated by the Hox transcription factors LIN-39 and MAB-5, which are expressed in overlapping domains in both sexes. These domains define regions of cell fates in the ventral cord (delineated by vertical dashed lines), each marked by a characteristic pattern of reporter expression (gray boxes). Bold vertical lines demarcate boundaries of reporter expression that correspond to Hox expression boundaries. The Pn.aap lineage is sexually dimorphic, with different patterns of survival, division, and differentiation in each sex. Cells in the Pn.aap lineage are indicated as circles, with X indicating programmed cell death. Functional requirements for LIN-39, MAB-5, and the TALE class homeodomain proteins CEH-20 and UNC-62 are indicated by colored shading. B: Effect of lin-39 and mab-5 loss-of-function on VCN fates. MAB-5 and LIN-39 reciprocally control fates in the boxed region, with these cells assuming more posterior fates in lin-39(-) and more anterior fates in mab-5(-).

Survival of Pn.aap

In hermaphrodites, P3-P8.aap cells survive and differentiate into the six VC neurons. This survival requires lin-39, unc-62, and ceh-20, which prevent transcription of the pro-apoptotic BH3 domain gene egl-1 (Clark et al., 1993; Conradt and Horvitz, 1998; Liu et al., 2006; Potts et al., 2009). Loss of function of lin-39, ceh-20, or unc-62 leads to egl-1-dependent cell death of P3-P8.aap and thus lack of VC neurons. In wild-type hermaphrodites, P2.aap and P9-P12.aap, lacking lin-39, undergo programmed cell death (Sulston and Horvitz, 1977). In males, P2.aap-P11.aap survive to produce CAs and CPs. This survival requires lin-39 and mab-5 in their expression domains, with P2-P6.aap undergoing apoptosis in lin-39 mutants, and P2-P11.aap undergoing apoptosis in lin-39 mab-5 double mutants. The pro-survival role of mab-5 in posterior cells is not entirely clear, however, as P9-P11.aap survive in mab-5 single mutants, additionally requiring a loss of lin-39 for apoptosis (Kenyon, 1986). If Pn.aap survival in males were analogous to Pn.aap survival in hermaphrodites, one might predict that ceh-20 and unc-62 would be required. Instead we find that knockdown of ceh-20 and unc-62 by RNAi does not affect survival and ida-1::gfp expression in Pn.aap in males (Fig. 5). Thus, one interpretation of our results is that regulation of Pn.aap survival is sexually dimorphic, with ceh-20 and unc-62 required to promote survival in hermaphrodites, and other (or no) Hox cofactors required in males. Further studies will be aimed at identifying potential male-specific regulators of Pn.aap cell fate.

Division and Differentiation

In males, the generation of CAs and CPs requires that Pn.aap cells undergo an additional round of cell division, with each daughter assuming distinct neuronal traits. Previous studies have demonstrated that division requires either lin-39 or mab-5, with lin-39 further required for expression of serotonin by the posterior daughter of P3-P8.aap (Clark et al., 1993). Consistent with this, we find that expression of four markers of serotonergic fate (tph-1::mCherry, cat-1::gfp, cat-4::gfp, bas-1::gfp) is absent in lin-39(n1760) mutants. While mab-5 can compensate for lin-39 in mediating cell division, it cannot compensate at this serotonergic specification step (Clark et al., 1993). Our results indicate that unc-62 and ceh-20 are required with lin-39 to promote serotonergic fate of CPs 1-6 as well as flp-22::gfp expression in CPs 1-4. In contrast, lin-39, ceh-20, and unc-62 appear not to be required for at least one aspect of CA fate, ida-1::gfp expression. Significantly, ida-1::gfp expression is a trait shared by hermaphrodite VCs and male CAs, suggesting that perhaps the non sex-specific “default” fate specification of Pn.aap includes ida-1 expression, with sex-specificity imposed separately. Persistence of ida-1::gfp expression in the lin-39; ced-3 double mutant further supports this idea.

unc-3 appears to act at a non-sex-specific step as well, acting in both sexes to prevent Pn.aaa, which generates neurons that control forward and backward movement (the VA and VB motor neurons, see Fig. 1), from assuming Pn.aap fates (Prasad et al., 2008; Kratsios et al., 2012). We find that lin-39 is required for serotonergic fate even outside Pn.aap descendants, suggesting that lin-39 may confer serotonergic competence on all Pn.aa-derived cells, with this competence limited by other regulators of cell fate such as unc-3.

We find that the sex determination pathway is necessary during L1 to promote serotonergic fate. In contrast, previous studies using a lin-39 heat shock transgene suggest that promotion of serotonergic fate by LIN-39 can occur as late as the L4 larval stage (Hunter and Kenyon, 1995). We observe strong expression of lin-39::gfp in CAs and CPs during L4, a time that coincides with the onset of serotonin immunoreactivity and of reporters for serotonin synthesis genes such as tph-1 and bas-1 in CPs 1-6 in late L4 and adult males (Sze et al., 2000; Hare and Loer, 2004). These findings are consistent with serotonergic specification by lin-39 occurring after sex determination in the Pn.aap lineage.

Beyond the role of lin-39, it is not yet clear what makes CAs and CPs different from one another. One candidate for this role is the Wnt pathway, as most asymmetric divisions, including many asymmetric neuronal divisions, deploy a Wnt pathway culminating in asymmetric localization of POP-1/TCF (Bertrand and Hobert, 2009; Phillips and Kimble, 2009; Hobert, 2010). Notably, sex-specific divisions of neuronal precursors in the C. elegans male tail involve interactions between Wnt pathway genes and Hox genes (Hunter et al., 1999; Maloof et al., 1999).

Regional Specification

Although C. elegans is not a segmented animal, the segmental structure of the ventral nerve cord suggests an underlying control of regional fates along the A-P axis by Hox genes (Minelli and Fusco, 2004). Despite the iterative pattern of motor neuron descendants of P3-P11.a, overt regionalization along the A-P axis by Hox genes is only apparent in the sex-specific descendants of the Pn.aap lineage (Fig. 7). This regionalization differs in the two sexes. In hermaphrodites, survival of VCs and death of distal Pn.aap cells reveal a region defined by LIN-39 as “midbody.” In males, regionalization is more complex. The overlapping functions of LIN-39 and MAB-5 are such that differentiation of P3-P6.aap (CA/CPs 1-4), P7-P8.aap (CA/CPs 5-6), and P9-P11.aap (CA/CPs 7-9) are controlled separately, resulting in three distinct regions, each with its characteristic program of Pn.aap fate. CPs 1-4 express flp-22::gfp and relatively less intense tph-1::mCherry, and CA/CPs 1-4 express lin-11::gfp. CPs 5-6 intensely express tph-1::mCherry and express lin-11::gfp. CAs 7-9 express high levels of ida-1::gfp and CPs 7-9 express flp-21::gfp, and lin-11::gfp.

From this and previous work, it is clear that in males lin-39 controls the fates of P3-P6.aap and mab-5 the fates of P9-P11.aap (Kenyon, 1986; Costa et al., 1988; Clark et al., 1993; Salser et al., 1993; Wang et al., 1993). Of interest, we find P7-P8.aap appear to be reciprocally controlled by lin-39 and mab-5 (Fig. 7b). In lin-39 mutants, CA/CPs 5-6 resemble CA/CPs 7-9, whereas in mab-5 mutants CA/CPs 5-6 resemble CA/CPs 1-4. Thus, the loss of lin-39 shifts the Pn.aap fates to more posterior ones and loss of mab-5 to more anterior ones. This is reminiscent of the control of cell fusion state in Pn.p cells, where P7-P8.p normally fuse to hyp7 while P9-P11.p remain unfused. In lin-39 mutants P7-P8.p inappropriately remain unfused and take on posterior fates, whereas in mab-5 mutants they also remain unfused but take on anterior fates (Clark et al., 1993). Our observations of P7-P8.aap are similar, suggesting that Hox combinatorial regulation acts both in anterior and posterior branches of the P cell lineage.

While regionalization is obvious only in Pn.aap-derived VCNs, this does not rule out the possibility that other VCNs are subtly regionally specialized, and that this regionalization is Hox-mediated. For example, posterior core motor neurons, such as those of the VD and AS classes, synapse with male-specific neurons (Jarrell et al., 2012). It is reasonable to speculate that regional specificity of these sexually dimorphic synapses might be Hox-mediated. lin-39 and mab-5 are good candidates for such a role, given their broad expression and function in the P lineages.

The Sex Determination Overlay

Differences between the sexes are apparent at virtually every step of Pn.aap development. At what point do global sexual regulators exert their influence in the ventral cord? Our temperature shift experiments demonstrate that the sex determination pathway is important during the L1 larval stage, a time that corresponds to the birth of the Pn.aap cell. Although this does not rule out an ongoing role for sexual regulation, our data suggest that sexual specification of VCNs is not reversed by altering sex determination pathway activity after the L1 molt. Thus, the fate of Pn.aap, which does not divide until the L3 larval stage in males, appears to be determined despite this latent period. Furthermore, activity of the sex determination pathway during embryogenesis does not affect VCN sexual differentiation.

TRA-1 has been shown to act as a transcriptional repressor to bring about sexual differentiation in diverse tissues. For example, in hermaphrodites, TRA-1 ensures survival of hermaphrodite-specific neurons (HSNs) by repressing transcription of egl-1 and ensures death of male-specific neurons (CEMs) by repressing the anti-apoptotic gene ceh-30 (Conradt and Horvitz, 1999; Peden et al., 2007; Schwartz and Horvitz, 2007). Outside of the nervous system, TRA-1 interacts with cell cycle regulators to mediate sex-specific asymmetric division in the gonad and has been reported to regulate fusion of vulval precursor cells by influencing expression of lin-39 (Tilmann and Kimble, 2005; Szabó et al., 2009). It is, therefore, likely that TRA-1's role in ventral cord neuron specification involves the repression of a male-specific program of development in the hermaphrodite ventral nerve cord. Given that the Hox genes are expressed and functional in the ventral cord in both sexes, we favor a model in which Hox transcription factors and TRA-1 (directly or indirectly) regulate a common set of target genes to bring about sex-specific differentiation of ventral cord neurons. Likely candidates include genes that regulate apoptosis, the cell cycle, or terminal fates such as neurotransmitter phenotype, axonal trajectory, or synaptic partners.

The suite of reagents described here are a powerful tool, as they allow the visualization of sex, region, and daughter-specific differences in ventral cord lineages. Aspects of VCN fate specification identified here, combined with future analyses, will shed light on the interactions of Hox transcription factors and sexual regulators that govern fates of sexually dimorphic neurons.

EXPERIMENTAL PROCEDURES

Worm Culture, Alleles, and Transgenes

Nematodes were cultured as described (Stiernagle, 2006). All strains, except the strain containing tra-2, contained a him-5 or him-8 high incidence of males mutation. The following alleles and transgenes were used:

Mutant alleles

LGII: tra-2(ar221); LGIII: lin-39(n1760), mab-5(e1239), ceh-20(ay9); LGIV: ced-3(n1286), him-8(e1489); LGV: him-5(e1490); LGX: lin-15B(hd126), nre-1(hd20), unc-3(e151).

Integrated transgenes

LGII: ida-1::gfp(inIs179) (Zahn et al., 2004); LGIII: lin-39fosmid::gfp(wgIs18) (Zhong et al., 2010; Sarov et al., 2012), cat-1::GFP(otIs221) (Flames and Hobert, 2009); LGIV: tph-1::gfp(zdIs13) (Clark and Chiu, 2003), flp-22::gfp(ynIs50) (Kim and Li, 2004); LGV: tph-1::mCherry(cccIs1), tph-1::cfp(bxIs16) (Yang et al., 2007), bas-1::GFP(otIs226) (Flames and Hobert, 2009); LG unknown: flp-21::gfp(ynIs80), lin-11fosmid::gfp(wgIs62) (Zhong et al., 2010; Sarov et al., 2012), cat-4::gfp(otIs225).

cccIs1 was made using a tph-1prom::mCherry transcriptional fusion (Pocock and Hobert, 2010). The construct was injected into N2 worms and integrated using a Stratagene UV Stratalinker 1800 at a setting of 300 microjoules × 100.

Temperature Shift Assays

tra-2(ar221ts); zdIs13(tph-1::gfp) worms were grown to gravid adults at 16°C and treated with a hypochlorite solution to release eggs (Stiernagle, 2006). Eggs were dropped onto NGM plates with no bacteria and allowed to hatch at the appropriate temperature to synchronize the population. These synchronized L1 hatchlings were washed onto NGM plates seeded with E. coli OP50-1. For upshift experiments, eggs were allowed to hatch at 16°C and then shifted to 25°C when moved to food (“hatch”), L1/L2 molt, L2/L3 molt, L3/L4 molt, or L4/adult molt. They were then allowed to grow into young adults. Developmental staging was verified by viewing a sample of worms under Nomarksi optics at 400x, using gonadal migration as a reference (Kimble and Hirsh, 1979). For downshift experiments, eggs were hatched at 25°C and shifted to 16°C at each of the above stages, and allowed to grow into young adults. Young adults were scored for the number of tph-1::gfp(+) cells in the ventral nerve cord. Nerve cords were scored as “male-like” if there were five or more tph-1::gfp(+) cells in an individual and “hermaphrodite-like” if there were four or fewer tph-1::gfp(+) cells.

RNAi

The lin-39, ceh-20, unc-62, and ceh-40 RNAi constructs were isolated from the C. elegans transcription factor RNAi library (Source Bioscience), a subset of the RNAi library from (Kamath et al., 2003). Clones were verified by sequencing. To generate ceh-60 and psa-3 RNAi constructs, polymerase chain reaction (PCR) fragments were generated using primers from (Kamath et al., 2003) with overhanging KpnI sites. PCR fragments were digested with KpnI and ligated into the RNAi feeding vector L4440. Ligated plasmids were transformed into E. coli HT115 and verified by sequencing.

Bacterial RNAi clones were grown overnight in 100 μg/ml ampicillin and 50 μl of culture was seeded onto NGM plates containing 25 μg/ml carbenicillin and 1 mM IPTG. Strains containing tph-1::mCherry; ida-1::gfp; him-8; nre-1 lin-15b or flp-22::gfp; him-8; nre-1 lin-15b were grown to gravidity and treated with hypochlorite solution to release eggs. Eggs were dropped onto seeded plates and allowed to grow to young adulthood at 20°C or 22°C. Controls for each RNAi experiment included lin-39(RNAi) and L4440 empty vector. Plates for double RNAi experiments were seeded with 50 μl of a mixture of overnight cultures of ceh-20 and unc-62 RNAi clones.

Genetic Screen

tph-1::gfp(zdIs13); him-8 hermaphrodites were mutagenized with EMS as previously described (Sulston and Hodgkin, 1988). F2 male progeny of individual self-fertilized F1 hermaphrodites were scored on a fluorescence stereo dissecting microscope for changes in tph-1::gfp expression. Homozygous mutant lines were established by propagating hermaphrodite siblings of affected males. Mutations of interest were mapped to chromosomal region by linkage to previously described SNPs (Wicks et al., 2001; Davis et al., 2005).

ACKNOWLEDGMENTS

We thank Oliver Hobert, Paschalis Kratsios, and Roger Pocock for their generous sharing of strains and reagents and the following students for their contributions: Brittany Ganser, Ryan Kast, Sonya Krishnan, Joel Martin, Rachel Stephenson, Maria Sterrett, and current and former members of the Wolff lab. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We also thank Shawn Galdeen and Sabrice Guerrier for their careful reading of this manuscript and David Zarkower and Mary Kroetz for helpful discussions. J.R.W. was funded by NSF Research at Undergraduate Institutions (RUI) and C.M.L. received an endowment from the Fletcher Jones Foundation.

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