
==== Front
bioRxiv
BIORXIV
bioRxiv
Cold Spring Harbor Laboratory

38766005
10.1101/2024.05.08.593207
preprint
1
Article
Experience-dependent, sexually dimorphic synaptic connectivity defined by sex-specific cadherin expression
w Chien-Po 1*
Majeed Maryam 12
http://orcid.org/0000-0002-7634-2854
Hobert Oliver 1*
1 Department of Biological Sciences, Columbia University, Howard Hughes Medical Institute, New York, NY 10027, USA
2 Present address: Allen Institute for Brain Science, Seattle, USA
* Correspondence: cl4102@columbia.edu and or38@columbia.edu
08 5 2024
2024.05.08.593207https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
nihpp-2024.05.08.593207.pdf
We describe here the molecular mechanisms by which juvenile experience defines patterns of sexually dimorphic synaptic connectivity in the adult nervous system of the nematode C. elegans. We show that starvation of juvenile males disrupts serotonin-dependent activation of the CREB transcription factor in a nociceptive sensory neuron, PHB. CREB acts through a cascade of transcription factors to control expression of an atypical cadherin protein, FMI-1/Flamingo. During postembryonic development, FMI-1/Flamingo has the capacity to promote and maintain synaptic connectivity of the PHB nociceptive sensory to a command interneuron, AVA, in both sexes, but the serotonin transcriptional regulatory cassette antagonizes FMI-1/Flamingo expression in males, thereby establishing sexually dimorphic connectivity between PHB and AVA. A critical regulatory node in this process is the CREB-target LIN-29, a Zn finger transcription factor which integrates four different layers of information – sexual specificity, past feeding status, time and cell-type specificity. Our findings provide the mechanistic details of how an early juvenile experience defines sexually dimorphic synaptic connectivity.
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pmcINTRODUCTION

Early life experiences, such as stress and depression, can affect later developmental processes and can also impact on the manifestation of neurological disorders (Lupien et al. 2009; Herringa et al. 2013). Notably, these past experiences can generate outcomes in a sex-dependent manner. For example, clinical studies indicate that females have a higher chance of developing anxiety or depression when experiencing early-life adversity (Hiscox et al. 2023). However, how past experiences influence later aspects of nervous system function in a sex-dependent manner remains elusive in humans. In rodents, a recent study has shown that juvenile adversity alters corticolimbic connectivity in traumatized female rat but not the male counterpart, and consequently, female rats are more likely to display depression behavior (Honeycutt et al. 2020). Here again, the molecular mechanisms that drive such sexually dimorphic outcomes are unknown.

Using the nematode C. elegans as a model, we have previously shown that the experience of early juvenile starvation affects the establishment of sexually dimorphic synaptic connectivity during sexual maturation (Bayer and Hobert 2018). Specifically, the phasmid sensory neuron PHB generates en passant synapses onto the command interneuron AVA in both sexes at early juvenile stages; however, upon sexual maturation this synaptic connection is sex-specifically maintained and progressively strengthened in hermaphrodites but eliminated in males (White et al. 1986; Jarrell et al. 2012; Oren-Suissa et al. 2016; Bayer and Hobert 2018; Cook et al. 2019). Early starvation increases the expression of the invertebrate norepinephrine analog octopamine to inhibit serotonin release from a pair of head sensory neurons (Bayer and Hobert 2018). This transient serotonin depletion and consequent lack of activation of the metabotropic SER-4 receptor in PHB, results in the failure of the PHB>AVA synaptic pruning and instead promotes growth of PHB>AVA synaptic connectivity in males, thereby eliminating sexually dimorphic connectivity (Bayer and Hobert 2018).

Serotonin-mediated activation of metabotropic receptors can affect a wide range of signaling pathways (Bockaert et al. 2006). We show here that the relevant read-out in sex-specific serotonin-mediated synaptic pruning is a multilayered transcriptional response, in which serotonin signaling first activates the CREB transcription factors. CREB then directly activates the Zn finger transcription factor LIN-29A, a key regulatory node in this process. Transcription of the lin-29a locus bookmarks feeding status via CREB activation, which cooperates with a cell-type specific terminal selector to direct lin-29 expression to specific neuron types. Activation of lin-29a transcription is antagonized in hermaphrodites by the TRA-1 master regulatory of sexual identity. lin-29a transcripts are translationally inhibited by the LIN-41 RNA binding protein until sexual maturation. Once LIN-29A protein is produced at the right place and time, it directs sexually dimorphic PHB>AVA connectivity by repression of another transcription factor, the Doublesex transcription factor, DMD-4, which we had previously found to be expressed in PHB of hermaphrodites, but not males (Bayer et al. 2020a). We identify the non-conventional cadherin fmi-1/Flamingo as the terminal effector of the CREB>LIN-29A>DMD-4 transcription factor cascade. We show that FMI-1 is normally required in hermaphrodites to promote the increase in the number of PHB>AVA en passant synapses. Male-specific repression of FMI-1 via serotonin/CREB-mediated LIN-29A induction (and DMD-4 repression) therefore leads to a failure to sustain and expand PHB>AVA synapse number. Hence, we have discovered a mechanism whereby food perception in early life is translated into the control of the later development of a sexually dimorphic synaptic connectivity.

RESULTS

Visualization of sexually dimorphic PHB>AVA connectivity and neurite contact length and its differential dependence on past experience

Juvenile starvation (starving animals at the L1 stage for 24 hours and transferring them back to food) affects sexually dimorphic PHB>AVA synaptic number decrease at the later sexual maturation stage via temporally mediating serotonin (5-HT) signaling in the sensory neuron PHB (Bayer and Hobert 2018). The molecular pathway underlying such experience-dependent, sex-specific synaptogenesis remains unknown. We had previously visualized the effect of feeding state and 5-HT signaling on PHB>AVA synaptic connectivity through the use of “GFP-reconstitution across synaptic partner” (GRASP) technology which exploits the synaptically localized neuroligin protein NLG-1 (Feinberg et al. 2008; Bayer and Hobert 2018). We validated and expanded our previous results through the establishment and usage of additional reagents. First, an independently generated PHB>AVA GRASP reporter transgene, otIs839 confirmed our previous results in experience-dependent sexual dimorphic connectivity of PHB and AVA (Bayer and Hobert 2018)(Figure 1A,B). Second, we confirmed that NLG-1-based GRASP is indeed a proper indicator of PHB>AVA synaptic connectivity by using a GFP-tagged synaptic active zone marker, CLA-1/Clarinet, expressed specifically in PHB, as well as a postsynaptic marker, the ionotropic glutamate receptor AVR-14 which is expressed in the AVA neurons, the postsynaptic target of the glutamatergic PHB neurons (Gat et al. 2023; Li et al. 2023). While GFP::CLA-1 alone labels all synaptic outputs of PHB, including those to many male-specific neurons, adjacent localization of PHB-expressed GFP::CLA-1 and AVA-expressed AVR-14::TagRFP signals represents an indicator of PHB>AVA connectivity (Figure 1C-F). We found that either in isolation or in combination, the number of GFP::CLA-1 and AVR-14::TagRFP signals corroborate the conclusions based on the GRASP constructs: The number of en passant PHB>AVA synaptic signals show (a) no dimorphisms in juvenile stages, (b) display dimorphisms in after sexual maturation and (c) starvation results in aberrant en passant synapse number increase in males (Figure 1E-F, Figure S1A,B).

Our previous global analysis of neurite adjacency and en passant synapse formation throughout the C. elegans nervous system indicates that the extent of adjacency of two neurites is a sufficient predictor of the number of synapses formed between adjacent neurons (Cook et al. 2023). Since the PHB>AVA synapses are generated en passant along the PHB and AVA neurites, we considered the possibility that the extent of neurite adjacency is also sexually dimorphic and regulated by juvenile experience. We visualized PHB/AVA neurite contact length by using the transmembrane CD4 protein to direct the two halves of GFP (GFP1-10 and GFP11) to the surface of the PHB and AVA membranes, respectively. As previously demonstrated in other C. elegans cell types (Feinberg et al. 2008), GFP should only reconstitute when the two neurite membranes contact each other. We measured GFP-positive neurite length to indicate the length of the PHB/AVA contact site (Figure 1G). We found that in juvenile animals, the PHB/AVA contact length was comparable between both sexes; however, in day 1 adults, the contact length was significantly increased in hermaphrodites but not in males (Figure 1G, H). This altered neurite contact length is confirmed by simply examining cytoplasmic reporters that fill AVA and PHB axons (data not shown), but due to the limits of resolution, it is only through the use of split GFP technology, that we can confirm that such adjacency is in the molecular range. Unexpectedly, while juvenile starvation affects the manifestation of sexually dimorphic synapse number, it did not affect sexually dimorphic neurite contact length (Figure 1G, H). Hence, the extent of sexually dimorphic neurite contact and sexually dimorphic synapse can be uncoupled. Below, we define a molecular pathway that is dedicated toward controlling sexually dimorphic synaptogenesis, without affecting sexually dimorphic extent of neurite contact.

CRH-1/CREB functions downstream of juvenile serotonin signals to control male-specific synaptic remodeling upon sexual maturation

We have previously shown that juvenile starvation operates via the disruption of serotonin signaling through the metabotropic serotonin receptor SER-4, to then control PHB>AVA en passant synapse number growth and elimination (Bayer and Hobert 2018). To dissect the serotonin-triggered signaling cascade in the PHB neuron, we expressed specifically in the PHB neurons a gain-of-function version (gof) of the G-alpha protein, GOA-1, that we hypothesized to act downstream of the SER-4 G-protein-coupled serotonin receptor (Gurel et al. 2012). We found that the loss of PHB>AVA synaptic dimorphisms in adult males that were starved at the L1 stage were rescued in such transgenic animals (Figure 2A), indicating that GOA-1 signaling in male PHB neurons promotes the male-specific diminishment of PHB>AVA en passant synapses. This result is further substantiated by genetic removal of the 5-HT synthesizing enzyme, TPH-1. In tph-1 mutant males, sexually dimorphic PHB>AVA connectivity was disrupted, based on GRASP, CLA-1 and AVR-14 punctae (Figure 2B, S1C, D) and these defects were rescued by PHB-specific GOA-1gof expression (Figure 2C).

Among the many downstream effector pathways of metabotropic 5HT signaling is the activation of CREB via protein phosphorylation (Lonze and Ginty 2002; Bockaert et al. 2006; Oury et al. 2010; Zhang et al. 2016). We analyzed two independent crh-1 loss of function alleles, tz2 and ot1342, and found that PHB>AVA sexually dimorphic synaptic patterning was lost in these animals (Figure 2D). PHB-specific GOA-1gof failed to rescue ectopic PHB>AVA synaptic defects in crh-1 mutants (Figure 2E), confirming that CRH-1 functions downstream of 5-HT-GPCR signaling to mediate adult male specific PHB>AVA remodeling. We also made use of the fact that CREB proteins, including C. elegans CRH-1, are activated by upstream G-protein signaling via a defined phosphorylation site, serine 48 (S48) in C. elegans (S133 in mammalian CREB)(Kimura et al. 2002; Lonze and Ginty 2002). A phosphorylation-deficient mutation (S48A) is predicted to inactivate the protein, while a phosphomimetic mutation (S48E) is predicted to make CRH-1 independent of an upstream-activating input (in this case, loss of serotonin signaling). Indeed, we found that restoration of wild-type and CRH-1S48E but not CRH-1S48A in the PHB rescued PHB>AVA defects in the crh-1 mutant males (Figure 2F). Moreover, only restoration of phosphomimetic CRH-1S48E but not wild-type or CRH-1S48A into the PHB neuron rescued the PHB>AVA synaptic defects of serotonin-deficient tph-1 mutant males (Figure 2G), consistent with CRH-1 acting downstream of serotonin (Figure 2I).

CRH-1/CREB controls feeding state-dependent male-specific LIN-29A expression

We identified a functionally relevant transcriptional target of CRH-1/CREB by turning to the Zn finger transcription factor LIN-29A, which we had previously shown to be expressed in several neuron classes, including PHB and AVA, only in males, but not hermaphrodites (Pereira et al. 2019). We found that juvenile experience impacts proper LIN-29A expression in sexually mature males by starving animals at the L1 stage, transferring them back to food, and examining the expression of a CRISPR/Cas9-engineered reporter allele of lin-29a. We observed that 80% of the animals show an obvious decrease (reduction or complete elimination) in LIN-29A protein expression (Figure 3A, B). We supplemented well-fed animals with octopamine and found that such treatment reduced LIN-29A expression, hence recapitulating the effect of starvation (Figure S2A). Corroborating the previously reported critical window period at which starvation affects PHB>AVA synaptic remodeling (Figure S1E)(Bayer and Hobert 2018), we observed that octopamine exposure at L1 but not the L3 stage results in reduced LIN-29A protein expression (Figure S2A). On the other hand, exogenously supplying 5-HT during L1-starvation rescued the LIN-29A expression deficiency (Figure S2A). This result is further confirmed by the demonstration that genetic removal of endogenous 5-HT by using animals that lack the 5-HT synthesizing enzyme, TPH-1, diminishes LIN-29A expression in PHB (Figure 3C). PHB-specific GOAgof overexpression rescued the L1-starvation LIN-29A expression defects (Figure 3D).

Male-specific LIN-29A expression in PHB was also diminished in two independent crh-1 alleles (Figure 3E). PHB-specific GOAgof overexpression restored LIN-29A expression defects in tph-1 mutants but not in crh-1 or tph-1; crh-1 double mutant males, consistent with GOA-1 acting downstream of serotonin, but upstream of CRH-1 activation (Figure 3F). Restoration of wild-type and CRH-1S48E but not CRH-1S48A CRH-1 rescued LIN-29A expression defects in the crh-1 mutant males (Figure 3G). Moreover, only restoration of phosphomimetic CRH-1S48E but not wild-type or CRH-1S48A rescued the reduction of LIN-29A expression in serotonin-deficient tph-1 mutant males (Figure 3H). The effect of CRH-1/CREB in LIN-29A expression is likely to be direct, since deletion of putative “CREB Responsive Elements” (CRE) in the third intron of the lin-29a gene also phenocopied defective LIN-29A expression in crh-1 mutants (Figure 3I).

Sexual, spatial and temporal specificity of LIN-29A expression

The feeding state-dependent control of LIN-29A protein appearance in the male PHB neurons illustrates a fascinatingly complex regulation of LIN-29A protein expression and, importantly, raises the question of how a signal perceived at the L1 stage is translated into male-specific LIN-29A protein appearance at later larval stages. We further investigated all axes of LIN-29 regulation, i.e. its cell-type/spatial specificity (PHB neuron), its sexual specificity (in males), its temporal specificity (protein occurrence during sexual maturation) and feeding state-dependence. We find that the cell-type specificity of induction of LIN-29A in PHB requires the terminal selector of PHB identity, the ceh-14 LIM homeobox gene (Kagoshima et al. 2013; Serrano-Saiz et al. 2013)(Figure 4A). The sexual specificity of LIN-29A induction in male PHB and not hermaphrodite PHB is, in turn, specified by the global master regulator of sexual identity, the Zn finger transcription factor TRA-1, since removal of TRA-1 selectively in PHB results in LIN-29A depression in hermaphrodite PHB (Pereira et al. 2019)(Figure 4B). CREB activation cannot overcome TRA-1-dependent, sex-specific repression since the PHB::GOA-1gof transgene is insufficient to induce ectopic LIN-29A protein expression in hermaphrodites (Figure 4C). We, therefore, surmise that the activity of CREB is directed to lin-29a by the presence of neuron-type specific cofactors, CEH-14, and this activation is antagonized in hermaphrodites by the master regulator of hermaphroditic sex, TRA-1.

Previous work has shown that the temporal aspect of LIN-29A protein accumulation is controlled by the global heterochronic pathway, such that the translational inhibitor LIN-41 represses lin-29a translation in all cells until the fourth larval stage (Aeschimann et al. 2017; Pereira et al. 2019). We therefore surmised that the feeding state is bookmarked by CREB on the level of lin-29a transcription at earlier larval stages, to then set the stage for translational inhibition by the heterochronic pathway. To probe this issue, we measured lin-29a gene transcription by generating an SL2-based transcriptional lin-29a reporter through CRISPR/Cas9 genome engineering (Figure 4D). We found that lin-29a transcription in PHB was induced in both sexes after animals were exposed to food for 12 hours (late L1 stage) and peaked at 24 hours (late L2 stage) (Figure 4E,F, S3). lin-29a transcription in another neuron that expresses LIN-29a protein in the adult, AVA, is not yet observed during these stages (Figure S2B,C, S3). In the hermaphrodite, lin-29a transcription began to be inhibited at the L3 stage, coinciding with the time when neuronal TRA-1 expression increased (Bayer et al. 2020b). The onset of lin-29a transcription after 12 hours of feeding in the late stage is reduced if animals have been starved prior to food exposure (Figure 4G). Similarly, animals lacking either tph-1, crh-1, or the CRE site in the lin-29a transcriptional reporter allele show reduced lin-29a transcription in PHB (Figure 4H). Taken together, LIN-29A in the PHB acts as a hub by integrating not only temporal (heterochronic pathway), sexual (TRA-1), and spatial (i.e. cell-type specific) information (CEH-14), but also an environmental axis that bookmarks past feeding status via CREB activation.

LIN-29A is required to specify sexually dimorphic PHB>AVA synaptic connectivity

Having shown that early-life serotonin signaling regulates LIN-29A expression, we next asked if lin-29a mutant males phenocopied L1-starvation effects on sexually dimorphic synaptic connectivity. We found that the sexually dimorphic nature of PHB>AVA synapses was indeed abolished by two independent lin-29a null alleles, xe38 and xe40. These defects can be measured with a GRASP transgene, as well as presynaptic CLA-1 and postsynaptic AVR-14 markers (Figure 5A, B, C; Figure S4). Sexually dimorphic connectivity of other LIN-29-expressing neurons is not affected (Figure S5). Consistent with the L1 starvation results, PHB/AVA neurite contact length was not affected in lin-29a mutant animals (Figure 5D,E).

Since a lin-29a reporter allele is expressed in both presynaptic PHB and postsynaptic AVA (Pereira et al. 2019), we addressed the cellular focus of action of LIN-29A through cell-specific rescue experiments and found that restoration of LIN-29A in only the PHB neurons but not the AVA neurons restored proper synaptic elimination in lin-29a mutant males (Figure 5G). Moreover, overexpressing LIN-29A in the PHB neuron in wild-type hermaphrodites caused ectopic PHB>AVA synaptic loss (Figure 5H). We corroborated that lin-29a functions in PHB downstream of early juvenile food experience by showing that the rescuing effect of GOA-1gof in starved males genetically depends on lin-29a (Figure 5I). Furthermore, genetic removal of lin-29a in serotonin-deficient tph-1 mutant males did not further increase the synaptic defects compared to that of either tph-1 or lin-29a single mutant animals (Figure 5J), Overexpression of either GOA-1gof or LIN-29A in the PHB rescued the defects in serotonin-deficient tph-1 males (Figure 5K), while overexpression of LIN-29A, but not GOA-1gof rescued the defects in tph-1 lin-29a double mutants (Figure 5L). Lastly, the effect of masculinization of PHB through PHB-specific TRA-1 removal on PHB>AVA connectivity (Oren-Suissa et al. 2016) is suppressed by lin-29a removal (Figure S6A) but not the effect of AVA masculinization (Figure S6B). Taken together, our results suggest that LIN-29A in the PHB sensory neurons acts downstream of early juvenile food experience and is required and sufficient to promote synaptic elimination in males.

We investigated the structural requirements of the LIN-29A function. We found that the DNA binding activity of LIN-29A was required for its function since LIN-29A with Zn finger domain deletion failed to rescue the lin-29a defects and was insufficient to induce ectopic synaptic elimination (Figure S7A,B). We also found that the human homolog of LIN-29A, ZNF362, is able to rescue the synaptic elimination defects in lin-29a mutants (Figure S7C). Moreover, ZNF362 is also able to induce ectopic synaptic elimination in wild-type hermaphrodites, just as LIN-29A (Figure S7D).

We assessed the behavioral consequences of lin-29a by considering the physiological function PHB>AVA en passant synapses, which control C. elegans avoidance response to noxious chemicals such as SDS. This response is sexually dimorphic in day 1 adult animals such that hermaphrodites avoid SDS less than their male counterparts due to sex-specific PHB>AVA connectivity. We quantified the avoidance response in lin-29a mutant males and found that it was indeed feminized (Figure 5F).

LIN-29A represses DMD-4 in PHB to promote sexually dimorphic PHB>AVA connectivity

Several sex-specific features are mediated by phylogenetically conserved Doublesex/Mab-3-related transcription factors (DMRTs). We had previously shown that the DMD-4 protein, one of several C. elegans Doublesex homologs, is initially expressed in juvenile PHA and PHB neurons in both sexes but becomes selectively degraded in male PHA and PHB neurons upon sexual maturation (Bayer et al. 2020a). The mutually exclusive expression pattern of LIN-29A (males) and DMD-4 (hermaphrodites) in PHB led us to investigate whether lin-29a may control DMD-4 degradation in male PHB. We find that DMD-4::GFP protein fails to be degraded in PHB in a lin-29a mutant background (Figure 6A, B). Since LIN-29A expression is feeding state-dependent, we predicted that L1 starvation (which leads to loss of LIN-29A expression) might stabilize DMD-4 protein expression in PHB and found this to be indeed the case (Figure 6C,D). Consistent with the implication of tph-1 and crh-1 in promoting lin-29a expression, DMD-4 expression in PHB was also stabilized in well-fed day 1 tph-1 and crh-1 mutant males (Figure 6E-G). Cell-specific rescue experiments showed that lin-29a functioned cell-autonomously in PHB to degrade DMD-4 in males (Figures 6H, I). We also find that PHB-specific expression of the human homolog of LIN-29A, ZNF362, rescues the effect of lin-29a on DMD-4 protein expression (Figure S7E).

As expected from DMD-4 expression in hermaphrodites (but not males), loss of dmd-4 does not affect the lack of PHB>AVA synapse number growth in males (Figure 6J). However, it is conceivable that it is the absence of dmd-4 that accounts for PHB>AVA synapse number increase in males and that, hence, the derepression of DMD-4 in lin-29a mutants is responsible for the ectopic PHB>AVA synapses in males. To test this notion, we generated lin-29a; dmd-4 double mutant animals and found that the synaptic defects found in lin-29a mutants were indeed suppressed (Figure 6J). Similarly, overexpressing DMD-4 in PHB neurons promoted the formation of en passant PHB>AVA synapses in males (Figure 6K). Loss of lin-29a did not further enhance the ectopic synaptic defect in males overexpressing DMD-4 in PHB, consistent with the epistatic relationship of these genes. Taken together, lin-29a acts through DMD-4 to specify the sexually dimorphic nature of PHB>AVA en passant synapses.

LIN-29A represses DMD-4 to inhibit fmi-1 expression in adult male PHB

We identified a functionally relevant effector gene for the CREB>LIN-29>DMD-4 regulatory cassette through a nervous system-wide expression pattern analysis of putative synaptogenic molecules, including all members of the cadherin gene family (MM, CPL and OH, in prep.). We found that the unconventional cadherin protein FMI-1, the C. elegans homolog of the Drosophila Flamingo and vertebrate CELSR proteins (Steimel et al. 2010), showed a sexually dimorphic expression pattern in the PHB neurons of adult animals (Figure 7A, B). At juvenile stages, an SL2::GFP::H2B-based fmi-1 reporter allele, generated by CRISPR/Cas9 genome engineering, showed non-dimorphic expression in PHB neurons, but upon sexual maturation, it was sex-specifically downregulated in males. Other neurons in vicinity to PHB show no sex-dependent difference (Figure 7A). Sexually dimorphic fmi-1 expression is not only apparent at the transcriptional level (as measured with our SL2-based reporter allele) but can also be observed on the protein level. To visualize endogenous FMI-1 protein specifically in PHB, we engineered six copies of split GFP11(6xGFP11) at the C-terminus of fmi-1 at the endogenous locus and overexpressed the other half of GFP with myristoylation peptide (myri-GPF1-10) in PHB (Figure S8A,B). We found that reconstituted FMI-1::GFP intensity was significantly lower in day 1 males compared to their hermaphrodite counterparts, consistent with decreased fmi-1 transcription in males upon sexual maturation (Figure S8C,D).

Male-specific downregulation of fmi-1 gene and FMI-1 protein expression was lost in lin-29a mutants (Figure 7A,B; S8). Expression of either LIN-29A in PHB or its vertebrate homolog, ZNF362, rescued the ectopic expression of FMI-1 in male PHB (Figure S6F). Ectopic expression of LIN-29A in hermaphrodite PHB shows that LIN-29A is not only required but also sufficient to downregulate fmi-1 expression (Figure 7C). The feeding state-dependence of LIN-29A expression predicts that the downregulation of fmi-1 should also be dependent on the feeding state. Indeed, we find that fmi-1 expression in male PHB is derepressed upon either L1 starvation, or in well-fed serotonin-deficient tph-1 or crh-1/CREB mutants (Figure 7D-F). The effect of lin-29a and feeding state on fmi-1 expression is mediated by the dmd-4 gene since the ectopic expression of fmi-1 in male PHB in lin-29a mutants or after L1 starvation is suppressed by removal of dmd-4 (Figure 7D). These findings indicate that dmd-4 normally acts to promote fmi-1 expression. Indeed, hermaphrodite-enriched PHB expression of fmi-1 is reduced in dmd-4 mutants and, conversely, overexpression of dmd-4 in male PHB promotes fmi-1 expression (Figure 7H,I).

Separable functions of FMI-1 in controlling neurite contact length and synaptogenesis

The regulation of fmi-1 by the serotonin>CREB>LIN-29A>DMD-4 axis, together with the documented role of vertebrate fmi-1 orthologs in synaptogenesis (Zou 2020), made us hypothesize that fmi-1 may act as a synaptogenic molecule in the PHB>AVA context and that its serotonin/LIN-29A mediated downregulation suppresses PHB>AVA synapse number increases in males, hence generating synaptic sexual dimorphisms. Indeed, we found that a fmi-1 null mutant allele that we generated by CRISPR/Cas9 genome engineering, results in decreased PHB>AVA GRASP puncta and presynaptic CLA-1 and postsynaptic AVR-14 puncta (Figure 8A; Figure S9A-F). Moreover, the PHB>AVA synaptic defects were rescued when FMI-1A was expressed in the PHB. In males, FMI-1A ectopic expression in the PHB also induced ectopic PHB>AVA synapses (Figure 8B). However, we also noted that the extent of PHB and AVA neurite contact, measured with CD4-based GRASP is significantly reduced as well (Figure 8C), already at the first larval stage (Figure 8D). The defect can be rescued by cell-specific re-expression in the PHB neurons (Figure 8C,D). This indicates that FMI-1 has, consistent with its role in other parts of the C. elegans nervous system (Steimel et al. 2010; Najarro et al. 2012), a role in neurite pathfinding and/or fasciculation of PHB during embryonic development, therefore preventing us from concluding that these synaptic defects are indeed the result of sexually dimorphic synaptogenic defects during postembryonic development.

To separate embryonic from possible later roles of fmi-1, we employed a conditional gene removal strategy in which we inserted loxP sites into the reporter-tagged fmi-1 locus (Figure S8A). We first confirmed that continuous removal of fmi-1 using pan-neuronal and PHB-specific but not AVA-specific Cre driver lines recapitulated synaptic defects in fmi-1(ot1291) null mutant (Figure 8E). We removed fmi-1 in a temporally controlled removal manner using a heat-shock inducible Cre driver line. Embryonic induction of Cre expression recapitulated the neurite contact length defects, while fmi-1 removal at any postembryonic stage had no effect on PHB/AVA contact length (Figure 8F). In contrast, eliminating fmi-1 at postembryonic stages caused a decrease in PHB>AVA synapses in hermaphrodites compared to those of counterparts without transgene (Figure 8G). Moreover, we found that removal of fmi-1 at the adult stage resulted in a reduction of PHB>AVA synapses, demonstrating that fmi-1 is not only required for synapse number growth during sexual maturation, but is also continuously required to sustain synaptic connectivity in hermaphrodites.

Lastly, we asked whether ectopic en passant synapses in lin-29a mutant males result from derepressed synaptogenic fmi-1 function in PHB. To this end, we removed fmi-1 postembryonically in lin-29a mutant to bypass its critical embryonic neurite placement function. We found that removal of fmi-1 in lin-29a mutant animals before sexual maturation (L3) and adulthood (day 1) significantly decreased the PHB>AVA synaptic puncta compared to the respective non-transgenic siblings (Figure 8H and I).

Taken together these experiments demonstrate that FMI-1 has two separable functions, one during embryonic development in neurite outgrowth and placement and a synaptogenic one during postembryonic development. Since postembryonic expression of FMI-1 is sexually dimorphic, depending on the feeding state of the animal, we conclude that FMI-1 is the key synaptogenic effector gene of the CREB>LIN-29A>DMD-4 regulatory cascade. In hermaphrodites, this synaptogenic function is DMD-4-dependent and unimpeded by feeding state, while in male animals, feeding state and LIN-29A-dependent suppression of FMI-1 expression results in increase of sexually dimorphic en passant synapses.

DISCUSSION

Early-life experience impacts later brain development or neurological disorders in a sex-specific manner, but the cellular and molecular mechanisms that lead to sex-specific vulnerability remain largely unknown. Here, we uncover the molecular basis of how the juvenile feeding-state experience affects the generation of sex-specificity of synaptic connectivity between a nociceptive sensory neuron and a command interneuron target.

A key regulatory bottleneck in this process is an evolutionarily conserved Zn finger transcription factor, LIN-29A, whose expression integrates four dimensions of specificity (Fig.S10A). Transcription of the lin-29a locus is directed to a subset of neuron cell types (like PHB) via terminal selectors, such as CEH-14 (shown here). The male-specificity of lin-29a transcription is imposed by the global sex identity regulator TRA-1, which antagonizes lin-29a transcription in hermaphrodites, while the temporal specificity of LIN-29A protein accumulation during sexual maturation is controlled by translational repression of lin-29a transcripts through the global heterochronic regulator lin-41. This repression is relieved upon sexual maturation by miRNA-mediated downregulation of lin-41. The fourth dimension of lin-29a regulation is conferred by the feeding state of the animal. A serotonin- and CREB-dependent input is required specifically in the L1 stage to enable terminal selector-dependent induction of lin-29a transcription that is then translated into protein expression during sexual maturation via the relief of translational repression. Once the initial lin-29a transcription activation has been bookmarked, the locus becomes independent of a requirement for a feeding input (as evidenced by post-L1 starvation having no effect on LIN-29A expression). Taken together, our studies tie an early transcriptional event, mediated by stimulus-dependent transcription factor, CREB, to the sustained expression of a locus, lin-29a, that later in life relays this input into the modulation of synaptic connectivity and, hence, information flow in the nervous system.

Our study here provides not only novel insights into the mechanistic basis of sculpting the sexually dimorphic nature of synaptic connectivity but reveals two distinct components of the establishment of sexually dimorphic connectivity: a sexually dimorphic increase in the adjacency of two neurites and a sexually dimorphic increase in the number of en passant synaptic connections. These two processes can be mechanistically uncoupled by by fmi-1/Flamingo, which does not affect the postembryonic increase in neurite adjacency, but only affects the increase in en passant synapse number and, postdevelopmentally, maintains this synaptic connectivity. Considering the early function of fmi-1 in controlling initial PHB/AVA neurite fasciculation during embryonic development, it is intriguing to note the lack of a role of fmi-1 in controlling the sex-specific adjacency increase of the PHB and AVA neurites during postembryonic sexual maturation. This observation suggests that neurite contacts of the same two neurons can be regulated by distinct means during distinct stages of development. Moreover, the distinct functions of fmi-1 in embryonic fasciculation and postembryonic synaptogenesis and maintenance are a likely reflection of context-dependent association of FMI-1 proteins with distinct interaction partners.

In vertebrates, the Flamingo orthologs CELSR1/2/3/4 have been implicated in axon outgrowth as well as synapse formation, but a function in maintaining synaptic structure had not been described before (Tissir et al. 2005; Lewis et al. 2011; Feng et al. 2012; Chai et al. 2014; Thakar et al. 2017; Zou 2020). Our work uniquely places FMI-1 function, as well as its apparent dependence on past experience, in the context of sexually dimorphic synaptic connectivity. Vertebrate brains are thought to display sexually dimorphic features on multiple levels (Simerly 2002; Morris et al. 2004; Dulac and Kimchi 2007; Yang and Shah 2014; Gegenhuber and Tollkuhn 2020), even though the cellular complexity of vertebrate brains has hampered the definition of such dimorphisms on a single neuron/single synapse level. We hope that our work will motivate a careful analysis of sexually dimorphic Flamingo expression in vertebrate brains, which may provide a critical entry point to not only identify vertebrate sexual dimorphisms but also understand their genetic specification.

STAR methods

RESOURCE AVAILABILITY

Lead contact

For further detailed information and requests for resources and reagents, please contact Chien-Po Liao (cl4102@columbia.edu) and Oliver Hobert (or38@columbia.edu)

EXPERIMENTAL DETAILS

Caenorhabditis elegans strains and handling

Worms were grown at 20°C on nematode growth media (NGM) plates seeded with E. coli (OP50) bacteria as a food source unless otherwise mentioned. Worms were maintained according to standard protocol. him-8(e1489) and him-5(e1490) were used as wild-type in this study to generate sufficient males. The key resources table lists a complete list of strains and transgenes generated and used in this study.

CRISPR/Cas9-based genome engineering

To generate tph-1(ot1274), two guide crRNA (5’catcggatatctaaaagagg3’ and 5’ acctctcttcatctcaatat 3’) and ssODN (5’gtgccgaattccagaagcaccacgccatcggatatctaaaagaggccaacacaaagacacgttttcctgcagaagaggaa 3’) were used to remove the whole tph-1 locus. To generate crh-1(ot1343), two guide crRNA (5’ taaggagattagttttccaa3’ and 5’ ttggagatttcttgttgagg 3’) and ssODN (5’ gtgtttgtttttcaaagaatagcttatatatatgatgaaatctcgtttttatttttatttcctaattttt 3’) were used to remove the whole crh-1 locus. To generate lin-29(ot1396), two guide crRNA (5’ atttgaacccaatattgaat3’ and 5’ gagttcattttgatttcacg 3’) and ssODN (5’ agtggtcaaagaaatttgagagaaaaagtgcggagcgtgaaatcaaaatgaactcggctatatttcggcc 3’) were used to remove the potential CRE site in the lin-29a locus. To generate fmi-1(ot1090) and fmi-1(ot1291), two guide crRNA (5’ ttgaatgtgaatgtcagtgg3’ and 5’ TGATGCGTATTACACATATA 3’) were used to remove the whole fmi-1 locus. To generate fmi-1(ot1349), crRNA (5’aagaactgaccagctgccaa3’) and ssODN (5’ ctgatacaaccctttgctcttttcacctcatatgtATAACTTCGTATAGCATACATTATACGAAGTTATcccgggttggcagctggtcagttcttcttccaaagagacgc3’) were used to insert the first LoxP site at 5’UTR region and second crRNA(5’ atcggaacaatgaacaagta 3’) and homemade LoxP::GFP::H2B ssODN via PCR and exonucleuase were used to insert the second LoxP site. To generate fmi-1(ot1429), crRNA (5’ TACCACATCTACATTCAACA3’) and codon-optimized 6XGFP11 ssODN were used to remove the original stop codon and insert GFP11 at the C terminus of fmi-1 gene locus. To generate, lin-29(ot1482), crRNA (5’ ttatcggaatatgtgagttc3’) and homemade SL2::GFP::H2B ssOND were used.

Molecular cloning

To generate pCPL1(srab-20p::goa-1gof::SL2::TagRFP) and pCPL4(srab-20p::goa-1gof::SL2::3XNLS::GFP) , upstream ~1.2kb promoter from srab-20, which was amplified from (srab-20p::GFP), the synthetic goa-1gof DNA fragment, and the SL2-backbone amplified from pEAB42 (srg-13p::ser-4::SL2::tagRFP) were ligated. To generate pCPL2(gpa-6p::lin-29a::SL2::3XNLS::GFP) and pCPL3(srab-20p::lin-29a::SL2::3XNLS::GFP), srab-20p and gpa-6p amplified from pCPL4 and pEAB3 (2.6 kb of the gpa-6 promoter fused to GFP) respectively, and lin-29a cDNA (1.4kb) and the SL2-base backbone amplified from pCPL4 were ligated. To generate pCPL5 (srab-20p::crh-1WT::SL2::3XNLS::GFP), pCPL6 (srab-20p::crh-1S48E::SL2::3XNLS::GFP) and pCPL7 (srab-20p::crh-1S48A::SL2::3XNLS::GFP), crh-1 variants subcloned from gcy-8p::crh-1WT, gcy-8p::crh-1S48E, and gcy-8p::crh-1S48A (gifts from Dr.Chun-Liang Pan) and backbone amplified from pCPL4 were ligated. To generate pCPL8 (srab-20p::crh-1WT::SL2::TagRFP), pCPL9 (srab-20p::crh-1S48E::SL2::TagRFP) and pCPL10 (srab-20p::crh-1S48A::SL2::TagRFP), crh-1 variants subcloned into pCPL1 backbone. To generate pCPL11(srab-20p::lin-29a::SL2::TagRFP) and pCPL12(srg-13p::lin-29a::SL2::TagRFP), lin-29a was subcloned into the backbone amplified from pCPL1 and pEAB42 (srg-13p::ser-4::SL2::TagRFP) respectively. To generate pCPL13(srab-20p::dmd-4::SL2::TagRFP), dmd-4 cDNA (0.8 kb) was amplified and subcloned into pCPL1. To generate pCPL14(srab-20p::fmi-1a::SL2::3XNLS::GFP), fmi-1a cDNA was amplified from ser2prom3::fmi-1a (gift from Dr. Chun-Liang Pan) and subcloned into pCPL4. To generate pCPL15 (UPN::3xNLS::Cre), pCPL16(srab-20p::3xNLS::Cre) pCPL17 (flp-18p::3XNLS::Cre) and pCPL23(hsp16.2::3xNLS::Cre), UPN, srab-20p, flp-18p (~1.4kb) and hsp16.2p (~0.4 kb) were subcloned into pCC301(rab-3p1::3xNLS::Cre). To generate pCPL18 (srab-20p::gfp::cla-1(s)), srab-20p were digested from pCPL1 by SphI and XmaI restriction enzymes and ligated into the backbone of pMM13(cat-4p::gfp::cla-1(s)). To generate pCPL19 (flp-18p::avr-14::TagRFP), avr-14 was amplified from avr-14 plasmid (gift from Dr. Meital Oren) and ligated into pCC45(flp-18p::TagRFP). To generate pCPL20(srab-20p::znf362::SL2::3XNLS::GFP) and pCPL21(srab-20p::znf362::SL2::TagRFP), synthetic codon-optimized human znf362 cDNA was subcloned into pCPL4 and pCPL1 respectively. To generate pCPL24(srab-20p::myriGFP1-10::SL2::TagRFP), split GFP1-10 was cloned with myristylation sequences and ligated with pCPL1 backbone. To generate pCPL25 (srab-20p::CD4::GFP1-10) and pCPL26(flp-18p::CD4::GFP11), srab-20p and flp-18p were digested from pCPL16 and pCPL17 respectively, and ligated to the backbone from ser2prom3p::CD4:: GFP1-10 and unc-17p::CD4:: GFP11 (gifts from Dr. Chun-Liang Pan), respectively.

L1 starvation assay

Animals with indicated genotype were synchronized by hypochlorite treatment of gravid adults followed by 12 hours in M9 at 20°C for embryos to hatch. Synchronized L1 animals were released onto the unseeded NGM plates for another 24 hours. L1-starved animals were then washed and transferred to seeded NGM plates.

Heat shock assay

OH18506, him-5(e1490) fmi-1(ot1349); otIs839, otEx8084[hsp-16.2p::3xNLS::Cre::p10UTR], OH19023, him-5(e1490) fmi-1(ot1349)/V;otEx8084[hsp-16.2p::3xNLS::Cre::p10UTR],otEx8152[srab-20p::TagRFP, srab-20p::CD4::GFP1-10,flp-18p::TagRFP, flp-18::CD4::GFP11], OH19231, lin-29(xe38);fmi-1(ot1349) him-5(e1490); otIs839; otEx8084[hsp-16.2p::3xNLS::Cre],animals were synchronized by hypochlorite treatment of gravid adults followed by 12 h ours in M9 at 20°C for embryos to hatch. Synchronized L1 animals were released onto the seeded NGM plates, and heat shock was performed 6, 20, 40 and 60 hours after releasing onto the seeded plates (indicates as hours after hatching the figures). Animals were heat shocked at 34°C for 20 minutes, followed by 20 minutes of resting at 20°C three times to induce sufficient heat shock response. PHB>AVA GRASP puncta and PHB/AVA adjacency were analyzed at day 1 stage for groups that received heat shock at 6, 20, or 40 hours post-hatching. For groups that received heat shock at 60 hours post-hatching, analyses of PHB>AVA GRASP puncta and PHB/AVA adjacency were conducted 24 hours after heat shock.

SDS avoidance behavior

The SDS avoidance assay was based on procedures as described. To deliver the testing droplets, we pulled 10-μl glass capillary pipette (VWR international) by hand on the flame to reduce the diameter of the tip and mounted the capillary pipette on a rubber tubing and operated by mouth. We delivered a small drop of solution containing either the repellent (0.1% SDS in M13 buffer) or buffer (M13 buffer: 30 mM Tris-HCl pH 7.0, 100 mM NaCl, 10 mM KCl) to near the tail of an animal while it moves forward. Once in contact with the tail, the drop surrounded the animal by capillary action and reached the anterior head region. Assayed worms were transferred individually to fresh and unseeded NGM plates. Each assay started by testing the animals with drops of M13 buffer alone. The response to each drop was scored as reversing or not reversing. The avoidance index is the number of reversal responses divided by the total number of trials. An interstimulus interval of at least two minutes was used between successive drops of the same animal.

Microscopy

Worms were anesthetized in 100 mM of sodium azide on the 5% agarose on glass slides. All images were acquired using a Zeiss confocal microscope (LSM 880 or LSM980).For synaptic GRASP and gene expression experiments, animals were imaged using 63 X objective and with a fixed imaging setting. For CLA-1 and AVR-14 puncta experiments, animals were imaged using 40 X objective and with a fixed imaging setting. For PHB-AVA adjacency CD4-GRASP experiments, animals were 40 X objective and with a fixed imaging setting.

QUANTIFICATION AND STATISTICAL ANALYSIS

Quantification of synaptic GRASP puncta

For all the synaptic GRASP, the images were acquired using 63 X objective and with a fixed imaging setting either with LSM880 or LSM980. The raw images were unbiasedly analyzed with PysQi (Majeed et al. 2024), the automatic puncta quantification software.

Quantification of synaptic iBLINC puncta

For iBLINC experiments, animals were imaged using a 63 X objective with a fixed imaging setting with LSM880, and puncta were quantified by scanning the original full Z-stack for distinct dots in the area where the processes of the two neurons overlap.

Quantification of GFP::LIN-29A, DMD-4::GFP, and lin-29a and fmi-1 expression

For expression of translational and transcriptional lin-29a reporter constructs, images of lin-29(xe63[gfp::lin-29a]) and lin-29(ot1482[lin-29::SL2::GFP::H2B]), animals with different mutant or transgene overexpression backgrounds were acquired using 63 X objective with fixed imaging settings with either LSM880 or LSM980. The expression level is categorized into three tiers: on, dim, and off. Cells with GFP fluorescent intensity lower than 50% of the normal “on” cells are identified as “dim.”

For DMD-4::GFP quantification, images of dmd-4(ot935) animals with different mutant or transgene overexpression backgrounds were acquired using 63 X objective with fixed imaging settings with either LSM880 or LSM980.

For fmi-1 gene expression quantification, images of fmi-1(syb4563) animals with different mutant or transgene overexpression backgrounds were acquired using 63 X objective with fixed imaging settings with either LSM880 or LSM980. Cells with GFP fluorescent intensity lower than 50% of the normal “on” cells are identified as “dim.”

Quantification of PHB and AVA contact CD4 GRASP

The contact site length between PHB and AVA processes in the CD4 reporter was quantified in Fiji ImageJ (Schindelin et al. 2012). Briefly, the entire Z-stack was scanned while tracing over the GFP+ region (where the PHB and AVA processes overlap) with a segmented line and then measuring the overall line length. In cases where the contact and resulting GFP signal was discontinuous, multiple lines were drawn, measured independently, and summed to yield the overall contact site length. For visualization purposes, figures contain a representative subset of the Z-stack reconstructed as maximum intensity projection using Zeiss Zen software to display the maximal PHB-AVA contact site.

Quantification of CLA-1 and AVR-14 puncta

GFP::CLA-1 and AVR-14::TagRFP puncta in the PHB and AVA, respectively, were quantified manually in Fiji by scanning the entire Z-stack and only scoring puncta co-localizing with cytoplasmic AVAp::RFP and cytoplasmic PHBp::GFP, respectively.

Quantification of the juxtaposition of the CLA-1 and AVR-14 puncta

For scoring the juxtaposition of the PHB GFP::CLA-1 and AVA AVR-14::RFP puncta, each Z-stack was first scanned in the region of interest to quantify all GFP::CLA-1 puncta. Next, AVR-14::TagRFP puncta directly adjacent to with CLA-1 puncta were scored. The juxtaposition index is calculated as follows:

AVR-14::TagRFP juxtaposed with CLA-1::GFP/Total GFP::CLA-1)*100%.

Supplementary Material

Supplement 1

ACKNOWLEDGEMENTS

We thank Chi Chen for generating transgenic strains and members of the Hobert lab, as well as Nathan Harris for comments on the manuscript. We thank Dr. Chun-Liang Pan for providing constructs for fmi-1a cDNA, crh-1 variants and CD4-GRASP constructs and Dr. Meital Oren for the avr-14 plasmid. Some of the strains were provided by the CGC, which is supported by the NIH (P40 OD010440). This work was supported by the NIH (R37NS039996) and the HHMI. Chien-Po Liao was supported by the Postdoctoral Research Abroad Program sponsored by the Ministry of Science and Technology from Taiwan and the Charles H. Revson Senior Fellowship in Biomedical Science (Grant No. 23-16).

Figure 1. Juvenile serotonin signaling patterns sexually dimorphic synaptic connectivity.

(A,B) Representative images (A) and quantification (B) of PHB>AVA synaptic GRASP(otIs839) in L3 and day 1 well-fed and L1-starved animals in both sexes.

(C)(top) Schematic illustration of and (bottom) Representative images of AVA-juxtaposed GFP::CLA-1 in PHB (otIs883;otEx8040) in L3 and day 1 well-fed and L1-starved animals in both sexes.

(D) Quantification of AVA-juxtaposed GFP::CLA-1 in PHB in L3 and day 1 well-fed and L1-starved animals in both sexes. Note for panel C and D that the number of AVA-juxtaposed CLA-1 puncta remained sexually dimorphic in the L1-starved adult male, which is consistent with electron micrographic data that shows that PHB generates many sex-specific synapses, in addition to AVA (COOK et al. 2019).

(E) (top) Schematic diagram and (bottom) representative images of PHB-juxtaposed AVR-14::TagRFP in in L3 and day 1 well-fed and L1-starved animals in both sexes.

(F) Quantification of AVR-14::TagRFP in AVA (otIs902; him-8(e1489)) in L3 and day 1 well-fed and L1-starved animals in both sexes.

(G) (top) Schematic diagram and (bottom) representative images of PHB>AVA neurite CD4-GRASP (otEx8152) in L3 and day 1 well-fed and L1-starved animals in both sexes. We measure the GFP-positive length to indicate the PHB/AVA contact site.

(H) Quantification of CD4-GRASP (otEx8152) in L3 and day 1 well-fed and L1-starved animals in both sexes.

Statistics: (B,D,F,H) Two-way ANOVA followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. + indicates the mean value. Scale bar = 10 μm.

Figure 2. Juvenile starvation controls sexually dimorphic PHB>AVA synaptic contacts via CRH-1/CREB.

(A) Quantification of PHB>AVA synaptic GRASP(otIs839) in well-fed wild-type animals and L1-starved wild-type and animals expressing PHB::LIN-29A (otEx7915 ) or PHB::GOA-1gof(otEx7925 and otEx8158).

(B) Quantification of PHB>AVA synaptic GRASP (otIs839) in wild-type and tph-1(ot1274) in both sexes.

(C) Quantification of PHB>AVA synaptic GRASP (otIs839) in wild-type and tph-1(ot1274) males expressing PHB::GOA-1gof(otEx7925 and otEx8158).

(D) Quantification of PHB>AVA synaptic GRAPS (otIs839) in wild-type and crh-1(tz2) and crh-1(ot1342) animals of both sexes.

(E) Quantification of PHB>AVA synaptic GRASP (otIs839) in wild-type and crh-1(tz2) males expressing PHB::GOA-1gof(otEx7925 and otEx8158).

(F,G) Quantification of PHB>AVA synaptic GRASP(otIs839) in crh-1(tz2) (F) and tph-1(ot1274) (G) males with overexpressing CRH-1 missense allele transgenes (otEx8045 for CRH-1WT, otEx8046 for CRH-1S48E, and otEx8082 for CRH-1S48A) in the PHB neurons.

(I) Schematic diagram indicates juvenile food experience acts through serotonin-GPCR-GOA-1 to activate CRH-1 to secure LIN-29A expression upon sexual maturation to establish PHB>AVA sexually dimorphic connectivity.

Statistics: (A,C,E,F,G) One-way ANOVA and (B,D,I) two-way ANOVA followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. Scale bar = 10 μm. + indicates the mean value.

Figure 3. Male-specific LIN-29A expression in PHB is controlled by juvenile serotonin experience via CRH-1/CREB.

(A) lin-29(xe63[gfp::lin-29a]) expression of well-fed and L1-starved day 1 males in the AVA and PHB neurons. lin-29(xe63[gfp::lin-29a]) expression is not affected in the AVA but is dim or lost in the PHB neurons when males undergo L1 starvation.

(B) Quantification of the percentage of neurons expressing lin-29(xe63[gfp::lin-29a]) in AVA or PHB under well-fed or L1 starvation conditions.

(C) Representative images (top) and quantification of (bottom) PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in wild-type and tph-1(ot1274).

(D) Representative images (top) and quantification of (bottom) PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in males that undergo L1-starvation with transgene overexpressing GOA-1gof (otEx8037) in the PHB neurons.

(E) Representative images (top) and quantification of (bottom) PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in wild-type, crh-1(tz2) and crh-1(ot1342).

(F) Quantification of PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in animals with or without PHB::GOA-1gof transgene (otEx8037) overexpression in wild-type, tph-1(ot1274), crh-1(tz2) and tph-1(1274); crh-1(tz2) background.

(G) Quantification of PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in crh-1(tz2) mutants with overexpressing CRH-1 missense allele transgenes (otEx8053 for CRH-1WT, otEx8157 for CRH-1S48E, and otEx8113 for CRH-1S48A) in the PHB neurons.

(H) Quantification of PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in tph-1(ot1274) mutants, overexpressing distinct types of crh-1 transgenes (otEx8053 for CRH-1WT, otEx8157 for CRH-1S48E, and otEx8113 for CRH-1S48A) in the PHB neurons.

(I) (top) Schematic illustration of “CREB Responsive Element” (CRE) sites in the lin-29a locus. The lin-29(ot1396) allele is designed to delete the potential CRE sites at the intron 3 of the lin-29a locus. (bottom) Representative images (left) and quantification of (right) PHB neurons expressing GFP::LIN-29A in wild-type and lin-29(ot1396).

Statistics: chi-squared tests followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. The red dashed circle indicates PHB. Scale bar = 5 μm.

Figure 4. Early larval lin-29a transcription is regulated by serotonin>CREB signaling.

(A) Representative images (top) and quantification of (bottom) PHB neurons expressing lin-29(xe63[gfp::lin-29a]) in wild type and ceh-14(ot900) males.

(B) Representative images (top) and quantification of (bottom) PHB expressing lin-29(xe63[gfp::lin-29a]) with transgene masculinizing PHB (PHB::FEM-3)(otEX7916) in hermaphrodites.

(C) Representative images (top) and quantification of (bottom) PHB expressing lin-29(xe63[gfp::lin-29a]) in hermaphrodites with transgene overexpressing GOA-1gof (otEx8037) in the PHB.

(D) Schematic illustration of lin-29(ot1482[lin-29::SL2::GFP::H2B]) and lin-29(ot1500). The lin-29(ot1500) allele is designed to delete the trunk of DNA elements, including the potential CRE site at the intron 3 of the lin-29a locus in lin-29(ot1482).

(E,F) Longitudinal analysis of lin-29(ot1482[lin-29::SL2::GFP::H2B]) expression in PHB neuron. Representative images (B) and quantification (C) of expression of PHB neuron expressing lin-29(ot1482[lin 29::SL2::GFP::H2B]) in different time points after food exposure.

(G) Representative images (left) and quantification of (right) PHB neurons expressing lin-29(ot1482[lin-29::SL2::GFP::H2B]) in males that undergo L1-starvation after 12 hours of food exposure.

(H) Representative images (left) and quantification of (right) PHB neurons expressing lin-29(ot1482[lin-29::SL2::GFP::H2B]) in wild type, tph-1(ot1274), crh-1(tz2), and lin-29(ot1500) males after 12 hours of food exposure.

Statistics: chi-squared tests followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. The red dashed circle indicates PHB. Scale bar = 5 μm. + indicates the mean value.

Figure 5. LIN-29A in PHB is required and sufficient to establish PHB>AVA sexual dimorphic connectivity upon sexual maturation.

(A,B) Representative images (A) and quantification (B) of PHB>AVA synaptic GRASP(otIs839) in day 1 wild-type, lin-29(xe38) and lin-29(xe40) in both sexes.

(C) Developmental analyses of PHB>AVA synaptic connectivity in wild-type and lin-29a mutants. N > 10 for each genotype and sex at any given time point.

(D) Representative images of PHB>AVA neurite CD4-GRASP(otEx8152) in lin-29a mutants in both sexes.

(E) Quantification of CD4-GRASP(otEx8152) lin-29a mutants in both sexes.

(F) Quantification of SDS-avoidance assay in wild-type, lin-29(xe38) and lin-29(xe40).

(G) Quantification of PHB>AVA synaptic GRASP in lin-29(xe38) males with transgenes expressing LIN-29A cDNA in either AVA(otEx7763) or PHB (otEx7790 for gpa-6p and otEx7915 for srab-20p).

(H) Quantification of PHB>AVA synaptic GRASP in wild-type hermaphrodite with transgenes expressing LIN-29A cDNA in either AVA(otEx7763) or PHB (otEx7790 for gpa-6p and otEx7915 for srab-20p).

(I) Quantification of PHB>AVA synaptic GRASP(otIs839) in well-fed wild-type animals and L1-starved wild-type and lin-29(xe38) animals expressing PHB::LIN-29A (otEx7915 ) or PHB::GOA-1gof(otEx7925 and otEx8158).

(J) Epistasis analysis of tph-1 and lin-29a for PHB>AVA synaptic GRASP (otIs839) in males.

(K,L) Quantification of PHB>AVA synaptic GRASP in tph-1(ot1274) (K) and tph-1(ot1274) lin-29(xe38) (L) males with transgene overexpressing GOA-1gof (otEx7925) and LIN-29A(otEx7915 ) in the PHB.

Statistics: (B,E,F,I) Two-way ANOVA , (C) three-way ANOVA, and (G,H,J,K,L) one-way ANOVA followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. Scale bar = 10 μm. + indicates the mean value.

Figure 6. LIN-29A represses DMD-4 in PHB to control sexually dimorphic PHB>AVA connectivity.

(A,B) Representative images (A) and (B) quantification of PHB neurons expressing dmd-4(ot935) in wild-type hermaphrodite and male and lin-29(xe38) male.

(C,D) Representative images of and (D) quantification of PHB neurons expressing dmd-4(ot935) in well-fed and L1-starved day 1 males.

(E,F,G) Representative images (E) and (F,G) quantification of PHB neurons expressing dmd-4(ot935) in wild-type, tph-1(ot1274) and crh-1(1342) males.

(H) (top) Representative images and (bottom) quantification of PHB neuron expressing dmd-4(ot935) in lin-29(xe38) with transgenes that express LIN-29A cDNA in either PHB (otEx7961 and otEx7964) or PHA(otEx8159 and otEx8160).

(J) Epistasis mutant analysis of lin-29a and dmd-4 for PHB>AVA synaptic GRASP (otIs839) in males.

(K) Quantification of PHB>AVA synaptic GRASP (otIs839) in wild-type and lin-29(xe38) males with transgene overexpression DMD-4 in either PHB (otEx7984) or PHA(otEx7983).

Statistics: (B,D,F,G,I) Two-proportion Z test, and (J,K) one-way ANOVA followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. The red dashed circle indicates PHB. Scale bar = 5 μm.

Figure 7. LIN-29A inhibits fmi-1/Flamingo expression via repressing DMD-4

(A,B) Representative images (A) and quantification (B) of fmi-1(syb4563) expression in the PHB in early L4 animals and day 1 animals with various genotypes.

(C) Representative images (top) and quantification (bottom) of fmi-1(syb4563) expression in the PHB in wild-type hermaphrodites with transgene overexpressing LIN-29A cDNA(otEx7961) in the PHB neurons.

(D) Representative images (left) and quantification (right) of fmi-1(syb4563) expression in the PHB in males that underwent L1 starvation.

(E,F,G)Representative images (E) and quantification (F,G) of fmi-1(syb4563) expression in the PHB in wild-type, tph-1(ot1274) and crh-1(ot1342) males.

(H) Representative images (top) and quantification (bottom) of fmi-1(syb4563) expression in the PHB in wild-type males with transgene overexpressing DMD-4 cDNA (otEx8083) in the PHB neurons.

(I) Representative images (top) and quantification (bottom) of fmi-1(syb4563) expression in the PHB in dmd-4(ot957ot935) hermaphrodites.

Statistics: (B,C,D,F,G,H,I) Two-proportion Z test, followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. The red dashed circle indicates PHB. Scale bar = 5 μm. + indicates the mean value.

Figure 8. FMI-1 acts in PHB to promote the formation of en passant PHB>AVA synapses.

(A,B) Quantification of PHB>AVA synaptic GRASP(otIs839) in fmi-1(ot1291) hermaphrodites (A) and wild-type males (B) with transgenes expressing FMI-1A cDNA (otEx8032 and otEx8033)in the PHB neurons.

(C) Quantification of CD4-GRASP(otEx8152) in L3 and day 1 wild-type, fmi-1(ot1291) and fmi-1(ot1291); PHB::FMI-1A.

(D) Quantification of CD4-GRASP(otEx8152) in L1 wild-type, fmi-1(ot1291) and fmi-1(ot1291); PHB::FMI-1A.

(E) Quantification of PHB>AVA synaptic GRASP (otIs839) in fmi-1(ot1349) hermaphrodite with transgenes expressing Cre in pan-neuronally (otEx8063) or in PHB (otEx8062 and otEx8161) or AVA(otEx8064 and otEx8162). fmi-1(ot1349) is a fmi-1 allele, which fmi-1 locus is flanked with LoxP site and a GFP::H2B tagged at the C-terminus region.

(F) Quantification of PHB>AVA synaptic GRASP (otIs839) in fmi-1(ot1349) hermaphrodites with transgenes expressing Cre in heat-shock promoter (otEx8084) and heat shock was performed by indicated time point.

(G) Quantification of PHB>AVA synaptic GRASP (otIs839) in fmi-1(ot1349) hermaphrodites with transgenes expressing Cre in heat-shock promoter (otEx8084) and heat shock was performed by indicated time point.

(H) Schematic illustration of fmi-1 expression in the lin-29(xe38); fmi-1(ot1349) in heatshock fmi-1 removal experiment.

(I) Quantification of PHB>AVA synaptic GRASP (otIs839) in lin-29(xe38);fmi-1(ot1349) males with transgenes expressing Cre in heat-shock promoter (otEx8084) and heat shock was performed by indicated time point.

Statistics: (A,B,D,E) One-way ANOVA and (C,F,G,I) two-way ANOVA followed by Bonferroni multiple comparisons test. p-value and N numbers are indicated on the graph. + indicates the mean value.

KEY RESOURCE TABLE

Bacterial and virus strains	Identifier	Resource	
E. coli	WormBase: OP50 WBStrain00041969	CGC	
Chemicals, peptides, and recombinant proteins			
Alt-R S.p. Cas9 Nuclease V3	Cat#1081059	IDT	
Alt-R CRISPR-Cas9 tracrRNA	Cat#1072533	IDT	
Experimental models: Organisms/strains			
Alleles			
lin-29(xe38)			
lin-29(xe40)			
lin-29(xe63)			
lin-29(ot1396)			
lin-29(ot1482)			
lin-29(ot1500)			
tph-1(ot1274)			
crh-1(tz2)			
crh-1(ot1342)			
him-8(e1489)			
him-5(e1490)			
fmi-1(ot1090)			
fmi-1(ot1291)			
fmi-1(ot1349)			
fmi-1(ot1429)			
fmi-1(syb4563)			
dmd-4(ot935)			
dmd-4(ot957ot935)			
Strains	Identifier	Resource	
lin-29(xe63); him-5(e1490); otEx8037[srab-20p:::goa-1gof::SL2::TagRFP]	OH18905	This Study	
tph-1(ot1274) lin-29(xe63)/II; him-5(e1490)/V;otEx8037[srab-20p::goa-1gof::SL2::TagRFP]	OH18389	This Study	
tph-1(ot1274) lin-29(xe63)/II; crh-1(tz2); him-5(e1490)/V;otEx8037[srab-20p::goa-1gof::SL2::TagRFP]	OH18390	This Study	
lin-29(xe63)/II; crh-1(ot1342)/III;him-5(e1490)/V; otIs839	OH18363	This Study	
lin-29(xe63)/II; crh-1(tz2)/III; him-5(e1490)/V; otEx8037[srab-20p::goa-1gof::SL2::TagRFP]	OH18413	This Study	
tph-1(ot1274) lin-29(xe63)/II; him-5(e1490)/V; otEx8053[srab-20p::crh-1WT::SL2::TagRFP ]	OH18439	This Study	
tph-1(ot1274) lin-29(xe63)/II; him-5(e1490)/V;otEx8157[srab-20p::crh-1S48E::SL2::TagRFP ]	OH18906	This Study	
tph-1(ot1274) lin-29(xe63)/II; him-5(e1490)/V; otEx8113[srab-20p::crh-1S48A::SL2::TagRFP ]	OH18636	This Study	
lin-29(xe63)/II; crh-1(tz2)/III; him-5(e1490)/V; otEx8053[srab-20p::crh-1WT::SL2::TagRFP ]	OH18907	This Study	
lin-29(xe63)/II; crh-1(tz2)/III; him-5(e1490)/V; otEx8157[srab-20p::crh-1S48E::SL2::TagRFP ]	OH18908	This Study	
lin-29(xe63)/II; crh-1(tz2)/III; him-5(e1490)/V; otEx8113[srab-20p::crh-1S48A::SL2::TagRFP]	OH18909	This Study	
lin-29(ot1396[ΔCRE])/II; him-5(e1490)/V; otEx8119[AVA::TagRFP, PHB::TagRFP]	OH18669	This Study	
lin-29(ot1482[lin-29::SL2::GFP::H2B])/II; him-5(e1490)/V;otIs839	OH19115	This Study	
tph-1(ot1274) lin-29(ot1482)/II; him-5(e1490)/V;otIs839	OH19168	This Study	
lin-29(ot1482)/II; crh-1(tz2)/III;him-5(e1490)/V;otIs839	OH19169	This Study	
lin-29(ot1500[ΔCRE]ot1482)him-5(e1490)/V;otIs839	OH19177	This Study	
him-5(e1490)/V;otIs839	OH17170	This Study	
lin-29(xe38)/II; him-5(e1490)/V;otIs839;	OH18925	This Study	
lin-29(xe40)/II; him-5(e1490)/V;otIs839;	OH18926	This Study	
otIs883; otEx8040[flp-18p(AVA)::TagRFP]	OH18393	This Study	
in-29(xe38)/II; him-8(e1489)/IV; otIs883; otEx8040[flp-18p(AVA)::TagRFP]	OH18394	This Study	
him-8(e1489)/IV; otIs902	OH18703	This Study	
lin-29(xe38)/II; him-8(e1489)/IV; otIs902	OH18756	This Study	
lin-29(xe38)/II; him-5(1490)/V; otIs839; otEx7763[flp-18p::lin-29a::SL2::3xNLSGFP]	OH17309	This Study	
lin-29(xe38)/II; him-5(e1490)/V; otIs839; otEx7915[srab-20p::lin-29a::SL2::3xNLS::GFP]	OH17828	This Study	
lin-29(xe38)/II; him-5(1490)/V; otIs839; otEx7790[gpa-6p::lin-29a::SL2::3XNLS::GFP]	OH18927	This Study	
him-5(1490)/V; otIs839; otEx7790[gpa-6p::lin-29a::SL2::3XNLS::GFP]	OH17411	This Study	
him-5(1490)/V; otIs839; otEx7763[flp-18p::lin-29a::SL2::3xNLSGFP]	OH18928	This Study	
him-5(e1490)/V; otIs839; otEx7915[srab-20p::lin-29a::SL2::3xNLS::GFP]	OH18929	This Study	
him-5(e1490)/V; otIs839; otEx7925[srab-20p::goa-1gof::SL::3xNLS::GFP ]	OH17867	This Study	
him-5(e1490)/V; otIs839; otEx8158[srab-20p::goa-1gof::SL::3xNLS::GFP ]	OH18911	This Study	
lin-29(xe38)/II; him-5(e1490)/V; otIs839; otEx7925[Psrab-20::goa-1(gof)::SL::3xNLS::GFP ]	OH18930	This Study	
lin-29(xe38)/II; him-5(e1490)/V; otIs839; otEx8158[srab-20p::goa-1gof::SL::3xNLS::GFP ]	OH18931	This Study	
tph-1(ot1274); him-5(e1490)/V; otIs839	OH18147	This Study	
tph-1(ot1274) lin-29(xe38)/II; him-5(e1490)/V; otIs839	OH18932	This Study	
crh-1(tz2)/III ; him-5(e1490)/V; otIs839	OH18217	This Study	
crh-1(ot1342)/III; him-5(e1490)/V; otIs839	OH18933	This Study	
tph-1(ot1274); him-5(e1490)/V; otIs839; otEx7925[srab-20p::goa-1gof:SL::3xNLS::GFP ]	OH18934	This Study	
tph-1(ot1274); him-5(e1490)/V; otIs839; otEx7915[srab-20p::lin-29a::SL2::3xNLS::GFP]	OH18935	This Study	
tph-1(ot1274) lin-29(xe38)/II; him-5(e1490)/V; otIs839; otEx7925[srab-20p::goa-1gof::SL::3xNLS::GFP ]	OH18936	This Study	
tph-1(ot1274) lin-29(xe38)/II; him-5(e1490)/V; otIs839; otEx7915[srab-20p::lin-29a::SL2::3xNLS::GFP]	OH18937	This Study	
tph-1(ot1274); otIs839; him-5(e1490); otEx8045[srab-20p::crh-1WT::SL2::3xNLS::GFP]	OH18415	This Study	
tph-1(ot1274); otIs839; him-5(e1490); otEx8046[srab-20p::crh-1S48E::SL2::3xNLS::GFP]	OH18416	This Study	
tph-1(ot1274)/II; otIs839; him-5(e1490)/V; otEx8082[srab-20p::crh-1S48A::SL2::3xNLS::GFP ]	OH18504	This Study	
crh-1(tz2) otIs839; him-5(e1490); otEx8045[srab-20p::crh-1WT::SL2::3xNLS::GFP]	OH18912	This Study	
crh-1(tz2) otIs839; him-5(e1490); otEx8046[srab-20p::crh-1S48E::SL2::3xNLS::GFP]	OH18913	This Study	
crh-1(tz2) otIs839; him-5(e1490)/V; otEx8082[srab-20p::crh-1S48A::SL2::3xNLS::GFP ]	OH18914	This Study	
him-5(e1490)/V;dmd-4(ot935)/X; otIs839	OH18938	This Study	
lin-29(xe38)/II; him-5(e1490)/V;dmd-4(ot935)/X; otIs839	OH18939	This Study	
tph-1(ot1274)/II; him-5(e1490)/V;dmd-4(ot935)/X; otIs839	OH18372	This Study	
crh-1(ot1342)/III; him-5(e1490)/V;dmd-4(ot935)/X; otIs839	OH18940	This Study	
him-5(e1490) V; dmd-4(ot957ot935) X; otIs839	OH18084	This Study	
lin-29(xe38)/II; him-5(e1490) V; dmd-4(ot957ot935) X; otIs839	OH18085	This Study	
lin-29(xe38); him-5(e1490)/V; dmd-4(ot935)/X;otEx7961[srab-20p::lin-29a::SL2::TagRFP]	OH18094	This Study	
lin-29(xe38); him-5(e1490)/V; dmd-4(ot935)/X; otEx7964[srab-20p::lin-29a::SL2::TagRFP]	OH18097	This Study	
lin-29(xe38)/II; him-5(e1490)/V; dmd-4(ot935)/X; otEx7997[srab-20p::znf-362::SL2::TagRFP]	OH18228	This Study	
lin-29(xe38)/II; him-5(e1490)/V; dmd-4(ot935)/X;otEx7998[srab-20p::znf-362::SL2::TagRFP]	OH18231	This Study	
lin-29(xe38); him-5(e1490)/V; dmd-4(ot935)/X; otEx8159[srg-13p::lin-29a::SL2::TagRFP ]	OH18915	This Study	
lin-29(xe38); him-5(e1490)/V; dmd-4(ot935)/X; otEx8160[srg-13p::lin-29a::SL2::TagRFP ]	OH18916	This Study	
lin-29(xe38)/II; him-5(1490)/V; otIs839; otEx7984[gpa-6p::dmd-4::GFP]	OH18186	This Study	
lin-29(xe38)/II; him-5(1490)/V; otIs839; otEx7983[srg-13p::dmd-4::GFP ]	OH18185	This Study	
him-5(1490)/V; otIs839; otEx7984[gpa-6p::dmd-4::GFP]	OH18941	This Study	
otIs839; otEx7983[srg-13p::dmd-4::GFP]	OH18942	This Study	
him-8(e1489)/IV; fmi-1(syb4563)/V; otIs839	OH18943	This Study	
lin-29(xe38)/II; him-8(e1489)/IV; fmi-1(syb4563)/V; otIs839	OH18924	This Study	
him-8(e1489); fmi-1(syb4563)/V; dmd-4(ot957ot935)/X; otIs839	OH18272	This Study	
lin-29(xe38); him-8(e1489); fmi-1(syb4563)/V; dmd-4(ot957ot935)/X; otIs839	OH18982	This Study	
him-8/IV; fmi-1(syb4563)/V; otEx7961[srab-20p::lin-29a::SL2::TagRFP]	OH18944	This Study	
tph-1(ot1274)/II; him-8(e1489)/IV; fmi-1(syb4563)/V; otIs839	OH18945	This Study	
crh-1(ot1342)/III; him-8(e1489)/IV; fmi-1(syb4563)/V; otIs839	OH18946	This Study	
lin-29(xe38)/II; him-8/IV; fmi-1(syb4563)/V; otEx7961[srab-20p::lin-29a::SL2::TagRFP]	OH18985	This Study	
him-8/IV; fmi-1(syb4563)/V; otEx8083[srab-20p::dmd-4::SL2::TagRFP ]	OH18505	This Study	
him-8/IV(e1489); fmi-1(ot1291)/V; otIs839	OH18271	This Study	
lin-29(xe38)/II; him-8(e1489)/IV; fmi-1(ot1291)/V;otIs839	OH18387	This Study	
otIs839;him-5(e1490); otEx8032[srab-20p::fmi-1a::SL2::3xNLS::GFP]	OH18370	This Study	
otIs839;him-5(e1490); otEx8033[srab-20p::fmi-1a::SL2::3xNLS::GFP]	OH18371	This Study	
him-8/IV(e1489); fmi-1(ot1291)/V; otIs839; otEx8032[srab-20p::fmi-1a::SL2::3xNLS::GFP]	OH19030	This Study	
him-8/IV(e1489); fmi-1(ot1291)/V; otIs839; otEx8033[srab-20p::fmi-1a::SL2::3xNLS::GFP]	OH19031	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V	OH18441	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V; otEx8062[srab-20p::3xNLS::Cre ]	OH18461	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V; otEx8063[UPN::3xNLS::Cre]	OH18467	This Study	
otIs839; him-5(1490)fmi-1(ot1349)/V;otEx8064[flp-18p::3xNLS::Cre]	OH18468	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V;otEx8084[hsp-16.2p::3xNLS::Cre]	OH18506	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V;otEx8161[srab-20p::3xNLS::Cre]	OH18917	This Study	
otIs839; him-5(1490)fmi-1(ot1349)/V;otEx8162[flp-18p::3xNLS::Cre]	OH18918	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V;otEx8084[hsp-16.2p::3xNLS::Cre]	OH18506	This Study	
him-8(e1489)/IV;otEx8152[srab-20p::TagRFP, srab-20p::CD4::GFP1-10,flp-18p::TagRFP, flp-18::CD4::GFP11]	OH18858	This Study	
him-8(e1489)/IV; fmi-1(ot1291)/V;otEx8152[srab-20p::TagRFP, srab-20p::CD4::GFP1-10,flp-18p::TagRFP, flp-18::CD4::GFP11]	OH18860	This Study	
him-8(e1489)/IV; fmi-1(ot1291)/V;otEx8152[srab-20p::TagRFP, srab-20p::CD4::GFP1-10,flp-18p::TagRFP, flp-18::CD4::GFP11], otEx8032[srab-20p::fmi-1a::SL2::3xNLS::GFP]	OH18979	This Study	
him-5(e1490) fmi-1(ot1349)/V;otEx8084[hsp-16.2p::3xNLS::Cre::p10UTR],otEx8152[srab-20p::TagRFP, srab-20p::CD4::GFP1-10,flp-18p::TagRFP, flp-18::CD4::GFP11]	OH19023	This Study	
lin-29(xe38)/II; otIs839;him-5(e1490)/V; otEx7916[gpa-6p::fem-3::SL2::2xNLS::TagRFP-T]	OH17830	This Study	
otEx6829	OH14590	Cook et al., 2019.	
lin-29(xe38)/II; otIs614; him-5(e1490)/V	OH18553	This Study	
otIs614	OH13577	Oren-Suissa et al., 2016	
otIs630	OH14099	Oren-Suissa et al., 2016	
him-8(e1489)/IV;otEx8176[srab-20p::gfp::cla-1 15ng/ul, flp-18p::avr-14::TagRFP]	OH18983	This Study	
him-8(e1489)/IV; fmi-1(ot1291)/V;otEx8176[srab-20p::gfp::cla-1 15ng/ul, flp-18p::avr-14::TagRFP]	OH18984	This Study	
lin-29(xe38)/II; him-8(e1489)/IV;otEx8176[srab-20p::gfp::cla-1 15ng/ul, flp-18p::avr-14::TagRFP]	OH19008	This Study	
lin-29(xe38)/II; otIs839;him-5(e1490)/V; otEx7916[gpa-6p::fem-3::SL2::2xNLS::TagRFP-T]	OH17830	This Study	
otIs839; him-5(e1490)/V; otEx8164[gpa-6p::fem-3::SL2::2xNLS::TagRFP-T ]	OH18920	This Study	
otIs839; him-5(e1490)/V; otEx8165[flp-18p::fem-3::SL2::2xNLS::TagRFP-T]	OH18921	This Study	
lin-29(xe38);otI839; him-5(e1490); otEx7929[srab-20p::lin-29a(delZn)::SL2::3xNLS::GFP]	OH17984	This Study	
otIs839; him-5(e1490); otEx7928[srab-20p::lin-29a(delZn)::SL2::3xNLS::GFP]	OH17881	This Study	
lin-29(xe38);otI839; him-5(e1490); otEx7929[srab-20p::lin-29a(delZn)::SL2::3xNLS::GFP]	OH17984	This Study	
otIs839; him-5(e1490)/V; otEx7930[srab-20p::znf362::SL2::3xNLS::GFP]	OH17895	This Study	
otIs839; him-5(e1490)/V; otEx7931[srab-20p::znf362::SL2::3xNLS::GFP]	OH17896	This Study	
lin-29(xe38);otI839; him-5(e1490); otEx7929[srab-20p::lin-29a(delZn)::SL2::3xNLS::GFP]	OH17984	This Study	
lin-29(xe38)/II; him-5(e1490)/V; dmd-4(ot935)/X; otEx7997[srab-20p::znf-362::SL2::TagRFP]	OH18228	This Study	
lin-29(xe38)/II; him-5(e1490)/V; dmd-4(ot935)/X;otEx7998[srab-20p::znf-362::SL2::TagRFP]	OH18231	This Study	
tph-1(ot1274)/II;; him-8(e1489); otIs902	OH18769	This Study	
him-5(e1490) fmi-1(ot1429)[fmi-1::6xGFP11]	OH18838	This Study	
him-5(e1490) fmi-1(ot1429)[fmi-1::6xGFP11];otEx8148[srab-20p::myriGFP::SL2::TagRFP]	OH18839	This Study	
lin-29(xe38)/II; him-5(e1490) fmi-1(ot1429)[fmi-1::6xGFP11];otEx8148[srab-20p::myriGFP::SL2::TagRFP]	OH18887	This Study	
otIs839; him-5(e1490) fmi-1(ot1349)/V;otEx8084[Phsp-16.2::3xNLS::Cre]	OH18506	This Study	
lin-29(xe38)/II;fmi-1(ot1349) him-5(e1490)/V; otIs839; otEx8084[hsp-16.2p::3xNLS::Cre]	OH19231	This Study
==== Refs
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