Cross-Tissue Coordination between SLC Nucleoside Transporters Regulates Reproduction inCaenorhabditis elegans

32 Metabolism is fundamental to organism physiology and pathology. From the intricate network of 33 metabolic reactions, diverse chemical molecules, collectively termed as metabolites, are 34 produced. In multicellular organisms, metabolite communication between different tissues is 35 vital for maintaining homeostasis and adaptation. However, the molecular mechanisms 36 mediating these metabolite communications remain poorly understood. Here, we focus on 37 nucleosides and nucleotides, essential metabolites involved in multiple cellular processes, and 38 report the pivotal role of the SLC29A family of transporters in mediating nucleoside 39 coordination between the soma and the germline. Through genetic analysis, we discovered that 40 two Caenorhabditis elegans homologs of SLC29A transporters, Equilibrative Nucleoside 41 Transporter ENT-1 and ENT-2, act in the germline and the intestine, respectively, to regulate 42 reproduction. Their knockdown synergistically results in sterility. Further single-cell 43 transcriptomic and targeted metabolomic profiling revealed that the ENT double knockdown 44 specifically affects genes in the purine biosynthesis pathway and reduces the ratio of guanosine 45 to adenosine levels. Importantly, guanosine supplementation into the body cavity/pseudocoelom 46 through microinjection rescued the sterility caused by the ENT double knockdown, whereas 47 adenosine microinjection had no effect. Together, these studies support guanosine as a rate 48 limiting factor in the control of reproduction, uncover the previously unknown 49 nucleoside/nucleotide communication between the soma and the germline essential for 50 reproductive success, and highlight the significance of SLC-mediated cell-nonautonomous 51 metabolite coordination in regulating organism physiology. 52 53 54 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 3 Author Summary 55 Metabolism is essential for life, involving a complex network of chemical reactions that requires 56 a well-organized system to maintain efficiency. This includes the optimal allocation of resources 57 and the dynamic exchange of metabolic products between various compartments within an 58 organism. Solute carriers (SLCs) are the largest family of transporters for metabolic products 59 across the animal kingdom. In our research, we investigated how specific SLC transporters 60 collaborate to move key metabolic products between different tissues. We identified two SLC 61 transporters, Equilibrative Nucleoside Transporter ENT-1 and ENT-2, which are vital for 62 transporting guanosine, a purine nucleoside, to support successful reproduction in the nematode 63 Caenorhabditis elegans. We discovered that ENT-2 acts in the gut to export guanosine to the 64 surrounding body cavity, while ENT-1 functions in the germline to import guanosine from the 65 body cavity. When both transporters are disrupted, the animals experience significant 66 reproductive defects. Our study underscores the importance of coordinated activity between SLC 67 transporters in different tissues to maintain organism health. A breakdown in this communication 68 can result in metabolic imbalances and physiological dysfunction. 69 70 71 72 73 74 75 76 77 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 4

78 In multicellular eukaryotic organisms, a complex network of small metabolites, encompassing 79 amino acids, glucose, lipids, and nucleotides/nucleosides, serves as the foundational framework 80 for cellular homeostasis. Perturbations in this intricate metabolic network have been linked to 81 various chronic diseases such as diabetes, cancers, and neurodegenerative disorders (Diamanti et 82 al., 2022; Pothiwala et al., 2009; Procaccini et al., 2016). In particular, nucleosides and 83 nucleotides are essential for DNA replication and RNA synthesis to enable cell growth and 84 division. Imbalances in nucleotide species can disrupt genome stability, mitochondrial activity, 85 cell proliferation, muscle integrity, germline maintenance, and organism development (Chi et al., 86 2016; Diehl et al., 2022; Marsac et al., 2019; Pai and Kearsey, 2017). 87 88 The synthesis of DNA and RNA precursors, nucleotides, primarily involves de novo and salvage 89 pathways. In the de novo pathway, nucleotides are synthesized from various substrates such as 90 amino acids, PPRP (Phosphoribosyl pyrophosphate), and tetrahydrofolate (Sato et al., 2006), 91 through a series of enzymatic steps. On the other hand, the salvage pathway, known for its higher 92 energy efficiency, directly produces nucleotides from free purine/pyrimidine nucleobases and 93 nucleosides. While nucleobases and nucleosides are preferentially salvaged within the cell, their 94 uptake from the extracellular space also plays crucial roles in regulating nucleotide balance, 95 especially in tissues with high demand. In C. elegans, the germline, which carries cells 96 undergoing rapid proliferation and division, requires a large amount of nucleotide synthesis. Our 97 recent findings found that the mitochondrial GTP but not ATP level in the germline regulates 98 reproductive activities during aging, which is coupled with bacterial inputs from the intestine 99 (Lee et al., 2023). 100 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 5 101 Solute carrier (SLC) transporters are one of the two major transporter superfamilies responsible 102 for the transport of a diverse range of small molecules across the plasma membrane and 103 subcellular organelle membranes. These transporters are essential for cellular homeostasis, 104 regulating the uptake and efflux of various vital nutrients such as glucose, amino acids, fatty 105 acids, vitamins, and ions (Zhang et al., 2019). They also play a significant role in whole-body 106 physiology, as many are expressed in a tissue-specific manner (Lin et al., 2015). Equilibrative 107 Nucleoside Transporter (ENT) family are encoded by SLC29 family genes, and mediate the 108 sodium independent transportation of different nucleobases and nucleosides across membranes 109 (Baldwin et al., 2004; Griffith and Jarvis, 1996). Despite the relatively well-studied role of ENT 110 transporters in the cell-autonomous regulation of nucleosides, understanding whether and how 111 they coordinate to mediate nucleoside transport across tissues remains limited. In this work, we 112 found that C. elegans ENT-1 and ENT-2 transporters control nucleoside transport between the 113 soma and the germline. Specifically, ENT-2 transports nucleosides from the intestine to the body 114 cavity/pseudocoelom, while ENT-1 facilitates their transportation from the pseudocoelom to the 115 germline. Their synergistic action is essential for reproduction. Moreover, our studies suggest the 116 substrate specificity of ENT-1/2 towards guanosine, through the integration of single-cell 117 transcriptomic profiling, targeted metabolomic profiling, and chemical screening. These findings 118 serve as a pioneering example of SLC transporters functioning in different tissues to coordinate 119 the cell non-autonomous communication of vital metabolites, thereby supporting organism 120 physiology. 121 122 123 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 6

and Discussion 124 ENT-1 and ENT-2 work together to regulate reproduction 125 In human, four ENT members exhibit varying tissue abundance and subcellular distribution 126 (Baldwin et al., 2004). C. elegans possesses seven ENT family members: ENT-1 to ENT-7. 127 Despite significant separation in the phylogenetic tree and limited amino acid sequence similarity 128 between C. elegans ENTs and human ENTs (Fig 1A), the predicted protein structures of ceENTs 129 based on AlphaFold2 are highly conserved with human ENT1 (Fig 1B) (Wright and Lee, 2019). 130 Among C. elegans ENTs, ENT-6 appears relatively divergent from others, while ENT-4, ENT-5, 131 and ENT-7 are relatively close to each other (Fig 1A). ENT-1 and ENT-2 exhibit a notably close 132 relationship (Fig 1A) and share 84% identity in exon sequences and 94% in amino acid 133 sequences (Appleford et al., 2004), which has been previously suggested to result from a 134 relatively recent gene duplication event (Sankar et al., 2002). 135 136 In C. elegans adulthood, only germline cells continue to undergo proliferation and division. To 137 investigate whether successful reproduction depends on nucleoside transport from other tissues 138 into the germline, we examined reproduction in worms where ENTs were knocked down by 139 RNA interference (RNAi). We found that RNAi knockdown of either ent-1 or ent-2 but not other 140 ents reduces the total progeny number (Figs 1C-E). RNAi knockdown of either ent-1 or ent-2 141 reduced the number of progenies in the first two days of the reproductive period, but increased 142 the number afterward, leading to an extension in the duration of the reproductive process 143 (reproductive lifespan) (Figs 1F and 1G). 144 145 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 7 Our previous studies revealed that C. elegans exhibits different reproductive strategies when 146 exposed to different types of bacteria (Lee et al., 2023; Sowa et al., 2015). Specifically, wild-147 type worms grown on OP50 E. coli have extended reproductive lifespan, in comparison with 148 those grown on HT115 E. coli that is commonly used for RNAi. We thus examined the effect of 149 ent-1 and ent-2 RNAi knockdown on reproduction in the background of OP50 E. coli. We 150 observed a similar extension of reproductive lifespan, however the reduction in the total progeny 151 number did not reach significance (S1A and S1B Figs). 152 153 Next, to confirm the result of RNAi inactivation, we utilized the CRISPR-Cas9 technique 154 (Dickinson and Goldstein, 2016) to generate knockout mutant strains for both ent-1 (ent-1KO) and 155 ent-2 (ent-2KO) (S1C Fig). We found that in the background of either OP50 or HT115 E. coli, the 156 reproductive pattern of the ent-1KO mutant is indistinguishable from WT, while the ent-2KO 157 mutant slightly increased the progeny number on the third day of the reproductive period (S1D- 158 S1G Figs). Further RT-qPCR analysis revealed elevated ent-2 mRNA levels in the ent-1 KO 159 mutant, and increased ent-1 mRNA levels in the ent-2 KO mutant (S1H, and S1I Figs), which 160 suggests that the loss of one transporter leads to the compensatory induction of the other one. In 161 supporting this idea, we found that the double mutant of ent-1 KO and ent-2 KO exhibited complete 162 sterility with developmental delay and vulva protrusion, which is consistent with previous 163 observations (Appleford et al., 2004). We also knocked down ent-2 by RNAi in the ent-1 164 knockout mutant (ent-1KO;ent-2KD) or knocked down ent-1 by RNAi in the ent-2 knockout 165 mutant (ent-1KO;ent-2KD). We found that under both conditions, the brood size is largely reduced 166 (Figs 1H and 1I). The phenotype associated with ent-1KO;ent-2KD is much stronger, close to 167 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 8 being sterile (Fig 1H, S2A Fig). These results suggest that ENT-1 and ENT-2 transporters 168 function together to regulate reproduction. 169 170 Germline ENT-1 and intestinal ENT-2 coordinate to regulate reproduction 171 To identify the functional tissues where these two ENTs act to regulate reproduction, we first 172 examined their expression pattens. We generated two CRISPR knock-in lines in which 173 endogenous ENT-1 and ENT-2 is tagged with mNeonGreen and wrmscarlet at the C-terminus, 174 respectively (S2B and S2C Figs). Using these lines, we revealed that ENT-1 expresses in the 175 germline and intestine, and ENT-2 expresses in gonadal sheath cells and the intestine (Figs 2A, 176 2B and S2D, S2E Figs). Next, we restored the expression of either ent-1 or ent-2 specifically in 177 the intestine of the ent-1KO or ent-2 KO mutant, and then performed RNAi knock down of either 178 ent-2 or ent-1, respectively. We found that the intestine-specific restoration of ent-2, but not ent-179 1, increases the brood size in the worms with both ent-1 and ent-2 knockdown (Figs 2C, 2D). In 180 parallel, we restored the expression of ent-2 specifically in gonadal sheath cells of the ent-2 KO 181 mutant and knocked down ent-1 by RNAi. We found this restoration does not rescue the 182 reduction in the brood size (Fig 2E). We also attempted to restore the expression of ent-1 in the 183 germline of the ent-1 KO mutant but failed to obtain a stable line. 184 185 We thus applied an auxin-inducible degradation (AID) system (Zhang et al., 2015) to deplete 186 ENT-1 proteins selectively in the germline. The AID system utilized a plant-specific F-box 187 protein that recognizes degron-tagged substrates upon auxin binding and induces their 188 degradation by proteasome (Fig 2F). We generated a CRISPR knock-in line in which 189 endogenous ENT-1 is tagged with degron and V5 tag at the N-terminus (S2F Fig), and 190 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 9 subsequently crossed this line with TIR1 tissue-specific expressing lines, germline (gld-191 1p::TIR1::mRuby) and intestine (ges-1p::TIR1::mRuby) (Fig 2F). In these crossed lines, the 192 auxin treatment resulted in selective degradation of ENT-1 in the germline or intestine (S2H 193 Fig). With RNAi knockdown of ent-2, we observed that auxin-induced germline-specific 194 depletion of ENT-1 resulted in a 2-fold reduction in brood size compared to the control without 195 the auxin treatment (Fig 2G). In contrast, the intestine-specific depletion of ENT-1 did not 196 reduce the brood size compared to the control (Fig 2H), which is consistent with the result using 197 the intestine-specific rescuing strain (Fig 2C). 198 199 We have also generated the degron-tagged ent-2 CRISPR knock-in line and crossed it with the 200 ent-1KO mutant (Fig S2G). This strain was further crossed with the TIR1 tissue-specific 201 expressing lines, gonadal sheath cell (lim-7p::TIR1::mRuby) and intestine (ges-202 1p::TIR1::mRuby) (Fig 2F). In these crossed lines, the treatment of auxin induces specific 203 degradation of ENT-2 either in the gonadal sheath cells or intestine (Fig S2I). Consistent with 204 the findings using tissue-specific rescuing strains, we found that the depletion of ent-2 205 specifically in the intestine led to a largely reduced brood size (Fig 2I), while the depletion in 206 gonadal sheath cells does not affect brood size (Fig 2J), when compared to the controls. 207 Together, these results demonstrated that ENT-1 and ENT-2 function in the germline and 208 intestine, respectively to regulate reproduction in a coordinated manner. 209 210 snRNA-seq profiling reveals the effect of ENTs on purine metabolism 211 Following our identification of the synergistic effect between ENT-1 and ENT-2 and their tissue-212 specificity, we next sought to understand how they coordinate the intestine and the germline to 213 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 10 regulate reproduction. To this end, we conduced single-nuclei RNA sequencing (snRNA-seq) 214 analysis and profiled cell type-specific transcriptomic changes in the ent-1KO, ent-2KO, and ent-215 1KO;ent-2KD worms. Approximately 3,000 worms were collected for each condition, and nuclei 216 were isolated using fluorescence-activated cell sorting (FACS) and profiled using the 10X 217 Genomics platform (Gao et al., 2024). Following pre-processing, cell filtering, and quality 218 control, we obtained 56,992 single-nucleus transcriptome profiles, which were subsequently 219 annotated into 14 distinct cell types (Fig 3A). Analysis of cell composition across these 220 conditions revealed a notable decrease in the proportion of germline cells and a higher proportion 221 of somatic cells collected from the ent-1KO;ent-2KD worms (Fig 3B), which is consistent with the 222 reduced brood size. 223 224 We further conducted germ cell pseudotime inference analysis (Gao et al., 2024), to examine 225 changes in the germline composition. This analysis allowed us to construct a pseudotemporal 226 order of germ cells (S3A Fig), where the x-axis signifies pseudotime denoting the progression 227 from germline stem cells (GSCs) through mitotic cells and meiotic cells to mature oocytes. We 228 found that the GSC number is increased in the ent-1KO single mutant but decreased in the ent-2KO 229 single mutant (S3A Fig), suggesting distinctive alterations in germline homeostasis despite the 230 absence of brood size changes in these single mutants. Moreover, in the ent-1KO;ent-2KD worms, 231 we observed an increased proportion of germ cells in proliferation stages, including GSC and 232 those in the mitotic zone (Fig 3C). In contrast, the proportion of cells in the meiosis stage was 233 decreased (Fig 3C), while the transition zone remained unchanged. These results suggest a 234 defect in germ cell differentiation associated with the loss of both ENT-1 and ENT-2. 235 236 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 11 We also performed the differential expressed gene (DEG) analysis to reveal molecular changes 237 in the germline upon the loss of ENT-1 and ENT-2. 716 DEG genes were identified through the 238 comparison between ent-1KO;ent-2KD worms and their controls, with a significance cutoff of p 239 adjust value 0.5. Among these genes, 302 were upregulated, while 240 412 were downregulated (Fig 3D). The Gene Ontology (GO) pathway analysis of the 241 downregulated genes in the germline of the ent-1KO;ent-2KD worms highlighted pathways 242 associated with cell cycle, chromosome organization, and mRNA processing, which is likely 243 resulted from the loss of differentiated germ cells in these worms (S3B Fig). When analyzing the 244 upregulated genes, we found that GO terms related to purine metabolism are overrepresented in 245 the ent-1KO;ent-2KD germline (Fig 3E). 246 247 A series of purine metabolic genes in both de novo and recycling pathways are conserved in C. 248 elegans (Marsac et al., 2019) (Fig 3F). When analyzing their expression levels using the snRNA-249 seq data, we found that most purine metabolic genes showed trends of up-regulation in the ent-250 1KO;ent-2KD germline (Fig 3G), while genes encoding enzymes involved in the de novo pathway 251 showed trends of down-regulation in the intestine (Fig 3H). However, no clear trends of changes 252 detected in other somatic tissues such as hypodermis and muscle (S3C and S3D Figs). 253 Furthermore, when analyzing conserved metabolic genes in the pyrimidine pathway (Fig 3I), we 254 did not observe an obvious trend of alterations in their expression in either the germline or 255 intestine of the ent-1KO;ent-2KD worms (Figs 3J and 3K). 256 257 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 12 Together, these results suggest that the level of purine, rather than pyrimidine, is likely altered in 258 the germline and intestine upon the loss of both ENT-1 and ENT-2, leading to compensatory 259 transcriptional alterations of purine biosynthesis genes. 260 261 ENT-mediated guanosine transport regulates reproduction 262 To directly assess whether purine and pyrimidine nucleoside levels change in association with 263 the ENT deficiency, we employed targeted metabolomic analysis by liquid chromatograph 264 coupled with mass spectrometry (LC/MS). We compared guanosine, adenosine, inosine, and 265 cytidine levels in ent-1KO, ent-2KO, and ent-1KO;ent-2KD worms with their controls. The levels of 266 uridine and thymidine are below the detection sensitivity. Interestingly, we observed that the 267 proportion of guanosine is decreased by ~20% and ~30% in the ent-1KO and ent-2KO single 268 mutants, respectively (Fig 4A). In the ent-1KO;ent-2KD worms, the decrease is close to be 3-fold 269 (Fig 4A). For the other two purine nucleosides, adenosine and inosine, their proportion exhibited 270 the opposite trend (Figs 4B and 4C). On the other hand, the proportion of cytidine did not show 271 significant alterations in either ent-1KO, ent-2KO, or ent-1KO;ent-2KD worms compared to their 272 controls (Figs 4D). It is worth noting that no reduction in the brood size was observed in the ent-273 1KO or ent-2KO single mutant, despite the decrease in guanosine. Hence, the germline is tolerant 274 to a certain level of reduction in guanosine levels while sustaining normal reproduction, but 275 exceeding this threshold would result in reproductive defects. 276 277 These results support the predominant role of ENT-1/2 in regulating purine especially guanosine 278 homeostasis. When integrating the tissue-specificity of ENT-1/2, the germline-specific 279 transcriptional up-regulation of purine metabolic genes, and the guanosine reduction, we 280 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 13 hypothesized that ENT-2 functions in the intestine to export guanosine into the body 281 cavity/pseudocoelom, while ENT-1 is responsible for the uptake of guanosine into the germline 282 (Fig 4E). The loss of ENT-2 in the intestine results in reduced guanosine availability in the 283 pseudocoelom, while the loss of ENT-1 in the germline compromises the uptake of guanosine 284 from the pseudocoelom. With the single mutation of either ent-1 or ent-2, the reduction of 285 guanosine in the germline does not reach the threshold for severe disruption of reproduction. 286 However, this reduction threshold will be met with the simultaneous loss of ENT-1 and ENT-2 287 (Fig 4F). 288 289 In supporting this hypothesis, we confirmed that ENT-2::wrmscarlet predominantly localizes at 290 the basolateral plasma membrane of the intestine (S2E Fig), while ENT-1::mNeonGreen is 291 positioned at the plasma membrane in the germline (S2D Fig). We also injected guanosine or 292 adenosine into the pseudocoelom at the fourth larval (L4) stage to increase their levels in the 293 pseudocoelom. We found that the microinjection of 1mM guanosine can increase the brood size 294 in the ent-1KO;ent-2KD worms, but adenosine microinjection showed no effect in restoring 295 reproduction (Fig 4G). However, feeding of either guanosine or adenosine was not able to rescue 296 the brood size in the ent-1KO;ent-2KD worms (Fig 4H). We also tried directly injecting 297 nucleosides into the germline of the ent-1KO;ent-2KD worms; however, the severe disruption in 298 germline morphology prevented this attempt. These results highlighted the importance of 299 guanosine levels in the pseudocoelom in the regulation of reproduction (Fig 4F). 300 301 In summary, our study underscores the significance of guanosine communication between the 302 soma and germline in the control of reproduction, as well as the pivotal role played by specific 303 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 14 SLC transporters in mediating this cell non-autonomous metabolite communication. SLC 304 transporters are highly conserved in humans and exhibit diverse tissue-specificity, and their 305 mutations have been associated with human diseases. Current research of these mammalian 306 homologs focuses on identifying their substrates and understanding their cell-autonomous 307 regulatory functions. It would be interesting to explore how their cell non-autonomous 308 coordination contributes to mammalian physiology and pathology in future studies. 309 310

311 C. elegans strains and maintenance 312 The strains N2, CA1352 (ieSi64 [gld-1p::TIR1::mRuby::gld-1 3 UTR + Cbr-unc-119(+)] II), 313 and CA1209 (ieSi61 [ges-1p::TIR1::mRuby::unc-54 3'UTR + Cbr-unc-119(+)] II), and 314 MQD2383 (hqSi11 [lim-7p::TIR1::mRuby::unc-54 3' UTR + Cbr-unc-119(+)] II; daf-315 2(hq363[daf-2::degron::mNeonGreen]) unc-119(ed3) III), were obtained from CGC. 316 317 Knockout mutants MCW1244 (ent-1(rax74) IV) and MCW1245 (ent-2(rax75)X) were generated 318 in our lab by using CRISPR/Cas9 technology, as previous outlined by Chen et al. (Chen et al., 319 2014), Paix et al.(Paix et al., 2015) and Arribere et al.(Arribere et al., 2014) with modifications. 320 Briefly, a mixture containing tracrRNA (1μg/μl), crRNAs (0.5μg/μl each for one on the 5' and 321 one on the 3' of the target gene), dpy-10 crRNA (0.16μg/μl), and Cas9 protein (0.05μg/μl) was 322 microinjected into the gonads of N2 young adult animals. Each injected worm was then placed 323 on individual plates, and the non-Dpy F1 progenies from the plates which contains animals with 324 Dpy phenotypes were individualized to single plates for further analysis. After confirming the 325 deletion region through PCR and Sanger sequencing of F1 animals, homozygous F2 animals 326 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 15 carrying the knockout mutation were selected and individualized. The knockout deletion strains 327 were backcrossed to wild-type N2 for at least four times before running reproductive 328 experiments. 329 330 Strains that carry extrachromosomal arrays: MCW1589(ent-2(rax75)X; raxEx624[lim-7p::ent-331 2cds::sl2RFP::tbb-2 3'UTR; lin-44p::GFP], MCW1634(ent-1(rax74) IV; raxEx628[ges-1p::ent-332 1cds:sl2RFP::unc-54 3'UTR; lin-44p::GFP], MCW1636(ent-2(rax75)X; raxEx630[ges-1p::ent-333 2cds:sl2RFP::unc-54 3'UTR; lin-44p::GFP] were generated by microinjecting DNA mixture 334 containing linearized expression construct and co-injection marker lin-44p::GFP into the 335 corresponding gonad of ent-1 or ent-2 knockout young adult animals. 336 337 PHX4218(ent-1(syb4218) ent-1::mNeonGreen IV), PHX5055(ent-2(syb5055) ent-338 2::wrmscarlet::3Xflag X), PHX7376(ent-1(syb7376) 3XV5::degron::ent-1 IV), PHX7457(ent-339 2(syb7457) 3XHA::degron::ent-2 X) were generated via CRISPR/Cas9 genome editing by 340 SunyBiotech (Fuzhou, China). 341 342 MCW1638 (syb7376[3XV5::degron::ent-1]IV; ieSi61 [ges-1p::TIR1::mRuby::unc-54 3'UTR + 343 Cbr-unc-119(+)]II) and MCW1639 (syb7376[3XV5::degron::ent-1]IV; ieSi64 [gld-344 1p::TIR1::mRuby::gld-1 3'UTR + Cbr-unc-119(+)] II) were generated by crossing PHX7376 345 with CA1209 or CA1352. 346 347 MCW1640 (syb7457[3XHA::degron::ent-2]X; ieSi61 [ges-1p::TIR1::mRuby::unc-54 3'UTR + 348 Cbr-unc-119(+)] II and MCW1641 (syb7457[3XHA::degron::ent-2]X; ent-1(rax74) IV; hqSi11 349 II[lim-7p::TIR1::mRuby::unc-54 3' UTR + Cbr-unc-119(+)] II) were generated by crossing 350 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 16 PHX7457 with CA1209 or the strain hqSi11 [lim-7p::TIR1::mRuby::unc-54 3' UTR + Cbr-unc-351 119(+)] II which were obtained from crossing MQD2383 and N2. 352 353 MCW1643 (syb7457[3XHA::degron::ent-2]X; ent-1(rax74) IV; ieSi61 [ges-354 1p::TIR1::mRuby::unc-54 3'UTR + Cbr-unc-119(+)] II and MCW1644 355 (syb7457[3XHA::degron::ent-2]X; ent-1(rax74) IV; hqSi11 II[lim-7p::TIR1::mRuby::unc-54 3' 356 UTR + Cbr-unc-119(+)] II) were generated by crossing MCW1640 or MCW1641 with 357 MCW1244. 358 359 All the C. elegans strains were grow and maintained non-starved for at least three generations at 360 20˚C on NGM agar plates seeded with OP50 E.coli using standard protocols (Stiernagle, 2006) 361 before experiments. The E. coli strain HT115 (DE3) and OP50 RNAi strain (OP50 bacteria 362 [rnc14::DTn10 laczgA::T7pol camFRT] generated by our lab (Neve et al., 2020) were used for 363 RNAi experiments. 364 365 RNA interference (RNAi) experiments 366 RNAi libraries from Dr. Julie Ahringer's lab were utilized in the study (Kamath and Ahringer, 367 2003). RNAi clones for ent-1, ent-2, ent-3, ent-5, and ent-6 were obtained from the Ahringer 368 library. RNAi clones for ent-4 and ent-7 were created in our lab using L4440 as the vector 369 backbone and ent-4 or ent-7 transcript fragments as inserts. For OP50 RNAi experiments, RNAi 370 plasmids were transformed into the genetically modified competent OP50 bacteria 371 [rnc14::DTn10 laczgA::T7pol camFRT], generated by our lab (Neve et al., 2020). All RNAi 372 colonies were selected for resistance to both 50 µg ml−1 carbenicillin and 50 µg ml−1 373 tetracycline. RNAi bacteria were cultured for 14 hours in LB with 25 µg ml−1 carbenicillin, then 374 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 17 seeded onto RNAi agar plates containing 1 mM IPTG and 50 µg ml−1 carbenicillin. Each RNAi 375 bacteria clone was allowed to dry on the plates before overnight incubation at room temperature 376 to induce dsRNA expression. 377 378 Molecular cloning of expression construct 379 All the expression plasmids were generated via the Gibson Assembly (NEB). The ent-1 and ent-380 2 coding sequences were PCR-amplified from C. elegans cDNA and then fused together with 381 sl2-RFP sequence by fusion PCR. The ent-1::sl2RFP and ent-2::sl2RFP fragments were then 382 ligated into the tissue-specific promoter vectors. 383 384 Measure the brood size 385 Synchronized L1 worms obtained from egg preparation were placed onto 6cm NGM plates, each 386 seed with corresponding bacteria for the experimental condition, and then incubated at 20 °C. 387 Upon reaching the L4 stage, individual worms were transferred to new plates. Subsequently, they 388 were transferred to new plates every day until reproduction ceased. Plates with progenies were 389 stored at 20°C until progeny reached the L4 stage for counting. Total brood size was determined 390 by summing viable progeny produced daily by each worm. 391 392 Structural stimulation by AlphaFold2 393 AlphaFold2 predictions for the structures of ceENT-1 (UniProt: G5EDJ3) and ceENT-2 394 (UniProt: Q93871) were retrieved from The AlphaFold Protein Structure Database 395 (https://alphafold.ebi.ac.uk/) (Jumper et al., 2021; Varadi et al., 2022). Molecular graphics for 396 human ENT1, ceENT-1, and ceENT-2 were generated using UCSF Chimera (Pettersen et al., 397 2004). 398 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 18 399 Auxin treatment 400 The L1 worms were places on the NGM plates seeded with bacteria containing auxin 4mM 401 indole-3-acetic acid (IAA), following the protocol outlined by Zhang et al.(Zhang et al., 2015). A 402 400 mM stock solution of IAA in ethanol was prepared, filtered through a 0.22µm filter, and 403 stored at 4°C for up to 1 month. This stock solution was diluted into NGM plates at a ratio of 404 1:100. Control plates were prepared by diluting ethanol into NGM plates at the same ratio. Fresh 405 bacteria were then seeded onto the plates and stored at room temperature for 1 day to allow 406 bacterial growth. The plates containing auxin were kept in a dark place to prevent photolysis. 407 408 RT-qPCR 409 Total RNA was extracted from approximately 3000 age synchronized D1 worms using Trizol 410 homogenization, chloroform phase separation, isopropanol precipitation, and subsequent 411 washing with 75% ethanol. 412 cDNA synthesis utilized the amfiRivert Platinum cDNA Synthesis Master Mix (GenDEPOT), 413 followed by quantitative PCR with the Kapa SYBR fast qPCR kit (Kapa Biosystems) in a 96-414 well Eppendorf Realplex 4 PCR machine (Eppendorf). 415 All presented data are from at least six independent biological samples and were normalized to 416 rpl-32 as an internal control. 417 418 Fluorescent microscopy 419 C. elegans were immobilized in 1% sodium azide in M9 buffer and positioned on a 2% agarose 420 pad between a glass microscopic slide and coverslip for imaging. Endogenous ENT-421 1::mNeonGreen and ENT-2::wrmscarlet expression patterns were visualized using a Nikon CSU-422 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 19 W1 spinning disk confocal microscopy system equipped with a 20x objective, oil immersion 60x 423 and 100x objective. Degron-mediated protein degradation was assessed using a laser scanning 424 confocal FV3000 (Olympus, US) with an oil immersion 60x objective. 425 426 Injection of nucleoside into the pseudocoelom 427 Stock solutions of nucleosides are stored at 100mM in DMSO at -20°C for up to 1 month. Fresh 428 1mM nucleoside injection solutions are prepared before each injection by dissolving the 100mM 429 stock solution in egg buffer [118 mM NaCl, 48 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 25 430 mM Hepes (pH 7.3)] at a ratio of 1:100. The control injection solution consists of DMSO 431 dissolved at a ratio of 1:100. Mid-stage L4 animals were injected with either the nucleoside 432 solution or DMSO control solution into the pseudocoelom , with the injection needle inserted 433 into the pharyngeal region. Successful injection is confirmed by the observation of liquid flow in 434 the pseudocoelom. 435 436 Immunostaining 437 Approximately 200 Adult animals were initially transferred to unseeded NGM plates to 438 minimize bacterial presence. M9 solution containing 0.4μM levamisole was then added to 439 immobilize animals on the plates. Subsequently, these animals, along with the M9 solution 440 containing 0.4μM levamisole, were transferred to a glass dissection plate for dissection. 441 Dissection involved using two 25 gauge syringe needles to extrude the gonad arm and intestine 442 completely. The dissected worms were fixed with 4% PFA for 10 minutes at room temperature 443 in darkness, followed by washing twice with PBST and post-fixation in −20°C methanol for 1 444 hour. After three washes with PBST, the specimens were then blocked with 5% BSA in PBST 445 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 20 for 30 minutes. Following blocking, a 200μL volume of primary antibody solution (in PBST) 446 [anti-HA antibody (1:200): HA-Tag (C29F4) Rabbit mAb #3724; anti-V5 antibody (1:200): V5 447 Tag Monoclonal Antibody (SV5-Pk1) (R960-25)] was applied and incubated overnight at 4°C. 448 The cut worms were then washed three times with PBST and incubated with secondary antibody 449 [1:500 dilution of Alexa 488 conjugated antibodies from Invitrogen] for 1 hour at room 450 temperature. After three additional washes with PBST, the specimens were resuspended in a 451 glycerol antifade reagent and mounted on agarose pads before imaging. 452 453 Nucleoside dietary supplementation 454 For dietary nucleoside supplementation, nucleoside powder was dissolved directly to a final 455 concentration of 100mM in standard NGM liquid medium immediately prior to pouring into 456 plates. These plates were then seeded with RNAi E. coli and allowed to grow overnight before 457 use. 458 459 Single-nucleus RNA sequencing analysis 460 Nuclei Isolation 461 We followed the nuclei isolation protocol similar to that described by Gao et al.(Gao et al., 462 2024). Briefly, worms were washed three times with PBS, collected in 1.5 mL tubes, and 463 homogenized in 100 μl of homogenization buffer (Li et al., 2022) using a pestle motor on ice. To 464 prevent nuclei adhesion, all equipment was pre-coated with homogenization buffer or PBS. The 465 homogenate was further processed in an autoclaved Dounce tissue grinder, with sequential 466 strokes using loose and tight pestles to ensure thorough disaggregation without foaming. After 467 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 21 filtering through cell strainers, the nuclei were pelleted by centrifugation, resuspended in PBS 468 with additives, and prepared for sorting. 469 Nuclei Sorting 470 The nuclei were stained with Hoechst 33342 for DNA content visualization and sorted using a 471 Sony MA800 sorter. Gating of sorting was the same as describe by Gao et al. (Gao et al., 2024). 472 Post-sorting, nuclei were checked for concentration and morphology before proceeding with 10X 473 Chromium Controller. 474 Library Preparation and Sequencing 475 The quality-checked nuclei were encapsulated using a 10X Chromium Controller, and libraries 476 were prepared according to the 10X Chromium Single Cell 3’ v2/v3 Solution protocol, selecting 477 appropriate indexing options. Sequencing was performed on a NovaSeq 6000 system, using 26 478 cycles for Read 1, 8 cycles for the i7 index, and 98 cycles for Read 2, as recommended. 479 Single-nucleus RNA-seq Data Preprocessing 480 Raw sequences were processed using Cell Ranger 6.0 (10x Genomics), aligned to the C. elegans 481 genome (WS282), and assembled into feature-barcode matrices. Doublet exclusion was 482 performed per 10X Genomics guidelines, utilizing Doubletfinder for accurate detection. 483 Subsequent analyses, including data integration, dimensional reduction, and clustering, were 484 conducted using Seurat 4.0.5 to identify and characterize cell populations. 485 Cell Type Annotation and Analysis 486 For cell type annotation, we predominantly employed a reference-based mapping approach using 487 the SingleR package (Aran et al., 2019), which allowed us to automatically match the expression 488 profiles of our cells to those from established reference datasets. This analysis predominantly 489 linked cell clusters to specific tissues, as evidenced by previous single-nucleus RNA sequencing 490 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 22 datasets produced by Gao et al. (Gao et al., 2024). We further enhanced cell type specificity by 491 employing the FindMarkers function within Seurat to identify distinctive marker genes for each 492 cluster. These markers were then cross-referenced with microscopy-based expression profiles 493 and literature-reported tissue markers to confirm cell type assignments. For enrichment analysis 494 and further validation of our findings, we utilized tools available on Wormbase (Angeles-495 Albores and Sternberg, 2018). 496 In addition to broad classification, we also focused on the germline cells, isolating them into a 497 separate Seurat object. This subset underwent a similar analysis pipeline, where we identified 498 highly variable genes and performed CCA integration followed by dimensional reduction and 499 clustering to determine germline subclusters. This refined analysis enabled us to explore cell 500 development within the germline specifically. 501 Germline Trajectory Analysis 502 Utilizing the refined germline cluster data, we applied Slingshot for trajectory analysis, which 503 utilized cluster labels and UMAP embeddings to construct cell lineages and ascertain pseudotime 504 trajectories. This analysis revealed the developmental progression from germline stem cells, 505 showing low pseudotime values, to mature oocytes, which displayed high pseudotime values. 506 We correlated these pseudotime findings with nuclei counts reported in the literature (Diag et al., 507 2018), allowing us to map each cluster to specific stages of germline development. This dual 508 analysis of pseudotime distribution and developmental status percentage provided a 509 comprehensive view of germline cell differentiation. 510 Differential Expression and Gene Ontology Analysis 511 To identify differentially expressed genes (DEGs) across various cell states and conditions, we 512 used Seurat's FindMarkers function, employing a Wilcoxon rank-sum test with thresholds set for 513 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 23 adjusted p-values below 0.05 and |log2 fold changes| greater than 0.5. Subsequent gene ontology 514 analysis was performed using the ClusterProfiler package, which helped to understand the 515 biological processes and pathways significantly associated with the identified DEGs. To visually 516 compare gene expression across different samples and conditions, we used the DotPlot function 517 within Seurat, facilitating an intuitive display of data that highlights specific gene expression 518 changes. 519 520 Targeted metabolic analysis of nucleosides 521 Sample collection: 522 Around 5000 C. elegans in each condition were collected in the bead beater tubes (Sarstedt Inc 523 Screw Cap Micro tube 2ml) with 250 µL RNAlater Stabilization Solution (Invitrogen: AM7021) 524 and 10µg/ml of tetrahydrouridine to prevent rapid deamination of the nucleosides (Persaud et al., 525 2023) and then snap freezing in liquid nitrogen which were stored at -80°C until use. 526 Nucleosides extraction and purification 527 Nucleosides were extracted from the samples following a previously outlined method (He et al., 528 2019) with some modifications. Briefly, RNAlater Stabilization Solution was removed from 529 thawed samples, and 250 µL LC-MS H2O with RNase Inhibitor and beads were added to the 530 samples for homogenization with a bead beater. After homogenization, the sample was 531 transferred to a 2 mL tube. pH adjustment to 8.5 was done using ammonium in methanol, 532 followed by addition of methanol at a ratio of 1:4 (v:v). The mixture was then centrifugated at 533 14,000 rpm for 20 min at 4 °C after vortexing for 3 min. The supernatant was collected and dried 534 using SpeedVac. Each dried sample was dissolved then in 250 μL water. 535 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 24 For nucleoside purification, each sample was loaded onto an OASIS HLB cartridge (Waters 536 Corp., Milford, MA, USA) activated 3 times with water and methanol. After loading, cis-diol 537 compounds were eluted with 250 μL 2.8% ammonium hydroxide (NH4OH) in methanol, 538 repeated 3 times. Eluates were then dried using SpeedVac 539 Dried samples were reconstituted in 100 μL water, centrifuged at 14,000 rpm for 10 min at 4 °C, 540 and the clear upper solution was transferred to an LC vial for LC-MS analysis. 541 LC-MS/MS Analysis 542 The experiments were performed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer 543 equipped with Ion Max API source housing with HESI-II probe and Vanquish UHPLC System 544 (ThermoFisher Scientific). The separation was performed on an ACQUITY UPLC HSS T3 545 column (100 × 2.1 mm i.d., 1.8 μm) (Waters Corp., Milford, MA, USA) at a flow rate of 0.2 546 mL/min with column temperature of 40 °C (mobile phase A: 0.1% FA in water, mobile phase B: 547 0.1% FA in acetonitrile). The gradient was as follows: 0-3 min, 0-2.8% B; 3-9 min, 2.8%-10% 548 B; 9-9.5 min, 10-30% B; 9.5-9.8 min, 30-60% B; 9.8-9.9, 60% B; 9.9-10 min, 60-0% B (He et 549 al., 2019). The targeted mode (parallel reaction monitor, PRM) was applied with Orbitrap MS 550 acquired a full-scan survey in positive mode (m/z: 140-1300, automatic gain control target: 551 standard, maximum injection time mode: auto, resolution at m/z 200: 120,000, the default charge 552 state: 1, followed by tMS2 with precursor ions list of nitrogenous bases (mass range: normal, 553 automatic gain control target: standard, maximum injection time mode: auto, Orbitrap with 554 resolution at m/z 200: 60,000, collision-induced dissociation (CID) collision energy (%): 30 with 555 activation time: 10 ms. Maximum injection time: 118 ms). 556 LC-MS/MS Data analysis 557 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 25 The raw files were analyzed by Skyline (Adams et al., 2020; Henderson et al., 2018; MacLean et 558 al., 2010). The transition list was generated, applied and peak area was extracted and calculated 559 by Skyline. All the samples were normalized based on protein concentrations. The sample 560 concentration was calculated based on the standard curve. 561 562 Quantification and statistical analysis 563 Data were expressed as mean ± standard error of the mean (s.e.m.) and analyzed using GraphPad 564 PRISM. Student’s t-test (unpaired) compared the means of two groups. One-way ANOVA or 565 two-way ANOVA followed by Holm–Sidak’s or Holm-Bonferroni’s corrections, as indicated in 566 the figure legends, were applied. Statistical significance in figure legends is denoted by asterisks: 567 ns (not significant, p > 0.05), *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001. Detailed 568 information on sample size, biological replicates, and statistical analysis for each experiment is 569 provided in the figure legends. Figures and graphs were generated using BioRender, GraphPad 570 Prism 10 (GraphPad Software), and Illustrator (CC 2019; Adobe). The researchers were not 571 blinded during experiments or outcome assessment. 572 573

574 We thank A. Dervisefendic and P. Svay for their assistance with worm strain maintenance. We 575 thank I. Neve for the great help in generating CRISPR knockout strains. Several strains were 576 obtained from the Caenorhabditis Genetics Center (CGC), which is supported by the NIH Office 577 of Research Infrastructure Programs (P40 OD010440). M.C.W. receives current support from 578 Howard Hughes Medical Institute. 579 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 26 580 Author Contributions 581 Conceptualization: Youchen Guan and Meng C. Wang 582 Data Curation: Youchen Guan 583 Formal analysis: Youchen Guan 584 Funding acquisition: Meng C. Wang 585 Investigation: Youchen Guan, Yong Yu, Shihong M. Gao, Lang Ding Qian Zhao, and Meng C. 586 Wang 587 Methodology: Youchen Guan and Meng C. Wang 588 Project administration: Meng C. Wang 589 Supervision: Meng C. Wang 590 Validation: Youchen Guan 591 Writing – original draft: Youchen Guan 592 Writing – review & editing: Meng C. Wang 593 594 595 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 27 Figure Legends 596 Fig 1. Specific ENT transporters regulate reproduction 597 (A) Phylogenetic analyses of human and C. elegans ENTs using protein sequences obtained from 598 Uniprot. 599 (B) Comparison between human ENT1 structure (Wright and Lee, 2019), and C. elegans ENT-1 600 (ceENT-1) and ENT-2 (ceENT-1) structures predicted by AlphaFold2. 601 (C) RNA inference (RNAi) knockdown of ent-1 reduces brood size compared to the empty 602 vector (EV) control. n = 22 (EV), n = 26 (ent-1 RNAi). 603 (D) RNAi knockdown of ent-2 reduces brood size compared to the EV control. n = 28 (EV), n = 604 31(ent-2 RNAi). 605 (E) RNAi knockdown of ent-3, ent-4, ent-5, ent-6, or ent-7 does not affect brood size compared 606 to the EV control. n = 17 (EV), n = 17 (ent-3 RNAi), n = 15 (ent-4 RNAi), n = 16 (ent-5 607 RNAi), n = 15 (ent-6 RNAi), n = 16 (ent-7 RNAi). 608 (F) RNAi knockdown of ent-1 alters the daily progeny number compared to the EV control. n = 609 22 (EV), n = 26 (ent-1 RNAi). 610 (G) RNAi knockdown of ent-2 alters the daily progeny number compared to the EV control. n = 611 28 (EV), n = 31(ent-2 RNAi). 612 (H) The ent-1 knockout (ent-1KO) mutant with ent-2 RNAi knockdown (ent-2KD) largely reduces 613 the brood size and daily progeny number, compared to the control. n = 20 (ctrl), n = 22 (ent-614 1KO; ent-2KD). 615 (I) The ent-2 knockout (ent-2KO) mutant with ent-1 RNAi knockdown (ent-1KD) reduces the 616 brood size and daily progeny number, compared to the control. n = 11 (ctrl), n = 10 (ent-2KO; 617 ent1KD). 618 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 28 RNAi condition: HT115 E. coli (C-G); Statistics: *p<0.05; **p<0.01, ***p<0.001, 619 ****p0.05. Student’s t-test (unpaired, two-tailed) was applied for C-E, and the 620 brood size graphs in H, I. Two-way ANOVA with Bonferroni’s post hoc test for F-G, and the 621 daily progeny graphs H, I. Data shown as mean ± S.E.M. 622 623 Fig 2. Germline ENT-1 and intestinal ENT-2 cooperate in regulating reproduction 624 (A) (B) The expression patterns of endogenously labeled ENT-1::mNeonGreen and ENT-625 2::wrmscarlet visualized in their CRISPR knock-in strains. Scale bar: 100 µm. White 626 arrowheads indicate intestine; hollow white arrowhead indicates germline, and white arrows 627 designate gonadal sheath cells. 628 (C) Intestine-specific restoration of ent-1 expression does not rescue the reduced brood size in 629 the ent-1KO mutant with ent-2 RNAi knockdown. 630 (D) Intestine-specific restoration of ent-2 expression rescues the reduced brood size in the ent-631 2KO mutant with ent-1 RNAi knockdown. 632 (E) Restoration of ent-2 expression in somatic gonadal sheath cells does not rescue the reduced 633 brood size in the ent-2KO mutant with ent-1 RNAi knockdown. 634 (F) Illustration of auxin-induced degradation of endogenous ENT-1 tagged with 3XV5 tag and 635 degron and ENT-2 tagged with 3XHA tag and degron in the intestine (ges-1 promoter), in 636 the germline (gld-1 promoter) and gonadal sheath cell (lim-7 promoter). 637 (G) Auxin-induced germline-specific degradation of ENT-1 together with ent-2 RNAi 638 knockdown reduces the brood size. n = 23 (-Auxin), n = 23 (+Auxin). 639 (H) Auxin-induced intestine-specific degradation of ENT-1 together with ent-2 RNAi 640 knockdown does not reduce the brood size. n = 23 (-Auxin), n = 23 (+Auxin). 641 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 29 (I) Auxin-induced intestine-specific degradation of ENT-2 in the ent-1KO mutant decreases the 642 brood size. n = 30 (-Auxin), n = 30 (+Auxin). 643 (J) Auxin-induced intestine-specific degradation of ENT-2 in the ent-1KO mutant does not affect 644 the brood size. n = 30 (-Auxin), n = 30 (+Auxin). 645 RNAi condition: HT115 E. coli (C, D, E, G, H). Statistic: *p<0.05; **p<0.01, ***p<0.001, 646 ****p0.05. Student’s t-test (unpaired, two-tailed) between control group and 647 experimental group was applied for the brood size graphs shown in C, D, E, G, H, I, J. Data 648 were shown as mean ± S.E.M. 649 650 651 Fig 3. SnRNA-seq profiling reveals the regulation of purine metabolism by ENT-1/2 652 (A) The Uniform Manifold Approximation and Projection (UMAP) plot displays fourteen cell 653 types in ctrl, ent-1KO, ent-2KO, and ent-1KO; ent-2KD worms. 654 (B) The percentage of different cell types in total captured cells shows decreased germline nuclei 655 in ent-1KO; ent-2KD worms. 656 (C) The proportion of nuclei within various regions of the germline among total germline nuclei 657 is compared between control and ent-1KO; ent-2KD worms 658 (D) The Volcano plot shows differentially expressed genes in the germline when comparing ent-659 1KO; ent-2KD worms vs. their controls. 660 (E) Gene ontology (GO) enrichment analysis of the up-regulated genes in the germline of ent-661 1KO; ent-2KD worms compared to their controls. The red box highlights GO terms involved in 662 purine metabolism. 663 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 30 (F) Schematic illustration of C. elegans homologous genes involved in purine metabolism, both 664 de novo and salvage pathways, based on the previous study (Marsac et al., 2019). 665 (G, H) Dot plots show the relative expression levels of genes associated with purine metabolism 666 in the germline (G) and intestine (H). The size of dots indicates the percentage of nuclei 667 expressing the gene, and the color indicates the average expression level. 668 (I) Schematic illustration of C. elegans homologous genes involved in pyrimidine metabolism 669 pathways. 670 (J, K) Dot plots show the relative expression levels of genes associated with pyrimidine 671 metabolism in the germline (J) and intestine (K). 672 673 674 Fig 4. Guanosine specifically contributes to the regulation of reproduction by ENT-1/2 675 (A-D) The percentages of guanosine (A), adenosine (B), inosine (C), and cytidine (D) among all 676 nucleosides measured by LC/MS are compared among control, ent-1KO, ent-2KO, and ent-677 1KO;ent-2KD worms. n = 4 (ctrl), n = 5 (ent-1KO), n = 5 (ent-2KO), n = 4 (ent-1KO;ent-2KD). 678 (E) A summary model illustrating the coordinated regulation of the reproductive process by 679 ENT-2 mediated export of nucleosides from the intestine to the pseudocoelom and ENT-1 680 mediated uptake of nucleosides from the pseudocoelom to the germline. 681 (F) Illustration represents possible changes in guanosine levels in the intestine, pseudocoelom, 682 and germline under different conditions, including ctrl, ent-1KO, ent-2KO, ent-1KO;ent-2KD, 683 and ent-1KO;ent-2KD with pseudocoelom guanosine microinjection. 684 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 31 (G) Microinjection of guanosine but not adenosine partially rescues the reduced brood sizes of 685 ent-1KO;ent-2KD worms. n = 22 (DMSO vehicle), n = 34 (Guanosine), n = 22 (1mM 686 Adenosine). 687 (H) Oral supplementation of neither guanosine nor adenosine rescues the reduced brood sizes of 688 ent-1KO;ent-2KD worms. n = 20 (DMSO vehicle), n = 20 (Guanosine), n = 20 (Adenosine). 689 690 Statistic: *p<0.05, ***p<0.001, ****p0.05. One-way ANOVA with Holm–Sidak 691 correction (A-D). Student’s t-test (unpaired, two-tailed) between control group and experimental 692 group (G-H). Data shown as mean ± S.E.M. 693 694 Supporting information 695 S1 Fig. ENT-1 and ENT-2 regulate reproduction independent of bacterial inputs. 696 (A) RNAi knockdown of ent-1 on OP50 bacteria alters the daily progeny number compared to 697 the EV control. n = 17(EV), n = 16(ent-1 RNAi). 698 (B) RNAi knockdown of ent-2 on OP50 bacteria alters the daily progeny number compared to 699 the EV control. n = 17(EV), n = 16(ent-2 RNAi). 700 (C) Genome illustrations of ent-1KO and ent-2KO mutants. 701 (D) ent-1KO mutants on OP50 bacteria show no significant alteration of daily progeny number or 702 brood size compared to wild type (WT) worms. n = 23(N2), n = 22(ent-1KO). 703 (E) ent-2KO mutants on OP50 bacteria show no significant alteration of daily progeny number or 704 brood size compared to WT. n = 18(N2), n = 18(ent-2KO). 705 (F) ent-1KO mutants on HT115 bacteria show no significant alteration of daily progeny number or 706 brood size compared to WT. n = 28(N2), n = 27(ent-1KO). 707 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 32 (G) ent-2KO mutants on HT115 bacteria show no significant alteration of daily progeny number or 708 brood size compared to WT. n = 22(N2), n = 22(ent-2KO). 709 (H) (I)RT-qPCR analysis shows that the ent-2 mRNA level is upregulated in the ent-1KO mutant 710 (H), and the ent-1 mRNA level is elevated in ent-2KO (I). n = 6 biologically independent 711 samples in each condition. 712 Statistic: **p<0.01, ***p<0.001, ****p0.05. Student’s t-test (unpaired, two-713 tailed) was applied for the brood size graphs in A, B, D, E, F and G, and RT-qPCR results in H 714 and I. Two-way ANOVA with Bonferroni’s post hoc test for the daily progeny graphs in A, B, 715 D, E, F and G. 716 Data shown as mean ± S.E.M. 717 718 S2 Fig. Germline ENT-1 and intestinal ENT-2 cooperate in regulating reproduction 719 (A) Bright-field images show sterile ent-1KO; ent-2KD mutants and their controls. Scale bar: 100 720 µm. 721 (B, C) Diagrams showing CRISPR knock-in strains: ent-1::mNeonGreen (B), ent-2::wrmscarlet 722 (C). 723 (D, E) Endogenous localization of ENT-1::mNeonGreen in the germline and intestine (D) and 724 ENT-2::wrmscarlet in gonadal sheath cells and the intestine (E). Scale bar: 50 µm. 725 (F, G) Diagrams showing CRISPR knock-in strains: degron::ent-1 (F) and degron::ent-2 (G). 726 (H)(I) Immunostaining results show the efficiency of tissue-specific degron-mediated protein 727 degradation induced by the Auxin treatment in the degron::ent-1 strain (H) and in the 728 degron::ent-2 strain (I). Scale bar: 50 µm. 729 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint 33 S3 Fig. Changes in the germline and other tissues in association with the loss of ENT-1 and 730 ENT-2 731 (A) Pseudotime density plots illustrate the distribution of germ cell nuclei across different 732 pseudotime points for control, ent-1KO, ent-2KO, and ent-1KO; ent-2KD worms. 733 (B) Gene ontology enrichment analysis of biological processed was performed on the down-734 regulated expressed genes in the germline of ent-1KO; ent-2KD worms compared to their 735 controls, revealing pathways related to cell cycle and division are overrepresent. 736 (C) (D) Dotplots show the relative expression levels of genes that are associated with purine 737 metabolism of control, ent-1KO, ent-2KO, and ent-1KO; ent-2KD animals in the hypodermis (C) 738 and muscle (D). 739 740 741 742 743 Adams, K.J., B. Pratt, N. Bose, L.G. Dubois, L. 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It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted September 16, 2024. ; https://doi.org/10.1101/2024.09.12.612591doi: bioRxiv preprint