In vivo reprogramming ofCaenorhabditis elegansleads to heterogeneous effects on lifespan

37 In vivo reprogramming, C. elegans, development, aging 38 39

40 Reprogramming somatic cells into pluripotency by forced expression of the Yamanaka 41 factors Oct4, Sox2, Klf4, and c-Myc (OSKM) has led to exciting developments of high 42 therapeutic interest1-3. Importantly, these reprogramming factors have been shown to act 43 at the epigenetic level by r eversing the epigenetic landscape towards embryonic and 44 younger states4. Consequently, multiple studies in rodents and human cells have used 45 these or other factors to reprogram differentiated cells towards new cell fates 5, recover 46 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint stemness, and slow or reverse age -associated phenotypes both in vitro and in vivo 2,4,6-47 9. In mice, c ontinuous induction of in vivo reprogramming leads to organ dysfunction, 48 teratoma formation, and premature death. Importantly, these effects can be avoided by 49 partial reprogramming using short-term cyclic expression of OSKM , resulting in the 50 reversal of age -associated phenotypes in multiple organs and extens ion of lifespan in 51 progeroid and aged wildtype mice 2,10-12. However, in vivo reprogramming is still 52 associated with adverse effects such as organ dysfunction, teratoma formation , and 53 premature death7. 54 55 To date, m ost reprogramming studies have been performed in human cell culture or in 56 mice2. However, induction of in vivo reprogramming has never been tested in C. elegans. 57 In this line, in vivo studies in mammals possess multiple disadvantages, such as a longer 58 lifespan, high costs, ethical constraints, and complexity. On the other hand, C. elegans 59 has a short mean lifespan of only three weeks when cultured at 20°C. C. elegans is a 60 widely used model organism that is particularly suited for epigenetic and cell fate studies 61 due to multiple advantages, including fully identified cell lineages, amenability for genetic 62 screens, small size, and transparency, allowing microscopy acquis itions of whole 63 organisms13. In addition to being one of the most commonly used model organism for 64 aging studies, C. elegans also provides the benefits of cost efficiency, lower organismal 65 complexity, and conservation of many signaling pathways14. 66 67 Regarding the feasibility of inducing in vivo reprogramming on C. elegans, several genes 68 share a certain degree of homology with the reprogramming factors in mammals15-22. 69 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Although the induction of in vivo reprogramming has never been described in C. elegans, 70 natural trans-differentiation events and ectopic expression of tissue-specific transcription 71 factors in embryos have been reported17-20. Moreover, during natural trans-differentiation 72 events, members of the NODE (Nanog and Oct4 associated deacetylase) complex and 73 SOX2 promote the initiation of a natural cellular reprogramming event where a fully 74 differentiated rectal cell transdifferentiates into a neuronal cell15. In addition, two more 75 natural trans -differentiation events during development have also been reported in C. 76 elegans20,23. Regarding the induction of direct reprogramming, ectopic expression of a 77 single C. elegans transcription factor has been shown to directly convert mitotic germ 78 cells into specific neuron al types24. In addition, another study showed that brief 79 expression of a single transcription factor (ELT-7 GATA) can convert fully differentiated, 80 highly specialized non-endodermal cells of the pharynx into fully differentiated intestinal 81 cells in adult C. elegans25. All these findings suggest a certain degree of cellular plasticity 82 and collectively propose C. elegans as a valuable model to study in vivo reprogramming. 83 84 Toward the goal of inducing in vivo reprogramming in C. elegans, we first conducted a 85 bioinformatic analysis to identify orthologs of the most commonly used transcription 86 factors to induce cellular reprogramming , including Oct4, Sox2, Klf4, c-Myc, Lin28, and 87 Nanog (OSKMLN)10. Subsequently, we selected the C. elegans orthologs of OSKL and 88 cloned them into vectors under the control of a heat -inducible promoter to generate 89 transgenic animals with multi -copy arrays to reprogram worms. After optimizing the 90 induction protocol, we characterized the effect of in vivo reprogramming at different 91 stages of C. elegans development and aging . Surprisingly, the i nduction of in vivo 92 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint reprogramming resulted in aberrant phenotypes such as developmental abnormality, 93 bagging, and premature death. These findings suggest C. elegans as a promising model 94 to study in vivo reprogramming and its effects on development, epigenetics, and aging. 95 96

97 Generation of heat-inducible reprogrammable worms and optimization of induction 98 protocol 99 To generate C. elegans reprogramming strains, we first identified the orthologs of mouse 100 reprogramming factors Oct4, Sox2, Klf4 , and cMyc ( OSKM). The four C. elegans 101 orthologs, ceh-6, sox-2, klf-1, and lin-28, were cloned into individual plasmids and 102 microinjected as a pool of plasmids. After recombination, heritable extrachromosomal 103 arrays containing multiple copies of the reprogramm ing factors were generated, and 104 reprogrammable worm strains (4F) were selected (Figure 1A and Figure S1A ). As the 105 transgenes were under the control of a heat shock promoter (Figure S1A), we first 106 optimized the conditions for the induction and assessed the effects of heat shock on C. 107 elegans lifespan26. Towards this goal, we tested the effect of different induction 108 temperatures and duration s by analyzing ceh-6 and sox-2 mRNA expression levels . 109 Induction at 33°C for 3 hours led to higher expression levels of the ceh-6 and sox-2 110 (Figure 1B and Figure 1C). Next, following this protocol, we analyzed the duration of 111 expression over time and observed a peak of expression 4 hours post-induction, which 112 completely subsided within 24 hours (Figure 1 D). Based on these observations , we 113 selected this protocol and analyzed the expression levels of all the reprogramming factors 114 4 hours after heat shock. Importantly, we detected significant levels of expression of all 115 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint four reprogramming factors in 4F induced worms compared to the control and 4F 116 uninduced worms (Figure 1E). Similar results were obtained by analysis of bulk RNA-seq, 117 where expression levels were higher 4 hours post-induction and decreased after 48- and 118 72-hours post -induction (Figure S1B) . In agreement with these observations, GFP 119 reporter expression was also detected throughout the body upon heat shock (Figure 1F). 120 These observations suggest the successful generation of 4F C. elegans and optimization 121 of the induction protocol. 122 123 Reprogramming at different developmental stages causes morphological 124 abnormality and premature death 125 To identify the effect of reprogramming during different developmental stages of C. 126 elegans, reprogramming was induced at different developmental stages (Figure 2A) . 127 First, the induction of reprogramming in embryos resulted in embryos that were non -128 viable, with a significant 80% reduction in survival compared to the control group (Figure 129 2B and Figure 2C). Next, we induced in vivo reprogramming in L2 larval worms, observing 130 morphological abnormalities as well as the inability to develop into adults (Figure 2 D). 131 Further characterization of these morphological defects showed a significant reduction of 132 60% in body size (Figure 2E), together with a significant reduction in median lifespan 133 (Figure 2F). Lastly, the induction of in vivo reprogramming in L4 larval worms induced 134 morphological alterations such as bagging (eggs retained inside the parental body) and 135 internal hatching (eggs hatched inside the parental body) (Figure 2G), reduction in size 136 (Figure 2H), and a significant reduction in the survival rate compared to control -induced 137 worms (Figure 2I). Altogether, these results demonstrate that the induction of in vivo 138 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint reprogramming during different developmental stages lea ds to high toxicity and 139 developmental abnormalities, ultimately compromising the survival of worms. 140 141 Reprogramming at reproductive stages causes morphological and behavioral 142 abnormalities leading to premature death 143 Since the induction of reprogramming during development led to extreme toxicity, we 144 decided to induce in vivo reprogramming in C. elegans during post-developmental stages. 145 Towards this goal, we first induced the factors in young adults of age day 1 (D1), a time 146 when worms are reproductively active 27, and followed them for several days post -147 induction (Figure 3A). Importantly, D1 worms showed morphological abnormalities such 148 as a significant size reduction and increased bagging upon induction of in vivo 149 reprogramming (Figure 3B-D, Figure S2 A). In addition, a significant reduction in egg -150 laying was observed in reprogrammed worms compared to their controls (Figure 3 E). 151 Moreover, behavioral abnormalities, including food avoidance (Figure 3F) and decreased 152 motility, were also observed (Figure 3G). Finally, we observed a significant reduction in 153 the survival rate following the expression of the reprogramming factors compared to 154 control worms (Figure 3H). 155 Next, in order to gain insight into the effects of in vivo reprogramming in C. elegans, we 156 performed global transcriptome analysis by bulk RNA-sequencing. Analysis of RNA-seq 157 showed a higher number of differentially expressed genes ( DEGs) at 4 hours post -158 induction that were reduced over time at 48 and 72 hours post-induction (Figure 3I). Gene 159 ontology (GO) analysis showed upregulation of genes related to sensory perception in 4F 160 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint reprogrammable worms, which could explain food avoidance behavior (Figure S3A), and 161 downregulation of genes related to immune response (Figure S3B). 162 In addition, functional enrichment analysis showed DEGs enrichment in transcription 163 factor motifs related to development, such as hlh-2, cnd-1, lin-14, che-1, and elt-3 (Figure 164 3J and Figure S3C -D). Altogether, these results demonstrated that the induction of 165 reprogramming factors during post-developmental stages leads t o toxicity and 166 morphological abnormalities, ultimately compromising the survival of worms as well as 167 the expression of developmental genes. 168 169 Reprogramming of adult worms causes loss of proliferation and germ cell identity 170 in the embryos and increased apoptosis without affecting somatic cell identity 171 Since the induction of in vivo reprogramming in D1 worms affect ed their reproductive 172 capacity, we decided to focus on the study of embryos and the germline. First, we 173 investigated the tissues where the reprogramming factors were expressed using the heat-174 inducible GFP marker. Upon inducing a D1 adult worm, w e detected GFP expression 175 corresponding to the four-factor expression in the embryos (Figure S4A). Importantly, no 176 GFP expression was detected in the germline of the adult worm (Figure S4B). In addition, 177 GFP was also detected in all major somatic tissues, such as head ganglions, intestine, 178 and body wall muscle (Figure S4C-E). Next, to further characterize the effects of 179 reprogramming at the cellular level, we crossed 4F non-GFP hermaphrodite worms with 180 male worms carrying reporters for proliferation, germ cell, apoptosis, intestine, body wall 181 muscle, and somatic cell identity (Figure S4F). 182 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint To test the effect of 4F induction on the proliferation rate, we induced reprogramming on 183 4F worms carrying a GFP proliferation reporter (4F.pr ol) on D1. Subsequently, w e 184 monitored the GFP signal for several days post-induction and observed a significant loss 185 of proliferation at day 2 post-induction (Figure 4A-B), suggesting that 4F induction inhibits 186 the proliferation of the embryos in adult worms. In order to further understand the effects 187 of reprogramming in the embryos, we generated 4F worms with GFP germ cell reporter 188 (4F.germ) to identify whether 4F induction c ould lead to an increase in germ cells. 189 Subsequently, we induced the expression of the factors on the D1 4F.germ worms and 190 detected the loss of germ cell identity in the embryos upon 2 days of reprogramming 191 induction (Figure 4C). In addition, quantification of the germ cell identity signal showed a 192 significant loss in the embryos of adult C. elegans upon reprogramming compared to their 193 induced controls (Figure 4D). 194 Since reprogramming could also lead to loss of cell identity and apoptosis28, we generated 195 4F worms carrying a GFP apoptotic reporter (4F.apop) to test the effect of 4F induction 196 on the apoptosis rate. Next, we induced the reprogramming factors at D1 in the 4F.apop 197 worms and observed a significant increase in apoptosis ( Figure 4F and Figure 4G). In 198 addition, to investigate whether reprogramming could also affect somatic tissues , we 199 generated 4F worms with GFP intestinal reporter (4F.inte), TOM20 body wall muscle 200 reporter (4F.bwm), and wrmScarlet somatic reporters (4F.soma). Subsequently, we 201 induced reprogramming in these worms and monitored them for several days post -202 induction (Figure S4G-I). Importantly, compared to induced control worms, we did not 203 detect significant differences in reporter signal associated with either the intestine, body 204 wall muscle, or somatic tissue, suggesting that reprogramming did not affect the intestine, 205 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint body wall muscle, or somatic cell identity (Figure S4J-L). Altogether, these results 206 indicated that in vivo reprogramming of C. elegans leads to loss of proliferation and germ 207 cell identity in the embryos of adult worms together with an increase in apoptosis 208 throughout the body, while cell identity of post-mitotic tissues remains unaffected. 209 210 Cyclic induction of reprogramming at post -reproductive stages leads to mild 211 toxicity 212 As induction of in vivo reprogramming results in severe side effects during developmental 213 stages as well as in the embryos of young adults, we decided to induce the expression of 214 the reprogramming factors at post -reproductive stages. Towards this goal , C. elegans 215 were subjected to heat shock at young adult on day 5 (D5), middle-aged adult on day 10 216 (D10), and old adult on day 15 (D15) (Figure 5A) . Interestingly, the induction of 217 reprogramming factors led to a 13% reduction in median lifespan at D5 and had no 218 significant effects on survival at D10 and D15 compared to the induced controls (Figure 219 5B). Importantly, analysis of mRNA expression of the reprogramming factors at D10 220 showed a significantly lower expression of the factors compared to D1 (Figure 5C). In 221 addition, and similar to D1, bulk RNA-seq analysis showed that the level of expression of 222 the reprogramming factors was higher 4 hours post -inductions and decreased after 48 223 hours post -induction (Figure S5A) . A similar trend was observed for DEGs, with a 224 significantly lower number of DEGs in D10 compared to D1-induced worms 4 hours post-225 induction (Figure S5B). For this reason, we decided to test the effect of cyclic protocols 226 for the induction of the reprogramming factors. Cyclic induction every 3 days or every 2 227 days did not have a significant impact on the survival rate compared to the induced 228 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint controls. However, cyclic induction every day resulted in mild toxicity compared to 229 uninduced controls, with a 10% reduction in median lifespan (Figure 5D). Together, these 230

suggest that C. elegans post-reproductive stages might be more resistant to the 231 toxic effects of in vivo reprogramming than reproductive and developmental stages due 232 to their lower plasticity and post-mitotic nature. 233 234

235 C. elegans as organism to study in vivo reprogramming 236 Cellular reprogramming has become increasingly important to investigate the interplay 237 between epigenetics, cellular identity, and various biological processes, including 238 regeneration and aging, yet numerous questions remain to be answered. Along this line, 239 induction of in vivo reprogramming in mice is toxic and leads to organ dysfunction and 240 tumor development resulting in premature death28. To date, no other organism has been 241 used to study 4F in vivo reprogramming, raising the question of whether the effects 242 observed in the mouse model could be similar in another organism. Importantly, C. 243 elegans has been widely used as a model organism for research on development and 244 aging. Here, we generated , for the first time, reprogrammable 4F worms to study the 245 effects of in vivo reprogramming in vivo. The novel transgenic 4F C. elegans strains 246 generated in this study allow the efficient induction of the reprogramming factors in adult 247 somatic tissues, larvae, and embryos. 248 249 Reprogramming at different developmental stages induces heterogeneous toxicity 250 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Importantly, the induction of reprogramming factors at different stages of C. elegans 251 development led to different degrees of toxicity. Specifically, reprogrammed embryos 252 were non -viable, while reprogrammed L2s were unable to develop into reproductively 253 active adults . Similarly, reprogrammed L4s became reproductive adults but showed 254 bagging phenotype and premature death. In addition, L2 reprogramming worms displayed 255 a stronger reduction in size compared to L4 and a more significant reduction in survival. 256 These results demonstrate milder toxicity for the induction of in vivo reprogramming at 257 advanced stages of development. 258 259 Reprogramming at reproductive stages leads to bagging and food avoidance 260 During reproductive stages, we observed GFP expression corresponding to the 4F 261 expression in the developing embryos, but no expression was achieved in the germline . 262 Although germline expression at low levels can be achieved from single -copy transgenes 263 (REF), transgene arrays30, including hsp16 promoter-bearing ones31, have been previously 264 shown to be vulnerable to silencing in the germline. 265 266 This might be due to the tight epigenetic regulation, which has been reported and is linked 267 to the silencing of transgenes in the germline29. Although germline expression at low 268 levels can be achieved from single-copy transgenes30, transgene arrays31, including 269 hsp16 promoter-bearing ones32, have been previously shown to be vulnerable to silencing 270 in the germline. Reprogramming of C. elegans at D1 led to strong morphological and 271 behavioral abnormalities such as bagging, reduced egg laying, reduced motility, and food 272 avoidance. Interestingly, the RNA-seq analysis showed downregulation of genes related 273 to vulval hlh-2 and embryonic development cnd-1, potentially explaining the bagging and 274 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint dysfunctional embryonic development. In addition, genes related to sensory perception 275 were upregulated in 4F reprogrammable worms , potentially explaining food avoidance 276 behavior. 277 278 Reprogramming at reproductive stages adversely affects the developing embryos 279 Previous studies have demonstrated that cellular reprogramming of mammalian cells 280 induces cell proliferation and leads to the expression of pluripotency markers1. However, 281 due to the lack of a pluripotency state in adult C. elegans, we focused on the study of 282 both germline and proliferation reporters. Surprisingly, r eprogramming in C. elegans 283 resulted in the loss of germ cell identity and a decrease in proliferation in developing 284 embryos in the adult worms . In addition, functional enrichment analysis showed DEGs 285 enrichment in transcription factors related to development. One of the transcription factor 286 motifs that was upregulated was lin-14, a temporal regulator of postembryonic 287 developmental events. Importantly, this gene is only expressed until the L2 stage during 288 normal development. Another transcription factor motif that was upregulated is zinc finger 289 transcription factor che-1, which has been previously used for reprogramming germ cells 290 into neurons24. Interestingly, the expression of the GATA transcription factor motif elt-3 291 was also affected. Importantly, previous studies have shown that the ectopic expression 292 of GATA transcription factor elt-7 can reprogram the pharynx or somatic gonad into 293 intestinal fate25. Our findings highlight the potential of in vivo reprogramming to influence 294 cellular plasticity in C. elegans . Nevertheless, somatic cell identity remained largely 295 unaffected upon induction of reprogramming at later adult stages, likely due to the limited 296 plasticity of the post-mitotic cell stage. 297 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint 298 Reduced toxicity of reprogramming at post-reproductive stages following a cyclic 299 induction protocol 300 Induction of in vivo reprogramming at post-reproductive stages, such as day 5, 10, and 301 15 worms, did not affect survival. This could be mainly attributed to two reasons: 1) lower 302 expression levels of the reprogramming factors or 2) absence of embryos at these post-303 reproductive stages. Moreover, the absence of embryos could explain the lower levels of 304 expression at D10 compared to D1 induction. In addition, RNA analysis on D10 worms 305 showed a lower number of DEGs compared to D1-induced worms. However, the cyclic 306 induction of reprogramming factors every day led to a mild reduction in the median 307 lifespan, which could be caused by the continuous expression of the reprogram ming 308 factors in the somatic tissues of adult worms. 309 In summary, these results demonstrate that the induction of in vivo reprogramming results 310 in different degrees of toxicity that are inversely correlated with development and, most 311 likely, cellular plasticity. Overall, this study represents the first attemp t to induce in vivo 312 reprogramming by expressing Yamanaka-like factors in C. elegans. Like previous studies 313 in mice, induction of in vivo reprogramming is highly toxic and lethal, especially during 314 development. Using invertebrate model organisms such as C. elegans to study in vivo 315 reprogramming might increase our understanding of the interplay between epigenetics 316 and cellular identity during development, adulthood, and aging. 317 318

of the study 319 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Induction of in vivo reprogramming predominantly affected embryos, larvae, and 320 reproductively active young adults, where cells are characterized by a higher degree of 321 plasticity. Nevertheless, no expression was achieved in the germline, possibly due to the 322 silencing of transgenes in the germline . In addition, w e are aware that the post -mitotic 323 nature of C. elegans cells represents a major limitation for studying cellular 324 reprogramming in this organism. In this line, previous studies have shown that some 325 epigenetic factors can hinder somatic cell reprogramming17,18. Further studies focused on 326 removing these epigenetic barriers in combination with the expression of the 327 reprogramming factors might allow a higher degree of cellular plasticity upon induction of 328 cellular reprogramming in adult C. elegans. 329 Importantly, the generation of 4F worms using CRISPR integration led to high lethality, 330 most probably due to the leakiness or induction of the reprogramming factors during the 331 process of genetic modification and their high toxicity during development. For this 332 reason, an extrachromosomal multi-copy array, where the copy number of factors cannot 333 be controlled, was used to generate reprogramming C. elegans strains. Lastly, despite 334 the common use of heat shock promoters in C. elegans, heat shock itself can have toxic 335 effects and be lethal beyond a certain temperature or duration. 336 Multiple studies have previously demonstrated the induction and need for cellular 337 proliferation during cellular reprogramming to pluripotency. Since our data does not 338 demonstrate complete reprogramming of C. elegans cells to stemness or pluripotency, 339 future experiments, including analysis of tissue-specific or cell -specific transcriptional 340 changes, will be necessary to understand better the effect of in vivo reprogramming on 341 cellular identity. Moreover, tissue-specific reprogramming could also be a better way to 342 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint understand this33. This analysis might provide a better understanding of the differences 343 in toxicity observed upon induction of in vivo reprogramming at the D1 and D10 stages. 344 Moreover, germline-less 4F worms lacking germ cells will allow us to dissect the role of 345 germ cells and embryos on the toxic effect of the reprogramming factors in young adults. 346

347 Generation of transgenic 4F strains 348 To generate reprogrammable C. elegans, orthologs of the murine reprogramming factors 349 Oct4, Sox2, Klf4, and Lin28 were identified using bioinformatic analysis. The 350 corresponding orthologs ceh-6, sox-2, klf-1, and lin-28 in C. elegans were codon -351 optimized (Supplementary Table 1 ). These factors were cloned under the control of a 352 heat-shock (hsp-16.2) promoter34. In addition to all four factors, a heat -inducible GFP 353 marker under the hsp-16.2 promoter, a constitutive mCherry marker under the myo-2 354 promoter, and a hygromycin resistance selection marker were also cloned (Figure S1A). 355 Subsequently, reprogramming factors ceh-6, sox-2, klf-1, and lin-28 were co-injected with 356 selection markers into the C. elegans germline resulting in the formation of 357 extrachromosomal multicopy array by homologous recombination of the plasmids, and a 358 final selection of hygromycin resist ant 4F transgenic worms (Supplementary Table 2). 359 Finally, three types of transgenic worms were generated: a reprogrammable C. elegans 360 strain containing the four factors with an inducible GFP marker as 4F, a strain lacking the 361 four factors that serve as a control, and a strain without an inducible GFP marker as 4F 362 non-GFP. 363 Induction protocols 364 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint The standard induction protocol for reprogramming factors in 4F C. elegans involved 365 incubating the embryos or worms in an incubator (Memmert) for a 3 -hour heat shock of 366 33°C or the duration and tem peratures indicated in the manuscript . Following the 367 induction, C. elegans was moved back to the maintenance temperature of 20°C. 368 Generation of 4F reporter strains 369 The reporter strains used in this study , DQM662 (Proliferation), JH3269 (Germline), 370 ZH231 (Apoptosis), SJ4143 (Intestine), PS6192 (Body wall muscle), and WBM136 371 (Somatic), were obtained from the Caenorhabditis Genetic Center ( CGC). To generate 372 4F reporter strains, the 4F non-GFP, carrying mCherry reporter strain, was crossed with 373 each of the above-mentioned reporters. Briefly, male progeny reporters were generated 374 by heat shocking L4 stage larvae at 30°C for 6 hours. For the crossing, a total of 5 L4 375 stage hermaphrodite 4F non-GFP worms were placed in a P60 NGM plate with 5 young 376 male reporter worms. Worms showing both reporters were selected and maintained 377 (Supplementary Table 3). 378 Survival experiment 379 Worms were grown on solid NGM plates (P60 plate , Falcon, 353004) seeded with UV -380 killed OP50 bacteria as food (150ul of 120 mg/ml UV killed bacteria per P60 plate with 10 381 ml NGM) at 20°C. Hygromycin selection plates were seeded with 4 mg/mL hygromycin B 382 (Hygromycin B Gold, InvivoGen, HGG -44-04). Worm populations were synchronized by 383 hypochlorite treatment (Bleach solution:2.8% bleach, 0.8N NaOH). Pelleted worms were 384 treated with bleach solution for up to 12 minutes until all the worms rapture and eggs were 385 released. Immediately, eggs were washed with ddH20 up to 2 times. The washed eggs 386 were seeded onto an NGM plate without food for 24 hours. After seeding OP50 bacteria, 387 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint we waited until the worm reached the L4 stage. For survival experiments, L4 worms were 388 sterilized by 5-fluorodeoxyuridine treatment (CAS number 50-91-9, Acros organics), and 389 lifespan was measured by testing the worm movement with ge ntle poking using a worm 390 pick. Worms were counted as dead when they showed no movement at all. 391 Confocal Microscopy 392 For confocal image acquisitions, worms were mounted on a fresh 4% low-melting agarose 393 (NuSieveTM GTGTM Agarose, Lonza, 50081 ) pad between glass slides (Epredia, 394 Superfrost Plus TM Gold Adhesion Microscope Slides, K5800AMNZ72) . First, 10 -15 395 worms were individually picked and transferred into an agar layer containing 10 mM 396 levamisole, used as a paralyzing agent. All the confocal microscopic worm images were 397 captured using a Nikon Ti2 Yokogawa CSU-W1 spinning disk confocal microscope with 398 NIS Elements software. The images were analyzed using FIJI Version: 2.14.0/1.54f. 399 Behavioral analysis 400 The food avoidance test was done on synchronized 4F -induced worms by counting the 401 number of worms that stayed out of the food compared to those that stayed within the 402 food. The movement analysis was done by capturing a minute video of worms using a 403 Nikon SMZ800N microscope with a coupled camera. The videos acquired were analyzed 404 using MBF bioscience Worm Tracker. 405 Morphological analysis 406 The morphological analysis and size measurements were performed on 10x images of 407 the worms acquired with Nikon Ti2 Yokogawa CSU -W1 spinning disk confocal 408 microscopy. Later, the quantification of worm length was measured using FIJI Version 409 2.14.0/1.54f. Bagging was measured by manual counting, by manually looking at worms 410 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint that have bagging phenotypes, and those without using 2x objective in a stereo 411 microscope Motic SMZ-171. The egg-laying rate was measured by acquiring images of 412 NGM plates with the worms using a Motic SMZ-171 stereo microscope with an objective 413 of 2x. 414 Quantitative real-time PCR 415 Total RNA was extracted from synchronized worm populations by TRIzol reagent 416 (Invitrogen, 15596018) and chloroform (Roth, 280298902) treatment. RNA was then 417 purified using Monarch Total RNA miniprep Kit (New England Biolabs, T2010S) according 418 to the manufacturer’s instructions. Samples were treated with DNase (Qiagen, 79254) for 419 15 minutes (1:8 in DNase buffer). Total RNA concentrations were determined using the 420 Qubit RNA BR Assay Kit (Thermofisher, Q10211). cDNA synthesis was performed by 421 adding 4 μL of iScript™ gDNA Clear cDNA Synthesis (Biorad, 1725035BUN) to 500ng of 422 RNA sample and run in a Thermocycler (Biorad, 1861086) with the following protocol: 5 423 min at 25°C for priming, 20 min at 46°C for reverse transcription, and 1 min at 95°C for 424 enzyme inactivation. Final cDNA was diluted 1:5 using autoclaved water and stored at - 425 20°C. qRT-PCR was performed using SsoAdvanced SYBR Green Supermix (Bio -Rad, 426 1725272) in 384 well PCR plates (Thermofisher, AB1384) using the QuantStudio™ 12K 427 Flex Real -time PCR System instrument (T hermofisher). Forward and reverse primers 428 (1:1) were used at a final concentration of 5 µM with 1 µL of cDNA sample (Supplementary 429 Table 4). 430 RNA-seq alignment and quantification 431 Data was processed using nf -core/rnaseq v3.14.0 432 (doi: https://doi.org/10.5281/zenodo.1400710) of the nf -core collection of workflows 35, 433 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint utilising reproducible software environments from the Bioconda 36 and Biocontainers 37 434 projects. The pipeline was executed with Nextflow v23.10.038 with the following command: 435 nextflow run 'https://github.com/nf-core/rnaseq' -params-file 'https://api.tower.nf/ephemer436 al/lEwfZ9yoEKPOtK_aMS3aCA.json' -with-tower -profile ethz_euler 437 The RNA -seq reads were aligned to the Caenorhabditis elegans reference genome 438 WBcel235 (GCA_000002985.3), and gene annotation was obtained from the Ensembl 439 release 111. 440 To align and quantify the reads assigned to the extrachromosomal array sequence, the 441 nf-core/rnaseq pipeline version 3.14.0 was used, having has inp ut the unmapped reads 442 generated from the pipeline run described above. Prior to alignment, rRNA contaminants 443 were removed using the 'SortMeRNA' package39. 444 The pipeline was executed with Nextflow v23.10.1 with the following command: 445 nextflow run 'https://github.com/nf -core/rnaseq' -params-file 446 'https://api.tower.nf/ephemeral/5dPm0oQkPbF3-HwuEKQ7sQ.json' -with-tower -r 3.14.0 447 -profile ethz_euler 448 449 RNA-seq analysis 450 Transcript read counts were imported into R and converted to gene counts using the 451 Bioconductor package 'tximport' 40. Normalization was conducted using the Bioconductor 452 package 'DESeq2'41, and dimensionality reduction was performed using the reads counts 453 after variance stabilizing transformation (VST). 454 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Differential expression analysis was carried out using the 'DESeq2' package with the 455 parameter modelMatrixType set to "standard" and default sett ings for other parameters. 456 Genes were considered differentially expressed between conditions if they exhibited an 457 adjusted p-value below 0.05 and an absolute log2 fold change exceeding 2. 458 Gene ontology analysis was conducted using the 'compareCluster' func tion from the 459 Bioconductor package 'clusterProfiler'42. The analysis utilized the org.Ce.eg.db database, 460 focusing solely on Biological Process (BP) ontology. Benjamini-Hochberg adjustment was 461 applied for p -values, with significance thresholds set to 0.05 for both p -values and q -462 values. 463 Statistical analysis 464 Statistical analysis was performed using GraphPad Prism 9.4.1 (GraphPad Software). 465 For the comparison of two independent groups, a two-tailed unpaired t-Student’s test 466 (data with normal distribution) was executed. The corresponding p and n values are 467 presented in the indicated figures, and the levels of significance are denoted as follows: 468 ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, and ns indicates not significant. n values represent 469 the number of animals. Data shown mean ± standard error mean. 470 471 ACKNOWLEDGMENTS 472 The authors thank all members of the Ocampo laboratory, especially Alba Vílchez-Acosta, 473 Gabriela Desdín-Micó, and María del Carmen Maza, for their valuable feedback and 474 support. In addition, we would also like to thank the UNIL Cellular Imaging Facility and 475 Rosa Chiara Paolicelli (Assistant Professor, UNIL, Switzerland) and Anne-Claire 476 Companion (Postdoc, UNIL, Switzerland) for their valuable support with microscopy and 477 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint imaging. We thank Pierre Gönczy (Professor, EPFL, Switzerland) for his valuable 478 support. Some strains were provided by the CGC, which is funded by the NIH Office of 479 Research Infrastructure Programs (P40 OD010440). 480 481 FUNDING 482 This work was supported by the Milky Way Research Foundation (MWRF), the 483 Eccellenza grants from the Swiss National Science Foundation (SNSF), the University of 484 Lausanne, and the Canton Vaud. 485 486 AUTHOR CONTRIBUTIONS 487 A.O. and N.K. designed the study. N.K. was involved in all experiments, data collection, 488 analysis, and interpretation. A.V.A. and S.P. revised the manuscript and prepared the 489 figures. Y.M. contributed to survival experiments , confocal microscopy, and qRT–PCR 490 analysis under the supervision of N.K. M.P ., C.F.J., and S.E.M. generated the transgenic 491 4F worms. V.P., F.v.M. and J.A.S performed bioinformatic analysis and analysis of RNA-492 seq data. A.O. directed and supervised the study and designed the experiments. N.K. 493 and A.O wrote the manuscript with input from all authors. 494 495 DECLARATION OF INTERESTS 496 A.O. is co -founder and shareholder of EPITERNA SA (non -financial interests) and 497 Longevity Consultancy Group (non-financial interests). The rest of the authors declare no 498 competing interests. 499 500 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint

501 1. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from 502 mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-503 676. 10.1016/j.cell.2006.07.024. 504 2. Ocampo, A., Reddy, P., Martinez-Redondo, P., Platero-Luengo, A., Hatanaka, F., 505 Hishida, T., Li, M., Lam, D., Kurita, M., Beyret, E., et al. (2016). In Vivo Amelioration 506 of Age -Associated Hallmarks by Partial Reprogramming. Cell 167, 1719 -507 1733.e1712. 10.1016/j.cell.2016.11.052. 508 3. Abad, M., Mosteiro, L., Pantoja, C., Cañamero, M., Rayon, T., Ors, I., Graña, O., 509 Megías, D., Domínguez, O., Martínez, D., et al. (2013). Reprogramming in vivo 510 produces teratomas and iPS cells with totipotency features. Nature 502, 340-345. 511 10.1038/nature12586. 512 4. Browder, K.C., Reddy, P., Yamamoto, M., Haghani, A., Guillen, I.G., Sahu, S., 513 Wang, C., Luque, Y., Prieto, J., Shi, L., et al. (2022). In vivo partial reprogramming 514 alters age-associated molecular changes during physiologi cal aging in mice. Nat 515 Aging 2, 243-253. 10.1038/s43587-022-00183-2. 516 5. Kurita, M., Araoka, T., Hishida, T., O'Keefe, D.D., Takahashi, Y., Sakamoto, A., 517 Sakurai, M., Suzuki, K., Wu, J., Yamamoto, M., et al. (2018). In vivo reprogramming 518 of wound -resident c ells generates skin epithelial tissue. Nature 561, 243 -247. 519 10.1038/s41586-018-0477-4. 520 6. Rodríguez-Matellán, A., Alcazar, N., Hernández, F., Serrano, M., and Ávila, J. 521 (2020). In Vivo Reprogramming Ameliorates Aging Features in Dentate Gyrus 522 Cells and Imp roves Memory in Mice. Stem Cell Reports 15, 1056 -1066. 523 10.1016/j.stemcr.2020.09.010. 524 7. Lu, Y., Brommer, B., Tian, X., Krishnan, A., Meer, M., Wang, C., Vera, D.L., Zeng, 525 Q., Yu, D., Bonkowski, M.S., et al. (2020). Reprogramming to recover youthful 526 epigenetic information and restore vision. Nature 588, 124-129. 10.1038/s41586-527 020-2975-4. 528 8. Chondronasiou, D., Gill, D., Mosteiro, L., Urdinguio, R.G., Berenguer -Llergo, A., 529 Aguilera, M., Durand, S., Aprahamian, F., Nirmalathasan, N., Abad, M., et al. 530 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint (2022). M ulti-omic rejuvenation of naturally aged tissues by a single cycle of 531 transient reprogramming. Aging Cell 21, e13578. 10.1111/acel.13578. 532 9. Gill, D., Parry, A., Santos, F., Okkenhaug, H., Todd, C.D., Hernando -Herraez, I., 533 Stubbs, T.M., Milagre, I., and Re ik, W. (2022). Multi -omic rejuvenation of human 534 cells by maturation phase transient reprogramming. Elife 11. 10.7554/eLife.71624. 535 10. Sarkar, T.J., Quarta, M., Mukherjee, S., Colville, A., Paine, P., Doan, L., Tran, C.M., 536 Chu, C.R., Horvath, S., Qi, L.S., et al. (2020). Transient non-integrative expression 537 of nuclear reprogramming factors promotes multifaceted amelioration of aging in 538 human cells. Nat Commun 11, 1545. 10.1038/s41467-020-15174-3. 539 11. Zhang, W., Qu, J., Liu, G.H., and Belmonte, J.C.I. (2020). The ageing epigenome 540 and its rejuvenation. Nat Rev Mol Cell Biol 21, 137 -150. 10.1038/s41580 -019-541 0204-5. 542 12. Macip, C.C., Hasan, R., Hoznek, V., Kim, J., IV, L.E.M., Sethna, S., and Davidsohn, 543 N. (2023). Gene Therapy Mediated Partial Reprogramming Extends Lifespan and 544 Reverses Age-Related Changes in Aged Mice. bioRxiv, 2023.2001.2004.522507. 545 10.1101/2023.01.04.522507. 546 13. Tissenbaum, H.A. (2015). Using C. elegans for aging research. Invertebr Reprod 547 Dev 59, 59-63. 10.1080/07924259.2014.940470. 548 14. Olsen, A., Vantipalli, M.C., and Lithgow, G.J. (2006). Using Caenorhabditis elegans 549 as a model for aging and age-related diseases. Ann N Y Acad Sci 1067, 120-128. 550 10.1196/annals.1354.015. 551 15. Kagias, K., Ahier, A., Fischer, N., and Jarriault, S. (2012). Members of the NODE 552 (Nanog and Oct4 -associated deacetylase) complex and SOX -2 promote the 553 initiation of a natural cellular reprogramming event in vivo. Proc Natl Acad Sci U S 554 A 109, 6596-6601. 10.1073/pnas.1117031109. 555 16. Jänes, J., Dong, Y., Schoof, M., Serizay, J., Appert, A., Cerrato, C., Woodbury, C., 556 Chen, R., Gemma, C., Huang, N., et al. (2018). Chromatin accessibility dynamics 557 across C. elegans development and ageing. Elife 7. 10.7554/eLife.37344. 558 17. Kolundzic, E., Ofenbauer, A., Bulut, S.I., Uyar, B., Baytek , G., Sommermeier, A., 559 Seelk, S., He, M., Hirsekorn, A., Vucicevic, D., et al. (2018). FACT Sets a Barrier 560 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint for Cell Fate Reprogramming in Caenorhabditis elegans and Human Cells. Dev 561 Cell 46, 611-626.e612. 10.1016/j.devcel.2018.07.006. 562 18. Rahe, D.P., and Hobert, O. (2019). Restriction of Cellular Plasticity of Differentiated 563 Cells Mediated by Chromatin Modifiers, Transcription Factors and Protein Kinases. 564 G3 (Bethesda) 9, 2287-2302. 10.1534/g3.119.400328. 565 19. Kazmierczak, M., Farré, I.D.C., Ofenbauer, A., Herzog, S., and Tursun, B. (2021). 566 The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies 567 RBBP-5 as a germ cell reprogramming barrier in C. elegans. Nucleic Acids Res 568 49, e22. 10.1093/nar/gkaa1171. 569 20. Riva, C., Hajduskova, M., Gally, C., Suman, S.K., Ahier, A., and Jarriault, S. (2022). 570 A natural transdifferentiation event involving mitosis is empowered by integrating 571 signaling inputs with conserved plasticity factors. Cell Rep 40, 111365. 572 10.1016/j.celrep.2022.111365. 573 21. Zuryn, S., Ahier, A., Portoso, M., White, E.R., Morin, M.C., Margueron, R., and 574 Jarriault, S. (2014). Transdifferentiation. Sequential histone -modifying activities 575 determine the robustness of transdifferentiation. Science 345, 826 -829. 576 10.1126/science.1255885. 577 22. Hajduskova, M., Baytek, G., Kolundzic, E., Gosdschan, A., Kazmierczak, M., 578 Ofenbauer, A., Beato Del Rosal, M.L., Herzog, S., Ul Fatima, N., Mertins, P., et al. 579 (2019). MRG-1/MRG15 Is a Barrier for Germ Cell to Neuro n Reprogramming in 580 Caenorhabditis elegans. Genetics 211, 121-139. 10.1534/genetics.118.301674. 581 23. Jarriault, S., Schwab, Y., and Greenwald, I. (2008). A Caenorhabditis elegans 582 model for epithelial -neuronal transdifferentiation. Proc Natl Acad Sci U S A 105, 583 3790-3795. 10.1073/pnas.0712159105. 584 24. Tursun, B., Patel, T., Kratsios, P., and Hobert, O. (2011). Direct conversion of C. 585 elegans germ cells into specific neuron types. Science 331, 304 -308. 586 10.1126/science.1199082. 587 25. Riddle, M.R., Weintraub, A., Nguyen, K.C., Hall, D.H., and Rothman, J.H. (2013). 588 Transdifferentiation and remodeling of post-embryonic C. elegans cells by a single 589 transcription factor. Development 140, 4844-4849. 10.1242/dev.103010. 590 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint 26. Yashin, A.I., Cyps er, J.R., Johnson, T.E., Michalski, A.I., Boyko, S.I., and 591 Novoseltsev, V.N. (2001). Ageing and survival after different doses of heat shock: 592 the results of analysis of data from stress experiments with the nematode worm 593 Caenorhabditis elegans. Mech Ageing Dev 122, 1477 -1495. 10.1016/s0047 -594 6374(01)00273-1. 595 27. Scharf, A., Pohl, F., Egan, B.M., Kocsisova, Z., and Kornfeld, K. (2021). 596 Reproductive Aging in Caenorhabditis elegans: From Molecules to Ecology. Front 597 Cell Dev Biol 9, 718522. 10.3389/fcell.2021.718522. 598 28. Parras, A., Vilchez-Acosta, A., Desdin-Mico, G., Pico, S., Mrabti, C., Montenegro-599 Borbolla, E., Maroun, C.Y., Haghani, A., Brooke, R., Del Carmen Maza, M., et al. 600 (2023). In vivo reprogramming leads to premature death linked to hepatic and 601 intestinal failure. Nat Aging. 10.1038/s43587-023-00528-5. 602 29. Gleason, R.J., and Chen, X. (2023). Epigenetic dynamics during germline 603 development: insights from Drosophila and C. elegans. Curr Opin Genet Dev 78, 604 102017. 10.1016/j.gde.2022.102017. 605 30. Zeiser, E., Frøkjær-Jensen, C., Jorgensen, E., and Ahringer, J. (2011). MosSCI 606 and gateway compatible plasmid toolkit for constitutive and inducible expression 607 of transgenes in the C. elegans germline. PLoS One 6, e20082. 608 10.1371/journal.pone.0020082. 609 31. Kelly, W.G., Xu, S., Montgomery, M.K., and Fire, A. (1997). Distinct requirements 610 for somatic and germline expression of a generally expressed Caernorhabditis 611 elegans gene. Genetics 146, 227-238. 10.1093/genetics/146.1.227. 612 32. Stringham, E.G., Dixon, D.K., Jones, D., and Candido, E.P. (1992). Temporal and 613 spatial expression patterns of the small heat shock (hsp16) genes in transgenic 614 Caenorhabditis elegans. Mol Biol Cell 3, 221-233. 10.1091/mbc.3.2.221. 615 33. Bacaj, T., and Shaham, S. (2007). Temporal control of cell -specific transgene 616 expression in Caenorhabditis elegans. Genetics 176, 2651 -2655. 617 10.1534/genetics.107.074369. 618 34. Dahlstrom, E.K., and Levine, E. (2019). Dynamics of Heat Shock Detection and 619 Response in the Intestine of Caenorhabditis elegans. bioRxiv, 794800. 620 10.1101/794800. 621 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint 35. Ewels, P.A., Peltzer, A., Fillinger, S., Patel, H., Alneberg, J., Wilm, A., Garcia, M.U., 622 Di Tommaso, P., and Nahnsen, S. (2020). The nf-core framework for community-623 curated bioinformatics pipelines. Nat Biotechnol 38, 276-278. 10.1038/s41587 -624 020-0439-x. 625 36. Grüning, B., Dale, R., Sjödin, A., Chapman, B.A., Rowe, J., Tomkins -Tinch, C.H., 626 Valieris, R., and Köster, J. (2018). Bioconda: sustainable and comprehensive 627 software distribution for the life sciences. Nat Methods 15, 475 -476. 628 10.1038/s41592-018-0046-7. 629 37. da Veiga Leprevost, F., Grüning, B.A., Alves Aflitos, S., Röst, H.L., Uszkoreit, J., 630 Barsnes, H., Vaudel, M., Moreno, P., Gatto, L., Weber, J., et al. (2017). 631 BioContainers: an open -source and community -driven fra mework for software 632 standardization. Bioinformatics 33, 2580-2582. 10.1093/bioinformatics/btx192. 633 38. Di Tommaso, P ., Chatzou, M., Floden, E.W., Barja, P.P., Palumbo, E., and 634 Notredame, C. (2017). Nextflow enables reproducible computational workflows. 635 Nat Biotechnol 35, 316-319. 10.1038/nbt.3820. 636 39. Kopylova, E., Noé, L., and Touzet, H. (2012). SortMeRNA: fast and accurate 637 filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211-638 3217. 10.1093/bioinformatics/bts611. 639 40. Soneson, C., Love, M.I., and Robinson, M.D. (2015). Differential analyses for RNA-640 seq: transcript-level estimates improve gene-level inferences. F1000Res 4, 1521. 641 10.12688/f1000research.7563.2. 642 41. Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change 643 and dispersion for RNA -seq data with DESeq2. Genome Biol 15, 550. 644 10.1186/s13059-014-0550-8. 645 42. Wu, T., Hu, E., Xu, S., Chen, M., Guo, P., Dai, Z., Feng, T., Zhou, L., Tang, W., 646 Zhan, L., et al. (2021). clusterProfiler 4.0: A universal enrichmen t tool for 647 interpreting omics data. Innovation (Camb) 2, 100141. 648 10.1016/j.xinn.2021.100141. 649 650 651 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint FIGURE LEGENDS 652 Figure 1: Generation of reprogrammable C. elegans. A) Schematic representation of 653 the generation of 4F C. elegans. B-C) Relative mRNA levels of ceh-6 and sox-2 on day 654 1 (D1) induced 4F worms after heat-shock induction at different temperatures (B) and at 655 different durations of 33 °C heat-shock (C). D) Relative mRNA levels of ceh-6 on day 1 656 induced 4F worms at different times post-induction at 33°C for 3 hours. E) Relative mRNA 657 levels of the four reprogramming factors on day 1 (D1) induced control, D1 uninduced 4F, 658 and D1 induced 4F worms with an optimized induction protocol of heat shocking at 33°C 659 for 3 hours . F) Representative confocal microscopic images of uninduced and induced 660 D1 4F worms four hours post-induction at 33°C for 3 hours. Inducible GFP was used as 661 a marker of 4F induction, while mCherry defined a constitutive marker indicating the 662 presence of all 4 factors. Data show mean ±  SEM. An ordinary two -way ANOVA test 663 determined statistical significance. n=3 for all q-PCR samples. 664 Figure 2: Reprogramming during developmental stages causes morphological 665 abnormalities and premature death. A) Schematic representation of C. elegans 666 developmental stages from embryos to larval L1, L2, L3, and L4 stage. Arrowheads 667 indicate the developmental stages selected for induction. B) Representative confocal 668 microscopic images of control and 4F worms were taken two days after induction at 669 embryonic stage. The red arrow points to nonviable embryos. C) Analysis of survival of 670 control and 4F worms two days after induction at embryonic stage. D) Representative 671 confocal microscopic images of control and 4F worms two days post-induction at L2 larval 672 stage. E) Length measurement of control and 4F worms two days post -induction at L2 673 stage. F) Analysis of survival of control and 4F worms after i nduction of reprogramming 674 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint at the L2 stage. G) Representative confocal microscopic images of control and 4F worms 675 two days post -induction at L4 larval stage. H) Length measurement of control and 4F 676 worms at two days post -induction at L4 stage. I) Analysis of survival of control and 4F 677 worms after induction of reprogramming at L4 stage. Data show mean ± SEM. An ordinary 678 two-way ANOVA test determined statistical significance. n=3, for body size measurement. 679 Figure 3: Induction of in vivo r eprogramming during reproductive stages leads to 680 morphological defects and premature death. A) Schematic representation of C. 681 elegans reproductive stages. Arrowheads indicate the reproductive stage selected for 682 induction. B) Representative confocal microscopic images of C. elegans control and 4F 683 worms after induction at day 1 (D1) stage, on the day of induction (0 days), one day post-684 induction (1 day), and two days post -induction (2 days). Red arrow head indicates 685 bagging. Red arrow points to internal hatching. C) Length of control and 4F worms two 686 days post-induction at D1. D) Percentage of bagging in 4F uninduced and induced worms 687 over several days post-induction at D1. E) Analysis of number of eggs laid per worms of 688 4F uninduced and induced worms at two days post-induction at D1. F) Percentage of food 689 avoidance of 4F uninduced (purple) and 4F induced (red) over several days post -690 induction at D1. G) Analysis of motility of 4F uninduced and induced worms measured 691 two days post -induction at D1 . H) Analysis of s urvival of control and 4F worms after 692 induction on D1. I) Quantification of differentially expressed genes in 4F compared to the 693 controls in the uninduced and induced at 4 hours, 48 hours, and 72 hours post-induction. 694 J) Functional enrichment analysis of downregulated genes in 4F worms 48 hours post 695 induction at D1. Data show mean ± SEM. An ordinary two-way ANOVA test determined 696 statistical significance. 697 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Figure 4: In vivo reprogramming at reproductive stages leads to loss of embryonic 698 proliferation and germ cell identity together with increased apoptosis. A ) 699 Representative fluorescent images of 4F worms uninduced (top panel) and induced 700 (bottom panel) at D1 stage, carrying the GFP proliferation reporter. Images were acquired 701 on the day of induction (0 days) and 1 - and 2 days post -induction. The yellow outline 702 represents the region of interest for the GFP reporter within the worm. B) Quantification 703 of embryonic proliferation in 4F worms uninduced and induced 2 days post -induction at 704 D1 stage. C) Confocal fluorescent microscopy images of 4F worms uninduced (top panel) 705 and induced (bottom panel) at D1 stage, carrying the GFP germ cell reporter, on the day 706 of induction and 1- and 2-days post-induction. The yellow outline represents the region of 707 embryos within the worms. D) Quantification of germ cell reporter in 4F worms 2 days 708 post-induction at day 1 stage . F) Confocal fluorescent microscopy images of 4F worms 709 uninduced (top panel) and induced (bottom panel) at D1 stage, carrying the GFP 710 apoptotic reporter, at the day of induction and 1-day post-induction. G) Quantification of 711 apoptotic GFP reporter fluorescence in 4F worms 2 days post -induction at day 1 stage . 712 The white box represents a zoomed in portion of the region of interest . MFI: Mean 713 fluorescent intensity. Data show mean ±  SEM. An ordinary two -way ANOVA test 714 determined statistical significance. 715 Figure 5: Reprogramming by cyclic induction of the reprogramming factors at post-716 reproductive stages results in mild toxicity. A) Schematic representation of post -717 reproductive stages of C. elegan s. Arrowheads indicate the post -reproductive stages 718 selected for induction. B) Analysis of survival of controls and 4F worms induced at day 5 719 (D5), day 10 (D10), and day 15 (D15) with single -shot induction. C) Relative mRNA 720 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint expression levels of ceh-6, sox-2, klf-1, and lin-28 in controls and 4F worms induced at 721 D1 and D10. D) Analysis of survival of control and 4F worms induced at D10 stage with 722 several cyclic induction protocols, once every three days, every 2 days, and every day. 723 Data show mean ±  SEM. An ordinary two -way ANOVA test determined statistical 724 significance. 725 Figure S1: Generation of 4F C. elegans strains. A) Schematic representation of 726 plasmid constructs used in the injection mixture for the generation of 4F worms. All four 727 factors w ere cloned under the control of the heat shock promoter hsp-16.2. GFP was 728 used as an inducible marker under the control of the hsp-16.2 promoter, mCherry as a 729 constitutive marker under the pharynx-specific myo-2 promoter, and hygromycinR as a 730 selection marker under the rps-0 promoter. B) Heatmap from RNA-seq analysis showing 731 the relative expression of ceh-6, sox-2, klf-1, and lin-28 in control and induced and 732 uninduced 4F worms 4 hours, 48 hours, and 72 hours post-induction at D1. 733 Figure S2: Bagging in D1-induced 4F worms. A) Representative confocal microscopic 734 images of the head, midbody, and tail region of 4F D1 induced and uninduced worms at 735 0 days , 1 day, and 2 days post -induction. The arrowhead indicates the bagging 736 phenotype. Scale bar 100 µm. 737 Figure S3: Upregulation of genes related to sensory perception and transcription 738 factors lin-14, che-1, and elt-3 in D1-induced 4F worms. A-B) Gene ontology analysis 739 showing upre gulated (A) and down regulated (B) pathways of 4F D1 -induced and 740 uninduced worms. C-D) Functional enrichment of upregulated (C) and downregulated (D) 741 genes in 4F induced 48 hours post-induction. 742 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Figure S 4: Preservation of cell identity in D1 -induced 4F worms . A-E) Confocal 743 fluorescent microscopy images of 4F worms induced at the D1 stage carrying the GFP 744 reporter for heat-shock corresponding to the 4F expression in the embryos (A), germline 745 (B), head ganglions (C), intestine (D), and body wall muscle (E), 4 hours post-induction. 746 F) Schematic representation of the generation of 4F reporter worms by crossing 4F non-747 GFP hermaphrodites with males carrying fluorescent reporters for proliferation, germline, 748 apoptosis, intestine, body wall muscle, and somatic cell identity. G) Confocal images of 749 4F GFP intestinal reporter worms (4F.inte) were imaged on 0d PI and 2d PI. The yellow 750 outline represents the region of interest for the GFP reporter within the worm. H) Confocal 751 images of TOM20 body wall muscle reporter worms (4F.bwm) imaged on 0d PI and 2d 752 PI. I) Confocal images of wrmScarlet somatic reporter worms (4F.soma) imaged on 0d 753 PI and 2d PI. The top panel indicates the somatic cells, and the bottom panel shows 4F 754 expression on 0d PI and 2d PI. J) Quantification of confocal fluorescent images of 4F.inte-755 HS and 4F.inte+HS on 2d PI. K) Quantification of confocal fluorescent images of 4F.bwm-756 HS and 4F.bwm+HS on 2d PI. L) Quantification of confocal fluorescent images of 757 4F.soma-HS and 4F.soma+HS on 2d PI. The white box represents a zoomed portion of 758 the region of interest. MFI: Mean fluorescent intensity. Data show mean ± standard mean. 759 An ordinary two-way ANOVA test determined statistical significance. 760 Figure S 5: Decreased number of differentially expressed genes in 4F worms 761 induced at D10. A) Heatmap from RNA-seq analysis showing the relative expression of 762 ceh-6, sox-2, klf-1, and lin-28 in controls and 4F induced and uninduced worms 4 hours 763 and 48 hours post-induction at day 10. B) Analysis of differentially expressed genes in 4F 764 uninduced and induced worms 4-hour and 48-hour post-induction at day 10 worms. 765 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint A E F Constitutive mCherry Inducible gfp 4F GFP induction reporters ceh-6 sox-2 0 200 400 600Relative mRNA levels Induction temperature 20°C 25°C 30°C 33°C ns *** ** ceh-6 sox-2 0 200 400 600Relative mRNA levels Duration 0 hour 1 hours 2 hours 3 hours *** ** ** * *** *** B C D D1 Control D1 4F uninduced D1 4F induced ceh-6 sox-2 klf-1 lin-28 0 50 100 150 200Relative mRNA levels 100 μm *** *** *** ** P = 0.007 P = 0.0019 P = 0.0013 P = 0.008 P = 0.04 P = 2.6 x 10-5 P = 3.4 x 10-7 P = 2.3 x 10-6 P = 2.4 x 10-6 ceh-6 0 200 400 600Relative mRNA levels 0 hour 4 hours 24 hours ceh-6 expression P = 4 x 10-4 *** P = 3 x 10-4 *** P = 8 x 10-4 *** P = 3 x 10-4 P = 2 x 10-4 *** P = 3 x 10-4 P = 1.6 x 10-4 Microinjection of factors 1 2 3 4 Homologous recombination Extrachromosomal plasmid multicopy arrays Hygromycin selection 4F C. elegans generation 4F expression 100 μm Figure 1 ceh-6 sox-2 klf-1 lin-28 Uninduced Induced Constitutive mCherry Inducible gfp 4F C. elegans .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Control 4F Embryo induction B E C D F G H I L2 induction L4 induction 0 5 10 15 20 25 0 25 50 75 100 Days Survival (%) 0 5 10 15 20 25 0 25 50 75 100 Days Survival (%) L2 - Survival L4 - Survival Control 4F 0 500 1000 1500Length (μm) L2 - Body size 0 500 1000 1500 *** ** P = 0.001 P = 1.6 x 10-5 0 50 100% of survived embryos ** P = 0.0082 *** P = 1.9 x 10-5 *** ** P = 0.0039 P = 1.6 x 10-5 *** *** Control (n=26) 4F (n=34) P = 2 x 10-15 P = 4.6 x 10-10 Control 4F Control 4F 100 μm 100 μm 100 μm Control uninduced 4F uninduced Control induced 4F induced Embryo survival Length (μm) Control (n=32) 4F (n=47) L4 - Body size Control 4F100 μm 100 μm 100 μm A Induction protocol at developmental stages Embryos Embryos Larval stage L1 L2 L3 L4 Developmental - Embryo survival - L2, L4 body size - L2, L4 survival After 2 days post-induction: Figure 2 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint B 0 days 1 day 2 days C D 4FControl E F G H D1 days post-induction 0 1 2 3 4 5 6 0 20 40 60 80 100 * * ** ** Control 4F 0 500 1000 1500 P = 0.008 4F Unind. 4F Induced 0 2 4 6 8 Number P = 0.01 P = 0.01 P = 0.002 P = 0.002 P = 3.19 x 10-8 *** 0 1 2 3 4 5 6 0 20 40 60 Days Worms outside food (%) 4F Unind. 4F Induced * *** ** *** *** P = 0.02 P = 0.001 P = 2.9 x 10-5 0 200 400 600Centre point speed (um/s) P = 0.02 * 0 5 10 15 20 25 0 20 40 60 80 100 Days % Control (n = 36) 4F (n = 25) *** P = 6.12 x 10-5 100 μm Body size ** Food Avoidance P = 4 x 10-4 P = 1 x 10-4 Length (μm) Egg laying Days Survival 4F Unind. 4F Induced Bagging % Speed 100 μm 100 μm100 μm 100 μm 100 μm Figure 3 A Induction protocol at reproductive stage Young adult D1 Reproductive - body size - food avoidance - egg laying and bagging - speed - survival Post-induction: D2 D3 D4 D5 D6 D7 D8 D9 Functional enrichment Downregulad genes in 4F - 48h post-induction 4F Differentially expressed genes 4h 48h 72h 4h 48h 72h Uninduced Induced I J 4F Unind. 4F Induced Middle adult .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint 0 days 1 day 2 days C D 4F nonGFP pcn-1::gfp Pmyo-2:mCherry 4F.prol Unind. (n = 10) 4F.prol Induced (n = 10) 0 2000 4000 6000MFI 4F.germ Unind. (n = 7) 4F.germ Induced (n = 7) 0 2000 4000 6000MFI P = 4.01 x 10-7 P = 1.32 x 10-6 0 days 1 day 2 days Embryonic germ cell post-induction Embryonic proliferation post-induction *** Embryonic proliferation 2 days post-induction *** Germ cell identity 2 days post-induction 100μm 100μm 0 200 400 600 800MFI Apoptosis 1 days post-induction P = 0.04 * Figure 4 0 days 1 day A B Apoptosis reporter post-induction UninducedInducedUninducedInduced 4F nonGFP ced-1p::2xFYVE::gfp Pmyo-2:mCherry 4F nonGFP pgl-1::gfp Pmyo-2:mCherry UninducedInduced F G 30 μm 30 μm 100μm 30 μm 4F.apop Unind. (n = 2) 4F.apop Induced (n = 3) .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint B 0 10 20 30 40 0 25 50 75 100 Days Survival (%) Every 3 days 0 10 20 30 40 0 25 50 75 100 Days Survival (%) Every 2 days 0 10 20 30 40 0 25 50 75 100 Days Survival (%) Everyday 0 5 10 15 20 25 0 25 50 75 100 Days Survival (%) 0 5 10 15 20 25 0 25 50 75 100 Days Survival (%) 0 5 10 15 20 25 0 25 50 75 100 Days Survival (%) *** ns ns ns D5 D10 D15 * P = 0.02 Control (n = 39) 4F (n = 36) Control (n = 36) 4F (n = 32) Control (n = 36) 4F (n = 25) Control (n = 37) 4F (n = 38) ns Control (n = 38) 4F (n = 36) Control (n = 39) 4F (n = 37) P = 3 x 10-4 D C ceh-6 0 50 100 150 Relative mRNA levels D1 4F D10 Control D10 4F sox-2 0 20 40 60 80 klf-1 0 20 40 60 lin-28 0 2 4 6 8 *** P = 1.28 x 10-7 P = 0.04 * *** * P = 0.02 *** P = 1.36 x 10-5 P = 0.032 * P = 0.005 ** P = 1.0 x 10-4 n = 3 3 2 n = 3 3 2 n = 3 3 2 n = 3 3 3 Figure 5 A Induction protocol at adult stage Young D1 Reproductive - survival - factors expression D10 Post-induction: D5 D10 D11 D12 D13 D14 D15 Single induction Post-reproductive Middle Old D10 Cyclic induction (+ cyclic induction) P = 3.73 x 10-3 ** P = 3.92 x 10-2 * P = 1.01 x 10-2 * .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Figure S1 A 4 factorsFour factors Inducible marker Constitutive marker Selection marker ceh-6 sox-2 klf-1 lin-28 Control GFP (CFJ248) 4F GFP (CFJ244) 4F non GFP (CFJ254) Phsp-16.2 Pmyo-2 Prps-0 GFP mCherry HygromycinR + + + B D1 OSKM RNA-seq normalized counts ceh-6 sox-2 klf-1 lin-28 4h 48h 72h 4h 48h 72h 4h 48h 72h 4h 48h 72h Control Uninduced UninducedInduced Induced 4F .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint 0 days Head region Mid body region Tail region Figure S2 UninducedInduced 1 day UninducedInduced 2 days UninducedInduced 100 μm A .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint Figure S3 A B Upregulated in 4F Downregulated in 4F Gene ontology analyses 4F vs. Control 4F Uninduced 48h (7) 4F Induced 48h (101) 4F Uninduced 4h (1) 4F Induced 4h (88) Functional enrichment Upregulad genes in 4F - 48h post-induction Downregulad genes in 4F - 48h post-induction Gene ontology analyses 4F vs. Control C D Functional enrichment .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint A Embryonic 4F GFP hsp-16::gfp Pmyo-2:mCherry Germline G 0 days 2 days 4F nonGFP myo-3p::TOM20 Pmyo-2:mCherry 2 days0 days 4F GFP eft3p::wrmScarlet Pmyo-2:mCherry hsp-16::gfp H I J K L hsp-16:gfp eft3p:wrmScarlet 0 days 2 days 4F nonGFP ges-1::gfp Pmyo-2:mCherry 0 1000 2000 3000 4000MFI D1 - Somatic 0 50 100 150MFI 0 2 106 4 106 6 106 100 μm 100 μm 100 μm Intestine 2 days post-induction Body wall muscle 2 days post-induction Soma 2 days post-induction MFI 100 μm Head ganglions Intestine Body wall muscle Intestine post-induction Body wall muscle post-induction Soma post-induction F Figure S4 30 μm 30 μm 30 μm B C D E 4F.inte Unind. (n = 2) 4F.inte Induced (n = 3) 4F.bwm Unind. (n = 4) 4F.bwm Induced (n = 4) 4F.soma Unind. (n = 4) 4F.soma Induced (n = 7) Genetic approach mCherry 4F nonGFP GFP reporters TOM20 reporter wrmScarlet reporter Proliferation Germline Apoptosis Intestine Body wall muscle Somatic 4F hermaphrodite Reporter males 4F.prol 4F.germ 4F.apop 4F.inte 4F.bwm 4F.soma 4F Reporter hermaphrodite .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint D10 OSKM RNA-seq normalized counts 4h 48h 4h 48h 4h 48h 4h 48h Control Uninduced Induced 4F Uninduced Induced A B 4F Differentially expressed genes 4h 48h 4h 48h Uninduced Induced Figure S5 .CC-BY-NC-ND 4.0 International licenseavailable under a 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 preprint (whichthis version posted May 4, 2024. ; https://doi.org/10.1101/2024.05.03.592330doi: bioRxiv preprint