The transcriptional corepressor CTBP-1 acts with the SOX family transcription factor EGL-13 to maintain AIA interneuron cell identity in C. elegans

Cell identity is characterized by a distinct combination of gene expression, cell morphology and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here we show that the gene ctbp-1, which encodes the transcriptional corepressor C-terminal binding protein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.


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Over the course of animal development, complex networks of transcription 40 factors act and interact to drive the division and differentiation of progenitor cells 41 towards terminal cell identities [1][2][3][4][5][6][7][8]. These networks of transcriptional activity 42 often culminate in the activation of master transcriptional regulators that are 43 responsible for directing the differentiation of a diverse range of cell and tissue 44 types [4, [9][10][11][12]. Examples of such master transcriptional regulators include the 45 mammalian bHLH transcription factor MyoD, which specifies skeletal muscle In previous studies, we screened for and characterized mutations that 85 prevent the programmed cell death of the sister cell of the C. elegans M4 neuron 86 [58,59]. For these screens, we used the normally M4-specific GFP transcriptional 87 reporter P ceh-28 ::gfp and identified isolates with an undead M4 sister cell, which 88 expresses characteristics normally expressed by the M4 cell, on the basis of 89 ectopic GFP expression. In addition to mutants with an undead M4 sister cell, we 90 isolated 18 mutant strains that express P ceh-28 ::gfp in a manner uncharacteristic 91 of M4 or its undead sister. These mutants express P ceh-28 ::gfp in a bilaterally 92 symmetric pair of cells located near the posterior of the C. elegans head, far from 93 both M4 and the single M4 sister cell (Fig. 1A).  (Fig. S1C-D), 105 similar to our ctbp-1 isolates. These findings demonstrate that loss of ctbp-1 106 function is responsible for P ceh-28 ::gfp misexpression. 107 To determine the identity of the cells misexpressing the normally M4-108 specific marker P ceh-28 ::gfp, we examined reporters for cells in the vicinity of the 109 observed misexpression in ctbp-1 mutants. The AIA-neuron reporter nIs843[P gcy-110 28.d ::mCherry] showed complete overlap with misexpressed P ceh-28 ::gfp, indicating 111 that the cells misexpressing the M4 reporter are the two bilaterally symmetric and 112 embryonically-generated AIA interneurons (Fig. 1B). 113

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The penetrance of ceh-28 reporter misexpression in the AIA neurons 115 increases with age 116 While characterizing ctbp-1 mutants, we noticed that fewer young worms 117 misexpress P ceh-28 ::gfp in the AIAs than do older worms (Fig. 1D). To investigate 118 the temporal aspect of this phenotype, we scored ctbp-1 mutants for P ceh-28 ::gfp 119 misexpression throughout the four worm larval stages (L1-L4) and into the first 120 day of adulthood ("early" and "day 1" adults). ctbp-1 mutants rarely misexpressed 121 P ceh-28 ::gfp at early larval stages, but displayed an increasing penetrance, though 122 invariant expressivity, of this defect as worms transitioned through larval 123 development, such that by the last larval stage (L4) nearly all worms exhibited 124 reporter misexpression specifically and solely in the AIAs (Fig. 1E). A similar 125 stage-dependent increase in reporter expression in ctbp-1 mutants occurred in 126 mutants carrying a second independently-generated ceh-28 reporter, nIs348[P ceh-127 28 ::mCherry] (Fig. S1E) as in day 1 adults (Fig. 3A). We found that L1 ctbp-1 mutant AIAs appeared 174 grossly wild-type in morphology (Fig. 3A). However, L4 and adult ctbp-1 mutant 175 AIAs had ectopic neurite branches that extended from both the anterior and 176 posterior ends of the AIA cell body (Fig. 3A). The penetrance of these ectopic 177 branches increased progressively in later larval stage and adult mutants (Fig. 3B-178 C). Older ctbp-1 mutant AIAs also appeared to have an elongated cell body 179 compared to wild-type AIAs. Quantification of this defect revealed that L4 and 180 adult mutant AIA cell bodies, but not those of L1s, were significantly longer than 181 their wild-type counterparts (Fig. 3D). To assess if this increase in AIA length was 182 a consequence of an increase in AIA size, we measured the maximum area of 183 the AIA cell body from cross-sections of these cells. We found that the maximum 184 area of the AIA cell body did not significantly differ between wild-type and mutant 185 AIAs at any stage (Fig. S4A), indicating that mutant AIAs were misshapen but not 186 enlarged. To confirm that we were not biased by an awareness of genotype while 187 measuring AIA lengths, we blinded the wild-type and ctbp-1 AIA images used for 188 length measurements and scored the blinded images as either "normal" or 189 "elongated" (Fig. S4B). Again, at the L1 larval stage both wild-type and ctbp-1 190 mutant AIAs appeared overwhelmingly "normal," whereas at both the L4 larval 191 stage and in day 1 adults ctbp-1 mutant AIAs were scored as "elongated" at a 192 consistently higher rate than their wild-type counterparts. Collectively, these 193 results demonstrate that ctbp-1 mutant AIAs display abnormal morphology and 194 that the severity of the observed morphological defects in ctbp-1 mutants 195 increases from L1 to L4 to adulthood. Furthermore, the relative lack of AIA 196 morphological defects in L1 ctbp-1 mutants suggests that ctbp-1 is not required 197 for the establishment of proper AIA morphology but instead acts to maintain AIA 198 morphology over time. found that heat shock at the L4 stage did not restore ctbp-1 mutant AIA 208 morphology in day 1 adults back to wild-type, nor was there any significant 209 difference in the frequency of morphological defects between heat-shocked and 210 non-heat-shocked adults carrying the rescue construct ( Fig. 3I-L). We speculate 211 that the lack of restoration of morphology in late-stage worms might be a 212 consequence of the defects being irreversible, and that ctbp-1 might be 213 continuously required to prevent such defects from occurring. From these data 214 we conclude that ctbp-1 can act cell-autonomously, and possibly continuously, to 215 maintain aspects of AIA morphology in a manner similar to AIA gene expression. Consistent with previous studies [66], we found that worms that had been 228 briefly starved with 90 minutes of food deprivation and had no prior experience 229 with butanone (so-called "naïve" worms) were generally attracted to the odor, 230 while worms that were briefly starved in the presence of butanone ("conditioned" 231 worms) adapted to the odor and exhibited mild repulsion to it (Fig. 4A-E). We 232 next compared wild-type and ctbp-1 mutant worms for their ability to adapt to 233 butanone. We found that while L1 ctbp-1 worms showed an ability to adapt to 234 butanone roughly similar to that of their wild-type counterparts, conditioned L4 235 ctbp-1 mutants displayed a significant decrease in repulsion from butanone 236 relative to wild-type L4 animals, indicating a decrease in their ability to adapt to 237 the odor (Fig. 4B-E). As a control, we assayed a strain carrying a transgenic 238 construct that genetically ablates the AIA neurons, JN580. As expected, JN580 239 worms displayed decreased butanone adaptation at both the L1 and L4 larval 240 stages. Thus, ctbp-1 mutant worms displayed a defect in butanone adaptation 241 similar to that of an AIA-ablated strain and did so only at a later larval stage, 242 suggesting a potential loss of AIA function in L4 ctbp-1 mutants. However, while 243 ctbp-1 mutant L4s exhibited weaker butanone adaptation than their wild-type 244 counterparts, this defect was not as severe as that of JN580 L4s, indicating that 245 ctbp-1 mutant AIAs might retain some function. Additionally, the lack of a 246 butanone adaptation defect in L1 ctbp-1 mutants similar to that of L1 JN580 247 worms further suggests that loss of ctbp-1 does not disrupt early AIA function 248 and shows that ctbp-1 is not required for the establishment of functional AIA 249

neurons. 250
We next asked if ctbp-1 can act cell-autonomously in the AIAs and in older 251 worms to regulate butanone adaptation. We assayed ctbp-1 mutants carrying the 252 AIA-specific rescue construct nIs743[P AIA ::ctbp-1(+)] for butanone adaptation 253 While conducting these assays, we observed that naïve ctbp-1 mutant 267 worms displayed a mildly weaker attraction to butanone than did their wild-type 268 counterparts at both the L1 and L4 larval stages (Fig. 4B,D). AIA-specific rescue 269 of ctbp-1 did not rescue this mild chemotaxis defect -naïve ctbp-1 mutants

ctbp-1 mutant AIAs have additional defects in gene expression 284
To better characterize the genetic changes occurring in mutant AIAs, we 285 performed a single, exploratory single-cell RNA-Sequencing (scRNA-Seq) 286 experiment comparing wild-type and ctbp-1 mutant worms. We sequenced RNA 287 from the neurons of wild-type and ctbp-1 L4 worms and processed the resulting 288 data using the 10X CellRanger pipeline to identify presumptive AIA neurons 289 based on the expression of several AIA markers (gcy-28, ins-1, cho-1) shown 290 above to be expressed in both wild-type and ctbp-1 mutant AIAs (Fig. 2B). 291 Confirming that these data captured changes in the AIA transcriptional profiles, 292 we found that ctbp-1 mutant AIAs showed high levels of expression of ceh-28, 293 while wild-type AIAs showed no detectable ceh-28 expression (Fig. S6). 294 We analyzed AIA transcriptional profiles to identify genes that appeared to 295 be either expressed in ctbp-1 mutant AIAs and not expressed in wild-type AIAs 296 (similar to ceh-28) or expressed in wild-type AIAs but not expressed in ctbp-1 297 AIAs. To confirm candidate genes, we crossed existing reporters for those genes 298 to ctbp-1 mutants or, in cases for which reporters were not readily available, 299 generated our own transgenic constructs. We identified and confirmed one gene 300 that, similar to ceh-28, was not expressed in wild-type AIAs but was 301 misexpressed in ctbp-1 mutant AIAs: acbp-6, which is predicted to encode an otIs123[P sra-11 ::gfp] and the glr-2 reporter ivEx138[P glr-2 ::gfp] in wild-type and 308 ctbp-1 L4 worms and confirmed that acbp-6 was absent in wild-type AIAs but 309 misexpressed in ctbp-1 mutants (Fig. 5A-B), while both sra-11 and glr-2 were 310 consistently expressed in wild-type AIAs but not expressed in the AIAs of ctbp-1 311 mutants ( Fig. 5C-F). We also visualized these reporters in L1 wild-type and ctbp-312 1 worms and found that both P acbp-6 ::gfp and P sra-11 ::gfp displayed a time-313 dependence to their expression similar to that of P ceh-28 ::gfp − P acbp-6 ::gfp was 314 rarely detectible in the AIAs of either wild-type of ctbp-1 AIAs at the L1 stage but 315 was consistently expressed in ctbp-1 mutant L4 AIAs (Fig. 5A-B), while P sra-316 11 ::gfp was rarely detectible in the AIAs of either wild-type or ctbp-1 mutant L1 317 worms but was expressed in the AIAs of most wild-type worms by the L4 stage 318 while remaining off in the AIAs of most L4 ctbp-1 mutants (Fig. 5C-D). These 319 observations suggest that, like ceh-28 expression, acbp-6 and sra-11 expression 320 is regulated by ctbp-1 primarily in the AIAs of late-stage larvae and adults. By 321 contrast, glr-2 was expressed in wild-type but not ctbp-1 AIAs in both L1 and L4 322 larvae ( Fig. 5E-F). 323 These data demonstrate that mutant AIAs fail to turn on and/or maintain 324 the expression of genes characteristic of the adult AIA neuron (sra-11 and glr-2) 325 while misexpressing at least two genes uncharacteristic of AIA (ceh-28 and acbp-326 6). That the majority of these abnormalities in AIA gene expression occurred long 327 after the AIAs are generated during embryogenesis further supports the 328 conclusion that ctbp-1 does not act to establish the AIA cell identity. 329 Collectively, our findings concerning AIA gene expression, morphology 330 and function demonstrate that ctbp-1 acts to maintain the AIA cell identity, plays 331 little to no role in the initial establishment of the AIA cell fate, and can act cell-332 autonomously and in older worms to maintain these aspects of the AIA identity. 333

egl-13 mutations suppress the ctbp-1 mutant phenotype 335
To investigate how ctbp-1 acts to maintain AIA cell identity, we performed 336 a mutagenesis screen for suppression of P ceh-28 ::gfp misexpression in the AIAs of 337 L4 ctbp-1 mutants (Fig. 6A)  We assayed the loss-of-function allele of egl-13 with the highest 355 penetrance of suppression, n5937, for its ability to suppress P ceh-28 ::gfp 356 misexpression over the course of larval development and into adulthood of ctbp-357 1 mutant worms (Fig. 6C). egl-13(n5937) strongly suppressed ctbp-1 at all 358 stages, resulting in little to no misexpression of P ceh-28 ::gfp in the AIAs of egl-13 359 ctbp-1 double mutants at any larval stage or in day 1 adults. 360 To determine if, like ctbp-1, egl-13 can act cell-autonomously in the AIAs, 361 we generated a transgenic construct that drives expression of a wild-type copy of the difference was no longer significant in adults). These data demonstrate that 378 loss of egl-13 has little consistent effect on the AIA morphological defects caused 379 by a loss of ctbp-1 activity, suggesting that ctbp-1 maintains AIA morphology 380 primarily through egl-13-independent pathways. 381 We next assayed the ability of egl-13(n5937) to suppress AIA functional 382 defects. We tested egl-13 ctbp-1 double mutants for butanone adaptation and 383 found that, at the L1 larval stage, this double mutant strain displayed a detectable 384 response to butanone similar to ctbp-1 single mutants (Fig. 6H-I We next asked if mutation of egl-13 could suppress other ctbp-1 mutant 398 AIA gene expression defects besides that of ceh-28. We crossed in acbp-6, sra-399 11 and glr-2 reporters to egl-13 ctbp-1 double mutants and visualized reporter 400 expression at the L1 and L4 larval stages. We found that mutation of egl-13 401 suppressed P acbp-6 ::gfp misexpression in the AIAs (Fig. 7A,D, compare to Fig. 5), 402 just as egl-13 mutation suppressed P ceh-28 ::gfp misexpression. By contrast, 403 mutation of egl-13 had no effect on the loss of P sra-11 ::gfp or P glr-2 ::gfp expression 404 in ctbp-1 mutants (Fig. 7B-D). We speculated that EGL-13 might directly regulate 405 expression of ceh-28 or acbp-6. To test this hypothesis, we examined the ceh-28 406 and acbp-6 promoter regions for possible EGL-13 binding sites. We failed to 407 identify any promising candidates, suggesting that regulation of these genes by 408 EGL-13 is likely indirect. These results demonstrate that some, though not all, of 409 the AIA gene expression defects seen in ctbp-1 mutants are regulated through 410 egl-13. 411 As egl-13 is required for misexpression of both ceh-28 and acbp-6 as well 412 as for disruption of AIA function in ctbp-1 mutants, we hypothesized that 413 misexpressed ceh-28 or acbp-6 might be contributing to the observed AIA 414 functional defect in ctbp-1 mutants. If so, we expected that mutations that 415 eliminated the functions of these ectopically expressed genes should restore AIA 416 function in ctbp-1 mutants. To test this hypothesis, we crossed mutant alleles of 417 ceh-28 (cu11) or acbp-6 (tm2995) (both deletion alleles spanning greater than 418 half their respective genes) to ctbp-1(n4784) mutants and assayed the resulting 419 double mutants for butanone adaptation in L1 and L4 worms. We found that 420 acbp-6; ctbp-1 double mutants were nearly identical to both naïve and 421 conditioned ctbp-1 single mutants at both the L1 and L4 larval stages (Fig. 7E-H), 422 indicating that misexpressed acbp-6 is likely not responsible for the observed AIA 423 functional defect. Conditioned ctbp-1 ceh-28 double mutants appeared similar to 424 both the wild type and ctbp-1 single mutants at the L1 stage (Fig. 7I-J). These 425 double mutants display an minor, though not statistically significant, difference in 426 adaptation at the L4 larval stage when compared to either wild-type or ctbp-1 427 animals (Fig. 7K-L). We speculate that overexpression of ceh-28, caused by a This motif is 100% conserved in C. elegans EGL-13. We speculate that CTBP-1 464 interacts with EGL-13 through its PLNLS motif to regulate EGL-13 activity as part 465 of AIA cell-identity maintenance. Specifically, we propose (Fig. 8)

CTBP-1 likely utilizes additional transcription factors besides EGL-13 to 480 maintain the AIA cell identity 481
Our understanding of how CTBP-1 acts to maintain the AIA cell identity is 482 incomplete. While we have identified a few genes with expression that changes 483 in the absence of ctbp-1 (ceh-28, acbp-6, sra-11, glr-2), none of these genes 484 seems to individually account for the full range of AIA defects seen in older ctbp-485 1 mutants. We speculate that there are many more unidentified transcriptional 486 changes occurring in ctbp-1 mutant AIAs that contribute to the observed AIA 487 morphological and functional defects.

C. elegans strains and transgenes 549
All C. elegans strains were grown on Nematode Growth Medium (NGM) plates 550 seeded with E. coli OP50 as described previously [80]. We used the N2 Bristol 551 strain as wild type. Worms were grown at 20°C unless otherwise indicated. 552 Standard molecular biology and microinjection methods, as previously described 553 [81], were used to generate transgenic worms. 554

555
The following strains were used in this study: 556  All images were obtained using an LSM 800 confocal microscope (Zeiss) and 596 ZEN software. Images were processed and prepared for publication using FIJI 597 software and Adobe Illustrator. 598

Heat-shock assays 600
Rescue of AIA defects in day 1 adult worms was assayed using the 601 AIAs were identified and tested as described in the text. 645

Morphology scoring 647
We assayed AIA morphology by visualizing and imaging AIAs expressing nIs840 648 using an LSM 800 confocal microscope (Zeiss) and a 63x objective. AIA cell 649 body length and area were quantified using FIJI software.     Tukey's correction. 1248