The proper function of animal nervous systems must accurately execute developmental programs that generate and specify neuronal cells and circuits to properly form and function. These processes allow neuronal cells to acquire the correct temporal and spatial identity and cell-type specific functions. Dysregulation of these programs leads to multiple neuro-developmental and neuro-psychiatric diseases (1). Although it has been well established that transcriptional control via trans- and cis-acting regulatory networks mediates neuronal differentiation and specification (2, 3), the molecular and neuronal mechanisms underlying transcription-mediated neuronal development are still not completely understood.

Caenorhabditis elegans has a relatively simple nervous system with 302 neurons categorized into 118 morphologically distinct classes (4, 5). Developmental programs to generate and specify these neurons have been well-characterized; distinct transcription regulatory networks result in cell-specific expression of pan-neuronal and terminal features such as synaptic vesicle components and neurotransmitters, respectively (3, 6-8). Hundreds of C. elegans transcription factors, including 102 homeobox genes, play roles in cell-fate determination, and many of them provide necessary and sufficient conditions to specify neuronal identity by acting as terminal selectors, which directly or indirectly regulate the expression of most terminal differentiation genes (2, 3, 9). However, the complete molecular mechanisms of how individual neurons achieve cell-type specific determination and specification are still not understood.

C. elegans innter labial neuron type 1 (IL1) is multimodal sensory/inter/motor neurons. Three pairs of radially symmetric neurons, including two dorsal (IL1 dorsal left [IL1DL] and right [IL1DR]), two lateral (IL1 lateral left [IL1L] and right [IL1R]), and two ventral pairs of neurons (IL1 ventral left [IL1VL] and right [IL1VR]). IL1s innervate the head muscles with their cell bodies located in the head (Fig. 1A) (5, 10, 11). Their processes run anteriorly to the tip of the nose, and their function may play roles in head locomotion (12). Several trans-acting factors are expressed in IL1s, including homeodomain (ceh-43/Dlx, ceh-32/Six3, ceh-18/POU, vab-3/Pax6, and zfh-2/zinc-finger protein 2) and non-homeodomain (lin-32/Atonal bHLH and sox-2/SoxB1 HMG) types of transcription factors (5, 8, 13-15). The lin-32 gene regulates both pan-neuronal and terminal differentiation via the ceh-43 and ceh-32 homeobox genes, which are also regulated by sox-2 and vab-3 genes (13-15). However, transcriptional regulation underlying the IL1 neuronal specification needs to be further understood.

Expression of a neuropeptide gene in the lateral and dorsal neurons of IL1 is abolished in nfya-1 mutants

To identify factors that specify the neuronal cell-fate of IL1s, we performed a non-biased genetic screen to isolate mutants where the expression. The expression pattern of a flp-3p::gfp reporter was disrupted in IL1s (Fig. 1B). flp-3 encodes an FMRFamide-related neuropeptide and is expressed in all six IL1 neurons as adult (16). Among mutants isolated from this screen, one mutant allele (lsk53) showed weak expression of flp-3, specifically in the lateral and dorsal IL1s, but the relatively normal expression in the ventral IL1s (Fig. 1C, D and Supplementary Fig. 1). We quantitated the expression phenotype of the flp-3 gene in IL1s of lsk53 mutants and found that lsk53 mutants showed significantly lower flp-3 expression in the lateral and dorsal IL1 neurons than wild-type animals. The average intensity for lsk53 mutants (IL1L: 50.03 ± 13.98 A.U., n = 50; IL1R: 58.06 ± 11.36 A.U., n = 50) was only about 38% (IL1L) or 46% (IL1R) of that of wild-type animals (IL1L: 132.32 ± 9.36 A.U., n = 50; IL1R: 127.08 ± 4.56 A.U., n = 50). The flp-3 expression in dorsal IL1 of lsk53 was also significantly reduced; however, the flp-3p::gfp intensity in the ventral IL1s was comparable to that of wild-type animals (Fig. 1D).

From the genetic mapping and whole-genome sequence analysis, we identified a molecular lesion of lsk53 mutant allele in the nfya-1 gene (Fig. 1E). nfya-1 encodes a NF-YA subunit that constitutes the NF-Y trimer complex with NF-YB and NF-YC subunits and has been shown to regulate the transcription of the egl-5 and tbx-2 transcription factors (17-19). nfya-1 has a conserved CCAAT-binding factor (CBF) domain which is highly conserved through evolution (20) (Fig. 1E). The lsk53 mutant allele carries a nonsense mutation in the second exon that results in a premature translation stop (R117Opal), suggesting that this mutation is a null allele (Fig. 1E). Then we tested an additional null allele (ok1174) of nfya-1 (21), in which the expression of flp-3 was decreased similarly to the lsk53 allele (Fig. 1F). These findings indicate that NFYA-1 plays a role in regulating flp-3 neuropeptide gene expression in the lateral and dorsal, but not ventral IL1 neurons. It is noteworthy that we had not observed ectopic flp-3 expression in other cell-types (Supplementary Fig. 2). This result indicates that nfya-1 may act as a positive regulator, in contrast to its negative role for egl-5 or tbx-2 expression (18, 19).

Expression of the IL1-specific marker genes is differentially affected in nfya-1 mutants

The IL1 neurons are both peptidergic and glutamatergic (16, 22). To determine the extent to which nfya-1 regulates gene expression in IL1s, we assessed the expression of IL1-specific terminal differentiation genes, such as the glutamatergic marker genes. We tested eat-4 vesicular glutamate transporter and unc-8 DEG/ENaC cation channel protein, of which reporter genes are expressed in IL1s (22, 23). We first confirmed that eat-4 (481 bp sequence located downstream of the translation start site: eat-4p12) (22) and unc-8 (293 bp sequence located ∼197 bp upstream of the translation start site: unc-8D7p) were indeed expressed in all six IL1 neurons (Supplementary Fig. 3). We next examined the expression pattern of eat-4 and unc-8 in nfya-1 mutants. The expression of eat-4 was abolished in all six IL1 neurons, whereas that of unc-8 was not affected (Fig. 1G, H and Supplementary Fig. 4), suggesting that nfya-1 differentially regulates gene expression of terminal differentiation markers in the IL1 neurons.

Next, to test whether other neuronal characteristics are retained in IL1s of nfya-1 mutants, we examined the expression of the pan-neuronal gene markers, rgef-1 Ras guanine nucleotide releasing protein and unc-119 chaperone (Fig. 1I, J and Supplementary Fig. 3, 4) (24, 25), and found that the GFP expression of the rgef-1p::gfp and unc-119p::gfp reporters was not changed in IL1s of nfya-1 mutants, confirming that nfya-1 regulates the expression of specific and limited genes, including flp-3 and eat-4, in IL1s.

The IL2 (inner labial neuron type 2) neurons, which are also embedded in the inner labial sensilla, are structurally similar to IL1s and closely related in cell lineage, where the sisters of the IL1 precursor cells are the IL2 neurons (4) (Supplementary Fig. 5). To examine whether nfya-1 also affects the characteristics of IL2, we tested the expression pattern of the tba-6 α-tubulin gene, an IL2-specific marker gene (26) in nfya-1 mutants, and found that the expression of tba-6 was not altered in IL2s of nfya-1 mutants (Supplementary Fig. 5), indicating that nfya-1 may play specific roles in the differentiation of IL1s.

nfya-1 is expressed and functions in the IL1 neurons to regulate flp-3 and eat-4 expression

nfya-1 has previously been expressed in most cell-types, including neuronal, intestinal, and germline cells of developing larvae and adults (17, 18). To determine whether nfya-1 is expressed in the IL1 neurons, we generated a transgenic animal expressing the nfya-1p::gfp transgene that includes 2.1 kb of an upstream sequence of the nfya-1 gene (Fig. 1E) and examined its expression pattern (Fig. 2A and Supplementary Fig. 6A, B). Consistent with previous reports, nfya-1 was expressed in many neurons in the head of worms (17, 18). To identify nfya-1 expression in IL1s, we generated a transgenic line expressing the nfya-1p::gfp transgene together with the flp-3p::mCherry transgene. We found that nfya-1 was co-expressed with flp-3 in all IL1s at either the adult or larval developmental stage animals, indicating that nfya-1 is indeed expressed in IL1s throughout development (Fig. 2A and Supplementary Fig. 6A, B). Moreover, GFP-tagged NFYA-1 driven under the control of the nfya-1 promoter was localized to the nucleus of all IL1 neurons, supporting its predicted role as a transcription factor (Fig. 2B and Supplementary Fig. 6C).

To determine whether nfya-1 acts cell-autonomously within IL1, we expressed a wild-type nfya-1 cDNA under the control of the nfya-1 promoter or unc-8D7 IL1-specific promoter in nfya-1 mutant backgrounds. The nfya-1 expression from these transgenes fully rescued the flp-3 expression defects in IL1s of nfya-1 mutants (Fig. 2C and Supplementary Fig. 7), indicating that nfya-1 acts in IL1s to regulate flp-3 expression. Moreover, defects in the eat-4 expression of nfya-1 mutants were also restored by the nfya-1 expression under the control of nfya-1 promoter as well as IL1-specific promoter (Fig. 2D). Together, nfya-1 is expressed in and acts in the IL1 neurons to regulate the expression of a subset of IL1 marker genes.

Expression of nfya-1 is partially sufficient to induce flp-3 expression in other cell-types

To address when nfya-1 is required for flp-3 expression in the IL1 neurons, we tried to trigger a temporal nfya-1 expression under the control of an inducible, ubiquitously expressed heat-shock promoter (hsp16.2) (27). Although we tried to heat-shock animals in several contexts, temporal expression of the nfya-1 gene at the egg, larval, or adult stage did not rescue the defects in nfya-1 mutants, suggesting that acute, post-developmental expression of nfya-1 does not induce appropriate flp-3 expression in IL1s (Fig. 2E).

To further investigate whether the expression of nfya-1 can induce flp-3 expression in other cell-types, we forced nfya-1 expression under the control of the flp-7 neuropeptide promoter, which is normally expressed in several head neurons, including a I5 pharyngeal neuron, but not in IL1s (Fig. 2F) (16, 28, 29). We found that nfya-1 expression under the control of the flp-7 promoter did not affect flp-3 expression in IL1s but induced ectopic flp-3 expression in the I5 neuron in about 11% of the transgenic worms (Fig. 2F), indicating that expression of NFYA-1 can induce flp-3 expression in other cells that do not relate to IL1. These results suggest that ectopically-expressed nfya-1 can drive the expression of the IL1 marker in non-IL1 cells.

Identification of cis-regulatory motifs of terminally differentiated IL1 markers

To identify cis-regulatory motifs required to drive the expression of the IL1 marker genes, flp-3, eat-4, and unc-8, in the IL1 neurons, we performed a promoter analysis in which DNA sequences within the promoters were serially deleted, and the resultant transgenic animals were examined for expression in IL1s. We found that deletions in several regions of the flp-3 promoter caused decreased flp-3 expression in IL1s; a 101 bp sequence between D3 and D4, a 27 bp sequence between D4 and D5, and a 78 bp sequence between D5 and D6 (Fig. 3A). We tried further to narrow down the regions by point-mutating DNA sequences and identified two DNA sequences or motifs; CCTCAATTTAT for flp-3 expression in the lateral IL1s and CCCAACACTCCTT for flp-3 expression in the dorsal and ventral IL1s (Fig. 3A). The lateral IL1 motif appears to be phylogenetically conserved in the promoters of the flp-3 orthologs of related Caenorhabditis species and shares core sequences with the mammalian NF-YA binding motif (CCAAT). However, the dorsal and ventral motif was not found in the flp-3 promoters of related Caenorhabditis species and did not contain an NF-YA binding motif (Fig. 3A).

Additionally, we analyzed the eat-4 and unc-8 gene promoters. Previously, a minimal region within the eat-4 promoter for eat-4 expression in IL1s was identified (22). We further mutated DNA sequences in this region which are highly conserved within related Caenorhabditis species. We identified the DNA sequence (AAGGCACACGGCCGTGA), and mutations in this sequence resulted in an almost complete loss of eat-4 expression in all IL1s (Fig. 3B). We next identified a minimal region in the unc-8 promoter by serially deleting DNA sequences and mutated conserved DNA sequences within a 293 bp sequence between D5 and D6 (Fig. 3C). This four bases (GGAA) DNA sequence is crucial to the expression of the unc-8 gene in all IL1s. We noted that these DNA sequences for the IL1 expression of eat-4 and unc-8 appeared unrelated to the conventional mammalian NF-Y binding motif of CCAAT (Fig. 3B, C). Taken together, these results suggest that distinct cis-regulatory motifs play roles in expressing the terminally expressed IL1 genes.

To test whether these IL1L/R motifs act to drive gene expression in the IL1 neurons, we inserted three copies of the IL1L/R motifs in the promoter of flp-7 (28) (Supplementary Fig. 8). Transgenic animals expressing a flp-7p-IL1L/R motif::gfp reporter construct still exhibited GFP expression in flp-7 expressing neurons, and we observed ectopic expression of flp-7 in the head. This result indicates that the insertion of IL1L/R motif into the flp-7 promoter does not drive a reporter in IL1s, but does in other cells, which do not usually express flp-7. These results suggest that inserted motifs may change flp-7 promoter activity to drive gene expression in other cells, possibly by interacting with NF-Y.

Sequence similarity between the lateral IL1 motif of the flp-3 gene promoter and the mammalian NF-Y binding motif prompted us to test whether the C. elegans NFYA-1 can directly bind this DNA sequence in the flp-3 gene promoter in vivo. Previously, the C. elegans NF-Y complex has been shown to bind directly to a CCAAT site identified in the egl-5 gene promoter via an electrophoretic mobility shift assay (EMSA) (18). We performed a chromatin immunoprecipitation (ChIP) assay coupled to a quantitative real-time PCR using an antibody specific to GFP and extracts derived from transgenic animals expressing the nfya-1p::gfp (GFP only) or nfya-1p::NFYA-1::gfp (GFP-tagged NFYA-1) transgenes. We selected five loci, including the flp-3, egl-5, eat-4, unc-8, and flp-12 genes. The flp-12 gene promoter, which is expressed in the SMB neuron specifically, was not altered in nfya-1 mutants, served as a negative control (16, 28) (Supplementary Fig. 9). We found significant enrichment of flp-3 fragments but not egl-5, eat-4, unc-8, or flp-12 fragments in extracts from transgenic animals expressing NFYA-1-GFP (Fig. 3D), indicating that NFYA-1 can directly bind to the CAAT sequences identified in the flp-3 promoter sequences.

nfya-1 and nfya-2 function redundantly, with nfyb-1 and nfyc-1, to regulate the expression of the IL1 markers

In contrast to the mammal, which has a single NF-YA gene, the C. elegans has two nfya genes, nfya-1 and nfya-2 (17, 18). These two nfya genes share similar DNA sequences and expression patterns. However, it has been reported that only nfya-1, but not nfya-2, regulates egl-5 and tbx-2 repression, and this result has raised questions about the roles of nfya-2 (17, 19). Therefore, we examined whether nfya-2 regulates the gene expression of IL1 markers and found that flp-3 expression was affected in nfya-2 (tm4194) mutants, in a similar manner observed in nfya-1 mutants (Fig. 4A and Supplementary Fig. 10); flp-3 expression was significantly decreased in the dorsal and lateral IL1s but not ventral IL1s (Fig. 4A, B). The flp-3 expression in nfya-2;nfya-1 double mutants was decreased even in the ventral IL1s, whose flp-3 expression was not affected in any single mutant background of nfya (Fig. 4A, C). Moreover, the flp-3 expression in all six IL1s was decreased in NF-YB nfyb-1 (cu13), or NF-YC nfyc-1 (tm4541) mutants, (Fig. 4A, B and Supplementary Fig. 10). These data indicate that nfya-1 and nfya-2 act in a partially redundant fashion together with other NF-Y components, nfyb-1 and nfyc-1, to regulate flp-3 expression in all IL1s.

Gene expression of eat-4 was decreased in IL1s of nfya-1 mutants, but not that of unc-8. Interestingly, the nfya-2 mutation did not affect gene expression of either eat-4 or unc-8 in IL1s (Fig. 4D, E and Supplementary Fig. 11). However, the unc-8 expression was strongly compromised in nfya-2;nfya-1 double mutants, indicating the redundant functions of nfya-1 and nfya-2 in regulating unc-8 expression in IL1s. In addition, the eat-4 and unc-8 expressions were significantly decreased in nfyb-1 or nfyc-1 mutants (Fig. 4D, E). These data, together with the flp-3 expression results, indicate that the NF-Y complex plays distinct roles in regulating the IL1 marker expression.

nfya-2, nfyb-1, and nfyc-1 have been previously shown to be expressed in many head neurons in adults (17, 18). We generated transgenic animals expressing gfp under the control of a 1.2 kb sequence upstream of nfya-2, 2 kb sequence upstream of nfyb-1, or 495 bp sequence upstream of nfyc-1 (Supplementary Fig. 10) and examined their expression pattern. Although our nfya-2p::gfp or nfyc-1p::gfp transgenic animals did not exhibit any detectable gfp expression, the nfyb-1p::gfp transgenic animals showed strong gfp expression in all six IL1s (Fig. 4F), supporting that the NF-Y complex acts in IL1s to regulate IL1 marker expression.

Due to the evolutionary conservation of NF-Y, understanding the molecular and neuronal mechanisms of the highly conserved NF-Y complex underlying neuronal specification in C. elegans will help understand what the function of NF-Y is in cell differentiation, which is the pivotal process in developmental of multicellular organisms (30).

The heteromeric NF-Y complex consisted of the three subunits, NF-YA, NF-YB, and NF-YC, is an evolutionarily conserved transcription factor in many organisms, including yeast, plants, Drosophila, and mammals (31). NF-Y has been shown to regulate various biological processes, including metabolism, cell-cycle progression, and neuronal maintenance (32). In Drosophila, it is reported that dNFY, the NF-Y homolog, positively regulates sevenless, a receptor tyrosine kinase, and negatively controls a senseless transcription factor during photoreceptor differentiation (33). These results indicated that dNFY acts as both an activator and a repressor. In C. elegans, it is shown that NF-Y negatively regulates the expression of the transcription factors, egl-5 hox and tbx-2 T-box, which are ectopically expressed in nfy mutants (18, 19). egl-5 and tbx-2 mediate tail morphogenesis and muscle development, respectively (18, 19, 34). These results suggest that C. elegans NF-Y acts as a repressor to regulate the expression of transcription factors. In this study, we report that the NF-Y transcription complex is required and responsible for the differentiation and specification of a distinct neuronal class, the IL1 neurons of C. elegans (Fig. 4G). The expression of terminally differentiated markers, including the flp-3 neuropeptide gene, was abolished in nfy mutants. In addition, a cis-regulated motif found in the flp-3 gene promoter, which resembles the CCAAT-box identified in other systems (31, 35, 36), appeared to interact with NF-Y directly. These data, together with previous reports, suggest that the C. elegans NF-Y also acts as an activator as well as a repressor to mediate developmental processes.

Although nfya-1 regulates a set of terminally differentiated markers in IL1s, such as flp-3 neuropeptide and eat-4 vesicular glutamate transporter, nfya-1 may not be a usual terminal selector for IL1 specification (8). First, the expression of another terminally differentiated marker, unc-8 DEG/ENaC cation channel, was not altered in nfya-1 mutants. Second, the cis-regulatory motifs identified in the eat-4 and unc-8 promoters were not similar to the CCAAT-box and directly interacted with NFYA-1 proteins. Third, forced expression of NFYA-1 using the heat-shock promoter in developing larvae and adults did not rescue the phenotype of nfya-1 mutants. These results, together with previous reports (18, 19), indicate that nfya-1 may play a role in neuronal differentiation and specification of C. elegans not only as a terminal selector but as a proneuronal gene.

See supplementary information for Materials and Methods.

We thank the Caenorhabditis Genetics Center (NIH Office of Research Infrastructure Programs, P40 OD010440) and the National BioResource Project (Japan) for strains. We also thank Jinmahn Kim and Ji-Won Lee for technical supports and K. Kim lab members for helpful comments and discussion on the manuscript. This work was supported by the National Research Foundation of Korea (NRF-2020R1A4A1019436, NRF-2021R1A2C1008418) (K.K.) and (2022R1F1A1071248) (J.L.).

The authors have no conflicting interests.