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. 2020 Jan 21;37(5):1350–1361. doi: 10.1093/molbev/msaa009

Evolution of Transcriptional Repressors Impacts Caenorhabditis Vulval Development

Helen M Chamberlin m1,✉, Ish M Jain m2, Marcos Corchado-Sonera m1, Leanne H Kelley m1, Devika Sharanya m2, Abdulrahman Jama m1, Romy Pabla m2, Adriana T Dawes m1,m3, Bhagwati P Gupta m2,✉
Editor: Ilya Ruvinsky
PMCID: PMC7182219  PMID: 31960924

Abstract

Comparative genomic sequence analysis has found that the genes for many chromatin-associated proteins are poorly conserved, but the biological consequences of these sequence changes are not understood. Here, we show that four genes identified for an Inappropriate Vulval cell Proliferation (ivp) phenotype in the nematode Caenorhabditis briggsae exhibit distinct functions and genetic interactions when compared with their orthologs in C. elegans. Specifically, we show that the four C. briggsae ivp genes encode the noncanonical histone HTZ-1/H2A.z and three nematode-specific proteins predicted to function in the nucleus. The mutants exhibit ectopic vulval precursor cell proliferation (the multivulva [Muv] phenotype) due to inappropriate expression of the lin-3/EGF gene, and RNAseq analysis suggests a broad role for these ivp genes in transcriptional repression. Importantly, although the C. briggsae phenotypes have parallels with those seen in the C. elegans synMuv system, except for the highly conserved HTZ-1/H2A.z, comparable mutations in C. elegans ivp orthologs do not exhibit synMuv gene interactions or phenotypes. These results demonstrate the evolutionary changes that can underlie conserved biological outputs and argue that proteins critical to repress inappropriate expression from the genome participate in a rapidly evolving functional landscape.

Keywords: developmental system drift, transcriptional repression, chromatin, developmental evolution

Introduction

The genome of eukaryotes is assembled into chromatin, which allows for regulated packing and processing of the genetic material throughout the cell cycle. Especially important in chromatin are histones, which are highly conserved proteins that assemble with DNA into nucleosomes, the first-order structural feature of chromatin. In addition to chromatin’s important role in packaging the genome, structural alterations by histone modifying and chromatin remodeling enzymes play a regulatory role and constrain the access other factors have to DNA (reviewed by Bracken et al. [2019]). Although many of the proteins that modulate or are part of chromatin are highly conserved across all eukaryotes, sequence analysis has identified other proteins that are less conserved or subject to positive selection, arguing that sequence conservation is not a uniform constraint (Calvo-Martín et al. 2016; Hemmer and Blumenstiel 2016; Ponte et al. 2017; Reddy et al. 2017; Baird 2018). Despite accumulating sequence evidence, it has not been demonstrated how these sequence changes relate to biological function.

One important chromatin modulating complex is the dREAM/MuvB complex, which recruits repressive cofactors to gene promoters (Burkhart and Sage 2008; van den Heuvel and Dyson 2008; Sadasivam and DeCaprio 2013). As the name suggests, founding members of this complex are encoded by “synMuvB” genes in Caenorhabditis elegans. Genes in this group were originally identified for their function with a second set of genes, termed synMuvA (Ferguson and Horvitz 1989; Fay and Han 2000; Fay and Yochem 2007). Double mutants between a synMuvA and a synMuvB gene exhibit inappropriate expression of the lin-3/EGF gene and give rise to a multivulva (or Muv) phenotype due to ectopic proliferation of cells that produce parts of the egg-laying apparatus, the vulval precursor cells (VPCs) (Cui et al. 2006). Single mutants for either type of gene are not Muv, indicating each class of genes can compensate for the other. This genetic redundancy arises from the participation of synMuvA and synMuvB genes in distinct compensatory processes, rather than direct (e.g., enzymatic) substitution, as they do not encode proteins with overt sequence similarity (Fay and Yochem 2007). Whereas many synMuvB genes encode nuclear proteins with clear human orthologs, canonical synMuvA genes encode worm-specific proteins, and how they modulate transcription is not known.

To better understand the evolution of developmental regulation, we are using genetics in the nematode Caenorhabditis briggsae, a species that diverged roughly 30 Ma from the better characterized C. elegans (Cutter 2008). The cell divisions and morphological processes underlying the development of the egg-laying system are essentially identical in these two species (Sulston and Horvitz 1977; Félix 2007), but a number of experimental studies have identified differences between the two when signaling cells or pathway components are perturbed (Félix 2007; Sharanya et al. 2015; Mahalak et al. 2017). Using forward genetic screens, we isolated mutations in four genes that confer a Muv phenotype in C. briggsae, but that affected genes not known to participate in vulval development signaling processes in C. elegans (Sharanya et al. 2015). We have characterized these genes and find that although one encodes the noncanonical histone H2A.z (Cbr-HTZ-1), the other three are members of worm-specific gene families. For three of the four genes, comparable disruption in C. elegans does not confer a Muv phenotype, demonstrating functional evolution for these genes. RNAseq, site of action, and RNAi-knockdown experiments argue that these genes exhibit biological similarities to C. elegans synMuv genes, in that they function nonautonomously and alter Cbr-lin-3/EGF transcript accumulation. Our data support a model in which H2A.z/HTZ-1 coordinates with a rapidly evolving collection of proteins to repress inappropriate gene expression.

Results

Caenorhabditis briggsae Inappropriate Vulval Cell Proliferation Genes Encode Predicted Nuclear Proteins

To identify C. briggsae genes that impact vulval development, we performed a set of unbiased genetic screens for mutants with an Inappropriate Vulval cell Proliferation (ivp) phenotype. Our screens identified seven genes that, when mutant, confer the Muv phenotype to C. briggsae animals. Three of these genes were found to be orthologs of well-characterized C. elegans genes that participate in EGF or Wnt signaling pathways, but four mapped to regions without clear candidates (Sharanya et al. 2015). To molecularly identify these four genes, we used whole-genome sequencing, paired with polymorphism mapping of a representative mutant for each gene and focused on genes with sequence changes predicted to disrupt biological function. This approach identified a candidate gene affected in each mutant (fig. 1A and B); targeted sequencing of DNA from animals bearing additional alleles confirmed that they also affected each candidate. Gene identification was validated using transgenic rescue with wild-type DNA (fig. 1C). Based on sequence similarity to C. elegans, the four genes are predicted to encode nuclear proteins, including the noncanonical histone H2A.z, Cbr-HTZ-1, and three proteins exhibiting sequence similarity only within the Caenorhabditis genus. Two of these genes (Cbr-spr-4 and Cbr-gon-14) have functionally characterized orthologs in C. elegans (Lakowski et al. 2003; Chesney et al. 2006). The third gene (Cbr-ivp-3/CBG03376) is orthologous to the C. elegans gene Y67D8C.3, which has been assigned the name ivph-3 (ivp-3 homolog). Although none of these genes have clear orthologs outside Caenorhabditis, gon-14 and ivp-3/ivph-3 from both species share sequence similarity and are classified as part of the same, relatively large gene family (Ruan et al. 2008).

Fig. 1.

Fig. 1.

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Molecular cloning of Caenorhabditis briggsae ivp genes. (A) Gene models for C. briggsae ivp genes, based on Genome Browser annotation validated with publicly available RNAseq data. Each gene is represented to scale, with 5′ to the left. gu163 (tgG [W] to tgA [stop]) is a nonsense mutation in Cbr-spr-4. gu236 (Cga [R] to Tga [stop]) and sy5216 (tgG [W] to tgA [stop]) are nonsense mutations in Cbr-ivp-3. gu167 (gCc [A] to gTc [V]) is a missense mutation in Cbr-htz-1. gu168 (Cga [R] to Tga [stop]) is a nonsense mutation, and gu102 (G to A) affects the 5′ splice site of intron 8 in Cbr-gon-14. gu102 results in transcripts which retain intron 8, leaving an in-frame stop codon. Genomic context of each mutation is listed in supplementary table 2, Supplementary Material online. Images modified from Wormbase.org (Lee et al. 2018). (B) Alignment of the predicted protein product of Cbr-HTZ-1 with orthologs from Caenorhabditis elegans (Cel-HTZ-1) and human (Hsa-H2AZ), and the canonical human histone H2A2 (Hsa-H2A2). The gu167 mutation results in a substitution of Valine (V) for Alanine (A) in the a2 domain of the histone fold, a structural domain conserved across histones. (C) Transgenes (identified as guEx) including PCR fragments from wild-type DNA corresponding to each gene rescue the corresponding mutant but fail to rescue other mutants, even if the genes are from the same group (Cbr-ivp-3 and Cbr-gon-14 share sequence similarity and are from the same TreeFam group, TF317492). n ≥ 50 for all genotypes. Full genotypes are listed in supplementary table 1, Supplementary Material online. Full data, exact sample sizes, and data from additional strains are listed in supplementary table 4, Supplementary Material online.

Caenorhabditis elegans Mutants Exhibit Distinct Phenotypes Compared with C. briggsae

Since there are identified orthologs for all four C. briggsae genes in C. elegans, we asked whether C. elegans mutants exhibit a phenotype similar to those of C. briggsae (fig. 2). Loss-of-function Cel-gon-14 mutants have been reported previously to exhibit a Muv phenotype (Chesney et al. 2006), but no such phenotype has been shown for the remaining orthologs. Cel-htz-1 is an essential gene, and homozygous null mutants maternally rescued to adulthood do not exhibit a Muv phenotype (Whittle et al. 2008). However, Cbr-htz-1(gu167) is a missense mutation that may alter gene function in an unpredictable manner, and its Muv phenotype is likewise maternally rescued (Sharanya et al. 2015). Thus, we used CRISPR-mediated genome editing to engineer the orthologous change into Cel-htz-1. This mutation can be maintained in a homozygous strain, indicating that it is nonnull, and allowing mutant offspring from mutant parents to be evaluated. We likewise used CRISPR-mediated genome editing to generate a deletion of Cel-sprp-4, a paralog of Cel-spr-4 that others have interpreted might compensate for some functions when Cel-spr-4 is disrupted (Lakowski et al. 2003). A Cel-ivph-3 deletion allele (also generated by CRISPR) was provided by the lab of Dr Don Moerman. We find that in contrast to C. briggsae, Cel-htz-1 and Cel-ivph-3 single mutants, and Cel-spr-4; Cel-sprp-4 double mutants all exhibit normal vulval development (fig. 2). Thus, the phenotypic consequences of these mutations are distinct in the different species background. We also note that, whereas Cbr-gon-14 and Cbr-ivp-3 mutants are fertile, similarly disruptive alleles of Cel-gon-14 or Cel-ivph-3 result in a sterile phenotype. To ask whether this might reflect incomplete disruption of gene function or redundancy between the two genes, we constructed Cbr-ivp-3(sy5216); Cbr-gon-14(gu102) double mutants and find that doubles remain viable and fertile (supplementary table 5, Supplementary Material online). These results suggest that there are functional differences between the species for these genes in addition to variation in the vulval development process.

Fig. 2.

Fig. 2.

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Caenorhabditis briggsae and C. elegans mutants exhibit distinct phenotypes. Normal C. briggsae (A) and C. elegans (F) vulval development is very similar, with an identical pattern of cell divisions from three VPCs resulting in a single vulval structure in the L4 larval stage (white arrowhead). Caenorhabditis briggsae mutants of htz-1, gon-14, ivp-3, and spr-4 (B–E) frequently exhibit the division of more than three VPCs (K), resulting in additional vulval-like structures (gray arrowheads). Although similar mutations in Cel-gon-14 can induce the division of supernumerary VPCs (H), they result in little or no change to vulval development for Cel-htz-1, Cel-ivph-3, or Cel-spr-4; Cel-sprp-4 (G, I, J, L). n ≥ 40 for each genotype, except Cel-ivph-3, where n = 24. Full genotypes are listed in supplementary table 1, Supplementary Material online. Full data and exact sample sizes are listed in supplementary table 7, Supplementary Material online. Error bars correspond to the standard deviation. Scale bar, 20 μm.

Due to these observed genetic distinctions in the two species, we asked whether the four genes exhibit any sequence features consistent with rapid evolution or positive selection. First, we tested for accelerated evolution using the codon substitution model of PAML (Yang 2007) and detected no difference in selection (ω) in the C. briggsae branch for these genes compared with the orthologs (supplementary table 6, Supplementary Material online). We then evaluated changes specifically between C. elegans and C. briggsae using the nonsynonymous substitution rate (KA), which offers a signature for evolution of protein encoding genes (Wang et al. 2011). Compared with the genome-wide KA of 0.121 (Cutter et al. 2019), KA for htz-1 is lower (0.007), whereas the values for spr-4, gon-14, and ivp-3/ivph-3 are notably higher (0.608, 0.887, and 0.554, respectively). Altogether, we conclude that these sequence analyses do not predict the functional differences observed in vivo, as the identical mutation affecting the highly conserved htz-1 confers a distinct vulval development phenotype in the two species, whereas mutation of the more divergent gon-14 confers a Muv phenotype in both species.

Evolution of synMuvA Genes Alters the In Vivo Function of htz-1 but Not Other ivp Genes

We sought to test the hypothesis that the ivp genes have functional similarities between C. elegans and C. briggsae, but that their disruption is compensated by other genes in the C. elegans genome. We focused on the synMuv system, as synMuvB genes include Cel-lin-35/Rb, which has been shown to function with Cel-htz-1 to repress transcription (Latorre et al. 2015). Since our Cbr-htz-1 mutants exhibit a Muv phenotype but Cel-htz-1 mutants do not, we asked whether this might result from changes to synMuvA genes. Consistent with this idea, the C. briggsae genome appears to lack a lin-15A and a lin-56 ortholog, based on Blast search, WormBase annotation (www.wormbase.org; Lee et al. 2018), and synteny (fig. 3A and B). Cel-LIN-15A and Cel-LIN-56 physically interact, and each protein is destabilized in mutants for the other, arguing they function together (Davison et al. 2011). Thus, we asked whether differences between the C. elegans and C. briggsae phenotypes can be explained by the absence of these synMuvA genes from the genome. Previous studies evaluated the functional consequences of disrupting Cel-lin-15A in the Cel-gon-14 background and found no difference compared with Cel-gon-14 alone (Chesney et al. 2006). We constructed double mutants with Cel-htz-1 and Cel-ivph-3, and triple mutants for Cel-spr-4 (fig. 3C–G). We find that Cel-htz-1; Cel-lin-15A double mutants indeed exhibit division of additional VPCs compared with single mutants, consistent with the idea that the presence of the lin-15A gene in the C. elegans genome can compensate for the htz-1 mutation. However, a similar effect was not observed for Cel-ivph-3 or Cel-spr-4 (fig. 3C), indicating that the apparent loss of lin-15A from the C. briggsae genome is not sufficient to explain the functional differences for these genes.

Fig. 3.

Fig. 3.

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Loss of synMuvA genes alters the in vivo function of Cbr-htz-1. (A) The Caenorhabditis elegans lin-15 locus comprises an operon of Cel-lin-15B (upstream) and Cel-lin-15A (downstream). The Caenorhabditis briggsae genome lacks an annotated lin-15A ortholog, based on wormbase.org annotation and Blast search. The Chromosome X genomic region including the Cbr-lin-15B gene is not syntenic with the Cel-lin-15B region, but rather flanked by sequences syntenic with C. elegans Chromosome I. Cbr-lin-15B is flanked by two annotated psuedogenes, neither of which bears sequence similarity to Cel-lin-15A (ψ1 = CBG13630, annotated ortholog and top BlastX hit: T26E3.6; ψ2 = CBG13633, no ortholog annotated. Top BlastX hits: Y52B11A.12, sdz-15) (B) The Cel-lin-56 locus is on Chromosome II. The C. briggsae genome lacks an annotated lin-56 ortholog, based on wormbase.org annotation and Blast search. The syntenic region on C. briggsae Chromosome II likewise lacks a gene where Cel-lin-56 is found. (A, B). Images modified from Wormbase.org (Lee et al. 2018). For C. briggsae genes not yet named based on orthology, each gene is followed by the C. elegans ortholog identified in wormbase.org, in parentheses. (C–G) Cel-htz-1; Cel-lin-15A double mutants exhibit increased numbers of induced VPCs, whereas no increase in induction is observed in Cel-ivph-3; lin-15A doubles and Cel-spr-4; Cel-sprp-4 Cel-lin-15A triples. n ≥ 40 for each genotype, except Cel-ivph-3; lin-15A, where n = 29. Full genotypes are listed in supplementary table 1, Supplementary Material online. Full data and exact sample sizes are listed in supplementary table 7, Supplementary Material online. Error bars correspond to the standard deviation. Scale bar, 20 μm.

Caenorhabditis briggsae ivp Mutants Exhibit Broad Misregulation of Transcripts, Including Germline Genes

Given that our C. briggsae genes are predicted to encode nuclear proteins, we evaluated gene expression changes in mutants using RNAseq. RNA was collected from two mutants, Cbr-htz-1(gu167) and Cbr-spr-4(gu163), both synchronized to mid-L3 larval stage, and differentially expressed (DE) genes were compared with those from similarly staged wild-type animals (fig. 4A and B). The analysis revealed that in both mutants, twice as many DE genes exhibit an increased transcript abundance compared with those that exhibit a decreased transcript abundance (fig. 4C and supplementary workbook 1, Supplementary Material online). Furthermore, overlap between the Cbr-htz-1(gu167) and Cbr-spr-4(gu163) data sets was prevalent among the genes with increased abundance, but negligible for those with decreased abundance. We conclude that an important normal function for these genes is to reduce transcript accumulation, such as through repression of inappropriate transcription from the genome.

Fig. 4.

Fig. 4.

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Caenorhabditis briggsae ivp mutants exhibit increased transcript abundance of many genes. MA plots of DE genes in Cbr-htz-1(gu167) (A) and Cbr-spr-4(gu163) (B). DE genes in each strain are more likely to be increased (835 vs. 397 for Cbr-htz-1(gu167) and 328 vs. 168 for Cbr-spr-4(gu163)). Red dots represent transcripts with an FDR P-adj of <0.05, whereas black dots represent transcripts with FDR P-adj of >0.05. (C) DE genes with increased abundance (“up”) exhibit overlap between the two data sets. Specifically, 211/952 (22%) of genes exhibiting increased abundance are shared between the two genotypes, whereas 7/558 (1.2%) of those with decreased abundance overlap. Also see supplementary workbook 1, Supplementary Material online. (D) Comparison of Cbr-htz-1(gu167) and Cbr-spr-4(gu163) DE gene orthologs with genes that are enriched in the germline of Caenorhabditis elegans (“germline genes” [Wang et al. 2009]). Also see supplementary workbook 2, Supplementary Material online. Sequence data submitted to GEO (GSE133769).

We evaluated Gene Ontology (GO) categories enriched in DE genes in Cbr-htz-1(gu167) and Cbr-spr-4(gu163) mutants, and noted that these included several gene categories involved in meiosis. Since one defect in C. elegans synMuvB mutants is dysregulation of germline genes and related processes (Petrella et al. 2011; Rechtsteiner et al. 2019), this observation prompted us to evaluate how germline genes are represented among those altered in the C. briggsae mutants. From the genes with altered abundance, we identified those with direct (1:1) orthologs in C. elegans, and then looked for germline-expressed genes among these orthologs (Wang et al. 2009). Both Cbr-htz-1(gu167) and Cbr-spr-4(gu163) DE gene sets exhibit a significant enrichment of germline genes (fig. 4D and supplementary workbook 2, Supplementary Material online). For Cbr-htz-1(gu167), the vast majority are upregulated compared with wild type, as is observed in C. elegans synMuvB mutants (Petrella et al. 2011) (supplementary workbook 2, Supplementary Material online). In contrast, the DE germline genes in Cbr-spr-4(gu163) include genes with both increased and decreased transcript abundance.

Caenorhabditis briggsae ivp Genes Function Nonautonomously to Repress Inappropriate Expression of the EGF Ligand Gene, Cbr-lin-3

Although the C. briggsae ivp mutants alter the transcript abundance of many genes, we wanted to better understand the specific contribution to vulval development. First, we asked where gene function is required by introducing cDNA under control of heterologous, cell-specific promoters. We found that introduction of either Cbr-htz-1 or Cbr-ivp-3 under control of a non-VPC, hypodermis-specific promoter can rescue each respective mutant (fig. 5A and supplementary fig. 1, Supplementary Material online). We then reasoned that one way to nonautonomously increase the number of VPCs that divide to produce vulval cells is to ectopically express genes that encode signals that promote vulval cell fates. Since the EGF, Notch, and Wnt pathways play an important role in vulval development in both species (Sternberg 2005), we evaluated the set of genes exhibiting increased transcript abundance in both Cbr-htz-1(gu167) and Cbr-spr-4(gu163) for ones encoding ligands for these three pathways. Only Cbr-lin-3/EGF (CBG05996) exhibited a significant increase in abundance in both strains, which is notable as the Muv phenotype in C. elegans synMuv mutants also results from an abnormal increase of Cel-lin-3/EGF (Cui et al. 2006). We used Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) to evaluate Cbr-lin-3/EGF gene transcripts and found that levels are elevated in all four mutant strains compared with wild-type animals (fig. 5B). Since the increase is modest (and not always detected [Sharanya et al. 2015]), we asked whether this observed difference is functionally relevant using RNAi to knock down Cbr-lin-3 in each mutant. The vulval induction was found to be reduced in response to RNAi treatment, indicating that abnormal expression of this gene contributes to the observed Muv phenotype (fig. 5C and D).

Fig. 5.

Fig. 5.

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The Caenorhabditis briggsae Muv mutant phenotype results from inappropriate expression of Cbr-lin-3/EGF. (A) Expression of cDNA for Cbr-htz-1 or Cbr-ivp-3 under control of a non-VPC hypodermis-specific promoter (dpy-7) rescues the Muv phenotype associated with mutants. Full genotypes are in supplementary table 1, Supplementary Material online. n ≥ 40 for all genotypes. Full data and exact sample sizes are listed in supplementary table 8, Supplementary Material online. Additional transgenes tested are in supplementary figure 1, Supplementary Material online. (B) Cbr-lin-3 transcripts exhibit increased abundance in the Muv mutants. * indicates statistically different from wild type (Student’s t-test, P < 0.01). (C, D) RNAi-knockdown of Cbr-lin-3 in the Muv mutants results in a decrease in the number of VPCs that divide to produce vulval tissue. Data are reported separately as the wild-type background strain is different in (C) (includes Cel-sid-2 transgene mfIs42) compared with (D) (includes Cel-sid-2 transgene mfEx32). Full genotypes are listed in supplementary table 1, Supplementary Material online. Data represent three independent trials for each, with n ≥ 30 animals for each genotype in each trial. Full data and exact sample sizes are listed in supplementary table 9, Supplementary Material online. * indicates statistically different from wild type (Student’s t-test, P < 0.01). Error bars correspond to the standard deviation.

Discussion

Here, we have characterized a set of genes in the nematode C. briggsae that act to prevent inappropriate proliferation and development of specialized epithelial cells, the VPCs. Altogether, the data support a model in which the gene products function in the nucleus to repress inappropriate transcription from the genome. The mutants exhibit increased transcript abundance of lin-3/EGF and of germline-associated genes, functionally similar to a class of genes defined in C. elegans as synMuv genes (Petrella et al. 2011). However, comparable genetic mutants in C. elegans result in a synMuvB phenotype for the conserved noncanonical histone gene htz-1/H2A.z, but not for the other genes that exhibit a similar phenotype in both species (gon-14) (Chesney et al. 2006) or distinct phenotypes (spr-4 and ivp-3/ivph-3). Thus, even though the development, anatomy, and life history traits are similar between the two species, there are functional differences in the underlying networks that promote normal gene expression and development.

Nematode vulval development has been a focus of study by many evolutionary biologists and provides several examples of developmental system drift wherein there is evolutionary change in the underlying cellular and genetic processes that result in the production of overtly similar biological outcomes (True and Haag 2001; Sommer 2012). For example, nematodes across the Rhabditidae family share the common feature in which the vulva is formed from the offspring of three specialized epidermal cells (Sommer and Sternberg 1995). However, species differ with respect to the underlying developmental processes, such as the dependence of vulval development on cells in the gonad, and the molecular signals that are critical (Sommer and Sternberg 1994; Sigrist and Sommer 1999; Tian et al. 2008; Vargas-Velazquez et al. 2019). Specifically within Caenorhabditis, vulval development is identical in C. elegans and C. briggsae, but cell ablation experiments demonstrate that premature removal of the anchor cell can result in distinct patterning defects (Félix 2007). Likewise, genetic and pharmacologic experiments show that whereas the EGF signaling pathway is essential for C. elegans vulval development, C. briggsae animals still produce some vulval tissue under comparable conditions (Mahalak et al. 2017). Our current findings relate to the signaling processes that promote vulval development in that expression of a key signaling gene, lin-3/EGF, is affected. However, our findings are distinct from these other studies in that they uncover evolution of the processes that constrain transcription from the genome, rather than specific features of the underlying signaling networks that influence vulval development.

Importantly, our results demonstrate functional evolution of HTZ-1/H2A.z and the other ivp proteins. One important role for chromatin is in managing the genomic material from generation to generation, during which products of the maternal genome interact with those of the paternal genome in the one-cell zygote, and in the embryo following initiation of transcription from the zygotic genome. This process can result in genomic conflict, wherein there is competition between the maternal genome to repress expression of particular genes or genetic elements in the paternal genome, and the paternal genome to bypass those repressive effects (Johnson 2010; Crespi and Nosil 2013). Such conflict could drive rapid change in sequence and in the relative importance of different proteins in maintaining silenced chromatin states, in an effort to limit the genomic risks of outcrossing. Indeed, genome-wide sequence analysis identifies proteins predicted to localize to the nucleus or participate in transcription as significantly enriched among rapidly evolving nematode genes (Castillo-Davis et al. 2004), and this is especially true for genes that are most abundant during early embryonic development (Cutter et al. 2019). Broadly, our results indicate that the function of proteins that impact access to the genome can evolve as part of developmental system drift. We anticipate that these may include critical regulatory factors that are understudied or yet to be discovered, as such proteins are refractive to canonical research approaches that emphasize sequence conservation and orthology to predict functional importance.

Materials and Methods

Worm Maintenance and Genetics

Methods for culturing and maintaining nematodes were as described for C. elegans (Brenner 1974; Stiernagle 2006). Specifically, worms were grown on NGM or NG-Agar plates seeded with Escherichia coli OP50 as a food source. All experiments were carried out at 20 °C, unless stated otherwise. The wild-type C. briggsae was AF16, and the wild-type C. elegans was N2. Specific strains and genotypes used are listed in supplementary tables 1 and 2, Supplementary Material online.

Generation of CRISPR Alleles

New alleles of C. elegans genes were generated using CRISPR-mediated genome editing methods. Standard procedure following recovery is to back-cross the strain 2× to wild type prior to phenotypic analysis.

Cel-htz-1(gu242) and Cel-htz-1(gu243) were generated using the method of Arribere et al. (2014). sgRNA targeting Cel-htz-1 was introduced into pDD162 (Dickinson et al. 2015) and coinjected into the germline of wild-type C. elegans with a single-stranded repair template and a fluorescent reporter (myo-2::gfp) as a transformation marker (primers provided in supplementary table 3, Supplementary Material online). GFP-positive F1 animals were selected and allowed to self-cross. After each plate that was founded by an F1 parent depleted the bacteria, a portion was saved, and the remainder prepared to recover DNA and subject to PCR for genotyping. Animals were recovered from plates heterozygous for the introduced (silent) restriction enzyme site alteration and allowed to self to recover homozygotes.

Cel-sprp-4(gu241) was generated using the method and plasmids of Dickinson et al. (2015), with two sgRNAs each in pDD162 (one targeting the 5′ end of the gene, and one targeting the 3′ end of the gene), and a repair template in pDD285 consisting with flanking homology arms corresponding to sequences upstream and sequences downstream of the coding exons, resulting in a mutant allele in which the coding exons are replaced with the intervening sequence (mKate > SEC > FLAG). These plasmids were coinjected into the gonad of wild-type C. elegans animals together with a negative selection fluorescent marker (myo-2::gfp). Candidate repair events were selected (hygromycinR GFP-), subjected to SEC excision, and validated with PCR and Sanger sequencing.

The C. elegans ivph-3(gk3691) allele was provided by the Moerman lab (UBC), and was also generated using a CRISPR/Cas9-based homology-directed repair and drug selection technique. Briefly, a GFP cassette (∼5.4 kb) containing myo-2p::GFP and rps-27p::Neo selection marker was inserted into the genomic region of Y67D8C.3 using two ∼500-bp homology arms that flanked the first exon of Y67D8C.3a. The insertion of the cassette resulted in the deletion of the entire first exon and 9 bp of the second exon of Y67D8C.3a. These exons are common to all Y67D8C.3 isoforms. The gk3691 mutation is homozygous sterile and Pvl so it requires picking GFP fluorescing animals to maintain it. The strain was validated by PCR and Sanger sequencing.

Whole-Genome Sequencing, Candidate Gene Identification, and Mutation Confirmation

Genomic DNA was prepared for whole-genome sequencing from Cbr-gon-14(gu102), Cbr-spr-4(gu163), Cbr-htz-1(gu167), and Cbr-ivp-3(sy5216) homozygotes and sequenced using the Illumina HiSeq platform by the Ohio State University Comprehensive Cancer Center Genomics Shared Resource (for gu102) and McGill University - Génome Québec Innovation Centre Montreal (for the remaining three strains). In general, paired read FASTQ files were aligned to the C. briggsae cb4 reference sequence using the Burrows–Wheeler Aligner BWA (Li and Durbin 2009) (version 0.6.2). SAMtools (Li et al. 2009) (version 0.1.19) was used to create the pileup file. Potential candidates with sufficiently high coverage (minimum four reads) were identified in the pileup file using custom Python scripts. Variants affecting genes on the chromosome to which each gene had been genetically mapped (Sharanya et al. 2015) were considered as prospective candidate genes. Although each mutation had been mapped against polymorphisms to particular chromosomal regions, we found that limiting our candidates to those regions excluded the (ultimately confirmed) affected gene in some cases, perhaps due to assembly errors in the reference genome (also supported by the data of others [Bi et al. 2015; Ren et al. 2018]). Nonsense mutations, splicing mutations, and alterations that disrupted reading frame were prioritized for further testing. No nonsense mutation was identified that affected genes in the Cbr-htz-1(gu167) strain. Cbr-htz-1 was selected for further analysis based on the impact of the missense mutation on a highly conserved amino acid. The DNA mutations in strains evaluated by WGS, and those of strains not subject to WGS, were confirmed or identified by Sanger sequencing of PCR fragments (generated from DNA of each strain) that covered all exons and exon/intron boundaries. The sequence alteration for each allele is listed in supplementary table 2, Supplementary Material online.

RT-qPCR, Whole-Genome RNA Sequencing, and Data Analysis

RNA was recovered from synchronized L3 animals using a standard TRIZOL method (Invitrogen). Animals were staged prior to harvesting by mounting a sample and observing them under the Nomarski microscope. RNA was extracted just prior to the stage when vulval precursors (Pn.p cells) initiate division. At this stage, vulval cells are responsive to Cbr-lin-3 levels (Félix 2007). RT-qPCR used to validate abundance of Cbr-lin-3 transcripts was carried out using the protocol described previously (Sharanya et al. 2015). For RNA sequencing (RNAseq) experiments, three independent biological replicates were prepared from AF16, Cbr-htz-1(gu167), and Cbr-spr-4(gu163) strains, and the samples were processed, depleted for rRNA, and sequenced using Illumina HiSeq 2000 V3 Single Read system by Génome Québec.

RNAseq data were analyzed as follows. The package cutadapt/trimgalore was used to trim the adapters, and reads with QC values (Phred score) <30 bases were discarded (Martin 2011). The processed sequencing reads were mapped to the C. briggsae reference genome (cb3 assembly, UCSC genome browser) using STAR aligner v2.5 (Dobin et al. 2013). More than 95% of sequencing reads were successfully mapped to the genome. Transcript-level abundance was estimated using quantMode, a HTSeq-count package incorporated in the STAR 2.5 (Dobin et al. 2013). Gene counts were quantile normalized to avoid bias between samples (Bullard et al. 2010; Martin 2011). DE genes were called at a false discovery rate (FDR) of 0.05% using the R package DESeq2 that is based on the negative binomial distribution model (Love et al. 2014). The results revealed 1,241 DE genes in Cbr-spr-4(gu163) and 505 in Cbr-htz-1(gu167) animals. RNAseq data are deposited with GEO (GSE133769).

GoAmigo (Carbon et al. 2009) was used with default settings to perform GO analysis of candidate genes. A GO-term containing at least three genes with a P value (adjusted for multiple comparisons) of <0.05 (Benjamini–Hochberg method) was considered as significant (Carbon et al. 2009).

Cbr-spr-4(gu163) and Cbr-htz-1(gu167) transcriptomes were analyzed for germline, oogenesis, and spermatogenesis genes using available C. elegans data sets (Reinke et al. 2000; Wang et al. 2009). Sequence comparisons revealed a total of 865 and 354 1:1 unique C. elegans orthologs of DE genes for each genotype, respectively (supplementary workbook 2, Supplementary Material online). Germline and oogenesis genes were significantly overrepresented in the transcriptomes of each mutant (supplementary workbook 2, Supplementary Material online).

Generation of Transgenes and Plasmids

Genomic rescue DNA fragments were generated by PCR amplification of each gene, plus at least 500-bp flanking DNA, from AF16 (wild-type C. briggsae) genomic DNA. PCR fragments were confirmed based on size using gel electrophoresis, incorporated into injection mixes (5 ng/μl myo-2::gfp, 15 ng/μl Cel-unc-119(+), and 50 ng/μl PCR product), and microinjected into the gonad of Cbr-unc-119(st20000) hermaphrodites using standard C. elegans methods (Mello et al. 1991). Males were induced in stable transgenic lines and crossed with hermaphrodites doubly homozygous for Cbr-unc-119(st20000) and the mutant to be tested for rescue. F1 non-Unc animals were selected individually and allowed to self. Animals homozygous for the Muv mutation were selected from among the offspring of the F1. Homozygotes were identified based on their Muv phenotype, or on the capacity of the non-Muv non-Unc parent to produce only non-Muv non-Unc or Muv Unc offspring. DNA from mutant rescued strains was recovered, subjected to PCR, and sequenced to evaluate the region including the mutation to verify that reversion of the mutant phenotype correlates with introduction of wild-type sequences into the genome and to confirm the identity of the DNA in the PCR product.

Plasmids for tissue-restricted rescue were generated in the pPD49.26 backbone (a gift from Dr A. Fire). Caenorhabditis elegans gene promoters that drive expression in the VPCs (lin-31), gonad (ddr-2), anchor cell (lin-3 ACEL), and hypodermis (dpy-7) were used (Hwang and Sternberg 2004; Myers and Greenwald 2005; Liu et al. 2017). cDNAs for Cbr-htz-1 and Cbr-ivp-3 were generated by harvesting RNA from mixed-stage AF16 animals with TRIZOL, synthesizing first strand cDNA using random hexamers and Superscript III RT (Invitrogen), followed by PCR using gene-specific primers into which appropriate restriction enzyme sequences were incorporated to allow for cloning into the plasmid. Candidate plasmids were confirmed by restriction enzyme digestion and sequencing. Plasmids were incorporated into injection mixes (5 ng/μl myo-2::gfp, 15 ng/μl Cel-unc-119(+), and 50 ng/μl PCR product(s)), and microinjected into the gonad of Cbr-unc-119(st20000) hermaphrodites using standard C. elegans methods (Mello et al. 1991). Transgenes were identified and tested for rescue activity using the methods outlined above for the genomic DNA rescue.

To create the Cbr-lin-3 RNAi plasmid, a 885-bp Cbr-lin-3 fragment was PCR amplified from genomic DNA using primers GL1242 and GL1243. The fragment was ligated into the L4440 (pPD129.36; a gift from Dr A. Fire) vector using restriction enzyme sites SalI and SphI. Escherichia coli HT115 cells were transformed, and the plasmid was confirmed by restriction digestion. RNAi in C. briggsae animals was performed as described earlier (Seetharaman et al. 2010). mfIs42 and mfEx32 transgenes containing Cel-sid-2 (necessary to introduce RNAi by feeding) (Nuez and Félix 2012) were introduced into mutant backgrounds through genetic crosses.

Microscopy and Phenotypic Scoring

Vulval development and VPC proliferation were assessed using two methods. Adult animals were evaluated for whether they exhibit two or more protrusions or growths on their ventral surfaces. To score adults, L4 worms were transferred on to a fresh plate and allowed to grow overnight at 20 °C. The next day, they were scored under a dissecting stereomicroscope for morphology and the presence of any ventral protrusions. Animals with two or more protrusions (or one protrusion in addition to the normal vulval opening) were scored as Muv.

Vulval induction was scored in early to mid-L4 animals under Nomarski optics. Animals were mounted on agar pads, with 1 mM Sodium Azide used to immobilize them. The overall morphology of the vulval cell pattern and the number of Pn.p cell progeny were counted, allowing an inference of whether each precursor cell divided multiple rounds to produce vulval cell types, or only once, as previously described (Sternberg and Horvitz 1986). In wild-type C. elegans and C. briggsae animals, normally three cells (P5.p, P6.p, and P7.p) divide to produce vulval progeny, termed “induced.” Muv mutants exhibit induction of greater than three cells.

Supplementary Material

Supplementary data are available at Molecular Biology and Evolution online.

Supplementary Material

msaa009_Supplementary_Data
Click here for additional data file. (312.2KB, zip)

Acknowledgments

We thank Ryan Johnson, Jennifer Patritti-Cram, Ayush Ranawade, Bavithra Thillainathan, Nagagireesh Bojanala, and members of the Sternberg lab (Caltech), in particular Shahla Gharib and Keith Brown, for technical assistance in the early phases of this project. We are grateful to Don Moerman and Mark Edgley for ivph-3(gk3691), and Stephane Flibotte for assistance with genome sequence analysis to identify Cbr-ivp candidate genes. Wen Tang provided comments on a previous draft of the manuscript. Some strains were supplied by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure programs (P40 OD010440). Some plasmids were provided by Addgene.org. Some work was done with the OSU CCC Genomics Shared Resource, which is funded by the National Cancer Institute (P30 CA016058). We thank Wormbase.org for data and images. M.C.-S. was supported by the CMBP predoctoral training grant from the National Institutes of Health (T32-GM086252) and the Pelotonia Cancer Research Graduate Fellowship. This work was supported by the National Science Foundation (DMS-1361251) and NSERC Discovery (RGPIN-2014-05153) grants. RNAseq data are submitted to GEO (GSE133769).

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