Developmental toolkit genes constitute the genomic repertoire that underlies the pathways patterning the organism (Carroll, 2008). These genes are generally involved in signaling transduction modules and transcription factors (TFs) (Degnan et al., 2009; Gerhart, 1999). Their assembly into specific temporal and spatial gene regulatory pathways leads to the specification of cell fates in the embryo. For example, the Caenorhabditis elegans endoderm is specified by a cascade of TFs operating sequentially (Maduro and Rothman, 2002). Identifying the principles of gene regulation has been a central endeavor in developmental biology.

John Sulston and colleagues elucidated the 1340 cells generated throughout the embryogenesis of the nematode C. elegans, leading to a hatched worm with 558 cells, each with a described name, lineage, and fate (Sulston et al., 1983). The cell lineage is delineated into a germ-line lineage – P – and five ‘founder’ somatic cell lineages – AB, MS, E, C, and D – that each have characteristic cell cycle timings (Deppe et al., 1978) and cell fates (Figure 1a). All founder cell lineages are present by the 28-cell stage, when gastrulation initiates, and by the 102-cell stage the progeny of most of the cells have a single fate.

Figure 1

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The invariant cell lineage of the C. elegans embryo provides an opportunity to systematically study developmental gene pathways. Overall, there are 934 annotated TFs in C. elegans, with 40% having experimentally studied DNA-binding specificities (Narasimhan et al., 2015). The overall set can be classified into families according to their DNA-binding domain, including the homeodomain, bHLH, bZIP, T-box, C2H2 zinc finger, and the very large nuclear hormone receptor domain family. While mapping the location of signaling pathways is difficult from gene expression data, effects of signaling on the transcriptional output are detectable and consequently studying TF expression provides an entry point into gene expression pathway characterization.

Here, we use single-cell RNA-Seq to identify the transcriptomes of each of the cells up to the 102-cell stage. We find that homeodomain genes are expressed in stripes along the anterior-posterior axis, as early as the 28-cell stage. Interestingly, each founder cell lineage – AB, MS, C, and E – establishes its own regionalization code. These Drosophila-like stripe patterns suggest a deep homology in cell fate specification between the embryogenesis of non-segmented and segmented animals, despite differences in syncytium-based and cell-cleavage-based modes of development.

Previous studies analyzed cells from a mixed suspension of cells from different C. elegans embryos (Packer et al., 2019). However, this approach does not ensure sampling of each of the individual cells of the early stages, where it is difficult to dissociate the cells. A complementary approach is to manually collect cells by mouth pipette thus ensuring that all cells are collected. Other works employed this approach to study the early stages, however only arrived at the 15-cell stage (Hashimshony et al., 2016; Tintori et al., 2016). Up to this stage, few new TFs are induced and the transcriptome is dominated by the maternal deposit. As we were interested in studying early specification events in the embryo, we thus manually isolated individual cells from dissociated embryos and processed them for scRNA-Seq (Figure 1b). Overall, we studied 840 cells from 38 embryos up to the 102-cell stage, collecting all or most cells of each embryo (Figure 1b and Supplementary file 1).

Analyzing the transcriptomes, we found that embryo-to-embryo variation could be efficiently normalized by standardizing each gene’s expression across all cells collected from the same embryo (see Methods), and this is another benefit of the manual collection approach. A dimensional reduction map of the examined cells (Figure 1b) reveals the unfolding of development with cells from the early stages occupying the center while cells from later stages occupy the periphery. We found that cells followed developmental trajectories according to their founder cell origin, each identified by the expression of genes known to be expressed in a lineage-specific manner: ceh-51 (MS), elt-7 (E), pal-1 (C,D and P), pes-1 (D), and nos-2 (P) (Figure 1c; Broitman-Maduro et al., 2009; Hunter and Kenyon, 1996; Lee et al., 2017; Maduro and Rothman, 2002). As also observed by other studies (Packer et al., 2019), the founder cell lineage trajectories are oriented according to cell fates, with similar cell fates converging between lineages (Figure 1b and d): the cells of the ecto-mesoderm producing C lineage formed a bi-lobed cloud with the muscle part adjacent to that of the MS and D lineages. Similarly, MS cells that will form the pharynx orient toward the pharynx producing portion of the AB lineage.

In order to infer the precise identity of each cell, we first organized the embryos into eight developmental stages: the 1-, 2-, 4-, 8-, 15-, 28-, 51-, and 102-cell stages (Figure 1a). For each stage, we studied cells from multiple embryos and identified clusters of cells according to differential gene expression. Figure 2a demonstrates this analysis for the 15-cell stage and Figure 2—figure supplement 1 for all other stages. The identified clusters contain cells from each of the individual embryos, thus confirming our underlying assumption that embryo to embryo gene expression variation is minimal relative to the coherence of the cell states within an embryo. Finally, we inferred the cell identity of each cluster with reference to known gene markers from the literature (Supplementary files 2 and 3). Figure 2b illustrates our approach for the cells of the 28-cell stage (see Figure 2—figure supplements 2–7 for all other stages). We required a new marker to distinguish some of the cell identities. Studying F19F10.1 by single-molecule fluorescent in situ hybridization (FISH), we found that this previously uncharacterized ets-factor is expressed in the posterior daughter cell through 2-cell cycle divisions (Figure 2c). We further tested for the robustness of the cell identity assignments and ensured that each cell is most similar to its assigned identity (computed with itself excluded; Figure 2—figure supplements 2–7, bottom of each subfigure). We identified 5433 genes as being differentially expressed within a stage, collectively across all of the stages (see Methods). Of these, 395 are TFs during this period of cell specification (Supplementary file 6).

Figure 2 with 9 supplements see all

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For some cell identities within a stage we were not able to distinguish distinct transcriptomes, and we thus denoted these as composing ‘equivalence groups’. These may either be attributed to daughter cells whose transcriptomes have yet to diverge, such as cells Ca and Cp in the 15-cell state, or to sets of cousins whose sub-lineages form repeating units, such as cells ABplp and ABprp (also of the 15-cell stage) (Sulston et al., 1983). In the first case, this indicates that sister cells are transcriptomically very similar shortly after division. Altogether we describe 119-cell identities with distinct transcriptome profiles, which by stage, are as follows: 1 (1-cell), 2 (2-cell), 4 (4-cell), 6 (8-cell), 11 (15-cell), 20 (28-cell), 38 (51-cell), and 37 (102-cell), with 64 of these representing equivalence groups of at least two cell states (Figure 2d, Supplementary file 5, and Figure 2—figure supplement 8). For each cell state that we assigned, we have an average of seven replicate cells (Supplementary file 4). We validated our annotations by imaging GFP reporters and found good correspondence, as illustrated for ceh-43, dmd-4, and unc-30 (Figure 2e and Figure 2—figure supplement 9). The delay between the GFP expression and the mRNA detected here is expected as previously described (Murray et al., 2012).

We next asked if the genes that are members of the same gene family have similar spatial and temporal expression arrangements. To test for this mode of expression, we considered groups of genes with the same DNA binding domain, as annotated by the PFAM database (Finn et al., 2016). For each cell, we queried for enrichments or depletions of genes of specific domain families, among its expressed TFs. As an example, the ‘ABplppp’ cell differentially expresses 14 TFs according to our analysis, including seven homeobox domain (HD) TFs, though overall HDs only account for 11% of the TFs (Figure 3a). We used the hypergeometric distribution to compute a significance of p=0.00015 for this overlap, given the overall number of TFs and the number of HD and ‘ABplppp’ TFs. Conversely, only two of the 69 TFs expressed by the Eala cell are HDs, a significant depletion (p=0.01, hypergeometric distribution). Figure 3b shows the overall pattern of enrichments and depletions of HDs across all of the examined cells, indicating enrichment of HDs in the 51-cell stage, in particular in the ABp lineage, and depletion in the E lineage. For the GATA type zinc factors we found enrichment in the E lineage, as well as some of the AB cells (Figure 3c).

Figure 3

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Examining the pattern of such biases, we found that TF families showed significant enrichments across lineages and developmental stages (Figure 3d–e). Four TF gene families are enriched in the AB lineage: the T-box domain, the helix-loop-helix (HLH) DNA-binding domain, the HD domain, and the high mobility group (HMG) box domain (Figure 3d). Each of these families also has enrichments for specific AB stages, from early expression (T-box and HLH) to later expression (HD and HMG).

From Figure 3 analysis we concluded that the homeodomain (HD) TF family in particular exhibited lineage-specific expression. Studying this in more detail, we observed that in the 28-cell stage, each of the 16 AB cells contains expression of at least one homeodomain gene, with 8 distinct cell state patterns (Figure 4a). This is mostly also apparent in the 51- and 102-cell stages. The expression patterns of all homeodomain genes across the lineage are not mono-phyletic. For example, ceh-43 is expressed in nine of the sixteen AB cells at the 28-cell stage in a complex pattern (Figure 4a). Thus, continuous symmetry breaking (Zacharias and Murray, 2016) does not appear to be a mechanism by which differential expression is generally established.

Figure 4

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We asked if the relative spatial location of the cells within the embryo can provide insight into the observed homeodomain expression patterns. For this analysis, we mapped the binarized expression of genes onto the known three-dimensional locations of cells in the embryo. Studying the 28-cell stage, we found that the homeodomain genes expressed in each lineage revealed dorsal-ventral stripes along the anterior-posterior axis (Figure 4b). For example, the ceh-43-expressing cells, deriving from distinct AB sub-lineages, are physically proximate. Clustering the AB lineage cells into eight groups according to their binarized expression, we mapped the gene expression combinations of each stripe. One stripe, for example, includes the expression of unc-30, ceh-36, and ceh-13; the adjacent stripe has expression of unc-30 and ceh-36 (Figure 4b). At the 51-cell stage, 4 new homeodomain genes are expressed in the AB lineage, and again position along the anterior-posterior axis correlates with gene expression. In the 102-cell stage, expression becomes more complicated as the AB lineage forms an external shell around the gastrulating non-AB cells. Generalizing this approach, we also found distinct positional systems for the MS and C lineages (Figure 4b and c). For the MS lineage, ceh-32 and ceh-51 are in the anterior and posterior, respectively, while for the C lineage vab-7 (even-skipped) and hmbx-1 are in the anterior and posterior, respectively. We thus provide evidence that distinct regionalization codes occur across the embryonic sub-lineages.

To compare the patterning code elucidated for C. elegans embryogenesis to the well-studied patterning pathway in Drosophila, we also studied the expression of C. elegans orthologous genes. Drosophila development proceeds through a series of molecular events which partition the syncytial embryo along the anterior-posterior axis (Wolpert et al., 2019). We found that the C. elegans genes such as nos-1,2 (nanos) and puf genes (pumilio) which are maternal genes in both systems are restricted to the primary germ cell lineage in C. elegans (Figure 5). Moreover, gap and pair-rule genes, such as pal-1 (caudal) and cwn-1 (wg), show C lineage and sub-lineage restriction, respectively (Figure 5). Thus, it is evident that while Drosophila patterning follows in a lineage-independent manner, in C. elegans, lineage dependency is coincident with specific expression of patterning genes across the founder cell lineages.

Figure 5

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Collectively, our analysis reveals that during embryogenesis, in between the early signaling period – involving Notch (Priess, 2005) and Wnt (Rocheleau et al., 1997; Thorpe et al., 1997) signaling – and the later cell fate differentiation period – involving myogenic factors such as myoD/hlh-1 (Chen et al., 1992) and GATA/elt-2 (Fukushige et al., 1998) – there is a period of lineage-specific patterning. Three of the homeodomain genes whose expression we have described – ceh-13, php-3, and nob-1 (Figure 4) – are members of the HOX sub-family, known to provide regionalization during embryogenesis in bilaterians (Carroll et al., 2004). Indeed, we observed that expression of these HOX genes transcends the founder lineages and patterns the posterior region in multiple lineages (Figure 4). However, we find 51 other homeodomain genes that likely pattern the lineages and set up locations in the embryo. This is a large genetic repertoire relative to Drosophila (97 genes in C. elegans, relative to 104 in Drosophila) despite the higher complexity of the latter in terms of cell types and total number of cells. Thus, in species where regionalization is under lineage control, as opposed to morphogen gradients, there is apparently a greater necessity for distinct positional systems across distinct sub-lineages – here the founder cell lineages. We find lineage-specific gene expression stripes (Figure 4), suggesting that such a developmental patterning mechanism may not be restricted to segmented organisms but is rather a universal feature of animal embryogenesis. The centrality of homeodomain genes to this system highlights the adaptability of this TF family to both lineage-dependent and position-dependent functions. It will be interesting to further compare other species at the single-cell level in order to trace the evolutionary origin of gene expression developmental patterning codes.

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Author details

1. Alison G Cole

   1. Department of Molecular Evolution and Development, University of Vienna, Vienna, Austria
   2. University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Data curation, Investigation

   Contributed equally with

   Tamar Hashimshony

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7515-7489

2. Tamar Hashimshony

   Department of Biology, Technion – Israel Institute of Technology, Haifa, Israel

   Contribution

   Conceptualization, Data curation, Visualization, Methodology

   Contributed equally with

   Alison G Cole

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4786-838X

3. Zhuo Du

   State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

   Contribution

   Resources, Data curation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6322-4656

4. Itai Yanai

   Institute for Computational Medicine, NYU School of Medicine, New York, United States

   Contribution

   Conceptualization, Formal analysis, Writing – original draft

   For correspondence

   Itai.Yanai@nyulangone.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8438-2741

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