Postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus: Description and comparison with those of Caenorhabditis elegans

The postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus are described and compared with those of Caenorhabditis elegans. The newly hatched larvae of P. redivivus females and males and C. elegans hermaphrodites and males are very similar. An almost identical set of blast cells divides postembryonically in P. redivivus and C. elegans to produce similar changes in the neuronal, muscular, hypodermal, and digestive systems. Most of these cell lineages are invariant; however, there is substantial variability in the number of cell divisions in the relatively extensive lineages of the lateral hypodermis of P. redivivus. Typically, in P. redivivus females, 55 blast cells generate 635 surviving progeny and 29 cell deaths; in P. redivivus males, 59 blast cells generate 758 surviving progeny and 35 cell deaths. The lineages generating the cells of the male tails of P. redivivus and C. elegans are almost identical; thus, the grossly different characteristics of these structures must reflect differences in the morphogenesis of cells equivalent in lineage history. Laser ablation experiments demonstrate that the gonad induces vulva development and that cell-cell interactions are important in specifying the fates of hypodermal precursor cells. The lateral hypodermal lineages provide striking examples of the apparent construction of complex lineages from modular sublineages; one simple pattern of cell divisions and cell fates occurs 70 times in the P. redivivus female. The differences in cell lineage between P. redivivus and C. elegans are relatively minor, and many appear to have involved two types of evolutionary change: the replacement of sublineages, and the modification of sublineages by the four classes of lineage transformations previously proposed based on a comparison of P. redivivus and C. elegans gonadal cell lineages (Sternberg and Horvitz, 1981). These types of differences suggest that the genetic programming of cell lineage includes instructions specifying where and when a particular sublineage is utilized, and other instructions specifying the nature of that sublineage.     

DSH-2 regulates asymmetric cell division in the early C. elegans somatic gonad

Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, as neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. Here we show that the C. elegansdsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. We suggest that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ.     

The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans

The ancestry of the cells in the hermaphrodite and male gonadal somatic structures of C. elegans has been traced from the two gonadal somatic progenitor cells (Z1 and Z4) that are present in the newly hatched larvae of both sexes. The lineages of Z1 and Z4 are essentially invariant. In hermaphrodites, they give rise to a symmetrical group of structures consisting of 143 cells, and in males, they give rise to an asymmetrical group of structures consisting of 56 cells. The male gonad can be distinguished from the hermaphrodite gonad soon after the first division of Z1 and Z4. However, the development of Z1 and Z4 in hermaphrodites shares several features in common with their development in males suggesting that the two programs are controlled by similar mechanisms. In the hermaphrodite lineage, a variability in the positions of two cells is correlated with a variability in the lineages of four cells. This variability suggests that cell-cell interaction may play a more significant role in organisms that develop by invariant lineages than has hitherto been considered. None of the somatic structures (e.g., uterus, spermatheca, vas deferens) develops as a clone of a single cell. Instead, cells that arise early in the Z1–Z4 lineage generally contribute descendants to more than one structure, and individual structures consist of descendants of more than one lineage.     

Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans

The postembryonic cell lineage of the somatic gonad is essentially invariant in Caenorhabditis elegans (J.E. Kimble and D. Hirsh, 1979, Develop. Biol.70, 396–417). The two exceptions to this rule of invariance involve a natural ambiguity in the ancestry of certain cells such that each of two precursor cells assumes one of two alternative fates in a given animal. In this paper, experiments are reported in which laser microsurgery is used to kill individual cells in the developing somatic gonad. Such intervention perturbs the normal environment of the remaining cells; a change observed in the expected behavior of these cells suggests that extrinsic cues may normally play a role in controlling that behavior. Several different lineage alterations have been observed after laser microsurgery in the somatic gonad. These include switches in the type of lineage followed by a given precursor cell, reversals in lineage polarity, duplications of a lineage, and alteratiions in the number of cells produced in the lineage. The only cases in which cells switch from one lineage type to another involve pairs of cells which exhibit natural ambiguity. In most cases, the interactions inferred from these changes seem to occur between neighboring somatic gonadal cells. In one case, induction of the vulva, the interaction occurs between a single somatic gonadal cell, the anchor cell, and the precursors to the vulva in a neighboring tissue, the hypodermis. The roles of intrinsic and extrinsic cues in controlling normally invariant cell lineages are discussed.     

Nematode Postembryonic Cell Lineages

The complete postembryonic ceil lineages of the free-living nentatodes Caenorhabditis elegans and Panagrellus redivivus are known. Postembryonic cell divisions lead to substantial increases in the number of cells and, in most cases, in the number of types of cells in the neuronal, muscular, hypodermal, and digestive systems. The patterns of postembyronic cell divisions are essentially invariant and generate a fixed number of progeny cells of strictly specified fates. Cell fates depend upon both lineage history and cell-cell interactions: lineage limits the developmental potential of each cell and, for certain cells, cell-cell interactions specify which of a small number of alternative potential fates is acquired. Relatively simple differences in cell lineage account for some of the striking differences in gross morphology both between sexes and between species. Genetic studies indicate that these cell lineage differences reflect one or a few relatively simple mutational events. Interspecific differences in cell lineage are likely to be good indicators of evolutionary distance and may be helpful in defining taxonomic relationships. Both the techniques utilized in, and the information acquired from, studies of cell lineages in C. elegans and P. redivivus may prove useful to other hematologists.     

gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans

The gon-4 gene is required for gonadogenesis in the nematode Caenorhabditis elegans. Normally, two precursor cells, Z1 and Z4, follow a reproducible pattern of cell divisions to generate the mature somatic gonadal structures (e.g., uterus in hermaphrodites, vas deferens in males). In contrast, in gon-4 mutants, the Z1/Z4 cell lineages are variably aborted in both hermaphrodites and males: Z1 and Z4 divide much later than normal and subsequent divisions are either absent or severely delayed. In gon-4 adults, normal somatic gonadal structures are never observed, and germ-line and vulval tissues, which depend on somatic gonadal cues for their development, are also aberrant. In contrast, nongonadal tissues and the timing of other developmental events (e.g., molts) appear to be normal in gon-4 mutants. The gon-4 alleles are predicted to be strong loss-of-function or null alleles by both genetic and molecular criteria. We have cloned gon-4 in an attempt to learn how it regulates gonadogenesis. The gon-4 gene encodes a novel, acidic protein. A GON-4::GFP fusion protein, which rescues a gon-4 mutant to fertility, is expressed in somatic gonadal cells during early gonadal development. Furthermore, this fusion protein is nuclear. We conclude that gon-4 is a regulator of the early lineage of Z1 and Z4 and suggest that it is a part of a genetic program common to the regulation of both hermaphrodite and male gonadogenesis.     

Gon-2, a Gene Required for Gonadogenesis in Caenorhabditis Elegans

The gonad of the Caenorhabditis elegans hermaphrodite is generated by the postembryonic divisions of two somatic precursors, Z1 and Z4, and two germline precursors, Z2 and Z3. These cells begin division midway through the first larval stage. By the end of the fourth larval stage, Z1 and Z4 produce 143 descendants, while Z2 and Z3 give rise to ~1000 descendants. The divisions of Z2 and Z3 are dependent on signals produced by Z1 and Z4, but not vice versa. We have characterized the properties of five loss-of-function alleles of a newly described gene, which we call gon-2. In gon-2 mutants, gonadogenesis is severely impaired; in some animals, none of the gonad progenitors undergo any postembryonic divisions. Mutations in gon-2 have a partial maternal effect: either maternal or zygotic expression is sufficient to prevent the severe gonadogenesis defects. By cell lineage analysis, we found that the primary defect in gon-2 mutants is a delay (sometimes a complete block) in the onset and continuation of gonadal divisions. The results of upshift experiments using a temperature-sensitive allele suggest that zygotic expression of gon-2 begins early in embryogenesis, before the birth of Z1 and Z4. The results of downshift experiments suggest that Z1 and Z4 can generate the full complement of gonadal tissues even when gon-2 function is inhibited until the end of the second larval stage. Thus, gon-2 activity is probably not required for the specification of gonadal cell fates, but appears to be generally required for gonadal cell divisions.     

hlh-12, a gene that is necessary and sufficient to promote migration of gonadal regulatory cells in Caenorhabditis elegans,evolved within the Caenorhabditis clade

Specialized cells of the somatic gonad primordium of nematodes play important roles in the final form and function of the mature gonad. Caenorhabditis elegans hermaphrodites are somatic females that have a two-armed, U-shaped gonad that connects to the vulva at the midbody. The outgrowth of each gonad arm from the somatic gonad primordium is led by two female distal tip cells (fDTCs), while the anchor cell (AC) remains stationary and central to coordinate uterine and vulval development. The bHLH protein HLH-2 and its dimerization partners LIN-32 and HLH-12 had previously been shown to be required for fDTC specification. Here, we show that ectopic expression of both HLH-12 and LIN-32 in cells with AC potential transiently transforms them into fDTC-like cells. Furthermore, hlh-12 was known to be required for the fDTCs to sustain gonad arm outgrowth. Here, we show that ectopic expression of HLH-12 in the normally stationary AC causes displacement from its normal position and that displacement likely results from activation of the leader program of fDTCs because it requires genes necessary for gonad arm outgrowth. Thus, HLH-12 is both necessary and sufficient to promote gonadal regulatory cell migration. As differences in female gonadal morphology of different nematode species reflect differences in the fate or migratory properties of the fDTCs or of the AC, we hypothesized that evolutionary changes in the expression of hlh-12 may underlie the evolution of such morphological diversity. However, we were unable to identify an hlh-12 ortholog outside of Caenorhabditis. Instead, by performing a comprehensive phylogenetic analysis of all Class II bHLH proteins in multiple nematode species, we found that hlh-12 evolved within the Caenorhabditis clade, possibly by duplicative transposition of hlh-10. Our analysis suggests that control of gene regulatory hierarchies for gonadogenesis can be remarkably plastic during evolution without adverse phenotypic consequence.     

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.     

Control of cell cycle timing during C. elegans embryogenesis

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P₃ (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.     

Quantitative Analysis of Cytokinesis In Situ during C. elegans Postembryonic Development

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.     

The sys-1 gene and sexual dimorphism during gonadogenesis in Caenorhabditis elegans

In wild-type Caenorhabditis elegans, the hermaphrodite gonad is a symmetrical structure, whereas the male gonad is asymmetric. Two cellular processes are critical for the generation of these sexually dimorphic gonadal shapes during early larval development. First, regulatory "leader" cells that control tube extension and gonadal shape are generated. Second, the somatic gonadal precursor cells migrate and become rearranged to establish the adult pattern. In this paper, we introduce sys-1, a gene required for early organization of the hermaphrodite, but not the male, gonad. The sys-1(q544) allele behaves genetically as a strong loss-of-function mutant and putative null. All hermaphrodites that are homozygous for sys-1(q544) possess a grossly malformed gonad and are sterile; in contrast, sys-1(q544) males exhibit much later and only partially penetrant gonadal defects. The sys-1(q544) hermaphrodites exhibit two striking early gonadal defects. First, the cell lineages of Z1 and Z4, the somatic gonadal progenitor cells, produce extra cells during L2, but the regulatory cells that control gonadal shape are not generated. Second, somatic gonadal precursor cells do not cluster centrally during late L2, and the somatic gonadal primordium typical of hermaphrodites is not established. In contrast, the early male gonadal lineage is asymmetric as normal, the somatic gonadal primordium typical of males is established correctly, and the male adult gonadal structures can be normal. We conclude that the primary role of sys-1 is to establish the shape and polarity of the hermaphrodite gonad.     

gem-1 Encodes an SLC16 Monocarboxylate Transporter-Related Protein That Functions in Parallel to the gon-2 TRPM Channel During Gonad Development in Caenorhabditis elegans

The gon-2 gene of Caenorhabditis elegans encodes a TRPM cation channel required for gonadal cell divisions. In this article, we demonstrate that the gonadogenesis defects of gon-2 loss-of-function mutants (including a null allele) can be suppressed by gain-of-function mutations in the gem-1 (gon-2 extragenic modifier) locus. gem-1 encodes a multipass transmembrane protein that is similar to SLC16 family monocarboxylate transporters. Inactivation of gem-1 enhances the gonadogenesis defects of gon-2 hypomorphic mutations, suggesting that these two genes probably act in parallel to promote gonadal cell divisions. GEM-1GFP is expressed within the gonadal precursor cells and localizes to the plasma membrane. Therefore, we propose that GEM-1 acts in parallel to the GON-2 channel to promote cation uptake within the developing gonad.     

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.     

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.     

Changing of the cell division axes drives vulva evolution in nematodes

Vulval epithelial tubes invaginate through concerted cell migration, ring formation, stacking of rings and intra-ring cell fusion in the nematodes Caenorhabditis elegans, Oscheius tipulae and Pristionchus pacificus. The number of rings forming the invaginations is invariantly seven, six, and eight, respectively. We hypothesize that each ring is formed from pairs of symmetrically positioned primordial vulval cells following three premises: If the final cell division is left-right, the daughters will fuse, migrate and form only one ring. If these cells do not divide, one ring will form. If the final division is anterior-posterior, two rings will form. We test the ring hypothesis and found coincidence between the patterns of vulva cell divisions and the number of rings for 12 species. We find heterochronic variations in the timing of division, migration and fusion of the vulval cells between species. We report a unique ring-independent pathway of vulva formation in Panagrellus redivivus. C. elegans lin-11(n389) mutation results in cell fate transformations including changes in the orientation of vulval cell division. lin-11 animals have an additional ring, as predicted by the ring hypothesis. We propose that the genetic pathway determining how vulval cells invaginate evolves through ring-dependent and ring-independent mechanisms.     

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.