Control of vulval cell division number in the nematode Oscheius/Dolichorhabditis sp. CEW1

Spatial patterning of vulval precursor cell fates is achieved through a different two-stage induction mechanism in the nematode Oscheius/Dolichorhabditis sp. CEW1 compared with Caenorhabditis elegans. We therefore performed a genetic screen for vulva mutants in Oscheius sp. CEW1. Most mutants display phenotypes unknown in C. elegans. Here we present the largest mutant category, which affects division number of the vulva precursors P(4-8).p without changing their fate. Among these mutations, some reduce the number of divisions of P4.p and P8.p specifically. Two mutants omit the second cell cycle of all vulval lineages. A large subset of mutants undergo additional rounds of vulval divisions. We also found precocious and retarded heterochronic mutants. Whereas the C. elegans vulval lineage mutants can be interpreted as overall (homeotic) changes in precursor cell fates with concomitant cell cycle changes, the mutants described in Oscheius sp. CEW1 do not affect overall precursor fate and thereby dissociate the genetic mechanisms controlling vulval cell cycle and fate. Laser ablation experiments in these mutants reveal that the two first vulval divisions in Oscheius sp. CEW1 appear to be redundantly controlled by a gonad-independent mechanism and by a gonadal signal that operates partially independently of vulval fate induction.     

Microevolutionary studies in nematodes: a beginning

Comparisons between related species often allow the detailed genetic analysis of evolutionary processes. Here we advocate the use of the nematode Caenorhabditis elegans (and several other rhabditid species) as model systems for microevolutionary studies. Compared to Drosophila species, which have been a mainstay of such studies, C. elegans has a self-fertilizing mode of reproduction, a shorter life cycle and a convenient cell-level analysis of phenotypic variation. Data concerning its population genetics and ecology are still scarce, however. We review molecular, behavioral and developmental intraspecific polymorphisms for populations of C. elegans, Oscheius sp. 1 and Pristionchus pacificus. Focusing on vulval development, which has been well characterized in several species, we discuss relationships between patterns of variations: (1) for a given genotype (developmental variants), (2) after mutagenesis (mutability), (3) in different populations of the same species (polymorphisms) and (4) between closely related species. These studies have revealed that evolutionary variations between sister species affect those characters that show phenotypic developmental variants, that are mutable and that are polymorphic within species.     

Control of vulval competence and centering in the nematode Oscheius sp. 1 CEW1

To compare vulva development mechanisms in the nematode Oscheius sp. 1 to those known in Caenorhabditis elegans, we performed a genetic screen for vulva mutants in Oscheius sp. 1 CEW1. Here we present one large category of mutations that we call cov, which affect the specification of the Pn.p ventral epidermal cells along the antero-posterior axis. The Pn.p cells are numbered from 1 to 12 from anterior to posterior. In wild-type Oscheius sp. 1 CEW1, the P(4-8).p cells are competent to form the vulva and the progeny of P(5-7).p actually form the vulva, with the descendants of P6.p adopting a central vulval fate. Among the 17 mutations (defining 13 genes) that we characterize here, group 1 mutations completely or partially abolish P(4-8).p competence, and this correlates with early fusion of the Pn.p cells to the epidermal syncytium. In this group, we found a putative null mutation in the lin-39 HOM-C homolog, the associated phenotype of which could be weakly mimicked by injection of a morpholino against Osp1-lin-39 in the mother's germ line. Using cell ablation in a partially penetrant competence mutant, we show that vulval competence is partially controlled by a gonadal signal. Most other mutants found in the screen display phenotypes unknown in C. elegans. Group 2 mutants show a partial penetrance of Pn.p competence loss and an abnormal centering of the vulva on P5.p, suggesting that these two processes are coregulated by the same pathway in Oscheius sp. 1. Group 3 mutants display an enlarged competence group that includes P3.p, thus demonstrating the existence of a specific mechanism inhibiting P3.p competence. Group 4 mutants display an abnormal centering of the vulval pattern on P7.p and suggest that a specific mechanism centers the vulval pattern on a single Pn.p cell.     

Evolution of vulva development in the Cephalobina (Nematoda)

Ventral cord and vulva development are analyzed in a large sample of nematode species of the suborder Cephalobina. We find a specific range of evolutionary variations at distinct developmental steps. (1) Unlike Caenorhabditis elegans and relatives, the vulva is formed from the four precursor cells P(5-8).p or, exceptionally, from P(6, 7).p only. (2) The vulval competence group is restricted to these four cells or is larger. (3) The fates of more anterior and posterior Pn.p cells vary between closely related species (mostly cell death versus epidermal fate). (4) The mechanism of vulval cell fate patterning varies within a single genus, even between strains of the same species. (5) We describe the first example of a vulval cell lineage that is asymmetric between the anterior and the posterior sides of the vulva. For a selection of the investigated taxa, phylogenetic trees were constructed in order to map vulval characters and infer evolutionary polarities. We can conclude that in this group, death of the Pn.p cells probably constitutes a derived character state compared to a syncytial fate. Rhabditophanes sp. and Strongyloides ratti are placed as sister taxa, probably sharing an exclusive common ancestor in which the number of precursor cells forming the vulva was reduced from four to two.     

Comparative genetics: a third model nematode species

Recent studies have introduced Oscheius sp. CEW1 as a third nematode species accessible to genetic analysis, joining the better known Caenorhabditis elegans and Pristionchus pacificus. A group of vulva-defective mutants in Oscheius has been identified, with defects not seen in C. elegans.     

The embryonic cell lineage of the nematode Rhabditophanes sp

One of the unique features of the model organism Caenorhabditis elegans is its invariant development, where a stereotyped cell lineage generates a fixed number of cells with a fixed cell type. It remains unclear how embryonic development evolved within the nematodes to give rise to the complex, invariant cell lineage of C. elegans. Therefore, we determined the embryonic cell lineage of the nematode, Rhabditophanes sp. (family Alloionematidae) and made detailed cell-by-cell comparison with the known cell lineages of C. elegans, Pellioditis marina and Halicephalobus gingivalis. This gave us a unique data set of four embryonic cell lineages, which allowed a detailed comparison between these cell lineages at the level of each individual cell. This lineage comparison revealed a similar complex polyclonal fate distribution in all four nematode species (85% of the cells have the same fate). It is striking that there is a conservation of a 'C. elegans' like polyclonal cell lineage with strong left-right asymmetry. We propose that an early symmetry-breaking event in nematodes of clade IV-V is a major developmental constraint which shapes their asymmetric cell lineage.     

Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans.

The nematode Pristionchus pacificus (Diplogastridae) has been described as a satellite organism for a functional comparative approach to the model organism Caenorhabditis elegans because genetic, molecular, and cell-biological tools can be used in a similar way in both species. Here we show that P. pacificus has three juvenile stages, instead of the usual four found in other nematodes. Embryogenesis is lengthened and many developmental events that take place during the first juvenile stage in C. elegans occur during late embryogenesis in P. pacificus. Video imaging and transmission electron microscopy revealed no embryonic moult. The timing of later developmental events relative to the moults differs between P. pacificus and C. elegans. In addition, the post-embryonic blast-cell divisions display a specific change in timing between the two species, resulting in heterochrony between different cell lineages, such as vulval and gonadal lineages. Developmental events appear to come into register during the last larval stage. Thus, differences in developmental timing between P. pacificus and C. elegans represent a deep heterochronic change. We designate the three juvenile stages of P. pacificus as J1 to J3. Comparison with other species of the family Diplogastridae indicates that this pattern represents an apomorphic character for the monophylum Diplogastridae.     

The two steps of vulval induction in Oscheius tipulae CEW1 recruit common regulators including a MEK kinase

The cell interactions that specify the spatial pattern of vulval precursor cell (VPC) fates differ between the nematodes Oscheius tipulae CEW1 and Caenorhabditis elegans. In the former, the centered pattern of fates is obtained by two successive inductions from the gonadal anchor cell, whereas in the latter, a single inductive step by the anchor cell (EGF-Ras-MAP kinase pathway) can act as a morphogen and is reinforced by lateral signaling between the vulval precursors (Notch pathway). We performed a genetic screen for vulva mutants in O. tipulae CEW1. Here we present the mutants that specifically affect the vulval induction mechanisms. Phenotypic and epistatic analyses of these mutants show that both vulval induction steps share common components, one of which appears to be MEK kinase(s). Moreover, the inductive pathway (including MEK kinase) influences the competence of the vulval precursor cells and more strikingly their division pattern as well, irrespective of their vulval fate. Finally, a comparison of vulval mutant phenotypes obtained in C. elegans and O. tipulae CEW1 highlights the evolution of vulval induction mechanisms between the two species.     

Evolutionary diversification of specification mechanisms within the O/P equivalence group of the leech genus Helobdella

Developmental fates and cell lineage patterns are highly conserved in the teloblast lineages that give rise to the segmental ectoderm of clitellate annelids. But previous studies have shown that the pathways involved in specification of the ventrolateral O lineage and the dorsolateral P lineage differ to some degree in distantly related clitellate species such as the leeches Helobdella and Theromyzon, and the sludgeworm Tubifex. To examine this developmental variation at a lower taxonomic level, we have explored the specification pathways of the O and P lineages in the leech genus Helobdella. In leech, the O and P lineages arise from a developmental equivalence group of O/P teloblasts. In this study, we demonstrate that the cell-cell interactions involved in cell fate specification of the O/P equivalence group differ among three laboratory colonies of closely related species. In two populations, the Q lineage is necessary to specify the P fate in the dorsalmost O/P lineage, but in the third population the P fate can be specified by a redundant pathway involving the M lineage. We also observe interspecific variation in the role played by cell interactions within the O/P equivalence group, and in the apparent significance of extrinsic signals from the micromere cell lineages. Our data suggest that cell fate specification in the O/P equivalence group is a complex process that involves multiple cell-cell interactions, and that the developmental architecture of the O/P equivalence group has undergone evolutionary diversification in closely related species, despite maintaining a conserved morphology.     

The Caenorhabditis elegans sqv genes and functions of proteoglycans in development

In the nematode Caenorhabditis elegans, the vulva is a simple tubular structure linking the gonads with the external cuticle. In this review we summarize knowledge of inter- and intracellular signaling during vulval development and of the genes required for vulval invagination. Mutants of one set of these genes, the sqv genes, have a normal number of vulval precursor cells (VPCs) with an unperturbed cell lineage but the invagination space, normally a tube, is either collapsed or absent. We review evidence that the sqv genes are involved in glycosaminoglycan synthesis and speculate on ways in which defective glycosaminoglycan formation might lead to collapse of the vulval structure.     

The nematode even-skipped homolog vab-7 regulates gonad and vulva position in Pristionchus pacificus

In free-living nematodes, developmental processes like the formation of the vulva, can be studied at a cellular level. Cell lineage and ablation studies have been carried out in various nematode species and multiple changes in vulval patterning have been identified. In Pristionchus pacificus, vulva formation differs from Caenorhabditis elegans with respect to several autonomous and conditional aspects of cell fate specification. To understand the molecular basis of these evolutionary changes, we have performed a genetic analysis of vulva formation in P. pacificus. Here, we describe two mutants where the vulva is shifted posteriorly, affecting which precursor cells will form vulval tissue in P. pacificus. Mutant animals show a concomitant posterior displacement of the gonadal anchor cell, indicating that the gonad and the vulva are affected in a similar way. We show that mutations in the even-skipped homolog of nematodes, vab-7, cause these posterior displacements. In addition, cell ablation studies in the vab-7 mutant indicate that the altered position of the gonad not only changes the cell fate pattern but also the developmental competence of vulval precursor cells. Investigation of Cel-vab-7 mutant animals showed a similar but weaker vulva defective phenotype to the one described for Ppa-vab-7.     

Formation of the egg-laying system in Pristionchus pacificus requires complex interactions between gonadal, mesodermal and epidermal tissues and does not rely on single cell inductions

The invariant cell lineage of nematodes allows the formation of organ systems, like the egg-laying system, to be studied at a single cell level. The Caenorhabditis elegans egg-laying system is made up of the vulva, the mesodermal gonad and muscles and several neurons. The gonad plays a central role in patterning the underlying ectoderm to form the vulva and guiding the migration of the sex myoblasts to their final position. In Pristionchus pacificus, the egg-laying system is homologous to C. elegans, but comparative studies revealed several differences at the cellular and molecular levels during vulval formation. For example, the mesoblast M participates in lateral inhibition, a process that influences the fate of two vulval precursor cells. Here, we describe the M lineage in Pristionchus and show that both the dorsal and ventral M sublineages are involved in lateral inhibition. Mutations in the homeotic gene Ppa-mab-5 cause severe misspecification of the M lineage, resembling more the C. elegans Twist than the mab-5 phenotype. Ectopic differentiation of P8.p in Ppa-mab-5 results from at least two separate interactions between M and P8.p. Thus, interactions among the Pristionchus egg-laying system are complex, involving multiple cells of different tissues occurring over a distance.     

The Caenorhabditis elegans EGL-26 protein mediates vulval cell morphogenesis.

In screens for Caenorhabditis elegans mutants defective in vulval morphogenesis, we isolated multiple mutants in which the uterus and the vulva fail to make a functional connection, resulting in an egg-laying defective phenotype. Two of these connection of gonad defective (Cog) mutants carry alleles of the egl-26 gene. We demonstrate that vulval lineages in egl-26 mutant animals are normal, but one vulval cell, vulF, adopts an abnormal morphology. This results in formation of an abnormally thick layer of vulval tissue at the apex of the vulva and a physical blockage of the exit to the vulva from the uterus. egl-26 was cloned and is predicted to encode a novel protein. Mosaic analysis indicates that egl-26 activity is required in the primary vulval lineage for vulF morphogenesis. Expression of a functional translational fusion of EGL-26 to GFP was observed within the primary vulval lineage only in vulE, which neighbors vulF. EGL-26 is localized at the apical edge of the vulE cell. It is thus possible that vulE acts to instruct morphological changes in the neighboring cell, vulF, in an interaction mediated by EGL-26.     

Phylogeny and taxonomy of the North American clade of the Ceratocystis fimbriata complex

Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C. caryae and is described as C. smalleyi.     

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.     

Pattern formation during vulval development in C. elegans

Previous studies have shown that the development of the vulva of the C. elegans hermaphrodite involves six multipotential hypodermal cells as well as the gonadal anchor cell, which induces vulval formation. Our further examination of the interactions among these seven cells has led to the following model. Each hypodermal precursor cell becomes determined to adopt one of its three potential fates; each of these fates is to generate a particular cell lineage. In the absence of cellular interactions each precursor cell will generate the nonvulval cell lineage; an inductive signal from the anchor cell is required for a precursor cell to generate either of the two types of vulval cell lineages. The inductive signal is spatially graded, and the potency of the signal specifies which lineage is expressed by each of the tripotential precursor cells.     

Competence and commitment of Caenorhabditis elegans vulval precursor cells.

Multipotent Caenorhabditis elegans vulval precursor cells (VPCs) choose among three fates (1 degrees, 2 degrees, and 3 degrees ) in response to two intercellular signals: the EGF family growth factor LIN-3 induces 1 degrees fates at high levels and 2 degrees fates at low levels; and a signal via the receptor LIN-12 induces 2 degrees fates. If the level of LIN-3 signal is reduced by a lin-3 hypomorphic mutation, the daughters of the VPC closest to the anchor cell (AC), P6.p, are induced by the AC. By expressing LIN-3 as a function of time in LIN-3-deficient animals, we find that both VPCs and the daughters of VPCs are competent to respond to LIN-3, and VPC daughters lose competence after fusing with the hypodermis. We also demonstrate that the daughters of VPCs specified to be 2 degrees can respond to LIN-3, indicating that 2 degrees VPCs are not irreversibly committed. We propose that maintenance of VPC competence after the first cell cycle and the prioritization of the 1 degrees fate help ensure that P6.p will become 1 degrees. This mechanism of competence regulation might have been maintained from ancestral nematode species that used induction both before and after VPC division and serves to maximize the probability that a functional vulva is formed.