Evolution of development in nematodes related to C. elegans^*

In C. elegans hermaphrodites, the two gonad arms develop nearly symmetrically from two somatic precursor cells, Z1 (anterior arm) and Z4 (posterior arm). Each of these cells gives rise to a distal tip cell (DTC; Z1.aa and Z4.pp), which promotes the growth and migration of the arm and later, the proliferation of the germ line (see Introduction to the germ line).

The first species with a monodelphic female gonad that was studied in greater detail was Panagrellus redivivus (Sternberg and Horvitz, 1981). Cell lineage analysis revealed that monodelphy results from the programmed cell death of the posterior dtc Z4.pp (Figure 1). Thus, the programmed cell death of a single cell has a major influence on overall nematode morphology.

Within the Rhabditidae, didelphy is thought to represent the ancestral character state. Monodelphy has evolved repeatedly, such as in the genera Mesorhabditis, Teratorhabditis and Cruznema. Felix and Sternberg analyzed representatives of these genera and additional species within the genera Cephalobus, Acrobeloides and Panagrolaimus (Felix and Sternberg, 1996). In all these cases, the first asymmetry between the anterior and posterior part of the gonad was already seen at the time of Z1 and Z4 division. The lineage that produces Z4.pp dies in all these species, albeit, at different time points in development (Felix and Sternberg, 1996; Figure 1).

Finally, it should be noted that the evolutionary diversification resulting in female monodelphy is distinct from one-armed gonads in males. C. elegans male gonad development does not involve programmed cell death and the posterior elongation requires a novel cell type, the linker cell (see Introduction to the germ line; Figure 1).

Later in development, the female/hermaphrodite gonad generates a unique cell type, the anchor cell (AC). The AC is morphologically distinct from all other cells of the somatic gonad (Sherwood and Sternberg, 2003). This cell is important for signaling to other ventral uterine cells, is involved in vulva induction and finally generates the connection between the uterus and the outside environment (Newman and Sternberg, 1996). The AC is determined by stochastic events, in that Z1.ppp and Z4.aaa have an equal chance of adopting the AC fate. lin-12 signaling is instrumental in AC specification (see LIN-12/Notch signaling in C. elegans). Recent studies suggest that the birth order of Z1.ppp and Z4.aaa is crucial for AC specification (Karp and Greenwald, 2003). Females/hermaphrodites of all nematode species studied to date generate an AC. Interestingly, AC specification is highly biased or even fixed in most monodelphic species (Felix and Sternberg, 1996).

As will be discussed below, monodelphic species always form a posterior vulva, rather than a vulva that is localized in the center of the animal. However, the degree to which the vulva is shifted to the posterior is species-specific and varies between 60-90% body length. In this context, it is noteworthy that vulva induction can be performed by cells other than the AC. In Mesorhabdits sp. PS1179, the AC is no longer involved in vulva induction (Sommer and Sternberg, 1994). However, Z1.apr becomes the AC in Mesorhabditis and assumes a new function: it leads the elongation of the gonad towards the posterior vulva (Felix and Sternberg, 1996) demonstrating the evolutionary plasticity of individual cells.

Hermaphrodite gonad development has also been compared between didelphic species. In particular, the Pristionchus pacificus hermaphrodite gonad has been studied in some detail by electron and fluorescent microscopy (Rudel et al., 2005; see section 3.2.4. for more detailed species overview). Although also didelphic, the P. pacificus gonad shows numerous differences in comparison to C. elegans (Figure 2). Among these differences are heterochronic changes, novel gonad arm-migrations, novel cellular composition of some tissues, the absence of a tissue (i.e. the spermathecal valve cells), a novel architecture for the sheath, and changes in the cellular and sub-cellular morphology of the individual sheath cells. Additionally, cell ablation experiments in P. pacificus indicate extensive cell communication between the somatic gonadal tissues and the germ line and some of which show significant differences in comparison to C. elegans (Rudel et al., 2005).

Taken together, the nematode gonad shows enormously evolutionary diversity, both, at the level of the overall morphology and cell type specificity. Future work will allow the dissection of the molecular mechanisms involved in this evolutionary diversification by studying selected examples in more detail.

Vulva development was initially chosen for comparative studies because of the detailed genetic and molecular understanding of the process in C. elegans and the ease with which the vulva cell lineage could be determined and manipulated in novel species. First investigations concentrated on different rhabditid nematodes (Ambros and Fixsen, 1987; Sommer and Sternberg, 1994; Sommer and Sternberg, 1995; Sommer and Sternberg, 1996) with a specific emphasis on nematodes with posterior vulva formation (Sommer and Sternberg, 1994). Most nematodes that develop a monodelphic gonad form their vulva in the posterior body region (Figure 3; Table 1). In some species, such as Mesorhabditis sp. PS1179, vulva formation occurs at 80% body length, whereas in others, such as Teratorhabdits palmarum, it occurs at 90% body length in immediate proximity to the rectum (Figure 3). Surprisingly, in all studied cases the vulva developed from P(5-7).p with a 2°-1°-2° pattern very similar to C. elegans. P(5-7).p are born in the central body region and migrate in the L2 stage, together with the other cells of the vulva equivalence group, towards the posterior. Migration stops at a species-specific position and cell divisions occur at the late L3 and early L4 stage (Figure 3).

Besides the involvement of cell migration, most species with posterior vulva formation differ from C. elegans in that the gonad no longer provides an inductive signal (Figure 4, Table 1; Sommer and Sternberg, 1994). If the gonad is ablated at hatching, a normal vulva forms in the absence of any gonadal tissue. This pattern has been observed in Mesorhabditis sp. PS1179 and T. palmarum (Sommer and Sternberg, 1994) of the Rhabditidae, in Brevibucca sp., SB261 of the Brevibuccidae (Felix et al., 2000) and in Diplogastrellus gracilis of the Diplogastridae (Sommer, unpublished observation; Table 1). Thus, posterior vulva formation in monodelphic species has evolved several times independently by convergent evolution and always requires cell migration of VPCs that were born in the central body region. In most cases - and again this feature has evolved independently by convergent evolution - posterior vulva species do not require an inductive signal by the gonad. One exception is Cruznema tripartitum, in which P(3-8).p migrate towards the posterior in the L2 stage, as in the other species, but vulva formation requires induction by the AC.

Initial comparative studies on vulva formation in clade V and clade IV species concentrated on patterning differences. Surprisingly, the vulval fate pattern of the equivalence group is relatively constant, and only three, basically “taxa-specific” patterns, for the Rhabditidae, Diplogastridae and Cephalobina, have been identified:

Rhabditidae pattern: P(4-8).p form a vulva equivalence group with the pattern 3°-2°-1°-2°-3° (Sommer and Sternberg, 1994; Sommer and Sternberg, 1995). In this regard, C. elegans is itself an exception in that P3.p is a member of the equivalence group in 50% of the individuals. Non-vulval cells in the anterior (P1.p to P3.p) and the posterior (P9.p to P11.p) body region fuse with the surrounding hypodermis (Figures 3, 5, 6A).

Diplogastridae pattern: In Pristionchus pacificus and all other species of the Diplogastridae studied to date, non-vulval cells in the anterior and posterior body region die by programmed cell death. These include P(1-4).p and P(9-11).p (see section 3.2.4. below). The vulva is formed by P(5-7).p with a 2°-1°-2° pattern (Figure 6A; Table 1; Sommer and Sternberg, 1996; Sommer, 1997).

Cephalobina pattern: In contrast to all species of the Rhabditidae and Diplogastridae, Cephalobina species form the vulva from the four precursor cells P(5–8).p (Sternberg and Horvitz, 1982; Sommer and Sternberg, 1996; Felix and Sternberg, 1997, Felix and Sternberg 1998; Felix et al, 2000). P(5,8).p have a 2° fate and P(6,7).p have a mixed 1°/2° fate with a UUTT and TTUU division pattern, respectively. The AC resides between P6.pp and P7.pa, whereas the AC resides above P6.p in species of the Rhabditidae and Diplogastridae. Interestingly, if a single VPC is isolated (that is, all other VPCs are ablated) the remaining cell will move beneath the AC and will have a TTTT pattern as is known from C. elegans. Thus, the wild type pattern UUTT and TTUU is a mixed 1°/2° fate. For simplicity, we consider the total pattern of the Cephabolina 2°-1°-1°-2° (Table 1). Cephalobina species differ in the fate of the non-vulval cells in the anterior and posterior body region. These cells fuse with the hypodermis in some species, but die by programmed cell death in others (Table 1; Sommer and Sternberg, 1996; Felix et al., 2000).

Although the vulva cell lineage patterns differ between most species, the final vulva fate pattern is surprisingly conserved with the only major alteration being the 2°-1°-2° pattern (clade V) vs. 2°-1°-1°-2° pattern (clade IV; Table 1). At the same time however, the exact cellular mechanisms of vulva induction vary greatly among species. Besides the absence of an inductive signal from the gonad in species with a posterior vulva, many of the didelphic species also differ strongly from C. elegans. Often, vulva induction occurs as a two-step process. In Oscheius tipulae and Rhabditella axei, the AC first provides a signal that specifies vulval fates. Later, after the first division of the VPCs, the AC provides a second, temporarily distinct signal that specifies the 1° cell fate (Felix and Sternberg, 1997; Figures 4 and 5).

In clade IV nematodes, similar two-step inductions are common, but with variations in individual species. In Panagrolaimus sp. PS1732, the first signal comes from the gonad early in larval development, whereas the second signal is provided by the AC (Felix and Sternberg, 1997). In P. redivivus, both signals come from the gonad and in Halicephalobus sp. JB128 three signals are provided by the gonad and the AC, respectively (Felix et al., 2000; Figure 4). In the latter, an additional survival signal from the gonad is required to prevent the programmed cell death of the VPCs (Felix and Sternberg, 1998).

Taken together, these comparative studies reveal an enormous amount of evolutionary variability, mostly at the level of vulva induction. Although these cell ablation studies do not indicate which molecular signaling systems are at work in species with altered vulva induction, they provide important information for C. elegans itself. The phylogenetic interpretation in Figure 4 reveals that it is currently impossible to determine the induction mechanism of the last common ancestor from the nematodes compared so far. It is clear however, that the one-step mechanism seen in C. elegans is a highly derived character. Thus, the textbook example of AC signaling in C. elegans is not a common nematode pattern. Rather, it represents a derived feature that has most likely evolved relatively recently within the genus Caenorhabditis. At the same time, genetic analysis in O. tipulae and P. pacificus starts to provide insight into the evolution of molecular mechanisms of vulva induction (see below). In particular in P. pacificus, recent studies suggest that the molecular mechanisms of vulva induction changed during nematode evolution and that Wnt signaling has opposite functions in C. elegans and P. pacificus (Zheng et al., 2005).

O. tipulae is a hermaphroditic species, has a fast life cycle, is culturable on E. coli OP50 and can be frozen using standard protocols. Genetic analysis is straightforward and mutations can readily be discovered and maintained. Morpholino knockdown experiments have been introduced as a reverse genetic tool (Louvet-Vallee et al., 2003). Given that the Oscheius vulva is generated by a two-step mechanism (see above). Large-scale mutagenesis screens for vulva defective mutants have been performed to obtain insight into the mechanisms of vulva induction in this species (Dichtel et al., 2001; Louvet-Vallee et al., 2003; Dichtel-Danjoy and Felix, 2004; Figure 5). Surprisingly, most mutants in O. tipulae display phenotypes unknown from C. elegans. For example, the largest group of mutants affects the cell division pattern of the vulva precursor cells without changing their fate (Dichtel et al., 2001). Similar mutants have only rarely been observed in C. elegans (Kipreos et al., 1996; Fay and Han, 2000). This very interesting example shows that vulval cell cycle and fate can readily be genetically dissected in one species (O. tipulae) but not the other (C. elegans) and that the outcome of genetic analyses is species-specific and depends on sometimes unpredictable circumstances.

A second group of O. tipulae vulva mutants affects the competence of the VPCs P(4-8).p. This group includes the lin-39 Hox gene, which seems to have a conserved role in the determination of the vulva equivalence group in nematodes (Louvet-Vallee et al., 2003). The third group of mutants specifically affects the vulva induction mechanism (Dichtel-Danjoy and Felix, 2004). These mutants show hypo - or hyperinduced vulval phenotypes. The hypoinduced iov-1 mutant reduces the level of both vulval induction steps indicating that both steps of induction share common molecular components (Dichtel-Danjoy and Felix, 2004). A MEK kinase inhibitor mimics the iov-1 mutation arguing that the RAS/MAPK pathway is involved in vulva induction in O. tipulae (Dichtel-Danjoy and Felix, 2004).

P. pacificus has been established as a satellite organism in evolutionary developmental biology in the last ten years. Also P. pacificus propagates as self-fertilizing hermaphrodites, has a 4-day life cycle (20°C) and can be cultured on OP50. P. pacificus is amenable to various cellular, genetic and molecular techniques successfully used in C. elegans. In addition to forward genetics, morpholino knockdown experiments provide a reverse genetic tool (Pires-daSilva and Sommer, 2004; Zheng et al., 2005).

In recent years, developmental and molecular studies have been complemented by a genomic approach. An integrated genome map of P. pacificus contains a genetic linkage map of approximately 400 molecular markers and a physical map of nearly 10.000 fingerprinted BAC clones (Srinivasan et al., 2002, Srinivasan et al.; 2003). A whole-genome sequencing project is ongoing (http://www.nhgri.nih.gov/12511858). A database, AppaDB, compiles the genomic information of P. pacificus in a comprehensive manner.

Vulva development in P. pacificus involves a set of evolutionary modifications when compared to C. elegans (Figure 6):

P. pacificus vulva defective mutants have been isolated in large-scale mutagenesis screens (Eizinger, 1999) and the phenotype of mutations in the three homeotic genes Ppa-lin-39 (Eizinger and Sommer, 1997; Sommer et al., 1998; Grandien and Sommer, 2001), Ppa-mab-5 (Jungblut and Sommer, 1998; Jungblut et al, 2001) and Ppa-vab-7 (Jungblut and Sommer, 2000) have helped elucidating the molecular mechanisms of evolutionary change. Interestingly, Ppa-lin-39 is not required for vulva induction and thereby differs from Cel-lin-39 (Sommer et al., 1998). Ppa-mab-5 mutants show ectopic vulva differentiation, whereas Cel-mab-5 mutants form a normal vulva (Jungblut and Sommer, 1998). These results indicate that homologous homeotic genes changed their exact function during nematode evolution.

Recent studies in P. pacificus have shed first light on the molecular basis of vulva induction in nematodes other than C. elegans. Mutations in Ppa-lin-17/Frizzled and morpholino knockdowns of Ppa-lin-44/Wnt, Ppa-egl-20/Wnt, Ppa-mig-5/Dsh, Ppa-apr-1/APC and Ppa-bar-1/ß-cat result in gonad-independent vulva differentiation and a multivulva phenotype indicating a role of Wnt signaling in a negative signaling process (Zheng et al., 2005). In contrast in C. elegans, Wnt signaling has a positive role in vulva induction and mutations in Cel-bar-1/ß-cat result in a hypoinduced phenotype (Eisenmann et al., 1998). Thus, the genetic control of vulva induction has diversified during evolution and differs strongly between P. pacificus and C. elegans.