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Comparison of Gonadogenesis in Hermaphrodite and Male Worms
The two gonadal somatic progenitor cells present in the newly hatched worm can either follow a hermaphrodite or a male developmental pathway. If the hermaphrodite pathway is selected, the structures that are made display a twofold rotational symmetry, and 143 cells are generated. If the male pathway is selected, the structures made are asymmetrical, and only 56 cells are generated. The hermaphrodite and male developmental programs may be compared as follows.
(1) The arrangement and morphological appearance of the four cells in the gonadal primordium present in newly hatched worms is identical in both sexes. The somatic progenitor cells, Z1 and Z4, are located at the anterior and posterior tips of the primordium, respectively. The germ line progenitor cells, Z2 and Z3, occupy the region between Z1 and Z4. The primordium exhibits twofold rotational symmetry.
(2) Z1 and Z4 develop according to a temporal pattern that is essentially identical in hermaphrodites and males. During the second half of L1, Z1 and Z4 undergo a series of divisions that gives rise to 12 cells in the hermaphrodite and 10 cells in the male. During L2, these cells increase significantly in size without dividing further. By late L2, a somatic primordium takes shape due to migration and enlargement. During L3 and the first part of L4, the cells in this somatic primordium undergo extensive divisions. Cells that have stopped dividing differentiate morphologically to the characteristic adult appearance.
(3) The number and orientations of divisions in the early mitotic period are nearly the same in hermaphrodites and males. This lineage differs in the two sexes only by a single division of each somatic progenitor cell. Thus, in hermaphrodites, Z1.a and Z4.p undergo a division that usually does not occur in males. However, in one male, we observed that Z1.a divided in a hermaphrodite fashion. Since extra divisions of this sort have never been observed at any other point in the Z1-Z4 lineages of either sex, this extra division of Z1.a suggests that the hermaphrodite and male early lineages share a common program of control. For example, Z1 and Z4 may be programmed to divide in both sexes according to a single pattern with repression of one division in males.
An important difference in the early events of hermaphrodite and male gonad development occurs after the first division of Z1 and Z4. In the male, Z4.a begins its anterior migration, disrupting the twofold rotational symmetry of the primordium. This is the first morphological indication of the asymmetry that characterizes the rest of male gonad development. By contrast, hermaphrodites maintain the primordial symmetry throughout gonadogenesis.
(4) The three cells in the Z1-Z4 lineages that do not divide after the period of early divisions arise from equivalent positions in the early lineage trees and have similar development fates in hermaphrodites and males (Fig. 18). In both sexes, two of these cells become distal tip cells, and the third cell, either the anchor cell or linker cell, serves to join the gonadal lumen with the animal's exterior. The anchor and linker cells also share the characteristic of variable ancestry. Either Z1.ppp or Z4.aaa can become the anchor cell in hermaphrodites, and either Z1.paa or Z4.aaa can become the linker cell in males.
FIG. 18. Comparison of the hermaphrodite and male Z1 and Z4 early division patterns. The arrows indicate asymmetric divisions, and point to the larger of the two daughters in each case. The numbers refer to the developmental fates of the cells that arise from the early divisions: 1, sheath-spermatheca lineage; 2, dorsal uterus-spermatheca lineage; 3, 4, and 5, ventral uterus lineages; 6, vas deferens lineage; 7, seminal vesicle lineage; dtc, distal tip cell; ac, anchor cell; lc, linker cell. See text for further explanation.
In hermaphrodites, the distal tips move away from the original primordium site, and the most proximal point, marked by the anchor cell and developing vulva, is located where the primordium had been in the L1. In males, the future proximal end moves away, led by the linker cell, and the distal tip is found at the primordium site. Thus, the polarity of the distal-proximal axis of the hermaphrodite is opposite that of the male with respect to the direction of elongation of the gonad. However, in both sexes the spatial and temporal polarity of gamete maturation is toward the somatic structures, and in this sense, they are equivalent. The distal tip cells and the proximal anchor or linker cell might be a reflection of the early establishment of the distal-proximal axes, or they themselves might function to establish the distal-proximal axes of the gonad.
(5) A homologous relationship can be drawn between individual hermaphrodite and male somatic structures by similarities in their positions, lineages, and functions. The most distal somatic structures in both sexes consist of thin, flat cells which encase maturing gametes: the sheath in hermaphrodites and the seminal vesicle in males. In both cases four cells in the somatic primordium each contribute five cells toward that structure, albeit through different lineage patterns. The male vas deferens and the hermaphrodite spermatheca are the next structures in line from distal to proximal. Since the spermatheca houses sperm, and can be considered a male component in the hermaphrodite, and since the lineages are very similar that give rise to these structures, they may be homologous structures. Electron microscopic studies have also revealed an ultrastructural similarity between the vas deferens and the spermatheca (Wolf and Hirsh, unpublished observations). The uterus has no obvious counterpart in the male lineage.
Comparison of gonadogenesis in hermaphrodites and males leads to the striking conclusion that similar developmental programs are followed to generate structures that differ in their properties of symmetry and terminal differentiation. Thus, the decision to make a hermaphrodite or male gonad probably pivots on a limited number of critical modifications in a fundamental program. This hypothesis is reminiscent of Ohno's hypothesis that the genetic control of mammalian sex differentiation is simple and depends on the presence or absence of testosterone during a particular period of development (Ohno, 1971). The genetic control of sex differentiation in C.elegans is also simple. After intensive searching for mutants that alter sex differentiation, only three genes have been identified that transform XX individuals into males (Hodgkin and Brenner, 1977), and only two genes have been identified that transform XO individuals into hermaphrodites (Nelson et al., 1978; Hodgkin, personal communication).
The temporal control of the decision to make either a male or a hermaphrodite gonad has been investigated in a temperature-sensitive transformer mutant that produces males in XX worms (Klass et al., 1976). The critical period of temperature sensitivity in this mutant extends from 3 hr before hatching to 12 hr after hatching (25°C). Thus, 12 hr after hatching is the first point in development at which a shift to restrictive temperature cannot cause any of the worms to make a male gonad. Since the first sign of sexual dimorphism occurs at about 9 hr (25°C) after hatching, the morphological divergence in the two programs may indicate a point of irreversible commitment to one developmental pathway.
Comparison of the Z1-Z4 Lineages to Other Lineages in C.elegans
Sulston and Horvitz (1977) discuss several standard lineage patterns that were observed in the nongonadal lineages. These patterns are also seen in the gonadal lineages. In fact, the entire male lineage can be described in terms of these basic patterns of division.
One of the simpler patterns is characterized by a stem cell repeatedly dividing asymmetrically to give rise to another stem cell and a differentiated cell that does not divide again. An archetypal example of stem cell logic is seen in the lineage of the seminal vesicle. Stem cell logic is also found in the early embryonic lineage of C.elegans (Deppe et al., 1978) and in the postembryonic lateral hypodermal lineages (Sulston and Horvitz, 1977).
A more complex type of stem cell pattern has been suggested from histological studies of neuroblast development in Drosophila (Poulson, 1950). In this variation, the stem cell gives rise to another stem cell and a "pre-differentiated cell" that will divide one more time before differentiating. The vas deferens lineage in C.elegans provides an example of this pattern. The spermathecal lineage of the hermaphrodite begins to divide according to this pattern, but then diverges from it. A similar pattern is seen in the ventral nerve cord lineage of C.elegans (Sulston and Horvitz, 1977). Thus, this pattern may represent a basic mechanism of increasing cell number in different tissues and different organisms.
The early lineage patterns of hermaphrodites and males represent a third basic, but more variable lineage type. A pattern identical to the male early lineage gives rise to each of the rays in the male tail, and similar patterns are seen in the lineages of the posterior lateral ganglia and the lumbar ganglia (Sulston and Horvitz, 1977). Sulston and Horvitz (1977) compare this type of pattern to an insect pattern (Lawrence, 1966) and suggest that it reflects a developmental program common to both organisms.
The standard patterns of cell division observed in C.elegans imply the existence of simple programs by which cell number is increased. These programs may or may not direct a differential segregation of developmental potential to the daughters that arise in the lineages as will be discussed under Mechanisms of Determination.
Temporal control of division. Z1 and Z4 follow a temporal pattern of divisions that is also found in other postembryonic lineages. This pattern is characterized by two periods of divisions separated by a period when no divisions occur. Two nongonadal lineages follow the same pattern (Sulston and Horvitz, 1977). The mesoblast cell, M, undergoes a series of divisions during the last half of L1 followed by a migration of certain of its early descendants during L2 (hemaphrodites) or L3 (males). These cells divide further in L3 to generate the sexual musculature of the vulva or the male tail. The P precursor cells of the ventral nerve cord follow a similar pattern in which a few of the descendants which arise in L1 divide again in L3.
The feature common to cells that divide first in L1 and then in L3 is their involvement in the development of the reproductive system. The function of this delay in development is open to speculation. A full elaboration of the sexual structures might be deferred until after the decision is made in L2 (Cassada and Russell, 1975) to bypass the dauer larval pathway and complete the sexually mature form. Fewer gonadal cells would be economical of the resources necessary to maintain the dauer larva until it finds a new source of nutrients. On the other hand, the L3 developmental delay may serve to ensure the coordinate development of the sexual tissues.
Mechanisms of Determination
Ancestry vs position. A fundamental question that arises out of lineage studies is how cells are led to diverge in their capacity for differentiation. Two mechanisms were proposed by Roux (1888). Cells might become committed to a particular pathway by "self-differentiation" (or cell ancestry) or by "correlative dependent differentiation" (or cell-cell interaction). If a cell is determined by ancestry, cell divisions must mediate a segregation of developmental potential to daughter cells. If a cell is determined by position, cell divisions might only serve to increase the number of cells. A third possibility is that both cell ancestry and cell position play significant roles in the determination of cells in development. For example, a segregation of cytoplasmic determinants during early cleavages in the embryo might restrict a cell's ability to differentiate to one subset of pathways, and cell position might convey to a cell which specific program it must follow among the available choices. Or, the determinants sequestered to one daughter rather than the other might allow that cell to respond differentially to external signals.
Invariance in cell lineages is regarded as a principle of nematode development, both classically (Boveri, 1899) and currently (Sulston, 1976; Sulston and Horvitz, 1977; Deppe et al., 1978). Such invariance only demonstrates that the developmental program is rigidly controlled. The inability of cells to alter their program after manipulation, such as isolation of blastomeres (e.g., Wilson, 1925) or ablation of neighboring cells (Sulston and Horvitz, 1977) shows that cells are irreversibly committed, but does not reveal how that commitment was established. In most lineages, the invariance observed in division pattern and cell fate also corresponds to an invariance in cell position. When both lineage and position of descendant cells are invariant, the influence of cell ancestry and/or position on cell fate is extremely difficult to assess. However, the limited variability that is observed in essentially invariant developmental programs can be utilized to explore the significance of position and ancestry to cell determination.
The most common pattern of variability seen in the Postembryonic lineages of C.elegans involves two cells, each of which is capable of following one of two alternative lineages. The two cells occupy one of two alternative positions, but once a position is assumed, the cell follows a lineage pattern that corresponds to that position.
The nongonadal lineages provide several examples of such variability (Sulston and Horvitz, 1977). Two pairs of cells in the male tail exhibit alternative lineages correlated with position. Certain pairs of ventral cord precursor cells follow different lineages depending on their anterior-posterior position after migration into the cord from the left and right sides of the worm. In addition, two cells in the male gonadal lineage exhibit alternative lineages. The more anterior of the pair of cells becomes the linker cell, whereas the more posterior follows a typical vas deferens lineage pattern.
In hermaphrodites, a more complex situation is found. The positions of only two cells (Z1.ppp and Z4.aaa) are variable, but the lineages of four cells are altered (Fig. 10). Concurrent with the establishment of the hermaphrodite somatic primordium at the end of L2, the four cells that give rise to the ventral uterus assume one of two configurations. These four cells, as a group, follow one of two alternative lineage patterns which corresponds to the configuration in the individual primordium. This group phenomenon suggests that neighboring cells in some way can influence each other's fates.
Another explanation of the hermaphrodite variability might be that the two somatic progenitor cells can randomly assume the anterior or posterior positions during the embryonic formation of the four-celled gonadal primordium. If only one of the somatic progenitor cells were capable of producing an anchor cell, for example, and if that cell could become either Z1 or Z4, the observed variability would result.
A third explanation of the 5R and 5L alternatives might have been that a random population consists of two subpopulations, one programmed to follow the 5R pattern and one programmed to follow the 5L pattern. This cannot be the case because these programs are not clonally inherited; individuals whose developmental pattern was recorded as 5L or 5R and were then cloned gave rise to progeny that followed both 5L and 5R patterns.
Thus, two hypotheses remain that might explain the unique variability observed in the hermaphrodite Z1-Z4 lineage. Either two somatic progenitor cells are irreversibly committed during embryogenesis and can interchangeably become Z1 or Z4, or the interaction of the cells in the somatic primordium affects their subsequent lineage pattern. It may be possible to distinguish between these two explanations by laser ablation of Z1 or Z4 in a large number of animals. If Z1 and Z4 are irreversibly committed cells, an anchor cell should arise in approximately one half of such experimental animals. If Z1 and Z4 are not irreversibly committed, an alteration in the development of the remaining cell may be seen. If some form of regulation occurs and cell interaction proves to be the most reasonable explanation for the variability in the lineages of four of the cells in the somatic primordium, one might postulate that the rearrangement of cells to form a somatic primordium represents a change in position for all the cells involved, and that cell-cell interaction might influence the developmental fate of all the cells concerned.
Coordinates and developmental fates. Whether two daughters are determined because they have acquired a specific cytoplasm or a specific position, some kind of coordinate system is an absolute requirement for information to be conveyed differentially to daughter cells. An anterior daughter might follow a different pathway from its posterior sibling due to a differential segregation of cytoplasmic determinants to the two daughters. However, the difference might just as reasonably be due to a different environment imposed on the daughter cells, whether that is mediated through interaction with its neighbors or an externally imposed gradient. Thus, lineage data do not reveal the mechanisms by which the fate of one daughter becomes different from the fate of its sibling. Yet, lineage data demonstrate that the fate of a cell does correspond to the spatial relationship in which the siblings arise, and therefore, shows that the coordinate exists and is significant to the fate of the daughter cells.
The male Z1-Z4 lineage provides evidence that at least the last half of male gonadal development proceeds according to its own coordinate system rather than the organismic coordinate system. The late divisions of the male begin just before the gonad makes its 180°turn. Since there is some variability in the correlation between when a cell divides and when the gonad makes its bend, a cell may divide before the turn, in the turn, or after the turn. The daughter nearer the leading or proximal edge would be an anterior daughter in the first case, a dorsal daughter in the second, and a posterior daughter in the last case. Yet, that daughter does not vary with respect to further divisions or differentiation. Thus, distal-proximal coordinates along the gonadal axis seem to be more relevant to these divisions than the coordinates of the worm. Since the initial growth of the male gonadal primordium is only anterior and never posterior, it seems likely that the organismic coordinate system influences this first developmental step in the male. The twofold rotational symmetry exhibited by the hermaphrodite gonad does not contradict the idea that the gonadal coordinate system may be independent of the organismic coordinate system.
One principle that has emerged from the information on position and cell fate in the lineages of Z1 and Z4 is that the symmetry of the structure in which a particular division takes place is correlated with the symmetrical relationship between the fates of Z1 descendants and the fates of Z4 descendants (Fig. 18). In both hermaphrodites and males, the fates of the daughters of the first division follow the twofold rotational symmetry of the four-celled gonadal primordium. Thus, Z1.a is equivalent to Z4.p, and Z1.p is equivalent to Z4.a. A comparison of the fates of Z1 descendants with the fates of Z4 descendants shows that, in hermaphrodites, cells that are equivalent in developmental fate occupy positions that are related by twofold rotational symmetry (Fig. 18). The morphology of the hermaphrodite developing gonad also retains the twofold rotational symmetry. By contrast, the male developing gonad becomes morphologically asymmetrical soon after the first division. And, in males, equivalent cells in the Z1 and Z4 lineages are related asymmetrically along the anterior-posterior axis after the first division (Fig. 18).
In hermaphrodites, the only cells in the somatic primordium that are not positioned according to twofold rotational symmetry have lineages that are not related to each other by twofold rotational symmetry. So, Z1.ppa is not lineally equivalent to Z4.aap, and Z1.ppp is not equivalent to Z4.aaa. However, these four cells can assume one of two configurations, 5R and 5L, that are related by twofold rotational symmetry, and the lineages followed by the four cells in 5L are related to the lineages of the four cells in 5R by twofold rotational symmetry.
The significance of the correlation observed between the morphological symmetry displayed by the developing gonad and the developmental symmetry exhibited by the fates of Z1 and Z4 descendants is purely a matter of speculation at the present time. The correlation might be coincidental, it might be a reflection of two independent responses to an underlying coordinate system, or it might involve a direct cause and effect relationship.
A second principle that emerges from the Z1-Z4 lineages is that no structure (e.g., uterus, anterior spermatheca, vas deferens) develops as a cell clone. Clonal development is defined by two criteria (Crick and Lawrence, 1975). First, a cell must contribute all its descendants to a single structure. Second, a structure must consist of only the descendants of a single clone. In the development of the gonadal somatic structures, each structure consists of descendants from more than one cell in the somatic primordium, and in most cases, each cell in the somatic primordium contributes to more than one structure. Such nonclonal development of structures is also typical of organisms whose lineages were followed in classical studies. For example, most structures in the trochophore larvae of the annelid Nereis (Wilson, 1892) and the mollusk Crepidula (Conklin, 1897)-apical rosette, prototroch, gut-are derived from descendants of all four progenitor cells that arise from the first two divisions of the egg.
In conclusion, the Z1-Z4 lineages are particularly advantageous for addressing
certain key developmental questions:
(1) The unique variability in the hermaphrodite lineage strongly suggests that cell-cell interaction affects cell fate during gonadogenesis. Alternatively, the two somatic progenitor cells might be irreversibly commited during embryogenesis and assume the Z1 or Z4 position interchangeably in the gonadal primordium. The laser ablation of Z1 or Z4 will test the commitment of the unablated cell and therefore may distinguish between the two models.
(2) The two developmental programs available to Z1 and Z4 raise questions about how the genetic control of the programs is organized, and how differences in symmetry are established. The genetic control is accessible since C.elegans is amenable to mutational analysis and since gonadogenesis defective mutants can be obtained (Hirsh and Vanderslice, 1976). However, the necessity for a selection method for mutants that specifically affect morphogenesis has become apparent (Kimble, 1978). The establishment of symmetry is a much more elusive problem, but the morphological change in symmetry that is reflected in the behavior of four cells in the male may provide a key to how that change is mediated.
(3) The significance of nonclonal development of the gonadal somatic structures may also be approachable in this system. One guess is that cell-cell interaction is important to the evolution of complexity during development. It should be possible to alter the composition of the cells of the somatic primordium by laser ablation so that only one precursor cell is left for a particular structure. If cell interaction between two precursor cells is critical to differentiation, such a change should cause a change in the pattern of differentiation of the precursor cell.
The Z1-Z4 lineages provide a model of morphogenesis and differentiation that is sufficiently complex so the principles involved will probably be applicable to the development of most higher eukaryotes. Yet, the system is simple enough that the behavior of individual cells can be studied as they proceed through the sequential steps leading to the adult form. Our current goal is to understand the extents to which ancestry and cell interaction play a role in the determination of individual cells. Ultimately, we hope to exploit this system to elucidate the genetic control of the cells' progression through each step in gonadogenesis.
Adapted by Yusuf KARABEY for WORMATLAS, 2003