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Hirsh et al. (1976) and Klass et al. (1976) have described the major features of post-embryonic gonadogenesis in hermaphrodites and males, respectively. Figure 1 summarizes these findings. In both sexes, gonadal development starts from a four-celled gonadal primordium present at hatching and proceeds through all four larval stages (L1, L2, L3, and L4). Hermaphrodite gonadogenesis is characterized by twofold rotational symmetry, whereas male gonadogenesis is asymmetrical. Both hermaphrodite and male gonads display a distal-proximal axis with proximal toward the vulva in hermaphrodites and toward the cloaca in the male. The maturation of gametes occurs from distal to proximal. The somatic structures are located most proximally in the reproductive system.
FIG. 1. Overview of gonad development in hermaphrodites and males. At the top, the four celled gonadal primordium is shown in its midventral position in the newly hatched worm. In hermaphrodites (left column), the developing gonad elongates anteriorly and posteriorly during L1, L2, and L3. The growing tips reflex around the time of the L3-L4 molt. In males (right column), the developing gonad initially grows only in an anterior direction, and reflexes at about the L2-L3 molt. The main somatic structures of the adult hermaphrodite and male are shown schematically in the bottom two drawings, dtc, distal tip cell; ac, anchor cell; lc, linker cell; a. sp., anterior spermatheca; p. sp., posterior spermatheca.
The gonadal primordium in the hermaphrodite is indistinguishable morphologically from that of the male. In each sex, the four primordial cells are located mid-ventrally along the anterior-posterior axis (figure 2A). These four cells are named Z1, Z2, Z3, and Z4 from anterior to posterior. Z1 and Z2 are located to the right of the worm's midsagittal plane, and Z3 and Z4 are to the left of that plane (figure 2B). Thus, the primordium exhibits twofold rotational symmetry. Although the hermaphrodite and male primordia are equivalent structures, newly hatched worms can be easily sexed according to three secondary sex characteristics recognized by Sulston and Horvitz (1977).
The organization of the somatic and germ line progenitor cells in the four-celled gonadal primordium of nematodes was revealed many years ago (reviewed in Chitwood and Chitwood, 1950). In both hermaphrodite and male worms, cells Z1 and Z4 give rise to the somatic structures of the gonad, and Z2 and Z3 give rise to the germ line cells. The primary focus of this paper is the Z1-Z4 somatic lineage. The cell lineages of Z2 and Z3 are variable in both sexes with respect to the planes of cell division and the times at which particular divisions occur. Z2-Z3 divisions occur continuously from L1 through adulthood. In hermaphrodites, the anterior and posterior gonadal arms each contain descendants of both Z2 and Z3. The plane of cell division can be left-right, dorsal-ventral, anterior-posterior, or oblique along any axis. A particular cell can divide relatively soon after its birth or it can be considerably delayed. No migrations have been observed among the germ line cells.
FIG. 2. Gonadal primordium in young L1 hermaphrodite; Nomarski optics. A. Lateral view. This worm is unusual in that Z1, Z2, Z3, and Z4 are all visible in the same focal plane. B. Ventral view.
In contrast to the germ line cells, the somatic progenitor cells, Z1 and Z4, follow a nearly invariant pattern of cell divisions, migrations, and differentiation. The timing of events and the orientation of cleavages are essentially the same from worm to worm. The lineages of Z1 and Z4 are characterized by two periods of mitoses. The earlier period occurs during the first larval stage (L1) and the later one spans L3 and extends into L4.
Hermaphrodite Z1-Z4 Lineage (See figure 3)
Early Mitotic Period
Z1 and Z4 start dividing midway during the first larval stage (L1), and give rise to 12 cells by the L1-L2 molt, or shortly thereafter. After the first division, the anterior daughter of Z1, Z1.a, and the posterior daughter of Z4, Z4.p, occupy respectively the anterior and posterior tips of the primordium, and Z1.p and Z4.a lie ventral to the germ line cells (figure 4a). When Z1.a and Z4.p divide, Z1.aa and Z4.pp remain at the anterior and posterior tips of the gonad, and their siblings, Z1.ap and Z4.pa become positioned more dorsally in the primordium (figure 4b). Z1.p and Z4.a divide asymmetrically, and each gives rise to one larger daughter (Z1.pa and Z4.ap) and one small daughter (Z1.pp and Z4.aa) (figure 4b). Z1.pa, Z1.pp, Z4.aa, and Z4.ap, lying ventrally in the primordium, each divide to complete the early divisions (figure 4c). By the time of this final round of divisions, Z1.pa has moved to the left side, and Z4.ap has moved to the right side of the developing gonad. The daughters arising from these cells remain on the side to which the mother cell moved previously (figure 4d). Thus, four descendants of Z1 now lie on the opposite side that Z1 originally occupied and similarly for the descendants of Z4 (cf. figure 2 and figure 4d). Thus, the division pattern and cell movements carried out by Z1 are related by twofold rotational symmetry to the division pattern and cell movements of Z4.
FIG. 3. Hermaphrodite lineage of gonadal somatic progenitor cells Z1 and Z4. Only the invariant lineages are shown here; the variant lineages are shown in figure 10. Divisions give rise to an anterior daughter and a posterior daughter unless otherwise indicated. Anterior is to the left. The fates of descendants are indicated either individually or in groups; a, anterior; p, posterior; dtc, distal tip cell; sp, spermatheca; jn, spermathecal-uterine junction.
FIG. 4. Spatial arrangement of hermaphrodite Z1 and Z4 descendant nuclei during the early mitotic period. The space occupied by Z2 and Z3 descendants is depicted by stippling, (a), (b), and (c) give lateral views after each round of divisions; (d) is a dorsal view of the same stage as (c) and shows that the positions of Z1 descendants are related to the positions of Z4 descendants by twofold rotational symmetry. The axis of symmetry is depicted as a dot, and the rotational operation is indicated by an arrow.
The somatic cells of the gonad do not divide again until late L2 or early L3, but the germ line cells approximately quadruple in number during L2. Thus, the somatic cells become separated from each other by an increasing number of germ line cells. Z1.aa remains at the anterior tip and Z4.pp at the posterior tip. Since the growing tips are the future distal tips in hermaphrodites, Z1.aa and Z4.pp are called distal tip cells. These two cells appear to serve a leader function, preceding the extending gonadal arms throughout gonadogenesis (figure 5). They do not divide again and they remain at the gonadal tips even in the adult. During L2, the gonadal primordium elongates from 25-30 to 50-70 micrometer.
FIG. 5. Hermaphrodite developing gonad after formation of the somatic primordium; Nomarski optics. This focal plane shows the anterior arm (Z2 and Z3 descendants), the anterior distal tip cell (Z1.aa), and three cells in the somatic primordium: 1, Z4.apa; 2, Z4.aap; 3, Z4.aaa.
The second half of L2 is characterized by a marked growth without cell division and a positional change of ten Z1 and Z4 descendants (Z1.aa and Z4.pp remain at the distal tips). Just before the L2-L3 molt, these ten cells, five Z1 descendants and five Z4 descendants, move to the center of the gonad and overlap each other. During this process, the germ line cells are completely displaced from the central region. This rearrangement of somatic cells forms the hermaphrodite somatic primordium (figure 5). This primordium represents a new association of all the cells that will divide further to generate the individual somatic structures of the hermaphrodite gonad.
The ten cells arrange themselves in one of two alternative configurations in the somatic primordium (figure 6). The positions of eight of the somatic primordial cells are invariant. However, two cells, Z1.ppp and Z4.aaa, assume one of two alternative positions. Late in L2, either Z1.ppp or Z4.aaa moves into the midsagittal plane on the ventral surface of the gonad. Whichever cell acquires this midsagittal position is fated to become the anchor cell-a small, round cell which divides no further, and seems to be involved in the formation of the vulva. If Z1.ppp moves in from the left side of the gonad, only four cells remain on the left, whereas five cells are present on the right. This is called the 5R configuration (figure 6a). Conversely, if Z4.aaa moves in from the right side, four remain on the right and five on the left. This is the 5L configuration (figure 6b). Individual worms that developed by passing through a 5R configuration were observed to give rise to 5L and 5R progeny. Similarly, progeny of 5L worms can develop through either the 5L or 5R pathway.
FIG. 6. 5R and 5L somatic primordia, dorsal view. The two alternate cell arrangements are related to each other by a 180° rotation around the dorsal-ventral axis passing through the anchor cell (ac). Anterior is to the left.
Late Mitotic Period
During L3 and L4, nine somatic cells (all the cells in the somatic primordium except the anchor cell) divide to generate 140 cells for a total of 143 cells in the L4, including the distal tip cells and the anchor cell. These cell divisions and subsequent differentiation give rise to five somatic structures: the anterior and posterior sheaths which encapsulate the germ line component of the gonad, the anterior and posterior spermathecae which store sperm from each gonadal arm, and a central uterus (figure 7).
FIG. 7. Hermaphrodite somatic structures at the mid-L4 stage; Nomarski optics, a. sp., anterior spermatheca; p. sp., posterior spermatheca, ut., uterus; v., vulval lumen.
a) Sheath lineage. Two cells (Z1.ap and Z1.paa) in the somatic primordium each contribute five descendants to the anterior sheath, and two (Z4.pa and Z4.app) contribute five descendants to the posterior sheath (figure 8). Thus, each sheath consists of 10 cells. The sheath precursor cells also give rise to a major portion of the spermatheca.
The future sheath cells arise next to the developing spermatheca. They become flat elongated cells and spread over the surface of the germ line tube. They either migrate distally or are dragged along as the germ line arm elongates. Unless the nuclei stay along the visible edge of the gonadal arm, the sheath cells are very difficult to see. Therefore, 1-micrometer serial sections were cut from dissected gonads embedded in Epon, and stained with toluidine blue. The sheath cell nuclei, easily visible in cross section, were counted to make sure that no further division had occurred, and to ascertain the adult positions of these cells (figure 8).
FIG. 8. Correlation of lineage with adult position of the hermaphrodite sheath cell nuclei. Z1.paa and Z1.ap both follow the indicated lineage pattern to give rise to the anterior sheath. Z4.pa and Z4.app give rise to the posterior sheath. Thus, two descendants, one from each precursor cell, are found at each position (1, 2, 3, 4, and 5). After a sheath cell arises, it becomes flat and begins to migrate distally onto the germ line arm. The positions marked in the diagram were ascertained by a combination of direct observation of this migration and tracing the positions of cells in serial 1-micrometer sections of gonads embedded in Epon. The identity of the cells in positions 4 and 5 is known by direct observation. The identity of cells in position 1 is assigned because the distal daughters of the sheath precursor cells arise early in the lineage and their positions are already quite distal along the germ line arm when the other sheath cells arise. Identity of cells in positions 2 and 3 simply follows the positions of these cells for the first few hours after they arise. An exchange in their positions at a later time is possible, but has not been observed.
(b) Spermathecal lineage. The anterior and posterior spermathecae each arise from four cells of the somatic primordium (figure 9). Two cells each contribute nine descendants, and two cells each contribute three descendants.
FIG. 9. Schematic of spermatheca development. Four cells contribute descendants to each spermatheca. Two cells (A) each contribute nine, and two cells (B) each contribute three. The formation of only half of the spermatheca (either the right half or the left half) is depicted diagrammatically. The shaded circles represent A descendants, and the stippled circles represent B descendants. The starred cells form the core of the spermathecal-uterine junction.
The spermathecae develop as spiral structures that join the developing uterus to the anterior or posterior sheath. Each spermatheca has a single left-handed twist in it along its longitudinal axis. These two structures are related by twofold rotational symmetry. As the spermathecal cells differentiate, the nuclei become small and irregular. The cytoplasm takes on a granular appearance and it becomes impossible to see the cytoplasmic boundaries. A lumen forms as the last divisions occur in the developing spermatheca. The adult spermatheca is a flexible tube that is continually contorted by the muscular contractions of the proximal arm sheath and the uterus.
(c) Uterine lineage. The dorsal portion of the uterus arises invariantly from two cells in the somatic primordium (figure 3). The ventral uterus can arise according to either of two alternative lineages (figure 10). These two lineages correspond to the two alternative configurations of the somatic primordium. One lineage is followed if the cells assume the 5R configuration (figure 10a), whereas another is followed from the 5L configuration (figure 10b). These two lineages, expressed in different animals, are related by twofold rotational symmetry.
FIG. 10. Alternate lineages of hermaphrodite lineage. Z1.ppa, Z1.ppp, Z4.aaa, and Z4.aap follow one lineage from the 5R configuration (a) and another lineage from the 5L configuration (b). The two alternate lineages are related by twofold rotational symmetry, a. jn., anterior spermathecal-uterine junction; p. jn., posterior spermathecal-uterine junction; v. ut., ventral uterus.
The difference in the ventral uterine lineage in 5L and 5R worms provides the only example of variability in the hermaphrodite lineage of Z1 and Z4. The 5R and 5L configurations differ only in the positions of two cells, but the lineages of four cells are affected. Figure 11 shows diagrammatically how the ancestry of cells in specific locations in the ventral uterus differs depending on whether the 5R or the 5L pathway is followed.
FIG. 11. Comparison of development of the ventral uterus from 5R and 5L primordia. Dorsal view. Cell 1, Z4.aaa; cell 2, Z4.aap; cell 3, Z1.ppa; cell 4, Z1.ppp. Descendants of these cells are also marked by the same number. (a) Position of cells 1, 2, 3, and 4 in 5R and 5L primordia. 2 divides left-right from 5R (arrow), but 3 divides left-right from 5L (arrow). (b) In both cases, symmetry is restored. (c) One round of divisions later. The left row of cells has shifted posteriorly somewhat with respect to the right row of cells. Cells divide left-right and restore bilateral symmetry. The cells not included in triangles divide dorso-ventrally in the next round of divisions, and are therefore left out of the next figure. (d) Similar structures arise from 5R and 5L primordia, but the ancestry of cells that occupy equivalent positions in the structure is different.
A uterine lumen begins to form during the last divisions of the uterine lineage. At this stage, the uterine cells are cuboidal with round nuclei and clear cytoplasm (figure 7). During the latter half of L4, the uterine cells become very thin. During this differentiation of the uterine epithelium, the uterine muscle cells, arising from a different lineage (Sulston and Horvitz, 1977), become attached to the uterus. The uterus begins to undergo massive distortion due to contractions, so the individual cells have not been traced through this period.
(d) Spermathecal-uterine junction lineage. Six cells contribute to the formation of the anterior, and six to the posterior, spermathecal-uterine junction. Their identities are indicated in figure 3 and figure 10. The adult junction consists of a junctional core that is surrounded by the nuclei of four cells (figure 12).
FIG. 12. Spermathecal-uterine junction; Nomarski optics. This focal plane shows the junctional core and the nucleus (arrowhead) of one of the two cells that appear to form the core. ut, uterus; sp, spermatheca.
Two cells combine to make the junctional core. Because these two cells arise in what otherwise would be a spermathecal lineage, their identities are shown in Fig. 9. During L4, these two cells enlarge to form a plug between the lumens of the uterus and the spermatheca. During the L4 to adult molt, they differentiate into the core of the junction. The nuclei protrude into the uterine lumen as the main bulk of their cytoplasm elongates and takes on the shape of the junction's core (figure 12). These two nuclei are present until the first fertilized egg squeezes through the junction into the uterus. Their subsequent fates have not been determined.
The four other cells that contribute to the junction arise from what otherwise would be uterine lineages. These cells separate themselves from the developing uterus with which they had been associated and surround the junctional core to complete formation of the junction.
(e) Anchor cell. The anchor cell occupies a midventral position in the gonad during L3. The vulval precursor cells are located in the hypodermis underlying the gonad and are centered around the anchor cell. (See Sulston and Horvitz, 1977, for a more complete account of vulva formation.) As invagination of the developing vulva begins, the anchor cell assumes a position at its apex. During invagination, the anchor cell appears round and exhibits a prominent nucleolus and a granular cytoplasm. Once the invagination is complete, small vacuoles appear in the anchor cell cytoplasm ventral to the nucleus. The anchor cell then elongates, and its nucleus moves off center, leaving a cytoplasmic membrane spanning the vulval orifice. The anchor cell seems to be missing in the adult, but its fate is not known.
The Male Z1-Z4 Lineage (See figure 13)
Early Mitotic Period
In males, the early period of divisions generates 10 cells by early L2. As in hermaphrodites, Z1 and Z4 begin to divide about halfway through L1, and Z1.a and Z4.p occupy tip positions in the primordium, while Z1.p and Z4.a lie ventrally to the germ cells. Shortly after this first division, a fundamental difference in symmetry emerges between hermaphrodite and male developing gonads. The first indication of this difference is the anterior migration of Z4.a which disrupts the twofold rotational symmetry of the male primordium (figure 14a). As Z4.a pushes forward, Z1.p displaces Z1.a at the anterior end of the primordium. The position of Z1.a changes from anterior to Z1.p, to dorsal to Z1.p, and then to a more posterior dorsal position (figure 14b). Cells Z1.a and Z4.p do not divide again in the male. As the male gonad elongates, Z1.a moves posteriorly along the dorsal margin of the primordium, until it lies just anterior to Z4.p at the posterior or distal tip of the gonad. These two cells become the male distal tip cells. In one animal, Z1.a did divide in the hermaphrodite fashion, but both daughter cells subsequently moved posteriorly to the distal tip to join Z4.p. In this case, Z1.aa and Z1.ap both acquired distal tip cell character; neither divided again, and both exhibited the small, oval nucleus which is characteristic of distal tip cells in males.
FIG. 13. One of the two alternate male lineages of gonadal somatic precursor cells Z1 and Z4. In the other lineage, Z4.aaa and Z1.paa exchange lineages so that Z4.aaa becomes the linker cell, and Z1.paa gives rise to 10 vas deferens cells and one seminal vesicle cell. Divisions give rise to an anterior daughter and a posterior daughter unless otherwise indicated. The fates of descendants are shown either individually or in groups: dtc, distal tip cell; lc, linker cell; s.v., seminal vesicle.
FIG. 14. Spatial arrangement of male Z1 and Z4 descendant nuclei during the early mitotic period. All diagrams are lateral views with anterior to the left and dorsal above. The space occupied by Z2 and Z3 descendants is depicted by stippling, (a) position of nuclei just after the first division of Z1 and Z4, (b) position of same nuclei after migration has started, (c) and (d) positions of nuclei after subsequent rounds of divisions. Note that Z1.a moves along the dorsal margin of the gonad toward the posterior pole.
Z1.p and Z4.a undergo two rounds of divisions to generate a cluster of eight somatic cells at the anterior tip of the developing gonad. The first round is asymmetrical in that the anterior daughters are larger than the posterior daughters. These daughters all divide symmetrically in the second round. The eight resulting daughters form the male somatic primordium (Fig. 15).
FIG. 15. Male somatic primordium; Nomarski optics. The linker cell (arrow) occupies the anterior tip of the primordium. Posterior to the linker cell is a large vas deferens precursor cell (3) and posterior to that are two smaller seminal vesicle precursor cells (1 and 2). This anterior to posterior arrangement is invariant. However, since the positions of the three vas deferens precursor cells, and of the four seminal vesicle precursor cells, are variable around the long axis of the developing gonad, the numbered cells cannot be lineally identified unless their lineage had been followed from the L1 stage.
The male somatic primordium exhibits a variability in the positions of two cells. Either Z1.paa becomes the linker cell, and Z4.aaa becomes a vas deferens precursor cell, or vice versa (Fig. 16). The linker cell occupies the anterior tip position in the developing male gonad. The linker cell appears to serve a leader function during male gonadogenesis, preceding the developing gonad throughout its elongation. Three vas deferens precursor cells lie just posterior to the linker cell. The positions of these cells around the anterior-posterior axis are not fixed for any of the cells, and are interchangeable among the cells as they enlarge during L2. Four seminal vesicle precursor cells lie just posterior to the anterior block of the three cells described above. The positions of these four cells are also not fixed around the anterior-posterior axis as they enlarge during L2. The germ line component of the developing male gonad lies posterior to the somatic primordium.
FIG. 16. Alternate male somatic primordia. In (a) Z1.paa has become the linker cell (1c), whereas in (b) Z4.aaa has become the linker cell. Thus, Z1.paa and Z4.aaa have alternate fates.
During the second larval stage, the cells of the somatic primordium grow significantly in size, but they do not divide. Growth of the developing gonad proceeds anteriorly by continuous divisions of the germ line cells. No posterior elongation is apparent. The developing gonad elongates from 25 to about 65-70 micrometer long during this period.
Late Mitotic Period
Seven somatic cells generate a total of 53 cells during the late mitotic period of the male. Divisions begin at the end of L2 continue through L3, and finish shortly after the L3-L4 molt. The cells gradually assume their adult appearance during L4. The somatic structures of the male include the seminal vesicle which holds mature sperm and the vas deferens which provides a passage for the sperm to the exterior via the cloaca (Fig. 17).
FIG. 17. Male somatic structures in late L4 stage; Nomarski optics. Anterior is to the right, and dorsal is above.
(a) Seminal vesicle lineage. Four cells in the male somatic primordium each contribute five cells to the seminal vesicle. The divisions of these precursor cells follow a simple pattern of four serial asymmetrical divisions that produce one larger and one smaller daughter in each round. The smaller cells do not divide further. The resultant 20 cells constitute the inner, apparently secretory layer of the seminal vesicle. Their cytoplasm becomes granular during L4 and small blebs appear at the lumenal surface as they mature. The outer layer of the seminal vesicle comprises three large, very thin cells. These cells are the most distal daughters of the three cells that give rise to the vas deferens (Fig. 13). They arise just proximal to the developing seminal vesicle. After these three cells arise, they begin to enlarge, flatten, and spread over the developing seminal vesicle to encapsulate it.
(b) Vas deferens lineage. Three cells in the male somatic primordium each contribute 10 descendants to the vas deferens and one descendant to the seminal vesicle. These three cells undergo a series of asymmetrical divisions, each of which generates one larger and one smaller daughter. In this lineage, the smaller cells each undergo one mitosis before differentiating. The vas deferens is a complex secretory tube consisting of a variety of cell types. Preliminary studies show at least three cell types based on morphological differences in secretory granules (Wolf and Kimble, unpublished observations). However, a detailed elucidation of the cellular anatomy is not complete, and study of the correlation between adult cells, cell types, and lineal descendants within the vas deferens is still in progress.
(c) Linker cell. During L4, the linker cell joins the vas deferens to two E cell descendants (Sulston and Horvitz, 1977) in the male tail. Just prior to the final molt, the linker cell undergoes the morphological changes typical of cell death. This cell death opens the passageway between the lumens of the vas deferens and the cloaca.
Adapted by Yusuf KARABEY for WORMATLAS, 2003