Fig 1.
Diagram of the C. elegans gonad.
(A-C) Images shown in this report are longitudinal optical sections (A), cross sections (B), or tangential sections (C). All images are oriented with distal left and proximal right. The gonad contains germ cells and somatic sheath cells (green), and is surrounded by a basal lamina (see panel D). Sheath cells cover most of the gonad, except for a region near the distal end. Germ cells are born in the mitotic zone and enter meiosis in the transition zone, where chromosomes pair. Germ cells move in the proximal direction as they progress through various stages of meiosis, and slowly and intermittently contribute cytoplasm to the core (grey arrows). Cells nearing the gonad loop undergo apoptosis (black cells), or rapidly intercalate and enlarge as they take up core cytoplasm (purple arrows). The germ nucleus outlined in bold marks the point where intercalation begins to create a single file of germ cells. (D) Schematic view of two compartments (“cells”) in the germline syncytium; note that a single plasma membrane surrounds both compartments. A germ cell has an apical pole that faces the gonad core, and an opposite or basal pole that contacts either a sheath cell as shown, or contacts the basal lamina where sheath cells are absent (see transition zone in panel A). The extracellular matrix protein HIM-4/hemicentin (red) is highly enriched along the apical faces of germ cells [115]. Note that microtubules (MTs) are oriented radially with respect to the long axis of the gonad. (E) Summary diagram of cytological changes during apoptosis, as described in this report. The full sequence occurs in engulfment-defective mutants such as ced-1, but sheath cell-mediated degradation in wild-type animals interrupts the late stages of the sequence at variable points. The apoptotic cell first loses mitochondria, and then shrinks while the ring channel remains open. PGL-1 (magenta) is lost rapidly from P granules, but P granules remain visible by TEM. After the ring channel closes, microtubules are remodeled into a cage-like array, and cofilin-actin rods begin to form. These changes are followed by further cell shrinkage, involving both cytoplasmic and nuclear compaction.
Fig 2.
(A) Video sequence of a ced-1(e1735) gonad expressing PGL-1::GFP (green) and a reporter for germ cell membranes (red); strains for the various transgenic reporters used here and elsewhere are listed at the ends of the respective figures legends. The images compare a non-apoptotic cell (labeled 1) with a cell (X) that underwent apoptosis during the recording. At 14 mins and at earlier timepoints the X cell is slightly larger than the non-apoptotic cell, because all germ cells are increasing in size as they move proximally (to the right). Immediately after the 14 min timepoint, PGL-1 diminishes in the X cell and the cell decreases in size. Note that two cells adjacent to, and above, the X cell also undergo apoptosis in the recording. (B) Video sequence of a wild-type gonad expressing a reporter for PGL-1 (red puncta) and a reporter for membranes (also red). Two cells (X1 and X2) underwent apoptosis during the recording, and are compared with two adjacent, non-apoptotic cells (1 and 2). The perimeters of the apoptotic cells and non-apoptotic cells are indicated by solid and dashed outlines, respectively. Both apoptotic cells begin to lose PGL-1 and decrease in size immediately after 28 mins. Note that both apoptotic cells are initially similar in size to the adjacent, non-apoptotic cells (see also S1 Fig). (C) The top row is a video sequence comparing a shrinking apoptotic cell (X) with two non-apoptotic cells (1 and 2). The bottom row shows the same cells combined with a sheath cell reporter (green). The optical plane is 1.5 microns below the outermost germ cell surface, and shows transient protrusions from the sheath cell that extend for short distances between some non-apoptotic germ cells. (D) Orthogonal view of the same gonad shown in panel C, taken through a plane that bisects the ring channel of the apoptotic cell (X). Shrinkage appears to be completed by 22 mins, but the ring channel appears to remain open until at least 26 mins. (E) Video sequence showing an enlarged oogonium (X) undergoing apoptotic shrinkage. The complete video sequence, including sheath cell engulfment, is shown in S2 Video. (F) Transformed fog-1(q253) mutant male gonad with oogonia, including three, shrunken apoptotic cells (X). The panel at right (F’) shows a 3 micron optical projection of the DNA channel: Engulfed apoptotic cells quickly show significant chromatin compaction, but there is relatively minor compaction in the absence of engulfment, as shown here. We use 3 micron projections where listed throughout this report to accentuate chromatin compaction. Note the high level of F-actin staining (red, phalloidin) in the shrunken apoptotic cells compared to the large, non-apoptotic cells (dashed outline). (G) F-actin staining of non-apoptotic cells. The high magnification in panel G’ indicates fine, cytoplasmic actin filaments extending between the nucleus and plasma membrane (short arrow). The intensity of staining in the cytoplasm is much less than at the cell periphery (long arrow), which is saturated in this image. (H) Comparison of F-actin staining at the perimeter of a non-apoptotic cell (solid arrow), and two apoptotic cells (open arrows) in a ced-1(e1735) gonad. Note that the level of F-actin is higher in the older apoptotic cell (right) than in the younger apoptotic cell (left). Bars = 5 microns. Fluorescent reporters: (A) JJ2100 + OD70, (B) JJ2212 + OD70, (C, D) OD70 + MD701.
Fig 3.
Mitochondrial exit from apoptotic cells.
(A) Video sequence showing shrinkage of an apoptotic cell (X) and ring channel (rc) closure; the t = 0 timepoint here and elsewhere is the last timepoint collected before visible shrinkage. The lower row shows an orthogonal view of the same germ cells, imaged at the focal plane of the ring channels. The apoptotic cell is fully shrunken by 6 mins, but the ring channel appears to remain open until 12 mins. (B) Video sequence showing a large aggregate of cytoplasmic PGL-1 (arrow) exiting a shrinking apoptotic cell (X). The small arrowheads in the first frame mark perinuclear PGL-1 on P granules. (C) Mitochondrial ATP synthase beta (green) and F-actin (red) in a wild-type gonad. The image shows two shrunken apoptotic cells (X1 and X2) and a non-apoptotic cell (left) for comparison. X2 is younger than X1; it has less chromatin compaction, and retains an open ring channel. However, both X1 and X2 lack mitochondria. (D) Mitochondrial ATP synthase beta (green) and F-actin (red) in a ced-1 mutant gonad. Two apoptotic cells are visible (X1 and X2); X2 lacks mitochondria similar to most apoptotic cells, but X1`contains a small clump of mitochondria (arrow). (E) Images from a live gonad expressing transgenic COX-4::GFP (green). Each panel shows a different apoptotic cell taken from the video in S3 Video. Note that the exiting mitochondria (arrows) are elongated radially with respect to the long axis of the gonad. (F) TEM micrograph of two germ cells in a wild-type gonad. Both cells appear to have similar cytoplasmic volumes and open ring channels, and neither cell is engulfed. However, the cell at right lacks mitochondria. (G) TEM micrograph of a germ cell in a wild-type gonad. This cell appears full size, but lacks mitochondria except for the two mitochondria near the ring channel. Panel G’ is a high magnification of the boxed region, and shows a microtubule (white arrowheads) next to the two mitochondria. Bars = 5 microns (A-E), 2.5 microns (F,G). Fluorescence reporters: (A) OD70 + BT24 (GFP::HIM-4, false colored red), (B) OD70 + JJ2208, (E) JJ2586.
Fig 4.
MT cytoskeleton during germ cell apoptosis.
(A,B) Apoptotic cells (X) showing the MT cytoskeleton during mitochondrial exit. Both cells appear shrunken relative to neighboring cells, but both cells have open ring channels and a few mitochondria (magenta, ATP synthase beta). (A-A”) The arrowhead in panel A’ indicates a mitochondrion that appears to be exiting the apoptotic cell. Panel A” shows a high magnification of MTs (arrow) in the same focal plane. The position of this apoptotic cell is indicated in the gonad diagram in Fig 1A by the nucleus with a bold outline. Cells at this position have fewer neighbors than more distal germ cells, which simplifies the identification and tracing of single MTs associated with the cell. From 124 gonads examined, a total of three cells at this position were identified as apoptotic, and all had MTs emerging from the ring channel as shown. (B-B”) Panel B shows a focal plane through the nucleus of the apoptotic cell, and panels B’ and B” show the cortex of the same cell. (C) Wild-type gonad containing apoptotic cells (X), as indicated by their engulfment by a sheath cell reporter (green, CED-1::GFP). In the tangential optical plane shown, MTs in normal cells appear in cross section as small dots (see also Fig 1C). Note that many MTs in the apoptotic cells are parallel with the optical plane (arrows and arrowheads), and thus orthogonal to MTs in normal cells. Some of the MTs in the apoptotic cells appear closely associated with the nuclear envelope (arrowhead), while others are in the cortex (arrows). (D) Video sequence of EBP-1::GFP comets in a ced-1(e1735) gonad. The arrow tracks a comet in a non-apoptotic cell that is adjacent to an apoptotic cell (X). Note that the apoptotic cell has no visible comets. (E) Longitudinal optical section through a nocodazole-treated ced-1(e1735) mutant gonad; panel E’ shows a 5 micron projection of the same region. The dashed line indicates the boundary between the germ cells and the gonad core (compare with Fig 1A). A few, radially aligned MTs (arrow in panel E’) persist in germ cells after nocodazole treatment, but most are depolymerized. By contrast, there are numerous stable, nocodazole-resistant MTs that line the gonad core, outside of the germ cells (arrowhead). (F-H) Tangential planes of a nocodazole-treated ced-1 gonad (panel F) and wild-type gonads (panels G and H). All MT panels show 3 micron projections, revealing most of the MTs in the cell. All of the ced-1 apoptotic cells show a cage or cocoon-like array of stable MTs, except for a cell that appears necrotic (arrow in panel F). Engulfed wild-type apoptotic cells vary in their number of MTs (panel H): The apoptotic cell at top has compacted chromatin, few MTs, and lacks at least one nuclear epitope (magenta, NPP-9), suggesting that it has begun to degrade. Bars = 2.5 microns (A-C, F-H), 5 microns (D, E). Fluorescent reporter: (D) OD1359.
Fig 5.
Kinesin is required for mitochondrial exit.
(A) ced-1(e1735); unc-116(RNAi) adult gonad with a large cluster of apoptotic cells (X). The F-actin and PGL-1 signals are imaged in the same channel (red) to identify apoptotic cells by their small size and lack of PGL-1 on P granules (lower left panel). The top right panel shows mitochondria (green, ATP synthase beta) visible in the same, single optical, plane. Panel A’ shows the outlines of the apoptotic cells drawn on a 3 micron projection of the mitochondrial signal; the projection is comparable to the diameter of an apoptotic cell, and reveals most of the mitochondria within a cell. Note that the projection shows a large cluster of mitochondria in all but one of the apoptotic cells (outlined). (B) Germ cells in a miro-1(tm1966) null mutant, stained as for panel A. The arrow indicates the plasma membrane of an apoptotic cell (X), which lacks mitochondria. An adjacent, non-apoptotic cell is outlined for comparison. The DNA panel is a projection showing there is little or no chromatin compaction in the apoptotic cell. Thus, the apoptotic cell is at an early stage, and is unlikely to have contained, but degraded, any mitochondria. (C) Video sequence of an apoptotic cell in a miro-1(tm1966) null mutant expressing reporters for membranes (red) and for mitochondria (green, COX-4::GFP). Note that the exiting mitochondria (arrow) are aligned radially with respect to the gonad axis, similar to exiting mitochondria in wild-type apoptotic cells. (D) Germ cells in a ced-1(e1735); klc-1(RNAi) adult. The apoptotic cells (X) are outlined as for panel A. Note that all of the apoptotic cells contain clusters of mitochondria. (E) Video sequence of apoptosis in a klc-1(ok2609) null mutant; the complete video is provided as S4 Video. Panel E’ shows the apoptotic cell at high magnification; the ring channel is open at the first time point shown, but closes by 18 mins. Bars = 5 microns (A,D), 2.5 microns (B-C, E). Fluorescent reporter: (C, E) JJ2586 + OD70.
Fig 6.
Cofilin-actin rods in apoptotic germ cells.
(A) ced-1(e1735) mutant gonad stained for F-actin (green, phalloidin) and immunostained for mitochondria (red, ATP synthase beta). The image shows two apoptotic cells (X1 and X2) that are fully shrunken, closed, and lack mitochondria. X1 has little or no chromatin compaction, and thus appears younger than X2. However, the level of F-actin staining at the periphery of X2 is higher than X1, suggesting that the level of actin continues to increase after cell shrinkage and closure. Panel A’ shows an optical plane taken at the surfaces of the cells, and panel A” shows higher magnifications of these surfaces. Note that the actin in the older apoptotic cell, X2, appears concentrated in multiple, rod-like structures. (B) ced-1 mutant gonads immunostained for actin (green, anti-actin) and cofilin (red, anti-UNC-60A); examples of cofilin-actin rods are indicated by arrowheads. Panel B’ is a higher magnification, surface view of a single apoptotic cell. (C) Wild-type gonad immunostained for cofilin (red, anti-UNC-60/cofilin) and sheath-specific GFP (green, CED-1::GFP). Four apoptotic cells are visible, as indicated by their engulfment by a sheath cell. The apoptotic cell labeled X1 has a single, prominent cofilin-actin rod (arrowhead). The apoptotic cells at left lack obvious rods, but appear to have higher levels of cytoplasmic cofilin than neighboring, non-apoptotic cells. (D) Video sequence of a live, ced-1(e1735) gonad expressing a transgenic reporter for actin (ACT-1::GFP). Two apoptotic cells (X1 and X2) are visible; X2 has completed shrinkage at the first timepoint, and X1 completes shrinkage at about 48 mins. Cofilin-actin rods (arrowheads) begin to form in X2 at about 48 mins, and in X1 at about 80 mins; X1 shifted below the focal plane at t = 96 mins. (E) Azide-treated, wild-type gonad, immunostained for cofilin (red, anti-UNC-60A). The apoptotic cell (X) contains rods, as in untreated gonads, but azide has induced rod formation in the gonad core in the mitotic region. The lower panels show confocal images of rods induced in mitotic region (panel F) and the pachytene region (panel G). Note that the induced rods form in the gonad core, and not within germ cells. Rod induction in the mitotic region was observed in the following gonads after exposures to 20 mM and 50 mM azide: 2 hrs azide (0/12, 2/32 gonads), 3 hrs (3/16, 30/36), 4 hrs (2/9, 1/35), 5 hrs (4/9, 7/31), 6 hrs (1/15, 1/30), 7 hrs (4/9, 2/25), 8 hrs (0/24, 0/24). Rod induction was observed in the pachytene region after a 3 hr treatment with azide (1/16, 2/36). No cofilin-actin rods were induced in gonads treated with 10 mM azide for 30 mins, 1 hr, and 2 hrs (n = 12–22 gonads, each). About 30% of animals treated for 8 hrs with 50 mM azide recovered some locomotion after removal from azide. Bars = 2.5 microns (A-D), 5 microns (E). Fluorescent reporters: (C) MD701, (D) JJ1477.
Fig 7.
Microfilament bundles in ced-1 apoptotic cells.
(A-C) TEM micrographs of ced-1(e1735) apoptotic cells showing microfilament bundles. The plasma membrane (yellow arrowhead) and the nuclear envelope (white arrowhead) of the apoptotic cell are indicated in all panels. (A) Example of a single bundle; a higher magnification of the boxed region is shown in panel A’, and indicates a single microfilament (open arrows) and two MTs (open arrowheads). (B and B’) Apoptotic cell with multiple microfilament bundles (green arrows), visible as cross sections; note that the surface of the cell is relatively smooth. (C and C’) Older apoptotic cell, as indicated by the convoluted nuclear membrane and increased cytoplasmic density. Note that the surface has bumps or ridges overlying each of the microfilament bundles. Bar = 200 nm (A), 50 nm (A’), 1 micron (B,C); 0.5 micron (B’, C’).
Fig 8.
Binucleate apoptotic germ cells.
(A) ced-1(e1735) mutant gonad showing rod-containing cells throughout the mid-pachytene region, and one rod-containing cell near the border between the transition zone and the early pachytene region (arrow). (B) High magnification of the distal region of a ced-1 mutant gonad. The double arrow points to a binucleate, rod-containing cell in the transition zone. Note that this cell is only about 12 cell diameters away from a mitotic cell at metaphase (single arrow). Similar distal, rod-containing cells were found in the transition zones in 5/178 gonads. (C) Two examples of rod-containing cells (double arrows) in the mid-pachytene region of the gonad. Note that both cells have two nuclei, as indicated by the chromatin patterns and nuclear envelopes (magenta, anti-NPP-9). (D) Mid-pachytene region of a ced-1 gonad showing multiple binucleate cells (double arrows). Cofilin-actin rods are visible in all of the binucleate cells except for the distalmost cell (asterisk), which has a higher level of cytoplasmic cofilin than in neighboring, non-apoptotic cells (inset in Panel D’). Note that the two nuclei in many of the binucleate cells are aligned in the same, surface plane as other gonad nuclei. As described below, this alignment contrasts with the radial alignment of the nuclei when binucleate cells first form. (E-E”‘) Each combination of panels depicts apoptotic-specific features of the binucleate, rod-containing cells (double arrows); the cofilin-actin rods are indicated by either UNC-60/cofilin (red in E and E”‘) or by actin (green in E’ and E”). The rod-containing cells are small with high levels of peripheral F-actin (panel E), they lack PGL-1 on P granules (panel E’), they have closed ring channels (panel E”), and they lack mitochondria (panel E”‘). (F) Graph showing the percentage of binucleate, rod-containing apoptotic cells compared to the total number of apoptotic cells in ced-1 gonads. The data is from single gonad arms in 21 ced-1 adults at 24 hrs. (G) Diagram showing the range of positions of rod-containing, binucleate, apoptotic cells in ced-1 mutants at 48 hrs. Each horizontal bar represents one of 73 gonad arms analyzed; the left endpoint indicates the position of the distalmost, binucleate apoptotic cell, measured from the distal tip of the gonad. The right endpoint is the position of the proximal-most apoptotic cell that could be scored as binucleate; other apoptotic cells might also have been binucleate, but their chromatin was too compacted to score. In the set of gonads analyzed, the proximal boundaries of the transition zones (TZ) ranged from 80–115 microns, and the gonad loops were at 336 +/- 23 microns. (H) Engulfment of a binucleate, rod-containing apoptotic cell in the mid-pachytene region of a wild-type gonad. The gonad is from an MD701strain expressing the sheath reporter CED-1::GFP, and is here immunostained for GFP (green). (I-K) Binucleate, apoptotic cells in mutants that have physiological apoptosis, but lack other forms of apoptosis. Binucleate, apoptotic cells were present in the following 48 hr adults: cep-1(gk138) (7/21 gonads), cep-1(gk138) ced-1(RNAi) (26/26 gonads, panel I), ced-1(e1735); egl-1(n1084n3082) (32/32 gonads, panel J), egl-1(n1084n3082) (7/28 gonads), and pch-2(tm1458) (8/26 gonads, panel K). Bars = 20 microns (A, D, I), 10 microns (B), 2.5 microns (C, E, H), 5 microns (J-K).
Fig 9.
Binucleate apoptotic cells and MAP kinase.
(A) ced-1(e1735) gonad at 48 hrs immunostained for activated MAP kinase (red, anti-dpMPK-1) and F-actin (green, phalloidin). The boxed region contains a binucleate apoptotic cell (arrow), as shown at higher magnification in panel A’ (the nuclear envelope is shown in magenta, anti-NPP-9). Note that this cell is located before the peak of activated MAP kinase, although most apoptotic cells (bracketed region in panel A) occur after the peak. Additional examples of binucleate apoptotic cells and dpMPK-1 are presented in S7 Fig. (B and C) Cell deaths in a nos-3(oz231); mpk-1(ga117) null gonad (panel B), and in a cep-1(RNAi); nos-3(oz231); mpk-1(ga117) gonad (panel C). The presumptive apoptotic cells are small, have closed ring channels, and have compacted chromatin. Panel C’ shows an orthogonal view of the rod indicated in panel C (arrow). Bars = 20 microns (A), 5 microns (A’, B-C).
Fig 10.
Non-apoptotic, binucleate germ cells in ced-3 mutant gonads.
(A-A”) Gonad from ced-3(n3692) adult at 72 hrs stained for F-actin (green, phalloidin) and for the nuclear envelope (magenta, anti-NPP-9). The proximal half of the gonad contain numerous, abnormally small oocytes, but does not contain any binucleate oocytes. The two boxed regions from the distal half of the gonad are shown in panels A’ and A” at higher magnification. The numbers indicate binucleate (2) or trinucleate (3) germ cells, as inferred from the nuclear channel. (B and C) Gonads from mated ced-3(n3692) adults at 72 hrs, showing a binucleate oogonium (panel B) and a trinucleate oogonium (panel C). Both oogonia are enlarged, but are not full-size oocytes; note open ring channels (rc). (D) Gonad from a mated ced-3(n3692) adult at 72 hrs. Oogonia in the proximal half of the gonad have better organization than in the gonad in panel A, but they do not form the normal, single file. A full-size, binucleate oocyte is visible in the boxed region, and shown at higher magnification in the inset. Panel D’ is a 3D rendered view of an optical stack through the same binucleate oocyte. The second panel is rotated 45 degrees to show all six bivalents in each nucleus. (E and F) Adult progeny from a cross between a wild-type male and a ced-3(n3692) hermaphrodite. Each image shows three adjacent oocytes (separated by dashed white lines) in a gonad arm; each oocyte contains a single nucleus (outlined by a dashed magenta circle). The adult in panel E is diploid, as each oocyte contains six bivalent chromosomes; the panel at right shows a higher magnification of two bivalents. The adult in panel F is a triploid, as each oocyte contains six bivalents plus six univalents; the panel at right shows a higher magnification of one bivalent and one univalent. The low magnification images are 5 micron projections, such that all chromosomes are visible. (G) 2-cell embryo in the uterus of a triploid adult as in panel F. Each nucleus in the 2-cell embryo contains 18 chromosomes, as expected for the self-progeny of a triploid hermaphrodite. The image is a projection of an optical stack through the nuclei. Bars = 10 microns (A, B), 5 microns (C-F).
Fig 11.
(A-A”) Distal end of wild-type gonad stained for F-actin (red), HIM-4 (yellow), and DNA (blue). The chromatin in an interphase mitotic nucleus forms a symmetrical sphere around the large, central nucleolus. Chromatin in a transition state (TZ) nucleus has an asymmetric, crescent shape, caused by chromosomes pairing. Panel A shows a binucleate germ cell (double arrow) next to the transition zone; note that both nuclei in the binucleate cell resemble transition state nuclei. Panel A’ shows an optical section through the middle of the same gonad; note the enrichment of HIM-4 at the periphery of the gonad core (see also diagram in Fig 1D). Panel A” is a 3D rendered view of an optical stack through the gonad core; the ring channels appear as small ovals on the surface of the core. Note that the ring channel of the binucleate cell (dashed vertical line) is larger than in surrounding germ cells. Although most germ cells contact the periphery of the gonad, several appear folded into the gonad core (open arrows in panel A’). The folding pattern can be inferred from the 3D rendered view of the core, where grooves or depressions in the core correspond to folds. Note that the gonad core expands significantly as the folds disappear. (B-D) Each row of panels shows distal binucleate cells lacking features of apoptotic cells as follows: They have normal levels of peripheral F-actin (panel B), they contain mitochondria (panel C), and they have open ring channels (panel D). Note in panel D that both binucleate cells (double arrows) are at the bases of small folds (open arrows). (E) Map showing the positions of binucleate, non-apoptotic cells (dots) in the distal gonad; each horizontal bar represents one gonad. Importantly, binucleate cells could be distinguished easily from mitotic germ cells in anaphase or telophase (S8 Fig). (F-I) Each set of panels shows EdU (green) that was incorporated into replicated DNA (red). Panel F shows a ced-1(e1735) gonad fixed and stained immediately after 45 mins of labeling. Germ nuclei show H:L or L:H patterns as indicated and described in the text; paired X chromosomes are indicated by an arrow. Panels G-I show binucleate apoptotic cells (indicated by cofilin-actin rods) 20 hours after a pulse of EdU. Note that the two nuclei in a binucleate cell have different labeling patterns, as indicated. (J) Binucleate germ cells in an aff-1(tm2214) mutant gonad. Bars = 5 microns (A-E), 2.5 microns (F-I).
Fig 12.
Interpretative diagram of folding, eversion, and fusion in the germline syncytium.
(A) Diagram representing possible membrane topology during a fusion event. Germ “cells” are nuclear compartments divided by saddle-like bends in a single plasma membrane (bold line). A reduction of the saddle opens the compartments between cells 1 and 2. The lower diagram shows a perspective view of the same fusion. (B) Cell division (bars between cells) and growth causes the germline syncytium to fold or buckle inward. Many or most mitotic spindles are not aligned radially, so the folds do not appear to be created by directional divisions. Binucleate cells are found near the base, but not tip, of a fold. The fold can be considered to have a tip and a base, and cell fusions occur at the base. The folds develop in the gonad “bare zone” where sheath cells do not cover the gonad (see Fig 1A), and eversion occurs in a region with sheath cell contacts.
Fig 13.
Folds/remnants and binucleate germ cells.
(A) Wild-type gonad at 48 hrs. Immunostaining for HIM-4 (yellow) outlines the core, and shows membranous, anuclear material (open arrow) extending between what appear to be two sides of an incompletely resolved fold; we refer to the anuclear, membranous material as a remnant. Cells bordering the remnants often appear elongated or stretched, as shown in the panel. Panels A’ and A” show rendered views of an orthogonal projection through the gonad; panel A’ is a rope-like remnant, and panel A” is a remnant that resembles a perforated sheet. (B) Wild-type gonad showing the typical region of core expansion. A fold (vertical arrow), and two remnants (open arrows 1 and 2) persist into the early and mid-pachytene regions of the gonad, respectively. A higher magnification of the remnants is shown in panel B’ (left); note that binucleate cells appear at the bases of both remnants. The second panel in B’ (right) is a higher optical section of the same region, showing additional binucleate germ cells at the bases of the remnants. (C-E) Gonads from 72 hr wild-type adult, showing remnants (open arrows) in several regions of the gonad core. Panels D and E, and the corresponding high magnifications in panels D’ and E’, show apoptotic binucleate cells associated with the bases of the remnants. (F) Graph showing the positions of the proximal-most fold (open circles) in the indicated gonads, measured from the distal tip of the gonad; Student’s t test. For example, folds typically are cleared by 100 microns in a 72 hr wild-type adult, although remnants persist much further proximally. (G) Fold in a ced-3(n3692) gonad at 48 hrs. Similar to wild-type gonads, binucleate cells appear by the bases of folds. The diagram at right is a projection of every cell in this region of the gonad, overlaid on a cartoon of the middle focal plane. Note that binucleate cells appear concentrated by the fold. (H) Fold in a ced-3(n3692) gonad as in panel G, but containing trinucleate germ cells. (I and J) ced-3(n3692) gonads at 72 hrs, showing folds (vertical arrows), rather than remnants, that persist through the pachytene region.
Fig 14.
Nuclear stacking in binucleate germ cells.
(A) ced-3(n3692) gonad at 48 hrs. The complete optical stack through the region shown contains a total of 11 binucleate cells, one of which is indicated by a vertical arrow. The remaining 10 occur with the boxed region surrounding a remnant (open arrow). (A’) Orthogonal views of numbered planes as indicated in panel A; the remnant appears in the central plane (numbered 0). Each of the 10 binucleate cells is indicated by a double arrow; note that the nuclei in each binucleate cell are stacked radially. Bar = 5 microns.