arrested apoptosis of nurse cells during hydra oogenesis ... · to resume after the primary polyp...

Arrested apoptosis of nurse cells during Hydra oogenesisand embryogenesis

Ulrich Technau,a,* Michael A. Miller,b,1 Diane Bridge,b,2 and Robert E. Steeleb

a Molecular Cell Biology, Darmstadt University of Technology, Schnittspahnstrassc 10, 64287 Darmstadt, Germanyb Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine, CA 92697-1700, USA

Received for publication 22 May 2002, revised 18 April 2003, accepted 21 April 2003

Abstract

During Hydra oogenesis, an aggregate of germ cells differentiates into one oocyte and thousands of nurse cells. Nurse cells display anumber of features typical of apoptotic cells and are phagocytosed by the growing oocyte. Yet, these cells remain unchanged in morphologyand number until hatching of the polyp, which can occur up to 12 months later. Treatments with caspase inhibitors can block oocytedevelopment during an early phase of oogenesis, but not after nurse cell phagocytosis has taken place, indicating that initiation of nurse cellapoptosis is essential for oocyte development. The genomic DNA of the phagocytosed nurse cells in the oocyte and embryo showslarge-scale fragmentation into 8- to 15-kb pieces, but there is virtually none of the internucleosomal degradation typically seen in apoptoticcells. The arrested nurse cells exhibit high levels of peroxidase activity and are prevented from entering the lysosomal pathway. Afterhatching of the polyp, apoptosis is resumed and the nurse cells are degraded within 3 days. During this final stage, nurse cells becomeTUNEL-positive and enter secondary lysosomes in a strongly degraded state. Our results suggest that nurse cell apoptosis consists ofcaspase-dependent and caspase-independent phases. The independent phase can be arrested at an advanced stage for several months, onlyto resume after the primary polyp hatches.© 2003 Elsevier Science (USA). All rights reserved.

Keywords: Apoptosis; Hydra; Oogenesis; Embryogenesis; Nurse cell; Cnidaria; Peroxidase; Caspase; Lysosome

Introduction

Oocyte growth in animals frequently depends on nutri-tion by surrounding nurse cells. In many cases these nursecells are derived from the germline, as is the oocyte. InDrosophila, for example, a cyst forms around 16 germ cellsthat remain interconnected by cytoplasmic bridges, one ofwhich is determined to form the oocyte, while the remaining15 cells differentiate into nurse cells and provide nutritivesubstances to the oocyte (King, 1970). In the course ofoocyte development, the nurse cells undergo apoptosis and

are finally resorbed by the oocyte (Spradling, 1993; Cava-liere et al., 1998; McCall and Steller, 1998).

Comparative analyses have shown that apoptosis ofgermline-derived nurse cells is widespread in the animalkingdom and often a necessary requirement for the properdevelopment of the oocyte (for review see Matova andCooley, 2001). In Drosophila, the caspase-3-deficient mu-tant dcp-1 does not form fertile oocytes (McCall and Steller,1998). In C. elegans, half of the female germ cells undergoapoptosis and are engulfed by the surrounding sheath cells(Gumienny et al., 1999). The programmed cell death ofthese cells appears to be required for the maintenance ofgermline homeostasis because full-size oocytes do not formin the apoptosis-deficient mutants ced-3 and ced-4 (Matovaand Cooley, 2001; Gumienny et al., 1999). Yet, both inDrosophila and C. elegans, the pathway for germ cell deathseems to differ from apoptosis in other cell lineages, assome of the crucial regulators involved in somatic cell

* Corresponding author. Molekulare Zellibiologie, TU Darmstadt,Schnittspahnstr. 10, 64287 Darmstadt, Germany, Fax: �49-6151-166077.

E-mail address: [email protected] (U. Technau).1 Present address: Department of Cell and Developmental Biology,

Vanderbilt University School of Medicine, Nashville, TN 37232; USA.2 Present address: Department of Biology, Elizabethtown College,

Elizabethtown, PA 17022-2298, USA.

R

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Developmental Biology 260 (2003) 191–206 www.elsevier.com/locate/ydbio

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apoptosis (i.e., EGL-1 in C. elegans and H99 genes inDrosophila) do not play a role in apoptosis of the germ cells(Foley and Cooley, 1998; Gumienny et al., 1999). More-

over, apoptosis of nurse cells in Drosophila involves highlyregulated intracellular rearrangements of the actin cytoskel-eton, which are important for the transfer of cytoplasmic

Fig. 1. Acridine orange (AO) staining during oogenesis and embryogenesis. (A–D) Oogenesis stages. (A) Oogenesis stage 2 showing a faint backgroundstaining due to the high cell density in the egg field of aggregated germ cells. At this stage the oocyte (arrow) becomes determined and nurse cells startdifferentiating. (B) At stage 3, when the oocyte starts phagocytosing the nurse cells, a small number of dispersed AO-positive nurse cells that have beenphagocytosed by the oocyte appear in the egg field. (C) At stage 5, the oocyte has phagocytosed most of the surrounding nurse cells, which are all brightlystained by AO. The arrows indicate the oocyte pseudopods which are being retracted at this stage. (D) Mature oocyte (stage 7) with thousands of incorporatedAO-positive nurse cells. (E, F) Embryogenesis. (E) Eight-cell cleavage stage embryo (F) gastrula stage embryo (G) polyp immediately after hatching (8 weeksafter fertilization) with a nearly equal density of AO-positive nurse cells as in oocytes and embryos. Note the orange fluorescence of AO-positive nurse cellsin the tentacles (arrow), indicating that the nurse cells are being degraded; (H) 2-day-old polyp showing a significantly reduced number of nurse cells relativeto a newly hatched polyp. Note the gradient of nurse cell disappearance from the tentacles (arrow) towards the aboral end of the polyp. (I) Close-up of a 2-dayembryo showing red (arrowheads) and green fluorescent AO-positive nurse cells (arrows). Bars, 200 (A–H), 150 (G, H), and 200 �m (I).

192 U. Technau et al. / Developmental Biology 260 (2003) 191–206

components into the oocyte (McCall and Steller, 1998;Mahajan-Miklos and Cooley, 1994a, 1994b; de Cuevas andSpradling, 1998; Matova et al., 1999). Such cytoskeletalrearrangements have not been reported in association withsomatic cell apoptosis.

In Hydra, a representative of the early-diverging meta-zoan phylum Cnidaria, thousands of germline-restricted in-terstitial stem cells aggregate to form the oocyte (Kleinen-berg, 1872; Nishimiya-Fujisawa and Sugiyama, 1995;Littlefield, 1991). Although many of these cells are compe-tent to form an oocyte, under normal conditions only onelocated in the middle of the aggregation is selected (Milleret al., 2000). The cells which are not selected differentiateinto nurse cells and are phagocytosed by the growing oo-cyte. During and following their phagocytosis, the nursecells begin a process in which they acquire a number offeatures of apoptotic cells (Honegger, 1989; Miller et al.,2000): the nuclear membrane becomes irregular, but retainsits integrity; the chromatin becomes condensed and concen-trates on one side of the cell; the cell rounds up and formsstrong interdigitations at the contact sites with adjacentnurse cells. Subsequently the volume of the cell decreases,presumably due to loss of cytoplasmic fragments. In apreceding study, we have shown that initiation of the nursecell apoptotic program is regulated by the oocyte (Miller etal., 2000). Apoptosis has been reported from Hydra andother Hydrozoa and attributed to growth regulation andmorphogenesis (Bosch and David, 1984; Cikala et al., 1999;Seipp et al., 2001). Recently, apoptosis has also been shownto occur during spermatogenesis in Hydra (Kuznetsov et al.,2001). Here, we have examined the process of nurse cellapoptosis during oogenesis on the cellular and molecularlevel. We show that the early but not the late stages ofoogenesis are caspase-dependent. After this caspase-depen-dent phase, the nurse cells are phagocytosed by the oocyteand arrest at an advanced stage of apoptosis. Arrested nursecells are characterized by the presence of high levels ofperoxidase activity and genomic DNA that is cleaved intolarge fragments. The internucleosomal cleavage pattern typ-ical of apoptotic cells and entry into the lysosomal pathwaydoes not occur until embryogenesis is completed.

Materials and methods

Hydra culture, and induction and staging of oogenesis

Animals were maintained as described previously (Tech-nau and Holstein, 1996). All experiments were carried outusing the AEP female strain of Hydra vulgaris (Martin etal., 1997). Fertilization of eggs was done with males of thePA2 strain. Stock cultures of AEP were fed with Artemianauplii five times a week, which kept them in a nonsexualstate. To induce gametogenesis, animals were starved forone week and thereafter fed two to three times a week forseveral weeks (modified after Hobmayer et al., 2001). The

Fig. 2. Phagocytosing early oocyte and morphology of incorporatednurse cells. (A) Macerated early stage 3 oocyte with the germinalvesicle in the center (arrow). (B) The same oocyte stained with DAPI,showing the nuclei of the phagocytosed nurse cells. (C) Phagocytosednurse cell in a mature oocyte double stained with AO and DAPIshowing that AO stains both nuclear (DNA; stained blue) and cytoplas-mic (RNA; stained green) material. Note the peripheral location of theDNA. The dotted line indicates the periphery of the nurse cell. Bars 100(A,B) and 20 �m (C), respectively.

193U. Technau et al. / Developmental Biology 260 (2003) 191–206

first stages of oogenesis became visible in such culturesabout 10–14 days after the initiation of starvation. Stagingof oocyte development was done according to Miller et al.(2000).

Acridine orange and DAPI staining

Live animals were incubated in 10 �g/ml acridine orange(AO) in Hydra medium for 3 min in the dark, followed byfive washes in Hydra medium (Miller et al., 2000). Theanimals were then relaxed by incubation in 0.03% heptanoleor 0.5% urethane in Hydra medium for the time of exami-nation under the microscope. Stained animals were mountedon glass slides and photographed immediately under epi-fluorescent illumination. DAPI staining was carried out onunfixed material (in the case of double staining of nursecells with AO) or on dissected egg fields which were mac-erated on glass slides and then fixed with formaldehyde.Maceration was done by incubating in maceration solution(acetic acid/glycerol/water; 1:1:26; David, 1973) for 30 minand fixed on gelatine-coated glass slides with 4% formal-dehyde.

TUNEL assay

The TUNEL assay (Gavrieli et al., 1992) was adaptedfrom Hensey and Gautier (1998). Animals were relaxed in2% urethane for 1 min, fixed in MEMFA (100 mM MOPS,pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde)for 1 h and washed several times in methanol. The methanolwas replaced stepwise with PBS, and then the animals werewashed twice for 15 min in PBS/0.2% Tween and once inPBS. The animals were then equilibrated in terminal de-oxytransferase (TdT) buffer for 1 h. End-labeling was car-ried out overnight at room temperature in 100 �l TdT buffercontaining 0.5 �M digoxygenin-dUTP and 150 U/ml TdT.The reaction was stopped by addition of PBS/EDTA at65°C (2�1 h), followed by four washes in PBS. The polypswere then washed in PBS/0.1% Triton X-100/2% BSA for15 min and incubated in blocking serum (Roche) for 1 h.Then the specimens were incubated in a 1:2000 dilution ofanti-digoxigenin antibody coupled to alkaline phosphatase(Roche) overnight at 4°C. Over the course of the followingday, the samples were washed with a number of changes ofPBS/Triton/BSA. For detection, the animals were equili-brated 2 � 5 min in alkaline phosphatase buffer (100 mMTris, pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween20, 1 mM Levamisol) and then the substrates NBT andBCIP were added. The color reaction was stopped with 95%ethanol and the animals were mounted in glycerin/PBS(9:1). In macerates (David, 1973) the TUNEL assay wasperformed essentially as for whole animals. Incubation ofthe enzyme and the antibody was done in a humid chamber;the enzyme reaction was stopped in a water bath. Colorreaction was stopped with PBS instead of ethanol and coun-

terstained with 0.05 �g/ml DAPI and the macerates weremounted in glycerol/PBS (9/1).

Hydroxyurea treatment

Animals were treated with 10 mM hydroxyurea in Hydramedium for 8 h, and then washed several times and ana-lyzed after 12–18 h.

Antibody staining

The monoclonal antibody H22.58 was generated in thelab of Charles N. David (Munich) and detects secondarylysosomes (Technau, David, and Holstein, in preparation).Animals were relaxed for 1 min in 2% urethane and fixedwith Lavdovsky’s fixative (4% formaldehyde, 10% aceticacid, 50% ethanol) overnight. Animals were washed threetimes with PBS and incubated overnight in undiluted mAbH22.58. After three washes in PBS the animals were incu-bated for 2 h in 1:400 anti-mouse IgG and IgM Alexa 488coupled secondary antibody (Molecular Probes). After threewashes in PBS the specimen were mounted in glycerol/ PBS(9:1). Double staining in macerates with TUNEL was per-formed after the TUNEL assay.

Caspase inhibitor treatment

The Caspase inhibitors z-VAD-FMK and Ac-DEVD-CMK (Calbiochem) were dissolved in 100% DMSO anddiluted immediately prior to use. Treatments were donewith inhibitor concentrations of 1, 10, 100, 300, and 600�M in 0.3–0.75% DMSO. The solution was replaced every6 h over the course of 48 h because of the short half-life ofthese inhibitors (Garcia-Calvo et al., 1998). As a control forunspecific protease effects and unspecific effects by thechloro-methyl-ketone group (CMK) we treated animalswith 600 �M of N-tosyl-lysine-CMK (Calbiochem), an in-hibitor of trypsin-like serine proteases.

Peroxidase staining

The peroxidase assay was carried out as previously de-scribed (Hoffmeister and Schaller, 1985; Technau and Hol-stein, 1996).

Isolation of genomic DNA

Fifty polyps or 50 embryos were incubated in lysis buffer(0.2 M Tris, pH 8.0, 100 mM EDTA, 0.2% SDS, 100 �g/mlProteinase K) for 2 h at 60°C; 2 �l of diethylpyrocarbonatewas added and incubation was continued for another 30 minat 60°C. Next, 50 �l of 5 M potassium acetate was addedand incubation was carried out on ice for 30 min. Thesamples were centrifuged for 5 min at 14,000 rpm and thesupernatant transferred to a fresh tube and again centrifugedat 14,000 rpm for 15 min. The supernatant was transferred


into a fresh tube and precipitated by adding 0.75 ml of 100%ethanol and centrifuging for 5 min. The dried DNA pelletwas resuspended in 350 �l of TE buffer, DNase-freeRNaseA was added to 100 �g/ml, and the samples wereincubated at 37°C for 1 h. The RNase was removed by aphenol–chloroform extraction. The DNA was precipitatedwith ethanol and resuspended in 50 �l of TE buffer.

Whole-mount in situ hybridization

In situ hybridization was carried out as previously de-scribed (Technau and Bode, 1999) except that detection wasdone with NBT/BCIP instead of BM Purple.

Results

Nurse cells phagocytosed by the oocyte are not degraded

Ultrastructural studies have shown that nurse cells dis-play several morphological features of apoptotic cells(Zihler, 1973; Honegger et al., 1989). We therefore stainedlive polyps at different stages of oogenesis with AO. AObinds to nucleic acids (RNA and DNA) in nonfixed apopto-tic cells. As the intracellular and nuclear environment be-come more acidic and chromatin condensation and degra-dation occur, AO fluorescence shifts from green to a yellow/red color under blue light excitation (Mpoke and Wolfe,1997).

In Hydra, the first AO-positive nurse cells can be de-tected at stage 3 of oogenesis, when the oocyte starts phago-cytosing the nurse cells (Fig. 1; Miller et al., 2000). Duringlater stages the number of AO-positive nurse cells rapidlyincreases, and results in a brightly stained oocyte withthousands of densely packed green nurse cells inside theoocyte (Figs. 1A–D). We followed the fate of the nurse cellsduring embryogenesis and found that the great majority ofthem can be detected throughout embryogenesis until hatch-ing of the polyp. During this time, their morphology andAO-staining pattern appears virtually unchanged (Figs. 1E–1). Hence, nurse cells are not degraded after phagocytosisby the oocyte and can be maintained in this nondegradedstate. This is remarkable, because hatching can occur up to12 months after fertilization and probably later (R.E.S.,unpublished observations). To further substantiate this ob-servation we macerated a developing oocyte from a femaleat oogenesis stage 3. When stained with DAPI we foundnumerous nuclei of the incorporated nurse cells in the oo-cyte (Figs. 2A and B). The chromatin of these nurse cells iscondensed, but the nucleus does not break down intosmaller fragments (Fig. 2B). In mature oocytes (stage 7),nurse cell chromatin frequently forms a ring that is dis-placed to the periphery of the nucleus (Fig. 2C), which ischaracteristic of cells displaying large-scale fragmentation(see below; Hunot and Flavell, 2001).

Caspase inhibitors can block the development of the oocyte

Caspases are key factors in the induction of apoptosis inmetazoans (Nicholson and Thornberry, 1997). In the canon-ical apoptotic pathway, activation of caspase 3 ultimatelyleads to the activation of endonucleases (e.g., DNase 1),which results in the final step in the degradation of apoptoticcells, the internucleosomal fragmentation of nuclear DNA.Two caspases have been isolated from Hydra, termedcaspase 3A and caspase 3B. These genes both encode class3-type caspases, i.e., potential effector caspases (Cikala etal., 1999). We analyzed the mRNA expression of these twocaspases during oogenesis. While caspase 3B does not ap-pear to be significantly expressed in the egg field or thedeveloping oocyte (data not shown), caspase 3A expressioncan be detected at elevated levels in the egg field from stage2 to stage 4 (Figs. 3A–C). At stage 2, expression is found inthe whole egg field (Fig. 3A). At this stage, oocyte selectionhas just occured (Miller et al., 2000) and the nurse cellshave not yet been phagocytosed by the oocyte. We thereforeconclude that at an early stage of oogenesis nurse cellsexpress caspase 3A. At oogenesis stage 4, when all nursecells have been taken up by the oocyte, strongest expressionis found in the center of the egg field (Fig. 3B). At stage 5expression starts to decrease and cannot be detected inoocytes or early embryos (data not shown). This suggeststhat type 3 caspases might play a role during early oogenesisin Hydra.

To test for a functional role of caspases in oogenesis, wetreated females at various stages of oogenesis with the broadspectrum caspase inhibitor zVAD-FMK and the caspase3-specific inhibitor Ac-DEVD-CMK (Garcia-Calvo et al.,1998). When we started the treatment at oogenesis stage 1or 2 with 0.3–0.6 mM inhibitor, oogenesis was blocked ina large fraction of females and no mature oocytes wereformed (Figs. 4C and D; Table 1). However, when westarted the treatment at stage 3 (or later), oogenesis pro-ceeded at the normal rate and the mature oocyte appearedunaffected (Table 1). Also, inhibitor concentrations rangingbetween 1 and 10 �M had no effect; at 100 �M they hadonly minor effects. A control treatment with 0.6 mM N-tosyl-lysine-CMK, an inhibitor of trypsin-like serine pro-teases, had no effect on the development of oocytes, al-though it had severe effects on other parts of the polyps(Figs. 4A and B). This suggests that the observed effects ofthe caspase inhibitors are not due to unspecific effects by thereactive CMK group. Upon early treatment with caspaseinhibitors virtually no TUNEL-positive cells could be foundin the body column (Figs. 4C and D; Table 1) nor couldAO-positive nurse cells be found in the egg field (data notshown). This suggests that caspase activity is necessary foroogenesis during early phases, before phagocytosis of nursecells, but not required for oocyte development after phago-cytosis. Further, the initiation of nurse cell apoptosis ap-pears to be an essential step for proper oocyte development.


Nurse cells phagocytosed by the oocyte are TUNEL-negative

To test for internucleosomal fragmentation of thegenomic DNA in nurse cells, we established a protocol forTUNEL staining in Hydra. As a positive control, we exam-ined hydroxyurea (HU)-treated polyps and polyps undergo-ing oogenesis. When apoptosis is induced by treatment ofpolyps with HU, numerous AO- and TUNEL-positive cellscan be detected in the body column of the polyp (data notshown). During late stages of oogenesis, a small number ofnurse cells located at the periphery of the egg field fail to bephagocytosed by the oocyte. Instead, they are phagocytosedand rapidly degraded by surrounding epithelial cells. Whenstained with AO, these nurse cells change from green to redfluorescence, indicating advanced DNA degradation (Fig.5A). Similarly, these nurse cells are TUNEL-positive(Figs. 5B and C). We then performed TUNEL assays duringall stages of oogenesis and all accessible stages of embry-ogenesis (i.e., stages prior to formation of the theca). Sur-prisingly, nurse cells incorporated by the oocyte wereTUNEL-negative at all stages examined, suggesting thatfragmentation of the genomic DNA does not occur (see, forexample, the postgastrula embryo in Fig. 6A). Thus, al-though nurse cells phagocytosed by the oocyte are brightlystained green by AO, they never become TUNEL-positive.Nurse cells were also negative for the TUNEL assay whenthe polyps and early embryos were cut before and afterfixation (data not shown), eliminating the possibility thatphagocytosed nurse cells are not accessible to the TUNELassay reagents.

Why are nurse cells not degraded inside the oocyte? Onepossibility is that the nuclear DNA of the nurse cells issomehow protected against cleavage by DNase I. To testthis, we treated various stages of oogenesis and embryogen-esis with DNase I after fixation, followed by a TUNELassay. In response to the DNase I treatment numerous epi-thelial cells of the parent polyp or nuclei of the blastomeresin embryos became TUNEL-positive, yet nurse cells re-mained TUNEL-negative (Figs. 6B–E). These results sup-port the hypothesis that the DNA of the phagocytosed nursecells is packed in a form distinct from that of somatic cells,a form which protects it from degradation.

DNA of phagocytosed nurse cells is cleaved into largefragments

Internucleosomal degradation of nuclear DNA in apo-ptotic cells is detected by the appearance of fragments inmultiples of 200 bp. We analyzed DNA from normal pol-yps, from polyps induced to undergo apoptosis by HUtreatment, from late oogenesis stages, and from early em-bryos. DNA from normal polyps migrates as a single bandat limiting mobility (Fig. 7A). The same band is found inHU-treated animals, presumably derived from unaffectedepithelial cells, in addition to bands of 200 and 400 bp,

Fig. 3. In situ hybridization of caspase 3A during oogenesis. (A) stage2 (B) stage 4 of oogenesis (C) sense control stage 4.


which are likely derived from strongly degraded genomicDNA of the interstitial cells undergoing apoptosis (Fig. 7A).DNA from late oogenesis stages does not differ from nor-

mal polyps and shows no obvious signs of degradation (Fig.7A). In early embryos, a slight laddering can be detected insome but not all preparations. Intriguingly, the largest sized

Fig. 4. Caspase inhibitors can block oocyte formation during the early phase of oogenesis. (A,B) live animals treated with N-tosyl-lysine-chloro-methyl-ketone (TL-CMK), a serine protease inhibitor, which leads to severe damage to the somatic portion of the polyp (A) but has no effect on oocyte formation(B). (C, D) treatment of animals with Ac-DEVD-CMK starting at stage 2 causes arrest of oocyte development.Fig. 5. Peripheral nurse cells that are not phagocytosed by the oocyte become degraded rapidly. (A) AO staining of a stage 7 polyp with a mature oocyte(arrowhead) shows orange-red AO-positive nurse cells at the periphery of the egg field that were not incorporated into the oocyte. (B) A view looking ontothe egg cup (the margin of the egg cup is marked by arrowheads) after detachment of the oocyte shows that peripheral nurse cells that were not phagocytosedby the oocyte have become TUNEL-positive (arrows). (C) Close-up of (B) showing TUNEL-positive nurse cells at the periphery of the egg field which havebeen phagocytosed by epithelial cells. Bars, 200 (A, B) and 60 �m (C).


DNA in the embryo preparations is always noticeablysmaller in size than is seen in adult polyps (Fig. 7A, lanes 8and 9). To analyze this size difference more accurately, wefractionated the DNA on a 0.7% agarose gel. We found thatmost of the embryo DNA is in fragments of 8–15 kb (Fig.7B). The early embryos used for this analysis consisted of8–100 blastomeres, but they have incorporated up to 10,000nurse cells. Hence, these preparations are enriched on theorder of 100- to 1000-fold for DNA from the nurse cells. Insupport of this conclusion, we found that DNA preparationsfrom older embryos with much higher numbers of zygoticcells gave two bands, one at limiting mobility (presumablyfrom the embryo nuclei) and one in the 8- to 15-kb region(presumably from the nurse cell nuclei) (data not shown).These results confirm the negative result of the TUNELassay. The number of free 3�OH DNA ends after the large-scale fragmentation is presumably too small to be detectedby TUNEL. In conclusion, the DNA in phagocytosed nursecells does become fragmented, but the fragments are muchlarger than the internucleosomal fragments seen in cellswhich are proceeding through the normal course of apopto-sis.

Persistent peroxidase activity in phagocytosed nurse cells

Peroxidases and catalases are known inhibitors of apo-ptosis, presumably by preventing oxidative stress to mito-chondria, a damage which can induce apoptosis (Zhang etal., 1997; Husbeck et al., 2001; Andoh et al., 2002;Petrosillo et al., 2001; Moon et al., 2002; Iwai et al., 2003;Imai et al., 2003; Hockenberry et al., 1993). Peroxidase istherefore potentially an agent responsible for preventingnurse cells from completing apoptosis until hatching. Totest this possibility, we performed a peroxidase activityassay at various stages of oogenesis and embryogenesis andon hatchlings. We found a high level of peroxidase activityin the nurse cells from early stages of oogenesis on through-out embryogenesis (Fig. 8A). Newly hatched polyps (Day0) also exhibit a high level of peroxidase activity (Fig. 8B),which is localized in the phagocytosed nurse cells (Fig. 8C).Interestingly, in many polyps we observe that peroxidaseactivity in newly hatched polyps decreases within 1–3 days

following hatching, starting at the apical end of the polypand spreading over the rest of the body (Figs. 8D–F). Thispattern of decrease in peroxidase activity is strikingly cor-related with the pattern of degradation and precedes thedisappearance of nurse cells after hatching (compare Figs.1G–1), suggesting a role for peroxidase in preventing apo-ptosis in nurse cells until hatching.

Nurse cells are prevented from entering the lysosomaldegradation pathway

Phagocytosis is an important step in the degradation ofapoptotic cells and has been shown to be necessary forcompletion of apoptosis (Reddien et al., 2001; Hoeppner etal., 2001, Hengartner, 2001). Since phagosomes normallyenter the lysosomal pathway by fusion with the acidic sec-ondary lysosomes, we stained oogenesis stages and oocyteswith H22.58, a monoclonal antibody specific for late sec-ondary lysosomes (Technau, David, and Holstein, unpub-lished results). While the antibody readily recognizes lyso-somes in the epithelial cells of the same specimen, it doesnot recognize phagosomes with incorporated nurse cells.H22.58� vacuoles are present in the entire body but absentfrom the oocyte (Fig. 9A). Similarly, during earlier phasesof oogenesis phagocytosed nurse cells are not located inH22.58� lysosomes (Figs. 9B and C). This suggests thatnurse cells incorporated by the oocyte do not enter thelysosomal pathway, and thereby escape complete degrada-tion. Although the nurse cells can persist for monthsthroughout embryogenesis in the unhatched polyp, the nursecells disappear within 3 days after the polyp hatches. Thedegradation of the nurse cells, which are now all located inthe endodermal cell layer in newly hatched polyps, appearsto begin in the apical part of the animal and progress downthe polyp, ending at the aboral end of the polyp (Figs.1G–I). Interestingly, this degradation pattern is correlatedwith a shift of green to orange-red fluorescence of the AO,similar to that seen with i cells that were induced by atreatment with HU to undergo apoptosis (Miller et al.,2000). Since the H22.58 antibody does not penetrate themesogloea in whole mounts, we prepared macerates fromDay 2 and Day 3 hatchlings and stained them with H22.58

Table 1Effect of caspase inhibitors on oogenesis

DMSO control Ac-DEVD-CMK z-VAD-FMK

Stage whentreatment wasstarted

Stage reachedafter 48 h oftreatment

Tunel� cells Stage after 48 h Tunel� cells Stage reachedafter 48 h oftreatment

Tunel� cells

Stage 2 5.6 � 1.4 220 � 111 2.5 � 0.6 7.2 � 3.7 1.9 � 0.7 11 � 9(n) 29 11 19 5 28 9Stage 3 7 � 0 n.d. 3.5 � 1.2 n.d. 5.3 � 1.8 131 � 42(n) 15 n.d. 11 n.d. 16 5

Note. Females at oogenesis stage 2 and 3 were selected and treated with DMSO (0.75%) or 300 �M Ac-DEVD-CMK and z-VAD-FMK. Solutions werechanged every 6 h. After 48 h the oogenesis stages were scored and the number of TUNEL-positive cells in the body column was determined.


antibody. In endodermal cells of these hatchlings about 10%of the nurse cells are found in the H22.58 secondary lyso-somes. DAPI counterstaining shows that all nurse cells inthe H22.58 lysosomes are highly degraded, with fragmentednuclei, and can be hardly detected morphologically as nursecells (Figs. 9D–G). Incorporated nurse cells that can beeasily recognized morphologically were never found in theH22.58 lysosomes. This suggests that nurse cells enter thesecondary lysosomes only shortly before or during completedegradation and that this step is prevented at earlier stagesof embryogenesis. To further confirm this result, we per-formed a TUNEL assay on macerates of hatchlings anddouble-stained them with H22.58 antibody. The resultsclearly showed that in Day 2–3 hatchlings about 99% of allnurse cells were TUNEL-positive, indicating advancedDNA fragmentation (Figs. 9H–J). However, we also stillfound a small fraction of TUNEL-negative nurse cells and asmall number of cells with only peripheral TUNEL activity(Fig. 9J). Interestingly, only TUNEL� nurse cells withhighly degraded and fragmented nuclei were found inH22.58 secondary lysosomes (Fig. 9H). These results sug-gest that fragmentation of the nurse cell DNA is resumed inhatchlings. Finally, the nurse cells enter the lysosomal path-way and are degraded rapidly.

Discussion

Apoptosis of nurse cells is arrested upon phagocytosis bythe oocyte

This study and previous work have shown that Hydranurse cells display a number of features characteristic ofapoptotic cells: the chromatin condenses to one side of thecell; the cells show membrane blebbing, become spherical,and produce membrane-enclosed fragments which are pre-sumably taken up by the growing oocyte (Zihler, 1972;Honegger et al., 1989). Fig. 10 summarizes the fate of thenurse cells during oogenesis and embryogenesis in Hydra.The oocyte becomes determined at oogenesis stage 2, whileall other germ cells differentiate into nurse cells (Miller etal., 2000). Shortly thereafter, nurse cells initiate apoptosis,are phagocytosed by the oocyte and become brightly stainedby acridine orange following phagocytosis (Figs. 10A andB). During the early stages of embryogenesis, nurse cellDNA becomes fragmented into large pieces about 8–15 kbin length. Despite all of these changes indicative of apopto-sis, the phagocytosed nurse cells are not TUNEL-positive.Further, they are not degraded and do not change much inappearance throughout embryogenesis until hatching, whichcan take as long as 12 months or longer. In hatchlings,however, they become TUNEL-positive and are eventuallyrapidly degraded in the secondary lysosomes of the newlyhatched polyp.

Thus, nurse cells enter the apoptotic pathway, possibly aspart of their differentiation program, but completion of

nurse cell apoptosis is arrested until hatching of the embryo.Since nurse cells which are not incorporated by the oocyteare phagocytosed and degraded by epithelial cells withinone day, we conclude that phagocytosis by the oocyte pro-tects nurse cells from further degradation. This shows thatthe apoptotic machinery can be stalled by appropriate ex-ternal signals. Completion of apoptosis, however, resumesquickly after hatching and might involve specific signalsfrom the phagocytes (i.e., the endodermal epithelial cells ofthe primary polyp).

During oogenesis in Drosophila, nurse cells are onlycompletely degraded and resorbed after maturation of theoocyte (McCall and Steller, 1998), although during a muchshorter time frame than that seen with Hydra. Anotherindication that apoptosis can be reversed at intermediatestages comes from studies in C. elegans, which showed thatprogrammed cell death of cells that have already activatedced-3 (caspase 3) can be reverted in mutants deficient forphagocytosis (Hoeppner et al., 2001; Reddien et al., 2001).Blocking phagocytosis in C. elegans leads to higher rates ofsurvival of cells destined to die, suggesting a recovery frominitial stages of cell death. In Hydra, phagocytosis by epi-thelial cells promotes degradation of apoptotic nurse cells.By contrast, phagocytosis by the oocyte leads to an arrest ofapoptosis.

Honegger et al. (1989) have suggested a cytoplasmictransfer between differentiated nurse cells and the earlyoocyte before initiation of phagocytosis (stage 3). Thisnutritional supply by the nurse cells might be necessary forthe proper development of the oocyte during early oogene-sis. Since degradation of nurse cells is stalled after phago-cytosis, what could be the role of nurse cells during lateoogenesis and embryogenesis? One possibility is thatphagocytosed nurse cells are still alive and continue tosynthesize RNA and/or proteins to provide them to theoocyte. However, how the molecules provided to the oocyteare transferred from the endosome into the cytoplasm of theoocyte is completely unclear. Also, it is not clear whetherthe fragmentation of the nuclear DNA into 8- to 15-kbpieces allows efficient transcription. We therefore favor theidea that nurse cells have only very limited metabolic ac-tivity, possibly just enough for survival inside the endo-somes. Their main role after phagocytosis would be to act asa source of nutrients for the newly hatched polyp.

A caspase-independent pathway of DNA fragmentation inHydra nurse cells?

Two caspase genes have been identified in Hydra andboth encode members of the caspase 3 class (Cikala et al.,1999). The level of caspase 3A RNA increases during earlyoogenesis, but drops in mature oocytes and embryos. Treat-ment with different caspase inhibitors during early oogen-esis can block oocyte formation at high doses but they haveno effect if applied during later stages of oogenesis. Thissuggests an early caspase-dependent phase of nurse cell


apoptosis that is necessary for proper oocyte development,and a later caspase-independent phase after phagocytosis,during which apoptosis is arrested.

Caspase 3 activation normally leads to the internucleo-somal degradation of nuclear DNA by activation of endo-nucleases such as DNase I. Despite their having manycharacteristics of apoptotic cells, nurse cells did not showinternucleosomal fragmentation even after a long period ofincorporation into the oocyte and embryos. Instead, wefound degradation into large fragments of about 8–15 kb. Inaddition to the formation of large DNA fragments, we alsofrequently observed the condensation of the chromatin atthe periphery of the nuclear envelope which is typical of thesituation when DNA degradation produces large fragments(Hunot and Flavell, 2001). Production of such large frag-ments has been described as an alternative route of apopto-sis (Oberhammer et al., 1993; Zhivotovsky et al., 1994;Lagarkova et al., 1995; Susin et al., 2000; Arnoult et al.,2001; Cipriani et al., 2001). This pathway is caspase-inde-pendent and involves an unknown nuclease (Hunot andFlavell, 2001). The endonucleases DNase II or DNase � andDNase � have been proposed as possible candidates for thispathway (Shiokawa et al., 1994; Barry and Eastman, 1993;Wu et al., 2000; McIlroy et al., 2000). Unlike DNase I, theseenzymes are cation-independent and require an acidic envi-

ronment (Shiokawa et al., 1994; Barry and Eastman, 1993).This fits the observation of a shift from green to orange-redfluorescence in AO-positive nurse cells, which indicates anintracellular acidification (Mpoke and Wolfe, 1997). On theother hand, the recently identified endonuclease G (Endo G)acts as a caspase-independent nuclease, which leads to thelate fragmentation of nuclear DNA into pieces of about 50kb (Parrish et al., 2001; Li et al., 2001; Widlak et al., 2001;Wang et al., 2002). Depletion of Endo G leads to an arrestof apoptosis or to less apoptotic cells, indicating, thatEndo G is necessary for the progression of apoptosis.This fits precisely our observation of the DNA cleavagepattern of nurse cells in Hydra. Initiation of the caspase-independent pathway in vertebrates seems to involve theapoptosis-inducing factor (AIF) that is released frommitochondria and translocates to the nucleus (Joza et al.,2001; Cande et al., 2002). However, AIF also appears toserve as a free radical scavenger, mediating anti-apoptotic functions in a manner similar to that of peroxi-dases (Klein et al., 2002; Lipton and Bossy-Wetzel,2002). Neither Endo G nor AIF has been reported fromHydra to date, but it will obviously be interesting toinvestigate the role of these proteins during Hydra oo-genesis.

The correlation between survival of the nurse cells and

Fig. 6. Nurse cells are TUNEL-negative and are not susceptible to DNase I treatment. (A) TUNEL assay on a postgastrula stage embryo shows that nursecells are TUNEL-negative. (B) TUNEL assay on a postgastrula embryo treated with DNase I after fixation. (C) Close-up view of an early cuticle embryonicstage showing that only embryonic nuclei (arrows) and not the nuclei of incorporated nurse cells (arrowheads) are TUNEL-positive. (D) Animal at oogenesisstage 4 treated with DNase I and showing numerous TUNEL-positive cells. (E) close-up of an egg field of a stage 4 female treated with DNase I shows thatonly epithelial cells (arrows) but not nurse cells (arrowheads) are TUNEL-positive. Bars, 200 (A, B), 300 (D), and 80 �m (C, E).


strong peroxidase activity suggests that peroxidases mightbe involved in the arrest of nurse cell apoptosis. Interest-ingly, peroxidases have been implicated as anti-apoptoticagents in other systems (Zhang et al., 1997; Husbeck et al.,2001; Andoh et al., 2002; Petrosillo et al., 2001; Moon etal., 2002; Imai et al., 2003; Iwai et al., 2003; for reviews seeAdrain and Martin, 2001; Salvesen and Renatus, 2002).Induction of apoptosis by oxygen radicals is thought to bean ancient mechanism, already present in yeast (Madeo etal., 1999). Hence, peroxidases might play a crucial role inregulating the arrest of nurse cell apoptosis in the basalmetazoan Hydra.

Final degradation of nurse cells in secondary lysosomes

A striking characteristic of the nurse cell apoptosis isthat nurse cells persist in phagosomes without beingdegraded. However, upon hatching, DNA fragmentationof nurse cells is resumed and the cells enter the secondarylysosomes within 1–3 days. The persistent peroxidaseactivity provides circumstantial evidence that nurse cellsmight even have a limited metabolic and synthetic activ-ity. This is reminiscent of a number of intracellular sym-bionts and pathogens. In Hydra viridissima, the greenHydra, endodermal cells phagocytose symbiotic Chlor-ella algae. These endosymbionts can proliferate insidethe cells and maintain a stable population. The endosym-bionts appear to prevent their degradation by secondarylysosomes (Technau, Hegen and Holstein, unpublishedresults). Likewise, many Anthozoa (corals and sea anem-

ones) harbor intracellular symbiotic Zooxanthella algae.Intracellular parasites and pathogenic bacteria such asLegionella also prevent acidification and fusion of thephagosomes with the lysosomes (Vogel and Isberg, 1999;Amer and Swanson, 2001). Thus, nurse cells in Hydramay use strategies similar to those of endosymbionts andpathogens to survive inside a host cell and may indeedhave many features in common with endosymbionts.

Evolution of apoptosis in germline-derived nurse cells

Given Hydra’s near basal position in the metazoantree, it is of obvious interest to try to place our findingsin an evolutionary context. Recently, it has been shownthat nanos and vasa, two germline-specific genes of Bi-lateria, are also expressed specifically in germ cells inHydra (Mochizuki et al., 2000, 2001). This suggests aconserved genetic control mechanism in germline differ-entiation of all animals (see also Hobmayer et al., 2001).Apoptosis of germline-derived nurse cells also appears tobe an ancestral feature of oogenesis (for review seeMatova and Cooley, 2001) and phagocytosis of nursecells by the oocyte has been reported from a number ofcnidarian species (Tardent, 1985). Interestingly, somesponges and Ctenophores also show a type of oogenesisvery similar to that seen in Hydra (Fell, 1969; Harrisonand Westfall, 1991), suggesting that apoptosis andphagocytosis of nurse cells is a basic feature of oogenesisin early metazoans.

Fig. 7. Analysis of DNA from eggs and embryos in double preparations; (A) 1.5% agarose gel showing DNA from normal polyps (lanes 1, 2),Hydroxyurea-treated polyps (lane 3, 4), oogenesis stages 3–5 (lanes 6, 7) and 8-cell to 100-cell embryos (lanes 8, 9); (B) 0.7% agarose gel of DNA fromnormal polyps and from early embryos which show degradation into fragments of 8–15 kb.


Fig. 8. Persistent peroxidase activity in nurse cells throughout oogenesis and embryogenesis. (A) Oogenesis stage 2/3. (B) Newly hatched polyp showing thehigh level of peroxidase activity. (C) Close-up of the same polyp showing that the peroxidase activity is limited to nurse cells (arrows) which are now alllocated in the endoderm; (D) 3-day-old hatching. (E, F) Close-ups of the apical and basal regions indicated in (D). Note that most peroxidase-positive nursecells have disappeared from the apical region, while still a few are found in the basal region of the animal. Bars, 200 (A, B, E, F), 400 (D), and 50 �m (C).

Fig. 9. Phagocytosed nurse cells are not located in H22.58-positive secondary lysosomes until hatching. (A) Whole-mount staining with mAb H22.58 offemale with mature oocyte (arrow). H22.58� vacuoles are present throughout the body column but not in the oocyte. (B, C) View on egg field of oogenesisstage 4 stained with mAb H22.58 (B) and visualized with Nomarski optics (C). Numerous H22.58� lysosomes can be detected in epithelial cells (arrowhead),but the underlying nurse cells (arrow) are not located in such vacuoles. (D–G) macerated epithelial cells of a 3-day-old hatchling stained for H22.58 (green)and DAPI (blue). Note, that only strongly degraded DNA can be found in H22.58 lysosomes (arrows), while more intact nurse cells (arrowheads) are notin lysosomes. (H) Macerated epithelial cells stained for H22.58, DAPI, and TUNEL (black), showing that only strongly degraded TUNEL-positive cells canbe found in the secondary lysosomes. Arrows indicate nurse cell remnants located in secondary lysosomes, while arrowheads show less strongly degradednurse cells which are not yet in the lysosome. The asterisks indicate the nucleus of the epithelial cell. (I–J) TUNEL-stained macerates of Day 3 hatchling,showing that most nurse cells are TUNEL-positive at that stage, but some are still negative (J, left) or appear to become TUNEL-positive at the peripheryof the nucleus (I, J, middle). Bars, 200 (A) and 50 �m (B, C).


Acknowledgments

We thank Charles N. David and Angelika Boettger forproviding the caspase clones and for fruitful discussions.This work was supported by a grant from the DFG to U.T.(TE 311/3-1) and NSF Grant IBN-9808828 to R.E.S.

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arrested apoptosis of nurse cells during hydra oogenesis ... · to resume after the primary polyp...

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