12 October 2000
Nature 407, 754 - 757 (2000) © Macmillan Publishers Ltd.
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Somatic control over the germline stem cell lineage
during Drosophila spermatogenesis
JOHN TRAN†, TAMARA J. BRENNER† & STEPHEN DINARDO
Department of Cell & Developmental Biology, University of Pennsylvania Medical Center, 1215 BRB II/III, 421 Curie Blvd,
Philadelphia, Pennsylvania 19104-6058, USA
† These authors contributed equally to this work.
Correspondence and requests for materials should be addressed to S.D. (e-mail: sdinardo@mail.med.upenn.edu).
Stem cells divide both to produce new stem cells and to generate daughter cells that can
differentiate1. The underlying mechanisms are not well understood, but conceptually are
of two kinds2. Intrinsic mechanisms may control the unequal partitioning of
determinants leading to asymmetric cell divisions that yield one stem cell and one
differentiated daughter cell. Alternatively, extrinsic mechanisms, involving stromal cell
signals, could cause daughter cells that remain in their proper niche to stay stem cells,
whereas daughter cells that leave this niche differentiate. Here we use Drosophila
spermatogenesis as a model stem cell system3 to show that there are excess stem cells
and gonialblasts in testes that are deficient for Raf activity. In addition, the germline
stem cell population remains active for a longer fraction of lifespan than in wild type.
Finally, raf is required in somatic cells that surround germ cells. We conclude that a
cell-extrinsic mechanism regulates germline stem cell behaviour.
The testis proliferation centre in Drosophila melanogaster includes the germline and
somatic stem cells that maintain spermatogenesis ( Fig. 1a)3, 4. As a germline stem cell
divides, one daughter becomes a gonialblast, while the other remains a stem cell. To
amplify the germline population, each gonialblast executes four divisions as 2°
spermatogonia, which exit the mitotic cycle and enter a meiotic and differentiation
programme as a clone of 16 spermatocytes. The gonialblast and its progeny are encysted
by somatic cells derived from cyst progenitor stem cells3. We previously characterized
mutants affecting 2° spermatogonia5, 6, and have begun to test signal transduction
pathways for possible involvement at earlier decision points in this germline stem cell
lineage. Null mutations in raf, encoding a serine/threonine kinase involved in receptor
tyrosine kinase pathways7, 8, are lethal in larvae, but such flies carrying a heat shock
(HS)-Raf transgene can survive to fertile adults by using daily heatshocks9. By
withdrawing heat shock when adults eclose, animals become progressively raf deficient
as the protein decays. Testes from such hypomorphic raf-deficient males 5 days after
eclosion were indistinguishable from controls. By day 7, however, 43 of 44 raf mutant
testes showed great proliferation centre expansion. The increase in cell number is due to
excess, early stage germ cells, and continues such that on day 15, raf -deficient testes
are filled with these cells at the expense of post-mitotic cells (compare Fig. 1b and c).
Figure 1 raf function restricts early germ cell number.
Full legend
High resolution image and legend (59k)
To identify the earliest defect in germ-cell progression, we examined the fusome, a
membrane and cytoskeletal organelle specific to the germline that is spheroid
throughout stem cells and gonialblasts, but branches extensively throughout
interconnected 2° spermatogonia (Fig. 2a )10-12. In raf-deficient testes, fusome structure
is not normal. Unbranched fusomes are found in many cells ( Fig. 2b), even those
located some distance from the hub, where, in wild-type testes, only branched fusomes
interconnecting 2° spermatogonia are found. Although branched fusomes do appear in
raf-deficient testes, unlike wild-type testes, these fusomes usually appear only after an
intervening region containing many excess germ cells that have only spheroid fusomes.
These excess germ cells do not result from an increased frequency of germline stem cell
divisions, as the M-phase index for the tier of cells adjacent to the hub in raf-deficient
testes was almost identical to that in the wild type (about 3 M phases in 14 testes on day
13).
Figure 2 Early stage germ cells accumulate in raf-deficient testes.
Full legend
High resolution image and legend (111k)
We next examined cytoplasmic Bag-of-marbles protein (Bam-C), which first
accumulates in 2° spermatogonial cells (Fig. 2c) and is required for their progression
into spermatocytes6, 13. In the wild type, the total population of germline stem cells and
gonialblasts comprises the few non-staining cells between the hub and the first rows of
2° spermatogonia (Fig. 2e). In raf-deficient testes, the first Bam-C-expressing germ
cells are located much further from the hub (Fig. 2f). Also, most of the intervening nonstaining cells contain unbranched fusomes (Fig. 2d), consistent with these cells
representing excess gonialblasts and/or germline stem cells.
To verify this, we examined M5-4 and S1-33, markers expressed in hub cells, germline
stem cells and gonialblasts, but not in 2° spermatogonia (Fig. 3a)6. In raf-deficient
testes, while hub cells appeared normal (Fig. 2d, 3b), the number of germ cells
expressing these markers was greatly increased (Fig. 3b). Furthermore, marker
expression persisted for a significantly longer fraction of adult lifespan. For instance, by
day 19, all control testes (n = 14) had M5-4 marker expression in fewer than three germ
cells (Fig. 3c), rather than the average 5 to 9 germline stem cells plus associated
gonialblasts contained in testes from young adults14. In contrast, only 17% of rafdeficient testes (n = 23) on day 19 showed loss of germ cell expression, whereas 48%
maintained expression at least equivalent to that of day 1, and a further 35% still
showed increased expression ( Fig. 3d). These data suggest that germline stem cells and
gonialblasts remain active for a significantly longer period than in the wild type. We
tested this further by directly counting the number of cycling germline stem cells, which
we judged to be cells that were located adjacent to the hub and contained a spherical
fusome and nuclear anillin, a late interphase marker. Whereas wild-type testes averaged
3.1 late interphase germline stem cells per testis (n = 10 testes; standard deviation, s.d. =
1.0) on day 1, on day 19 aged cohorts had only 0.8 (n = 10; s.d. = 1.0; P < 10-4; Fig. 3e).
This suggests an age-induced quiescence of the germline stem cell population. In
contrast to this, in raf-deficient testes late interphase germline stem cell numbers were
maintained over 19 days (2.5 per testis on day 1, n = 10, s.d. 1.0; 2.8 per testis on day
19, n = 10, s.d. = 1.7; Fig. 3f ) suggesting that germline stem cells as a population
remain active for a longer period than in wild-type.
Figure 3 Prolonged activity for the stem cell population in rafdeficient testes. Full legend
High resolution image and legend (70k)
To determine whether raf function is required in the germ line, which exhibits the
phenotypes described above, or in the surrounding somatic cells, we generated raf null
mutant clones. Persistent germline clones indicate the existence of a raf null germline
stem cell3, 6. In all cases (34% of testes; n = 50), progression through spermatogenesis is
normal as judged by groups of 16 mutant germ cells that are morphologically
indistinguishable from surrounding wild-type spermatocytes ( Fig. 4a). Thus, raf
function is dispensable in germ cells. In contrast, we recovered no cyst cell clones,
suggesting that raf is necessary for viability or proliferation of cyst progenitor cells. As
our previous analysis was under hypomorphic conditions for raf, we repeated the
mosaic analysis, introducing a HS–Raf transgene to provide a basal level of raf function
so that raf-deficient cyst cells might survive. Persistent raf-deficient cyst cell clones
(5% of testes; n = 204), indicate the existence of a raf-deficient cyst progenitor cell.
Such testes had excess early stage, raf+ germ cells ( Fig. 4b, and inset). Thus, raf is
required in the cyst cell lineage. In addition, in raf-deficient testes two cyst cell markers
normally expressed in later-stage cyst cells, LacZ600 (Fig. 4c)15 and Eyes absent (not
shown), were now expressed prematurely in somatic cells adjacent to the hub, probably
the cyst progenitor cells (Fig. 4d). This shows that raf function is required in somatic
cells surrounding germline stem cells.
Figure 4 raf is required in cyst progenitor cells. Full legend
High resolution image and legend (54k)
Germline stem cell divisions lead to a distinction between a self-renewing daughter cell
and a sister cell committed to differentiation as a gonialblast. Our data suggest that a
somatic signal influences this decision by limiting the self-renewing potential. We
cannot yet say how this potential is encoded. Our hypothesis is that in raf-deficient
testes, where cyst progenitor cell identity is disturbed, the signal is lost, and excess stem
cell potential is produced. Upon division, both daughters of the germline stem cell
inherit some stem cell character. At steady state, this increases the number of cells that
become gonialblasts, and somehow prolongs the active state of the stem cell population.
Thus, a somatic cell defect leads to a tumour in the germline stem cell lineage, which
suggests that some tumours of progenitor cell populations could be initiated by genetic
lesions in support cells, rather than in the tumorous cells themselves. In the testis, the
Raf-dependent signal may be delivered by cyst progenitor cells, or their cyst-cell
daughters. Our mosaic analysis shows that depletion of Raf from just one of the two
cyst progenitor cells surrounding a germline stem cell causes a defect. This may indicate
a dose effect, where one heterozygous somatic cell is not sufficient to allow normal
signalling. It is likely that the signal transducer Raf is engaged, owing to activation of
the Epidermal growth factor receptor pathway in somatic cells16. We note that in rafdeficient testes, differentiation of 2° spermatogonia is blocked as they do not transit to
the spermatocyte stage. We believe this is a secondary effect owing to the defective cyst
lineage, as we previously showed that a cyst cell signal governs this later transition from
2° spermatogonia to spermatocytes5.
Somatic signals have been postulated to affect germline stem cell behaviour in the
Drosophila ovary17, 18. However, the characterized signals are necessary to maintain
germline stem cells, rather than restrict their self-renewing potential, as we find here.
Additionally, raf-deficient ovaries exhibit no increases in early stage germ cells19. Thus,
despite the superficial similarities of early germ cell development in ovary and testis,
oogenesis and spermatogenesis are emerging as complementary systems from which
different principles of stem cell regulation will emerge.
Methods
The raf 11-29 or raf 400B6; HS-Raf/+ larvae were heat shocked for 1 hour at 38 °C, daily.
At eclosion (day 1), heat shocking was halted, and flies were aged with wild-type
FM6/Y siblings. Stains were carried out as in refs 3,11 and 15. Figure 3e, f shows wildtype and mutant cohorts that were aged and processed using Anti-Anillin for cell-cycle
stage20, Anti-Fasc III and Anti-1B1. Cells adjacent to the hub containing dot fusomes
and exhibiting nuclear anillin accumulation were scored as late interphase germline
stem cells. Wild-type and raf-deficient testes differed significantly at day 19 (P <
0.003). Figure 4a, b shows clones induced by a 1-hour heat shock at 38 °C in day 1
raf400B6Act> DRaf+>nuc-lacZ / Y; HS-Flp/+ or raf400B6Act5C>DRaf+>nuc- lacZ / Y;
HS-Raf /+; HS-Flp/+ males that were fixed and stained on day 10. The DRaf+ flip-out
is described in ref. 21. The continued production of persistent marked clones
demonstrates the marking of a stem cell6.
Received 2 June 2000;
accepted 28 July 2000
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Acknowledgements. We thank our laboratory staff, B. Calvi, A. Kiger, M. Fuller, E.
Matunis and S. Wasserman for comments and suggestions. Fly stocks and reagents were
contributed by the Bonini, Lipschitz, McKearin, Struhl, Wasserman and Xu labs, as
well as the Bloomington Stock center and Iowa Hybridoma bank. The NSF and the NIH
supports our work.
Figure 1 raf function restricts early germ cell number. a, Testis apex, where 5–9 germline stem
cells (one shown for clarity) and approximately twice as many somatic cyst progenitor cells are
anchored around somatic hub cells. The testis proliferation centre, comprising hub, germline and
somatic stem cells, gonialblasts and 2° spermatogonia (which undergo incomplete cytokineses;
white bars), is restricted to the tip as shown by bright DNA stain (b, arrowhead). Spermatocyte and
postmeiotic spermatids, which fluoresce more weakly owing to chromatin reorganization, fill the
remainder of the testis. c, raf-deficient testis on day 15. Scale bar, 100 µm.
Figure 2 Early stage germ cells accumulate in raf-deficient testes. Confocal projection of half testis
depth; DNA, purple (a, b, e, f). a, Wild-type (WT) testis fusome (mAb1b1) is spherical in stem
cells (small arrow) and gonialblasts, which are located only near the hub (arrowhead). Fusome
branches in 2° spermatogonia (larger arrows). b, raf-deficient testis, day 8. Spherical fusomes
(small arrows) are found both next to and far away from the hub (arrowhead; anti-Fasciclin (Fasc)
III). Fusome branching is detected (larger arrow), more so farther from the hub (not shown). c, WT
testis, day 6; Bam-C (red) is expressed only in 2° spermatogonial cells, four groups of which are
shown, all having branched fusomes (large arrow; white). Cells to the right of this are maturing
spermatocytes, in which Bam-C protein has decayed, and all of which possess fusomes branching
into and out of the focal plane. The hub is just below the focal plane (arrowhead). d, raf-deficient
testis, day 6; two of nine cells with unbranched fusomes are indicated (small arrows), none of
which accumulates Bam-C. The fusome is branched in the spermatogonial cluster expressing BamC (large arrow). The asterisk shows one of two 2° spermatogonial cysts with interconnected
fusomes that either did not accumulate Bam-C, or lost it precociously. The hub is just below focal
plane (arrowhead). e , WT testis, day 8; only a thin band of cells (line) separate Bam-C-expressing
2° spermatogonial cells (white) from the hub (arrowhead; white). f , raf-deficient testis, day 8;
many (line) germline stem cells and gonialblasts separate Bam-C-expressing 2° spermatogonial
cells (white) from the hub (arrowhead; white). Further along the testis, Bam-C-expressing 2°
spermatogonia accumulate in cysts of up to 35 cells, as shown by S- or M-phase synchrony (not
shown). Interspersed with these clusters are individual cells that cycle in isolation and do not
express Bam-C. Scale bar, 20 µm in a, b, e, f; 10 µm in c, d.
Figure 3 Prolonged activity for the stem cell population in raf-deficient testes. a, WT testis, X-gal
activity; M5-4 expressed in the hub (arrowhead), germline stem cells (large arrow), and
gonialblasts (smaller arrow). b , raf-deficient testis; M5-4 is expressed in many cells near hub
(arrowhead). c, WT testis, day 19; expression solely in hub. d, raf-deficient testis, day 19. e, WT
testis, day 19; branched fusomes (green) are visible further along the testis (arrow), but there are no
cells with spherical fusomes adjacent to hub (arrowhead). f, raf-deficient testis, day 19; an example
of late-interphase (nuclear Anillin, red) germline stem cell (arrow), as indicated by the presence of
a spherical fusome (green) in the cell adjacent to the hub (arrowhead). Scale bar, 40 µm.
Figure 4 raf is required in cyst progenitor cells. a, Normal spermatocytes ( -galactosidasepositive, arrow) produced 10 days after induction of a raf germline stem cell clone. Similar data
were obtained for null mutations in egfr and ras1. b, Deficiency of raf in somatic cyst cells
(arrowheads) leads to excess early stage germ cells (inset; DNA stain). Glutaraldehyde fixation for
LacZ activity meant that we could not assay germ cells for stem or gonial cell markers. c, WT
testis, day 18. LacZ600 (white; anti- -galactosidase) is expressed in the hub (arrowhead; green)
and in later cyst cells (arrow). d, raf-deficient testis, day 18. LacZ600, expressed in cyst progenitor
cells (arrows) adjacent to the hub (arrowhead); this is visible before day 9. Scale bar, 100 µm in a,
b (125 µm in inset); 20 µm in c, d