Fig. 3. H3 is asymmetrically distributed after the first GSC division after
heat shock. (A) Heat shock regime. (B to G) H3 is distributed asymmetrical-
ly in GSC versus GB (B to D) but symmetrically in two-cell spermatogonia
(E to G). (H to J) H3.3 is distributed symmetrically in GSC versus GB. (K
to N) A telophase GSC. Asterisk, HUB (anti–Fas III); arrow, spectrosome
(anti–a-spectrin). (O) Quantification of GFP and mKO fluorescence intensity
ratio (table S4). H3 GSC/GB GFP ratio > 1 (*P < 10−4), GSC/GB mKO ratio < 1
(*P < 10−4), N = 12. H3 two-cell spermatogonial (SG) SG1/SG2 GFP ratio
(#P = 0.225) and mKO ratio (#P = 0.365) are insignificantly different from 1,
N = 11. H3.3 GSC/GB GFP ratio (#P = 0.970) and mKO ratio (#P = 0.594)
are insignificantly different from 1, N = 13. Error bars: SE; P value: one-
sample t test.
Fig. 4. Loss of asymmetric H3 distribution pattern upon overexpression of upd. (A to C) In nanos-Gal4;
UAS-upd testis. (A), both H3-GFP (B) and H3-mKO (C) are symmetrically distributed in overproliferative
GSC-like cells. Asterisk, HUB (anti–Fas III).
tables S1 and S2: H3-GFP ratio in SG1/SG2 =
1.09; H3-mKO ratio in SG1/SG2 = 1.02).
In contrast to the asymmetric distribution
pattern for the canonical histone H3, the histone
variant H3.3 did not show this asymmetry during GSC divisions, by fluorescence images (Fig.
2, H to J) and by quantification (H3.3 GSC/GB
data in Fig. 2T, tables S1 and S2: H3.3-GFP
ratio in GSC/GB = 1.03; H3.3-mKO ratio in
GSC/GB = 1.03). The symmetry of the histone
variant H3.3 suggests that the asymmetric mode
is specific for canonical histone H3.
Fewer than 2% of all GSCs are undergoing
mitosis; thus, all analyses above were based on
postmitotic GSC-GB pairs. To further examine
the histone segregation pattern during mitosis,
we screened for mitotic GSCs. Indeed, old his-
tones were mainly associated with the chromatids
segregated to the GSC side at metaphase (fig. S2),
anaphase (Fig. 2, N to P, fig. S2, arrowheads),
and telophase (Fig. 2, Q to S, arrowheads). By
contrast, new histones were more enriched at the
chromatids segregated to GB side (Fig. 2, N, P,
Q, and S, and fig. S2, arrows). These results sug-
gest that the sister chromatids preloaded with old
histones are preferentially retained in GSCs and
that the ones enriched with new histones are par-
titioned to GBs during GSC mitosis.