Supplementary Figure Legends
Figure 1. β-galactosidase staining showing Umodl1expression in developing otic
vesicle (Ot) and thymic primordium (TP) at E10.5(a). In young adults, olfactory neurons
(ON) in adult olfactory bulb (b), olfactory epithelium (OE) in the nasal cavity (c), and the
arcuate nucleus of hypothalamus (ArN, d) are actively expressing the Umodl1-BAC
transgene. (e) Expression of Umodl1 in GnRH neurons (GnN) in young adult
hypothalamus was confirmed by in situ hybridization using digoxigenin-labeled Umodl1.
(f-h) High magnification photos of areas indicated by upper arrow (f), lower arrow (g),
and lower arrowhead (h) in the Figure 5n of the main text were shown. In the 4-month
old Tg ovary, absence of primordial follicles and excessive innervation of blood vessels
are apparent. Abbreviations: ArN, arcuate nucleus of hypothalamus; Bv, blood vessel;
Ge, germinal epithelium; GnN, GnRH-positive neuron; OE, olfactory epithelium; ON,
olfactory neuron ; Ot, otic vesicle; Pit, pituitary; and TP, thymic primordium. Scale bars,
100µm.
Figure 2. Disorganization of Tg follicles examined by lacZ-staining, and down-regulation
of AMH in a 4-month old Tg ovary by ISH. (a) a healthy control antral follicle; (b) an
mutant follicle showing disorganized granulosa cells and a malformed oocyte. The
mutant granulosa cells tended to dissociate from each other and from the theca layer.
Notably, the granulosa cells immediately adjacent to oocyte were lacZ-positive,
reminiscent of cumulus cells in the young Tg follicles after eCG stimulation (Figure 4). (c)
A representative photograph showing AMH expression in the granulosa cells of growing
WT follicles by ISH. (d) AMH expression in the 4-month old mutant ovaries is drastically
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down-regulated, characterized by activities slightly above background levels in the
cortical stromal cells. Age of each sample is indicated in the lower left corner of each
panel. Abbreviations: cGC, cumulus granulosa cell; CS, ovarian cortical stroma; GC,
graunulosa cell; mGC, mural granulosa cell; mo, months; and O, oocyte. Scale bar, 100
µm.
Figure 3. Western blots in the main text, showing aberrant expression of FSH and AMH
in sera, and Umodl1 in various ovarian cells of the Tg mice, were analyzed and
quantified by NIH ImageJ software. (a) A significant increase in serum FSH was
detected in Tg females at all ages examined. (b) A sharp decline in circulating AMH
started from 2-months of age and reached to a undetectable level in mice older than 6
months. (c) Expression of Umodl1 was ectopically initiated in cGCs at 2-months of age,
and reached the peak level by 3 months, followed by a drastic decline. (d & e) Overexpression of Umodl1 was maintained at elevated levels up to 3-months of age.
Reduction in Umodl1 levels coincides with the progression of ovarian dysfunction of the
Tg mice, likely due to the diminution of Umodl1-expressing cells in whole ovary or a
suicidal effect in oocyte. (f) Increased levels of Bax suggest apoptosis played a crucial
role in the progressive degeneration of the Tg ovaries. Data at each time point were
obtained from three experiments and presented as mean+SEM. Relative protein levels
were compared between age-matched WT and Tg groups. *p<0.05
Figure 4. Abnormal thymus involution in 4-month old pW224 Tg mice. (a) lacZ-stained
WT and Tg thymuses. (a) Significant size difference was seen between two
experimental groups. (b) Bar figure showing weight difference between WT and Tg
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thymuses. Error bars indicate standard deviations. (c) A representative photograph of a
WT thymus shows distinct cortex and medulla zones; whereas in the Tg thymus, only
tightly packed “cortex” zone was present (d). Lack of distinct thymic medulla may impair
the finalization of T-cell maturation in the 4-month old Tg mice. Abbreviations: Ctx,
thymic cortex; Me, thymic medulla. Scale bars, 100µm.
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