National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
JSPS Research Fellow:
Research Fellow:
SOKENDAI Graduate Student:
Technical Assistant:
Secretary:

KITADATE, Yu
HARA, Kenshiro
NAKAGAWA, Toshinori
MIZUGUCHI, Hiroko
HIRA, Seiji
NAKAMURA, Yoshiaki
NAKAMURA, Yoshiaki*
IKAMI, Kanako
TOKUE, Moe
NONAMI, Yuta
ISHIZAKA, Miho
HIRANO, Kodai
KON, Yayoi
MARUYAMA, Ayumi
NISHIMURA, KeikoKUBOKI, Yuko

Overview of our research

Production of numerous sperm for a long period in testis
is fundamental for continuity of life across generations.
This process of spermatogenesis is supported by the robust
function of “stem cells”, which both maintain the undifferentiated cells, while generating differentiated cells in a nicely
balanced manner. The Division of Germ Cell Biology aims
to fully understand the mouse sperm stem cell system under
the context of in vivo testicular tissue. Our particular interests have been laid on the “undifferentiated spermatogonia”,
which are responsible for the stem cell functions. Our study
has revealed several key properties of this interesting population.
First, we found that this stem cell system includes a functional hierarchy. It is comprised of an “actual” stem cellcompartment that is prone to self-renew, and a differentiation-primed, “potential” stem cell compartment. Regarding
the “actual” stem cells, we have been investigating theircellular identity, their in vivo behavior at a single-cell resolution, and the underlying mathematical principles. This leadto the discovery of neutral competition between the stemcells. We are currently investigating the mechanisms underlying the constant size of this population, and its connection
to its tissue environment. “Potential stem cells” are also of
our enthusiastic interest: In undisturbed, steady-state spermatogenesis, they largely contribute as transient amplifying
cells and differentiate rather than self-renew. However, whentissues are insulted or testicular cells are transplanted to ahost testes, their probability of self-renewal jumps up andthey effectively replenish the lost “actual” stem cells. Sucha flexible feature of stem cell dynamics has been found paradigmatic for many other stem cell-supported tissues.
Key references for these studies that are currently public
include Nakagawa et al., Dev. Cell 2007; Yoshida et al.,Science 2007; Nakagawa et al., Science 2010; Klein et al.,Cell Stem Cell 2010; and Hara et al., Cell Stem Cell 2014.

24I. The stem cell dynamics

Morphologically, the population of Aundiff includes singly
isolated cells (As), or syncytia consisting mainly of 2 (Apr), 4(Aal-4), 8 (Aal-8), or 16 (Aal-16) cells. The formation of syncytia
is due to incomplete cell division, a germline-specific celldivision process where cytokinesis does not complete andthe cytoplasmic connection between daughter cells persists
via intercellular bridges. The prevailing stem cell theory
proposed in 1971 states that stem cell activity is restricted
to the population of As cells, while interconnected Apr and
Aal syncytia are irreversibly committed to differentiation andno longer contribute to the stem cell pool (Huckins, 1971;Oakberg, 1971), known as the “As model”.
Figure 1 represents our latest model for the functional
structure of the spermatogenic stem cell system, which
indeed proposes an alternative for the “A s model”. Thisis the simplest interpretation of the results of our functional analyses of GFRα1+ spermatogonia, which act as the“actual” stem cells. These include intravital live-imaging
experiments, clonal fate analysis of pulse-labeled cells, andbiophysical modeling analysis of the results.

Figure 1. A proposed stem cell dynamics. On the top of the differentiation
hierarchy, GFRα1+ spermatogonia comprise a single stem cell pool, in

which cells continually and reversibly interconvert between states of As,

Apr and Aal spermatogonia through incomplete cell division (blue arrows)

and syncytial fragmentation (red arrows), while giving rise to Ngn3+cells. After leaving the GFRα1+ compartment, differentiation-destined

cells follow a series of transitions (GFRα1+→Ngn3+→Kit+; downward

black arrows) that accompany the extension of syncytial length (rightward black arrows). Ngn3+ and, to a lesser extent, Kit+ cells retain thecapacity to revert back into the GFRα1+ compartment in a contextdependent fashion (broken arrows). (Reprinted from Hara et al., Cell
Stem Cell 2014.)

As crystalized in this model, our results suggest that theGFRα1+ sub-population of A undff spermatogonia, which
include both As cells and syncytia (Apr and Aal) comprises
a single stem cell pool, in which cells continually interconvert between these morphologically heterogeneous states
through stochastic incomplete division and fragmentation ofsyncytia. The incomplete division and syncytial fragmentation causes the expansion of this population, while the excess
cells over a particular “quota” would overflow to become theNote: Those members appearing in the above list twice under different titles are members whose title changed during 2015. The former title isindicated by an asterisk (*).

Ngn3+ state of Aundff, which then further differentiate into Kit+
“differentiating” spermatogonia that are largely devoid ofself-renewing potential. Currently, we are investigating themechanism that determines the quota or tissue capacity of
GFRα1+ cells, as well as the detailed nature of the heterogeneity of this population.

II. M echanisms of the different behavior of“actual” and “potential” stem cells

In 2015, we challenged one key question: Why a particular“actual” stem cell population of Aundiff (i.e., GFRα1+ cells)
remain undifferentiated, while the other, “potential” stemcells (Ngn3+) differentiate, despite that both cells are locatedin the same tissue “open niche” environment and exposedto the same differentiation-inducing signal? (Ikami et al.,Development 2015)

2-1. Architecture of the mouse testis – the “open niche”

It was generally considered that the behavior of stem cells
is regulated by a tissue microenvironment, or the stem cellniche. In some tissues, like Drosophila gonads or mammalian small intestines, stem cells cluster in an anatomicallyspecialized (“closed” or “definitive”) niche that determines
the stem cell fates. Because niche-derived signals appear to
be spatially restricted, cells that are located within the niche
can be maintained in an undifferentiated state, and their displacement from the niche leads to differentiation. In mouse
testis, in contrast, spermatogenic stem cells appear to be distributed over the basal compartment and intermingled withdifferentiating

Figure 3. Intermingling of GFRα1+, Ngn3+, and KIT+ spermatogonia.
Representative images of triple immuno-stained whole-mount seminiferous tubules of an Ngn3-EGFP mouse. Scale bars = 50 μm. Reproduced
from Ikami et al., Development (2015)

progeny, designated as an “open” niche. Here, the details of
the mechanisms that determine whether stem cells differentiate or remain undifferentiated are unknown.
Mouse spermatogenesis occurs in seminiferous tubules andrepresents a typical example of an open niche-supported
stem cell system (Figure 2) (Russell et al., 1990; Stine andMatunis, 2013). Here, GFRα1+ (roughly corresponding to“actual” stem cells), Ngn3+ (nearly “potential” stem cells),and Kit + (“differentiating” spermatogonia that have lost
most stem cell potential) populations of spermatogonia areintermingled with each other in the basal compartment of theseminiferous tubules (Figure 3). These cells are uniformly
exposed to retinoic acid (RA), an essential differentiationinducing signaling molecule that is synthesized in a spatiallyubiquitous but temporally periodic manner. Intriguingly,
however, all of Aundiff cell do not differentiate, rather someAundiff always remain undifferentiated. Although this process
is extremely important for the long-term maintenance of thestem cells and the integrity of spermatogenesis, the underlying mechanism has been largely unknown.

2-2. H eterogeneous differentiation competence in
response to retinoic acid

Figure 2. Anatomy of seminiferous tubules and seminiferous epithelium.
GFRα1+ and Ngn3+ cells and KIT+ differentiating spermatogonia, residein the basal compartment (between the basement membrane and thetight junction of Sertoli cells). Modified from Ikami et al., Development(2015).

To address this issue, we tested the response of Ngn3 +and GFRα1+ spermatogonia to RA by pulse-labeling these
populations using the tamoxifen-inducible cre-loxP system.
The results indicated that Ngn3+ cells rapidly and efficientlydifferentiated into KIT+ cells in response to RA. In contrast,GFRα1+ cells did not show effective differentiation induced
by RA, but they generated Ngn3+ cells in an RA-independentmanner. Thus, GFRα1+ and Ngn3+ cells show distinctive differentiation competence in response to RA.
Next, through DNA microarray gene expression analysisof FACS-sorted GFRα1+ and Ngn3+ fractions, we found thatthe expression of Retinoic acid receptor gamma (RARγ) was
highly specific in Ngn3+ cells compared to GFRα1+ cells(Figure 4). In contrast, the levels of other transcripts related
to the RA signaling pathway were similar between thesecells, Further, we found that enforced expression of RARγ
provided the GFRα1+ cells with differentiation competence
to differentiate to KIT+ cells in response to RA.

25

National Institute for Basic Biology

Developmental Biology
Publication List:〔Original paper〕

K., Tokue, M., Sugimoto, R., Noda, C., Kobayashi, S., Hara,• Ikami,K., and Yoshida, S. (2015). Hierarchical differentiation competence in

response to retinoic acid ensures stem cell maintenance during mouse
spermatogenesis. Development 142, 1582-1592.

〔Review Article〕

S. (2015). From cyst to tubule: innovations in vertebrate• Yoshida,spermatogenesis. WIREs Developmental Biology, doi: 10.1002/
wdev.204

Figure 4. Predominant expression of RARγ in NGN3+ spermatogonia
compared with GFRα1+ cells.
Representative images of whole-mount seminiferous tubules from Ngn3EGFP mice stained for EGFP, GFRα1 and RARγ. Scale bars = 50 μm.
Reproduced from Ikami et al., Development (2015)

2-3. Differential dynamics of GFRα1+ and Ngn3+ spermatogonia

To summarize, these results inferred that, when exposed toa high concentration of RA, RARγ drives the Ngn3+ cells
to differentiation, while the absence of RARγ allows theGFRα1+ cells to remain undifferentiated. This heterogeneous
differentiation competence, combined with the periodicallybut ubiquitously distributed RA, appears to be importantfor homeostatic spermatogenesis in the open niche environment of seminiferous tubules (Figure 5). This scenario
would be paradigmatic for other open niche-supported tissue
stem cells, since it would allow the stem cell population tomaintain the undifferentiated pool while periodically producing differentiating progeny.

Figure 5. A model of stem cell dynamics during the seminiferous epithelial cycle.
Before the elevation of RA, the entire Aundiff population is composed of a
mixture of GFRα1+ (RARγ­–) and Ngn3+ (RARγ+) cells (top). When theRA amount increases, to which both GFRα1+ and Ngn3+ spermatogonia
appear to be equally exposed (right), only Ngn3+ cells that express RARγ
respond and differentiate into Kit + cells, while GFRα1+ cells remain
undifferentiated (bottom). Then, the GFRα1+ cells replenish Ngn3+ cells
through a RA-independent mechanism (left). Modified from Ikami et al.,Development (2015)26