National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:

KITADATE, Yu
NAKAGAWA, Toshinori
Technical Staff:
MIZUGUCHI, Hiroko
NIBB Research Fellow:
HIRA, Seiji
JSPS Researcher:
NAKAMURA, Yoshiaki
Postdoctoral Fellow:
IKAMI, Kanako
TOKUE, Moe
SOKENDAI Graduate Student: NONAMI, Yuta
ISHIZAKA, Miho
HIRANO, Kodai
Visiting Graduate Student: SATO, Toshiyuki
Technical Assistant:
KON, Yayoi
MARUYAMA, Ayumi
NISHIMURA, Keiko
Secretary:
KUBOKI, 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 cell pool, while generating differentiation-destined
cells in a well balanced manner. The Division of Germ Cell
Biology aims to fully understand the mouse sperm stem cellsystem under the context of in vivo testicular tissue. Ourparticular interests have been laid on the “undifferentiatedspermatogonia”, which are responsible for the stem cell functions. Our study has revealed several key properties of thisinteresting population.
First, we found that this stem cell system includes a functional hierarchy. It is comprised of an “actual” stem cell compartment that is prone to self-renew, and a differentiationprimed, “potential” stem cell compartment. Regarding the“actual” stem cells, we have been investigating their cellularidentity, their in vivo behavior at a single-cell resolution,and the underlying mathematical principles. This lead to thediscovery of neutral competition between the stem cells. We
are currently investigating the molecular mechanism underlying their control of self-renewal and differentiation, andtheir connection to tissue environment. “Potential stem cells”
are also of our enthusiastic interest: In undisturbed, steadystate spermatogenesis, they largely contribute as transient
amplifying cells and differentiate rather than self-renew.
However, when tissues are insulted or testicular cells aretransplanted to a host testes, their probability of self-renewal
jumps up and they effectively replenish the lost “actual” stem
cells. Such a flexible, and probabilistic, feature of stem celldynamics 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,and Ikami et al., Development 2015.

30I. The identity of spermatogenic stem cells andtheir 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 cell
division 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 proposed model for the functional structure of the spermatogenic stem cell system, whichindeed 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-imagingexperiments, 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 asingle stem cell pool, in which cells continually interconvert
between these morphologically heterogeneous states throughstochastic incomplete division and fragmentation of syncytia.
The incomplete division and syncytial fragmentation causes
the expansion of this population, while the excess cells overa particular “quota” would overflow to become the 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 internal
heterogeneity of the population of GFRα1+ cells.

2. Mechanisms underlying the balanced
differentiation and self-renewal of stem cells
in an “open” stem cell niche

In general, stem cells support tissue homeostasis throughcontinual production of differentiating progeny from a poolof undifferentiated cells. This is traditionally thought tobe dependent on a couple of paradigmatic mechanisms: 1)“asymmetric cell division”, which always gives rise to one
self-renewing cell and one differentiating cell; 2) control
by “an anatomically defined niche”, inside of which stem
cells remain undifferentiated, but they differentiate outside(Fuller and Spradling, 2007; Morrison and Spradling, 2008).
However, the mouse spermatogenic stem cell system does
not show a defined niche environment and does not rely on
asymmetric division; their fates are stochastic (Hara et al.,2014; Klein et al., 2010; Klein and Simons, 2011).
In 2017, we challenged a fundamental question: How sperm
stem cells follow different fates (to differentiate or to selfrenew) in response to homogeneously distributed extracellular signals, in a facultative (or open) niche environment? The
result is published in Tokue et al., Stem Cell Reports (2017).

2-1. Wnt/β-catenin signaling promotes differentiationof GFRα1+ to NGN3+ cells

To unveil the signal that promotes the GFRα1+ to NGN3+transition, we first compared the gene expression profiles
between GFRα1+ and NGN3+ cells by cDNA microarray.
From the microarray data, we focused on Wnt signaling. In
cultured spermatogonia (viz. GS cell), Wnt/β-catenin signaling induced Ngn3 expression (Figure 2A). Moreover, micecarrying a gain-of-function mutation of β-catenin showedspermatogenesis defects, in which the number of GFRα1+cells was reduced (Figure 2B), whereas no significant reduction in retinoic acid receptor gamma (RARγ)+ cells (largelycorresponding to NGN3+ cells) was observed. Consequently,the RARγ + cell-to-GFRα1 + cell ratio increased, consistent with the idea that GFRα1+ to NGN3+ differentiation
increased.

Figure 2. Wnt/β-catenin signal promotes the differentiation of GFRα1+
spermatogonia in vitro and in vivo. (A) Expression of Ngn3 mRNA inGS cells in the presence or absence of GDNF or WNT3a. GS cells werestimulated by the indicated conditions and analyzed by RT-qPCR for theNgn3 mRNA level. (B) Average numbers of GFRα1+ cells per tubule,observed in mice with indicated genotypes. Number of GFRα1+ cells wasreduced in Nanos3Cre/+;Ctnnb1 fl(ex3)/fl(ex3) mice, in which β-catenin was activated in a germ cell specific manner. Modified from Tokue et al., Stem
Cell Reports (2017),

the other Shisa family members have been reported as Wnt
inhibitors. Experiments using Xenopus laevis embryos andluciferase assays in HEK293T cells showed that SHISA6 isa novel Wnt inhibitor that acts autonomously (Figure 3A).
Moreover, Shisa6 expression was restricted to about 30% of
the GFRα1+ population in vivo (Figure 3B).

2-3. SHSIA6+ spermatogonia show stem cell charactersand contribute to maintenance of GFRα1+ cells

We found that, although Shisa6 KO or stabilized β-cateninheterozygous mutants show no apparent phenotype in thepool of GFRα1 + cells, synthetic heterozygotes for thesealleles showed a reduced GFRα1+ cell pool and spermatogenesis defects. Thus, SHISA6 plays a role in the maintenance of the stem cell pool by suppressing Wnt/β-cateninsignaling in a cell-autonomous manner (Figure 4A).
We then addressed the behavior of SHISA6+ cells conjectured from that of T (Brachyury)+ cells, which were found tolargely overlap with SHISA6+ cells. A pulse-label and chaseexperiment showed that T+ cells have the ability to continu-2-2. a cell-autonomous Wnt inhibitor, is expressed in a
subset of GFRα1+ cells

We found that Wnt6 is expressed in Sertoli cells, a supporting somatic cell type, in a spatially ubiquitous manner,suggesting that WNT6 is participating in the differentiationpromotion in seminiferous tubules, and that GFRα1+ cells
are uniformly exposed to WNT6 signals. These raised a nextquestion: How do stem cells persist without exhaustion if
they are equally bathed with Wnt ligand(s)?
To address this issue, we hypothesized that a Wnt
inhibitor(s) may confer resistance to Wnt/β-catenin signaling
in some fraction of GFRα1+ cells. In our microarray data, wefound Shisa6 tobe an interesting candidate, because Shisa6
was highly enriched in the GFRα1+ fraction and some ofFigure 3. Function and expression of SHISA6 (A) Cell-autonomous
inhibition of Wnt/β-catenin signaling by SHISA6. HEK293T cells
were transfected with a Wnt reporter luciferase construct and a Shisa6
expression plasmid either simultaneously (co-transfection), or separately(transfected cells were mixed afterword), followed by stimulation ofWnt/β-catenin signal. The Wnt-dependent Luciferase activity was suppressed only when Shisa6 was co-transfected. (B) Representative imageof dissociated testicular cells of adult mice doubly stained for Shisa6by FISH and for GFR (B) Representative image. Note the overlapped
expression of Shisa6 and GFRsa is modified from Tokue et al., Stem CellReports (2017),

31

National Institute for Basic Biology

Developmental Biology

ally produce progeny differentiating to sperm for at least 6months. Together, these results suggest that T+ (and probablySHISA6+) cells have stem cell-related characteristics (Figure4B).

Figure 4. SHISA6 and spermatogenic stem cells (A) SHISA6 maintains
the GFR S+ cell pool through inhibiting the Wnt/l pool through i. Average
number of GFRve+ cells per tubule section in testes with the indicatedgenotypes, based on double IF for GFR microtesticular sections. (B)(Upper) A schematic of the structure of the TnEGFP-CreERT2 allele, whichenables pulse-labeling of T+ cells using CreERT2. (Lower) A result ofpulse-labelling of T+ cells with 6 months of chase. Note that GFP (thelineage reporter) positive cells form a prominent patch in a particularsegment of seminiferous tubules. Modified from Tokue et al., Stem Cell
Reports (2017),

2-4. SHISA6 protects the stem cells from differentiation promoting Wnt/β-catenin signaling: A
proposal of a generic mechanism of stem cell regulation in facultative niches

To conclude, we propose a generic mechanism underlying the heterogeneous stem cell fates in facultative niche
environments. Different levels of cell-autonomous inhibitor(SHISA6, in this case) may confer heterogeneous resistanceto uniformly distributed extracellular signaling that promotes
differentiation (such as WNTs). Here, stem cells with higher
levels of inhibitors would remain in the undifferentiated cellpool with higher probabilities, while those with lower levels
of inhibitors are more inclined to differentiate (Figure 5).

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Figure 5. A conceptual diagram of differentiation promotion by Wnt/βcatenin signaling to become Ngn3+ and its cell-autonomous protection
by SHISA6 in a subset of GFRd + cells. The heterogeneous expression ofSHISA6 may confer different levels of differentiation probability in an
open niche environment in which differentiation-promoting Wnt ligand
appears to distribute uniformly. Modified from Tokue et al., Stem Cell
Reports (2017),

Publication List:〔Review Article〕

S. (2016). From cyst to tubule: innovations in vertebrate• Yoshida,spermatogenesis. WIREs Developmental Biology 5, 119-131.