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:
IKEDA, Tatsuro
TOKUE, Moe
ISHIZAKA, Miho
SOKENDAI Graduate Student: ISHIZAKA, Miho*
HIRANO, Kodai
Visiting Graduate Student: SATO, Toshiyuki
Visiting Undergraduate: YOSHIDO, Kana
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,and their connection to 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, 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 from this division related to these studies
that are currently public include Nakagawa et al., Dev. Cell
2007; Yoshida et al., Science 2007; Nakagawa et al., Science2010; Klein et al., Cell Stem Cell 2010; and Hara et al., Cell

Stem Cell 2014, Ikami et al., Development 2015, and Tokueet al., Stem Cell Reports 2017.

I. 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 mitotic
division process with which cytokinesis does not complete
and the cytoplasmic connection between daughter cells
persists via intercellular bridges. The prevailing stem celltheory proposed in 1971 states that stem cell activity isrestricted to the population of As cells, while interconnected
Apr and Aal syncytia are irreversibly committed to differentiation and no 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 “As model”. Thisis the simplest interpretation of the results of our funcWLRQDO DQDO\VHV RI *)5Ơ+ 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
KLHUDUFK\ *)5Ơ+ spermatogonia comprise a single stem cell pool, inwhich 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+FHOOV$IWHU OHDYLQJ WKH *)5Ơ+ compartment, differentiation-destined
FHOOVIROORZDVHULHVRIWUDQVLWLRQV *)5Ơ+ĺNGN3+ĺKIT+; downwardblack arrows) that accompany the extension of syncytial length (rightward black arrows). Ngn3+ and, to a lesser extent, KIT+ cells retain theFDSDFLW\ WR UHYHUW EDFN LQWR WKH *)5Ơ FRPSDUWPHQW LQ D FRQWH[W
dependent fashion (broken arrows). (Reprinted from Hara et al., Cell
Stem Cell 2014.)

As crystalized in this model, our results suggest that the*)5Ơ + 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 overNote: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

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National Institute for Basic Biology

Developmental Biology

a particular “quota” would overflow to become the NGN3+
state of Aundff, which then further differentiate into KIT+ “differentiating” spermatogonia that are largely devoid of selfrenewing potential.
Currently, we are investigating the mechanism that deterPLQHV WKH TXRWD RU WLVVXH FDSDFLW\ RI *)5Ơ+ cells, as wellas the detailed nature of the internal heterogeneity of theSRSXODWLRQ RI *)5Ơ+ cells. Further, it is also becoming
DSSDUHQWWKDWWKH*)5Ơ+ population is not totally homogeneous but associated with some heterogeneity in their gene
expression and fate behavior, as described by our group (seebelow) and others. Therefore, our next challenges include
understanding the overall scheme of the stem/progenitor cell
dynamics in which short-term heterogeneity and long-term
equipotency (randomness) are both observed in a compatible
manner.

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 outside of which they differentiate (Fuller and Spradling, 2007; Morrison and Spradling,2008). However, the mouse spermatogenic stem cell systemappears to not show a defined facultative niche environmentand does not rely on asymmetric division; their fates arefound to be 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).

:QWơFDWHQLQ VLJQDOLQJ SURPRWHV GLIIHUHQWLDWLRQ
RI*)5Ơ+ to NGN3+ cells++7R XQYHLO WKH VLJQDO WKDW SURPRWHV WKH *)5Ơ to NGN3transition. We first compared the gene expression profilesEHWZHHQ *)5Ơ+ and NGN3+ cells by cDNA microarray.
From the microarray data, we focused on Wnt signaling. In
FXOWXUHGVSHUPDWRJRQLD YL]*6FHOO :QWơFDWHQLQVLJQDOing induced Ngn3 expression (Figure 2A). Moreover, micecarrying a gain-of-function mutation of ơFDWHQLQ showed
VSHUPDWRJHQHVLV GHIHFWV LQ ZKLFK WKH QXPEHU RI *)5Ơ+cells was reduced (Figure 2B), whereas no significant reducWLRQ LQ UHWLQRLF DFLG UHFHSWRU JDPPD 5$5Ƣ + cells (largelycorresponding to NGN3+ cells) was observed. Consequently,
WKH 5$5Ƣ + FHOOWR*)5Ơ + cell ratio increased, consisWHQW ZLWK WKH LGHD WKDW *)5Ơ+ to NGN3+ differentiation
increased.

36)LJXUH  :QWơFDWHQLQ VLJQDO SURPRWHV WKH GLIIHUHQWLDWLRQ RI *)5Ơ+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 P51$ OHYHO % $YHUDJH QXPEHUV RI *)5Ơ+ cells per tubule,
REVHUYHGLQPLFHZLWKLQGLFDWHGJHQRW\SHV1XPEHURI*)5Ơ+ cells was
reduced in Nanos3Cre/+;Ctnnb1 fl(ex3)/fl(ex3) miceLQZKLFKơFDWHQLQZDVDFWLvated in a germ cell specific manner. Modified from Tokue et al., Stem
Cell Reports (2017),

2-2. SHISA6, a cell-autonomous Wnt inhibitor, is
H[SUHVVHGLQDVXEVHWRI*)5Ơ+ 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 differentiationSURPRWLRQ LQ VHPLQLIHURXV WXEXOHV DQG WKDW *)5Ơ+ 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
LQKLELWRU V  PD\ FRQIHU UHVLVWDQFH WR :QWơFDWHQLQ VLJQDOLQJLQVRPHIUDFWLRQRI*)5Ơ+ cells. In our microarray data,
we found Shisa6 an interesting candidate, because Shisa6ZDV KLJKO\ HQULFKHG LQ WKH *)5Ơ+ fraction and some ofthe 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 in about 30% ofWKH*)5Ơ+ population in vivo (Figure 3B).

2-3. SHISA6+ spermatogonia show stem cell characters
DQGFRQWULEXWHWRPDLQWHQDQFHRI*)5Ơ+ cells
We found that, although Shisa6 KO or stabilized ơFDWHQLQ
heterozygous mutants show no apparent phenotype in the
SRRO RI *)5Ơ+ cells, synthetic heterozygotes for these
DOOHOHV VKRZHG D UHGXFHG *)5Ơ+ cell pool and spermatogenesis defects. Thus, SHISA6 plays a role in the mainteQDQFH RI WKH VWHP FHOO SRRO E\ VXSSUHVVLQJ :QWơFDWHQLQ
signaling 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 continually 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 3. Function and expression of SHISA6 (A) Cell-autonomous
LQKLELWLRQ RI :QWơFDWHQLQ VLJQDOLQJ E\ 6+,6$ +(.7 FHOOV
were transfected with a Wnt reporter luciferase construct and a Shisa6expression plasmid either simultaneously (co-transfection), or separately(transfected cells were mixed afterword), followed by stimulation of:QWơFDWHQLQ VLJQDO 7KH :QWGHSHQGHQW /XFLIHUDVH DFWLYLW\ ZDV VXSpressed only when Shisa6 was co-transfected. (B) Representative imageof dissociated testicular cells of adult mice doubly stained for Shisa6by FISH and for GFR (B) Representative image. Note the overlapped
expression of Shisa6 and GFRsa is modified from Tokue et al., Stem CellReports (2017),)LJXUH $ FRQFHSWXDO GLDJUDP RI GLIIHUHQWLDWLRQ SURPRWLRQ E\ :QWơcatenin 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:〔Original Paper〕
M., Ikami, K., Mizuno, S., Takagi, C., Miyagi, A., Takada,• Tokue,R., Noda, C., Kitadate, Y., Hara, K., Mizuguchi, H., Sato, T., Taketo,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),M.M., Sugiyama, F., Ogawa, T., Kobayashi, S., Ueno, N., Takahashi,S., Takada, S., and Yoshida, S. (2017). SHISA6 confers resistanceto differentiation-promoting Wnt/beta-catenin signaling in mouse
spermatogenic stem cells. Stem Cell Rep. 8, 561-575.

2-4. SHISA6 protects the stem cells from differenWLDWLRQ SURPRWLQJ :QWơFDWHQLQ VLJQDOLQJ $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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