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:
IKEDA, Tatsuro
HIRA, Seiji*
JSPS Researcher:
NAKAMURA, Yoshiaki*
Postdoctoral Fellow:
IKEDA, Tatsuro*
HIRA, Seiji
Research Staff:
ISHIZAKA, Miho
SOKENDAI Graduate Student: HIRANO, Kodai
Visiting Graduate Student: SATO, Toshiyuki
Visiting Undergraduate: YOSHIDO, Kana
Visiting Scientist:
NAKAMURA, Yoshiaki
Technical Assistant:
KON, Yayoi
MARUYAMA, AyumiNISHIMURA, Keiko
FUJITA, Miyako
Secretary:KUBOKI, Yuko

Overview of our research

Production of numerous sperm over a long period in thetestis is fundamental for continuity of life across generations.
The Division of Germ Cell Biology aims to understand thekey processes of germ cell development that lead to robust
sperm formation.
Our emphasis has specifically been on the regulation andfunctional behavior of spermatogenic stem cells (SSCs) inthe testes of mice. SSCs play a crucial role, not only as theorigin of long-term sperm production ensuring the continuityof life, but also as the crucible of genetic and potentially epigenetic mutations, the underpinning of evolution as well ascongenital disease mechanisms. The principal and signature
strategy of our research is to directly observe the behavior
of individual germ cells in vivo in their natural (i.e. physiological) state and take advantage of intravital live-imaging,lineage analysis, and biophysical analysis.
Regarding the study of SSCs, first, we found that SSCs
include a functional hierarchy, comprised of an “actual” stem
cell compartment that is able to self-renew, and a differentiation-primed, “potential” stem cell compartment. We have been
also investigating the cellular identity of “actual” SSCs, theirin vivo behavior at a single-cell resolution, and the underlying mathematical principles, leading to the discovery of thedynamics of “population asymmetry” and subsequent “neutralcompetition” between SSCs. We are also highly interested in
the area of “potential stem cells”. In steady-state spermatogenesis, these largely contribute as transient amplifying cells
and differentiate rather than self-renew. However, when tissues
are insulted or testicular cells are transplanted into host testes,
their probability of self-renewal increases and they effectively
replenish the lost “actual” stem cells. Such flexible and probabilistic features of stem cells have been found to be paradigmatic for many other stem cell-supported tissues.
Our past key references related to these studies include
Nakagawa et al., Dev. Cell 2007; Yoshida et al., Science2007; Nakagawa et al., Science 2010; Klein et al., Cell StemCell 2010; Hara et al., Cell Stem Cell 2014, Ikami et al.,Development 2015, and Tokue et al., Stem Cell Reports 2017.

Mechanism of “mitogen competition” regulates thestem cell density homeostasis in an “open” niche

In 2018, we developed a novel hypothesis regarding themechanism of SSC maintenance in the mouse testis (Kitadate
et al., Cell Stem Cell 2019). In contrast to better-investigatedtissues such as Drosophila testes and ovaries, as well asmammalian intestinal crypts, where physical contact madeby stem cells with an anatomically-defined niche provides aplatform to maintain homeostasis, the question of how stem
cell homeostasis is achieved in the mouse testis remains
unknown. In the mouse testes’ seminiferous tubules, theplace where spermatogenesis proceeds, SSCs are motile
and dispersed among their progeny, thus representing aclass of microenvironment designated as an “open niche”(Yoshida, Dev. Growth. Diff. 2008). Intriguingly, the densityof SSCs (defined as GFRα1+ cells) is strikingly constantwhen averaged over a certain length (e.g., >1cm), despite
local fluctuations. This suggests a presence of an unknown
mechanism that regulates the SSC homeostasis in a mannerthat stabilizes their average density.

I. F GF5 expression near the vasculatureand its mitogenic function on GFRα1 +spermatogonia

In order to find the key factors regulating the GFRα1+ celldensity, we screened the genes expressed in the tubule area

Figure 1. (A) Outline of the screening for genes preferentially expressedin the vasculature-associated region. (B) Representative ISH images for
Fgf5 (blue) in testis sections, counterstained with nuclear fast red. (C)Representative image of an inter-tubular region of a GFRα1-GFP mouse
testis stained for GFP (green), FGF5 (magenta) and DNA (blue). Scalebars, 10 µm. (D) Mitogenic effect of FGF5 (red) or FGF2 (blue) oncultured spermatogonia. Fold increase in the number of GS cells cultured
with indicated concentration of FGF5 for 8 days. Shown in average ± SEM(n = 3 independent experiments). (E) Effects of FGF5 on gene expression.
GS cells depleted of FGF2 and GDNF for 3 days were supplemented withor without FGF5 (100 ng/ml) for 24 hours, followed by cDNA microarray
analyses. * intertubular arterioles/venules All figures are reproduced frommodifications taken from Kitadate et al., Cell Stem Cell (2019).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2018. The formertitle isindicated by an asterisk (*).

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National Institute for Basic Biology

Developmental Biologythat were facing the interstitium and vasculature, whereGFRα1+ cells preferentially locate (Figure 1A). From thisscreening, we focused on Fgf5, since it is located in peritubularly located large flattened cells (termed lymphatic endothelial cells) near the interstitium (Figures 1B and C). By
using an in vitro spermatogonial culture, we found that FGF5
showed mitogenic and anti-differentiation effects (Figures1D and E).

II. F GF5 controls GFRα1 + cell density in alinear dosage-dependent manner

We then investigated the in vivo role of FGF5 in micecarrying a null allele (Fgf5–) or an extra copy of BACmediated transgene (BAC-Fgf5 Tg). In mutant testes, theaverage density of GFRα1+ spermatogonia showed a strikingly linear correlation with Fgf5 dosage (Figure 2A). Thesemutants notably sustained steady-state spermatogenesis withdifferent density set points of GFRα1+ cells that correlate ina manner that depends linearly on Fgf5 dosage during adulthood (Figure 2B).

IV. GFRα1+ spermatogonia consume FGF5

To further develop this hypothesis, we then examined
whether GFRα1 + cells consume the extracellular FGF5
when they receive its signal in vivo, as this was probably
the simplest form of competition consistent with the generalmechanism of FGF signal reception by target cells. Wedetected speckled FGF5 signals inside GFRα1+ cells (Figure3A). Significant portions of FGF5 cytoplasmic signals wereco-localized with SDC4 as cytoplasmic puncta on LAMP1+lysosomes (Figures 3B). These observations indicated that
GFRα1+ cells consume extracellular FGF5, supporting theidea that GFRα1+ cells compete for extracellular FGF.

Figure 3. (A) Representative images of GFRα1-GFP+ (cyan) cells exhibiting the speckled cytoplasmic staining of FGF5 (magenta, white arrowheads). (B) Representative images of SDC4+ (magenta) cells co-stainedfor FGF5 (green) with LAMP1 (cyan). FGF5+ speckles (arrows) wereoften co-localized with LAMP1+ (arrowheads) foci in SDC4+ cytoplasmic
clamp. Scale bars, 10 µm.

V. H omeostatic stem cell density regulationf o l l o w s f ro m a m o d e l o f “ m i t o g e ncompetition”

Figure 2. (A) Average densities ± SEM of GFRα1+ cells and representative IF images of whole-mount seminiferous tubules for GFRα1 (right)of 2.5 month-old mice with indicated genotypes. Scale bar, 50µm. (B)Average densities ± SEM of GFRα1+ cells in mice with indicated genotypes and ages. (C–E) Indexes of proliferation (EdU+ and pH3+ fractions),
differentiation [quantified as the RARγ+/KIT– (≈NGN3+) over GFRα1+cell ratio], and death (cPARP+ fraction) in GFRα1+ cells of indicatedmice at 2.5 months of age.

III. Each GFRα1+ cell receives an unchanged
level of FGF signal in Fgf mutants
Given the mitogenic and differentiation-inhibiting functions of FGF5, we first considered whether GFRα1+ cells
receive altered levels of FGF signal in mutants, which in turnchanges their fate resulting in altered densities. However, therates of proliferation, differentiation and death of GFRα1+cells, as well as their gene expression profile, surprisingly
were not different between Fgf5–/–, BAC-Fgf5Tg/+ and WT
mice (Figures 2C–E; not shown), indicating conserved fatebehavior of GFRa1+ cells between mutants.
The above findings lead to a hypothesis that FGF supply
is a limiting factor that is competed for among the GFRα1+cells. In this case, the levels of FGF signal received by each
GFRα1+ cell would be equalized among Fgf mutants harboring different densities of GFRα1+ cells.

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To understand the mechanism of density regulation, wedeveloped a minimal theoretical model, in which GFRα1+cells are exposed to a steady supply of FGF from the lymphatic endothelial cells, which in turn elicits the concentration-dependent mitogenic and differentiation-inhibitingactivities, while also being consumed by, the GFRα1+ cells.
This model was found to be capable of capturing the counterintuitive observation that the fate behavior of GFRα1 +cells does not change in Fgf mutants (Figures 2C–E), as aconsequence of the steady-state FGF concentration being
always pinned and at which the increase (renewal) anddecrease (differentiation) of GFRα1+ cells is balanced. Given
that GFRα1+ cells effectively compete with each other forthe limited supply of FGFs, we refer to this mechanism as
the “mitogen competition ”.
We then questioned whether the model could also predict
quantitatively the dynamics of stem cells, if the sytem isstrongly perturbed from a steady state. Analysis of the model
predicted that the recovery of GFRα1+ cell density following
a strong perturbation from its steady-state (viz. uninjured)value would show decaying oscillations. To test this prediction, we examined the kinetics during regeneration following the reduction of GFRα1+ cells by injecting a cytotoxic
reagent, busulfan. Strikingly, decaying oscillations that converged into the steady-state value over several months wereindeed observed, with a profile that quantitatively matchedwith our theory (Figure 4).

Y., Takahashi S., *Simons, B.D., and *Yoshida, S. Competition formitogens regulates spermatogenic stem cell homeostasis in an open
niche. Cell Stem Cell. 2018 Dec 20. (*: Co-corresponding authors)〔Review Articles〕

S. (2018). Open niche regulation of mouse spermatogenic• Yoshida,stem cells: Develop. Growth Differ. 60, 542-552.
S. (2018). Regulatory mechanism of spermatogenic stem
• Yoshida,
cells in mice: their dynamic and context-dependent behavior, In

Figure 4. Observed kinetics of the GFRα1 + cell density following
busulfan treatment in WT (left), and examples of IF images of wholemount seminiferous tubules stained for GFRα1 (right). Model results(curves) compared to experimental measurements (dots) of the average
GFRα1+ cell density following busulfan treatment.

Reproductive and Developmental Strategies, K. Kobayashi, T. Kitano,Y. Iwao, and M, Kondo, eds. (Springer Japan), pp. 47-67.

To summarize, our results show that the in vivo fatebehavior of GFRα1+ cells is regulated by the mitogenic andanti-differentiation effects of FGFs released in proximity
to the vasculature (Figure 5). We propose that competition
for mitogens might be a paradigmatic mechanism that canexplain the regulation of stem cell homeostasis in a wide
range of tissues.

Figure 5. A conceptual diagram of mitogen competition leading to stem
cell density homeostasis. Limited supply of FGF from a subset of lymphatic endothelial cells in proximity to vasculature plays a key role instem cell density homeostasis.

Publication List:〔Original Papers〕

S., Thumkeo, D., Ohta, H., Zhang, Z., Huang, S.R.,• Sakamoto,Kanchenawong, P., Fuu, T., Watanabe, S., Shimada, K., Fujihara, Y.,•Yoshida, S., Ikawa, M., Watanabe, N., Saitou, M., and Narumiya, S.
(2018). mDia1/3 generate cortical F-actin meshwork in Sertoli cells
that is continuous with contractile F-actin bundles and indispensable
for spermatogenesis and male fertility. PLoS Biol. 16, e2004874.
Shawki, H.H., Oishi, H., Usui, T., Kitadate, Y., Basha, W.A., Abdellatif,A.M., Hasegawa, K., Okada, R., Mochida, K., El-Shemy, H.A.,
Muratani, M., Ogura, A., Yoshida, S., and Takahashi, S. (2018). MAFB
is dispensable for the fetal testis morphogenesis and the maintenance ofspermatogenesis in adult mice. PLoS ONE 13. e0190800.

〔Original Paper (E-publication ahead of print)〕

Y., Jörg, D.J., Tokue, M., Maruyama, A., Ichikawa, R.,• Kitadate,
Tsuchiya, S., Segi-Nishida, E., Nakagawa T., Uchida, A., KimuraYoshida, C., Mizuno, S., Sugiyama, F., Azami, T., Ema, M., Noda,C., Kobayashi, S., Matsuo, I., Kanai, Y., Nagasawa, T., Sugimoto,41