© 2016. Published by The Company of Biologists Ltd.

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis

Chang Liu1, Karina Rodriguez, and Humphrey H-C Yao*

Reproductive and Developmental Biology Group, National Institute of Environmental

Health Sciences, Durham, North Carolina, USA.

1Current address: Genetic and Developmental Biology Center, National Heart, Lung, and

Blood Institute, Bethesda, Maryland, USA

*Correspondence to:

Email: humphrey.yao@nih.gov

Phone: 919-541-1095

Key words: Testis, lineage specification, Sertoli cells, Leydig cells, Notch, Hedgehog

Summary statement:

Somatic progenitor cell populations in the testis are defined by progressive lineage-

specific acquirement of WT1, HES1, SOX9, and GLI1 beginning at the time of sex

determination.

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http://dev.biologists.org/lookup/doi/10.1242/dev.135756Access the most recent version at First posted online on 12 September 2016 as 10.1242/dev.135756

Abstract

Testis morphogenesis is a highly orchestrated process involving lineage determination

of male germ cells and somatic cell types including supporting (Sertoli cells) and

interstitial cells (Leydig cells and others). While the origin and differentiation of germ

cells are known, the developmental course specific for each somatic cell lineage has not

been clearly defined. Here we construct a comprehensive map of somatic cell lineage

progression in the mouse testis. Both supporting and interstitial cell lineages arise from

WT1+ somatic progenitor pool in the gonadal primordium. A subpopulation of WT1+

progenitor cells acquire SOX9 expression and become Sertoli cells that form the testis

cords, whereas the remaining WT1+ cells contribute to progenitor cells in the testis

interstitium. Interstitial progenitor cells further diversify through the acquisition of

HES1 expression, an indication of Notch activation, at the onset of sex determination.

Once Sertoli cells differentiate they produce Hedgehog signals that induce GLI1

expression in the HES1+ interstitial progenitor cells. The interstitial cells eventually

developed into two cell lineages: steroid-producing fetal Leydig cells and non-

steroidogenic cells. The steroid-producing fetal Leydig cell population is restricted by

Notch2 signaling from the neighboring somatic cells. The non-steroidogenic progenitor

cells retain their undifferentiated state during fetal stage and give rise to adult Leydig

cells in the post-pubertal testis. These results provide the first lineage progression map

that illustrates the sequential establishment of supporting and interstitial cell

populations in testis morphogenesis, and reveal the specific involvement of Notch2 in

controlling steroid-producing fetal Leydig cell population.

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Introduction:

Morphogenetic transformation of gonadal primordium into a testis begins

around embryonic day (E) 11.5 and 12.5 in mice (Brennan and Capel, 2004). Before this

period, gonadal primordium is morphologically indistinguishable between XX and XY

embryos (Brennan and Capel, 2004; DeFalco and Capel, 2009). Around E11.5, testis

morphogenesis is initiated by the Y chromosome-linked Sry gene (Gubbay et al., 1990;

Hawkins et al., 1992; Koopman et al., 1991; Lovell-Badge and Robertson, 1990), which

is expressed in the supporting cell lineage Sertoli cells of the XY gonads (Albrecht and

Eicher, 2001; Schmahl et al., 2000). SRY induces the differentiation of Sertoli cells

through a positive feedback loop between SOX9 and FGF9 (Chaboissier et al., 2004; Kim

et al., 2006; Palmer and Burgoyne, 1991; Schmahl et al., 2004; Willerton et al., 2004).

Sertoli cells then orchestrate formation of testis cords, a hallmark structure that

separates Sertoli cells and germ cells from the interstitium (Brennan and Capel, 2004).

The coelomic epithelium, which encloses the gonad and mesonephros, has been

described as one source of Sertoli cells and interstitial cells (Brennan and Capel, 2004;

Karl and Capel, 1998; Schmahl et al., 2000; Tanaka and Nishinakamura, 2014).

In contrast to Sertoli cells, which are a homogenous population within testis

cords, the cell types in the testis interstitium are diverse. The testis interstitium harbors

the steroidogenic Leydig cells, peritubular myoid cells, macrophages, vasculature, and

other uncharacterized cell types such as fibroblasts and vascular-associated cells

(Brennan and Capel, 2004; DeFalco et al., 2014) In the mouse, steroidogenic Leydig cells

consist of two populations based on the time of their appearance: fetal and or adult

Leydig cells (Benton et al., 1995; Huhtaniemi and Pelliniemi, 1992). Fetal Leydig cells

serve as the primary source of androgens that virilize the embryos. The population of

fetal Leydig cells declines after birth and is eventually replaced by the adult Leydig cells

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at puberty. Adult Leydig cells maintain androgen production throughout adulthood,

functionally replacing fetal Leydig cells (Griswold and Behringer, 2009; Habert et al.,

2001). Despite their similar functions in producing androgens, fetal and adult Leydig

cells exhibit many differences in their transcriptomes (Dong et al., 2007; Shima et al.,

2013), morphology (Haider, 2004) and regulation (Agelopoulou et al., 1984; Aubert et

al., 1985; Baker and O'Shaughnessy, 2001; Dong et al., 2007; El-Gehani et al., 1998;

Gangnerau and Picon, 1987; Ma et al., 2004; Majdic et al., 1998; O'Shaughnessy et al.,

1998; Patsavoudi et al., 1985; Zhang et al., 2001). These differences between fetal and

adult Leydig cells lead to the hypothesis that the two Leydig cell populations are in fact

distinct cell lineages arising from separate origins (Baker et al., 1999; Haider, 2004;

Kerr and Knell, 1988; Lording and De Kretser, 1972; O'Shaughnessy et al., 2003;

O'Shaughnessy and Fowler, 2011; Roosen-Runge and Anderson, 1959; Shima et al.,

2013). In fact, multiple origins of fetal Leydig cells have been suggested, including Sf1+

non-steroidogenic interstitial cells originated from the gonadal primordium (Barsoum

et al., 2013; Barsoum and Yao, 2010), mesonephros (Merchant-Larios and Moreno-

Mendoza, 1998; Val et al., 2006), neural crest (Mayerhofer et al., 1996), coelomic

epithelium (Karl and Capel, 1998), and cells residing in the border between gonad and

mesonephros (DeFalco et al., 2011). On the other hand, adult Leydig cells have been

suggested to stem from peritubular interstitial progenitor cells that arise in the adult

testis (Davidoff et al., 2004; Ge et al., 2006; Li et al., 2016; Odeh et al., 2014; Stanley et al.,

2012), or COUP-TFII-positive interstitial cells in the fetal testis (Kilcoyne et al., 2014).

Although significant advancement has been made in identifying the molecular

mechanisms underlying testis morphogenesis, how these molecular pathways integrate

and the progenitor cells respond to them remain unclear. This deficiency in the field is

largely attributed to a lack of clear definition of various cell types in the testis. In this

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study, we delineated the lineage progression of somatic progenitor cells in the mouse

testis by genetic lineage-tracing experiments in vivo. In addition, we explore the

mechanisms for the lineage assignment with a particular focus on the testis interstitium.

Results:

Wt1+ somatic cells are progenitors for both supporting and interstitial cells in the

testis

One of the earliest markers that define the somatic cell lineages in the gonads is

Wilms’ tumor 1 (WT1), a transcription factor essential for gonadogenesis (Armstrong et

al., 1993b; Kreidberg et al., 1993). At E11.5 when testis morphogenesis begins,

endogenous WT1 protein was present in somatic cells but absent in the PECAM-1

positive germ cells and endothelial cells of fetal testes (Fig. S1A-C). One day later at

E12.5, while its expression remained in coelomic epithelial cells, WT1 protein inside the

testis became mostly restricted to AMH-positive Sertoli cells within the testis cords and

its presence was significantly reduced in the interstitium surrounding testis cords (Fig.

S1D-F). This pattern of WT1 expression, which is consistent with previous observations

(Armstrong et al., 1993b), implies that WT1+ somatic progenitor cells in the gonadal

primordium could either give rise to only Sertoli cells, or its expression is extinguished

in the interstitial cell populations. To test these possibilities, we utilized a tamoxifen-

induced Rosa-LSL-tdTomato lineage-tracing model, in which Wt1+ cells were labeled

permanently with tdTomato fluorescent protein in the presence of tamoxifen (Liu et al.,

2012). We administered a single tamoxifen injection to pregnant mice carrying Wt1-

CreERT2; Rosa-LSL-tdTomato embryos at E10.5, before the onset of testis morphogenesis

(Brennan and Capel, 2004; Eggers et al., 2014). The dose (1 mg/mouse) and frequency

(one injection) of the tamoxifen treatment induced recombination for ~24 hours, so

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that all tdTomato-positive cells are derived specifically from the WT1+ cell population

between E10.5 to E11.5 (Liu et al., 2015). At E11.5, or 24 hours after tamoxifen

treatment, the lineage-labeled Wt1+ cells were present in the somatic compartment of

the fetal testis, co-localizing with the endogenous WT1 protein (Fig. 1A-D and Fig. S2A-

D). At E13.5, the lineage-labeled Wt1+ cells became localized to not only the testis cords

but also the interstitium (Fig. 1E, H & I). To identify what cell types the lineage-labeled

Wt1+ cells become, we performed immunofluorescence for Sertoli cell maker SOX9 and

Leydig cell marker CYP17A1. The lineage-labeled Wt1+cells within the testis cords were

positive for SOX9, confirming their identity as Sertoli cells (Fig. 1E-H and Fig. S2E-H). On

the other hand in the interstitium, the lineage-labeled Wt1+ cells gave rise to two

subpopulations: CYP17A1+ steroidogenic fetal Leydig cells (arrows in Fig. 1K & L) and

CYP17A1- non-steroidogenic interstitial cells (asterisk in Fig. 1K & L; Fig. S2I-L). This

pattern of Wt1+ cell lineage contribution persisted in the postnatal testis: the linage-

labeled Wt1+ cells derived from E10.5 fetal testis were located in both seminiferous

tubules and the interstitial compartment delineated by laminin staining (Fig. S2M-T). In

addition to becoming SOX9+ Sertoli cells within testis cords (Fig. 1M-P), lineage-labeled

Wt1+ cells in the interstitium were also positive for 3HSD, a marker for Leydig cells

(Fig. 1Q-T). 3HSD-positive adult Leydig cells all contained Wt1-tdTomato lineage

labeling, strongly suggesting that E10.5 Wt1+ progenitor cells give rise to all

steroidogenic cells including adult Leydig cells. These results demonstrate that Wt1+

progenitor cells contribute to the supporting cell linage (Sertoli cells), the steroidogenic

cell lineage (Leydig cells), and non-vasculature and non-steroidogenic interstitial cells

in fetal and adult testes.

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Wt1+ progenitor cells give rise to Hes1+ cells that contribute to steroidogenic and

non-steroidogenic interstitial cells in the testis

To further define the interstitial cell populations, we searched for factors that

are involved in cell fate specification and maintenance. One such candidate is HES1, a

downstream effector of Notch signaling (Kageyama et al., 2007) implicated in fetal

Leydig cell differentiation (Tang et al., 2008). Hes1 mRNA expression is enriched in the

interstitial cells in the differentiated fetal testis based on in situ hybridization (Tang et

al., 2008) and sorted cell microarrays (Jameson et al., 2012). By analyzing fetal testes of

Hes1-GFP reporter embryos, we uncovered that as early as the onset of testis

morphogenesis (E10.5-E11.5), Hes1-GFP expression (indicative of endogenous Hes1

expression) was already present in a subpopulation of Wt1+ somatic progenitor cells

but absent in PECAM1+ germ cell population (Fig. 2A-H and Fig. S3A-D).

Coimmunostaining with Sertoli cell marker SOX9 showed that Hes1-GFP was primarily

located in the SOX9-negative somatic cells (Fig. S3E-H). In E12.5 and E16.5 testes, Hes1-

GFP was predominantly expressed in the interstitium of the testis and located in mainly

CYP17A1- population (Fig. S3I-P). To investigate what Hes1+ cells at E10.5 become later

in development, we used a lineage-labeling approach similar to the one used in the Wt1+

model in Figure 1. Instead of the Wt1+ cells, we lineage-labeled the Hes1+ cells in Hes1-

CreERT2; Rosa-LSL-tdTomato embryos at the onset of gonadal formation (E10.5) before

the separation of testis cords and interstitium. One day after the lineage labeling at

E11.5, we found that the lineage-marked Hes1+ cells represented a subpopulation of

Wt1+ somatic progenitor cells, consistent with the observation from the Hes1-GFP

mouse model (Fig. 2I-L). At E15.5, we stained the lineage-labeled testes with Leydig cell

marker CYP17A1 and observed three cell populations in the interstitium (Fig. 2M-P and

Fig. S4): 1) Hes1+/CYP17A1+ fetal Leydig cells (arrow in the inset), 2) Hes1-/CYP17A1+

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fetal Leydig cells (empty arrowhead), and 3) Hes1+/CYP17A1- non-steroidogenic

interstitial cells (solid arrowhead). Although some of the fetal Leydig cells originated

from the Hes1+ progenitor cells (Hes1+/CYP17A1+), the majority of the fetal Leydig cells

did not (Hes1-/CYP17A1+; Fig. 2M-P). This observation implies the presence of multiple

sources for fetal Leydig cells. To rule out the possibility that the three cell populations

in the interstitium is a consequence of insufficient lineage labeling, we enhanced the

efficiency of lineage labeling of the Hes1+ cells by administering tamoxifen to embryos

from E10.5 to E13.5 for four consecutive days (Fig. S5). Similar patterns of three

interstitial cell populations were observed in the testis, confirming the presence of

heterogeneous interstitial populations delineated by HES1 and CYP17A1. At one month

of age, the Hes1+ cells derived from the E10.5 fetal testis were found in the interstitium

and were positive for CYP17A1, indicating that these cells have become adult Leydig

cells (Fig. 2Q-T).

Differentiation of steroidogenic cells in the interstitium is mediated by Notch2-

signaling

Steroidogenic fetal Leydig cell population is sensitive to the level of Notch

activation: Inhibition of general Notch signaling pathway with a γ-secretase inhibitor

resulted in an increase in the number of fetal Leydig cells (Tang et al., 2008). Notch2,

one of the Notch receptors, is a potential candidate for Notch activation based on its

expression in the testis interstitium (Tang et al., 2008). We inactivated Notch2 in the

testis using a gonadal somatic cell specific Sf1-Cre (hereafter referred as Notch2 cKO).

At E13.5, Sf1-Cre-mediated recombination decreased about 50% of the Notch2

expression (Fig. 3A). The reduced Notch2 expression did not affect the development of

Sertoli cells (Dhh expression in Fig. 3A and SOX9 immunolocalization in Fig. 3B & C) and

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germ cells (Ddx4 expression in Fig. 3A and PECAM-1 immunolocalization in in Fig. 3B &

C). However, the population of CYP17+ fetal Leydig cells was slightly expanded in the

cKO testis compared to the controls (Fig. 3D & E and Fig. S6). Leydig cell-specific genes

such as Hsd3b1 and Nr5a1 were also significantly elevated (Fig. 3A). In the newborn

testes, where expression of Notch2 was more effectively depleted (Fig. 3F), the number

of CYP17+ fetal Leydig cells was greatly increased compared to that of control testes (Fig.

3G & H and Fig. S6), implicating Notch2 as the major Notch receptor in suppressing fetal

Leydig cell differentiation.

Hes1+cells from the gonadal primordium become positive for Gli1 as a result of

Hedgehog activation

In addition to the Notch pathway, the Hedgehog (Hh) pathway is important for

fetal Leydig cell differentiation (Yao et al., 2002). Unlike Notch signaling, which is

activated soon after the gonadal formation around E10.5 (Fig. 2A-H and Fig. S3A-H)

(Jameson et al., 2012; Tang et al., 2008), the Hh pathway does not become functional

until after the onset of testis morphogenesis at E12.5 (Barsoum and Yao, 2011; Jameson

et al., 2012; Yao et al., 2002). The Hh pathway, induced by the Hh ligand Desert

hedgehog (DHH) from Sertoli cells, was activated in the interstitium, which

consequently expresses Hh-responsive gene Gli1 (Barsoum and Yao, 2011; Yao et al.,

2002). Inactivation of Dhh, or inhibition of Gli1 along with another Hh downstream

target Gli2, resulted in a decrease of fetal Leydig cells (Barsoum and Yao, 2011; Yao et al.,

2002). The observations that both Hh and Notch signaling regulate fetal Leydig cell

differentiation suggest that these signaling may target the same type of cells in the testis.

We developed the Hes1-CreERT2; Rosa-LSL-tdTomato lineage tracing model as described

in Fig. 2 to label Notch-responsive cells at E10.5. This Hes1-CreERT2; Rosa-LSL-tdTomato

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mice was then bred to the Gli1-LacZ mice, in which the Hh-responsive Gli1 expression is

indicated by the presence of beta-galactosidase. The resulting mice (Hes1-CreERT2; Rosa-

LSL-tdTomato; Gli1-LacZ) allowed us to visualize Notch (tdTomato)- and Hh (LacZ)-

responding cells at the same time. At E14.5, almost all lineage-labeled Hes1+ cells

became positive for Gli1-LacZ expression (Fig. 4A-C and Fig. S7A-C), indicating that

Hes1+ progenitor cells in the gonadal primordium give rise to Gli1+ cells in the fetal

testis. Because Hes1+ cells in the gonadal primordium represent a subpopulation of

Wt1+ progenitor cells (Fig. 2A-H), we anticipate that some Wt1+ cells would also give

rise to Gli1+ cells. The idea was tested by the generation of Wt1-CreERT2; Rosa-LSL-

tdTomato; Gli1-LacZ mice followed by lineage-labeling the Wt1+ cells. As expected, the

fetal gonad-derived Wt1+ cells in the E14.5 testis interstitium were positive for Gli1-

LacZ and negative for Sertoli cell marker AMH, indicating that a subpopulation of Wt1+

cells become Gli1+ as they differentiate into interstitial cell linage (Fig. 4D-G and Fig.

S7D-F).

Next we examined the composition of Gli1+ interstitial cell populations by

characterizing endogenous Gli1 expression in details with coimmunostaining of Gli1-

LacZ and various somatic cell markers (Fig. 5A-D and Fig. S8). Gli1-LacZ expression was

present in a subpopulation of CYP17A1+ fetal Leydig cells (empty arrowhead in Fig. 5C

& D insets) and CYP17A1- non-steroidogenic interstitial cells (arrow in the Fig. 5C & D

insets) in the fetal and newborn testes (Fig. 5A-D and Fig. S8E-H & M-P). In addition,

Gli1-LacZ+ cells in proximity to testis cords were positive for myoid peritubular cell

marker αSMA+, suggesting that these Gli1+ cells are myoid peritubular cells. This pattern

of Gli1 expression resembles the lineage contributions of the Hes1+ progenitor cells (Fig.

2M-P and Fig. S7A-C), further supporting the notion that Hes1 and Gli1 mark the same

interstitial cell populations.

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Fetal testis-derived Gli1+ cells give rise to adult Leydig cells in the testis

We and others have proposed that the non-steroidogenic interstitial cell

populations in the fetal testis could be a source of steroidogenic cells in the adult testis

(Barsoum and Yao, 2010; (Kilcoyne et al., 2014). To test this hypothesis, we took

advantage of Gli1 expression in the non-steroidogenic cell populations (Fig. S8) and

developed a lineage tracing model (Gli1-CreERT2; Rosa-LSL-tdTomato embryos), which

allowed us to follow the fate of fetal-derived Gli1+ cells to adulthood. We labeled the

Gli1+ cells by a single injection of tamoxifen at E12.5, when Gli1 expression just started

to appear in the testis interstitium (Barsoum and Yao, 2011). At the time of birth, or

about 7 days after the lineage labeling by tamoxifen, fetal testis-derived Gli1+ cells

contributed to CYP17A1- non-steroidogenic interstitial cells (Arrow in Fig. 5E, G and H)

and a subpopulation of CYP17A1+ fetal Leydig cells (Solid arrowhead in Fig. 5G-H). Fetal

testis-derived Gli1+ non-steroidogenic interstitial population also gave rise to the cells

that were tightly associated with testis cords (Empty arrowhead, Fig. 5E, G and H),

consistent with the endogenous Gli1-LacZ expression pattern in the fetal testis (Fig. 5A-

D). At 2 months and 4 months of age when adult Leydig cells occupied the testis, the

fetal testis-derived Gli1+ cells were positive for CYP17A1 in the testis interstitium,

indicating that they have become adult Leydig cells in the adult testis (Fig. 5I-L).

However, not all the adult Leydig cells were derived from the Gli1+ cells in this

experimental model, as the presence of CYP17A1+/GLI1- adult Leydig cells was evident

(Arrow in Fig. 5I-L). We suspect that a single tamoxifen treatment was not sufficient to

label all the Gli1+ cells. We therefore increased the efficiency of lineage labeling of the

Gli1+ cells by administering tamoxifen to embryos from E12.5 to E14.5 for three

consecutive days. Under this experimental scheme, all fetal testis-derived Gli1+ cells

were positive for CYP17A1 expression at 2 months of age, (Fig. 5M-P), indicating that

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the fetal testis-derived Gli1+ progenitor pool is the sole source for the entire adult

Leydig population in the testis.

Discussion

Using various lineage-tracing models, we construct the lineage map (Fig. 6) that

delineates the fate progression of heterogeneous somatic cell populations during testis

morphogenesis in mice. At the onset of testis morphogenesis at E10.5, all progenitor

cells in the gonadal primordium are positive for WT1 (Armstrong et al., 1993a;

Kreidberg et al., 1993; Wilhelm and Englert, 2002). At this stage, the WT1+ progenitor

cell pool is already heterogeneous, consisting of at least three subpopulations: HES1-

pre-Sertoli cells, HES1- interstitial progenitor cells, and HES1+ interstitial progenitor

cells (see model in Fig. 6). Between E10.5-12.5, WT1+ pre-Sertoli cells acquire SOX9

expression via the action of SRY, starting the organization of testis cords while

maintaining WT1 expression. Meanwhile, HES1+ and HES1- interstitial progenitor cells

lose WT1 expression and commit to interstitial cell lineages that are located outside

testis cords. Although both restricted to the testis interstitium, HES1+ and HES1-

interstitial progenitor cells exhibit distinct lineage contributions. HES1- interstitial

progenitor cells give rise to a subpopulation of GLI1-/CYP17A1+ fetal Leydig cells,

whereas HES1+ interstitial progenitor cells later acquire the expression of GLI1 via the

action of DHH and contribute to two subsequent cell lineages: GLI1+/CYP17A1+ fetal

Leydig cells and GLI1+/CYP17A1- non-steroidogenic cells. Thus, testis interstitium in the

fetal testis comprises at least three distinct cell lineages: GLI1-/ CYP17A1+ fetal Leydig

cells, GLI1+/CYP17A1+ fetal Leydig cells, and GLI1+/CYP17A1- non-steroidogenic cells.

Because fetal Leydig cells (GLI1-/CYP17A1+ and GLI1+/CYP17A1+) do not give rise to

adult Leydig cells (Shima et al., 2015), the GLI1+/CYP17A1- non-steroidogenic

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interstitial cells are likely the progenitor cells for the adult Leydig cell population (Fig.

6).

WT1+ populations in the gonadal primordium and/or mesonephros: progenitors

of Sertoli and Leydig cells

At E10.5, WT1 already exhibits a broad spatiotemporal expression pattern as it

expresses in the coelomic epithelium, the somatic compartment of the testis and the

mesonephros (Armstrong et al., 1993b; Kreidberg et al., 1993; Pelletier et al., 1991).

When these WT1+ cells are lineage-marked, they give rise to not only Sertoli cells but

also fetal and adult Leydig cells. It is unclear how a common WT1+ progenitor pool

adapts two distinct cell fates (supporting cell lineage and interstitial cell lineage) at the

bipotential stage (E10.5). The difference in the location of WT1+ positive cells (coelomic

epithelium in the testis and mesonephros) is unlikely to be the cause because coelomic

epithelial cells have been shown to give rise to both Sertoli cells and fetal Leydig cells

(DeFalco et al., 2011; Karl and Capel, 1998), and the mesonephros only contributes to

almost exclusively endothelial cell population in the testis (Combes et al., 2009).

Because endogenous WT1 expression in the somatic compartment of the testis becomes

restricted to Sertoli cells within the testis cords after E12.5, WT1 could be involved in

the maintenance of Sertoli cell identity. In line with this hypothesis, ablation of Wt1

gene in Sertoli cells results in the transformation of Sertoli cells into fetal Leydig–like

cells, whereas overexpression of Wt1 in fetal Leydig cells promotes the expression of

Sertoli cell-specific genes and suppresses steroidogenic genes (Wen et al., 2014; Zhang

et al., 2015). WT1 appears to mediate the cell fate determination of the common

progenitor cells to become either Sertoli cells or fetal Leydig cells. The continuous

presence of WT1 in the pre-Sertoli cells promotes the Sertoli cell fate, whereas the

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diminishing Wt1 expression in the interstitial progenitor cells leads to the

establishment of interstitial cells lineages. Although how WT1 delineates Sertoli cell fate

is not clear, several binding partners of WT1 have been suggested to regulate its

activities, such as transcriptional cosuppressor BASP1 and serine protease HtrA2

(Carpenter et al., 2004; Hartkamp et al., 2010). Interestingly, Basp1 and Htra2 are

expressed at higher levels in pre-Sertoli cells than in interstitial cells at the onset of

testis morphogenesis (Jameson et al., 2012). It remains to be determined how WT1

expression is extinguished in the Leydig cell lineage. Because the Sertoli cell lineage is

established earlier than that of the Leydig cell lineage, factors suppressing WT1

expression in the fetal Leydig cells might come from Sertoli cells.

Testicular interstitium consists of HES1+ and HES1- cell population that derive

from the WT1+ somatic progenitors

Among the WT1+ progenitor cell pool in the gonadal primordium, a

subpopulation of WT1+ cells acquires HES1 expression at the onset of sex determination.

The HES1+ progenitor cells further become positive for Leydig cell marker CYP17A1 in

the fetal and post-pubertal testes. Hes1 apparently marks a progenitor population

specific for not only fetal Leydig cells but also adult Leydig cells in the testis, suggesting

that both adult Leydig cells and at least a subpopulation of fetal Leydig cells

(GLI1+/CYP17A1+) could derive from a common progenitor population in the gonadal

primordium.

It is unclear how the HES1+ and HES1- populations are established at early testis

development. Because both populations are derived from WT1+ cells, the segregation of

HES1+ and HES1- lineages might be the consequence of asymmetric division of the WT1+

progenitor cells (Lai, 2004; Rhyu et al., 1994). Two daughter cells of the WT1+

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progenitor cells might inherit different signaling components that influence Notch

signaling and therefore adopt different fates: HES1- cells in which Notch signaling is

suppressed, and HES1+ cells in which Notch pathway is activated (Lai, 2004; Rhyu et al.,

1994).

Notably, both HES1+ and HES1- interstitial cells contribute to fetal Leydig cells,

indicating fetal Leydig cells are a heterogeneous population with progenitor cells pre-

determined in nascent testes. It was shown that fetal Leydig cells arise from two

different origins: the coelomic epithelium and the gonadal-mesonephric border region

(DeFalco et al., 2011). The precursor cells in these regions are associated with Mafb and

Arx, putative markers for fetal Leydig cell progenitors (DeFalco et al., 2011; Miyabayashi

et al., 2013). Interestingly, isolated Mafb-EGFP interstitial cells from fetal testes are

highly enriched for not only Arx, but also Hes1 expression (Jameson et al., 2012),

implying that MAFB+ cells, ARX1+ cells, and HES1+ cells likely represent a similar

progenitor population in fetal testes. In addition to MAFB+/ARX1+/HES1+ cells as

sources for fetal Leydig cells, another source of HES1- somatic progenitors also

contribute to fetal Leydig cells. The mechanism that defines the HES1- progenitor cells

remains to be determined.

Notch2 is the main Notch receptor responsible for the differentiation of fetal

Leydig cells

Although the Notch pathway is known to control fetal Leydig cell population in

the interstitium, the signaling components that facilitate its action is still not known. We

have provided genetic evidence that place Notch2 as the main Notch receptor. Among

the four mammalian Notch receptors, involvement of Notch3 in testes determination is

minimal because its expression is not detected until after E12.5 (Jameson et al., 2012;

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Tang et al., 2008), whereas strong expression of Hes1, the downstream target of Notch

signaling, is detected with Hes1-GFP reporter at E11.5. Notch1 and Notch4 are

specifically expressed in the endothelial cells in the gonads, whereas Notch2 is detected

in both supporting and interstitial progenitor cells in the gonadal primordium (Brennan

et al., 2002; Jameson et al., 2012). We inactivated Notch2 using the SF1-cre model, which

targets all WT1+ somatic cell populations (SF1 and WT1 co-localize to gonadal somatic

cells). This model did not induce deletion in endothelial cells that are enriched with

Notch2 expression (Jameson et al., 2012). In the Notch2 conditional knockout newborn

testes, the number of fetal Leydig cells is increased. Because Notch signaling is capable

of restricting progenitors cells from taking on a specialized fate (Lai, 2004), the

increased fetal Leydig cells are likely the consequence of compromised inhibitory Notch

signaling. A previous study of the Hes1-/- testis revealed loss of germ cells phenotype

associated with disrupted Notch signaling (Tang et al., 2008). While germ cells appeared

to be affected in the absence of Hes1, inhibition of the entire Notch signaling in cultured

testis did not affect neither germ cells nor Sertoli cells, suggesting that Notch signaling

has a specific role on fetal Leydig cells (Tang et al., 2008). The difference in the germ

cell phenotype between Notch2 KO and Hes1 KO could be due to the fact that the Notch2

conditional knockout only target Sf1+ somatic cells, whereas the Hes1 conventional

knockout not only targets the Sf1+ somatic cells in the gonadal primordium but also

other cell types such as germ cells and endothelial cells. It is also possible that HES1

plays a Notch-independent role as found in many other model systems (Ingram et al.,

2008; Katoh and Katoh, 2007). Our results provide genetic evidence that implicates

Notch2 as the major receptor of Notch signaling that restricts fetal Leydig cell

population in the fetal testis.

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GLI1+ non-steroidogenic interstitial cells in the fetal testis are the progenitor cells

for adult Leydig cell

In our previous work that focused on fetal Leydig cells, we uncovered a unique

cell population in the interstitium that are negative for any steroidogenic enzyme

expression (Barsoum et al, 2013). We speculate that this non-steroidogenic interstitial

population could be the progenitors for adult Leydig cells. Using the Gli1 lineage tracing

model, we provide evidence that the non-steroidogenic cells are positive for GLI1+ and

give rise to adult Leydig cells. The GLI1+ interstitial cells consists of two subpopulations:

GLI1+/CYP17A1+ fetal Leydig cells and GLI1+/CYP17A1- non-steroidogenic interstitial

cells. It is clearly demonstrated that fetal Leydig cells do not contribute to adult Leydig

cells in the postnatal testis (Shima et al., 2015). Instead the GLI1+/CYP17A1- non-

steroidogenic interstitial cells are the primary progenitor source for adult Leydig

population. The existence of such non-steroidogenic interstitial progenitor cells was

also observed in cell-sorting studies where interstitial cells with high Sf1 expression

represent fetal Leydig cells, whereas low Sf1-expressing interstitial cells are potential

non-steroidogenic cell progenitors (Inoue et al., 2015; McClelland et al., 2015). These

non-steroidogenic interstitial progenitor cells appear to be enriched for Ptch1,

suggesting that these cells respond to Hedgehog signaling (Inoue et al., 2015;

McClelland et al., 2015). Consistent with these observations, genetic manipulation of

Hedgehog signaling in the fetal testis significantly influences the adult Leydig cell

population(Barsoum et al., 2013), supporting the presence of a Hedgehog-responsive

GLI1+ progenitor population for adult Leydig cells. This idea of fetal progenitors for

adult Leydig cells was also noted in the studies that manipulations of COUTFII+

interstitial progenitor cells during the fetal stage result in changes in the number of

adult Leydig cells in adult testis (Kilcoyne et al., 2014; Qin et al., 2008). It is not clear

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whether COUPTFII and GLI1 mark a similar, or distinct interstitial progenitor cell

populations in the fetal testis. Because COUPTFII also appears to be a downstream

target of Hedgehog signaling (Krishnan et al., 1997a; Krishnan et al., 1997b; Lee et al.,

2006; Takamoto et al., 2005), it is likely that COUPTFII and GLI1 mark the same

interstitial progenitor cell population in the fetal testis.

In conclusion, in contrast to the static snapshots of cell types at a certain

developmental stage, our time course lineage tracing analyses capture the cell fate-

defining steps as somatic cells in the testis progress through the primordial stage to the

differentiated stage in adulthood. In the testis primordium, where all somatic

progenitor cells express WT1+, Sertoli cell lineage emerges as a result of SRY and SOX9

action. A distinct HES1+ cell population also appears at this primordial stage, and by

down-regulating WT1 they commit to interstitial cell linage. These HES1+ interstitial

progenitors gain GLI1 expression in response to Sertoli cell-derived DHH signal, and

destine to become a subpopulation of fetal Leydig cells and future adult Leydig cells.

Another subpopulation of fetal Leydig cells is also identified, as the descendants of

HES1-/GLI1- interstitial cell population. Although the processes that specify unique cell

populations (i.e. non-steroidogenic interstitial population and HES1-/GLI1- fetal Leydig

cells) remain to be determined, our findings provide a critical foundation for the

understanding of how various cell types gain their identities during testis organogenesis.

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Materials and Methods:

Animals

Gli1-LacZ (Jax #008211), Gli1-CreERT2 (Jax #007913), Wt1-CreERT2 (Jax #010912), Rosa-

LSL-tdTomato (Jax #007905), and Notch2 floxed mice (Jax #010525) were purchased

from the Jackson Laboratory (Bar Harbor, ME). Sf1-Cre mice (Bingham et al., 2006) were

provided by the late Dr. Keith Parker at UT Southwestern Medical Center. Hes1-CreERT2

mice (Kopinke et al., 2011) were provided by Dr. Charles Murtaugh at the University of

Utah. Hes1-GFP mice (Fre et al., 2011) and Hes1 f/f mice (Revollo et al., 2013) were

kindly provided by Dr. Spyros Artavanis-Tsakonas at Harvard College and Dr. John A.

Cidlowski at National Institute of Environmental Health Sciences, respectively. Female

mice were housed with male mice overnight and checked for the presence of a vaginal

plug the next morning. The day when the vaginal plug was detected was considered

embryonic day (E) 0.5. All animal procedures were approved by the National Institute of

Environmental Health Sciences (NIEHS) Animal Care and Use Committee and are in

compliance with a NIEHS-approved animal study proposal. All experiments were

performed on at least three animals for each genotype.

Tamoxifen treatment

CreERT2 activity was induced by I.P. injection of 1 mg tamoxifen (T-5648, Sigma-Aldrich,

St. Louis, MO) per mouse per day in corn oil. An equivalent volume of corn oil served as

vehicle control (Liu et al., 2015).

Immunohistochemistry and histological analysis

Testes were collected and fixed in 4% paraformaldehyde overnight at 4°C. The samples

were then dehydrated through a sucrose gradient, embedded in OCT compound and

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cryosectioned to 10 μm section as described (Liu et al., 2012). The frozen sections were

incubated with primary antibodies (Table S1) in PBS-Triton X-100 solution with 5%

normal donkey serum at 4°C overnight. The sections were then washed and incubated

in the appropriate secondary antibodies (1:500; Invitrogen, NY, USA) before mounting

in Vector Mount with DAPI (Vector Labs). Slides were imaged under a Leica DMI4000

confocal microscope. For histological analysis, the samples were fixed in 4%

paraformaldehyde in PBS at 4°C overnight, dehydrated through an ethanol gradient, and

embedded in paraffin wax. Sections were stained with hematoxylin/eosin, and were

scanned using Aperio ScanScope XT Scanner (Aperio Technologies, Inc., CA, USA) for

digital image analysis.

Gene expression analysis

Total RNA was isolated from E13.5 and newborn testes using the PicoPure RNA

isolation kit (Arcturus, Mountain View, CA) according to the manufacturer’s protocol.

The cDNA preparation was synthesized using random hexamers and the Superscript II

cDNA synthesis system (Invitrogen Corp., Carlsbad, CA) following manufacturer’s

instruction. Gene expression was analyzed by real-time PCR using Bio-Rad CFX96TM

Real-Time PCR Detection system (Liu et al., 2015). Taqman probes (ThermoFisher

Scientific. Waltham, MA), were used to examine the fold changes of the transcripts. The

following Taqman probes were used: Notch2 (Mm00803077), Ddx4(Mm00802445_m1),

Dhh (Mm01310203_m1), Ihh (Mm00439613_m1), Nr5a1 (Mm00446826_m1), Hsd3b1

(Mm01261921_mH), Hes1 (Mm01342805_m1), and 18S rRNA (Mm03928990_g1). Fold

changes in gene expression were determined by quantitation of cDNA from target

(knockout) samples relative to a calibrator sample (control). All real-time PCR analyses

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were performed in duplicates, and the results were analyzed from a minimal of 3

biological replicates for each experiment. The relative fold change of transcript was

normalized to 18S rRNA as an endogenous reference.

Statistical analysis

Data were analyzed using Prism (Version 6, GraphPad Software) by two-tailed Student’s

t-test. Values are expressed as mean ± s.e.m. A minimum of 3 biological replicates was

used for each experiment.

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Acknowledgments

We are grateful for Dr. Charles Murtaugh at the University of Utah for providing the

Hes1-CreERT2 mice, Dr. Spyros Artavanis-Tsakonas at Harvard College for the Hes1-GFP

mice, and Dr. Ken-Ichirou Morohashi at Kyushu University in Japan for the SOX9

antibody. We thank Dr. Kathryn McClelland for her critical comments on the

manuscript.

Competing interests

The authors declare no competing financial interests. Correspondence and requests

should be addressed to H.H.Y (humphrey.yao@nih.gov).

Author Contributions

C. L. and K. R. performed the experiments; C.L. and H.H.Y designed the study, analyzed

data and wrote the paper.

Funding

This work was supported by the Intramural Research Program (ES102965) of the NIH,

National Institute of Environmental Health Sciences and NIH Graduate Partnerships

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Revollo, J.R., Oakley, R.H., Lu, N.Z., Kadmiel, M., Gandhavadi, M., Cidlowski, J.A., 2013. HES1 Is a Master Regulator of Glucocorticoid Receptor-Dependent Gene Expression. Science Signaling 6. Rhyu, M.S., Jan, L.Y., Jan, Y.N., 1994. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477-491. Roosen-Runge, E.C., Anderson, D., 1959. The development of the interstitial cells in the testis of the albino rat. Acta Anat (Basel) 37, 125-137. Schmahl, J., Eicher, E.M., Washburn, L.L., Capel, B., 2000. Sry induces cell proliferation in the mouse gonad. Development 127, 65-73. Schmahl, J., Kim, Y., Colvin, J.S., Ornitz, D.M., Capel, B., 2004. Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination. Development 131, 3627-3636. Shima, Y., Matsuzaki, S., Miyabayashi, K., Otake, H., Baba, T., Kato, S., Huhtaniemi, I., Morohashi, K.I., 2015. Fetal Leydig Cells Persist as an Androgen-independent Subpopulation in the Postnatal Testis. Mol Endocrinol, me20151200. Shima, Y., Miyabayashi, K., Haraguchi, S., Arakawa, T., Otake, H., Baba, T., Matsuzaki, S., Shishido, Y., Akiyama, H., Tachibana, T., Tsutsui, K., Morohashi, K., 2013. Contribution of Leydig and Sertoli cells to testosterone production in mouse fetal testes. Mol Endocrinol 27, 63-73. Stanley, E., Lin, C.Y., Jin, S., Liu, J., Sottas, C.M., Ge, R., Zirkin, B.R., Chen, H., 2012. Identification, proliferation, and differentiation of adult Leydig stem cells. Endocrinology 153, 5002-5010. Takamoto, N., You, L.R., Moses, K., Chiang, C., Zimmer, W.E., Schwartz, R.J., DeMayo, F.J., Tsai, M.J., Tsai, S.Y., 2005. COUP-TFII is essential for radial and anteroposterior patterning of the stomach. Development 132, 2179-2189. Tanaka, S.S., Nishinakamura, R., 2014. Regulation of male sex determination: genital ridge formation and Sry activation in mice. Cell Mol Life Sci 71, 4781-4802. Tang, H., Brennan, J., Karl, J., Hamada, Y., Raetzman, L., Capel, B., 2008. Notch signaling maintains Leydig progenitor cells in the mouse testis. Development 135, 3745-3753. Val, P., Jeays-Ward, K., Swain, A., 2006. Identification of a novel population of adrenal-like cells in the mammalian testis. Dev Biol 299, 250-256. Wen, Q., Zheng, Q.S., Li, X.X., Hu, Z.Y., Gao, F., Cheng, C.Y., Liu, Y.X., 2014. Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis. Am J Physiol Endocrinol Metab 307, E1131-1143. Wilhelm, D., Englert, C., 2002. The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1. Genes & development 16, 1839-1851. Willerton, L., Smith, R.A., Russell, D., Mackay, S., 2004. Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse. Int J Dev Biol 48, 637-643. Yao, H.H., Whoriskey, W., Capel, B., 2002. Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis. Genes & development 16, 1433-1440. Zhang, F.P., Poutanen, M., Wilbertz, J., Huhtaniemi, I., 2001. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol Endocrinol 15, 172-183. Zhang, L., Chen, M., Wen, Q., Li, Y., Wang, Y., Wang, Y., Qin, Y., Cui, X., Yang, L., Huff, V., Gao, F., 2015. Reprogramming of Sertoli cells to fetal-like Leydig cells by Wt1 ablation. Proc Natl Acad Sci U S A 112, 4003-4008.

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Figures

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Fig. 1. Lineage-tracing analysis of the Wt1+ cells in the fetal testis. (A-T) Lineage

tracing of the fetal testis-derived Wt1+ cells in the Wt1-CreERT2; Rosa-LSL-tdTomato

embryos was induced by tamoxifen (TM) administration at E10.5. The testes were

analyzed at E11.5 (A-D), E13.5 (E-L) and 1 month of age (M-T) by fluorescent

immunohistochemistry for Wt1-td (red, tdTomato-labeled fetal testis-derived Wt1+

cells), WT1 (green, endogenous protein), Germ cell marker PECAM-1 (light blue), Sertoli

cell marker SOX9 (green), or Leydig cell marker CYP17A1 or 3HSD (green), and

nuclear counterstain DAPI (blue). The insets are higher magnification of outlined areas

in A-T. Arrows in the insets of K & L point to CYP17A1+ fetal Leydig cells. Asterisks in

the insets of K & L indicate CYP17A1- non-steroidogenic cells. Dotted lines outline the

testis cords. Scale bar: 25 μm.

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Fig. 2. Detection of Hes1 expression by Hes1-GFP reporter and Hes1 lineage-

tracing analysis. (A-H) Expression of Hes1-GFP in the E11.5 fetal testes was detected by

fluorescent immunohistochemistry for GFP (Hes1+ cells), WT1 (red), and PECAM-1

(grey) that marks germ cells and endothelial cells in low and high magnifications. The

insets are higher magnification of outlined areas in (E-H). Scale bar: 25 μm. (I-T)

Lineage-tracing of fetal testis-derived Hes1+ cells in the Hes1-CreERT2; Rosa-LSL-

tdTomato embryos was induced by tamoxifen (TM) administration of a single shot at

E10.5 (I-L), or at E10.5 and 11.5 for two consecutive days (M-T). The samples were

examined at E11.5 (I-L), E15.5 (M-P) and 1 month of age (Q-T) for Hes1-td (red,

tdTomato-labeled fetal testis-derived Hes1+ cells), WT1 (green), PECAM-1 (grey), Leydig

cell marker CYP17A1 (green), and nuclear counterstain DAPI (grey). The insets are

higher magnification of outlined areas in (I-T). Scale bar: 25 μm. Arrows in insets of O &

P point to Hes1+/CYP17A1+ fetal Leydig cells. Empty arrowheads in insets of O & P

indicate Hes1-/CYP17A1+ fetal Leydig cells. Arrowheads in insets of O & P represent

Hes1+/CYP17A1- non-steroidogenic interstitial cells.

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Fig. 3. Leydig cell phenotypes as a result of inactivation of Notch2 in Sf1-positive

somatic cells. (A & F) Quantitative PCR analysis of gene expression for Notch2, Ddx4

(germ cell marker), Dhh (Sertoli cell marker), Nr5a1 and Hsd3b1 (Fetal Leydig cells) and

Hes1 in control (n=5) and Notch2 cKO (n=3) E13.5 and newborn testes. *P<0.05.

Student’s t-test was used and values are presented as means ± s.e.m. (B-E & G-H)

Immunofluorescence of SOX9 (green), PECAM-1 (red), CYP17A1 (grey), in control

(Notch 2 fx/fx) and Sf1-Cre; Notch2 fx/fx or Notch2 conditional KO (cKO) at E13.5 (B-E) and

newborn (G-H) testes. Scale bar: 50 μm (B&C), 100 μm (D&E, G&H).

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Fig. 4. Contribution of Hes1+ and Wt1+ progenitor cells to the Gli1+ interstitial cell

population in the fetal testis. (A-C) Lineage tracing analysis of Hes1+ progenitor cells

in the Hes1-CreERT2; Rosa-LSL-tdTomato; Gli1-LacZ embryos was induced by tamoxifen

(TM) administration at E10.5 and E11.5. The samples were analyzed at E14.5 by

fluorescent immunohistochemistry for Hes1-td (red, tdTomato-labeled fetal testis-

derived Hes1+ cells), LacZ (green for Gli1+ cells). The insets are higher magnification of

outlined areas in (A-D). Scale bar: 25 μm. (D-G) Lineage labeling of Wt1+ progenitor

cells in the Wt1-CreERT2; Rosa-LSL-tdTomato; Gli1-LacZ embryos was induced by

tamoxifen (TM) administration at E10.5. The samples were analyzed at E13.5 by

fluorescent immunohistochemistry for Wt1-td (red. tdTomato-labeled fetal testis-

derived Wt1+ cells), LacZ (green for Gli1+ cells), and AMH (grey for Sertoli cells). The

insets are higher magnification of outlined areas in (D-G). Scale bar: 25 μm.

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Fig. 5: Detection of Gli1 in the fetal testis by Gli1-LacZ reporter and genetic

lineage-tracing analysis. (A-D) Expression of Gli1-LacZ in the E16.5 fetal testes was

detected by fluorescent immunohistochemistry for LacZ (red for Gli1+ cells), Leydig cell

marker CYP17A1 (green), and nuclear counterstain DAPI (grey). The insets are higher

magnification of outlined areas in (A-D). Scale bar: 25 μm. (E-L) Lineage-tracing analysis

of the Gli1+ cells in the Gli1-CreERT2; Rosa-LSL-tdTomato embryos was induced by

tamoxifen (TM) administration at E12.5. The testes were examined at different stages of

development for Gli1-td (red, tdTomato labeled fetal testis-derived Gli1+ cells), Leydig

cell marker CYP17A1 (green), and DAPI (grey). The insets are higher magnification of

the outlined areas in (E-L). Arrows in G-H insets: Gli1+/CYP17A1- interstitial cells.

Arrowheads in G-H insets: Gli1+/CYP17A1+ fetal Leydig cells. Empty arrowheads in G-H

insets: Gli1+/CYP17A1- peritubular myoid cells. Arrow in K-L insets: Gli1-/CYP17A1+

adult Leydig cells. Arrowhead in K-L insets: Gli1+/CYP17A1+ adult Leydig cells. Scale

bar: 25 m. (M-P) Lineage-tracing analysis of the Gli1+ cells in the Gli1-CreERT2; Rosa-

LSL-tdTomato embryos was induced by tamoxifen (TM) administration from E12.5 to

E14.5 for three consecutive days. The testes were examined for Gli1-td (red, tdTomato

labeled fetal testis-derived Gli1+ cells), Leydig cell marker CYP17A1 (green), and DAPI

(grey). The insets are higher magnification of outlined areas in (M-P). Scale bar: 25 m.

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Fig. 6. Model of the lineage progression of supporting cells and interstitial cells in

the testis. At E10.5, the WT1+ progenitor cell pool consists of at least three

subpopulations: HES1- pre-Sertoli cells, HES1- interstitial progenitor cells, and HES1+

interstitial progenitor cells. Between E10.5-12.5, the three WT1+ subpopulations in the

gonadal primordium give rise to SOX9+ Sertoli cells and SOX9- interstitial cells (HES1+

and HES1-) within and outside testis cords, respectively. While HES1- interstitial cells

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give rise to a subpopulation of fetal Leydig cells (GLI1+/CYP17A1+), HES1+ interstitial

cells further acquire expression of GLI1 at around E12.5 and differentiate into two

distinct cell lineages: GLI1+ progenitor cells that contribute to a subpopulation of fetal

Leydig cells (CYP17A1+), and GLI1+ non-steroidogenic interstitial cells that eventually

give rise to adult Leydig cells. This model is based on the results of this study. Markers

that were reported in other studies such as SF1, MAFB, and ARX are not included for the

sake of simplicity.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Supplemental Figures

Fig. S1 Detection of Wt1 in the fetal testis by endogenous protein expression. (A-F)

E11.5 and E12.5 fetal testes were immunostained for WT1 (red), germ cell and

endothelial cell marker PECAM-1 (green), or Sertoli cell marker AMH (green). Scale bar:

25 m.

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Development 143: doi:10.1242/dev.135756: Supplementary information

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S2. Lineage-tracing experiments for the Wt1+ cells in the testes. (A-T) Lineage

tracing of the fetal gonad-derived Wt1+ cells in the Wt1-CreERT2; Rosa-LSL-tdTomato

embryos was induced by tamoxifen (TM) administration at E10.5. The testes were

analyzed at E11.5, E13.5 and 1 month of age by fluorescent immunohistochemistry for

Wt1-td (red, tdTomato labeled fetal testis-derived Wt1+ cells), Germ cell marker

PECAM-1 (Cyan), Sertoli cell marker SOX9 (green), or Leydig cell marker CYP17A1

(green), or laminin (green; demarcates boundaries of testis cords), and nuclear

counterstain DAPI (blue). Scale bar: 25 μm.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S3. Detection of Hes1 expression by Hes1-GFP reporter. (A-P) Expression of

Hes1-GFP in the fetal testes (E10.5, E11.5, E12.5 and E16.5) was detected by fluorescent

immunohistochemistry for GFP (Hes1+ cells), WT1, Sertoli cell marker SOX9, fetal Leydig

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Development 143: doi:10.1242/dev.135756: Supplementary information

cell CYP17A1, and PECAM-1 that marks germ cells and endothelial cells. The insets are

higher magnification of outlined areas in (E-H). Scale bar: 25 μm.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S4 Lineage-tracing experiments for the Hes1+ cells in the testes. (A-D) Lineage-

tracing of fetal testis-derived Hes1+ cells in the Hes1-CreERT2; Rosa-LSL-tdTomato

embryos was induced by tamoxifen (TM) administration at E10.5 and 11.5 for two

consecutive days. The samples were examined at E15.5 for Hes1-td (red, tdTomato-

labeled fetal testis-derived Hes1+ cells), Leydig cell marker CYP17A1 (green), and

nuclear counterstain DAPI (grey). Scale bar: 25 μm.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S5. Lineage-tracing experiments for the Hes1+ cells in the testes. (A-D) Lineage

tracing of fetal testis-derived Hes1+ cells in the Hes1-CreERT2; Rosa-LSL-tdTomato

embryos was induced by tamoxifen (TM) administration from E10.5-13.5 for four

consecutive days. The samples were examined at E15.5 for Hes1-td (red, tdTomato-

labeled fetal testis-derived Hes1+ cells), Leydig cell marker CYP17A1 (green), and

nuclear counterstain DAPI (grey). The insets are higher magnification of outlined areas

in (A-D). Scale bar: 25 μm. Arrows in insets of C & D point to Hes1+/CYP17A1+ fetal

Leydig cells. Empty arrowheads in insets of C & D indicate Hes1-/CYP17A1+ fetal Leydig

cells. Arrowheads in insets of C & D represent Hes1+/CYP17A1- non-steroidogenic

interstitial cells.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S6. Quantification of the number of fetal Leydig cells. Graph shows the number

of CYP17A1+ cells per 1.5 x 1.5 mm2 Leica confocal optical section in control (Notch 2

fx/fx) and cKO (Sf1-Cre; Notch2 fx/fx) in E13.5 and newborn testes. Sections (n=5~10) for

each genotype/time point were obtained from at least 3 different gonads. Cells were

counted manually with Image J’s cell counter plugin.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S7. Contribution of Hes1+ and Wt1+ progenitor cells to the Gli1+ interstitial cell

population in the fetal testis. (A-C) Lineage tracing analysis of Hes1+ progenitor cells

in the Hes1-CreERT2; Rosa-LSL-tdTomato; Gli1-LacZ embryos was induced by tamoxifen

(TM) administration at E10.5 and E11.5. The samples were analyzed at E14.5 by

fluorescent immunohistochemistry for Hes1-td (red, tdTomato-labeled fetal testis-

derived Hes1+ cells), LacZ (green for Gli1+ cells). Scale bar: 25 μm. (D-F) Lineage labeling

of Wt1+ progenitor cells in the Wt1-CreERT2; Rosa-LSL-tdTomato; Gli1-LacZ embryos was

induced by tamoxifen (TM) administration at E10.5. The samples were analyzed at

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Development 143: doi:10.1242/dev.135756: Supplementary information

E13.5 by fluorescent immunohistochemistry for Wt1-td (red. tdTomato-labeled fetal

testis-derived Wt1+ cells) and LacZ (green for Gli1+ cells). Scale bar: 25 μm.

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Development 143: doi:10.1242/dev.135756: Supplementary information

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Development 143: doi:10.1242/dev.135756: Supplementary information

Fig. S8. Expression of Gli1 in the fetal and newborn testes. (A-S) Expression of Gli1-

LacZ in the testes was detected by fluorescent immunohistochemistry for LacZ (red),

Sertoli cell marker AMH (green), or Leydig cell marker CYP17A1 (green), or peritubular

myoid cell marker αSMA (Cyan), and nuclear counterstain DAPI (grey). The insets are

higher magnification of outlined areas in (M-S). Scale bar: 25 μm.

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Development 143: doi:10.1242/dev.135756: Supplementary information

Table S1. List of primary antibodies.

Primary antibodies Species Cat# Dilution Source

β-galactosidase Chicken ab9361 1:1000 Abcam, San Francisco, CA,

PECAM-1 Rat 550274 1:500 BD Biosciences, San Jose, CA

WT1 Rabbit ab89901 1:300 Abcam, San Francisco, CA

3 HSD Rabbit KO607 1:500 CosmoBio Co.Ltd, Japan

SOX9 Rabbit N/A 1:300 Dr. Ken-ichirou Morohashi, Japan

AMH Goat sc6886 1:500 Santa Cruz Biotechnology, Texas

CYP17A1 Goat sc46081 1:100 Santa Cruz Biotechnology, Texas

α-SMA Rabbit ab5694 1:500 Abcam, San Francisco, CA

COUPTFII Mouse PP-H7147-00 1:200 R&D systems, Minneapolis, MN

laminin Rabbit L9393 1:500 Sigma-Aldrich, St. Louis, MO USA

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