Desert Hedgehog/Patched 1 signaling
specifies fetal Leydig cell fate in
Humphrey Hung-Chang Yao,1Wendy Whoriskey,2and Blanche Capel1,3
1Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA;2Curis, Inc.,
Cambridge, Massachusetts 02138, USA
Establishment of the steroid-producing Leydig cell lineage is an event downstream of Sry that is critical for
masculinization of mammalian embryos. Neither the origin of fetal Leydig cell precursors nor the signaling
pathway that specifies the Leydig cell lineage is known. Based on the sex-specific expression patterns of
Desert Hedgehog (Dhh) and its receptor Patched 1 (Ptch1) in XY gonads, we investigated the potential role of
DHH/PTCH1 signaling in the origin and specification of fetal Leydig cells. Analysis of Dhh−/−XY gonads
revealed that differentiation of fetal Leydig cells was severely defective. Defects in Leydig cell differentiation
in Dhh−/−XY gonads did not result from failure of cell migration from the mesonephros, thought to be a
possible source of Leydig cell precursors. Nor did DHH/PTCH1 signaling appear to be involved in the
proliferation or survival of fetal Leydig precursors in the interstitium of the XY gonad. Instead, our results
suggest that DHH/PTCH1 signaling triggers Leydig cell differentiation by up-regulating Steroidogenic Factor 1
and P450 Side Chain Cleavage enzyme expression in Ptch1-expressing precursor cells located outside testis
[Key Words: Desert Hedgehog; Patched 1; Leydig; mesonephros; testis; organogenesis]
Received February 1, 2002; revised version accepted April 10, 2002.
A critical event in testis organogenesis is the specifica-
tion of somatic cell lineages including Sertoli cells, peri-
tubular myoid cells, and Leydig cells. Specification of
these lineages is crucial for the establishment of testis
morphology and the production of hormones. A single
gene on the Y chromosome, Sry (sex-determining region
of the Y chromosome), is believed to induce a cascade of
signaling pathways for the differentiation of these so-
matic cell lineages (Gubbay et al. 1990; Koopman et al.
1991). Autonomous expression of Sry in somatic cells in
the XY gonad leads to differentiation of Sertoli cells (Al-
brecht and Eicher 2001). Differentiating gonadal cells in-
duce migration of cells from the mesonephros into the
gonad. The migrating cells contribute to precursors of
the peritubular myoid and vascular cell lineages (Mar-
tineau et al. 1997; Capel et al. 1999; Tilmann and Capel
1999). Differentiation of peritubular myoid cells and the
consequent formation of testis cords are regulated by
Desert hedgehog (DHH), a signaling protein produced by
Sertoli cells (Clark et al. 2000; Pierucci-Alves et al.
2001). Fetal Leydig cells are first identifiable within the
interstitium of the XY gonad (between testis cords) when
they express P450 Side Chain Cleavage (Scc) enzyme
and other steroidogenic enzymes required for the produc-
tion of androgens.
The specification of adult Leydig cells has been stud-
ied extensively (Habert et al. 2001). Adult Leydig cells
are believed to be a separate population of steroidogenic
cells that arise from adult peritubular mesenchymal
cells (Ariyaratne et al. 2000). They are believed to be
completely independent of the population of fetal Leydig
cells responsible for initial masculinization of the em-
bryo. The origin of fetal Leydig cells is unknown. During
fetal life, Leydig cell precursors could arise from one or
both of two possible sources: the mesonephros or the
coelomic epithelium. When gonads from 11.5 days post-
coitum (dpc) embryos were grafted to mesonephroi from
mice carrying transgenic markers such as ?-galactosi-
dase (?-gal) or GFP, the markers were found in some of
the peritubular myoid cells and other interstitial cells of
the testis (Buehr et al. 1993; Merchant-Larios et al. 1993;
Nishino et al. 2000). Some migratory mesonephric cells
acquired ultrastructural features of steroidogenic Leydig
cells (Merchant-Larios and Moreno-Mendoza 1998). A
small population of these migrating cells differentiated
into Leydig cells when cultured in vitro (Nishino et al.
2001). However, when the XY gonad was separated from
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GENES & DEVELOPMENT 16:1433–1440 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 1433
the mesonephros at 11.5 dpc and cultured alone (Mer-
chant-Larios et al. 1993) or when XY gonads were grafted
to embryonic hind limbs at 11.5 dpc and subsequently
cultured (Moreno-Mendoza et al. 1995), differentiation of
Leydig cells proceeded normally. The results of these
two experiments suggest that most Leydig precursors are
already present in the gonad by 11.5 dpc. Another pos-
sible source of Leydig cell precursors is the coelomic
epithelium that covers the entire coelomic surface of the
gonad. Both proliferation studies (Schmahl et al. 2000)
and DiI lineage tracing experiments (Karl and Capel
1998) revealed that coelomic epithelial cells in XY go-
nads proliferate rapidly between 11.5 and 12.5 dpc and
contribute many interstitial cells to the developing tes-
tis. The fate of these cells has not been defined. The
signals that induce differentiation of fetal Leydig cells
are also unknown. At present only a negative regulator of
Leydig cell differentiation (Wnt4) has been identified
(Vainio et al. 1999). Expression of the hedgehog receptor,
Patched 1 (Ptch1), throughout the cells of the intersti-
tium in 12.5 dpc XY gonads suggested that DHH/PTCH1
signaling might function in Leydig cell differentiation in
addition to its role in signaling between Sertoli and peri-
tubular myoid cells (Bitgood et al. 1996). To determine
the role of DHH/PTCH1 signaling in Leydig cell differ-
entiation, we explored the temporal and spatial expres-
sion patterns of Dhh, Ptch1, and Scc, and analyzed go-
nads from Dhh−/−XY embryos. Here we show that dis-
ruption of DHH/PTCH1 signaling in Dhh−/−mice results
in defects of fetal Leydig cell differentiation, whereas it
has no effect on mesonephric cell migration or on the
establishment of the interstitial cell population. These
results suggest that DHH/PTCH1 signaling does not af-
fect the origin of fetal Leydig precursors, but instead,
operates later to specify the Leydig cell lineage by up-
regulating Steroidogenic Factor 1 (Sf1) and Scc expres-
sion in Ptch1-expressing precursor cells located outside
Temporal and spatial expression of Dhh, Ptch1,
and Scc in testis organogenesis
To determine whether fetal Leydig cells might be targets
of DHH signaling, we first detailed the expression pat-
terns of Dhh, its receptor, Ptch1, and a Leydig cell
marker Scc (Rouiller et al. 1990) in XY gonads from 11.5
to 13.5 dpc, the period during which the differentiation
of fetal Leydig cells occurs. Expression of Dhh began at
11.5 dpc and continued afterward in the Sertoli cell lin-
eage as previously described (Fig. 1; Bitgood et al. 1996).
(PtchLacZ) XY gonads, we found that PtchLacZwas not
expressed at 11.5 dpc XY gonads, but was prominently
expressed in the interstitial space between testis cords in
12.5 and 13.5 dpc XY gonads (Fig. 1). PtchLacZexpression
was also found around the mesonephric tubules in the
anterior part of the mesonephros from 11.5 to 13.5 dpc.
We compared PtchLacZexpression with Scc expression to
determine whether PtchLacZ-expressing cells became
Scc-positive. At 12.5 dpc, the majority of interstitial cells
were PtchLacZ-positive and only a small population of
them expressed Scc (Fig. 1). In 13.5 dpc XY gonads, a
much larger percentage of PtchLacZ-expressing cells were
also expressing Scc (Fig. 1, bottom panels). Neither
PtchLacZnor Scc was expressed in the coelomic epithe-
lium of XY gonads (Fig. 1, bottom panels) or in endothe-
lial cells of the vasculature (data not shown). Patched 2,
another mammalian hedgehog receptor (Carpenter et al.
(dark purple), Ptch1 (blue), and the Leydig
cell marker Scc (red) in XY gonads from
11.5 dpc to 13.5 dpc. Expression of Dhh
and Scc were detected by whole-mount in
situ hybridization. Ptch1 expression was
detected by analyzing ?-galactosidase ac-
tivity in the Ptchtm1MpsXY gonads. Sec-
tions of 13.5 dpc whole-mount samples are
shown at the bottom to confirm the cell-
specific expression patterns of Dhh, Ptch1,
and Scc in the gonads. CE, coelomic epi-
thelium; G, gonad; M, mesonephros; TC,
Expression patterns of Dhh
Yao et al.
1434GENES & DEVELOPMENT
1998), was not expressed in XY gonads during this time
period (data not shown). Other hedgehog genes such as
Sonic Hedgehog and Indian Hedgehog are not expressed
in the gonad (Bitgood and McMahon 1995).
Defects in differentiation of fetal Leydig cells
in Dhh−/−XY gonads
The expression patterns of Dhh and its receptor, Ptch1,
indicated that DHH signaling could be involved in the
early development of Leydig cells. To investigate
whether differentiation of fetal Leydig cells was affected
by loss of DHH signaling, we analyzed the expression of
Scc in 13.5–14.5 dpc Dhh+/+, Dhh+/−, and Dhh−/−XY go-
nads (Clark et al. 2000). No differences were noted be-
Dhh+/−samples are shown in Figures 2 and 3. At 13.5
dpc, expression of Scc appeared in the center of all
Dhh+/+and Dhh+/−gonads, whereas Scc expression was
completely absent in 70% (7/10) of Dhh−/−gonads (Fig.
2). By 14.5 dpc, Scc expression reached its peak in inter-
stitial cells in Dhh+/+and Dhh+/−gonads. However, only
sparse staining for Scc was seen in the majority of 14.5
dpc Dhh−/−gonads (Fig. 2). It is known that the expres-
sion of Scc is under the regulation of SF1 (Clemens et al.
1994; Hatano et al. 1994). We performed immunocyto-
chemistry for SF1 on 13.5 dpc XY gonads after in situ
hybridization for Scc to verify that Scc-expressing cells
were also SF1-positive. We found that all Scc-expressing
cells (Fig. 3A, red cells outside of testis cords) showed
strong nuclear staining for SF1 (Fig. 3A, green stain). In
Dhh−/−gonads, the number of interstitial Leydig cells
with strong nuclear SF1 staining was dramatically de-
creased compared to Dhh+/+and Dhh+/−gonads (Fig.
3B,C, arrows). However, interstitial cells with weak
nuclear SF1 staining were still present in Dhh−/−gonads
in normal numbers (Fig. 3C, arrowheads). Expression of
SF1 in Sertoli cells in testis cords was not affected by
disruption of DHH signaling (Fig. 3B,C, asterisks).
Normal mesonephric cell migration in Dhh−/−
One of the cellular events downstream of Sry is migra-
tion of interstitial cells from the mesonephros into the
gonad between 11.5 and 12.5 dpc (Capel et al. 1999; Til-
mann and Capel 1999). Because most interstitial cells
express PtchLacZat 12.5 dpc (Fig. 1), we investigated
whether Dhh signaling regulates mesonephric cell mi-
gration. PtchLacZexpression showed a unique pattern
The mRNA for the Leydig cell marker Scc (black stain) is pre-
sent at 13.5 and 14.5 dpc in Dhh+/+(data not shown) and Dhh+/−
but is reduced or absent in Dhh−/−XY gonads.
Expression of Scc in Dhh+/−and Dhh−/−XY gonads.
Dhh−/−XY gonads. (A) Colocalization of SF1 (green nuclear
staining) and Scc (red cytoplasmic staining) in Leydig cells in
normal 13.5 dpc XY gonads by immunocytochemistry for SF1
and in situ hybridization for Scc. (B) Strong nuclear SF1 staining
(arrows) in Leydig cells in Dhh+/−gonads. (C) Absence of strong
nuclear SF1 staining in Leydig cells in Dhh−/−gonads. Weak
nuclear SF1 staining was still present (arrowheads). SF1 staining
was also detected in Sertoli cells (asterisks) in testis cords
(TC, outlined by dotted lines). Germ cells and endothelial cells
were stained with an anti-PECAM antibody (blue staining in B
Expression of SF1 and Scc in 13.5 dpc Dhh+/−and
DHH/PTCH1 signaling in Leydig cell differentiation
GENES & DEVELOPMENT 1435
during the period when mesonephric cell migration oc-
curs. At 11.5 dpc, PtchLacZexpression was observed only
around the mesonephric tubules at the anterior part of
the mesonephros but not in gonads of either sex (Fig. 1).
As the development of gonads proceeded to 12.0 dpc,
PtchLacZexpression appeared in the interstitium in the
anterior part the XY gonad close to the mesonephric tu-
bules (Fig. 4A). At 12.25 dpc, PtchLacZexpression in the
XY gonad extended anteriorly and posteriorly (Fig. 4A).
By 12.5 dpc, the entire interstitium of the XY gonad ex-
pressed PtchLacZ, except for the most posterior tip of the
gonad (Fig. 1). No PtchLacZexpression was found in XX
gonads at any stage examined (data not shown).
This unique pattern of PtchLacZexpression (Fig. 4A)
suggested that the DHH/PTCH1 signaling pathway
might induce migration of Ptch1-expressing cells from
the mesonephros into the interstitium of the XY gonad,
beginning near the anterior end of the gonad. To test this
hypothesis, we assembled two different recombinant or-
gan cultures at 11.25 dpc. In the first recombinant cul-
ture (Fig. 4B), we assembled a wild-type gonad with a
PtchLacZmesonephros. We reasoned that if PtchLacZ-ex-
pressing cells derive from the mesonephros, we should
observe ?-gal-positive cells in the wild-type gonad after
migration has taken place. In the second recombinant
culture (Fig. 4C), we assembled the reciprocal combina-
tion with a PtchLacZgonad apposed to a wild-type me-
sonephros. After culture for 30 h (corresponding to ∼12.5
dpc in vivo), samples were stained for ?-gal. We found no
?-gal staining in the interstitium of the wild-type gonad
in the first recombinant culture (Fig. 4B), suggesting that
few if any cells that have migrated from the mesoneph-
ros during this period of culture express PtchLacZ. In the
second recombinant culture with a PtchLacZgonad and a
wild-type mesonephros, ?-gal staining appeared in the
interstitium of all PtchLacZgonads (Fig. 4C), suggesting
that PtchLacZexpression is induced in cells already pres-
ent in the gonad by 11.25 dpc.
To further test the possibility that DHH/PTCH1 sig-
naling was involved in mesonephric cell migration, we
assembled an 11.5 dpc Dhh+/+, Dhh+/−, or Dhh−/−XY
gonad apposed to an 11.5 dpc mesonephros expressing
GFP and compared the migration of GFP-expressing cells
in the presence and absence of DHH signaling. We found
that GFP-expressing cells migrated from the mesoneph-
ros into the XY gonad in a similar pattern in Dhh+/+(data
not shown), Dhh+/−, and Dhh−/−gonads (Fig. 4D, red ar-
rows). Analysis of Scc expression in these samples re-
vealed that despite normal mesonephric cell migration,
expression of Scc is completely absent in Dhh−/−XY go-
nads compared to Dhh+/+and Dhh+/−gonads (Fig. 4D, red
Stage-specific effects of the hedgehog inhibitor
cyclopamine on Leydig cell differentiation
To determine whether DHH/PTCH1 signaling regulates
the earliest stages of Leydig cell differentiation or later
responsible for induction of mesonephric
cell migration into XY gonads. (A) Analy-
sis of PtchLacZexpression in 12.0 and 12.25
dpc XY gonads. (B) Recombinant organ
culture using a wild-type gonad apposed to
a PtchLacZmesonephros. (C) Reciprocal or-
gan culture with a PtchLacZgonad apposed
to a wild-type mesonephros. Recombinant
organ cultures in both B and C were as-
sembled at 11.25 dpc, cultured for 48 h,
and assayed for PtchLacZexpression. (D)
Mesonephric cell migration and Scc ex-
pression in Dhh+/−and Dhh−/−gonads: Re-
combinant gonad cultures were assembled
with an 11.5 dpc Dhh+/−or Dhh−/−XY go-
nad apposed to an 11.5 dpc mesonephros
expressing GFP. Cell migration (green cell,
red arrows) was detected 48 h after culture.
Samples were fixed, and then expression of
Scc was detected by in situ hybridization
(red staining in gonads).
DHH/PTCH1 signaling is not
Yao et al.
1436GENES & DEVELOPMENT
maintenance or expansion of the Leydig cell population,
we examined Scc expression in gonad organ cultures in
the presence and absence of a DHH signaling inhibitor,
cyclopamine, introduced at 11.5 dpc or 12.5 dpc. Cyclo-
pamine inhibits hedgehog signaling by inactivating
Smoothened, the first downstream signaling molecule
after binding of hedgehog protein to its receptor, PTCH1
(Taipale et al. 2000). Scc was expressed normally in both
11.5 and 12.5 dpc gonads after 24-h culture in the ab-
sence of cyclopamine. When cyclopamine was added at
11.5 dpc, the expression of Scc in Leydig cells was com-
pletely inhibited. In contrast, addition of cyclopamine to
cultures at 12.5 dpc or 13.5 dpc had no effect on Scc
expression in Leydig cells (Fig. 5, black stain; 13.5 dpc
data not shown).
To determine whether the loss of DHH signaling af-
fected proliferation or maintenance of Leydig precursors,
we examined cell proliferation using an antibody against
phosphorylated Histone H3 (pHH3; Paulson and Taylor
1982; Hendzel et al. 1997; Saka and Smith 2001), and
apoptosis, using LysoTracker reagent (Zucker et al. 1998,
1999), in 11.5 dpc gonad explants cultured for 40 h in the
presence or absence of cyclopamine. We found a similar
total number of pHH3-positive cells (cell counts from 10
serial sections) in gonads cultured in the absence or pres-
ence of cyclopamine (Fig. 6, arrows). Although normal
apoptotic cells were detected in the Müllerian duct in
the mesonephros at this stage (Roberts et al. 1999), no
apoptotic cells were found in the gonadal region of
samples cultured in the presence or absence of cyclopa-
mine (Fig. 6, the gonad is outlined by a dotted line).
It has been more than five decades since Jost first dis-
covered that testosterone synthesized by the fetal testis
is essential for differentiation of the Wölffian duct and
development of male secondary sex characteristics (Jost
1947). Here we report that DHH/PTCH1 signaling is a
positive regulator of the differentiation of steroid-pro-
ducing Leydig cells in the fetal testis. Dhh is expressed
downstream of Sry, specifically in Sertoli cells inside tes-
tis cords (Bitgood et al. 1996), and is the only known
mammalian hedgehog protein expressed in the gonad be-
tween 11.5 and 13.5 dpc. One of the hedgehog receptors,
Ptch1, was known to be expressed in interstitial cell
populations (Bitgood et al. 1996). Original generation of
Dhh-null mice on a 129/Sv genetic background resulted
in defects in spermatogenesis but no defects in testis
organogenesis and Leydig cell differentiation despite
down-regulation of Ptch1 (Bitgood et al. 1996). However,
transfer of the Dhh mutation to another genetic back-
ground resulted in discrete defects in development of the
peritubular myoid cell lineage, leading to abnormal cord
organization and loss of adult Leydig cells (Clark et al.
2000; Pierucci-Alves et al. 2001). We show here that it
also results in a defect in differentiation of fetal Leydig
Ptch1 is first expressed around the mesonephric tu-
bules at the anterior end of the mesonephros. By 12.0
dpc, interstitial cells toward the anterior end of the go-
nad begin to express Ptch1 under the positive regulation
of DHH. Expression of Ptch1 gradually extends toward
both anterior and posterior ends of the gonad. Despite
the implications of this expression pattern, we find no
evidence that DHH is involved in signaling for meso-
nephric cell migration. Nor does loss of Dhh appear to
exert a detrimental effect on Sertoli differentiation, as
MIS and Sox9 expression in Dhh−/−gonads and in cyclo-
pamine treated gonads (Yao and Capel 2002) are normal.
Instead, this and previous data suggest that DHH is
involved in signaling proximal cells to differentiate
clopamine on expression of Scc mRNA in Leydig cells. XY go-
nads (11.5 or 12.5 dpc) were cultured in the presence or absence
of cyclopamine (25 µM) for 24 h followed by whole-mount in
situ hybridization for Scc (black staining in gonads).
Stage-specific effects of the hedgehog inhibitor cy-
proliferation and apoptosis in 11.5 dpc gonads. Gonads (11.5
dpc) were cultured for 24 h in the presence or absence of cyclo-
pamine (25 µM) followed by immunocytochemistry for phos-
phorylated Histone H3 (arrows) or LysoTracker staining for
apoptosis (arrows indicate position of the Müllerian duct). G,
gonad (outlined by a dotted line); M, mesonephros.
Effects of the hedgehog inhibitor cyclopamine on
DHH/PTCH1 signaling in Leydig cell differentiation
GENES & DEVELOPMENT1437
along specific pathways. For example, it has been shown
that DHH influences the differentiation of peritubular
myoid cells in Ptch1-expressing cells most proximal to
the DHH signal (Clark et al. 2000; Pierucci-Alves et al.
2001). Here we show that DHH signals the Ptch1-ex-
pressing cells located slightly further away from the
DHH-producing Sertoli cells to differentiate as Leydig
cells. Although it appears that all Leydig cells express
Ptch1, not all Ptch1-expressing cells differentiate as Ley-
dig cells. This likely means that other signals combine
with DHH signals to specify Leydig cell fate.
Leydig precursors responsive to the DHH signal may
be set aside earlier by their lineage origin, or they may be
specified among cells of the interstitium by the intersec-
tion of multiple signals. Some evidence suggests that
Leydig cells and steroid cells of the adrenal share a com-
mon origin at 10.5 dpc near the anterior end of the me-
sonephros (Hatano et al. 1996). If this is true, they must
move into the gonad prior to 11.25 dpc under the control
of signals other than DHH or they would have been de-
tected in our recombinant organ culture system. An-
other possibility is that Leydig cells do not have a dis-
crete lineage origin: pluripotent cells may derive from
the coelomic epithelium between 11.5 and 12.5 dpc
whose differentiation is under the control of combinato-
rial signals that intersect in the field of the gonad. This
type of paradigm could suggest that the interstitial cells
of the gonad are equivalent and plastic in the sense that,
regardless of where they originate, they may follow one
of several cell fates in the gonad. This decision could
depend not on their lineage origin, but on their distance
from other signaling cells or their spatial relationship to
the vasculature or to other structural features of the go-
nad. Hedgehog signaling effects related to distance from
the signal have been noted in many systems (Bumcrot
and McMahon 1996; Neumann and Cohen 1997; Strigini
and Cohen 1999; Vervoort 2000).
DHH does not regulate the size of the precursor popu-
lation. We found that interstitial cells with low SF1 ex-
pression were still present in the Dhh−/−gonads, which
may account for morphological identification of fetal
Leydig cells in electron micrographs in Dhh−/−gonads
(Clark et al. 2000). In previous work, we showed that low
SF1-expressing cells derived from a second wave of pro-
liferation in the coelomic epithelium (Schmahl et al.
2000). No difference in proliferation or apoptosis was
observed in gonads cultured with the hedgehog inhibitor
cyclopamine, suggesting that DHH/PTCH1 signaling
does not regulate proliferation or survival of fetal Leydig
cell precursors as has been shown to occur in other sys-
tems (Cann et al. 1999; Oppenheim et al. 1999; Charrier
et al. 2001). The time at which DHH affects Leydig dif-
ferentiation, based on in vitro experiments using cyclo-
pamine to block hedgehog signals, suggests that DHH/
PTCH1 signaling specifies Leydig cell fate by early up-
regulation of SF1 and its target, Scc.
The failure of fetal Leydig cell differentiation provides
an explanation for the feminized external genitalia phe-
notype of Dhh−/−XY mice (Clark et al. 2000) and a 46,XY
partial gonad dysgenesis patient with a Dhh mutation
(Umehara et al. 2000). Both cases developed premature
female external genitalia with a blind vagina. The inter-
nal accessory sex glands and ducts, whose development
depends upon the proper amount of testosterone from
fetal Leydig cells, are decreased in size, and the testes
were undescended. The appearance of a few Leydig cells
in Dhh−/−gonads at later stages is not sufficient to rescue
differentiation of secondary sex characteristics in Dhh−/−
mice; however, it does suggest that other signaling path-
ways may partially compensate for loss of the DHH/
PTCH1 signaling pathway. Alternatively, a subpopula-
tion of Leydig cells may derive independent of DHH/
PTCH1 signaling. We are conducting more experiments
to explore the origin of Leydig cell precursors and the
interaction between DHH/PTCH1 and other signaling
Materials and methods
The generation of Dhh-null mice was described previously, and
original breeding mice for the Curis colony were kindly pro-
vided by Dr. Andrew McMahon (Harvard University, Cam-
bridge, MA). Mice were bred on a mixed background of 129/Sv,
C57BL/6, and Swiss Webster. The Dhh genotype was deter-
mined by polymerase chain reaction (PCR) of tail DNA. CD1
random-bred mouse strains (Charles River) were used for organ
culture, immunocytochemistry, and in situ hybridization. GFP
transgenic mice (Hadjantonakis et al. 1998) were used for mi-
gration studies. The Ptchtm1Mpsmice were generated as de-
scribed by Goodrich et al. (1997) and were kindly provided by
Dr. Matthew Scott of Stanford University.
Genital ridges (gonad plus mesonephros) from 11.25–11.5 dpc
embryos (0.5 dpc represents noon of the day when the vaginal
plug was detected) were obtained for organ culture. To deter-
mine the sex of 11.25–12.5 dpc embryos, we used a staining
method (Palmer and Burgoyne 1991) to detect the presence of
XX-specific Barr bodies in the amnion of individual embryos.
Genital ridges were cultured at 37°C with 5% CO2/95% air on
a 1.5% agar block for 48 h in Dulbecco’s Minimal Eagle Medium
(DMEM), supplemented with 10% fetal calf serum (Hyclone),
and 50 µg/mL ampicillin. Cyclopamine (25 µM, TRC Biomedi-
cal Research Chemicals) was added to the culture medium to
inhibit the hedgehog signaling pathway. This concentration of
cyclopamine represented the minimal concentration resulting
in disruption of testis cord formation as determined previously
(Yao and Capel 2002). An equivalent volume of methanol (sol-
vent for cyclopamine) was added to other organ cultures as con-
Whole-mount in situ hybridization
Samples were fixed overnight in 4% paraformaldehyde in PBS at
4°C and processed according to the method of Henrique et al.
(1995). We used alkaline phosphatase-conjugated digoxigenin-
labeled RNA probes for Dhh and Scc. Two different alkaline
phosphatase substrates (NBT/BCIP for Dhh, Fast Red for Scc,
Boehringer Mannheim) were used for color development.
Yao et al.
1438 GENES & DEVELOPMENT
Double whole-mount in situ hybridization
To double-label Scc (mRNA) and SF1 (protein) in the gonads,
whole-mount in situ hybridization was performed as described
above using Fast Red as the substrate for alkaline phosphate
followed by immunocytochemistry against SF1. After fast red
color development (∼5 h at room temperature), samples were
washed in PBS for 10 min and blocked in the blocking solution
(10% heat-inactivated goat serum and 0.1% Triton X-100 in
PBS) for 1 h at room temperature. A rabbit polyclonal antibody
against SF1 (1:200) was added to the blocking solution and
samples were incubated overnight at 4°C. Samples were then
washed 3 times for 10 min each in washing solution (1% heat-
inactivated goat serum and 0.1% Triton X-100 in PBS) followed
by incubation in the blocking solution with the secondary an-
tibody (FITC-conjugated goat anti-rabbit antibody, 1:1000, Jack-
son Immunochemicals). Samples were washed 3 times for 10
min each in washing solution and mounted for confocal micros-
Gonads and mesonephroi from 11.5 dpc CD1 or GFP or PtchLacZ
embryos were separated. A CD1 XY gonad was assembled with
a GFP or a PtchLacZmesonephros and cultured on an agar block
for 48 h as described (Martineau et al. 1997). Images were ob-
tained using a Leica MZFLIII dissecting microscope with a GFP
Samples were washed in PBS and fixed in 2% paraformaldehyde
for 20 min at room temperature. Samples were then rinsed in
washing solution (2 mM MgCl2, 0.02% Nonidet P-40 in PBS),
incubated overnight at 37°C in ?-gal stain (1 mg/mL X-gal, 200
mM K3Fe(CN)6, 200 mM K4Fe(CN)6), washed, and postfixed in
Assay for proliferation and apoptosis
To assay proliferation, gonad explants were fixed overnight in
4% paraformaldehyde in PBS at 4°C immediately after culture.
Samples were processed and cut into 10-µm frozen serial sec-
tions as described (Karl and Capel 1998) and stained immuno-
cytochemically for a proliferation marker, phosphorylated His-
tone H3 (pHH3). The primary antibody was a rabbit polyclonal
antibody against pHH3 (1:1000; Upstate Biotechnology) and the
secondary was an FITC-conjugated goat anti-rabbit antibody (1:
500, Jackson Immunochemicals). pHH3-positive cells from 10
serial sections of each gonad (n = 5) were counted and subjected
to statistical analysis. To assay apoptosis, gonad explants were
cultured in 1 mL medium with 2 µL of LysoTracker Red DND-
99 (Molecular Probes) for an additional 30 min at the end of 24-h
of culture. Gonad explants were washed 3 times in PBS, fixed
overnight in 4% paraformaldehyde in PBS at 4°C, and mounted
for confocal imaging.
We sincerely thank Dr. Ann Clark, who initiated this collabo-
Schmahl, Andrea Ross, Jordan Bachvarov, and Leo DiNapoli,
who all contributed to useful discussions. For their generous
gifts of materials, we thank Harold Erickson (laminin antibody),
Ken-ichirou Morohashi (SF1 antibody), Keith Parker (Scc probe),
and Matthew Scott (Ptchtm1Mpsmice). This work was supported
by grants to B.C. from the NIH (HD39963-04) and a postdoctoral
fellowship from the Lalor Foundation to H.Y.
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section
1734 solely to indicate this fact.
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