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| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 16 August 2006
doi: 10.1242/dev.02542
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1 Department of Cell Biology, New York University School of Medicine, 550 First
Avenue, New York, NY 10016, USA.
2 Department of Dermatology, New York University School of Medicine, 550 First
Avenue, New York, NY 10016, USA.
* Author for correspondence (e-mail: cowinp01{at}med.nyu.edu)
Accepted 19 July 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mammary, Breast, Gli, Hedgehog, Wnt
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The Hh pathway was elucidated and is best understood in the development of
cuticular denticle belts of the fruitfly, Drosophila melanogaster
(Hammerschmidt et al., 1997).
Flies express a single Hh protein that binds to Patched (Ptc), a twelve-pass
transmembrane receptor, on neighboring cells. Hh-Ptc association relieves the
seven-pass transmembrane protein, Smoothened (Smo), from Ptc-mediated
repression. Smo then promotes phosphorylation of Cubitus interruptus (Ci), a
microtubule-bound transcription factor, inhibiting its proteolysis into a
transcriptional repressor, CiR, and converting it into a
full-length transcriptional activator, CiA
(Aza-Blanc et al., 1997;
Ohlmeyer and Kalderon, 1998).
Thus, the Hh morphogen gradient is translated into position-specific gene
expression by modulating the CiA:CiR ratio
(Aza-Blanc and Kornberg,
1999).
In mammals, the Hh pathway is far more complicated. Mammals express three
Hh genes [sonic (Shh), Indian (Ihh) and desert
(Dhh) hedgehog] and two patched genes (Ptc1 and
Ptc2) (Echelard et al.,
1993; Goodrich et al.,
1997; Lewis et al.,
1999a; Motoyama et al.,
1998; Pathi et al.,
2001; Pearse et al.,
2001). Moreover, the transcriptional activator and repressor roles
of Ci have been subdivided in complex ways among three homologues: Gli1, Gli2
and Gli3 (Hui et al., 1994).
Gli2 is expressed in the absence of Hh signals and, in this situation, is
either inactive or functions as a weak transcriptional repressor
(Aza-Blanc et al., 2000;
Bai and Joyner, 2001;
Sasaki et al., 1999;
Sheng et al., 2002). Hh
signals activate Gli2 initiating transcription of Hh target genes, including
Ptc1 and Gli1 (Bai et
al., 2002). Gli1 is strictly dependent on Hh signals for
its expression and thus is an excellent reporter of positive Hh signaling
(Bai et al., 2002;
Bai et al., 2004). It lacks a
transcriptional repressor domain and is a strong activator of Hh target genes,
including itself. It can effectively substitute for Gli2 and antagonize Gli3,
yet it appears to be dispensable for Hh signaling as demonstrated by the
normal phenotype of Gli1-/- mice
(Bai et al., 2002;
Bai and Joyner, 2001;
Dai et al., 1999;
Hynes et al., 1997;
Lee et al., 1997;
Park et al., 2000). Gli3 can
be expressed in the absence of Hh signals. However, Shh signaling suppresses
both Gli3 transcription and the N-terminal proteolytic processing
that produces the Gli3 repressor (Gli3R)
(Aza-Blanc et al., 2000;
Li et al., 2004;
Marigo et al., 1996;
Wang et al., 2000). Gli3 can
function as a transcriptional activator or repressor of Gli1 and other target
genes depending upon the cell context (Bai
et al., 2004; Wang et al.,
2000). In the simplest model, Gli2 acts at the top of the pathway
to induce expression of the amplifier Gli1, which antagonizes the repressor
activity of Gli3. However, cell context-specific roles of Gli2 and Gli3 mean
that activator and repressor functions cannot be assumed for these proteins
but must be determined empirically.
The Shh pathway plays a central role in the formation of many vertebrate
epidermal appendages (sweat, sebaceous, lachrymal and salivary glands, hair,
whiskers, feathers, scales, teeth and nails) that arise as a result of
epithelial-mesenchymal interactions
(Chuong et al., 2000;
Cobourne and Sharpe, 2005;
Dassule et al., 2000;
Gallego et al., 2002;
Michno et al., 2003;
Pispa and Thesleff, 2003;
Ting-Berreth and Chuong,
1996). The requirement for Shh during embryonic and adult hair
follicle development and downward growth has been particularly well documented
(Chiang et al., 1999;
Mill et al., 2003;
St-Jacques et al., 1998). Hair
follicles and mammary glands co-evolved and share many local inductive
pathways (Wnt, Fgf, Bmp and Pthlh) (Andl et
al., 2002; Chu et al.,
2004; Hens and Wysolmerski,
2005; Mailleux et al.,
2002; Oftedal,
2002; Wysolmerski,
2002; Wysolmerski et al.,
1998). Many similarities exist between the cyclical development of
the mammary gland during pregnancy, lactation and involution, and the rounds
of hair follicle growth (anagen), regression (catagen) and resting (telogen).
Mammary glands first form around embryonic day 10 (E10.5) as bilateral
ectodermal thickenings between the fore and hindlimbs, known as milk lines
(Veltmaat et al., 2003). At
about E11, the lines fragment and cells coalesce into five pairs of mammary
placodes that, within 1 day, form elevated mammary buds. Between E13-E14 the
mammary buds invaginate, forming a bulb below the surface of the epithelium.
This structure induces the underlying stroma to condense and differentiate
into mammary mesenchyme. In males, fetal androgens stimulate mammary
mesenchymal fibroblasts to constrict around the epidermal buds, choking
further development (Dunbar et al.,
1999). In females, epithelial buds elongate at E16, forming the
mammary sprout, which penetrates the underlying fat pad precursor and branches
to form 5-6 primary ductules by E18. Further development does not occur until
puberty, when estrogen and growth hormone induce stromal IGF secretion,
stimulating proliferation of cells at the tips of each duct within terminal
end buds, leading to ductal elongation
(Marshman and Streuli, 2002).
During each cycle of pregnancy, progesterone and prolactin stimulate several
local paracrine pathways that promote extensive ductal side-branching and
alveologenesis (Brisken et al.,
2000; Brisken et al.,
1998; Henninghausen and
Robinson, 1998; Robinson et
al., 2000). At the end of lactation, the mammary gland involutes
by a sequential process involving epithelial apoptosis, extensive matrix
remodeling and a wave of adipogenesis, which replenishes the mammary fatpad
(Lund et al., 1996).
Both Shh and Ihh mRNAs have been detected within
embryonic mammary bud epithelium by in situ hybridization, but elimination of
either gene has no effect on bud development
(Gallego et al., 2002;
Michno et al., 2003). Thus,
the function of Hh signaling within the mammary gland is obscure, and
questions remain as to whether signaling by Shh and Ihh is redundant or
dispensable for embryonic mammary gland development. To explore the role of
the Hh pathway further, we determined the expression of the three downstream
transcription factors, Gli1, Gli2 and Gli3, and examined the effects of
altering the Gli activator/repressor ratio during mammary development. Our
results demonstrate that, contrary to previous suggestion, and, in contrast to
other epidermal appendages, positive Hh signaling is absent throughout mammary
development. Furthermore, we show that Gli3-mediated transcriptional
repression is essential for the formation of two pairs of mammary buds, and
misactivation of the Hh pathway, by targeted expression of Gli1, induces bud
loss.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mice
carrying Gli1 or lacZ knocked into the Gli2 locus
(Gli21nki/+ and Gli2lzki/+) were
constructed as described (Bai and Joyner,
2001). Gli3xt/+, Ptc1lzki/+and
TOP-Gal mice were obtained from Jackson Laboratories (Bar Harbor, ME).
Ptc1lzki/+ mice were as described
(Goodrich et al., 1997).
Whole-mount analysis
For detection of lacZ expression, mammary glands or embryos were
fixed in 4% paraformaldehyde (PFA) diluted in phosphate-buffered saline (PBS)
for 1 hour, followed by three 1-hour washes in rinse buffer (2 mM
MgCl2, 0.1% sodium deoxycholate, 0.2% NP-40 in PBS). X-gal staining
was carried out overnight in staining buffer (50 µg/ml X-gal in rinse
buffer containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide).
Mammary glands were washed in PBS, post-fixed for 1 hour in 4% PFA, dehydrated
through a graded series of ethanol, cleared for 
30 minutes in Citrisolve
(Fisher Scientific, Pittsburg, PA) and mounted in Securemount (Fisher
Scientific) and viewed under a Leica dissecting microscope (Bannockburn,
IL).
Histology
For histological analysis, mammary glands and embryos were stained as above
with X-gal then post-fixed with 4% PFA overnight at 4°C. They were
embedded in paraffin and sectioned and in some cases processed further for
immunohistochemistry as described below.
Immunohistochemistry
Sections (4 µm) were deparaffinized with xylene and rehydrated through a
graded series of ethanol. Citric acid antigen retrieval was performed for all
antibodies by placing slides in 1.92 g/l of sodium citrate (pH 6.0) and
microwaving for 20 minutes. Rabbit anti-Keratin 14 (K14) (Covance, Berkeley,
CA) (1:400) primary antibody was detected by using the DAKO
EnVision+ Kit comprising horse-radish peroxidase (HRP) coupled
anti-rabbit IgG followed by diaminobenzidine (DAB) following the
manufacturer's protocol (DAKO, Carpinteria, CA). Mouse anti-p63 (Neomarkers,
Freemont, CA) (1:500) was detected using biotin labeled goat anti-mouse IgG
(Vector Labs, Burlingame, CA) and rabbit anti-Gli3 (Santa Cruz Biotechnology,
Santa Cruz, CA) (1:100) was detected using biotin labeled goat anti-rabbit IgG
(Vector Labs) (1:1000) followed by streptavidin-HRP, which was detected using
DAB.
Whole-mount in situ hybridization
Embryos were fixed overnight in 4% PFA diluted in PBS, dehydrated in
methanol and stored at -20°C. Before hybridization embryos were
rehydrated, bleached by incubating for 30 minutes in 6%
H2O2, treated with 6 µg/ml proteinase K for 10
minutes, washed in 2 mg/ml glycine, then fixed in 4% PFA for 20 minutes. All
solutions were made up in PBS-T (PBS, 1% Tween-20) and three 5 minute PBS-T
washes followed each step. Embryos were prehybridized for 2-3 hours in 50%
formamide 5x SSC, 50 µg/ml tRNA, 1% SDS, 50 µg/ml heparin then
hybridized overnight at 70°C in the same buffer containing 2 µg/ml of
digoxigenin (DIG)-labeled Gli3 probe. Following several washes, DIG was
detected by overnight incubation at 4°C in alkaline phosphatase (AP)
labeled anti-DIG Fab' fragments (Roche Indianapolis IN). Color was developed
with BM-purple AP substrate (Roche). Further protocol details are available at
http://saturn.med.nyu.edu/research/dg/joynerlab/protocols.html
Section in situ hybridization
Sections were dewaxed in xylene and rehydrated, fixed with 4% PFA, treated
with 1 µg/ml proteinase K for 15 minutes at 37°C, post-fixed in 4% PFA
and dehydrated. Sections were hybridized overnight at 55°C in 50%
formamide, 10% dextran sulfate, 1x Denhardt's solution, 300 mM NaCl,
0.02 M Tris-HCl pH 8.0, 5 mM EDTA, 0.01% sarkosyl, 250 µg/ml yeast tRNA
containing 1 µg/ml DIG labeled Gli3 probe. DIG was detected as above.
Northern analysis
Total RNA was isolated from mammary gland using the ToTALLY RNA kit
(Ambion, Austin, Texas) (Imbert et al.,
2001). mRNA was purified from 30 µg of total RNA using the
Poly(A)Pure kit (Ambion). Northern analysis was carried out on these mRNA
samples using the NorthernMax-Gly kit (Ambion). The Gli3 cDNA probe was
obtained from Dr Alexandra Joyner and the K18 cDNA probe was obtained from
Caroline Alexander (University of Wisconsin, Madison, WI).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Bai et al., 2002;
Sasaki et al., 1999;
Sheng et al., 2002). To
determine the activity of the Gli2 promoter during embryonic mammary
development, we examined expression of a lacZ reporter knocked into
the Gli2 locus (Gli2lzki/+)
(Bai and Joyner, 2001). In the
region of the developing mammary line, E11 Gli2lzki/+
embryo whole mounts and sections showed mesenchymal lacZ expression
in a diffuse arc between the fore and hind limb and in the underlying somites
(Fig. 1A). By E14, strong
expression was observed within all five pairs of mammary buds as well as in
hair follicles and whisker pads (Fig.
1B,C). Histological sections of E14 embryos revealed intense
Gli2-lacZ expression within the epidermis, the mammary bud epithelium
and the surrounding condensed mammary mesenchyme
(Fig. 1D). By E16.5, epithelial
Gli2-lacZ expression became restricted to the basal layer of the
epidermis and mammary sprout (Fig.
1E). At this stage, p63 and keratin 14 (K14) expression within the
mammary sprout epithelium is uniform (Fig.
1F,G), but becomes restricted, within the adult mammary gland, to
basal myoepithelial cell types.
|
). A reporter allele of Gli3 has not been described.
Therefore, we examined expression of Gli3 mRNA by in situ
hybridization. Gli3 mRNA was detected in E11 embryo whole mounts in a
diffuse arc between the fore and hind limb and the underlying somites, a
pattern similar to that observed for Gli2-lacZ
(Fig. 2A). This pattern is
consistent with recent reports of somitic and weaker dermal mesenchymal
Gli3 mRNA expression (Veltmaat et
al., 2006). E13.5 embryo wholemounts showed weak Gli3
expression in the vicinity of all mammary buds with stronger expression in
pair number 3 (Fig. 2B). Gli3
protein was localized by immunohistochemistry within the nuclei of mammary bud
epithelial cells and surrounding stromal cells
(Fig. 2C,D), in a similar
expression pattern to that of Gli2-lacZ.
|
;
Bai et al., 2004).
Gli1-lacZ expression in Gli1lzki/+ mice was
absent throughout embryonic mammary development yet was prominent in embryonic
hair follicles and whisker pads (Fig.
3A,B). The absence of Gli1-lacZ reporter expression in
mammary buds and robust activity in hair follicles strongly suggests that Hh
pathway activation and Gli-dependent gene expression diverges among distinct
types of epidermal appendages. To test this hypothesis further, we examined
the activity of a second Hh target gene, Ptc1
(Goodrich et al., 1997). Hh
signals upregulate Ptc1 expression as part of a negative feedback
mechanism. Although Ptc1 provides a receptor for Hh proteins, at high levels
it suppresses Hh activity by sequestering Smo
(Casali and Struhl, 2004).
Ptc1-lacZ expression was observed in hair follicles but was absent
throughout embryonic mammary development
(Fig. 3C,D). These results
confirm that the Hh pathway is active in epidermal appendages, such as hair
follicles, but is either inactive or repressed throughout embryonic mammary
development.
Postnatal mammary Gli2-lacZ expression is observed continuously in stromal cells and cyclically in myoepithelial cells
At birth Gli2-lacZ expression was found in both basal epithelial
and stromal layers of the mammary gland
(Fig. 4A,B). At the onset of
puberty, it was lost from the epithelial cells but was expressed in spikes
along the entire ductal system (Fig.
4C,E), and was concentrated around the neck of the terminal end
buds, giving a thistle-like appearance to these structures
(Fig. 4E, arrow).
Gli2-lacZ was also prominently expressed in mammary lymphatics
(Fig. 4E, asterisk).
Histological sections showed that Gli2-lacZ periductal spikes
comprise groups of tightly adherent stromal cells that triangulate between
adipocytes and the myoepithelium (Fig.
4D,F). Immunohistochemical analysis confirmed that in virgin
(Fig. 4G,H) and early pregnant
(P8) mice all Gli2-lacZ-positive cells lay beneath the
K14/p63-positive myoepithelial layer and thus were stromal. P14 glands,
however, showed additional myoepithelial expression surrounding alveoli as
revealed by Gli2-lacZ colocalization with K14 and p63
(Fig. 5A-D). This
Gli2-lacZ-positive myoepithelial population persisted during
lactation but was lost during involution (data not shown).
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;
Lewis et al., 1999b).
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|
).
Gli3xt/+ mice showed normal embryonic development of all
ten mammary glands and, as adults, could successfully feed their litters
(Table 1). By contrast, all
Gli3xt/xt embryo whole-mounts lacked bud pair number 5 and
the majority lacked bud number 3. A small percentage showed reduction and/or
misplacement of bud number 3 (Fig.
8B,D; Table 1). To
analyze which stage of mammary bud formation is affected by loss of Gli3
function, we examined the embryos of Gli3xt/+ mice crossed
to Gli3xt/+;TOP-Gal mice harboring the TOP-Gal transgenic
reporter of Lef/Tcf transcriptional activity
(DasGupta and Fuchs, 1999).
TOP-Gal is expressed during the earliest stages of mammary line formation and
later in the mammary placodes and buds
(Chu et al., 2004). At E11
TOP-Gal expression was seen in the developing placode pairs number 3 and
number 4 in all wild-type and Gli3xt/+ embryos
(Fig. 8A). By contrast, 80% of
Gli3xt/xt embryos lacked TOP-Gal expression from the
placode 3 region of the mammary line (Fig.
8B; Table 1).
Fourteen percent of embryos showed normal marker expression on one side and
absence of marker expression on the contralateral side. Similar results were
observed by in situ hybridization for Wnt10b, another early marker of
the mammary line and placodes (Veltmaat et
al., 2004) (data not shown). These data demonstrate that early
patterning of the mammary line and placode 3 formation is compromised in
Gli3xt/xt embryos. By E12.5, TOP-Gal was expressed in all
five pairs of mammary buds in wild-type and Gli3xt/+
embryos (Table 1;
Fig. 8C). TOP-Gal staining in
Gli3xt/xt embryos revealed that bud pair number 5 was
always missing and bud number 3 was absent 83% of the time or was small and/or
misplaced in the remaining 17% (Fig.
8D; Table 1). Thus,
Gli3 function is essential for the normal complement and positioning of
mammary buds. A similar percentage of absence of bud pairs number 3 and 5 was
observed in E14.5 Gli3xt/xt embryos. Histological sections
of Gli3xt/xt embryos showed that the remaining bud pairs
are normal at E14.5 (Fig.
2D).
|
). To
investigate whether the repressor or activator function of Gli3 is essential
for formation of mammary bud pairs number 3 and number 5, we examined a series
of double mutant mice (Table
1). Gli2 requires positive hedgehog signaling for activation and,
in the absence of signaling, Gli2 is present in an inactive or weakly
repressive state. Gli1, however, lacks a repressor domain and is a strong
amplifier of the pathway (Dai et al.,
1999; Park et al.,
2000). Thus, driving expression of the constitutive Gli1
activator under the control of the Gli2 promoter
(Gli21nki/+ and Gli21nki/1nki) tests
the effect of progressively increasing the activator to repressor ratio within
the Gli2 field of expression (embryonic mammary bud and mesenchyme). In
similar experiments, substitution of Gli2 by Gli1 has been
shown to exacerbate the Gli3xt/+ limb phenotype
(Bai and Joyner, 2001). In the
E13.5 mammary gland, substituting one or even two copies of Gli2 with
Gli1 (Gli21nki/+ and
Gli21nki/1nki) had no effect on mammary development
(Fig. 8E-G;
Table 1). Likewise, replacing a
single copy of Gli2 by Gli1 and at the same time lowering
Gli3 expression (Gli21nki/+;Gli3xt/+)
failed to produce a mammary phenotype. However, when both Gli2 copies
were replaced by Gli1, and Gli3 was simultaneously reduced
(Gli21nki/1nki;Gli3xt/+), mammary bud pairs
number 3 and number 5 were lost in the majority of embryos
(Fig. 8H-J;
Table 1). Thus, in the mammary
gland, two copies of the Gli1 activator expressed from the
Gli2 promoter are sufficient to antagonize Gli3 repressor expressed
from a single copy of Gli3. This establishes that the function of
Gli3, within the embryonic mammary gland, is to repress Hh target genes. Our
results further demonstrate that the Gli activator/repressor ratio of hedgehog
signaling is crucial for correct mammary gland patterning and that buds 3 and
5 are particularly susceptible to changes in this ratio.
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E10.5
prior to mammary anlagen formation. However, to test further whether Gli3
directly represses Gli2-mediated activation of Hh target genes we crossed
Gli1lzki/+ reporter mice to Gli3xt/+
mice and examined the effect of loss of Gli3 expression on the
Gli1-lacZ reporter. At E14.5 Gli1-lacZ was detected in the
stroma surrounding bud pairs 1 and 4 of Gli3xt/xt embryos,
n=4 (Fig. 8L-N) but
not wild-type embryos (Fig.
3A,B). As Gli1 expression is dependent on Gli2-mediated
transcriptional activation, we conclude that Gli2 is present in an activator
form but is fully antagonized by Gli3R in normal embryos. These experiments
further demonstrate that, in addition to affecting early patterning events,
which govern placode formation, Gli3R continues to repress Hh target genes
within the stroma surrounding mammary buds after their formation. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Positive Hh signaling is absent during embryonic mammary development
The Hh pathway is crucial for the patterning and growth of many epidermal
appendages (Chuong et al.,
2000). An absolute requirement for its activity has been
documented in the development of hair, teeth and feathers
(Hardcastle et al., 1998;
St-Jacques et al., 1998;
Ting-Berreth and Chuong,
1996). The fact that epidermal appendages have a common origin and
share many developmental pathways has prompted several recent investigations
into the role of Hh signaling in mammary development. These studies detected
Shh and Ihh mRNA within mammary bud epithelium, but,
nevertheless, showed that Shh-/- and
Ihh-/- embryos develop ten mammary buds, which undergo
normal postnatal development if transplanted into adult mice
(Gallego et al., 2002;
Michno et al., 2003). These
data suggest that Hh signaling is either redundant or dispensable for mammary
development. The presence of Ptc1 mRNA within
Shh-/- mammary glands was interpreted as evidence for Hh
redundancy (Michno et al.,
2003). However, in comparison with the hair follicle, where the Hh
pathway is unquestionably active, mammary Ptc1 mRNA levels are barely
detectable. Such basal levels are consistent with absence of Hh signaling
resulting in repression of Ptc1 transcription. Consistent with the
viewpoint that Shh and Ihh are dispensable for mammary bud development, our
results show that two well-characterized and sensitive reporters of positive
Hh signaling, Gli1-lacZ and Ptc1-lacZ, are absent during
embryonic mammary development. Further challenging the concept that positive
Hh signaling is required for mammary development, our analyses show that
Gli1and Gli2 mutants have no obvious defects in mammary bud
formation.
Gli3 acts as a repressor during embryonic mammary development
In stark contrast to the lack of mammary phenotypes in
Gli1lzki/lzki and Gli2lzki/lzki
embryos, the majority of Gli3xt/xt mutants lack mammary
placodes number 3 and 5, and the remainder show hypoplastic, asymmetric or
lateral displacement of placode number 3. Marker analysis indicates that
Gli3 is essential for the earliest stages of embryonic mammary
development, affecting the positioning and formation of mammary placodes. Gli3
is present in the absence of Hh signaling and thus can have Hh-independent
functions. However, Hh signals downregulate Gli3 transcription and
inhibit Gli3 proteolysis into Gli3R, a process referred to as
negative Hh signaling. Although Gli3 often acts as a potent repressor of the
Hh pathway, several studies have shown that in certain contexts it functions
as a weak activator (Gli3A). For example, Gli3 is essential for the
correct patterning of the ventral spinal cord where its activation of
Gli1 is involved in the development of motoneurons
(Bai et al., 2004). Additional
examples of Gli3A function are found in the development of the
glandular epithelium of the embryonic stomach and skeletal muscle
(Kim et al., 2005;
McDermott et al., 2005). To
determine whether Gli3 functions as an activator or repressor of Hh regulated
genes within the embryonic mammary gland, we conducted double Gli
mutant experiments. Targeted replacement of Gli2 by Gli1
within Gli3xt/+ mice resulted in loss of mammary bud pairs
number 3 and number 5. The ability of the constitutive Gli1 activator to
antagonize Gli3 reveals that Gli3 functions as a repressor in this
developmental context. Whether Gli3R functions independently of Hh
signals or is modulated by them during embryonic mammary development remains
to be determined. However, the induction of the Hh target gene Gli1
in the stroma of the remaining Gli3xt/xt E14.5 mammary
buds suggests that, in normal embryos, Gli3 is actively repressing Hh
signaling at this stage.
Mammary placodes arise in a distinct order: number 3, number 4, number 1 +
number 5, number 2. Pairs 3 and 4 form at the anterior and posterior ends of
the mammary line. Pairs 1 and 5 form from independent streaks of cells that
encircle the fore- and hindlimbs (Veltmaat
et al., 2004). Pair 2 develops last from streaks of Wnt10bpositive
cells extending from placodes 1 and 3. Wnt/catenin signaling is the earliest
known marker of embryonic mammary development, and mice misexpressing the Wnt
inhibitor Dkk within the epidermis fail to form any mammary placodes
(Andl et al., 2002;
Chu et al., 2004). Lack of
expression of the Wnt signaling reporter TOP-Gal in the central region of the
mammary line in E11 Gli3xt/xt embryos demonstrates that
Gli3 repression is required prior to these early patterning events that
precede mammary placode formation. It has been proposed that cells migrate
along the mammary line and perilimbal streaks and coalesce to form placodes
(Veltmaat et al., 2004).
Intriguingly, genetic interaction has been reported between Gli3 and Twist, a
regulator of epithelial-mesenchymal transitions that are critical for cell
migratory processes (O'Rourke et al.,
2002). The loss of only two pairs of mammary buds upon
misactivation of the Hh pathway in Gli3xt/xt mice
reinforces previous data showing that specific combinations of molecular cues
govern the formation of different pairs of placodes
(Veltmaat et al., 2003).
Analysis of inbred mouse strains and, more recently, of scaramanga (Ska) mice
implicates variable susceptibility of specific bud pairs to both loss and
supernumerary formation (Howard et al.,
2005; Little and McDonald,
1945; Veltmaat et al.,
2003). In these studies, correct regulation of morphogenic
interactions involved in mammary line and placode formation appears to be most
crucial for the formation of bud pair 3 and least crucial for that of bud pair
4. Further examples are found in studies on loss of Tbx3, which in humans
produces mammary hypoplasia and nipple loss, as well as in studies on Lef1 and
Fgf pathways. Mice lacking Tbx3 show loss of mammary buds but
occasional retention of bud number 2
(Bamshad et al., 1997;
Davenport et al., 2003;
Eblaghie et al., 2004).
Lef1-/- mice form small mammary placodes that degenerate
and occasionally retain bud 4 (van
Genderen et al., 1994). Mice that lack Fgf10, which is expressed
within the ventrolateral portion of somites, or its receptor Fgfr2b, which is
expressed in the mammary placodes, fail to develop mammary buds 1, 2, 3 and 5,
yet retain bud number 4 (Mailleux et al.,
2002). Recently, a genetic requirement for Gli3 in the ventral
somitic expression of Fgf10 has been described
(Veltmaat et al., 2006). Our
demonstration that Gli3 acts as a transcriptional repressor now shows that
this Gli3-mediated induction of Fgf10 expression must be indirect.
The studies described above suggest that different placodes vary in their
susceptibility to a crucial developmental threshold during the earliest stages
of mammary development (Veltmaat et al.,
2003). Our experiments reveal that the
GliA/GliR ratio provides such a crucial developmental
threshold for buds number 3 and number 5. Yet there are no reports of mammary
bud loss in mouse models or human syndromes (basal cell nevus syndrome) where
the Hh pathway is aberrantly activated
(Johnson et al., 1996;
Nilsson et al., 2000;
Sheng et al., 2002). However,
this is not surprising because, in mice, mammary bud loss occurs only under
conditions that result in embryonic lethality (complete loss of Gli3
or partial loss of Gli3 in conjunction with pathway misactivation)
and are likely to have the same outcome in humans. Nevertheless, there are
occasional reports of rare human syndromes with features suggestive of pathway
misactivation, such as polydactyly associated with hypoplastic nipples
(Teebi and Druker, 2001).
Epidermal appendages show distinct requirements and proliferative responses to Hh signaling: repression of hedgehog signaling is essential for mammary development
The results of this study show that, despite their common origin, mammary
glands differ from other epidermal appendages in their requirement for Hh
pathway repression rather than activation. Hh-positive activity is essential
for tooth and hair follicle development
(Cobourne et al., 2001;
Dassule et al., 2000;
Gritli-Linde et al., 2002;
Hardcastle et al., 1998;
Mill et al., 2003;
St-Jacques et al., 1998). For
example, hair follicles express high levels of Shh, show pathway
activation, as evidenced by Gli1-lacZ expression, and proliferate in
response. In the absence of positive Hh signaling, hair placodes form but fail
to undergo downward growth and arrest at the hair plug stage
(Chiang et al., 1999;
Mill et al., 2003;
St-Jacques et al., 1998). By
contrast, mammary buds express low levels of Shh and Ihh,
fail to activate the pathway, as evidenced by lack of Gli1-lacZ
expression, yet proliferate when signals are repressed. Repression of the Hh
pathway is essential for the correct morphogenesis of several other organs.
For example, in the limb, loss of Gli3 function in
Gli3xt/xt mutants results in polydactyly, resembling a Shh
gain-of-function phenotype (Hui and
Joyner, 1993; Litingtung et
al., 2002).
Hh target gene expression is absent during development of the adult mammary tree but is robust within lymphatics
In addition to defining the roles of Hh signaling in embryonic mammary
development, our results provide new insight into the role of the Hh pathway
during adolescent and adult mammary gland development. Gli2-lacZ
expression within the stroma encasing virgin mammary ducts is entirely
consistent with a previous report of Gli2 mRNA expression
(Lewis et al., 2001). However,
the greater sensitivity and clarity afforded by the lacZ reporter,
combined with immunohistochemical marker analysis, allows us to define that
Gli2 expression occurs cyclically in mid-pregnant mice within
myoepithelial cells and not within all epithelial cells as reported previously
(Lewis et al., 2001).
Furthermore, we show that although Gli3 is co-expressed with Gli2 in stroma
and myoepthelia, it is the only Gli found within the lumenal epithelium of the
adult gland. Lack of expression of Gli1-lacZ and Ptc1-lacZ
within mammary epithelia, myoepithelial, stroma or adipocytes leads us to
conclude that Hh signaling is absent or stifled within the parenchyma of the
adult gland yet is robust within lymphatics. This finding is at odds with a
previous report of Ptc1 mRNA expression in the epithelial layers of
virgin ducts and a haploinsufficient phenotype in Ptc1+/-
mice involving minor changes in terminal end bud clefting and transient ductal
hyperplasia that rectifies during pregnancy
(Lewis et al., 1999b). Of
note, our examination of Ptc1-lacZ expression employs the same mouse,
albeit on an outbred background. We have looked extensively and have seen no
evidence of these phenotypic aberrations. Moreover, our observations are
entirely consistent with other studies reporting lack of Gli1 expression in
normal human breast tissue (Kubo et al.,
2004). Intriguingly, these studies reported Gli1 protein
upregulation in 52/52 epithelial breast cancers and 4/6 epithelial breast
tumor cell lines probably resulting from epigenetic regulatory events, as
mutations in Hh pathway components are infrequent in breast tumors
(Chang-Claude et al., 2003;
Kubo et al., 2004;
Vorechovsky et al., 1999;
Wicking et al., 1998;
Xie et al., 1997). Our results
show that Gli3 is the only Gli expressed in normal lumenal epithelial cells.
Whether Gli1 misexpression in breast tumors results from loss of
Gli3R activity is an important issue for future investigation.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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