Crosstalk between the p190-B RhoGAP and IGF signaling pathways is
required for embryonic mammary bud development
Brandy M. Heckmana, Geetika Chakravartyb, Tracy Vargo-Gogolaa, Maria Gonzales-Rimbaua,
Darryl L. Hadsellc, Adrian V. Leed, Jeffrey Settlemane, Jeffrey M. Rosena,⁎
aDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
bDepartment of Molecular and Cellular Oncology, MD Anderson Cancer Center, Houston, TX 77030, USA
cU.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine,
Houston, TX 77030, USA
dThe Breast Center, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA
eMassachusetts General Hospital Cancer Center and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA
Received for publication 16 May 2006; revised 25 June 2007; accepted 3 July 2007
Available online 10 July 2007
P190-B RhoGAP (p190-B, also known as ARHGAP5) has been shown to play an essential role in invasion of the terminal end buds (TEBs) into
major defects in embryonic mammary bud development. Overall, p190-B-deficient buds were smaller in size, contained fewer cells, and displayed
characteristics of impaired mesenchymal proliferation and differentiation. Consistent with the reported effects of p190-B deletion on IGF-1R
signaling, IGF-1R-deficient embryos also displayed a similar small mammary bud phenotype. However, unlike the p190-B-deficient embryos, the
IGF-1R-deficient embryos exhibited decreased epithelial proliferation and did not display mesenchymal defects. Because both IGF and p190-B
signaling affect IRS-1/2, we examined IRS-1/2 double knockout embryonic mammary buds. These embryos displayed major defects similar to the
p190-B-deficient embryos including smaller bud size. Importantly, like the p190-B-deficient buds, proliferation of the IRS-1/2-deficient
mesenchyme was impaired. These results indicate that IGF signaling through p190-B and IRS proteins is critical for mammary bud formation and
ensuing epithelial–mesenchymal interactions necessary to sustain mammary bud morphogenesis.
© 2007 Elsevier Inc. All rights reserved.
Keywords: p190-B; ARHGAP5; IGF-1R; IRS-1; IRS-2; Mammary bud; Epithelial–mesenchymal
Mouse mammary gland development can first be detected as
a line of epithelial cells that thicken between the limb buds at
embryonic day 10.5–11.5 (E10.5–11.5) (DasGupta and Fuchs,
elevated domes, which then invaginate into the underlying
dermal mesenchyme to form bulb-shaped buds at E12.5
(Mailleux et al., 2002). At E13 the bud enters a resting phase,
and the mesenchyme of fibroblasts adjacent to the bud con-
denses into concentric rings that are separated from the bud by
basement membrane (Kratochwil, 1969; Kimata et al., 1985).
This process coincides with differentiation of the mesenchyme,
which is characterized by upregulation of fibronectin, tenascin-
C, and androgen and estrogen receptors. Expression of these
proteins distinguishes the mammary mesenchyme from the
surrounding dermis and the underlying mesenchyme (Chiquet-
Ehrismann et al., 1986; Durnberger and Kratochwil, 1980;
Inaguma et al., 1988). This mesenchyme has been shown to be
both permissive and instructive in mammary morphogenesis
(Hens et al., 2007; Sakakura et al., 1976; Veltmaat et al., 2003).
The cells underlying the mammary mesenchyme condense to
pad precursor becomes less compact at E15–16 forming a loose
connective tissue and producing fatty substances (Sakakura,
1991). During this time, the bud undergoes rapid proliferation
Developmental Biology 309 (2007) 137–149
E-mail address: firstname.lastname@example.org (J.M. Rosen).
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
leading to bud elongation. The distal end of the primary sprout
pad precursor where it branches to give rise to the rudimentary
ductal tree that is present at birth. Several studies have identified
signaling pathways that are critically involved in regulating
embryonic mammary bud development, and these studies
highlight the importance of the interactions between the
mammary bud and underlying mesenchyme (Hens and Wysol-
P190-B is a member of the RhoGTPase activating protein
(RhoGAP) family, which function as negative regulators of Rho
activity (Burbelo et al., 1995). P190-B and Rho are both
recruited to the plasma membrane following integrin cross-
linking where p190-B functions to inhibit Rho activity by
enhancing the intrinsic GTPase activity of Rho converting the
deletion of p190-B results in central nervous system defects
p190-B-deficient embryos and cells are smaller than their wild-
type counterparts, which is due to impaired IGF signaling.
Importantly, the p190-B and IGF signaling pathways have been
shown to directly interact and to be critically involved in
regulating both cell growth and differentiation (Sordella et al.,
The IGF-1 receptor (IGF-1R) functions through activation of
an intrinsic tyrosine kinase in its cytoplasmic domain (Sachev,
2001). Upon ligand binding, the receptor becomes autopho-
sphorylated and recruits intracellular substrates, including
insulin receptor substrate (IRS) proteins. The IRS proteins
(IRS-1, -2, -3, and -4), are adaptor molecules that organize
signaling complexes at sites of receptor activation .IRS-1 and -2
are ubiquitously expressed including in mammary epithelium.
IRS-3 and -4 are restricted in their localization, and are
predominantly found in adipose tissue and brain, respectively
(White, 1998). IRS-1 and IRS-2 play a critical role in IGF
signaling as well as insulin, interferon, growth hormone, and
The IGF signaling axis has been demonstrated to play a
and gain of IGF-IR disrupts postnatal ductal morphogenesis
(Bonnette and Hadsell, 2001; Carboni et al., 2005; Jones et al.,
2007). Loss of IRS-1 has been shown to reduce mammary fat
pad size. IRS-1 does not appear to be critical for normal ductal
development or pregnancy induced proliferation although it was
hypothesized that IRS-2 might compensate for the loss of IRS-1
expression (Lee et al., 2003). However, despite structural and
functional similarities, IRS-1 and IRS-2 are not interchangeable
in terms of IGF-stimulated gene expression and cell cycle
progression (Bruning et al., 1997).
Previous studies from our laboratory have demonstrated that
p190-B interacts with the IGF-1R signaling pathway to regulate
postnatal mammary ductal morphogenesis (Chakravarty et al.,
2003). Specifically, both deletion of IGF-1R or haploinsuffi-
ciency of p190-B resulted in decreased proliferation in the
terminal end buds (TEBs) and delayed ductal elongation.
Additionally, embryonic mammary transplantation studies
from p190-B- and IGF-1R-deficient mice have shown that
these pathways are essential for postnatal mammary gland
development, since the deficient transplants fail to grow out
(Chakravarty et al., 2003), as is the case with p190-B, or have
only limited outgrowth potential, as seen in IGF-1R-deficient
transplants (Bonnette and Hadsell, 2001). Overexpression of
Gogola et al., 2006). In these mice, IGF signaling was altered
further indicating a role for interaction of these pathways in
regulating postnatal mammary gland development. Although it
is clear that the IGF and p190-B pathways interact to regulate
postnatal mammary gland development, whether they also
contribute to embryonic mammary bud formation remained to
The studies presented here indicate a critical interaction of
p190-B with the IGF signal transduction pathway during
embryonic mammary morphogenesis and suggest that these
pathways are critical for migration of epithelial progenitors.
Furthermore, loss of either p190-B or IRS-1/2 results in major
mesenchymal defects implicating this signaling network in
establishment of the epithelial–mesenchymal interactions that
are necessary to promote mammary bud development. We
propose that these embryonic mammary defects underlie the
failure of the p190-B-deficient mammary glands to develop
postnatally in part through modulation of the IGF signaling
Materials and methods
Heterozygous females (p190-B C57Bl/6; IGF-1R FVB; IRS-1/IRS-2 FVB)
were mated with heterozygous males with detection of a plug marked as E1.
Pregnant females at E14.5 of pregnancy were injected with BrdU (100 mg/kg)
intraperitoneally. Four hours later, embryos were dissected by Cesarean section
in PBS and fixed overnight in 4% paraformaldehyde (PFA). Specimens were
dehydrated and embedded in paraffin. Serial sections were then taken in the
frontal plane at 7 μm on probe on plus slides (Fisher Scientific). Prior to
hybridization, the slides were deparaffinized in xylene, rehydrated, and fixed in
4% PFA for 30 min.
In situ hybridization
Riboprobes were labeled with [DIG]-UTP (Boehringer 1277073), using T7
transcription system from Stratagene. To generate antisense riboprobe template,
CAG and reverse primer 5′-GTAATTACTTTCCCAATTTCT. The sense ribo-
probe template was generated from the same cDNA by PCR using forward
primer 5′-ATGAGATTTATGTTGTCCCAG and reverse primer 5′-GGATCC-
For whole mounts: Embryos were dissected and fixed in 4% PFA overnight,
washedwith70%alcohol.To reducethe background theembryoswerebleached
with 4:1 mix of ethanol and 30% H2O2for 1 h and treated with 15 μg/ml
proteinase K (PK) for 15 min. Proteinase K treatment was blocked with a short
Embryoswere re-fixed with freshly prepared 0.2% glutaraldehyde/4%PFA/PBS
for 20 min. Fixative was replaced with pre-warmed (68 °C) hybridization buffer
(DAKO S3304) and rocked gently until the embryos sank (indicating that the
formamide had penetrated the embryos). This hybridization buffer was replaced
with fresh pre-warmed hybridization buffer containing the probe at a
concentration of 1 μg/ml and hybridized overnight at 68 °C. The embryos
138B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
were then washed two times for 30 min each with freshly prepared pre-warmed
of 10 min in a pre-warmed 1:1 mix of solutions I and solution II (0.5 M NaCl,
5 min each with solution II at RT. Nonspecific hybridization was reduced by
incubating the embryos for 30 min in 100 μg/ml RNase A in solution II at 37 °C.
Excess RNAse Awas removed with two 30 min washes with freshly prepared,
pre-warmedsolution III (50% deionized formamide, 2× SSC) at 65 °C. Embryos
FCS/2% blocking reagent (Roche 1096176)/2 mM levamisole in TBST for at
least 1 h. This was followed by overnight incubation of the blocked embryos at
4 °C with 1:1000 anti-digoxigenin-AP antibody (Roche 1093274) supernatant
solution diluted 1:1 in 10% FCS/2% blocking reagent/TBST/2 mM levamisole.
Subsequently they were washed with 2 mM levamisole/TBSTsolution for 5–6 h
and left overnight in fresh levamisole/TBST at 4 °C. Color development was
initiated by washing the embryos with freshly prepared NTMT (100 mM NaCl,
100 mM Tris–HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween 20)+2 mM levamisole
twice for 20 min and incubating them in pre-warmed BM purple (Roche
1442074) from 30 min to overnight in the dark at room temperature (RT). The
stained embryos were post fixed with freshly prepared 4% PFA.
For embryo sections: 5-μm paraffin sections of E14.5 embryos were
deparaffinized, rehydrated and washed in PBS. Then treated with PK (25 μg/ml)
for 1 h at 37 °C and fixed with 4% PFA for 30 min followed by washing with 2×
SSC.Pre-warmedhybridization buffercontainingthe probe at a concentrationof
1 μg/ml was added and hybridized overnight at 55 °C. Slides were washed with
washes with 2× SSC 20 min at RTand digestion with 40 μg/ml of RNase A for
15 min at 37 °C then washed with 0.1× SSC for 15 min at 42 °C and then 10 min
at RT. Slides were then washed with Buffer I (100 mM Tris, pH 7.5, 150 mM
NaCl) for 5 min at RTand blocked for 2 h in Buffer I with 3% sheep serum and
0.3% Triton X-100 at RT. The slides were then incubated overnight with 1:200
anti-digoxigenin-AP at 4 °C. For color development, slides were washed with
Buffer I for 10 min at RTand then washed with Buffer II (100 mM Tris, pH 9.5,
BM purple (Roche 1442074) from 30 min to overnight in the dark at RT. Slides
were counterstained with nuclear fast red, dehydrated, and mounted using
Permount (Sigma, St. Louis, MO).
5- to 7-μm sections were deparaffinized with xylene, and rehydrated with
ethanols. Heat-induced antigen retrieval was performed in 10 mM citrate by
1081-P(Ab1): 1:500 in M.O.M diluent buffer (Vector Laboratories); androgen
receptor (Upstate, 06-680): 1:500 in 5% BSA, 0.5% blocking buffer; estrogen
(Upstate, 06-248): 1:800 in 5% BSA, 0.5% blocking buffer; IRS-2 (Upstate 06-
506): 1:800 in 5% BSA, 0.5% blocking buffer; biotin-conjugated BrdU (BD
Pharmingen, 550803): 1:10 in 5% BSA, 0.5% blocking buffer all incubated
overnight at RT. ER-α was used in place of AR due to non-specific staining in
new lot of AR antibody. Sections were washed in PBS and incubated with anti-
rabbit (Oncogene Research) or anti-mouse (Oncogene Research) secondary
antibodies diluted 1:1000 in 5% BSA, 0.5% blocking buffer for 1 h at RT.
Vectastain Elite ABC and diaminobenzidine (DAB) substrate kits were used to
detect immunoperoxidase staining according to the manufacturer’s instructions
(Vector Laboratories). As a negative control, slides were incubated with purified
rabbit immunoglobulin (The Jackson Laboratory). Detection was achieved by
incubation with diaminobenzidine (DAKO) for 10 min or less. Slides were
counterstained with hematoxylin for 30 s, dehydrated, and mounted using
Permount. Changes in IRS-1 and IRS-2 staining were quantitated using
ImagePro software by adding the mean color values within the same size
ellipsis in both the wild-type and deficient p190-B embryos and converting the
raw number to a percentage followed by inverting this percentage because in
bright-field imaging more stain in the sample means that the camera sees less
subtracting the blue hematoxylin counterstain and then calculating the
percentage of brown staining within the same size ellipsis to correct for the
change in the area of the bud between the p190-B wild-type and deficient
p190-B is expressed in the developing mammary anlagen
Previously it was reported that p190-B is ubiquitously
expressed in most adult tissues (Burbelo et al., 1995). In
contrast, p190-B is developmentally regulated throughout
postnatal mammary gland development (Chakravarty et al.,
2000). Lack of ductal outgrowths from p190-B-deficient
transplants indicated that its expression might also be spatio-
temporally restricted during embryonic development. We
examined this by performing whole mount in situ hybridization
for p190-B expression on wild-type embryos. For this analysis,
we examined E8.5 embryos, prior to mammary development,
and E12.5 embryos, which is the stage in mammary bud
development where the spherical mammary placode differenti-
ates into an epithelial bud. Although p190-B mRNA shares only
57% homology with p190-A at the nucleotide level, the
specificity of p190-B antisense probe was further ascertained
by aligning the probe sequence with that of mouse p190-A. In
either case, no significant homology was detected between the
two sequences. The sense probe was included as a negative
control (Fig. 1d) and all hybridizations were performed under
highly stringent conditions.
Ubiquitous expression of p190-B was detectable as early as
in the brain, spinal cord, skin, and the limbs (Fig. 1a). At E12.5,
the p190-B transcript is detected throughout the mammary
epithelial bud compartment (Fig. 1b). This was further
confirmed by in situ hybridization in tissue sections at E14.5
where expression of p190-B is present in the epithelium and at a
lower level in the surrounding mesenchyme of wild-type
embryonic mammary buds (Fig. 1c) as compared to the sense
control (Fig. 1d). This expression pattern suggested p190-B
might play an essential role in mammary placode formation and
Loss of p190-B results in a smaller mammary bud size with a
While a number of signaling molecules have been shown to
be expressed within the epithelium or mesenchyme of the
development of the bud. Because loss of p190-B resulted in
complete failure of postnatal ductal development, we examined
whether p190-B deficiency also impacted formation and
differentiation of the mammary anlagen. For this analysis,
wild-type, heterozygous, and deficient E14.5 embryos were
isolated and the histology of hematoxylin and eosin (H&E)-
stained sections was analyzed. Because the buds are known to
form at different rates a bud-to-bud comparison was performed
(Veltmaat et al., 2003). The wild-type buds (Fig. 2a) had an
organized epithelial center surrounded by a dense mesenchyme.
The heterozygous buds displayed a variable intermediate
139B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
phenotype. Some buds were comparable to the wild-type, while
in others, the epithelial compartment was smaller and the
surrounding mesenchyme appeared disorganized. In contrast,
the buds from deficient embryos exhibited markedly fewer
epithelial cells and the mesenchyme surrounding the epithelium
appeared to be diminished and disorganized (Fig. 2c). Bud size
was determined by quantifying the number of sections through
which the bud is detected and, for this analysis, 3 buds were
counted from 3 independent animals. A significant decrease in
bud size is observed in the heterozygous (pb0.001) and
deficient (pb0.0001) embryos as compared to the wild-type
animals (Fig. 2d).
To gain further insight into the role of p190-B signaling in
placode formation, we examined the expression of markers of
mammary epithelium and mesenchyme in tissues from the three
genotypes (n=6). To evaluate possible alterations in progenitor
epithelial content, the expression of p63 was compared in wild-
type, heterozygous and p190-B-deficient E14.5 mammary
anlagen. p63 is a member of the p53 gene family that has been
demonstrated to be critical for the regulation of proliferation and
differentiation in epithelial progenitor cells (Koster et al., 2004).
in wild-type mice (Fig. 3a). Interestingly, a pronounced
reduction in the number of p63-positive cells (pb0.009) was
detected in the p190-B-deficient embryos suggesting that fewer
p63-positive epithelial progenitors had migrated into the bud
from the overlying epidermis (Fig. 3c). As seen in Fig. 3b, no
statistically significant change was seen in the heterozygous
group of embryos (Fig. 3g).
To further evaluate the functional status of the mesenchyme,
differentiation, was examined in tissues from the three
genotypes. In the wild-type embryonic anlagen, a large number
of AR-positive cells formed the condensed mesenchyme (Fig.
3d). However, the mammary anlagen from the p190-B
heterozygous (pb0.004) and deficient (pb0.0001) mice con-
sistently exhibited a reduction in the number of cells staining
positive for AR in the mesenchyme (Figs. 3e, f) as shown by the
quantitation (Fig. 3g). Taken together, these results suggest that
loss of p190-B function impairs mesenchymal condensation and
p190-B may interact with IGF-1R to affect migration of
Disruption of the IGF-1R gene has also been shown to retard
postnatal mammary development (Bonnette and Hadsell, 2001).
In particular, it has been reported that IGF-1R-deficient
embryonic mammary buds display reduced growth potential
when transplanted into syngeneic hosts. This phenotype is
reminiscent of p190-B heterozygous transplants. Furthermore,
our previous studies implicated interaction of these pathways in
the developing postnatal mammary gland (Chakravarty et al.,
2003; Vargo-Gogola et al., 2006). We therefore explored the
Fig. 1. p190-B is expressed throughout the differentiating mammary anlagen. Whole-mount in situ hybridization of wild-type, E14.5 embryos with p190-B antisense
riboprobe showing strong transcript expression in the developing mammary anlagen (a) low magnification (b) high magnification. Spatial localization of p190-B
mRNA in E14.5 mammary buds of wild-type mice using DIG-labeled antisense riboprobe. Shown are representative antisense (c) and sense (d) images with strong
transcript expression in the epithelial compartment of the mammary bud and lower expression in the mesenchyme. Scale bar: 50 μm (c, d).
140B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
possibility that IGF-1R might also inhibit mammary anlagen
formation and differentiation. To test this hypothesis, we
analyzed mammary buds from IGF-1R wild-type and deficient
embryos both histologically and through expression analysis of
markers of epithelial and mesenchymal differentiation.
As shown in Fig. 4, the H&E-stained sagittal sections from
E14.5 IGF-1R-deficient embryonic mammary buds indicated
that the smaller mammary bud phenotype of p190-B-deficient
mice was similarly observed in IGF-1R-deficient mice (Fig. 4b).
The wild-type buds (Fig. 4a) had an organized epithelial center
surrounded by a dense mesenchyme whereas the deficient buds
displayed very little epithelium (n=9). However, unlike the
p190-B-deficient mice, these buds did not show any apparent
mesenchymal defects. These observations were further con-
firmed by staining tissue sections from all three genotypes with
both p63 and estrogen receptor-α (ER) antibodies, another
marker of mesenchymal differentiation (n=9). The mammary
buds from IGF-1R-deficient mice had a significantly reduced
number of p63-positive cells (Figs. 4c, d), but did not appear to
exhibitdefects inthe number ofER-positive cells (Figs. 4e, f).A
significant decrease in bud size is observed in the IGF-1R-
deficient (pb1×10−7) as compared to the wild-type (Fig. 4g).
Quantification of marker staining (Fig. 4h) shows a decrease in
the number of p63-positive cells within the IGF-1R-deficient
buds (pb3×10−6) as compared to the wild-type, but no
significant difference in the number of intensely staining ER-
positive cells. These results suggests that IGF-1R signaling is
required for the recruitment of p63-positive progenitors into the
mammary bud, but that loss of IGF-1R does not effect the
epithelial–mesenchymal interactions that facilitate mesenchy-
mal condensation and differentiation.
IRS-1/IRS-2 expression is decreased within the
p190-B-deficient mammary buds resulting in inhibition of
We previously reported that IRS-1 and -2 expression was
decreased in TEBs of p190-B heterozygous mice (Chakravarty
et al., 2003). Fibroblasts from p190-B-deficient embryos
exhibited an increase in Rho kinase activity, which resulted in
inhibition of IGF/insulin signaling as well as the activity of
several downstream effectors including p38, JNK, and Akt
(Sordella et al., 2002). This modulation of IGF signaling was
shown to be due to phosphorylation of IRS-1 on Ser612, which
targets IRS-1 for degradation (Sordella et al., 2002). To examine
if the downstream effectors of the IGF signaling pathway are
affected in the p190-B-deficient mammary buds, we examined
the level of IRS-1 and IRS-2 (n=3). Quantitation of this staining
(Fig. 5e) demonstrated that the intensity of IRS-1 and IRS-2
staining was decreased (pb0.05) within the epithelial compart-
ment of the p190-B-deficient (Figs. 5b, d) as compared to the
wild-type mammary bud (Figs. 5a, c). Interestingly the IRS-1
Fig. 2. p190-B−/−mice do possess distinct embryonic mammary buds but have reduced epithelial content and exhibit marked reduction of the mammary mesenchyme.
Sagittal sections of E14.5 embryonic mammary buds were stained with hematoxylin and eosin (H&E) to demonstrate a bud to bud comparison of the reduced number
of epithelial cells and loss of a well-defined condensed mesenchyme around the p190-B-deficient (c) and heterozygous (b) buds compared to wild-type (a). Bud size is
significantly decreased as shown by quantitation (d). Scale bar: 50 μm.
141 B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
expression is also decreased in the mesenchyme. These results
areconsistentwithpreviousreports thatlossofp190-Balters the
To further examine the mechanism by which p190-B loss
leads to a decrease in IRS-1 and affects downstream signaling,
we assessed the activation of Akt by performing immunostain-
ing to detect phospho-Akt (Ser473) (n=3). Punctate nuclear
staining for phospho-Akt staining was detected in the p190-B
wild-type buds (Fig. 5f) and was decreased in the p190-B-
deficient buds (Fig. 5g). Quantitative analysis shows a decrease
in pAkt within the p190-B-deficient epithelium (pb0.04) as
compared to the wild-type (Fig. 5h). These results suggest that
loss of p190-B disrupts signaling downstream of IGF-1R, which
may affect formation of the mammary bud.
Loss of IRS-1/2 phenocopies loss of p190-B affecting migration
of epithelial progenitors and condensation of mammary
Because p190-B has been shown to regulate the level of IRS
proteins both during postnatal ductal development and within
test this, we analyzed mammary buds from IRS-1/2 double-
Fig. 3. Loss of p190-B results in reduced epithelial content and a marked reduction in mammary mesenchyme. p63 expression in the epithelium of E14.5 mammary
buds of (a) p190-B wild-type, (b) p190-B heterozygous and (c) p190-B-deficient mice. Androgen receptor (AR) is expressed in the condensed mesenchyme
surrounding the epithelial buds in (d) wild-type and (e) heterozygotes, however, the number of cells expressing AR are dramatically reduced in p190-B-deficient
embryonic mammary buds (f). Quantitation of these staining patterns (g) show a statistically significant decrease in the number of p63-positive cells within the bud of
the p190-B-deficient embryos as well as a statistically significant decrease in the number of AR-positive cells within both the p190-B heterozygous and deficient
anlagen as compared to the wild-type. Scale bar: 100 μm.
142 B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
deficient embryos both histologically and through expression
analysis of markers of epithelial and mesenchymal differentia-
tion (n=6). As shown in Fig. 6, the E14.5 IRS-1/2 double-
deficient embryos appeared to exhibit a similar smaller
mammary bud phenotype seen in the IGF-1R-deficient mice
and the p190-B-deficient mice. The wild-type buds (Fig. 6a)
displayed an organized epithelial center surrounded by a dense
mesenchyme, whereas the double-deficient buds displayed very
little epithelium (Fig. 6c), and the double heterozygous buds
manner as the p190-B-deficient buds and a similar decrease in
size was seen in the IRS-1/2 heterozygous (pb0.02) and
deficient (pb0.001) mammary buds (Fig. 6d). However, unlike
the IGF-1R-deficient mice, the mesenchyme surrounding the
epithelium appeared to be diminished and disorganized much
like the p190-B-deficient embryos. These observations were
further confirmed by staining tissue sections of all three
genotypes with both p63 and AR antibodies. The mammary
buds from both the IRS-1/2 heterozygous and deficient mice
exhibited a decrease in p63-positive cells (Fig. 7c), as well as a
marked reduction in the number of cells staining positive for AR
in the mesenchyme (Fig. 7f). Quantification of these staining
patterns (Fig. 7g) showed a statistically significant decrease in
the number of p63-positive cells within the buds of the IRS-1/2
Fig. 4. IGF-1R-deficient mice phenocopy the small mammary bud phenotype of p190-B−/−mice, but not the mesenchymal defect. Shown here are the sagittal sections
fromwild-typeandIGF-1R-deficientE14.5embryonicmammarybudsstainedwithH&E.Notethesmaller mammary budsin thedeficientembryos(b) ascomparedto
the wild-type (a) littermates. p63 expression in the epithelium of E14.5 mammary buds of (c) IGF-1R wild-type and (d) IGF-1R-deficient mice. ER expression can be
detected in the condensed mesenchyme of (e) wild-type and (f) IGF-1R-deficient embryonic mammary buds. In panel g, bud size is significantly decreased as shown
by quantitation.Quantitation of markersof each compartment (h) show a statistically significantdecreasein the numberof p63-positive cellswithin the bud of the IGF-
1R-deficient embryos but no statistically significant difference in the number of intense staining ER-positive cells between the IGF-1R-deficient anlagen as compared
to the wild-type. Scale bar: 100 μm (a–f).
143 B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
heterozygous (pb0.003) and deficient (pb0.0006) embryos as
well as a statistically significant decrease in the number of AR-
positive cells within the heterozygous (pb0.001) and deficient
(pb4×10−7) anlagen as compared to the wild-type. Taken
together, these results demonstrate that the IRS-1/2-deficient
mammary buds phenocopy the p190-B-deficient buds.
Loss of either p190-B or IRS-1/2 leads to a defect in
mesenchymal proliferation at E14.5
It is possible that p190-B affects the mesenchyme by either
interrupting pathways necessary for the mesenchymal differ-
entiation or affecting signaling pathways necessary for the
proliferation. Previous studies have pointed to a role of IGF-1R
in proliferation and migration of mammary epithelial and breast
cancer cells (Bonnette and Hadsell, 2001; Zhang et al., 2004,
2005). To investigate whether p190-B is required for mesench-
ymal proliferation, we examined cell proliferation by quantitat-
ing the number of BrdU-positive cells associated with the
mammary bud at day E14.5 of p190-B (Figs. 8a–c) and IRS-1/2
(Figs. 8d–f) embryos (n=6). As shown in Fig. 8g, loss of p190-
surrounding the E14.5 mammary bud (pb0.01). This decrease
was calculated by determining the percentage of BrdU-positive
cells that also stained positive for AR in paired serial sections.
The IRS-1/2-deficient E14.5 mammary buds exhibited a similar
decrease in the level of mesenchymal proliferation (pb0.01)
(Fig. 8h). A significant decrease was also detected in the
heterozygous IRS-1/2 mammary buds (pb0.03). Importantly,
there were no cleaved-caspase 3-positive cells detected in either
the p190-B or IRS-1/2 mammary buds in any of the genotypes
tested (data not shown), suggesting that the smaller bud size is
not due to increased apoptosis. Only a few of the epithelial cells
within the E14.5 bud were proliferating, and no difference was
observed in the epithelial proliferation between wild-type and
p190-B- or IRS-1/2-deficient mammary buds as calculated by
determining the percentage of BrdU-positive cells within the
mammary epithelium. Interestingly, in the IGF-1R embryonic
buds stained with BrdU (Figs. 9a–c), there was no statistically
significant change in the proliferation within the mammary
mesenchyme as determined by the percentage of BrdU-positive
cells that also stained positive for ER in a serial section (n=6).
However there was a significant decrease in the proliferation
within the mammary epithelium in both the heterozygous
to the wild-type. Furthermore, there was no change in the
Fig. 5. Decrease in IRS protein expression and the downstream IGF signaling component phospho-Akt in p190-B-deficient mammary buds. Sagittal sections of E14.5
embryonic mammary buds stained with IRS-1 (a, b); IRS-2 (c, d); pAkt(Ser473) (f, g). Note the obvious reduction in both IRS-1 and IRS-2 as well as the decrease in
pAkt in the p190-B-deficient buds (b, d, g) as compared to the wild-type (a, c, f). Quantitation of IRS-1 and IRS-2 staining (e) using ImagePro software shows a
statistically significant decrease in intensity of brown staining in the p190-B-deficient embryos as compared to the wild-type. Quantitation of the pAkt staining (h)
shows a statistically significant decrease in pAkt within the mammary bud of the p190-B-deficient embryos as compared to the wild-type. Scale bar: 100 μm.
144 B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
proliferation, ranging from 25% to 35%, of the overlying epi-
IGF-1R embryos (data not shown). These results coupled with
the altered expression of mesenchymal markers indicate that
p190-B and the IRS proteins play a critical role in regulating the
mesenchymal compartment within the developing mammary
anlagen and may play a role in migration of the mammary
epithelial cells from the overlying epidermis. However, our
findings suggest that IGF-1R is critical for proliferation of the
mammary bud, but is not critical for mesenchymal proliferation.
Previous genetic studies have implicated several signaling
pathways such as fgf/fgfr, Lef-1/Tcf, Tbx-3, PTHrP/PTHrPR,
and Eda/Edar in various stages of bud development. Most of
these pathways have been shown to play a role in epithelial–
mesenchymal interactions where the ligand is expressed in one
compartment and the receptor is expressed in the reciprocal
RhoGAP is another essential gene for mammary bud formation.
P190-B mRNA was detected in both the epithelium and
mesenchyme of the bud suggesting that it may regulate both
compartments. Accordingly, our results propose that p190-B
mediates two key processes in mammary bud formation
including the migration of mammary progenitor cells from the
epidermis into the mammary bud as well as mesenchymal
proliferation and condensation. Importantly, these data indicate
that defects in embryonic mammary bud development underlie
the failure of p190-B-deficient mammary glands to develop
The IGF signaling axis has been identified as a crucial
interacting pathway with p190-B. As evidence of this, p190-B-
deficient embryos and fibroblasts exhibit impaired cell growth
and differentiation as a result of altered IGF signaling (Sordella
et al., 2002; Sordella et al., 2003). Previously, we demonstrated
that both loss and gain of p190-B function affects IGF signaling
in the developing postnatal mammary gland (Chakravarty et al.,
2003; Vargo-Gogola et al., 2006). Furthermore, we have
demonstrated by immunohistochemical analysis that IRS-1
and IRS-2 expression as well as activation of the downstream
effector Akt are decreased in the p190-B-deficient mammary
To further investigate the role of IGF and p190-B signaling
interactions in mammary bud development, we examined the
effects of loss of either IGF-1R or IRS-1/2 expression on
Fig. 6. Defective epithelial and mesenchymal differentiation in IRS-1/2-deficient mice. Sagittal sections of E14.5 embryonic mammary buds stained with H&E to
demonstrate a bud to bud comparison of the reduced number of epithelial cells and loss of a well defined condensed mesenchyme around the IRS-1/2-deficient (c) and
heterozygous (b) buds compared to wild-type (a). Bud size is significantly decreased as shown by quantitation (d). Scale bar: 100 μm.
145 B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
mammary bud development. Strikingly, IRS-1/2-deficient
mammary buds phenocopy the small bud size and aberrant
mesenchyme observed in the p190-B-deficient buds. Although
IGF-1R loss resulted in a similar small bud phenotype, the
mesenchyme appeared unaffected suggesting that IGF-1R is not
required for proper formation of the mesenchyme. In support of
this, loss of p190-B or IRS-1/2 significantly inhibited mesench-
ymal proliferation whereas IGF-1R loss had no effect. These
data complement our previous loss and gain of function studies,
which suggested that cross-talk between the p190-B and IGF
signaling pathways plays a critical role in postnatal mammary
An important question arising from these studies is what is
absence of p190-B, IRS-1/2, or IGF-1R. Furthermore, why does
IRS-1/2 loss phenocopy the loss of p190-B, whereas IGF-1R
that there are distinct mechanisms that affect bud size in the
absence of p190-B and IRS-1/2 as compared to IGF-1R. It is
well known that mesenchymal condensation and differentiation
Fig. 7. Loss of IRS-1/2 expression results in reduced bud size and disrupted mesenchyme at E14.5. p63 expression in the epithelium of E14.5 mammary buds of
(a)IRS-1/2+/+,(b) IRS-1/2+/−,(c)IRS-1/2−/−mice.Notethe dramaticreductionin thenumber ofepithelial cellsthatstain for p63 bothin theheterozygous(b) andin the
IRS-1/2-deficient mammary placodes (c). Note the dramatic reduction of AR-positive cells within the condensed mesenchyme surrounding the epithelial buds in
(e) heterozygous and (f) deficient anlagen as compared to the wild-type. Quantitation of these staining patterns (g) show a statistically significant decrease in the
number of p63-positive cells with in bud of the IRS-1/2 heterozygous and deficient embryos as well as a statistically significant decrease in the number of AR-positive
cells within the p190-B heterozygous and deficient anlagen as compared to the wild-type. (f). Scale bar: 100 μm.
146B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
mesenchyme is both permissive and instructive and plays a
critical role in directing mammary epithelial cell fate (Hens and
Wysolmerski, 2005). We have demonstrated that the absence of
either p190-B or IRS-1/2 expression in the developing bud
profoundly affects mesenchymal proliferation and conden-
sation. We suggest that this aberrant mesenchyme is not capable
of directing migration of the p63-positive mammary progenitor
cells from the overlying epidermis into the developing bud
resulting in the small bud phenotype. In the case of IGF-1R loss,
our data indicate that the small bud size results from decreased
proliferation in the epithelial compartment of the bud, although
we cannot rule out the possibility of a compounding migratory
defect. We suggest that in these buds, signaling from the
respond. It is likely that the lack of any apparent proliferation
defects in the epithelial compartment of the p190-B- or IRS-1/2-
deficient buds is obscured by the mesenchymal defects.
Importantly, there was no change in proliferation of the
overlying epidermis in any of the models examined, supporting
p190-B also showed impaired epithelial–mesenchymal inter-
actions (Vargo-Gogola et al., 2006).
The mesenchyme may form properly in the IGF-1R-deficient
mammary anlagen through a variety of compensatory mech-
anisms. Signaling to IRS-1 and IRS-2 via other growth factor
receptors including other IGF-R family members, insulin
receptor, or through epidermal growth factor receptor
(EGFR) are possible mechanisms, which may explain the
observed effects on cell migration. EGF has been shown to
increase expression of both IRS-1 and IRS-2 in breast cancer
cell lines with IRS-2 being required for cell migration (Cui et
al., 2006). Many studies have also shown a link between
integrins and IRS-1 and IRS-2. However, there may also be
redundancy in this pathway as mammary glands from either
α3- or α6-integrin-deficient epithelium are fully developed and
Fig. 8. Loss of either p190-B or IRS-1/2 leads to a defect in mesenchymal proliferation at E14.5. Sagittal sections of E14.5 mammary buds stained for BrdU. Loss of
p190-B (c) leads to a reduced number of BrdU-positive cells in the mesenchyme as compared to wild-type (a) and heterozygous (b) mice. Loss of one (e) or two copies
(f) of IRS-1/2 also leads to a reduced number of BrdU-positive cells within the mammary mesenchyme as compared to the wild-type (d). Quantitation of BrdU
postitive staining revealed loss of p190-B (g) or IRS-1/2 (h) leads to a decrease in the percentage of proliferating mesenchymal cells, but no change in epithelial
proliferation. Scale bar: 100 μm.
147B.M. Heckman et al. / Developmental Biology 309 (2007) 137–149
functional (Klinowska et al., 2001). Loss of β1 integrin leads
to defects in alveolargenesis and lactation in the mammary
gland, while loss in the epidermis leads to defects in assembly
of basement membrane proteins resulting in defects in hair
follicle differentiation though altered proliferation as well as
impaired cytoskeletal dynamics leading to reduced migration
(Brakebusch et al., 2000; Grose et al., 2002; Naylor et al., 2005;
Raghavan et al., 2003).
The studies reported here enhance a growing understanding
of the molecular mechanisms that regulate embryonic mam-
mary gland formation. The roles of the p190 RhoGAPs are
diverse and include involvement in proliferation (Chakravarty
et al., 2003; Su et al., 2003), cell transformation (Ellis et al.,
1990), EMT (Ozdamar et al., 2005), gene transcription (Jiang
et al., 2005), integrin signaling (Burbelo et al., 1995),
cytoskeletal reorganization (Hall, 1998), vesicular trafficking
and development (Bernards and Settleman, 2005). Many of
these contrasting functions may be attributed to their large
molecular weight, the presence of multiple protein motifs, their
ability to act as a scaffold, and their capacity to interact with
various chromatin-remodeling complexes through its GAP
domain (Nagaraja and Kandpal, 2004). Further studies in-
cluding in vivo mammary bud specific gene arrays and
pathway-specific inhibitor studies will yield more insight into
the roles of p190-B and IRS-1/2 in embryonic mammary bud
These studies were supported through the CA030195-22
(JMR) and CA94118 (AVL). T. V-G. is supported by a Howard
Temin Pathway to Independence award (1K99CA127361-
01). BMH is supported by a Department of Defense Breast
Cancer Program Predoctoral Traineeship Fellowship (DAMD
W81XWH-06-1-0704). Thanks to Mr. Walter Olea for technical
assistance with the IRS knockout mice and Remigo Lopez from
the Breast Center Pathology Core for the embryo sectioning.
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