ErbB2 is required for ductal morphogenesis of the
Amy J. Jackson-Fisher*, Gary Bellinger*, Rajani Ramabhadran*, Jacqueline K. Morris†, Kuo-Fen Lee‡,
and David F. Stern*§
*Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023;†Department of Biology and Geology, Baldwin-Wallace College,
Berea, OH 44017; and‡The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037
Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved October 25, 2004 (received for review September 24, 2004)
The ERBB2?HER2?NEU receptor tyrosine kinase gene is amplified in
up to 30% of human breast cancers. The frequent and specific
selection of this receptor kinase gene for amplification in breast
cancer implies that it has important normal functions in the
mammary gland. To investigate the functions of ErbB2 during
normal mouse mammary gland development, we transplanted
mammary buds from genetically rescued ErbB2?/?embryos that
express ErbB2 in the cardiac muscle. ErbB2?/?mammary buds
transplanted to a wild-type mammary fat pad support outgrowth
of an epithelial tree that advances only slowly through the mam-
mary fat pad at puberty. This penetration defect is associated with
in body cell number, an increased presence of cap-like cells in the
prelumenal compartment, and the presence of large luminal
spaces. Lobuloalveolar development was not affected in glands
that developed from ErbB2?/?transplanted tissue. The results may
have implications for the aggressive phenotypes associated with
ERBB2-overexpressing mammary carcinomas.
development ? terminal end bud ? HER2 ? EGF
(EGFR) family, which includes EGFR?ERBB1?HER1, ERBB2?
HER2?NEunits, ERBB3?HER3, and ERBB4?HER4. ERBB2 is
amplified in up to 30% of human breast cancers (1). Amplifi-
cation and overexpression of ERBB2 are associated with poorer
prognosis of breast cancer patients (1–3). ERBB2 is the target
for the therapeutic antibody Herceptin (trastuzumab), which
is used for treatment of women with advanced breast cancer
that overexpresses ERBB2 (4). The preferential amplification of
ERBB2 in breast cancer, relative to other receptor tyrosine
kinase genes, and the association with poor prognosis imply
that activated ErbB2 has powerful and pleiotropic carcino-
genic properties. Indeed, in contrast to some oncogenes, ERBB2
promotes invasion and metastasis, in addition to excessive
proliferation (5, 6). Moreover, in mouse models, activated
ERBB2 is especially potent in induction of metastatic mammary
carcinoma (7, 8). The preferential association of ERBB2 ampli-
fication with mammary and ovarian carcinomas suggests that
these properties reflect important normal functions for ERBB2
in these endocrine-responsive tissues.
ErbB2 is an orphan receptor that is unable to bind conven-
tional growth factors on its own (reviewed in ref. 9). However,
ErbB2 forms heteromers with ligand-activated EGFR, ErbB3,
and ErbB4, which enables ErbB2 to respond to the ?13 EGF-
and neuregulin-like growth factors. The ErbBs are promiscuous
in their ability to form heteromeric complexes, but ErbB2 is a
other ErbBs (10–13). In this capacity, it may function as a
common ErbB subunit that augments signaling power and
diversity, through its ability to couple to specific substrates and
to alter down-regulation pathways of other ErbBs.
Despite the link of ERBB2 to tumorigenesis, the roles of
ERBB2 in normal mammary gland development are unknown.
he protooncogene ERBB2?HER2?NEU encodes a receptor
tyrosine kinase of the epidermal growth factor receptor
The overlapping expression pattern of the four ErbBs and
multiple ErbB ligands has made it difficult to interpret the
physiological function of any individual ErbB (reviewed in ref.
14). In pubescent female mice at 5 weeks of age, both ErbB2 and
EGFR proteins are expressed in the major cell compartments of
the mammary gland. By 8 weeks of age, ErbB2 is especially
prominent in the epithelium and reduced in the stroma, whereas
EGFR is localized to the stroma (15). Both EGFR and ErbB2
are Tyr-phosphorylated at puberty in the mouse mammary
gland, indicating that they are functionally activated (16). ErbB3
and ErbB4 are expressed at low levels before maturity but are
expressed at higher levels during pregnancy and lactation (15,
16). ErbB2 expression has been detected at later stages of
development as well. All four ErbBs were detected in mammary
glands of mice in late pregnancy and early lactation. EGFR and
ErbB2 proteins are expressed in the lobuloalveolar epithelium,
whereas ErbB3 and ErbB4 are enriched in the ducts (15).
The timing of expression and Tyr phosphorylation of ErbB2
during mammary development suggests roles for ErbB2 in the
nulliparous gland at puberty, during late pregnancy, and during
early lactation. The phenotype of transgenic mice expressing a
truncated dominant-negative ErbB2 in adult females, which
retards late mammary development and lactation, has suggested
an important role for ErbB2 in final stages of lactational
differentiation (17). However, the ability of ErbB2 to form
heteromers with other EGF family receptors means that the
dominant-negative effects may be mediated through inactivation
of endogenous ErbB2, other ErbBs, or some combination.
Determination of the ErbB2?/?mammary phenotype has
been hampered by early embryonic lethality due to a cardiac
defect (18, 19). This defect can be genetically rescued by
tissue-specific expression of a rat neu?ErbB2 transgene targeted
to cardiac muscle, yet the ErbB2?/?mice still die at birth because
of loss of innervation of the diaphragm (20, 21). We have
determined the consequences of inactivating murine ErbB2 by
transplanting embryonic mammary buds from the genetically
rescued ErbB2?/?transgenic mice into the cleared mammary fat
pads of immature female mice. Transplanted ErbB2?/?mam-
mary buds support outgrowth of an epithelial tree in a wild-type
mammary fat pad. However, there are substantial delays in
ductal penetration. The growth defect is associated with struc-
tural defects in terminal end buds (TEBs), characterized by a
decrease in body cell number, an increased presence of cap-like
cells in the prelumenal compartment, and the presence of large
Materials and Methods
Mammary Gland Transplants. Embryonic day (E) 12.5–E15.5 em-
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TEB, terminal end bud; EGFR, epidermal growth factor receptor; IHC,
immunohistochemistry; SMA, smooth muscle actin; MMP, matrix-metalloproteinase.
§To whom correspondence should be addressed. E-mail: email@example.com.
© 2004 by The National Academy of Sciences of the USA
December 7, 2004 ?
vol. 101 ?
no. 49 www.pnas.org?cgi?doi?10.1073?pnas.0407057101
and?or appearance of embryos) resulting from intercross of
compound heterozygotes for an ?-myosin heavy chain (?-MHC)
promoter rat neu?ErbB2 cDNA transgene and ErbB?/?in a
mixed 129?BALB?c?ICR strain background (20) were har-
vested by Caesarean section. The embryos were maintained in
DMEM?10% FCS overnight while sex (SRY gene in males) and
ErbB2 genotypes were determined by PCR. Three-week-old
epithelium in the no. 4 inguinal mammary fat pads by removing
the tissue between the nipple and the lymph node (22). The
excised portions of recipient glands were whole-mounted and
stained with carmine alum to verify clearing of the epithelium.
For each recipient mouse, one no. 4 inguinal mammary fat pad
was transplanted with a single mammary bud isolated from a
cardiac-rescued MHC-ErbB2 ErbB2?/?female, and the con-
tralateral no. 4 inguinal fat pad was transplanted with a mam-
mary bud from an ErbB2?/?or ErbB2?/?female littermate. The
fat pads containing transplants were harvested from virgin hosts
at 4, 7, or 13 weeks posttransplantation, or the recipients were
bred and the glands harvested at 1 day postpartum or after
various points after gland remodeling. The overall take rate for
transplanted buds was 46% and was not affected by the genotype
of the donor embryo. All animal work was approved by the Yale
University Institutional Animal Care and Use Committee.
Morphological Analysis. For whole-mount analysis, mammary
glands were fixed in acetic acid?ethanol and cleared with ace-
tone, and the ductal tree was visualized by staining with carmine
alum as described (23). The number of whole mounts analyzed
was six ??? or ??? and six ??? late pregnancy fetuses or
newborn pups, six ??? and two ??? gland outgrowths at 4
weeks after transplantation, four ??? and six ??? glands 7
weeks after transplantation, four ??? and four ??? glands at
13 weeks after transplantation, seven ??? or ??? and six ???
glands at 1 day postpartum (uniparous and multiparous), and
two ??? or ??? and two ??? parous nonpregnant adults.
For histological analysis, mammary glands were placed in
freshly prepared 4% paraformaldehyde for 3 h to overnight at
4°C, subjected to a series of ethanol washes with up to 70%
ethanol, and embedded in paraffin. Five-micrometer sections
were cut and stained with hematoxylin?eosin (H&E) (17).
Twelve ???, four ???, and seven ??? gland outgrowths were
analyzed 4 weeks posttransplant; six ??? or ??? and nine ???
glands at 7 weeks posttransplant; one ??? and two ??? glands
at 13 weeks posttransplant; and five ??? or ??? and four ???
glands at 1 day postpartum (uniparous and multiparous).
To determine total ductal outgrowth length, digital images of
each whole gland or half gland stained with carmine alum were
imported into NIH imaging software. A line was drawn along the
longest axis of ductal growth and measured in arbitrary units.
The measurements for all of the glands were averaged, and the
standard deviation was determined. To determine the branch
density, digital images of the whole gland or half gland stained
with carmine alum were analyzed by using NIH IMAGEJ. The
number of branch points was determined within a box of fixed
dimensions. The statistical significance was determined by Stu-
dent’s t test.
Immunohistochemistry. Mouse monoclonal anti-E-cadherin clone
36 (Transduction Laboratories, Lexington, KY), mouse mono-
clonal anti-? Smooth Muscle Actin clone 1A4 (Sigma Immuno
Chemicals), goat polyclonal P-cadherin (N-19) (Santa Cruz
Biotechnology), and rabbit polyclonal neogenin (H-175) (Santa
Cruz Biotechnology) were used. For E-cadherin immunohisto-
chemistry (IHC) (n ? 4 ??? and 6 ??? glands), the mouse-
on-mouse staining procedure (Vector Laboratories) was fol-
lowed, with microwave antigen unmasking in antigen unmasking
solution (Vector Laboratories) and a 1:300 dilution of primary
antibody. For smooth muscle actin (SMA) IHC (n ? 6 ??? and
6 ??? glands), the mouse-on-mouse staining procedure was
was used as a negative control for both E-cadherin and SMA
IHC. For P-cadherin IHC (n ? 6 ??? and 4 ??? glands), the
TSA Biotin System kits protocol for IHC (PerkinElmer) was
used, with a 1:400 dilution of primary antibody. Goat IgG was
used as a negative control for P-cadherin IHC. For neogenin
IHC (n ? 5 ??? and 5 ??? glands), the DAKO catalyzed signal
amplification (CSA) system, peroxidase, and the DAKO CSA
ancillary system were used (DAKO), with the provided anti-
mouse link replaced with the CSA Rabbit Link (DAKO) and
with a 1:1,000 dilution of primary antibody. Mammary gland
tissue from neogenin?/?mice was only very minimally stained
with anti-neogenin. Rabbit IgG was also used as a negative
control for neogenin IHC.
BrdUrd and TUNEL. For BrdUrd analysis (n ? 8 ??? and 4 ???
glands), mice were injected with cell proliferation labeling
reagent (Amersham Pharmacia) 2 h before killing. A 1:10
dilution of anti-BrdUrd-POD (monoclonal antibody to the thy-
midine analogue 5-bromo-2?-deoxyuridine, Fab fragments with
peroxidase conjugated) (Roche) was used as described (24). For
(Roche) was used according to the manufacturer’s instructions.
ErbB2?/?mice die mid-gestation (around day 10.5) because of
a failure of ventricular trabeculation (18, 19). The cardiac defect
can be bypassed by using a transgene with a cardiac-specific
?-myosin heavy chain promoter expression driving expression of
a rat ErbB2 cDNA in the myocardium (20, 21). These ‘‘cardiac-
rescued’’ ErbB2?/?mice die at birth from respiratory arrest. In
late-stage embryos and perinatally, cardiac-rescued ErbB2?/?
mammary glands are grossly normal (Fig. 1b). Hence, ErbB2 is
not absolutely required in the epithelium or stroma for prenatal
mammary gland development.
The contribution of ErbB2 to postnatal mammary develop-
ment was evaluated by transplanting mammary buds from
cardiac-rescued ErbB2?/?mice [embryonic day (E) 13.5–
E15.5] into cleared mammary fat pads of prepubescent
3-week-old immunocompromised Rag1?/?mice. The con-
tralateral fat pad received buds from homozygous (???) or
heterozygous littermates. In virgin animals, ductal outgrowth
of glands reconstituted with ErbB2?/?tissue was defective
(Fig. 2). ErbB2?/?outgrowths typically filled the fat pad within
4 weeks of transplantation (Fig. 2 a, c, and e), whereas ductal
penetration by ErbB2?/?tissue was incomplete, even after 7
weeks (Fig. 2 b and d). By 13 weeks, ductal penetration of
Shown are no. 4 inguinal mammary glands from female embryos harvested
attached to the nipple (left) with few branches. (b) The ErbB2?/?littermate
embryo has a ductal tree emanating from the nipple, with an increased
number of branches.
Whole-mount analysis of prenatal mammary gland development.
Jackson-Fisher et al.PNAS ?
December 7, 2004 ?
vol. 101 ?
no. 49 ?
ErbB2?/?mammary glands ranged from three-quarters full to
completely filled (Fig. 2f). When compared pairwise in the
same recipient females, outgrowths from ErbB2?/?donors
were, on average, 67% longer than outgrowths from ErbB2?/?
donors (Table 1 and Fig. 2). At 7 weeks posttransplantation,
the number of branch points per unit area was higher in the
ErbB2?/?transplants than the ErbB2?/?transplants, indicating
an increase in branching in the ErbB2?/?transplants (Table 1).
In the majority of the transplant recipients analyzed after
pregnancy, the ErbB2?/?and ErbB2?/?glands filled the fat pad
to the same extent and supported formation of alveoli (Fig. 3
a and b). The fat pads were filled in four of the five ErbB2?/?
mice and four of the six ErbB2?/?mice. Analysis of lactation
in transplants after 1 day postpartum is not possible because
the transplanted epithelium is not attached to the nipple;
apoptosis and remodeling commence rapidly after birth be-
cause of the lack of suckling. However, hematoxylin?eosin-
stained sections of the glands at 1 day postpartum show similar
distended alveoli with milk in the lumens and lipid droplets in
the cells, suggesting that both the differentiation and secretory
pathways are normal in the ??? and ??? glands (Fig. 3 c and
d). In addition, the proportion of epithelium relative to stroma
is similar in both the ??? and ??? glands (Fig. 3 c and d).
Both the reconstituted ??? and ??? glands remodeled
normally after pregnancy (Fig. 3 e and f).
TEBs are sites for proliferation and penetration of the
advancing duct into the fat pad at puberty, and they normally
regress once the nascent ducts have traversed the mammary fat
pad (25). A typical TEB is a bulbous structure at the end of the
duct consisting of an outer monolayer of cap cells at the
advancing edge that gives rise distally to the myoepithelium
surrounding the duct, and an inner compartment of prelume-
nal and luminal body cells that line the duct (Fig. 4 a and c)
(25, 26). In contrast, the ErbB2?/?TEBs, although similar in
number to the ErbB2?/?TEBs, seem to have a normal cap
cell?myoepithelial cell layer, but the body cell layer often
seems loosely packed, with large spaces, and severely dimin-
ished cell number (Figs. 4 b, d, and f and 5 b, d, f and h). This
day 13.5 embryos were transplanted into contralateral cleared mammary fat pads of 3-week-old Rag1?/?females. The glands were harvested 4 weeks (a and
Whole-mount analysis of ductal morphogenesis in transplanted glands from virgin female animals. Paired mammary buds from ErbB2?/?and ErbB2?/?
Table 1. Analysis of whole mounts, seven weeks after transplantation
Scored in same recipients* Scored in all recipients†
n ? 3
n ? 3
n ? 4
n ? 6
Length of total outgrowth in glands‡
Branch points per unit area§
8.41 ? 1.5
29.7 ? 17.2
5.04 ? 1.4
48.7 ? 9.3
8.41 ? 1.2
26.0 ? 13.3
5.41 ? 1.1
48.8 ? 8.4
*Results in animals in which both ??? and ??? transplants were successful, permitting direct comparison.
†Results for all successful transplants, regardless of status of contralateral gland.
‡The average end-to-end length of total ductal outgrowth at 7 weeks posttransplantation measured in arbitrary
units, with standard deviation (P ? 0.0449 for same recipient and P ? 0.0036 for all recipients).
§The average number of branch points within a defined area, with standard deviation (P ? 0.1678 for same
recipient and P ? 0.0174 for all recipients).
www.pnas.org?cgi?doi?10.1073?pnas.0407057101Jackson-Fisher et al.
aberrant TEB phenotype was evident in most or all TEBs in
hematoxylin?eosin-stained sections of every ErbB2?/?trans-
planted mammary gland and in none of the ErbB2?/?or
ErbB2?/?transplanted mammary glands. The poor outgrowth
of ErbB2?/?transplanted glands at 4 weeks made it difficult to
identify TEBs (Fig. 2b). Direct comparisons of TEBs from
ErbB2?/?transplants with those of ErbB2?/?or ErbB2?/?
transplants were difficult at later time points, e.g., 7 weeks,
because the wild-type TEBs regress at times when ErbB2?/?
ducts have only minimally penetrated the fat pad (e.g., Fig. 2
c, regressed, vs. d). Therefore, detailed comparisons of TEBs
were made between the 4-week ??? outgrowths and the
7-week ??? outgrowths.
Proliferating cells are normally enriched in the cap cell layer
and the outer layers of body cells (BrdUrd incorporation, Fig.
4e) (27). BrdUrd-positive cells are present in the ErbB2?/?
TEB cap cell and body cell layers (Fig. 4f). The percentage of
BrdUrd-positive cells did not differ significantly between the
ErbB2?/?and ErbB2?/?outgrowths, with 19.3 ? 9.4% Br-
dUrd-positive cells in ErbB2?/?TEBs and 17.2 ? 9.1% in
ErbB2?/?TEBs. However, the ErbB2?/?TEBs, with fewer
cells overall, had fewer BrdUrd-positive cells. Analysis of
apoptosis rates was inconclusive for ErbB2?/?TEBs, owing to
the small cell numbers.
SMA is expressed by cap cells and myoepithelial cells, but
not by body cells (28). ErbB2?/?TEBs display normal SMA
immunostaining (Fig. 5a), with the single outer layer of the
TEBs staining. The small number of SMA-positive cells lo-
cated interiorly are thought to be cap cells that have migrated
in and are destined for proliferation and?or differentiation
into luminal cells (26). In contrast, the ErbB2?/?TEBs had
many SMA-positive cells within the body cell compartment
(Fig. 5b). P-cadherin and E-cadherin are adhesion molecules
that are differentially expressed in the TEB cell compartments.
P-cadherin is expressed in the cap cells and myoepithelial cells,
whereas E-cadherin is expressed only by the body cells and is
required for integrity of the body cell compartment (29). In the
ErbB2?/?TEBs, the cap cells and myoepithelial cells express
P-cadherin (Fig. 5c, note nearly complete exclusion of inner
cell layers), resulting in a similar staining pattern to ErbB2?/?
TEBs stained for SMA (Fig. 5a). Conversely, only the body
cells expressed E-cadherin uniformly (Fig. 5e, note exclusion
of outer cell layer). The ErbB2?/?TEBs had many P-cadherin-
positive cells in the body cell compartment (Fig. 5d) and were
heterogeneous for E-cadherin immunoreactivity in the inter-
nal layers (Fig. 5f). Together, these results suggest an in-
creased body cell infiltration by cap-like progenitor cells in the
ErbB2?/?TEBs, or altered differentiation in the body cell
Compartmentalization of the TEB by E-cadherin and P-
cadherin status is stabilized through adhesion mediated by
binding of netrin-1, produced by prelumenal cells, to the
receptor neogenin on the cap cells, which promotes cap
cell?prelumenal cell interactions. Loss of netrin-1 or neogenin
results in migration of SMA-positive, P-cadherin-positive,
E-cadherin-negative cap-like cells into the prelumenal com-
partment, similar to the phenotype of ErbB2?/?TEBs (30). In
the ErbB2?/?TEBs, the cap cells and myoepithelial cells
express neogenin (Fig. 5g), resulting in a similar staining
pattern to ErbB2?/?TEBs stained for SMA and P-cadherin
(Fig. 5 a and c). The ErbB2?/?TEBs had neogenin-positive
cells in the cap cell and body cell compartment (Fig. 5h), in a
similar staining pattern to ErbB2?/?TEBs stained for SMA
and P-cadherin (Fig. 5 b and d). Neither the cap cell nor body
cell layers stained positively for neogenin in tissue from
c) TEBs in ErbB2?/?and ErbB2?/?transplants at 4 weeks posttransplantation
have a typical single cap cell?myoepithelial cell outer layer and multilayered
body cell compartment. (a and b) A direct comparison of TEBs from the
ErbB2?/?and ErbB?/?transplants from the same recipient mouse at 4 weeks
posttransplantation shows a disorganization of the body cells and a large
space between the cap cell and body cell layers. (d) The TEBs in ErbB2?/?
transplants at 7 weeks posttransplantation also show a decrease in the num-
ber of body cells and disorganization of the body cells. (e and f) BrdUrd
(e), but there are fewer BrdUrd-positive cells in the ErbB2?/?TEBs at 7 weeks
(f), concomitant with a smaller cell number (P ? 0.7246). H & E, hematoxylin?
Morphology and proliferation of TEBs in transplanted glands. (a and
ing of transplanted glands. Mammary buds from day 13.5 embryos (??? or
???) were transplanted into the cleared no. 4 inguinal fat pads of a 3-week-
old recipient mouse. The mouse was mated 4 weeks posttransplantation and
delivered a litter 7 weeks posttransplantation. The glands were harvested 1
from the same recipient mouse. The ErbB2?/?ductal tree (b) has filled the fat
differences in alveolar morphology; however, there is no difference in the
The ErbB2?/?gland (e) and the ErbB2?/?gland (f) are from separate parous
nonpregnant females. H & E, hematoxylin?eosin staining.
Whole mount analysis of lobuloalveolar development and remodel-
Jackson-Fisher et al. PNAS ?
December 7, 2004 ?
vol. 101 ?
no. 49 ?
neogenin?/?animals (kindly provided by P. Strickland and L.
Hinck, University of California, Santa Cruz) (data not shown).
However, the loss of ErbB2 did not impede production of
neogenin, suggesting that a different signaling system is im-
paired in ErbB2-null transplants. Similarly, immunostaining
patterns with anti-netrin were comparable in mammary glands
reconstituted with wild-type and ErbB2?/?epithelium (data
not shown). Hence, the dysregulation of compartmentalization
in TEBs reconstituted from ErbB2?/?tissue does not seem to
operate through the netrin?neogenin axis.
The lack of ErbB2 in the virgin mammary gland resulted in
delayed ductal growth during puberty and adolescence. This
phenotype was associated with structural defects of the TEBs.
Because in these experiments the recipient fat pad should
supply stromal functions, ErbB2 is apparently required in the
epithelium. The postnatal contribution of stromal ErbB2 was
not addressed in this study. Serial transplants of ErbB2?/?
glands harvested 7 weeks posttransplantation were successful,
suggesting that null stroma from the original transplant does
not contribute significantly to the ductal defect.
A similar ductal penetration defect was evident when floxed
ErbB2 was selectively deleted by using a mouse mammary
tumor virus (MMTV) transgene (31). However, a TEB defect
was not observed, perhaps owing to lack of MMTV-Cre
expression in cap cells, or other experimental differences.
At early stages after transplantation, branch points were
denser in ErbB2?/?epithelium. Other work has suggested a
positive role for ErbBs in branching (32), and TGF-? and
neuregulins promote branching (23, 33). The greater branch-
ing early after transplantation did not translate to a greater
overall number of TEBs. Loss of the most defective ErbB2?/?
TEBs might account for this discrepancy and also help to
explain the recovery of normal mammary gland morphogen-
esis as maturation continued.
Structural defects in the TEBs associated with ErbB2?/?
epithelium undoubtedly contribute to the ductal penetration
defect. Patency of cap?myoepithelial and body cell compart-
ments is maintained by P-cadherin- and E-cadherin-dependent
homophilic interactions (29). This compartmentalization is
disrupted in ErbB2?/?epithelium. This defect is not due to the
loss of the adhesion molecule neogenin, despite the similar
structural defects in neogenin-null TEBs (30).
The penetration defect could be caused by disruption of the
normal regulation of matrix-metalloproteinases (MMPs).
Mammary gland branching morphogenesis requires MMP-2 to
facilitate TEB invasion and repress precocious lateral branch-
ing in mid-pregnancy, whereas MMP-3 is required for second-
ary and tertiary lateral branching of ducts (34, 35). EGFR has
been shown to control branching morphogenesis of the lung by
regulating MT1-MMP?MMP14 and MMP-2, a known regula-
tor of normal lung morphogenesis (36). Likewise, ErbB2
signaling could contribute to mammary branching morpho-
genesis or TEB invasion through one or several MMPs.
ErbB2 is normally activated through growth factor-
dependent heteromerization with other ErbBs (14). EGFR
and ErbB2 are highly expressed, Tyr-phosphorylated, and
colocalized in major cell compartments during ductal mor-
phogenesis, and all four ErbB receptors are expressed and
localized to the epithelium during pregnancy and lactation in
the mouse mammary gland (15, 16). Of the several EGF family
growth factors expressed during mouse mammary develop-
ment, amphiregulin (AR) seems to be the foremost regulator
at puberty, because it is expressed in the virgin mammary gland
and promotes ductal morphogenesis when implanted, and
because AR knockout mice have a ductal penetration defect
that is stronger than disruption of EGF and TGF-? (37, 38).
However, TEBs were apparently normal in triple AR, EGF,
and TGF-? knockout mice (38). The finding that EGFR
knockouts have a limiting mammary function in the stroma,
not the epithelium (33), seems to contradict the notion that
EGFR?ErbB2 interactions are the most important route for
ErbB2 activation at puberty. This difference might be ex-
plained by the significant methodological differences between
the EGFR study and the present one. However, it is possible
that the minimal stromal signal can be supplied by EGFR?
EGFR homodimers, and that ErbB2 is dispensable in this
context. Alternatively, other ErbB receptors may be involved.
Because neuregulin-1, which binds to ErbB3 and ErbB4,
provokes a strong ductal outgrowth response when implanted
in the mammary gland, it is possible that one of these receptors
is the functional partner for ErbB2 in this context (23).
At puberty, systemic estrogen works in concert with growth
hormone to promote ductal outgrowth (reviewed in ref. 39).
Local mediators include insulin-like growth factor 1, fibroblast
growth factors, hepatocyte growth factor, and EGF family
growth factors (40–42). Further elucidation of the roles of
epithelial ErbB2 in integrity of the TEB, and possibly other
processes required for ductal elongation (proliferation, sup-
with anti-SMA of glands transplanted with ErbB2?/?donor tissue analyzed at
4 weeks posttransplantation (a) and ErbB2?/?donor tissue analyzed at 7
weeks posttransplantation (b). (c–h) Immunostaining with anti-P-cadherin of
glands transplanted with ErbB2?/?donor tissue analyzed at 4 weeks post-
transplantation (c) and ErbB2?/?donor tissue analyzed at 7 weeks posttrans-
plantation (d); immunostaining with anti-E-cadherin of glands transplanted
with ErbB2?/?donor tissue analyzed at 4 weeks posttransplantation (e) and
ErbB2?/?donor tissue analyzed at 7 weeks posttransplantation (f); and im-
munostaining with anti-neogenin of glands transplanted with ErbB2?/?do-
nor tissue analyzed at 4 weeks posttransplantation (g) and ErbB2?/?donor
tissue analyzed at 7 weeks posttransplantation (h).
Immunohistochemical analysis of TEBs. (a and b) Immunostaining
www.pnas.org?cgi?doi?10.1073?pnas.0407057101Jackson-Fisher et al.
pression of apoptosis, communication with the stroma for
regulation of metalloproteinase production), and the place-
ment of ErbB2 in the growth factor regulatory hierarchy will
enhance understanding of mammary development and carci-
nogenesis. The importance of ErbB2 in promoting invasive
penetration of the mammary fat pad is consistent with the
aggressive properties of mammary carcinoma with ERBB2
amplification. Better understanding of invasion-related func-
tions of ERBB2 in mammary development may help to explain
the frequent amplification of ERBB2 and lead to therapies
that target this process in cancer.
We thank Cathrin Brisken, John Wysolmerski, Jeffrey Rosen,
Phyllis Strickland, and Lindsay Hinck for protocols and helpful discussions,
with special thanks to John and Cathrin for training on transplant tech-
niques. This work was supported by U.S. Public Health Service Grant
Materiel Command Grant DAMD17-99-1-9459 (to A.J.J.-F.).
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Jackson-Fisher et al. PNAS ?
December 7, 2004 ?
vol. 101 ?
no. 49 ?