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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02394
1 The Saban Research Institute of Childrens Hospital Los Angeles/University of
Southern California, Developmental Biology Program, Los Angeles, CA 90027,
USA.
2 Institut Curie/CNRS-UMR144, Equipe de Morphogenèse Cellulaire et
Progression Tumorale, 75005 Paris, France.
3 Institut Pasteur/CNRS-URA 2578, Département de Biologie
Moléculaire, Génétique Moléculaire du
Développement, 75015 Paris, France.
4 King's College, Departments of Orthodontics and Craniofacial Development,
London SE1 9RT, UK.
5 Cancer Research UK, London Research Institute, Experimental Pathology
Laboratory, and Imperial College London, Department of Histopathology, London
WC2A 3PX, UK.
* Author for correspondence (e-mail: jveltmaat{at}chla.usc.edu and jmveltmaat{at}hotmail.com)
Accepted 4 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ectodermal patterning, Mammary gland, Somites, Placode individuality, FGF10/FGFR2B, GLI3, PAX3, WNT signaling, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
engaged in canonical WNT signaling (Chu et
al., 2004). The first streak forms the mammary line between the
forelimb and hindlimb, and mammary placode 3 develops first on this line at
E11.0-E11.5 (Mailleux et al.,
2002; Eblaghie et al.,
2004; Veltmaat et al.,
2004). Meanwhile, the inguinal and axillary streaks arise, from
which placodes 5 and 1, respectively, form. Placodes 4 and 2 emerge where the
mammary line abuts these respective streaks
(Veltmaat et al., 2004).
Few genes are known to be involved in early mammogenesis
(Mustonen et al., 2003;
Chu et al., 2004;
Mustonen et al., 2004;
Howard et al., 2005;
Jerome-Majewska et al., 2005).
Most of them are expressed in the ectoderm, yet factors from the underlying
mesenchyme initiate mammogenesis (Veltmaat
et al., 2003). We have previously shown that FGF10 signaling via
the FGF receptor isoform 2b (FGFR2B) is required for the formation of mammary
placodes 1, 2, 3 and 5. We proposed the ventral (hypaxial) dermomyotome of the
somites as the source of FGF10 (Mailleux
et al., 2002). Hypaxial FGF10 may be a mesenchymal initiator of
mammogenesis, as it could reach the ectodermal FGFR2B either via diffusion
through the thin lateral plate mesoderm or via delamination of hypaxial
cells.
The emergence of the mammary line as fragments overlying the hypaxial
somitic buds further suggested an involvement of hypaxial somitic signals in
induction (Veltmaat et al.,
2004). However, only the thoracic somites 11-24 between the
forelimb and hindlimb possess hypaxial buds (Eloy-Trinquet, 2000). Therefore,
we now hypothesize that hypaxial signals are important only for the formation
of the mammary line on which mammary placodes 2, 3 and 4 form
(Fig. 1), whereas other sources
of signals may be decisive for the formation of the streaks on which placodes
1 and 5 form. As placode 4 forms in the absence of FGF10/FGFR2B signaling, the
requirement for hypaxial FGF10 would thus be restricted to the formation of
placodes 2 and 3 only.
To test this hypothesis, we examined mammary development in Pax3
null mutants that lack the hypaxial somitic buds
(Relaix et al., 2003); in
Gli3 null and Fgf10 hypomorphic embryos, both expressing
reduced levels of somitic Fgf10; in Fgf10 and
Fgfr2b null embryos; and by applying recombinant FGF10. We identified
a signaling cascade involving somitic GLI3 upstream of FGF10 within the
somites, which in turn activates ectodermal FGFR2B, leading to Wnt10b
expression and canonical WNT signaling. This cascade is required for the
induction and correct positioning of the mammary line on the flank, and
indispensable for the formation of placode 3. We propose that correct
patterning is achieved through a combination of somitic elongation and somitic
Fgf10 gradients.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Gli3Xt-J/+ mice [Jackson
Laboratories, stock 00026; for genotyping see Maynard et al.
(Maynard et al., 2002)] were
maintained on a C57BL/6 background; Fgfr2b+/- mice
(De Moerlooze et al., 2000)
were maintained on a mixed C57BL/6xGK129 background; TOPGAL mice
(DasGupta and Fuchs, 1999) were
maintained on a CD1 background and backcrossed at least three generations with
the mutants above on their respective backgrounds;
Pax3ILZ/+ mice (Relaix
et al., 2003) were maintained on a mixed C57BL/6xBCA
background; and Fgf10Mlc1v-nLacZ-v24 (here
Fgf10mlcv) mice (Kelly
et al., 2001) were maintained on a mixed agouti background. The
latter were mated with Fgf10+/- mice to obtain
Fgf10mlcv/- hypomorphic embryos. Noon of the day of a
vaginal plug was considered E0.5. Mutant embryos were stage matched with their
littermate controls on the basis of somite counts.
X-gal staining
Pax3ILZ/ILZ, TOPGAL and Fgf10mlcv
embryos were fixed in 4% PFA/D-PBS and stained with 1 mg X-gal/ml
dimethylformamide in 5 mM K3Fe(CN)6/5 mM
K4Fe(CN)6/2 mM MgCl2 in D-PBS pH 7.4 to
reveal ß-galactosidase (lacZ) activity.
RNA in situ hybridization
Whole-mount in situ hybridization was performed as described
(Veltmaat et al., 2004) with
digoxigenin-labeled riboprobes of Fgf10
(Bellusci et al., 1997),
Wnt10b or Lef1. Paraffin sections (10 µm) were hybridized
with [35S]UTP labeled riboprobes of Fgfr1b, Fgfr1c, Fgfr2b,
Fgfr2c, Fgfr3b, Fgfr3c (Kettunen et
al., 1998) and Fgfr4
(Partanen et al., 1991) and
analyzed as described (Rice et al.,
2000).
Explant culture and application of recombinant FGF10
Eviscerated embryonic flanks were cultured as described for tooth anlagen
(Kratochwil et al., 2002).
Heparin-acrylic beads (Sigma) were incubated in D-PBS containing 100 ng
BSA/µl with or without 100 ng rFGF10 (R&D Systems)/µl, and implanted
underneath the surface ectoderm, at the hypaxial bud of somite 14-15 or 17-18.
After 2-3 days of culture, explants were processed for whole-mount in situ
hybridization or X-gal staining.
Histology
Specimens fixed in 60% methanol/30% CHCl3/10% HAc or 4%
PFA/D-PBS were embedded in paraffin wax, sectioned (6 µm) and stained with
Hematoxylin/Eosin or Nuclear Fast Red (Lab Vision Corporation),
respectively.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Somites
11-24 are located between the forelimb and hindlimb, underlying the mammary
line and placodes 2, 3 and 4 (Fig.
1, Fig. 2A). Whole-mount in situ hybridization shows that Wnt10b is expressed at reduced levels and in a narrower line on the flank of Pax3ILZ/ILZ embryos compared with control littermates (Fig. 2E-H). Placode 3 forms a day late, while the other placodes emerge in time (Fig. 2I,J, and not shown). Furthermore, Wnt10b expression is not attenuated between the placodes, and seems to be located slightly more dorsal in Pax3ILZ/ILZ mutants. Transverse sections demonstrate that the mammary line is located at the level of the notochord in Pax3ILZ/ILZ mutants, but more ventrally in control embryos (lines in Fig. 2K-N). The narrower width of the mammary line in Pax3ILZ/ILZ mutants, as inferred from Wnt10b expression (Fig. 2F), correlates with a narrower band of multilayered ectoderm (between asterisks in Fig. 2O,P), which moreover contains one cell layer less than in control embryos (Fig. 2O,P). This phenotype strongly suggests that hypaxial signals are required for the correct temporal formation and dorsoventral position of the mammary line and placode 3, but surprisingly, not for the formation of placodes 2 and 4, and as expected, not for placodes 1 and 5.
Our previous data had suggested that at E10.5, the hypaxial buds produce
the FGF10 required for mammary placode formation
(Mailleux et al., 2002).
However, by E11.25 (43 somites), Fgf10 expression extends throughout
the entire thoracic somites 12-18 spanning the region underlying mammary
placodes 2 and 3, while expression is highest in the hypaxial buds
(Fig. 2Q). This domain of
highest Fgf10 expression is absent in the somites of
Pax3ILZ/ILZ mutant embryos, while the level of
Fgf10 expression in the central part of the somites seems unaffected
(Fig. 2R). As mammary placode 3
is formed in Pax3ILZ/ILZ mutants, and Pax3 is
neither expressed in the surface ectoderm nor in the underlying mesenchyme
(Fig. 2A,C)
(Relaix et al., 2003), we now
hypothesize that both hypaxial and central somitic Fgf10 are required
for the formation of placode 3, and perhaps for the mammary line prior to
placode formation.
Defective initiation in mammogenesis in Fgf10-/- and Fgfr2b-/- mutants
To test this hypothesis, we examined ectodermal maturation in detail and
determined when the defect in mammogenesis becomes first apparent in
Fgf10-/- and Fgfr2b-/- embryos.
Normally, the surface ectoderm starts out as a single layer, the stratum
germinativum, of squamous cells (Fig.
3A,B). At the Wolffian ridge (i.e. the flank), these cells become
cuboidal and covered by a layer of squamous periderm by E8
(Fig. 3C)
(Sengel, 1976;
Stephens, 1982). By E11.5 (45
somites) the cells of the stratum germinativum become cylindrical at the flank
while remaining squamous ventral and dorsal to the flank
(Sengel, 1976)
(Fig. 3B). Subsequently, one or
two non-stratified layers of cylindrical cells form the stratum intermedium
between the stratum germinativum and the periderm
(Sengel, 1976). We show that
this multilayering occurs first ventrally on the flank, and co-localizes with
Wnt10b expression (Fig.
3D,G), indicative for the formation of the mammary line and the
individual placodes. In E11.5-E11.75 (44-47 somites)
Fgf10-/- and Fgfr2b-/- embryos, the
cells of the stratum germinativum of the flank are cuboidal instead of
cylindrical (Fig. 3E,F), and
formation of the periderm is impaired. Furthermore, the stratum intermedium
and Wnt10b expression are absent
(Fig. 3E,F,H,I), except in the
area of placode 4. There, a small streak of weak Wnt10b expression
coincides with the formation of a stratum intermedium composed of cuboidal
instead of cylindrical cells (not shown). As the role of WNT10B in mammary
line formation is unknown, but canonical WNT signaling is required
(Chu et al., 2004), we crossed
Fgf10+/- and Fgfr2b+/- mice with mice
carrying the TOPGAL transgenic reporter for canonical WNT signaling
(DasGupta and Fuchs, 1999),
used here as a functional marker for mammary line and placode formation
(Fig. 3Q,R). Like
Wnt10b expression, TOPGAL expression is weak, and restricted to the
area of placode 4 in E11.5 (46 somites) Fgf10-/- and
Fgfr2b-/- mutants (Fig.
3J-L). Accordingly, the expression pattern of Lef1,
another early marker for mammogenesis
(Mailleux et al., 2002)
demonstrates that all buds except 4 are absent at E12.5
(Fig. 3M-O). We conclude that
ectodermal maturation preceding mammary line formation requires FGF10/FGFR2B
signaling, except in the region of placode 4. If FGFR2B is activated by
somitic FGF10, then central somitic Fgf10 expression appears
sufficient for initiation, as seen in Pax3ILZ/ILZ mutants.
Additional hypaxial Fgf10 expression is required for the normal
formation of the stratum intermedium, a hallmark of the mammary line, and for
the correct temporal formation of placode 3.
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), which lie just posterior to the prospective mammary placode
3 (Eblaghie et al., 2004)
(J.M.V., unpublished). Using the more sensitive radioactive in situ
hybridization, we detected Fgf10 mRNA in the hypaxial buds and at a
lower level throughout the somites from E10.5 onwards, shown for E10.75 (37
somites) (Fig. 4C), but not in
the dermal mesenchyme and surface ectoderm. By E11.5, when placode 3 has
formed, Fgf10 expression has increased throughout the somites, and is
still relatively high in the hypaxial buds
(Fig. 4D). A radioactive signal
above background levels in the dermal mesenchyme suggests that
Fgf10-expressing cells delaminate from the somites. While the mammary
placodes transform into buds, somitic Fgf10 expression becomes
progressively reduced [data not shown at E12.0 (50 somites) and E12.5 (54
somites)]. These data support a role for hypaxial and central somitic
Fgf10 expression from around E10.5 onwards in the induction of a
mammary cell fate. Fgfr2b mRNA was detected in the surface ectoderm at E9.5 (26s) and during the onset of mammary line formation at E11.0 (41s), but not in the somites and dermal mesenchyme (Fig. 4E-H). No expression of any other Fgfr isoform was detected in the somites, dermal mesenchyme or ectoderm at these stages (not shown). Notably, Fgfr2b expression is relatively high in the surface ectoderm overlying the hypaxial buds at E11.5-E12.25 (45-50 somites) (Fig. 4G,H), corresponding to the area of the mammary line. These expression data support the hypothesis that somitic FGF10 acts via ectodermal FGFR2B in mammary line formation, prior to placode formation. However, it is still possible that Fgf10 expression in the lateral plate mesoderm at E9.5-10 makes the ectoderm receptive to later somitic signals involved in mammogenesis.
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In wild-type flanks without bead or with a BSA bead (Fig. 4I), we either found no, one, two or occasionally all three buds (2, 3 and 4) expected between the limbs (Table 1), as assessed by Wnt10b (Fig. 4I-L) or TOPGAL expression. Implantation of an rFGF10 bead in wild-type flanks (Fig. 4K) or a BSA bead in Fgf10-/- flanks (Fig. 4J) never resulted in the formation of a supernumerary mammary rudiment. Remarkably, in 30% of Fgf10-/- flanks cultured with an rFGF10 bead, we detected one bud in addition to the only bud (4) that normally forms in these mutants, by Wnt10b expression and histology (Fig. 4L-N). Although rFGF10 failed to induce extra placodes in wild-type flanks, the rescue of bud formation in Fgf10-/- explants of fairly advanced stages (E11.5 and even E12.5) indicates that the initiation of mammogenesis does not depend on Fgf10 expression prior to somitic Fgf10 expression.
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).
Gli3Xt-J/Xt-J embryos lack mammary bud 3 at E13.5
(Johnson, 1967). In the
wild-type mouse embryo, Gli3 is expressed in the somites
(McDermott et al., 2005), and
GLI and FGF family members genetically interact in organogenesis
(Brewster et al., 2000;
Aoto et al., 2002;
te Welscher et al., 2002;
Kuschel et al., 2003).
Therefore, we examined whether the bud defect is preceded by impaired
initiation of mammogenesis in Gli3Xt-J/Xt-J embryos, and
whether initiation of mammogenesis required an interaction between somitic
Fgf10 and Gli3. Wnt10b and TOPGAL expression are reduced at the level of the mammary line, and not elevated at the position of placode 3 in Gli3Xt-J/Xt-J embryos at E11.5 (44 somite) (Fig. 5A-D). Accordingly, Lef1 is expressed at the position of mammary buds 2 and 4, but not of bud 3 by E12.5 (Fig. 5E,F). The cells of the stratum germinativum are cylindrical, slightly enlarged along the width of the mammary line and, as in Pax3ILZ/ILZ mutants and wild-type embryos, covered with periderm. However, the stratum intermedium is absent (Fig. 5G,H), in accordance with the reduced expression levels of Wnt10b and TOPGAL, and absence of Lef1 expression. Thus, mammary line formation is indeed impaired in Gli3Xt-J/Xt-J embryos. In contrast to the delayed formation of placode 3 in Pax3ILZ/ILZ mutants, gland 3 was not found at all in histological sections or skin preparations of Gli3Xt-J/Xt-J embryos between E12.5 and term (not shown).
In situ hybridization revealed high Gli3 expression in all thoracic somites at E10.0-E11.0 (whole-mount data not shown; Fig. 5I). Gli3 is less expressed in the dermal mesenchyme, and not detected in the surface ectoderm. Gli3 expression is normal in the somites of E10.5-E11.5 Fgf10-/- embryos (Fig. 5J,K). Conversely, Fgf10 expression is not detected in the central somitic domain, and reduced in the hypaxial somitic domain of E10.5-E11.5 Gli3Xt-J/Xt-J embryos. Moreover, Fgf10 expression extends posteriorly to only the level of somite 16 instead of 18 in Gli3Xt-J/Xt-J embryos (Fig. 5L,M). Furthermore, hypaxial Fgf10 expression is highest in somites 15-16, underlying mammary placode 3 in control embryos, whereas it is barely detectable in those somites of Gli3Xt-J/Xt-J embryos. As patterning and histology of Gli3Xt-J/Xt-J somites seem normal (McDermott et al., 2004) (our data not shown), the reduced Fgf10 expression does most probably not reflect an absence of somitic tissue, but is due to an absence of Gli3 expression.
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Hypomorphic Fgf10 mutants lack placode 3
To test whether reduced Fgf10 expression is indeed responsible for
the mammary phenotype in Gli3Xt-J/Xt-J embryos, we
generated embryos carrying one Fgf10- allele and one
Fgf10mlcv allele
(Kelly et al., 2001), which
expresses reduced levels of Fgf10
(Mailleux et al., 2005). This
allelic combination results in a hypomorphic Fgf10 phenotype in the
embryonic lung (Mailleux et al.,
2005), limbs (arrows in Fig.
6D,F) and gut (F.G.S., J. L. Curtis, J.M.V., P. M. Del Moral, T.
Fairbanks, D. Warburton, K. Wang, R. C. Burns and S.B., unpublished). The
mammary line does form in these embryos, and so do mammary placodes 2 and 4.
However, we could neither detect placode 3 in E11.5 and E12.5 hypomorphs by
Wnt10b and Lef1 expression
(Fig. 6A-H), nor in
histological sections of embryos until term (not shown). Along the mammary
line, cells of the stratum germinativum are still cuboidal at E11.0 (40
somites) instead of cylindrical and covered by periderm (not shown). By E11.5
(45 somites) cells of the stratum germinativum are also enlarged cylindrical
in hypomorphs, and covered by periderm as in control embryos, but fail to
generate a stratum intermedium (Fig.
6I,J). The mammary phenotype of hypomorphs is thus less severe
than that of Fgf10-/- mutants and very similar to that of
Gli3Xt-J/Xt-J mutants, while the absence of rudiment 3
distinguishes this phenotype from that of Pax3ILZ/ILZ
embryos. These data strongly support the conclusion that reduced somitic
Fgf10 expression impairs mammary line formation and abolishes
formation of placode 3 in Gli3Xt-J/Xt-J embryos.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Stamataki
et al., 2005). GLI3 exerts an activator function in the
epaxial-medial domain of the somites, and a repressor function in the lateral
domain of the somites (McDermott et al.,
2005). GLI3 may therefore directly or indirectly activate
Fgf10 transcription in somitic subdomains. No GLI-binding sequence
has been found within 6.6 kb upstream of the Fgf10-coding sequence
(Ohuchi et al., 2005).
However, sequences further upstream, e.g. as far as 114 kb
(Kelly et al., 2001;
Mailleux et al., 2005), may
contain regulatory elements including GLI3-binding sites. Interestingly, null
mutants for sonic hedgehog form all mammary placodes
(Gallego et al., 2002;
Michno et al., 2003),
indicating that sonic hedgehog is not required for GLI3-mediated activation of
Fgf10 transcription.
We also show that Wnt10b expression and canonical WNT signaling
are activated downstream of FGF10/FGFR2B signaling in the surface ectoderm
(Fig. 7A), and thus identified
a genetic cascade from GLI3 via FGF10/FGFR2B to Wnt10b expression,
and to canonical WNT signaling required for the induction of a mammary cell
fate in the ectoderm. Wnt10b expression precedes TOPGAL expression,
and coincides with the acquisition of an enlarged cylindrical cell shape
(Fig. 5H,
Fig. 6J). Both Wnt10b
and TOPGAL expression increase with localized multilayering of the flank
ectoderm. Multilayering is initially restricted to the mammary line.
Previously, the enlargement of cells of the single-layered ectoderm was
suggested to indicate mammary line formation
(Turner and Gomez, 1933;
Sakakura, 1987). Although
enlargement of cylindrical cells coincides with low levels of Wnt10b
expression, it is not sufficient for the formation of a mammary placode, as
seen in Gli3Xt-J/Xt-J and hypomorphic Fgf10
mutants. Therefore, we postulate that multilayering is the first histological
manifestation of a mammary cell fate.
A model combining ventral elongation of the thoracic somites with gradients of somitic Fgf10 expression in the patterning of mammary epithelium in the interlimb region
Based on the similarity in mammary phenotype of
Fgf10-/- and Fgfr2b-/- mutants
(Table 2), and on the
complementary expression patterns of Fgf10 and Fgfr2b in
wild-type embryos, we conclude that somitic FGF10 binds to and activates
ectodermal FGFR2B, leading to a mammary cell fate in the ectoderm. An analysis
of transverse sections at the level of somites 14-18 of progressively older
wild-type embryos revealed that the prospective hypaxial buds of the thoracic
somites are located dorsal to the flank at E10.0 (30 somites). The somites
extend ventrally, growing into the lateral plate mesoderm of the flank between
E10.5 and E11.5 (35-45 somites). Elongating more rapidly than the dorsoventral
axis of the body, they reach the ventral side of the flank by E12.0 (50
somites). We observed that the ectodermal cell morphology changes from
cuboidal to cylindrical along the dorsoventral axis of the flank during
somitic elongation. Subsequently, the cylindrical cells enlarge, express
Wnt10b (Fig. 5B,H) and
give rise to the stratum intermedium. This enlargement and multilayering
occurs first at the ventral position of the flank between E11 and E11.5, where
it indicates mammary line formation. Somitic elongation also coincides with
the onset of somitic Fgf10 expression (pink in
Fig. 7B, based on
Fig. 4A-D and data not shown).
Therefore, somitic FGF10 may mediate ectodermal maturation even before the
differentiation into mammary epithelium. The cuboidal morphology of the
ectodermal cells in Fgf10-/- and
Fgfr2b-/- mutants (Fig.
3E,F) demonstrates that FGF10/FGFR2B signaling is not required for
the maturation of squamous ectoderm cells into cuboidal cells. However, it is
required for the progression to a cyclindrical ectodermal cell shape. In
Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, the
somites elongate normally, but express reduced levels of Fgf10. This
correlates with the acquisition of an enlarged cylindrical cell morphology and
low levels of Wnt10b expression, yet failure to form the stratum
intermedium by E11.5 (Fig. 5H,
Fig. 6J). Therefore, we
conclude that progressive maturation of the ectoderm requires increasing
amounts of somitic FGF10 (Fig.
7C).
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The absence of the ventral bud of the somites correlates with a more dorsal location of the mammary line in Pax3ILZ/ILZ mutants. As the line is located above the ventral-most edge of the somite, we conclude that the position of the mammary line is determined by the ventral edge of the thoracic somites, or ventral-most delivery point of somitic FGF10, rather than being predetermined in the ectoderm and waiting for FGF10 signals. In summary, we propose that the intra-somitic Fgf10 expression gradient and dynamics, combined with the rapid hypaxial elongation of the thoracic somites, determines the dorsoventral position of the mammary line and the progressive differentiation towards a mammary cell fate at the position of placode 3 on the anteroposterior aspect of this line.
Delaminating epaxial and central somitic dermomyotomal cells form the
dorsal dermis before E11 in the mouse
(Houzelstein et al., 2000).
Some of these cells may mix with the flank mesenchyme at the dorsolateral
boundary, as shown in chick
(Olivera-Martinez et al.,
2000; Nowicki et al.,
2003). Although it remains to be elucidated whether somitic FGF10
reaches the overlying ectoderm by a similar delamination of dermal precursors,
or by diffusion, or by both mechanisms, we provide evidence that somitic FGF10
is required for the earliest differentiation steps of the ectoderm, and
subsequently for the formation of mammary epithelium. To our knowledge, this
is the first evidence that the hypaxial and central domain of the somites are
implicated in the differentiation and patterning of the flank ectoderm. The
failure of cells of the stratum germinativum to become cylindrical and
generate a stratum intermedium in E11.5 Fgfr2b-/- and
Fgf10-/- mutants may underlie the hypoplasia of the
stratum granulosum [derived from the stratum germinativum and intermedium
(Sengel, 1976)] observed in
these mutants at birth, and the impaired hair follicle formation in
Fgfr2b-/- but not in Fgf10-/- mutants
(Suzuki et al., 2000;
Petiot et al., 2003).
Does FGF10 regulate epithelial migration during the initiation of mammogenesis?
The mammary line and placodes show an increased cell density compared with
the surrounding ectoderm. As this density seems to be established without
locally increased cell proliferation, it has been suggested that cells migrate
towards and along the mammary line
(Balinsky, 1949-1950). Although
cells have never been shown experimentally to migrate along the mammary line,
the observation of elongated, fibroblast-like morphology of cells along the
mammary line may indeed suggest migratory behavior of these cells
(Propper, 1978;
Chu et al., 2004). This view
may be supported by the fragmented Wnt10b expression domain on the
flank, fusing into one continuous mammary line
(Veltmaat et al., 2003), and
by the transition of a small stripe of Lef1 expression into a dot via
a comet-shaped intermediate at the level of placode 3
(Mailleux et al., 2002). It
now raises the interesting issue of whether FGF10 mediates cell migration
during mammary line and placode formation, as it does in eyelid development
(Tao et al., 2005) and lung
development (Bellusci et al.,
1997; Park et al.,
1998). In this scenario, cells along the flank would be pulled in
a ventral direction along with the elongating hypaxial buds. As Fgf10
expression is highest in the hypaxial buds of somites 15 and 16,
Wnt10b-expressing cells along the mammary line would be recruited to
the position above these somites to form placode 3. Such recruitment is
supported by the condensation of initially three fragments of high
Wnt10b expression somites 15, 16 and 17, to one continuous domain
above somite 15 and 16 (Veltmaat et al.,
2004). The shorter somites in Pax3ILZ/ILZ
mutants would draw ectodermal cells across a narrower, more dorsal domain,
resulting in a more dorsal position of the mammary line. In combination with
reduced production of total somitic FGF10, fewer ectodermal cells would be
recruited, leading to a narrower line and delayed multilayering. Subsequently,
the absence of the high hypaxial Fgf10 expression of somites 15 and
16 would then explain why Wnt10b expression remains present for a
prolonged period along the mammary line, instead of coalescing at the position
of placode 3.
Does mammogenesis depend on a unique molecular network for the induction of each pair of placodes?
Little is known about the initiation of mammogenesis, and less is known
about the sources of molecules used in the formation of the five individual
placode pairs. We show here that placode 3 is most sensitive to a reduction of
somitic Fgf10. Similarly, placode 3 is more sensitive than placodes 2
and 4 to loss of Gli3 (this report) or neuregulin 3
(Howard et al., 2005). Gland 3
is also the gland most frequently absent in wild-type mice
(Little and McDonald, 1945).
Furthermore, increased signaling through EDA/EDAR leads to formation of
supernumerary placodes only between placode 3 and 4
(Mustonen et al., 2004), while
Lef1-null and Tbx2/3 double heterozygous mutants have a more
severe placode induction or maintenance defect in the thoracic region than in
the inguinal region (van Genderen et al.,
1994; Jerome-Majewska et al.,
2005). We can therefore conclude that different molecular networks
regulate mammogenesis at different levels along the anteroposterior axis, and
different developmental thresholds exist for these networks along this axis.
This may explain the differences in number and position of glands along the
mammary line in different species, and the incidence of supernumerary nipples
and breasts found in 2-5% of the human population
(Schmidt, 1998;
Grossl, 2000). In particular,
with our findings that somitic signals are required for placode formation, we
may begin to understand the etiology of Poland's syndrome
(Poland, 1841), characterized
by compound hypoplasia of the breast and the somite-derived pectoral muscles
and thoracic skeletal structures.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Universitair Medisch Centrum Utrecht, Afdeling Metabole en
Endocriene Ziekten, Divisie Biomedische Genetica, 3584 EA Utrecht, The
Netherlands 
Present address: Harvard Medical School, Department of Cell Biology,
Boston, MA 02115, USA
|
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