Divisions of Developmental Biology and Ophthalmology, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
¶ Author for correspondence (e-mail: richard.lang{at}chmcc.org)
Accepted 3 June 2004
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SUMMARY |
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Key words: Bone morphogenetic protein, Bmp, Branching morphogenesis, Lacrimal gland
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Introduction |
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In previous studies (Govindarajan et
al., 2000; Makarenkova et al.,
2000
), it has been shown that fibroblast growth factor 10 (Fgf10)
is expressed in temporal periorbital mesenchyme and has activity as an inducer
of the lacrimal gland. In vitro inhibition studies show that Fgf10 promotes
proliferative responses in lacrimal gland epithelium through Fgfr2IIIb
(Makarenkova et al., 2000
), a
result consistent with the involvement of this pathway in many examples of
branching morphogenesis (Arman et al.,
1999
; Cancilla et al.,
1999
; Sekine et al.,
1999
; Ohuchi et al.,
2000
). The epithelial expression of Pax6 in lacrimal
gland (Kammandel et al., 1999
;
Makarenkova et al., 2000
) has
also implied a developmental role for this transcription factor and indeed,
Pax6+/Sey-1Neu mice show vestigial glands
(Makarenkova et al., 2000
).
The absence of any change in the pattern of expression of Fgf10 in
periorbital mesenchyme of Pax6+/Sey-1Neu mice suggests
that Pax6 might act as a lacrimal gland competence factor in
conjunctival epithelium (Makarenkova et
al., 2000
).
The process of branching morphogenesis
(Hogan, 1999) requires an
exchange of signals between epithelium and mesenchyme. The end result is the
reiterative production of a series of epithelial buds that have a
characteristic distribution. Signaling molecules of the bone morphogenetic
protein (Bmp) family have previously been implicated in the regulation of
branching morphogenesis. For example, Bmp4 has a role in development of the
lung (Bellusci et al., 1996
),
kidney (Raatikainen-Ahokas et al.,
2000
) and prostate gland (Lamm
et al., 2001
). In each case, Bmp4 appears to modulate branching by
suppressing proliferation of the epithelial component of the structure. Bmp7
has also been identified as an important regulator of branching morphogenesis.
In the kidney, Bmp7 promotes growth and survival of the metanephric mesenchyme
(Dudley et al., 1999
), a
result consistent with the effect of low doses of Bmp7 on the mIMCD-3 cell
model of collecting duct morphogenesis
(Piscione et al., 1997
).
Similarly, Bmp7-null mice have deficiencies in development of the
submandibular gland (Jaskoll et al.,
2002
). An assessment of the role of Bmps in branching
morphogenesis is complicated by their overlapping and dynamic expression
patterns, their ability to heterodimerize
(Suzuki et al., 1997
), the
variety of receptor heterodimers through which they can signal
(Massague, 1998
) and the
possibility of activity modulation by secreted antagonists
(Hemmati-Brivanlou et al.,
1994
; Piccolo et al.,
1996
; Zimmerman et al.,
1996
; Cho and Blitz,
1998
; Cancilla et al.,
2001
).
In the current study, we have investigated the function of Bmp7 in
branching morphogenesis of the lacrimal gland. We show that Bmp7 is
expressed with a complex pattern in the developing gland and that
Bmp7-null mice have distinctive reductions in lacrimal gland size and
branch number. Consistent with a role for Bmp7 in promoting branching is the
observation that whole gland explant cultures exposed to recombinant Bmp7 show
an increased number of lacrimal gland buds, while those exposed to the
inhibitors noggin and follistatin show decreased budding. Explants also show
that Bmp7 does not have a discernible effect on isolated epithelium but does
stimulate a response in mesenchymal cells in the form of increased cell
division, aggregation and upregulation of cadherins, connexin 43 and
-smooth muscle actin. These data suggest that mesenchyme is the primary
target of Bmp7 and, in turn, that the effect of Bmp7 on epithelial responses
is indirect. These data also suggest that through its activity in stimulating
mesenchymal condensation, Bmp7 may be important for formation of signaling
centers. This model can explain both the mesenchymal responses to Bmp7 and the
gland development deficiency of the Bmp7-null mice.
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Materials and methods |
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Lacrimal gland reporter gene visualization and explant cultures
Lacrimal glands were visualized in whole-mount embryos by dissecting away
the skin overlying the gland and then X-gal stained using established
techniques (Song et al.,
1996). Whole gland explant cultures were prepared from embryos
between embryonic days (E) 15.5 and 17.5. Lacrimal glands were excised and
placed into collagen gel in a four-well plate (Nunc). Glands were cultured at
37°C and 5% CO2 in growth medium consisting of CMRL-1066 (Gibco
BRL) supplemented with heat inactivated 10% fetal calf serum (FCS), glutamine,
non-essential amino acids and an antibiotic-antimycotic (Gibco BRL)
antibiotics. Explants were cultured in either serum-free defined medium
(Zuniga et al., 1999
) or in
the CMRL-1066+10% FCS supplemented with 100 ng/ml Bmp7 (R&D systems). For
experiments with Bmp inhibitors, explants were cultured with or without either
noggin-conditioned medium or 100 ng/ml recombinant noggin (R&D systems) or
follistatin. Xenopus noggin concentrated in conditioned medium was
used as described previously (Lamb et al.,
1993
). After 48 hours in culture, tissues were fixed and
photographed or stained with X-gal (Song
et al., 1996
).
Culture of lacrimal gland epithelium
Lacrimal gland epithelium was isolated and cultured as described previously
(Makarenkova et al., 2000).
Briefly, the isolated epithelium was placed in the center of one well of a
four-well plate and covered with Matrigel or collagen gel. Once the gel had
set, 300 µl of defined medium was added into the well. For some
experiments, an in vitro epithelial bud extension assay was performed as
previously described (Weaver et al.,
1999
). In these assays, heparin acrylic beads (Sigma) were loaded
with recombinant Fgf10 (R&D systems) and placed
100-150 µm from
the anterior part of the epithelial bud. Explants were cultured in defined
medium alone or in the same medium supplemented with either recombinant Bmp4
or Bmp7 (R&D systems).
Mesenchyme cultures
Lacrimal gland mesenchyme cultures were prepared in a similar manner to
limb bud micromass cultures (Vogel and
Tickle, 1993). Lacrimal glands were isolated from E15.5-16 embryos
and placed in 2% trypsin solution on ice for 1 hour. The tissue was then
placed in medium containing serum to stop the enzyme reaction and then the
epithelium was mechanically separated from the surrounding mesenchyme using
fine needles. The mesenchyme was triturated and then centrifuged at 400
g for 5 minutes. Cells were re-suspended in defined medium and
plated at a density of 10,000 cells in 10 µl on Poly-l-Lysine and
laminin-coated coverslips (Biocoat) in Nunc four-well plates and left to
attach for 1 hour. Once the cells had attached, 300 µl defined medium or
defined medium containing 1-100 ng/ml Bmp7 was added to each well. Cultures
were maintained for 6, 24, 48 or 72 hours in an incubator at 37°C with 5%
CO2 in air before being fixed and immunolabeled. For mesenchyme
cell proliferation assays, the culture medium was replaced after 24 hours with
DMEM containing 1% FCS and 10 µM BrdU with or without Bmp7 for 2 hours
prior to fixation. Cells were fixed and labeled as described previously
(Brennan et al., 2000
).
Quantification
The degree of lacrimal gland development in explants was quantified simply
by counting the number of terminal epithelial buds (acini). All error bars
shown represent standard errors. Statistical significance of data was
determined using the Student's t-test with the exception of
Fig. 4L, which required the
unpaired t-test, and of Fig.
5A, which was better suited to the Mann-Whitney U test. P
values of less than 0.05 were considered to be significant and this was the
case (as indicated in legends and Figures) for all data shown. For
Fig. 3, the length of the
lacrimal gland from the temporal fornix of the conjunctiva to the most distal
end of the epithelial component of the gland was measured in mm (by placing a
mm scale within the images of gland preparations). As budding of the lacrimal
first occurs at E13.5, we defined the length as zero and the acinus number as
one. In this case, statistical significance was determined using the unpaired
t-test.
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Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described
(Nieto et al., 1996). The
Fgf10 probe has been described previously
(Bellusci et al., 1997
).
Antibodies and immunostaining
For immunostaining, the following primary antibodies were used: rabbit
polyclonal antibody to Connexin 43 (Cx43) (GAP 15) (amino acids 131-142) was
diluted to 1:200; mouse anti-BrdU antibody (Dakopatts) diluted 1:100 in PBSBT
(PBS containing 1% BSA and 0.2% Triton); mouse anti--smooth muscle
actin (Clone 1A4 Sigma) diluted 1:500; rabbit polyclonal pan-cadherin (Sigma)
diluted 1:500; mouse anti human Ki-67 (Novocastra laboratories, Newcastle upon
Tyne, UK) diluted 1:250; mouse anti chick Pax6 (Developmental Hybridoma
Studies Bank, Iowa, USA) diluted 1:500. All antibodies were diluted in TBST
(TBS containing 1% Triton). All incubations with primary antibodies were
carried out at 4°C overnight except for BrdU antibody which was added for
1 hour at room temperature. Primary antibodies were revealed by either Alexa-,
Rhodamine-, Cy3 or Texas Red-conjugated goat anti-mouse or goat anti-rabbit
immunoglobulins (Molecular Probes Eugene, OR). Nuclei were counterstained with
either OliGreen (Molecular Probes Eugene, OR) at a dilution of 1:5000 for 5
minutes or with Hoechst 33258. Labeled preparations were viewed on a Zeiss
Axioplan microscope and digital images obtained with a Sony DKC5000 camera.
Confocal images were obtained on a BioRad laser-scanning confocal
microscope.
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Results |
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As development proceeds through E17.0, Bmp7lacZ
expression is observed in the lacrimal gland mesenchyme
(Fig. 1H) with the strongest
expression associated with the distal tips of the epithelial buds
(Fig. 1H, red arrows) and with
the midsection of the lacrimal duct (Fig.
1H, ld). Sectioning of whole X-gal-stained glands from
E17.5 Bmp7lacZ embryos showed that
Bmp7lacZ expression is observed not only in the mesenchyme
(Fig. 1I, broken line) but also
in some cells of the gland epithelium. This is apparent in the epithelium of
the lacrimal duct (Fig. 1I,
ld) and in the terminal epithelial buds
(Fig. 1I, e.g. ep).
Expression of Bmp7lacZ in condensing mesenchyme adjacent
to epithelial buds (Fig. 1I,
arrowheads) is consistent with the patterns observed in both E17.0
(Fig. 1H) and E17.5
(Fig. 1J)
Bmp7lacZ whole-mount glands and is confirmed by
higher-power magnifications of the lacrimal duct
(Fig. 1K), an extended region
of budding epithelium (Fig. 1L)
and a terminal epithelial bud (Fig.
1M). A higher magnification view of the
Bmp7lacZ/+ lacrimal gland at E18.5 in excised whole-mount
(Fig. 1N) also indicates that
cells at the tip of developing branches are more strongly positive than at the
branch points. This pattern of expression can be contrasted with the uniform
X-gal labeling observed throughout the epithelium of the P6 5.0-lacZ
mouse (Fig. 1O). Although
sporadic epithelial cells express Bmp7lacZ, the overall
pattern corresponds to the mesenchymal component of the so-called signaling
centers (Hogan, 1999).
Bmp7 is essential for normal branching morphogenesis of the lacrimal gland
To determine whether Bmp7 functions in development of the lacrimal
gland, we examined a second line of mice that carry a conventional targeted
allele (designated Bmp7m1Rob) of the Bmp7 gene
(Dudley et al., 1995). These
animals were crossed with the P6 5.0-lacZ reporter to the F2
generation to produce Bmp7m1Rob/m1Rob, P6
5.0-lacZ, and Bmp7m1Rob/+, P6 5.0-lacZ
embryos. These could be stained with X-gal for easy visualization of gland
structure (Makarenkova et al.,
2000
). Despite the microophthalmia that is characteristic of some
Bmp7-null mice (Dudley et al.,
1995
; Wawersik et al.,
1999
) formation of the first lacrimal bud occurred with normal
timing (E13.0-13.5) and in the normal location on the temporal side of the eye
(compare Fig. 2A with
2B). By E19.5, the wild-type
gland consists of two lobes: a small intraocular lobe derived from a single
branch of the proximal duct (Fig.
2C, io and blue arrowhead); and an extensively branched exorbital
lobe (Makarenkova et al.,
2000
) (Fig. 2C,
xo). By contrast, the lacrimal gland in Bmp7-null animals showed
varying degrees of deficiency. In some cases, both intraorbital
(Fig. 2D, blue arrowhead) and
exorbital lobes (Fig. 2D, xo)
were near normal in size while in others, the gland was severely vestigial
(Fig. 2E and F). The
distribution of buds and branches in the lacrimal glands of Bmp7-null
mice was also abnormal; they were often observed in the primary duct region
(Fig. 2F-H, red arrowheads).
With dissection of glands from Bmp7m1Rob/m1Rob, P6
5.0-lacZ, embryos, it was clear that absence of Bmp7 did not prevent
formation of the mesenchymal sac (Fig.
2H, arrows). The degree to which development of the lacrimal gland
was affected did not correlate with the variable degree of microophthalmia
observed in Bmp7-null mice (Fig.
2D-G, mi), indicating that the two structures develop
independently. Quantification of the gland length and the number of branches
or acini in the lacrimal gland indicated that in the Bmp7-null mouse,
while there was not a significant reduction in the overall length of the gland
(P=0.7, n=6), the number of branches and acini was
significantly reduced (P<0.01, n=4)
(Fig. 3A,B). These results
demonstrate that Bmp7 is required to establish the appropriate number and
position of lacrimal gland branches.
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A reduction in bud number in lacrimal gland explants treated with Bmp inhibitors is consistent with suppression of bud formation observed in the lacrimal gland of Bmp7-null mice. However, the morphology of acini in noggin/follistatin treated glands was very different from that observed in the Bmp7 null. Specifically, in the Bmp7 null, while there were fewer acini, those that existed were of normal size and shape. By contrast, the acini in noggin and follistatin-treated glands were much larger. We reasoned that this might be a consequence of the activity of additional Bmp family member ligands that could be inhibited by noggin and follistatin in explants but were unaffected in the Bmp7 null.
To investigate this possibility, we performed noggin inhibition studies on gland explants established at E16.5 from Bmp7m1Rob/m1Rob, P6 5.0-lacZ embryos. Again, whole gland explants were cultured for 48 hours in the presence or absence of noggin and as before, a quantitatively significant reduction in the number of acini was observed (Fig. 4L). Interestingly, the acini that did form in Bmp7m1Rob/m1Rob noggin-treated glands (Fig. 4K) were larger than those in the untreated controls (compare Fig. 4I with 4K) as had been observed in earlier inhibition experiments. When combined, these data indicate that at least one other Bmp family member functions in lacrimal gland development and is most probably involved in regulating epithelial cell proliferation.
Lacrimal gland mesenchyme is a target for Bmp7 activity
As it was possible that the lacrimal gland branching defect in
Bmp7lacZ mutant mice could reside in either epithelium or
mesenchyme, we performed a series of gain-of-function experiments using
explant cultures of whole glands or isolated gland epithelium and
mesenchyme.
When whole-gland explants were established at E15.5 and cultured for 48 hours in the presence or absence of recombinant Bmp7, we observed a modest but statistically significant increase in the number of branches formed following Bmp7 treatment (Fig. 5A). This was in agreement with assessment of the Bmp7-null mice indicating that the role of Bmp7 is to promote branching of the epithelium.
To assess the action of Bmp7 on the epithelial component of the gland, we
performed a bud extension assay that has been used previously to characterize
the responses of lung endoderm (Weaver et
al., 2000). In both the lung and the lacrimal gland, Fgf10 is an
endogenous stimulus for the epithelial proliferation and chemotaxis that drive
epithelial extension (Makarenkova et al.,
2000
; Weaver,
2000
). In the lung, Bmp4 can suppress Fgf10 mediated proliferation
and as a result, suppress epithelial extension
(Weaver et al., 2000
). We used
Fgf10-soaked heparin-sepharose beads to stimulate extension of epithelial
explants from the lacrimal gland, and assessed the effect of adding either
Bmp4 or Bmp7 to the media of these cultures.
E15.5 mesenchyme-free primary bud explants were established in collagen gel
100-150 µm from an Fgf10 bead and allowed to respond over a 48 hour period.
Control explants were grown in defined medium only and showed the expected
extension response (Fig. 5B).
The addition of Bmp4 to the media suppressed growth and extension of the
lacrimal gland epithelial explant in response to an Fgf10-soaked bead in the
same manner as has been observed with lung epithelium
(Weaver et al., 2000)
(Fig. 5B). Interestingly
however, addition of Bmp7 to media did not suppress extension or growth
towards the Fgf10-soaked bead (Fig.
5B).
These data, combined with the knowledge that Bmp7 is expressed in gland mesenchyme, suggested that mesenchyme might be the crucial target of Bmp7 activity. To examine this possibility, we isolated mesenchymal cells from wild-type embryos at E15.5 and determined whether there were distinctive responses to 100 ng/ml Bmp7 in low density micromass cultures. In control cultures, mesenchymal cells were evenly distributed over the plating area even after 48 hours (Fig. 6A). By contrast, Bmp7 treatment caused dramatic changes in cell morphology and distribution. After 12 hours, the cells became elongated and formed multiple small aggregates; in 24-48 hours, these small cell aggregates had formed larger clusters (Fig. 6B). As we did not observe increased levels of apoptosis or a decrease in cell number in the Bmp7-treated cultures (data not shown), this changed distribution presumably required migration.
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In a number of systems, mesenchymal condensation is associated with
increased expression of the cadherin adhesion molecule family as well as
junctional proteins such as connexin 43 (Cx43)
(Minkoff et al., 1994;
Haas and Tuan, 1999
). To
determine whether the Bmp7-stimulated aggregation of mesenchyme was associated
with formation of cell contacts, we immunolabeled mesenchymal cell cultures
for the gap junction protein Cx43 and for cadherins. This showed that the
cells found in aggregates upregulated Cx43 (compare
Fig. 6D with
6E) and cadherins (compare
Fig. 6F with G) in response to
Bmp7.
As many glandular structures contain cells that express smooth-muscle
-actin (
-SMA), we also determined whether Bmp7 might influence
expression of this marker. In Bmp7-stimulated mesenchyme cultures, we observed
an upregulation of
-smooth muscle actin (
-SMA) immunoreactivity
in most cellular aggregates (Fig.
6H,I) and positive cells showed a network of well organized stress
fibers (Fig. 6I). Only a few
-SMA-positive cells were detected in control cultures even after 72
hours in vitro and stress fibers were not observed
(Fig. 6H). Interestingly,
explanted lacrimal glands exposed to 100 ng/ml Bmp7 for 48 hours do not show a
changed distribution of
-SMA but do show an upregulation of this marker
in cells associated with the epithelium-mesenchyme boundary
(Fig. 6J,K).
These data implied that in the Bmp7 null gland, we might observe
reduced proliferation as well as reduced expression of cadherins and
-SMA. To test this, we performed immunofluorescence detection for
cadherins and
-SMA in wild-type and Bmp7 null glands
(Fig. 7). We also
double-labeled with anti-Pax6 antibodies to identify epithelial cells. So that
we could objectively compare levels of immunoreactivity, sections from
wild-type and mutant embryos were processed and labeled in the same
experiment, images were acquired digitally under identical lighting conditions
and figure panels were adjusted en masse in the same digital image file.
Cadherin immunoreactivity in wild-type glands shows the anticipated junctional
pattern in Pax6-positive epithelial cells
(Fig. 7A). By contrast,
although Pax6 nuclear labeling is easily detected, Bmp7-null glands
show a consistently reduced level of cadherin immunoreactivity
(Fig. 7B). Similarly, when
detecting
-SMA in both wild-type
(Fig. 7C,E) and
Bmp7-null glands (Fig. 7D and
F) Pax6 and
-SMA positive cells are found in smaller
numbers in the mutant (compare
7C with
7D, arrowheads). The
-SMA expression level in the positive cells of mutant glands is also
very much lower. This is obvious when observing wild-type
(Fig. 7E) and
Bmp7-null (Fig. 7F)
glands at higher magnification. Interestingly, the
-SMA positive cells
are also positive for the Pax6 epithelial marker. An assessment of
proliferation was performed using BrdU-labeling
(Fig. 7G). This showed that in
both the mesenchyme and epithelium of Bmp7-null glands, there were
statistically significant reductions in the level of proliferation. The
reduction was more dramatic in the mesenchyme. Given that Fgf10 has been
identified as a proliferative stimulus for gland epithelium, we performed in
situ hybridization for Fgf10 in both wild-type and Bmp7 mutant glands
(Fig. 7H,I). This indicated
that Fgf10 expression levels were not noticeably different in the absence of
Bmp7, suggesting an alternative explanation for reduced epithelial
proliferation levels.
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Discussion |
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Bmp7 is required for normal morphogenesis of the lacrimal gland
Several lines of evidence indicate that Bmp7 has an important function in
regulating the formation of buds and branches in the lacrimal gland.
Morphological assessment of gland development in wild-type and Bmp7
null mice supports this proposal as there are many fewer buds and branches in
Bmp7 null glands. Interestingly however, gland length is not
significantly affected by the absence of Bmp7, and this suggests that
Fgf10-driven primary and secondary branch extension occurs independently of
Bmp7. This is also consistent with unchanged Fgf10 expression observed in the
Bmp7 null. The appearance of primary lacrimal gland buds in all
Bmp7 null mice also indicates that Bmp7 is not required during the
inductive phases of lacrimal gland development and this, in turn, suggests
that there may be distinct bud induction mechanisms employed at different
stages of gland formation.
There are also likely to be multiple Bmp family members active during
lacrimal gland development. Exposure of whole gland explants to the Bmp
inhibitors noggin and follistatin produces fewer buds in a response that is
consistent with Bmp7 function. However, the buds that do form have an unusual
morphology in that they are larger than normal. This response is not a result
of inhibition of Bmp7 activity as it is also seen when Bmp7-null
glands are treated with noggin and follistatin and is a phenotype absent from
the Bmp7-null mice. This is a clear indication that other Bmp family
members are active in suppressing epithelial proliferation during lacrimal
gland development. We speculate, based on evidence from the lung
(Weaver et al., 2000), kidney
(Raatikainen-Ahokas et al.,
2000
) and prostate (Lamm et
al., 2001
) that one of them is Bmp4.
Previous studies have demonstrated the importance of Bmp7 for the
development of branched structures. In the kidney system, Bmp7 is involved in
the early developmental stages in mediating epithelial-mesenchymal
interactions and stimulating survival of metanephric mesenchyme
(Dudley et al., 1999).
Although our experiments do not reveal a function for Bmp7 in stimulating
mesenchymal survival, the positive effect of Bmp7 on lacrimal gland branching
may be analogous to its function in the kidney. It has also been recorded that
Bmp7-null mice have deficiencies in development of the submandibular
gland (Jaskoll et al., 2002
)
and this too is consistent with the general effect of Bmp7 in the lacrimal
gland system.
Bmp7 stimulates lacrimal gland branching by signaling mesenchymal responses
In attempting to understand the origin of the lacrimal gland development
defect, we performed a series of explants of whole glands and of isolated
mesenchyme and epithelium. Consistent with the phenotype of Bmp7-null
mice, whole lacrimal glands exposed to Bmp7 showed a statistically significant
increase in the number of epithelial buds. This indicates that Bmp7 is
necessary for the formation of normal bud number and in the context of the
explant assay, is sufficient to stimulate new bud formation. Furthermore, an
Fgf10-driven bud extension assay (Weaver
et al., 2000) was used to demonstrate that gland epithelium did
not discernibly respond to Bmp7 (but did to Bmp4). By contrast, exposure of
isolated mesenchyme to Bmp7 resulted in distinctive responses that included
proliferation, aggregation and differentiation. These data argue that the
primary target of Bmp7 in lacrimal gland development is the mesenchyme and
that deficient epithelial responses are an indirect consequence of defective
mesenchymal function.
The in vitro response of mesenchyme to Bmp7 is distinctive with
dose-dependent increases in proliferation and the formation of cellular
aggregates. Increased proliferation is presumably important for providing the
numbers of cells required for expansion of the gland. The formation of
aggregates is accompanied by upregulation of cadherin family adhesion
molecules and the junctional protein Cx43; both have been associated with
formation of condensed mesenchyme (Minkoff
et al., 1994; Haas and Tuan,
1999
). These data demonstrate that in response to Bmp7, gland
mesenchymal cells form close contacts characteristic of the tissue structures
found in the lacrimal gland.
However, the nature of the gland components that Bmp7-stimulated mesenchyme
represents is currently open to interpretation. Two models can be proposed. In
the first, we can suggest that the major function of Bmp7 is to facilitate the
formation of signaling centers (Fig.
8). In the context of branching morphogenesis
(Hogan, 1999) signaling
centers are defined as small groups of closely associated cells that are able
to secrete factors and respond to stimuli in a manner that directs the process
of branching. Close association of a critical number of cells in signaling
center-like structures is thought to be essential for providing the necessary
level of signaling [the so-called community effect
(Gurdon, 1988
)] required for
some developmental processes. Signaling centers are proposed to include both
epithelial and mesenchymal cells in the region of the developing bud tip.
Reiterative formation of signaling centers is crucial for the progression of a
single epithelial bud into a multi-branched mature organ. The shape of
signaling centers is also thought to influence the shape of the growing tissue
(Hogan, 1999
).
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In an alternative, and arguably more speculative, model, we can propose
that Bmp7 may function to stimulate a mesenchymal to epithelial transition.
This developmental strategy has precedents in the formation of branched
structures, notably in the kidney (Stark
et al., 1994), where epithelial cells of the ureteric bud produce
signals that stimulate mesenchymal cells to condense and form the epithelial
cells of the nephron. Suggestive evidence for this model in the context of the
lacrimal gland comes from the observation that markers expressed at high
levels in the epithelial component of the lacrimal gland (Connexin 43,
cadherins and smooth muscle actin, Fig.
7) are upregulated when isolated mesenchymal cells are stimulated
with Bmp7. In the wild-type gland, we also show, using the epithelial marker
Pax6, that cadherin and
-SMA-positive cells have epithelial character.
Thus, it is possible that the high expression level of Bmp7 found in
the signaling centers is crucial to stimulate proliferation and a
mesenchymal-epithelial transition that supplies the expanding gland with
epithelial cells. Clearly, an assessment of the mesenchymal-to-epithelial
transition model will require additional work developing strategies for
fate-mapping different components of the gland.
Localized Bmp7 may be crucial for patterning of the lacrimal gland
A striking feature of structures that arise through branching morphogenesis
is the reproducibility of the primary branching pattern and, as a consequence,
the placement of organ lobes. The lung is a good example
(Hogan et al., 1997), but this
is also true for the lacrimal gland where both intraorbital and exorbital
lobes arise and the timing and placement of branch initiation is highly
reproducible. Interestingly, our current analysis suggests a mechanism by
which Bmp7 can determine the location of the exorbital lobe of the gland
(Fig. 7).
In Bmp7lacZ embryos, it is clear that Bmp7 expression
is absent from the periorbital mesenchyme adjacent to the initial lacrimal
gland bud. However, strong Bmp7 expression is observed in a discrete patch of
mesenchyme located between the eye and the pinna of the ear. After
Fgf10-driven bud extension (Govindarajan
et al., 2000; Makarenkova et
al., 2000
), during which the lacrimal gland epithelium extends
away from the temporal side of the eye, the epithelium reaches and invades the
Bmp7-expressing region of mesenchyme. Only when the epithelial bud has invaded
does epithelial branching occur. When combined with evidence that Bmp7 is an
important stimulus for branching, this sequence of events suggests that
migration of the epithelial bud into a Bmp7 positive mesenchymal domain may
determine the timing and location of primary branching. Further supporting
this model is the loss of defined lobe structure in Bmp7-null mice.
Conceivably, other steps in lacrimal gland development, such as formation of
the intraorbital lobe and secondary branching, may be regulated by similar,
Bmp7-dependent mechanisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Molecular Biology Department, Sloan Kettering Institute,
East 67th Street, New York, NY 10021, USA
Present address: Department of Anatomy, National Defense Medical College,
3-2 Namiki, Tokorozawa, 359-8513, Japan
Present address: The Neurosciences Institute, 10640 John Jay Hopkins Drive,
San Diego, CA 92121, USA
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