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First published online 11 July 2007
doi: 10.1242/dev.005066
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,
1 Graduate Institute of Pathology, College of Medicine, National Taiwan
University, Taipei, Taiwan.
2 Department of Pathology, National Taiwan University Hospital, Taipei,
Taiwan.
3 Center for Neuroscience, University of California, Davis, CA 95618, USA.
Author for correspondence (e-mail:
phhuang{at}ntu.edu.tw)
Accepted 30 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Branching morphogenesis, Salivary gland, Cleft formation, Class 3 semaphorin, Plexin, Neuropilin (Npn1; Nrp1), Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Branching
morphogenesis involves growth and branching of the tubular epithelial cells
from their unbranched precursors and requires regulated interactions between
the developing epithelial cells and the surrounding mesenchyme
(Affolter et al., 2003;
Hogan and Kolodziej, 2002).
Several families of molecules have been implicated in regulating branching
morphogenesis, but they are thought to act in a tissue-specific manner
(Davies, 2002).
The mouse submandibular gland (SMG) has been used as a classical example
for studying branching morphogenesis
(Grobstein, 1953;
Hieda and Nakanishi, 1997).
Mouse SMG development begins at embryonic day 11 (E11). At E12, a single
epithelial bud surrounded by condensed mesenchyme is formed. By E12.5, small
clefts start to appear at the end of the epithelial bud (cleft formation).
Meanwhile, the cells in the bud continue to proliferate and cleave
repetitively, resulting in bush-like branching patterns. Several branching
buds and cords can be seen at E14. Lumenization of the solid cellular cords
and buds occurs at E17, and, by postnatal day 1 (P1), the salivary gland is
fully developed and starts to secrete mucin.
Molecular regulation of branching morphogenesis has been studied
extensively in the lung and the kidney, but the regulation of SMG development
has been relatively less well explored
(Affolter et al., 2003;
Cardoso and Lu, 2006;
Hogan, 1999;
Hogan and Kolodziej, 2002;
Kuure et al., 2000;
Lü et al., 2004;
Metzger and Krasnow, 1999;
Warburton et al., 2000). To
date, some molecules are implicated in the branching morphogenesis of the SMG.
For example, fibroblast growth factors (FGFs) and bone morphogenetic proteins
(BMPs) have been reported to mediate the formation of the SMG
(Hoffman et al., 2002;
Steinberg et al., 2005).
Interestingly, abnormal salivary gland morphology has also been observed in
Bmp7-null, Fgf10-null, Fgf8-null and
Fgfr2b-null mice (Jaskoll et al.,
2004b). In addition, sonic hedgehog (Shh) stimulates the
proliferation of branching epithelia by increasing Fgf8 expression,
and Shh-null mice have a hypoplastic SMG remnant
(Jaskoll et al., 2004a).
Despite these results, our understanding of the molecular mechanisms that
regulate the development of the salivary gland is still incomplete. In
particular, it is not known whether the cleft formation and epithelial
proliferation are regulated by the same sets of molecules and how the two
essential processes are coordinated to ensure proper branch formation.
Class 3 semaphorins are a family of conserved secreted molecules that play
roles in various developmental processes. In the developing nervous system,
class 3 semaphorins regulate axon repulsion, axon pruning and neuronal
migration (Fujisawa, 2004;
He et al., 2002;
Huber et al., 2003;
Raper, 2000;
Waimey and Cheng, 2006).
Interestingly, some of these semaphorins have been reported to regulate the
formation of the cardiovascular system and the branching formation of the lung
(Gitler et al., 2004;
Gu et al., 2005;
Ito et al., 2000;
Kagoshima and Ito, 2001;
Torres-Vazquez et al., 2004).
Neuropilin is the binding receptor for class 3 semaphorin, but neuropilin has
to form co-receptor with plexin to transduce the semaphorin signal inside the
cell. In vertebrates, seven class 3 semaphorins, two neuropilins and nine
plexins have been identified. In addition to semaphorin, neuropilin binds to
vascular endothelial growth factor (VEGF; also known as VEGF-A and Vegfa -
Mouse Genome Informatics) and regulates the formation of the vasculature
(Fuh et al., 2000;
Gu et al., 2003;
Soker et al., 1998). Here, we
report the role of class 3 semaphorins in branching morphogenesis of the
developing SMG. By examining members of vertebrate class 3 semaphorin,
neuropilin and plexin, we conclude that semaphorin signaling specifically
regulates cleft formation during the development of SMG.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For SMG co-culture experiment, COS cells in a six-well culture dish were
lipofectamin-transfected with Sema3-expressing plasmids 60 hours before the
dissection of SMGs (Zou et al.,
2000). The COS cell culture medium was then replaced with SMG
culture medium 12 hours before the dissection of SMGs. The SMG explants were
dissected as described above and cultured on the conditioned SMG culture
media.
Mesenchyme-free SMG explants were obtained by incubation of dissected E13 SMGs with Hanks' balanced salt solution containing 1.6 U/ml Dispase (Roche Molecular Biochemicals) at 37°C for 20 minutes. Epithelia were separated from mesenchyme with fine forceps in Hanks' solution containing 10% BSA. The SMG epithelia were placed on a Nuclepore filter, covered with growth factor-reduced Matrigel (prepared in 1:1 dilution with culture medium; BD Biosciences) and cultured in SMG culture medium.
For dissociated SMG epithelial culture, the mesenchymal-free SMGs (prepared as described above) of 10 to 15 E13 mouse embryos were pooled, minced and then dissociated into single cells by use of Ca2+-free Hank's balanced salt solution containing 0.07% collagenase Type II and Type III (1:1 v/v; Sigma) for 45 minutes at 37°C. Triturated cells were centrifuged at 300 g and resuspended with SMG culture medium. Cell clumps and tissue debris were removed by passing the cell suspension through a cell strainer (40 µm Nylon, BD Falcon). The yielded cell suspension was then seeded onto a 35 mm dish and cultured in SMG medium containing exogenous Fgf7 (100 ng/ml), Fgf10 (200 ng/ml) and Hgf (50 ng/ml).
Antisense oligodeoxynucleotides, recombinant peptides and antibodies
Antisense experiments were performed with 2 µM oligodeoxynucleotides
(ODNs) with phosphorothioate modification. The nucleotide sequences used in
this report were: Sema3A antisense (891-872),
3'-CCTGAAGTACCCTGCCCTGA-5'; Sema3B antisense (2068-2059),
3'-ACCGACTCCTCTCTCATCTC-5'; Sema3C antisense (589-571),
3'-ATACAGACACCCTCACCTCG-5'; Sema3D antisense (2359-2341)
3'-ATCTGCTCACAGTACTGGT-5'; Sema3E antisense (1962-1943),
3'-TGTCTCACCTACCTCCTTCA-5'; Sema3F antisense (549-530),
3'-CTACCGTTACCTCTCACACC-5'; plexin A1 antisense (5015-4996),
3'-CTGACCTTCTCCGACTTGTG-5'; plexin A2 antisense (5309-5290),
3'-CCTGTACCTAGACCTCACCG-5'; plexin B1 antisense (1751-1732),
3'-CCTTCTCTCCTCCCTCCAAA-5'; plexin B2 antisense (3247-3228),
3'-CTGTCACCACCTCTCCTACG-5'; plexin B3 antisense (4920-4901),
3'-CTACCACTCCTTCCACCTCA-5'; plexin C1 antisense (1329-1348),
3'-ACCCTTCCTCCACTCTTCTT-5'; plexin D1 antisense (4809-4790),
3'-CTCACCGACGACTCCCTCTT-5'; plexin D1 sense (4790-4809),
5'-GAGTGGCTGCTGAGGGAGAA-3'; scrambled sequence,
5'-CCGACTCTACCACTTGCCTC-3'.
Recombinant peptides or antibodies were added into SMG culture medium with concentrations indicated. The human recombinant SEMA3A peptide (R&D systems) was added at concentrations of 10, 25, 50, 100 or 150 ng/ml, respectively. Fgf10 (R&D systems) or Fgf7 (R&D systems) was added at concentrations of 250, 500 or 1000 ng/ml, respectively. The neutralizing antibodies to Npn1 (Calbiochem) or control IgGs (Calbiochem) were added at concentrations of 1, 2, 5 or 10 µg/ml, respectively.
Semi-quantitative RT-PCR
SMGs were dissected at E13, E17, P1, P4 or in adult mice, respectively.
DNase-free RNA was prepared by using an RNAquous-4 PCR kit and DNA-free DNase
removal reagent (Ambion). cDNA was generated with a reverse-transcriptase kit
(Invitrogen). Semi-quantitative PCR was performed using specific primers for
each transcript.
Whole-mount RNA in situ hybridization and antibody immunostaining
Whole-mount RNA in situ hybridization of the SMG explant cultured in vitro
was performed essentially as described
(Steinberg et al., 2005).
Riboprobes for plexins and semaphorins were used as described
(Cheng et al., 2001;
Zou et al., 2000). The
riboprobes for Npn1 and Npn2 were prepared from the mouse Npn1 cDNA
fragment (GenBank accession number BC060129, nucleotide 619-1030) and the
mouse Npn2 cDNA fragment (GenBank accession number NM_01093,
nucleotide 1578-2686), respectively. Whole mount immunostaining was performed
by application of primary antibodies in M.O.M. blocking reagent (Dako) for 3
hours at room temperature and then of secondary antibodies in PBS-Tween 20
(0.1%) for 2 hours. The antibodies used included Fgfr2 (1:200 dilution, Santa
Cruz), Flt1 (1:500, Santa Cruz), Flk1 (1:500, Santa Cruz), VEGF-A (1:1000,
AbCam), E-cadherin (1:100, BD Biosciences), fibronectin (1:100, BD
Biosciences), alkaline phosphatase-conjugated anti-digoxigenin antibody
(1:2000, Roche Molecular Biochemicals) and donkey F(ab)2 fragments
labeled with AlexaFluor 488 or AlexaFluor 594 (Molecular probe).
AP in situ hybridization, BrdU labeling and TUNEL assay
AP-fusion protein was prepared and AP in situ hybridization was performed
as described (Cheng and Flanagan,
2001). For labeling of proliferative cells, the cultured SMGs were
incubated with 10 µM 5-bromo-2'-deoxyuridine (BrdU) for 2 hours at
37°C, and were followed by three washes in PBS with 0.1% Tween-20. The
SMGs were then fixed with 0.5% Triton X-100 in ethanol/glycine/water
(70:20:10, v/v) at pH 2.0 for 1 hour, followed by five washes in PBS. A
monoclonal antibody (1:10 dilution) from a BrdU labeling kit (Roche Molecular
Biochemicals) was used to detect the BrdU labeling. Pictures were photographed
by fluorescence microscope (Leica) and analyzed by MetaMorph Software
(Universal Imaging).
Apoptotic cells in SMG explants were examined by performing TUNEL staining, essentially as described in the In Situ Cell Death Detection Kit, POD (Roche Molecular Biochemicals).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Chen et al., 1998). The SMG
treated with anti-Npn1 antibody prohibited cleft formation in a dose-dependent
manner. The effect could last for 72 hours, and a concentration of 5 µg/ml
completely blocked the activity (Fig.
2Aa,b). Second, we applied ODNs against Npn1 mRNA into
the culture to downregulate the expression of Npn1 in the SMG epithelial
cells. As clearly shown in Fig.
2B, the antisense ODNs specifically inhibited cleft formation
(Fig. 2Ba, upper panels, and
b). In situ hybridization and RT-PCR analysis confirmed the diminished
Npn1 transcript after addition of antisense ODNs
(Fig. 2Ba, middle panel, and
2C). In addition, we could not detect changes in the extent of BrdU labeling
(Fig. 2Ba, lower panels, and c)
and TUNEL activity (data not shown) in the SMG explants treated with Npn1
antisense ODNs compared with the control. Taken together, these data indicate
that Npn1 regulates the SMG branching morphogenesis mainly in the process of
cleft formation.
Sema3A and Sema3C promote SMG cleft formation
We next screened the expression patterns of class 3 semaphorins in the
developing SMG to identify the candidate semaphorins that were utilized in the
Npn1-mediated cleft formation. To our surprise, semi-quantitative RT-PCR
analysis revealed that almost all class 3 semaphorins were detectable in the
embryonic SMGs, although the amounts varied (see Fig. S1A in the supplementary
material). Interestingly, RNA in situ hybridization on E15.5 tissue sections
revealed that Sema3A and Sema3C were abundantly expressed at
this stage, whereas others were not (see Fig. S1B in the supplementary
material). Thus, expression pattern studies suggest that multiple class 3
semaphorins can be involved in SMG development.
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In the SMG co-culture assay, we found that Sema3A and Sema3C promoted branching morphogenesis in a concentration-dependent manner (Fig. 3Ca). At lower concentrations of proteins, Sema3A and Sema3C enhanced the ability of each other to induce the branching activity in an additive manner (Fig. 3Ca,c). In addition, simultaneous knockdown of endogenous Sema3A and Sema3C by application of antisense ODNs also additively reduced the SMG branching activity (Fig. 3B). Such additive effects were specific to Sema3A and Sema3C, because other class 3 semaphorins had no effects on Sema3A- or Sema3C-promoted branching activity (data not shown).
To address whether cell proliferation is required for Sema3A and/or
Sema3C-dependent branching activity, we tested their effects in the SMG
co-culture assay at the presence of tunicamycin, a translation inhibitor that
inhibits cell proliferation (Spooner et
al., 1989). We found that, when the sizes of the epithelial cords
were partially reduced, the cleft formation was not affected (see Fig. S2A in
the supplementary material). Moreover, the overall proliferation in the SMG
explants as measured by BrdU incorporation was not significantly changed in
the presence of overexpressed Sema3A (see Fig. S2B in the supplementary
material). These results indicate that Sema3A and Sema3C act together to
promote cleft formation during SMG branching morphogenesis without obvious
effects on cell proliferation.
Sema3A and Sema3C induce cleft formation through functional Npn1
If Npn1 mediated the Sema3A and Sema3C signals in the developing SMG,
blockage of Npn1 function should abolish the branching activity enhanced by
Sema3A or Sema3C in the SMG co-culture assays. Indeed, the SMGs treated with
anti-Npn1 antibodies or Npn1 antisense ODNs in the Sema3A (or Sema3C)
co-cultures exhibited reduced enhancement of branching morphogenesis that
would otherwise be promoted by Sema3A or Sema3C
(Fig. 4A). Again, at a
concentration of 5 µg/ml, the anti-Npn1 antibody completely blocked the
cleft formation of the SMGs, even though the epithelial cells still
proliferated, as evidenced by the expansion of the epithelial cords
(Fig. 4A). Two additional
fusion protein experiments were performed to corroborate the requirements.
First, we added a soluble protein containing the extracellular domain of
neuropilin into the SMG cultures to block the semaphorin-neuropilin
interactions. In the presence of Npn1-AP (alkaline phosphatase) fusion
proteins, Sema3A no longer enhanced the branching activity in the co-culture
assay (Fig. 4B, upper panels).
By contrast, the presence of Npn2 fusion proteins in the culture could not
inhibit the Sema3A-mediated branching activity
(Fig. 4B, lower panels). Note
that addition of Npn1-AP alone could suppress branching activity of the SMG,
presumably by disrupting the interactions between the endogenous semaphorin
ligands and Npn1 (Fig. 4B).
Second, to test the direct binding of Sema3A to the SMG, we incubated the SMG
explant with Sema3A-AP-conditioned media and showed that AP binding activity
was apparent in the epithelial buds. However, this binding activity
disappeared if the Sema3A-AP proteins in the conditioned media were depleted
by pre-incubating the conditioned media with COS cells expressing Npn1
(Fig. 4C). Taken together,
these results indicate that the SMG branching morphogenesis stimulated by
Sema3A and Sema3C is mainly mediated by Npn1.
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). To address
whether VEGF plays a role in the Npn1-mediated SMG branching morphogenesis, we
first examined the expression patterns of VEGF and its receptors, Flt1 and
Flk1 in SMGs. Immunostaining of the cultured SMG explants showed that VEGF was
only detectable in the mesenchyme near the stalk of the SMG, whereas Flt1 and
Flk1 were not expressed in the epithelial buds of developing SMGs. As a
control, Fgfr2 was shown mainly expressed in the SMG epithelial buds
(Fig. 5A).
The functional role of VEGF and VEGFR was further examined in the SMG
explant cultures. Addition of recombinant VEGF into the SMG cultured ex vivo
had no apparent effects on SMG development
(Fig. 5B). Likewise, blocking
the endogenous VEGF by adding anti-VEGF antibody also had no effects on the
SMG (Fig. 5B). As VEGF might
compete with Sema3A for Npn1 binding (Miao
et al., 1999), we tested whether adding VEGF could have effects on
the branching activity promoted by Sema3A in the SMG co-culture assay.
Addition of VEGF to the cultures, even at a very high concentration (1.75
µg/ml), had no detectable effects (Fig.
5C). Alternatively, we pre-treated the SMG explants with VEGF 1
day before the appliance of 2 µg/ml anti-Npn1 antibody, which only
partially inhibited the branching activity. If VEGF had any Sema3A-competitive
binding effect toward Npn1, the pre-added exogenous VEGF should enhance
Npn1-mediated inhibition in the assay. Again, no differences were observed
(Fig. 5D). These results taken
together, we conclude that VEGF plays no role in the Sema3A (3C)/Npn1-mediated
SMG branching morphogenesis.
Plexin A2 and plexin D1 are required for Sema3-mediated cleft formation
We then investigated which plexins are required for Npn1-mediated cleft
formation in the SMG. Semi-quantitative RT-PCR analysis showed that only
plexin A1, A2 and D1 were relatively abundantly expressed in the E13 SMG (see
Fig. S3A in the supplementary material). Other plexins were either absent in
the SMG or not expressed until late embryonic stages. RNA in situ
hybridization of plexins on the E15.5 SMG sections showed similar patterns
(see Fig. S3B in the supplementary material). In the SMG explant cultures, we
found that, among all plexins, only loss of plexin A2 or plexin D1 had a
significant reduction in the branching morphogenesis
(Fig. 6A). Additional
expression pattern studies on these two plexins revealed that their transient
high expressions in the SMGs from E13.5 to E17.5 corresponded well to the
period of active branching of the developing SMG (see Fig. S4A,B in the
supplementary material). Therefore, plexin A2 and plexin D1 are likely the
candidates for mediating semaphorin signals in the developing SMG.
We next asked whether plexin A2 and plexin D1 played roles in the branching activity enhanced by Sema3A or Sema3C in the SMG co-culture assay. Clearly, the effects of Sema3A (or Sema3C)-inducing SMG cleft formation were ameliorated by application of either anti-plexin A2 or anti-plexin D1 ODNs (Fig. 6Ba). It is interesting to note that application of the two antisense ODNs against plexin A2 and plexin D1 in the SMG co-cultures only partially enhanced the effects. In addition, the inhibitions reached by adding these two specific ODNs were not as complete as those caused by adding anti-Npn1 antibodies (Fig. 6Bb, Fig. 2Ab). These observations could be explained by the presence of residual plexin A2 or plexin D1 in the SMG. Alternatively, additional molecules might participate in the Npn1-mediated branching activity. However, it is sufficient to conclude that Npn1, at least partially, forms receptor complexes with either plexin A2 or plexin D1 to transduce the Sema3A or Sema3C signal to mediate SMG cleft formation.
Npn1-mediated semaphorin signaling acts in parallel with FGF signaling in the developing SMG
The FGF family members, including Fgf1, 7 and 10, are shown to stimulate
SMG branching morphogenesis through activation of their receptor - Fgfr2b
(Steinberg et al., 2005). To
test whether inactivation of Npn1 would affect FGF signaling in the developing
SMG, we first examined the expression level of FGFs and Fgfr2 in the cultured
SMG explants pre-treated with Npn1 antisense ODNs. Semi-quantitative RT-PCR
analysis showed that their expressions were not changed with the treatment
(Fig. 7A). Moreover, in the SMG
explant culture, Fgf7 or Fgf10 effectively stimulated SMG branching
morphogenesis in the presence of Npn1 antisense ODNs
(Fig. 7Ba,b). Interestingly,
Fgf7 specifically promoted SMG cleft formation with short ductile formation,
as has been reported (Steinberg et al.,
2005), even in the absence of Npn1
(Fig. 7Ba, upper panels).
Likewise, the specific effects of Fgf10 on ductal elongation and branch number
were independent of Npn1 (Fig.
7B, lower panels). These results indicate that the Npn1-mediated
semaphorin signaling pathway functions in parallel, perhaps in an independent
but cooperative manner, with the FGF signaling pathway to mediate the
development of salivary glands.
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). Interestingly, addition of Sema3A into this culture did not
stimulate branching of the SMG epithelia
(Fig. 8A), whereas application
of Fgf10 or Fgf7 did stimulate growth and branching of mesenchyme-free SMG
rudiments as reported before (data not shown)
(Steinberg et al., 2005).
These results indicate that the Sema3A signal per se does not stimulate the
proliferation of SMG epithelial cells. In addition, growth-promoting signals
from the mesenchyme are required for Sema3A-mediated cleft formation in the
developing SMG.
|
). The degree of migration could be quantified by
measuring the ratio of the final spreading area to the initial area covered by
the epithelial rudiment. Adding Sema3A (100 ng/ml) to the culture
significantly reduced the cell migration
(Fig. 8A,B). To examine the
effects of Sema3A on single epithelial cells, we added Sema3A to the
dissociated SMG epithelial cells and found that the cytoplasmic expansion in
each cell collapsed after the treatment of Sema3A for 24 hours
(Fig. 8C). In these
Sema3A-treated cells, partial dissolution of actin fiber was observed when
stained with rhodamine phalloidin (Fig.
8C). These data suggest that Sema3A may collapse the epithelial
cells locally and thus induce cleft formation of the developing SMG. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Specific roles for class 3 semaphorins in regulating SMG cleft formation
The roles of class 3 semaphorins in branching morphogenesis have recently
been studied in the lung, mammary gland and kidney
(Hinck, 2004). In particular,
Sema3A and Sema3C have been proposed to regulate the branching of the lung bud
via a push-pull mechanism: Sema3A is expressed in the mesenchyme to push (or
repel) the developing lung epithelial cells that express Npn1, while the
epithelial cells at the tip of the lung bud secrete Sema3C to pull (or
attract) themselves, perhaps through Npn2, in an autocrine fashion
(Hinck, 2004;
Ito et al., 2000). In
addition, other signaling pathways such as FGFs and Shh are involved in the
lung branching morphogenesis (Cardoso and
Lu, 2006), and all of these factors function together in a
complicated, yet coordinated, manner to ensure the normal development of the
lung.
Previous studies on the development of embryonic SMG have already shown
that FGFs, BMPs and Shh are involved in SMG branching morphogenesis
(Jaskoll et al., 2004b;
Steinberg et al., 2005) (see
discussion below), but the roles of semaphorins in this process have not been
demonstrated. In this report, we find that Sema3A and Sema3C are required in
the developing SMG, but, surprisingly, the way they work seems quite different
from that reported in the developing lung bud: these two semaphorins are
secreted from the developing epithelial cells and function additively, instead
of antagonistically, to promote SMG cleft formation. Moreover, both Sema3A and
Sema3C signals are mediated by Npn1. Only Npn1, but not Npn2, is detected in
the developing SMG epithelial cells, and we have demonstrated that the cleft
formation activity promoted by either semaphorin requires the presence of
functional Npn1. Our results reveal an unexpected functional interaction
between semaphorins and their receptors. The cooperative behavior of Sema3A
and Sema3C through the same binding receptor, Npn1, in SMG development may be
mechanistically different from the reported antagonistic effects of Sema3C and
Sema3A observed in the developing lung bud
(Kagoshima and Ito, 2001) or
in the repulsion of the dorsal root ganglion axons
(Takahashi et al., 1998).
The plexin receptors utilized in the developing SMG to mediate the
semaphorin signals are also unique. In the projections of peripheral sensory
neurons, plexin A3 and plexin A4 are required for Sema3A function
(Cheng et al., 2001;
Yaron et al., 2005). In the
embryonic vasculogenesis, plexin D1 directly binds Sema3E to mediate its
signal (Gu et al., 2005). But
in the development of the heart, plexin D1 is reported to form co-receptor
with Npn1 to mediate Sema3C function
(Gitler et al., 2004;
Torres-Vazquez et al., 2004).
In the developing SMG, however, we show that plexin D1 and plexin A2 are the
main plexin receptors that form co-receptors with Npn1 to mediate Sema3A and
Sema3C signals. Sema3E does not affect the plexin D1-mediated SMG branching,
indicating that direct functional binding between plexin D1 and Sema3E may not
occur during SMG development. The SMG explants from plexin A3-null or plexin
A4-null mutant mice grow normally, indicating that these two plexins are
dispensable. Taken all together, these results suggest that molecular
interactions within the same ligand-receptor families can be distinct,
depending on the cellular contexts and developmental stages. We still do not
know whether plexin D1 and plexin A2 function independently of each other as
separate Npn1-co-receptor complexes or whether they aggregate together with
Npn1 as huge protein complexes for downstream signaling. Given the abundance
and diversity of the semaphorin ligand-receptor families, one can expect that
many other specific interactions will be identified in different tissues or
developmental processes.
We have demonstrated in this study that Sema3A or Sema3C collapses and
constricts dissociated embryonic SMG epithelial cells, indicating that these
semaphorins act as autocrine inhibitory cues. Recently, an elegant study on
the branching morphogenesis of the mammary gland epithelia shows that the
branching of the epithelial buds is regulated by local concentration of
autocrine inhibitory factors (Nelson et
al., 2006). Interestingly, during the development of embryonic
SMG, the highest expression of Sema3A transcript is found at the
front tip of the epithelial buds. This expression pattern is consistent with
its role as a local regulator for cleft formation, as primitive indentations
for subsequent cleft formation are often initiated at the tip of the
epithelial bud. It is thus quite plausible that, in the developing SMG, the
autocrine semaphorins exert a pushing force locally to separate neighboring
epithelial cells through collapsing the cells that express the receptors, and
consequently promote cleft formation.
Multiple signaling pathways control SMG branching morphogenesis
Branching morphogenesis requires coordinated interactions between
epithelial and mesenchymal cells to promote cell proliferation,
differentiation and migration. Cleft formation is a very distinct event during
the early phase of branching morphogenesis. Essentially all the developing
epithelial buds repeatedly undergo cleft formation until the final tree-like
or bush-like structure is formed. Several morphogens, including FGFs, Hgf,
BMPs and Shh, are required for SMG branching morphogenesis. Previous studies
suggest that, during embryonic development, these factors are mainly derived
from the neighboring mesenchyme. They may stimulate cell proliferation and/or
regulate cleft formation (Steinberg et
al., 2005). Here we show that semaphorin signaling is also
required for SMG branching morphogenesis, but its action seems to be more
specific: semaphorins seem not to stimulate cell proliferation; they promote
cleft formation.
How semaphorins are coordinated with other factors to regulate SMG branch formation is still not clear, but our results suggest that semaphorins function in parallel with some FGFs without much crosstalk between their signaling pathways. Moreover, it has been reported that the SMG epithelial rudiments without mesenchyme developed normally in the presence of FGFs. In SMG co-culture assays, we observed that the autocrine function of semaphorins required additional signals from the mesenchyme. Therefore, it is likely that at the early stage of SMG morphogenesis, the surrounding mesenchymal cells secrete morphogens such as FGFs to stimulate the proliferation of the SMG epithelial cells. Later, when the growing epithelial bud is ready for cleft formation, the cells at the front end secrete semaphorins, perhaps with the existing FGFs, to promote the local cleft formation.
Cleft formation also requires conversion of cell-cell adhesive interactions
to cell-matrix interactions (Hosokawa et
al., 1999; Kadoya and
Yamashina, 2005). It has recently been shown that the stimulation
of integrin receptor on the epithelial cell by fibronectin deposited in the
matrix is essential for SMG cleft formation
(Sakai et al., 2003). It would
therefore be interesting to know whether semaphorin signaling interacts with
integrin signaling in the developing epithelial cells. Among all semaphorins,
Sema7A has been shown to directly bind to integrin subunit ß1
and promote neurite outgrowth (Pasterkamp
et al., 2003). However, as class 3 semaphorin proteins lack the
integrin-binding motif, RGD, it is unlikely that Sema3A (or Sema3C) can bind
integrin receptors. In addition, although semaphorin signaling has been
associated with integrin activities, the interactions seem to be diverse and
context-dependent. For example, Sema3C can increase integrin activity in
glomerular epithelial cells (Banu et al.,
2006), while semaphorin/plexin signaling can also disrupt
integrin-based adhesion, leading to inhibition of lamellipodia extension and
cell motility in NIH3T3 and COS cells
(Barberis at al., 2004). It has
also been shown that autocrine Sema3A can inhibit integrin function to control
morphogenesis of the vasculature (Serini
et al., 2003). Apparently, further experiments are required to
understand how semaphorin signals can contribute to the changes of cell
adhesion during SMG cleft formation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/16/2935/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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7). The number of terminal buds at the presence of Sema3A or
Sema3C was almost doubled. (B) Treatment of antisense ODNs against
Sema3A or Sema3C specifically reduced the number of terminal buds in the SMG
cultured ex vivo. Representative explants are shown. The bar graph summarizes
five independent experiments. The terminal bud number was significantly
further reduced when both Sema3A antisense ODNs and Sema3C antisense ODNs were
added together into the culture. Paired t-test: *,
P<0.05; **, P<0.01. (C) Sema3A and
Sema3C additively promoted bud formation in a concentration-dependent manner.
In the SMG co-culture experiments, Sema3-transfected COS cells were serially
diluted to test the synergistic effects. Fold dilution in the co-culture is
indicated as the Sema3-transfected COS cells diluted with mock-transfected COS
cells. (a) Additive effects of Sema3A and Sema3C were most obvious at
the lower concentrations of semaphorins. The effects were saturated at 1:1
dilution. (b) The amounts of semaphorin proteins present in each
condition were assayed by western blotting. Gradual decreases of Sema3A or
Sema3C in the serial dilutions were observed. Tubulin: internal loading
control. (c) The bar graph summarizes four independent experiments.
Scale bars: 100 µm. C, scrambled sequence.
Co-senior authors