Embryonic Submandibular Salivary Gland Development
Tina Jaskoll, Ph.D. and Michael Melnick, DDS, Ph.D.
Following a classic epithelial-mesenchymal interaction developmental program,
the mouse neonatal submandibular salivary gland (SMG) is comprised of large and
small ducts which terminate in lumen-containing, presumptive acini that express
embryonic mucin (Ball, 1974; Cutler and Gremski, 1991; Denny et al., 1997;
Gresik et al., 1998; Kashimata et al., 2000a, Redman, 1987; Wessells, 1977; Jaskoll
et al., 1998; Melnick and Jaskoll, 2000). Embryonic mouse SMG branching
morphogenesis is best conceptualized in stages: PreBud, Initial Bud,
Pseudoglandular, Canalicular, And Terminal Bud (Jaskoll and
Fig. 1). In the PreBud Stage, SMG development begins as a thickening of
the primitive oral cavity epithelium adjacent to the developing tongue. During
the Initial Bud Stage, this thickened epithelium grows down into the
first branchial (mandibular) arch mesenchyme to form the initial SMG bud. With
continued epithelial proliferation and downgrowth, the SMG primordium becomes a
solid, elongated epithelial stalk terminating in a bulb; this SMG primordium is
surrounded by condensed mesenchyme. The primordium branches by repeated
furcation at the distal ends of successive buds to produce a bush-like structure
comprised of a network of elongated epithelial branches and terminal epithelial
buds surrounded by loosely packed mesenchyme (the Pseudoglandular Stage).
These branches and buds hollow out by epithelial cell apoptosis during the Canalicular
and Terminal Bud Stages to form the ductal system and presumptive
acini (for details, see Jaskoll and Melnick, 1999;
Melnick and Jaskoll, 2000).
Epithelial cell proliferation is found in all stages, even after well-defined
lumen formation in the Terminal Bud Stage. Epithelial cell apoptosis
begins with the onset of lumen formation in the Canalicular Stage.
Moreover, our studies suggest that ductal canalization is primarily due to
caspase 8-mediated apoptosis whereas p53 primarily mediates terminal bud lumina
formation (Melnick and Jaskoll, 2000; Melnick et al.,
2001 a, b; Table 1).
In addition, although we found three pro-survival/anti-apoptotic proteins (NF-κB,
bcl2, and survivin) in lumen-bounding epithelial cells during luminization, the
presence of only nuclear-localized (activated) survivin in the layer of
surviving cells is seen after the completion of lumen formation (Jaskoll et
al., 2001). Based on our data, we conclude that apoptosis-mediated lumen
formation and cell survival are achieved through several different pathways.
Embryonic SMG cell proliferation, cell quiescence, apoptosis, and
histodifferentiation events are mediated by specific growth factors, cytokines,
and transcription factors which are expressed at specific times and locations
(Hardman and Spooner, 1994; Kashimata and Gresik, 1997; Kashimata et al., 2000a,
b, Jaskoll and Melnick, 1999; Melnick et al.,
2001a, b; Jaskoll et al., 2002).
Although it has conclusively been shown that SMG morphogenesis is dependent on
epithelial-mesenchymal interactions (see reviews, Wessells, 1977; Cutler and
Gremski, 1991), little was known about which molecules are involved in the
induction and regulation of early embryonic SMG branching. To begin to delineate
the key molecules and signaling pathways involved in the induction and
regulation of embryonic SMG morphogenesis, we have determined the stage-specific
distribution of multiple growth factors and cytokines (and their receptors) and
transcription factors, and correlated their expression pattern with cell
proliferation and apoptosis (Fig. 1; Jaskoll and
Melnick and Jaskoll, 2000; Jaskoll et al., 2002; Table 1). Functional
studies in our laboratory and others support this conclusion (Hardman and
Spooner, 1994; Kashimata and Gresik, 1997; Kashimata et al., 2000a, b, Jaskoll
and Melnick, 1999, Melnick and Jaskoll, 2000;
Melnick et al., 2001a, b,
Jaskoll et al., 2002, De Moerlooze et al., 2000; Ohuchi et al., 2000).
|Figure 1. Schematic representation of SMG development prior to and during embryonic development, as determined in our laboratory during the grant period 9/30/96-9/30/00.
The important cell-specific distribution patterns for multiple signal transduction molecules are shown in the boxes. * epithelial stalk localization;
factors or receptors only found in the center of epithelial terminal buds.
|Figure 2. Connections Map. This signaling map reflects the pathways investigated in our laboratory. Known and putative connections are based on work in ours and
The cellular and extracellular components of these key signaling pathways may
be visualized as a Connections Map (Fig. 2) which details the functional
relationships within and between pathways. The promotional and inhibitory,
synergistic and antagonistic, molecular interactions noted are supported by an
enormous experimental effort by numerous laboratories worldwide.
Analysis of Transgenic and Mutant Mouse SMG Phenotypes
The SMG phenotype in a wide variety of transgenic and mutant mice has
been analyzed (Table 2). Importantly, disruption of Fgfr2-IIIb and Fgfr2-IIIc
expression indicates that FGF-R2-IIIb and FGF-R2-IIIc signaling play key roles
during embryonic SMG development. The presence and subsequent death of
Fgfr2-IIIb-/- Initial Bud Stage SMG epithelial cells (Table 2;
DeMoerlooze et al., 2000) indicates that FGFR2-IIIb signal transduction is
essential for Pseudoglandular Stage development but not initial
bud formation. By contrast, our demonstration of hypoplastic SMGs in Fgfr2-IIIc
mice indicates that FGFR2-IIIc plays an essential role during branching
morphogenesis. Moreover, since sonic hedge hog (SHH) has been shown to be a
downstream target of FGFR2-IIIb-mediated signaling (Revest et al., 2001) and FGF
and SHH exhibit a feedback mechanism (Laufer et al., 1994), we evaluated the SMG
phenotype in Shh-/- mice in collaboration with Peter Carlsson
(Goteborg University, Sweden). Shh null SMGs are severely dysplastic.
Importantly, the observation of no SMGs in Fgfr2-IIIb null mice and
dysplastic SMGs in Shh null mice is consistent with the fact that
FGFR2-IIIb signals through multiple downstream pathways in addition to SHH.
Further, since the forkhead gene Foxf1 is a downstream target of SHH
signaling (Mahlapuu et al., 2001) and Foxf1+/- mutant mice
exhibit lung and foregut abnormalities, we have also investigated the SMG
phenotype in Foxf1 mutant mice in collaboration with Peter Carlsson.
[Note that the Foxf1 null mutation is embryolethal; thus we evaluated Foxf1+/-
SMGs]. Foxf1+/- SMGs are hypoplastic Given the above, we conclude
that both SHH/Foxf1 signaling play key roles during embryonic SMG development.
Moreover, our demonstration of dysplastic and hypoplastic SMGs in Shh-/-
and Foxf1+/- mice, respectively, is consistent with
fact that SHH signals through multiple downstream target genes, not only Foxf1 (Mahlapuu
et al., 2001; see review, Ingham and McMahon 2001).
Regarding other signaling pathways, our observations of an abnormal SMG
phenotype in Pax6 and Bmp7 null mice (Table 2) indicates that Pax6 and Bmp7are
essential for embryonic SMG branching morphogenesis, but not initiation. In
addition, given that one of the functions of TACE is the proteolytic activation
of TGF-α, the sole EGF-R ligand in pre-Canalicular
SMGs, our analyses of Tace and Egfr null mice provide solid
evidence that the EGF-R pathway is not essential for SMG initiation, but is
essential for normal post Canalicular Stage branching morphogenesis (Table 2).
By contrast, the presence of normal SMGs in Tgfβ2,
Tgfβ3, Msx2 and Hox-a5 null mice
indicates that TGF-β2, TGF-β3,
Msx-2, and Hox-a5 are not essential for SMG morphogenesis (Table 2). Finally,
since N-myc null mutations are embryo lethal (Stanton et al., 1992), we
evaluated N-myc's role in transgenic mice (N-myc9a/9a) which
underexpress the N-myc gene, exhibit marked lung abnormalities, and die at birth
(Moens et al., 1992). The similarity of SMGs in N-myc9a/9a
transgenic and wild-type mice indicates that N-myc is not a sine qua
non of embryonic SMG development.
Finally, given that: (1) mutations in ectodysplasin (EDA) or
its mouse homologue Eda give rise to the syndrome hypohidrotic ectodermal
dysplasia (HED), characterized by absence or hypoplasia of teeth, hair, and
sweat glands, and (2) a similar phenotype is seen in downless (Edardl)
mutant mice due to mutations in the ectodysplasin receptor (Edar) gene (Gruneberg,
1965, 1971; Kere et al., 1996; Srivastava et al., 1997; Monreal et al., 1998),
we postulated that Eda/Edar signal transduction may also play a key role during
SMG development. We evaluated the Eda-less Tabby (EdaTa)
and the Edar-less downless (Edardl)
mutant mouse strains and found abnormal SMG phenotypes in both mutant strains
(Table 2). EdaTa (Tabby) SMGs are hypoplastic, with a
marked decrease in intercalated ducts and acini, as well as a notable decline in
acinar-specific mucin expression. By contrast, Edardl (downless)
SMGs are dysplastic, characterized by an absence of ducts, acini, and mucin
protein. Significantly, our observation that EdaTa
(Tabby) and Edardl (downless) SMGs are abnormal indicates that Eda/Edar-mediated
signal transduction is essential to SMG development. Moreover, the presence of
SMGs in both Ta and dl mutant mice indicate that Eda/Edar signaling is important
for branching morphogenesis and histodifferentiation, but not initial gland
Functional Analysis of TNF, IL-6, NF-κB,
and TGF-β-RII Mediated Signaling
In our laboratory, we have investigated the morphoregulatory role during
embryonic SMG development for each of the following signaling pathways:
(1) TNF/TNF-R1 Signal Transduction. We utilized in vitro
experiments to investigate the phenotypic outcomes of enhanced and deficient TNF
ligand (Melnick et al., 2001a). TNF supplementation induced a significant
3-fold increase in cell proliferation and a highly significant 2-fold increase
in epithelial branching compared to controls. In addition, a 2/3 reduction in endogenous
TNF protein levels resulted in a highly significant 2/3 decline in SMG
epithelial cell proliferation and a significant 6-fold increase in apoptosis.
Our data indicates that the TNF/TNF-R1 signal transduction pathway plays an
important role in balancing cell proliferation and apoptosis during SMG duct and
presumptive acini formation.
(2) IL-6 Signal Transduction: We investigated the phenotypic outcomes of
enhanced and decreased IL-6 signaling in vitro (Melnick et al., 2001b)
and found: (1) IL-6 supplementation induces a substantial increase in overall
size and in number of ductal branches and terminal buds, as well as a highly
significant increase in epithelial cell proliferation and (2) SMG explants
cultured with anti-IL-6 neutralizing antibodies exhibited a marked decrease in
epithelial ducts and terminal buds, concomitant with a highly significant 60%
decline in cell proliferation and a highly significant 13-fold increase in
apoptosis. Our data indicates that IL-6 signaling is important to SMG
(3) NF-κB-Mediated Transcription and
Protein Expression: Since the TNF-R1/NF-κB
cascade is the primary pathway induced by TNF ligand/receptor binding, we then
investigated the role of NF-κB during
embryonic SMG morphogenesis (Melnick et al., 2001c). We interrupted NF-κB
activation using the cell permeable peptide SN50. SN50-inhibition of NF-κB
nuclear translocation in E15 SMG primordia cultured for 2 (E15+2) days results
in an highly significant 10-fold increase in apoptosis and 81% decline in cell
proliferation. The increase in apoptosis is associated with a highly significant
> 4-fold increase in activated p53.
Using transcriptome and proteome analyses, we identified those
transcripts and proteins in our cognate Connections Map that have altered
expression with NF-κB inhibition.
cDNA expression array analysis of 1176 genes revealed significant
changes in expression for 691 genes (-60%). Among
these, genes encoding other transcription factors and those genes associated
with signal transduction, cell cycle progression, cell adhesion, and apoptosis
exhibited 1.5 or greater-fold change in mRNA levels (Melnick et al., 2001c).
Unable to extrapolate from RNA abundance to relevant protein
levels, since this would ignore changes in translational efficiency or protein
stability, we analyzed NF-κB-inhibited
and control SMG explants using 2-D Western Multiprotein Arrays. This technique
allows for the analysis of - 600 signal
transduction and other proteins simultaneously in each independent sample.
Proteomic analyses demonstrated notable qualitative and quantitative differences
in protein expression between control and NF-κB
E15+2 explants (Melnick et al., 2001c). We detect 18 proteins with a 1.5
or greater-fold change in expression with NF-κB
inhibition and specifically related to the Connections Map. Importantly,
they include signal transduction, translation control (checkpoint), cell cycle,
and apoptosis proteins that are downstream from activation of the TNF, IL-6, EGF,
IGF, and FGF signaling pathways. PNN analyses of transcriptomic and proteomic
assays identified specific transcripts and proteins with altered expression that
best discriminate control from SN50-treated SMGs. These include PCNA, GR, BMP1,
BMP3b, Chk1, Caspase 6, E2F1, ERK1/2, and JNK, as well as several others of
lesser importance ( Melnick et al., 2001c).
Finally, we focused our attention on two particularly important pathways
relative to cell proliferation and apoptosis, ERK 1/2 and Caspase 3. Downstream of activated ERK
1/2 is an upregulation of cell proliferation proteins and potentially enhanced cell
division; downstream of activated Caspase 3 are the sequellae of apoptosis,
including PARP cleavage and inhibition of DNA repair, DNA fragmentation, and
nuclear membrane fragmentation. To determine if these key pathways were
activated in the absence of NF-κB
nuclear translocation, we quantitated the level of activated (phosphorylated/cleaved
) ERK 1, ERK 2, c-Raf, Caspase 3, and PARP (Melnick et al., 2001c); all
proteins except c-Raf are significantly (p<0.05) increased in SN50-treated
explants. Paradoxically, increased ERK 1/2 activation is associated with virtually no change in the antecedent activation
of Raf. Although we find an increase in total Raf protein, no increase in
activated c-Raf is found, indicating that increased total protein level is not always
indicative of increased protein activation.
Taken together, our results
indicate that NF-κB-mediated
transcription is critical to embryonic developmental homeostasis. Inhibition of
NF-κB nuclear translocation alters the
expression of a genetic network of broadly related, rather than independent,
components. Our results also demonstrate how informative it can be regarding
organogenesis to simultaneously evaluate the gene and protein expression of
multiple genes from multiple pathways within broad functional categories.
mediated signal transduction: Although our studies of null mice
indicate that neither TGF-β2 nor
TGF-β3 expression is essential
for embryonic SMG development (Jaskoll et al.,
1999), we could not
exclude the possibility that one TGF-β
isoform compensates for another in these null mice. To address this
question, we cultured embryonic SMG primordia in the presence of antisense
oligodeoxynucleotides (ODNs) designed against TGF-β-RII,
the cognate receptor for all TGF-β
isoforms (Fig. 3). Anti-TGF-β-RII
ODNs induced an - 40% increase in branching
compared to sense or nonsense ODNs (compare Fig. 3B to 3A.).To confirm
that TGF-β-RII signaling was
interrupted in this set of experiments, we cultured embryonic SMG
primordia in the presence of TGF-β2
supplementation or TGF-β2 +
antisense ODN supplementation (Fig. 2C, D). TGF-β2
supplementation induced an -50% reduction in
branch number (Fig.3C). By contrast, TGF-β2
supplementation failed to inhibit branching morphogenesis in the presence
of TGF-β-RII antisense ODNs (Fig.
3D). This result indicates that anti-TGF-β-RII
ODNs interrupted TGFβ/TGF-β-RII
|Figure 3. Antisence interruption of
TGF-β/ TGF-β-RII signal transduction results in a marked increase in branching. A marked increase in branching is seen between antisense (B) and nonsense (A)
ODN-treated E13 + 3 explants. TGF-β2 supplementation (0.5
ng/ml, R & D) of E13+3 SMG (C) decreases SMG branching. However, no decrease in branching is seen in the presence of
TGF-β2 + AS ODN (D), indicating that
TGF-β/TGF-β-RII signaling is interrupted.
Cloning, Sequencing and Functional Analysis of Embryonic SMG Mucin
Prior to investigating which factors regulate mucin expression during
embryonic SMG development, we determined embryonic SMG mucin mRNA and protein
expression (Jaskoll et al., 1998, Melnick et al., 2000d). Mucin
transcripts are localized throughout branching epithelia in Pseudoglandular
Stage and older SMGs, with increased hybridization signal being seen in terminal
bud and proacinar epithelial cells. By contrast, SMG mucin protein is not
immunodetected until Terminal Bud Stage, being primarily immunolocalized
to terminal bud and proacinar epithelial cell membranes. Our observation of
mucin transcripts throughout early embryonic SMG epithelia compared to mucin
proteinís restricted distribution in terminal bud and proacinar epithelia
suggests that mucin transcription and/or translation is likely downregulated in
nonterminal bud (ductal) epithelia.
Importantly, Northern and Western blot analyses demonstrated notable
differences between embryonic and adult SMG mucin mRNA and protein (Jaskoll
et al., 1998). E17 SMGs exhibited two unique mucin transcripts (0.85 and
1.20 kb) which are approximately 19% greater or smaller in size than the single
(1.01 kb) adult transcript; substantial protein differences were also seen. To
characterize embryonic SMG mucin, we cloned and sequenced the embryonic mouse
low molecular weight mucin (GenBank accession # AF247816; Melnick et
al., 2001d). The cDNA encodes a 885 bp sequence and a 220 aa protein (~
25 kDa), rich in potential O-glycosylation sites, and variably glycosylated.
Significantly, embryonic low molecular weight mucin is an alternately spliced
variant of adult Muc10. Consensus secondary structure prediction for
embryonic low molecular weight mucin is consistent with a molecule that is
anchored to the plasma membrane, directly or indirectly, and has a protein core
that serves as a scaffold for carbohydrate presentation. Given that saliva is
nonfunctional in utero and is only utilized after birth with the onset of
feeding, we also determined the timing of the transition from the embryonic to
adult form. Both embryonic and adult mucin protein variants are immunodetected
in newborn and 21 day SMGs, with a marked increase in adult forms being seen
between the newborn and 21-day old gland. The predominance of adult mucin
proteins is correlated with the appearance of acinar cell histodifferentiation
which occurs 2-3 weeks after birth (Srinivasan and Chang, 1979).
The specificity and expression pattern of embryonic mucin suggested that it
may play a morphogenetic role. Since lectins and other glycoproteins play
important roles in cell adhesion during organogenesis (Mann and Waterman, 1998),
and selectins bind mucins or mucin-like proteins (Lasky et al., 1992; Prakobphol
et al., 1998, 1999), we postulated that one possible function of the embryonic
low molecular weight mucin is to bind L-selectin. Immunoprecipitation and
Western blot analyses demonstrate that embryonic, but not adult, mucin is able
to bind L-selectin and does so endogenously (Melnick et al., 2001d).
Since the primary role of L-selectin is to mediate weak cell adhesion prior to
strong adhesion mediated by integrins (Kaltner and Stierstoffer, 1998; Shimizu
and Shaw, 1993) and its ligands are mucin-like glycoproteins, we suggest that
this low molecular weight embryonic mucin is a MucCam (mucin cell
Regulation of Embryonic Mucin Expression
Studies in our laboratory has demonstrated that exogenous glucocorticoids (CORT)
treatment in utero induces a significant increase in mucin mRNA and
protein expression (Melnick and Jaskoll, 2000). In addition, our
in vitro experiments also indicates that CORT is required for normal
embryonic mucin protein expression (Melnick and
Jaskoll, 2000).Given that
mucin transcripts are distributed throughout all epithelia in the Pseudoglandular
and Canalicular Stages whereas mucin protein is expressed only in Terminal
Bud Stage epithelia (Jaskoll et al., 1998), our results suggest
that CORT downregulates mucin transcription and/or translation in ductal
epithelia during SMG development. In addition, we also find that IL-6, but not
TNF, signaling modulates embryonic mucin expression in vitro (Melnick et al.,
2001a, b). As part of our ongoing research projects, we continue to
investigate which growth factor-mediated signaling pathways modulate embryonic
SMG mucin expression.
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