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DEVELOPMENTAL DYNAMICS 212:482–494 (1998)
Mesenchyme Specifies Epithelial Differentiation in
Reciprocal Recombinants of Embryonic Lung and Trachea
JOHN M. SHANNON,1,2* LARRY D. NIELSEN,1 SARAH A. GEBB,1 AND SCOTT H. RANDELL3
of Medicine, National Jewish Medical and Research Center, Denver, Colorado
2Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado
3Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina School of Medicine,
Chapel Hill, North Carolina
1Department
ABSTRACT
Normal lung morphogenesis and
cytodifferentiation require interactions between
epithelium and mesenchyme. We have previously
shown that distal lung mesenchyme (LgM) is
capable of reprogramming tracheal epithelium
(TrE) from day 13–14 rat fetuses to branch in a
lung-like pattern and express a distal lung epithelial phenotype. In the present study, we have
assessed the effects of tracheal mesenchyme (TrM)
on branching and cytodifferentiation of distal
lung epithelium (LgE). Tracheae and distal lung
tips from day 13 rat fetuses were separated into
purified epithelial and mesenchymal components, then recombined as homotypic (LgM 1 LgE
or TrM 1 TrE) or heterotypic (LgM 1 TrE or
TrM 1 LgE) recombinants and cultured for 5 days;
unseparated lung tips and tracheae served as
controls. Control lung tips, LgM 1 LgE, and LgM
1 TrE recombinants all branched in an identical
pattern. Epithelial cells, including those from the
induced TrE, contained abundant glycogen deposits and lamellar bodies, and expressed surfactant
protein C (SP-C) mRNA. Trachea controls, and
both TrM 1 TrE, and TrM 1 LgE recombinants
did not branch, but instead formed cysts. The
epithelium contained ciliated and mucous secretory cells; importantly, no cells containing lamellar bodies were observed, nor was SP-C mRNA
detected. Mucin immunostaining showed copious production of mucous in both LgE and TrE
when recombined with TrM. These results demonstrate that epithelial differentiation in the recombinants appears to be wholly dependent on the
type of mesenchyme used, and that the entire respiratory epithelium has significant plasticity in eventual phenotype at this stage in development. Dev.
Dyn. 1998;212:482–494. r 1998 Wiley-Liss, Inc.
Key words: lung; alveolar epithelium; trachea;
epithelial-mesenchymal interactions;
surfactant proteins
INTRODUCTION
Development of the rodent lung begins at midgestation as paired evaginations of the foregut endoderm
into mesenchyme derived from the splanchnic mesor 1998 WILEY-LISS, INC.
derm. Repetitive terminal and lateral branching of the
epithelium gives rise to the pulmonary tree, a process
commonly referred to as branching morphogenesis.
Concomitant with patterning in the developing lung is
the differentiation of several epithelial cell types, e.g.,
alveolar type II cells, nonciliated bronchiolar (Clara)
cells, ciliated cells, goblet cells, mucous cells, that will
play specific functional roles postnatally. Differentiation of these epithelial cell types within the lung occurs
in a spatially precise manner, such that proximal and
distal cell types arise in areas significant to their
postnatal function.
Interactions between epithelium and mesenchyme
have been shown to be critical for the morphogenesis of
many different organs (Grobstein, 1967), including the
lung. Studies in both avian (Dameron, 1961) and mammalian (Masters, 1976; Sampaolo and Sampaolo, 1959;
Spooner and Wessells, 1970; Taderera, 1967) species
have demonstrated that an interaction of the presumptive lung epithelium with lung mesenchyme is absolutely required for normal branching morphogenesis to
proceed. These epithelial-mesenchymal interactions
driving lung morphogenesis and epithelial differentiation most likely involve a complex interplay of hormones, growth factors, and extracellular matrix molecules (for reviews see Cardoso, 1995; Minoo and King,
1994; Shannon and Deterding, 1997). The inductive
potency of lung mesenchyme is most strikingly demonstrated by its ability to induce early tracheal epithelium that has been denuded of its own mesenchyme to
branch in a lung-like pattern (Alescio and Cassini,
1962). We extended these observations by showing that
lung branching morphogenesis induced in tracheal
epithelium by lung mesenchyme is accompanied by a
reprogramming of the tracheal epithelial cells to express an alveolar type II cell phenotype (Shannon,
1994), including the expression of the type II cellspecific marker surfactant protein C (SP-C).
The specific requirement for distal lung mesenchyme
to support branching morphogenesis of the pseudoglan-
Grant sponsor: National Heart, Lung, and Blood Institute of the
NIH; Grant numbers: HL45011 and HL58345.
*Correspondence to: John M. Shannon, Ph.D., Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson
Street, Denver, CO 80206. E-mail: shannonj@njc.org
Received 26 February 1998; Accepted 3 April 1998
MESENCHYMAL SPECIFICATION OF RESPIRATORY EPITHELIAL DIFFERENTIATION
dular stage lung epithelium is further emphasized in
experiments done by Wessells (1970), who observed
that grafting tracheal mesenchyme onto distal lung
epithelium that had been denuded of its own mesenchyme inhibited any further branching. Since these
experiments only examined the effects of tracheal mesenchyme on lung epithelial branching, the differentiated phenotype of the epithelial cells was unknown. It
is possible that although the epithelium adopts a
morphogenetic pattern specified by tracheal mesenchyme, lung epithelial cytodifferentiation persists. Such
a separation of tissue patterning and epithelial differentiation has been observed in recombinants of salivary
gland mesenchyme and mammary gland epithelium
(Sakakura et al., 1976). A second possibility is that lung
epithelium associated with tracheal mesenchyme does
not receive the appropriate signals to sustain lung
differentiation, and therefore dedifferentiates. A third
possibility is that tracheal mesenchyme produces signals specifying tracheal patterning and differentiation,
and reprograms lung epithelium to express a tracheal
epithelial phenotype. In the present study, we have
constructed reciprocal tissue recombinants between
day 13 fetal rat lung and trachea and evaluated them
for markers of lung and trachea epithelial differentiation to distinguish among these possibilities.
RESULTS
Culture of intact distal lung tips or bisected tracheal
rudiments for 5 days gave results essentially identical
to those previously reported (Shannon, 1994). The cut
edge of the distal lung tips healed within 24 hours, such
that the epithelium formed a dilated cyst within the
mesenchyme (Fig. 1B). Incipient branching was seen as
early as 48 hours after explanting, was clearly established by 72 hours (Fig. 1C), and continued until
harvest at 120 hours (Fig. 1D). In contrast, tracheal
explants did not branch. After the cut ends of the
epithelium had healed (Fig. 1F), the epithelium enlarged within the mesenchyme by 72 hours (Fig. 1G),
but did not branch by 120 hours (Fig. 1H), although the
epithelium appeared dilated.
Epithelial patterning within the tissue recombinants
was dictated by the type of mesenchyme that was used.
This effect was so pronounced that lung and tracheal
epithelium appeared grossly identical as soon as 24
hours after recombination with the same type of mesenchyme. When recombined with lung mesenchyme, both
lung and tracheal epithelium formed a dilated cyst by
24 hours (Fig. 1J and N). Lung epithelium recombined
with lung mesenchyme showed some branching by 48
hours, which was even more apparent by 72 hours (Fig.
1K). Recombination of lung mesenchyme with tracheal
epithelium also induced branching; as in the homotypic
lung recombinants, this was readily apparent by 72
hours (Fig. 1O). Branching of both lung and tracheal
epithelium in these recombinants initially appeared to
lag behind intact lung tip controls, but appeared identical by day 5 of culture (Fig. 1D, L, and P).
483
When recombined with tracheal mesenchyme, both
tracheal and lung epithelium initially formed a tight
mass of cells (Fig. 1R and V). These epithelia appeared
essentially unchanged at 72 hours (Fig. 1S and W). On
day 5 of culture, both the tracheal and lung epithelium
appeared dilated, similar to what was seen in intact
tracheal explants, but did not branch (Fig. 1T and X).
Thus, branching of the epithelium from the distal lung
tip, where active branching morphogenesis occurs, was
either suppressed or uninduced when associated with
tracheal mesenchyme.
Sections of intact lung tips, homotypic lung mesenchyme (LgM) 1 lung epithelium (LgE) recombinants,
and heterotypic LgM 1 tracheal epithelium (TrE) recombinants cultured for 5 days appeared similar by light
microscopy. Branching of both lung and tracheal epithelium in response to distal lung mesenchyme resulted in
a large lumen in the center of the explants and acinar
structures in the explant periphery (Figs. 2A–C, 9).
Cells constituting the central (proximal) area appeared
uniformly columnar, while those in the peripheral
(distal) region were cuboidal to cuboidal-columnar (Figs.
2A–C). In explants of intact lung tips, as well as both
types of recombinants, the cells exhibited significant
accumulations of glycogen, which in vivo serves as a
substrate for surfactant phospholipid biosynthesis in
the maturing type II cell during late gestation. Glycogen deposition was enhanced by addition of dexamethasone to the medium. The identity of these areas as
glycogen was confirmed by the abolition of periodic
acid-Schiff (PAS) staining by pretreating the sections
with diastase (data not shown). Darkly stained inclusions were observed in many of the epithelial cells as
well as the lumina (Figs. 2A–C, arrows) of the most
distal acinar structures.
Ultrastructural examination of cultured lung tips
and both homotypic and heterotypic LgM-containing
recombinants revealed that the epithelial cells in the
distal acinar structures exhibited an ultrastructure
typical of late gestation alveolar type II cells (Fig.
3A–C). The cells contained many osmiophilic lamellar
bodies, which are the storage organelles of pulmonary
surfactant. Lamellar bodies were also present in a
number of the lumina, demonstrating that the type II
cells not only synthesized pulmonary surfactant, but
also secreted it. These ultrastructural observations are
consistent with the reprogramming of tracheal epithelium to express a distal lung epithelial phenotype.
In contrast to tissues containing LgM, explants and
recombinants containing tracheal mesenchyme (TrM)
showed a strikingly different morphology. When viewed
by light microscopy, sections of intact trachea cultured
for 5 days revealed a multilayered epithelium of primarily columnar cells lining a large central lumen (Fig. 4A).
The basal portion of the epithelium sat on a dense
stroma that contained multiple layers of mesenchymal
cells. Many of the epithelial cells contained glycogen,
and ciliated cells were readily observed (Fig. 4A, arrows). Homotypic TrM 1 TrE recombinants appeared
484
SHANNON ET AL.
Fig. 1. Time course study of reciprocal recombinants made from
isolated day 13 fetal distal lung mesenchyme (LgM), distal lung epithelium
(LgE), tracheal mesenchyme (TrM), and tracheal epithelium (TrE). Intact
distal lung tips (Lung) and tracheae served as controls. Arrows (I, M, Q, U)
indicate epithelial rudiments surrounded by mesenchyme at the time of
recombinations. All panels are shown at the same magnification (original
magnification 5 563). This figure demonstrates that epithelial morphoge-
netic pattern is specified by the type of mesenchyme with which it is
associated. Epithelia recombined with LgM (I–L and M–P) show a pattern
of branching identical to that seen in cultured intact distal lung tips (A–D).
In contrast, TrM does not support branching when recombined with either
tracheal (Q–T) or lung (U–X) epithelium, instead inducing the formation of
an epithelial cyst identical to that seen in cultured intact trachea (E–H).
MESENCHYMAL SPECIFICATION OF RESPIRATORY EPITHELIAL DIFFERENTIATION
essentially identical to intact tracheal explants (Fig.
4B). Heterotypic TrM 1 LgE recombinants also appeared similar to intact tracheal explant controls, with
the exception that a greater number of epithelial cells
in these recombinants appeared to contain significant
485
glycogen deposits (Fig. 4C). Concentrations of cartilage
precursor cells were observed in the mesenchyme of
intact trachea and both homotypic and heterotypic
TrM-containing recombinants by the end of the culture
period (data not shown). No cartilage precursors, however, were observed in LgM 1 TrE recombinants. This
suggests that LgE produces a factor(s) that reciprocally
supports cartilage formation in TrM, and that this
factor acts permissively.
The ultrastructure of both homotypic and heterotypic
recombinants made with TrM appeared essentially
identical to that seen in cultured intact tracheal explants (Fig. 5A–C). A portion of the epithelial cell
population consisted of columnar cells containing glycogen; these cells exhibited no apparent differentiated
phenotype. A significant percentage of the epithelial
cells, however, had differentiated into the readily identifiable ciliated and mucous secretory cells. The ultrastructural evidence supported the conclusion that the
LgE had been reprogrammed to express a tracheal
epithelial cell phenotype. This interpretation was further supported by the fact that we never observed
osmiophilic lamellar bodies or tubular myelin in TrM 1
LgE recombinants, even when they were cultured in
the presence of dexamethasone.
The differentiated status of the epithelial cells in the
cultured explants and recombinants was evaluated by
their ability to express mRNAs for the surfactant
proteins. Poly A1 RNA was isolated from approximately equal amounts of tissue, reverse-transcribed,
and amplified using intron-spanning primers for SP-A,
SP-B, SP-C and b-actin. Because of the small amount of
starting tissue, no attempt was made to quantitate the
amount of mRNA in each sample. The results are
shown in Figure 6. Explants of lung tips showed strong
signals for all three surfactant proteins (Fig. 6, lane 4),
as did both homotypic (Fig. 6, lane 5) and heterotypic
(Fig. 6, lane 6) recombinations made using LgM. Notable in these results is the induction of SP-C mRNA in
TrE by LgM, confirming our earlier observation that
LgM grafted onto a stretch of trachea denuded of its
own mesenchyme resulted in expression of both SP-C
mRNA and immunodetectable SP-C pro-protein (Shannon, 1994, 1996). Intact tracheal explants (Fig. 6, lane
3), as well as heterotypic recombinants (Fig. 6, lane 8)
made with TrM expressed mRNAs for SP-A and SP-B.
Interestingly, the level of SP-B expression in TrM 1 LgE
Fig. 2. Representative light microscopy of intact day 13 lung tips (A),
LgM 1 LgE recombinants (B) and LgM 1 TrE recombinants (C) that have
been cultured for 5 days. Dexamethasone (1027M) was present for the
final 24 hrs in this experiment. The epithelium has undergone significant
branching and acini have formed under these conditions. The areas
shown here are cross-sections through the most distal regions of all three
tissue types. Note that the induced epithelia exhibit a similar morphology.
The constituent cuboidal epithelial cells contain many darkly-stained
inclusions and large amounts of pale-staining material, which was shown
by histochemistry to be glycogen. Acinar lumina also contain material
(arrows) that was shown by ultrastructural examination to be secreted
lamellar bodies. All panels are at the same magnification (original
magnification 5 5253).
486
SHANNON ET AL.
Fig. 3. Electron microscopy of epithelial cells from the distal regions of
intact lung tips (A), LgM 1 LgE recombinants (B), and LgM 1 TrE
recombinants (C) that have been cultured as in Figure 2. Note the
similarity in morphology of epithelial cells from all three tissues. Extensive
areas of cytoplasmic glycogen are seen in most cells, as are numerous
osmiophilic lamellar bodies. TrE recombined with LgM has assumed a
morphology indistinguishable from that seen in late-gestation distal lung
epithelium. Original magnifications: A 5 59503; B 5 61603; C 5 67203.
recombinants appeared greater than that seen in
either intact tracheal explants or TrM 1 TrE recombinants, but below that seen in explants or recombinants
containing LgM. Expression of SP-C was not detected
Fig. 4. Representative light microscopy of intact day 13 tracheae (A),
TrM 1 TrE recombinants (B), and TrM 1 LgE recombinants (C) that have
been cultured as in Figure 2. The epithelium appears similar in all three
tissues. Cells containing glycogen are seen, as well as cells with apical
cilia (arrows). Note the lack of inclusions in LgE when recombined with
TrM. All panels are at the same magnification (original magnification 5 5253).
in either intact explants (Fig. 6, lane 3) or homotypic
(Fig. 6, lane 7) or heterotypic (Fig. 6, lane 8) recombinants that contained TrM, even though distal tip lung
MESENCHYMAL SPECIFICATION OF RESPIRATORY EPITHELIAL DIFFERENTIATION
Fig. 5. Electron microscopy of intact day 13 trachea (A), TrM 1 TrE
recombinants (B), and TrM 1 LgE recombinants (C) that have been
cultured as in Figure 2. The appearance of the constituent epithelial cells
in all three types of tissues is essentially identical. Ciliated cells and
mucous-containing cells are readily observed. Lamellar bodies are
notably absent in TrM 1 LgE recombinants. Original magnifications: A 5
70003; B 5 67203; C 5 64403.
epithelium is positive for SP-C mRNA at the time of
isolation (Wert et al., 1993). Although we did not
perform quantitative polymerase chain reaction (PCR),
amplification with intron-spanning b-actin primers
showed that similar amounts of mRNA were present in
487
Fig. 6. Reverse transcription polymerase chain reaction (RT-PCR)
identification of surfactant protein mRNA expression in reciprocal recombinants, intact tracheae, and intact lung tips cultured for 5 days. Uncultured day 13 tracheae and lung tips served as controls. Poly A1 RNA was
isolated from approximately equal amounts of tissue, treated with RNasefree DNase, reverse-transcribed using an oligo(dT) primer, and subjected
to PCR using specific primers for rat surfactant proteins (SP-) A, B, and C.
PCR products were electrophoresed on an agarose gel, transferred to
nylon membranes by Southern blotting, then probed with 32P-labeled
full-length cDNAs for SP-A, SP-B, and SP-C. Lanes: 1, uncultured day 13
tracheae; 2, uncultured day 13 lung tips; 3, intact tracheae cultured for 5
days; 4, intact lung tips cultured for 5 days; 5, LgM 1 LgE recombinants
cultured for 5 days; 6, LgM 1 TrE recombinants cultured for 5 days; 7, TrM
1 TrE recombinants cultured for 5 days; 8, TrM 1 LgE recombinants
cultured for 5 days. SP-A mRNA is present in all samples, although the
level of expression is highest in cultured tissues that contain LgM. SP-B
mRNA is not detected in day 13 trachea (lane 1) or cultured TrM 1 TrE
recombinants (lane 7), and is faintly detectable in cultured intact trachea
(lane 3). TrM 1 LgE recombinants (lane 8) express SP-B mRNA, although
not at the level seen in cultures of either intact lung tips, LgM 1 LgE
recombinants, or LgM 1 TrE recombinants (lanes 4–6). SP-C mRNA is
not detectable in any tissues that contain TrM, notably TrM 1 LgE
recombinants (lane 8), where SP-C expression has been extinguished.
Day 13 lung tips (lane 2) are positive for SP-C mRNA expression, which
increases dramatically after 5 days culture (lane 4). Both LgM 1 LgE and
LgM 1 TrE recombinants are strongly positive for SP-C mRNA expression, indicating induction of a gene specific to the distal lung in tracheal
epithelium. Amplification using intron-spanning b-actin primers demonstrates that there is no genomic DNA contamination of the samples.
each sample, and that it was not contaminated with
genomic DNA.
We next examined the spatial expression of respiratory epithelial markers, using SP-C as a marker of
distal lung epithelial differentiation (Glasser et al.,
1991; Wert et al., 1993) and CC10, the 10-kDa Clara cell
488
SHANNON ET AL.
secretory protein, as a marker of the proximal lung
epithelium. Expression of SP-C mRNA in intact lung
tips cultured for 5 days was confined to the most distal
aspects of the explants (Fig. 7A, D). Although some
small proximal epithelial tubules expressed SP-C transcripts, the most proximal epithelial cells constituting
the large central lumen of the explants were invariably
SP-C-negative. In contrast, these proximal epithelial
cells exhibited strong CC10 mRNA expression (Fig. 7G,
J). Cells positive for CC10 mRNA extended into the
distal epithelium, which appeared more weakly positive than the proximal epithelium. Examination of
consecutive sections, however, clearly showed that some
cells in the distal epithelium expressed both SP-C and
CC10 (Fig. 7D, J, arrows). The spatial patterns of
expression of SP-C and CC10 mRNAs in both homotypic (Fig. 7B, E, H, K) and heterotypic (Fig. 7C, F, I, L)
recombinants made with LgM were identical to those
seen in cultured intact lung tips.
Confirming the results obtained with reverse transcriptase (RT)-PCR, in situ hybridization demonstrated
that tissues containing TrM did not express SP-C
mRNA, even after prolonged exposure of the autoradiograms. This was true for cultured intact tracheal
rudiments (Fig. 8A, D), as well as homotypic (Fig. 8B,
E) and heterotypic (Fig. 8C, F) recombinants constructed with TrM. Thus, SP-C mRNA expression was
extinguished when LgE was associated with TrM.
Expression of CC10 mRNA, however, was strong in
virtually all of the epithelial cells in cultured intact
tracheal explants (Fig. 8G, J), and in both homotypic
(Fig. 8H, K) and heterotypic (Fig. 8I, L) recombinants.
Differentiation to a tracheal phenotype was further
evaluated by immunostaining with monoclonal antibody RTE 11, which reacts with all rat tracheobronchial
mucins. As can be seen in Figure 9, intact tracheal
explants, as well as both homotypic and heterotypic
recombinants made with TrM, showed strong immunoreactivity in the central lumen, indicating mucin synthesis and secretion. RTE 11 immunostaining in homotypic and heterotypic recombinants made with LgM
was identical to that seen in cultured intact distal lung
tips: some immunoreactivity was present in the proximal lumina of the explants, but the most distal epithelial cells and lumina were negative.
DISCUSSION
The data presented here demonstrate that mesenchyme not only dictates the pattern of respiratory
epithelial morphogenesis, but also plays a preeminent
role in specifying the eventual phenotype of the epithelium. The entire day 13 respiratory epithelium, from
the trachea to the distal lung tips, has the potential to
express either a proximal or distal epithelial phenotype, depending on the type of mesenchyme with which
it is associated. Thus, both the tracheal and distal lung
epithelial phenotypes appear to require a specific inducing factor(s) provided by tracheal and distal lung
mesenchyme, respectively. This is further emphasized
by our observations that purified day 13 lung (Deterd-
ing and Shannon, 1995) and day 13 tracheal (Shannon,
1996) epithelium cease proliferation and differentiation
when cultured in the absence of mesenchyme.
We and others (Alescio and Cassini, 1962; Wessells,
1970) have demonstrated that mesenchyme isolated
from the distal tips of glandular stage lung can induce
tracheal epithelium of the same age to branch in a
lung-like pattern. The present and a previous (Shannon, 1994) study has extended these observations to
demonstrate that these changes in branching morphogenesis are accompanied by changes in epithelial differentiation. Under the influence of distal lung mesenchyme, tracheal epithelium was reprogrammed to
express both morphological and biochemical markers of
alveolar type II cell differentiation. Notable among
these are the presence of cytoplasmic and luminal
osmiophilic lamellar bodies, which are the storage
organelles for pulmonary surfactant, and the expression SP-C mRNA. Furthermore, SP-C expression was
restricted to the distal epithelium, as occurs in vivo,
indicating that parameters dictating spatial organization in the developing lung were conserved in the
recombinants. Because the tracheal epithelium has
been induced to express a new phenotype, we consider
this to be an instructive interaction. The potential for
tracheal epithelium to be reprogrammed by lung mesenchyme does not persist indefinitely, however, since we
have observed that day 16 tracheal epithelium was not
competent to respond to the inductive influence of day
13 lung mesenchyme (Shannon, 1994). The mechanism
by which this restriction occurs is unknown, although
our present results would suggest it occurs via signals
from the mesenchyme.
Our present results show for the first time that
tracheal mesenchyme is able to instructively reprogram distal lung epithelium to express a tracheal
epithelial cytodifferentiation. Wessells (1970) observed
that tracheal mesenchyme grafted onto the distal tip
epithelium of glandular stage mouse lungs from which
lung mesenchyme had been removed caused cessation
of epithelial branching. These results suggested that
branching was not a property intrinsic to the lung
epithelium, but instead required specific interaction
with lung mesenchyme. Our data confirm these observations on the effect of tracheal mesenchyme on branching morphogenesis, and demonstrate further that the
distal lung epithelial cells have undergone a change in
differentiated phenotype, as indicated by the morphological differentiation of ciliated cells and mucous secretory cells, which are not seen in the distal lung epithelium. Our observations indicate that LgM and TrM are
fundamentally different in their ability to induce epithelial morphogenesis and differentiation, even though
both share the same general spatial origin within the
embryo.
Unequivocal characterization of the tracheal epithelial phenotype is hindered by a lack of definitive
markers of differentiation among the different epithelial cell types. TrM 1 LgE recombinants did, however,
produce immunodetectable tracheobronchial mucins.
MESENCHYMAL SPECIFICATION OF RESPIRATORY EPITHELIAL DIFFERENTIATION
489
Fig. 7. In situ hybridization of cultured intact lung tips, LgM 1 LgE
recombinants, and LgM 1 TrE recombinants. Consecutive sections were
hybridized with 33P-labeled antisense probes for SP-C (A–F) and the 10
kDa Clara cell secretory protein, CC10 (G–L). Exposure times of the
autoradiographs are identical for each probe. Darkfield images were
photographed and printed identically. Cultured intact lung tips (A,D) and
LgM 1 LgE recombinants (B,E) express SP-C mRNA only in the most
distal epithelial cells. CC10 mRNA is expressed throughout the epithe-
lium, although the signal appears stronger in the proximal epithelium. LgM
1 TrE recombinants exhibit patterns of expression of both SP-C (C,F) and
CC10 mRNAs (I,L) in the branched epithelium that are identical to those
seen in tissues containing LgE. Some cells in the distal epithelium appear
to express both SP-C and CC10 mRNAs (arrows). Control preparations
using radiolabeled sense probes show no hybridization signal (not
shown). All panels are at the same magnification (original magnification 5 1323).
Although mucins are not completely specific markers of
tracheal differentiation, the distribution of strongly
immunopositive cells and the copious amount of mucin
present in the lumina were essentially identical among
intact tracheal explants and TrM-containing homotypic
and heterotypic recombinants. That the changes we
observed in TrM 1 LgE recombinants represent a
change in the developmental fate of lung epithelium is
further supported by the lack of lamellar bodies and the
extinguishing of SP-C mRNA expression. The mechanism by which SP-C mRNA is suppressed in the
presence of TrM is unknown. Tracheal mesenchyme
may produce some factor(s) that actively inhibits SP-C
expression. An alternative and equally plausible possi-
490
SHANNON ET AL.
Fig. 8. In situ hybridization of cultured intact tracheae, TrM 1 TrE
recombinants, and TrM 1 LgM recombinants. Consecutive sections were
hybridized with antisense probes for SP-C (A–F) and CC10 (G–L).
Exposure of the autoradiographs was identical for each probe, and
samples were photographed and printed identically. SP-C mRNA is not
detectable in any epithelium (arrows) that is associated with TrM, even
though LgE is SP-C mRNA positive at the time of isolation. In contrast,
CC10 mRNA is strongly expressed throughout the epithelium in all
TrM-containing tissues. Control preparations using radiolabeled sense
probes show no hybridization signal (not shown). All panels are at the
same magnification (original magnification 5 1323).
bility is that tracheal mesenchyme does not produce a
factor(s) whose continuous presence is required for
sustaining distal lung epithelial differentiation. Although our data demonstrate that several key characteristics of distal lung epithelium have been altered by
tracheal mesenchyme, the apparent increased amounts
of glycogen and SP-B mRNA in TrM 1 LgE versus TrM
1 TrE recombinants suggest the possibility of an
intermediate phenotype, which might occur if the lung
epithelium is already partially restricted to a lung
developmental fate on day 13. As a whole, however, our
data support the concept that tracheal mesenchyme
induces lung epithelium to initiate a new program of
gene expression resulting in tracheal differentiation.
The molecular nature of the inductive cues provided
by mesenchyme are undoubtedly complex, as evidenced
by observations documenting the roles of growth factors, hormones, and extracellular matrix molecules in
MESENCHYMAL SPECIFICATION OF RESPIRATORY EPITHELIAL DIFFERENTIATION
491
Fig. 9. Mucin immunostaining of cultured tissues. Intact tracheae (A),
distal lung tips (B), TrM 1 TrE recombinants (C), LgM 1 LgE recombinants (D), TrM 1 LgE recombinants (E), and LgM 1 TrE recombinants (F)
were cultured for 5 days, fixed, and processed for immunostaining with
monoclonal antibody RTE 11, which reacts against mucin. LgMcontaining tissues exhibit some positive luminal reactivity that is confined
to the proximal portion of the explant. Note that the most distal epithelial
acini are negative. Cultured intact trachea, as well as both types of
TrM-containing recombinants, exhibit strong mucin reactivity in epithelial
cells and the lumina. All panels are at the same magnification (original
magnification 5 2653).
specifying respiratory epithelial phenotype (Minoo and
King, 1994; Hilfer, 1996; Shannon and Deterding, 1997).
Observations from experiments in which epithelial and
mesenchymal rudiments were cultured with a filter
interposed between them demonstrate that the active
factor(s) are diffusible over a short distance (Taderera,
1967; Shannon, 1996). Although a number of growth
factors have been shown to be present in the developing
lung, recent experiments suggest that the fibroblast
growth factor (FGF) family may play a key role in
mediating epithelial-mesenchymal interactions in the
developing lung. FGF1, FGF7, and FGF10 have all
been shown to stimulate proliferation and differentiation of embryonic rat or mouse lung epithelium cultured in the absence of mesenchyme (Deterding and
Shannon, 1995; Nogawa and Ito, 1995; Deterding et al.,
1996; Cardoso et al., 1997; Bellusci et al., 1997). Antisense oligonucleotides to FGF7 have been shown to
inhibit branching morphogenesis in embryonic rat lung
explants (Post et al., 1997). Furthermore, when a
dominant negative form of the FGF7 receptor is targeted to the lung with the SP-C promoter, lung branch-
492
SHANNON ET AL.
ing does not occur at all (Peters et al., 1994). These
observations, combined with the fact that FGF7 (Mason
et al., 1994; Finch et al., 1995; Post et al., 1997) and
FGF10 (Bellusci et al., 1997) are produced in the
mesenchymal cells subtending the lung epithelium,
suggest a primary role for these growth factors in
mediating epithelial-mesenchymal interactions in lung
development. It should be noted, however, that transgenic mice null for FGF7 expression exhibit no abnormalities in lung development (Guo et al., 1996). This
suggests other members of the FGF family, perhaps
FGF10, can serve as functionally redundant molecules.
In summary, we have demonstrated that epithelium
from the entire respiratory tree of the early pseudoglandular stage rat remains competent to respond to induction by heterotypic respiratory mesenchyme. Precisely
when and, more importantly, how these epithelia become restricted in their developmental fates has yet to
be elucidated. We believe that the ability to reprogram
both tracheal and lung epithelium to express new
phenotypes will prove useful in determining the factors
involved in both the induction and restriction of respiratory tissues.
EXPERIMENTAL PROCEDURES
Animals
Timed-pregnant Sprague-Dawley rats were obtained
from Charles Rivers Laboratories (Raleigh, NC); the
day on which a sperm-positive vaginal plug was detected was considered day 0 of gestation. Pregnant
dams were sacrificed on day 13 of gestation, then the
fetuses were removed and weighed. Fetuses weighing
80–90 mg, whose lungs are at the pseudoglandular
stage of development, were used in these experiments.
Tissue Separation, Recombination, and Culture
Fetuses were decapitated and transferred to a Maximov depression slide containing ice-cold Hank’s balanced salt solution (HBSS; GIBCO/BRL, Gaithersburg,
MD) supplemented with 100 U/ml penicillin, 100 µg/ml
streptomycin, 2.5 µg/ml amphotercin B (all from GIBCO/
BRL), and 10 µg/ml gentamicin sulfate (Sigma Chemical Co., St. Louis, MO). The lungs and trachea were
dissected intact using Moria microsurgery knives (Fine
Science Tools Inc., Foster City, CA).
Separation of lung epithelium and mesenchyme was
performed as previously described (Shannon, 1994;
Deterding and Shannon, 1995). Briefly, distal lung tips
were cut off, then incubated with dispase (Collaborative
Biomedical Products, Bedford, MA) for 30 minutes at
37°C. The epithelium and mesenchyme were separated
using tungsten needles (Fine Science Tools Inc.), washed
four times with HBSS containing 10% fetal bovine
serum (FBS; GIBCO/BRL) to remove any residual
enzyme. Mesenchymal rudiments were transferred to
Waymouths 752/1 medium (GIBCO) containing 10%
FBS (hereafter culture medium) and stored on ice until
recombination. Epithelial rudiments were given a second incubation in dispase for 10 minutes to remove any
possible adherent mesenchymal cells, washed as above,
and stored on ice in culture medium; this method has
been shown to effectively eliminate contamination by
mesenchymal cells (Deterding and Shannon, 1995).
The trachea was removed from the lung/trachea
complex just above the bifurcation and bisected. Epithelial and mesenchymal tracheal rudiments were isolated
by incubating the tracheal halves in HBSS containing
0.05% collagenase (CLS IV; Worthington Biochemicals,
Freehold, NJ) and 1% FBS for 45 minutes at 37°C. The
tracheae were washed twice with HBSS 1 10% FBS,
treated briefly with 1–2 drops of a 1-mg/ml solution of
DNase I (Sigma) in HBSS, and separated into epithelial
and mesenchymal components with tungsten needles.
Mesenchymal rudiments were washed twice with HBSS
1 FBS, then placed on ice in culture medium. Epithelial rudiments were treated with dispase for 10 minutes, washed, and placed on ice in culture medium.
Epithelial and mesenchymal tissues were recombined on the surface of a semisolid medium consisting
of 0.5% agarose (Sigma) in culture medium. The appropriate epithelial and mesenchymal rudiments for each
experimental condition were transferred to 35-mm
dishes containing 2 ml of semisolid medium and excess
liquid medium removed with a flame-drawn Pasteur
pipette. Epithelial and mesenchymal rudiments were
teased into close apposition using microsurgery knives.
Typically 3–4 lung mesenchymes, or 2–3 tracheal mesenchymes, were used per epithelial rudiment in each
recombinant. After the positioning of the epithelial and
mesenchymal rudiments, a moat was created in the
agarose around each recombinant using a flame-drawn
Pasteur pipette; this prevented liquid from flooding the
rudiments and causing them to drift apart during the
initial culture period. Excess fluid was then removed
from around the recombinants and the tissues repositioned, if necessary. After culture overnight, the recombinants were transferred to 35-mm dishes containing
fresh semisolid medium. A few drops of culture medium
was added to each dish to maintain the recombinants at
an air-liquid interface. Cultures were maintained for a
total of 5 days, whereupon they were processed as
described below. Four different recombinants were constructed: TrM 1 TrE; TrM 1 LgE; LgM 1 LgE; and
LgM 1 TrE. Intact lung tips and bisected tracheae were
cultured as controls.
The ability of glucocorticoids to accelerate maturation of the surfactant system in the developing distal
lung epithelium has long been appreciated (for review
see Odom and Ballard, 1997). Therefore in some experiments dexamethasone (1027M; Sigma) was added to
the culture medium of all groups for the final 24 hours
to maximize the possibility of distal epithelial maturation within the 5-day culture period. This allowed us to
better assess the ability of LgM and TrM to induce and
suppress the distal lung epithelial phenotype, respectively.
MESENCHYMAL SPECIFICATION OF RESPIRATORY EPITHELIAL DIFFERENTIATION
Isolation of mRNA and Reverse Transcription
Polyadenylated RNA was isolated from recombinants
and control cultures using a Micro-Fast Track kit
(Invitrogen, San Diego, CA). Approximately equal
amounts of tissue from each condition were used for
each isolation. The mRNA was treated with 35 U of
RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) in the presence of 40 U of RNasin (Promega
Biotech, Madison, WI), then ethanol precipitated with 5
µg mussel glycogen present as a carrier. The mRNA was
reverse-transcribed in a final volume of 20 µl using a
cDNA Cycle kit (Invitrogen) and immediately used for
PCR.
PCR Amplification and Analysis of Products
Oligonucleotide primers were synthesized on an Applied Biosystems Model 381A DNA synthesizer (Foster
City, CA) in the Molecular Resource Center at the
National Jewish Medical and Research Center. The
primers were designed from the published sequences
for rat SP-A (Fisher et al., 1988), rat SP-B (Emrie et al.,
1989), and rat SP-C (Fisher et al., 1989). The sequences
for the SP-A primers were (1) 58-AGTCCTCAGCTTGCAAGGATC-38 coding sense and corresponding to bases
422–442, and (2) 58-CGTTCTCCTCAGGAGTCCTCG-38
coding antisense and corresponding to bases 549–569;
the predicted size of the SP-A product was 148 base
pairs (bp). The sequences of the SP-B primers were (1)
58-GAGCAGTTTGTGGAACAGCAC-38 coding sense and
corresponding to bases 997–1017, and (2) 5-TGGTCCTTTGGTACAGGTTGC-38 coding antisense and
corresponding to bases 1152–1172; the predicted SP-B
product size was 176 bp. The sequences of the SP-C
primers were (1) 58-CATACTGAGATGGTCCTTGAG-38
coding sense and corresponding to bases 202–222, and
(2) 58-TCTGGAGCCATCTTCATGATG-38 coding antisense and corresponding to bases 381–401; the predicted SP-C product size was 200 bp. The sequences for
the b-actin primers were (1) 58-GTATGGAATCCTGTGGCATCC-38 coding sense and corresponding to bases 2
683–2703 of the genomic DNA sequence, and (2) 58TACGCAGCTCAGTAACAGTCC-38 coding antisense
and corresponding to bases 3135–3155 of the genomic
DNA sequence. The predicted size of the b-actin product amplified from cDNA was 349 bp, whereas the size
of the product amplified from contaminating genomic
DNA was 473 bp.
Four microliters of each reverse-transcribed cDNA
were used for amplification of SP-A, SP-B, and SP-C,
and 2 µl for amplification of b-actin. The final 50 µl
reaction mixture contained 10 mM Tris-HCl (pH 8.3),
50 mM KCl, 1.5 mM MgCl2, 200 µM each of dATP,
dCTP, dGTP, and dTTP, 0.01% gelatin, 0.5 µM of the
appropriate upstream and downstream primers, and
2.5 U AmpiTaq DNA polymerase (Perkin-Elmer Cetus,
Norwalk, CT). Reaction mixtures were overlaid with
silicone oil and amplified for 30 cycles. The amplification profile consisted of denaturation at 94°C for 1
minute, annealing at 58°C for 2 minutes, and extension
493
at 72°C for 3 minutes; the reaction was terminated by
cooling to 4°C. After amplification the reaction products
were electrophoresed through a 1% agarose (Sigma)
gel, then transferred to Nytran (Schleicher and Schuell,
Keene, NH) for Southern blot analysis by standard
methods. Southern blots were probed with full length
cDNAs for SP-A, SP-B, and SP-C that were labeled to
high specific activity with 32P-dCTP (Amersham, Arlington Heights, IL) using a Redi-prime kit (Amersham).
In Situ Hybridization
Tissues and recombinants for in situ hybridization
were enrobed in a solution of neutralized rat tail
collagen (Emerman and Pitelka, 1977) prior to fixation.
Samples were transferred in a small volume of medium
to a piece of Parafilm (American National Can, Neena,
WI), then covered with 200–300 µl of cold collagen
solution. The explants were mixed into the collagen and
centered at the bottom of the drop. After gelation at
37°C oven for 15 minutes, the enrobed tissues were
inverted into a 35-mm dish containing 2 ml of 4%
paraformaldehyde in RNase-free phosphate-buffered
saline (PBS; pH 7.4) and fixed overnight at 4°C. After
fixation, the samples were washed once in PBS, dehydrated, and embedded in paraffin. The enrobement
procedure not only expedited locating the tissues when
sectioning, but also allowed several samples to be
evaluated in one section.
In situ hybridization was performed on 4-µm sections
as previously described (Deterding and Shannon, 1995),
with the exception that 33P-UTP (2,000–4,000 Ci/mmol;
NEN Life Science Products, Boston, MA) was used in
the transcription of RNA probes. Slides were dipped in
NTB-2 nuclear track emulsion (Eastman Kodak Co,
Rochester, NY) and developed after an exposure period
appropriate for each probe. Hybridization with radiolabeled sense RNA probes was done as a control in all
experiments.
As a marker of the distal lung epithelial phenotype,
we examined the expression of SP-C, which was transcribed from a full length rat cDNA (Fisher et al., 1989).
As a marker of the proximal respiratory epithelial cell
phenotype, we examined the expression of the 10-kDa
Clara cell secretory protein (CC10) using a 275-bp
fragment of the rat cDNA (generously provided by Dr.
Arun Rishi).
Electron Microscopy
Samples were fixed in 2% glutaraldehyde-1% paraformaldehyde, postfixed in 1.5% osmium tetroxide, stained
en bloc with uranyl acetate, and embedded in Lufts 3:7
mixture of LX-112 (Ladd Research Industries, Burlington, VT). Sections 1- to 1.5-µm-thick were stained with
Mallory’s azure II-methylene blue. Ultrathin sections
were stained with uranyl acetate and lead citrate and
examined in a Phillips 400 electron microscope.
494
SHANNON ET AL.
Immunocytochemistry and Histochemistry
Samples for immunocytochemistry were fixed with
Omni-Fix II and embedded in paraffin. As a marker of
the tracheal and proximal airway phenotype, sections
were stained as previously described with the mouse
monoclonal antibody RTE 11, which reacts with mucin
(Randell et al., 1993). The presence of glycogen was
detected in paraformaldehyde fixed tissues by comparing PAS staining in sequential sections that were or
were not pretreated with diastase (Sheehan and Hrapchak, 1980).
ACKNOWLEDGMENTS
This work was performed in the Lord and Taylor
Laboratory for Lung Biochemistry and the Anna Perahia Adatto Clinical Research Center. The authors gratefully acknowledge Lynn Cunningham for performing
the histology and histochemistry, Janet Leiber for
performing the electron microscopy, and Leigh Landskroner and Barry Silverstein for assistance in preparing the illustrations. We also thank Kathy Ryan Morgan for excellent assistance in preparing the manuscript.
This work was supported by the National Heart, Lung,
and Blood Institute of the NIH, grants HL45011 (J.M.S.)
and HL58345 (S.H.R.).
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