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DEVELOPMENTAL DYNAMICS 20R47-59 (1996)
Retinoic Acid Alters the Expression of Pattern-Related
Genes in the Developing Rat Lung
WELLINGTON V. CARDOSO, S. ALEX MITSIALIS, JEROME S. BRODY, AND MARY C. WILLIAMS
The Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
ABSTRACT
Exogenous retinoids alter pattern formation and differentiation in many developing systems, such as limb, vertebrae, and central nervous system. Many of these effects are
mediated by changes in expression of patterning
genes such as Hox genes and Sonic hedgehog. We
have previously shown that exogenous retinoic
acid, administered to the embryonic rat lung in
culture alters the structural pattern of the developing lung, suppressing formation of distal lung
and favoring growth of proximal tubules. To determine whether these retinoic acid-induced
changes in lung development were linked to alterations in pattern-related genes, we characterized
the expression of Hoxa-2, Hoxh-6, and Sonic
hedgehog mRNAs in vivo and in vitro, with or
without 10-'M retinoic acid, by in situ hybridization and quantitative polymerase chain reaction.
Each of these genes demonstrated unique timing
and distribution of expression that was similar in
vivo and in control cultured embryonic lungs.
Hoxb-6 and Sonic hedgehog mRNAs both decreased during lung development in vivo or in
vitro. From the patterns of mRNA expression we
propose that Hoxb-6 is involved in distal airway
branching while Homz-2 is involved in differentiation of proximal mesenchymal derivatives and
vasculogenesis in the lung. RA upregulated all
three genes, changing their developmental pattern of distribution and preventing the developmental decrease in Sonic hedgehog expression.
We propose that RA acts to maintain high levels of
expression of these and likely other pattern-related genes in a fashion that is characteristic of
the immature lung, promoting continued formation of proximal lung structures and preventing
formation of typical distal lung structures of the
mature lung. 0 1996 Wiley-Liss, Inc.
Key words: Retinoic acid, Hox genes, Sonic
hedgehog,Lung development
INTRODUCTION
Retinoic acid (RA) is largely known for its effects on
pattern formation during development and regeneration of structures such as vertebrae, limb, face, neural
tube, and other organs (Hill et al., 1993; reviewed in
Tabin, 1991, and Gudas, 1994). In the adult lung retinoids play an important role in maintenance of the difB 1996 WILEY-LISS,INC.
ferentiated state of the respiratory epithelium, preventing squamous metaplasia (reviewed in Chytil,
1992). The developing lung is responsive to exogenous
retinoids. Administration of retinoic acid (RA) to pregnant hamsters results in alterations in the lobation
pattern of the fetal lung (Shenefelt, 1972).
Retinoic acid interacts with a number of retinoic acid
receptors (RARs) which have been shown to be essential for normal lung formation. Double knock-outs of
RARs, such as FtARalp2, cause dramatic abnonnalities in the respiratory tract of transgenic mice. Such
abnormalities are similar to those described in vitamin
A deficient animals (Wilson et al., 19531, and include
developmental arrest and growth retardation (Mendelsohn et al., 1994). Short exposures to exogenous FtA
appear to stimulate branching in murine lung cultures
previously growth arrested with serum (Schuger et al.,
1993). Recently we have shown that longer exposure
(5-9 days treatment) of the 13.5 day embryonic rat
lung to RA (10-6-10-5M) alters its normal developmental pattern in vitro. RA suppresses formation of
the distal lung and induces a proximal-like phenotype
in the lung epithelial tubules (Cardoso et al., 1995).
Many of the retinoid effects on pattern formation are
associated with changes in expression of patterning
genes, such as Hox (Kessel and Gruss, 1991; Papalopulu et al., 1991) or Sonic hedgehog (Shh)(Riddle et al.,
19931,which are expressed in the developing lung (Riddle et al., 1993; Bitgood and McMahon, 1995; reviewed
in Cardoso, 1995). Hox genes encode transcriptional
regulators involved in specification of cell fate along
the anterior-posterior axis of the body (Krumlauf,
1994). Shh is a mouse homolog of the Drosophila segment polarity gene hedgehog that encodes a secreted
protein implicated in patterning of the central nervous
system, somites, and limb (Echelard et al., 1993;
Laufer et al., 1994; Johnson and Tabin, 1995). RA has
been shown to upregulate Hox mRNA in fetal lung
explants (Bogue et al., 1994). Although Shh is known
to be induced by exogenous RA in the limb mesenchyme, no information is available on how Shh responds to RA in the lung.
In the present work, we hypothesized that the proximalized phenotype induced by exogenous RA in culReceived December l, 1995;accepted March 18, 1996.
Address reprint requestskorrespondence to Wellington V. Cardoso,
MD, PhD, The Pufmonary Center, Boston University School of Medicine, 80 East Concord Street, R-304,Boston, MA 02118.
48
CARDOSO ET AL.
p-actin (20 ngheaction). The oligonucleotide primer sequences used for PCR amplification were as follows:
Hoxa-2: 5' oligo: AAA CCC AGT GCA AGG AGA A, 3'
oligo: GCT GAA ATA TCT ACA GGA CTG (465 bp,
Patel et al., 1992);Hoxb-6: 5' oligo: GAG AGC AAA
CTG CTC AGT GC, 3' oligo: GGA GAC ATC AAG TGA
GTC CG (354 bp, Shen et al., 1991); p-actinr 5' oligo:
ATG CCA TCC TGC GTC TGG A, 3' oligo: CAC ATC
TGC TGG AAG GTG G (552 bp, Nude1 et al., 1983).
Samples were PCR-amplified for 15 cycles as follows: 1
min at 95°C (melting), 1 min a t 55°C (hybridization),
and 1.5 min at 72°C (extension); this protocol was
shown previously to give a n optimal signal on a Southern blot within the linear range of amplification for the
genes studied (data not shown). Amplified DNA was
blotted onto nitrocellulose membranes (Hybond'" -N,
MATERIALS AND METHODS
Amersham, Arlington Heights, IL), and hybridized
Embryonic Lung Cultures
with random primer labelled probes (Feinberg and VoEmbryonic lungs were obtained from timed-preg- gelstein, 1983) a t 68°C overnight and washed at high
nant Sprague-Dawley specific pathogen-free rats stringency (0.3M NaCl). Each filter was hybridized
(Zivic-Miller, Zelionople, PA) and were sacrificed a t with probes for Hoxa-2 and Hoxb-6 and subsequently
gestational day 13.5 according to standard procedures reprobed for P-actin. Autoradiographic bands were an(Cardoso et al., 1995). Lung explants were cultured for alyzed by densitometry (ImageQuant, Molecular Dy12 hr to 9 days in either a control defined, serum-free namics, Eugene, OR). The values obtained from the
medium (BGJb, GIBCO, Gaithersburg, MI) with 0.2 densitometric measurements for each Hox gene were
mg of ascorbic acid and 50 U / I J .of
~ penicillidstrepto- normalized for loading efficiency of the PCR by the
mycin (GIBCO) (Slavkin et al., 1989), or the same me- corresponding p-actin band in the same lane of the
dia containing all-trans-retinaic acid (Sigma, St. Louis, same filter. Densitometric measurements were exMO), diluted in ethanol at lOV5M,a concentration that pressed as % of control values. A statistical analysis
has been previously shown to cause a marked change was performed (ANOVA, Fisher PLSDTest, Statview);
in lung patterning in vitro (Cardoso et al., 1995). Har- differences were considered significant a t P < 0.05.
vested cultures were photographed, and quick-frozen in Each experimental condition was tested several times
liquid nitrogen (mRNA extraction) or fixed in 4% para- with samples from different litters. The number of
samples "n" used in this procedure is shown in the
formaldehyde (in situ hybridization studies).
legend of Figure 4. Each sample (n) represents a pool of
Quantitation of Hox mRNA Abundance by
three lungs from a same litter. Reproducibility of the
Southern Blot Analysis of RT/PCR Products
method has been previously tested (Cardoso et al.,
Poly-A RNA was extracted from freshly isolated rat 1995).
lungs (gestational day 13.5, 15.5, 18.5, and 21.5) or
cultured lungs (control, RA-treated) which had been In Situ Hybridization Analysis
Paraffin sections (6 bm) were obtained from freshly
quick-frozen in liquid nitrogen (Micro-Fast Track
mRNA Isolation Kit, Invitrogen, La Jolla, CAI. To isolated (gestational days 13-14, 18.5, and 21.5) and
avoid contamination with genomic DNA, samples were 5-day cultured (control, RA-treated) lungs fixed in 4%
treated with DNase prior to RT/PCR analysis. Products paraformaldehyde. After PCR amplification, Hoxu-2
of the reverse transcriptase (RT) reaction were labelled (465 bp, nucleotides 784-1,249, Patel et al., 1992) and
with r3H]thymidine 5'-triphosphate, tetrasodium salt Hoxb-6 (354 bp, nucleotides 1,880-2,234, Shen et al.,
(f3H]-dTT,10 p,Ci/pl, specific activity of 100 Ci/mmol) 1991) rat cDNA fragments were cloned, respectively,
to allow loading of equal counts and to equalize the into a PCR- I1 vector (TA cloning kit, Invitrogen) and
amount of cDNA template used for each reaction. The an EcoRI site of a pBluescript KS+ vector. The Shh
final concentration of dATP, CTP, GTP, in the RT re- cDNA consisted of a 640 bp EcoR-I fragment cloned
action was 0.5 mM, whereas dTTP was 0.1 mM. Unin- into a pBluescript I1 SK of the chicken Shh gene that is
corporated [3H]-dTTwas separated from cDNA by eth- highly homologous to the mouse Shh. In addition to
anol precipitation. Approximately 4 ng of DNA were these genes we also assessed expression of surfactantused as template for PCR. For PCR we used 50 mM associated protein C (SP-C), a marker of distal lung
KC1, 10 mM Tris (pH 8.31, 1.5 mM MgCl,, 0.01%gel- differentiation. SP-C probe consisted of a 574 bp cDNA
atin, 1.25 M each of dATP, dCTP, dTTP, and dGTP, 2.5 (nucleotides 22-596, Fisher et al., 1989) cloned into a
U of recombinant Taq DNA polymerase (Perkin Elmer pBluescript KS+. Recombinant Hoxa-2, Hoxb-6, Shh,
Cetus, Norwalk, CT), and specific upstream and down- and SP-C plasmids were linearized to achieve transtream primers of Hoxa-2, Hoxb-6 (75 ngjreaction), and scription of sense and antisense templates. The tran-
tured lung explants is associated with changes in
pattern-related genes. Thus, we assessed mRNA expression of Shh and representative anterior and relatively posterior Hox genes Hoxa-2 and Hoxb-6 in vivo,
in vitro, and in lungs whose development was altered
by exogenous RA. We found that these genes are developmentally regulated in the lung, and that exposure
of lung explants to 10-6M RA consistently upregulates
Hoxu-2, Shh, and to a lesser extent Hoxb-6 mRNAs.
We propose that RA may act to maintain high levels of
expression of these and other pattern-related genes in
a fashion that is characteristic of the immature lung,
thereby promoting continued formation of proximal
lung structures and preventing formation of the distal
lung structures of the mature lung.
RETINOIC ACID AND PATTERNING OF THE LUNG
49
Fig. 1. Airway branching in the embryonic rat lung in culture for 5 days under control conditions (A), in the
presence of RA lO-%l (B), and after RA washout (C) (RA for 3 days, and in control medium for additional 2
days). RA treatment prevents formation of typical distal bud-like structures seen in controls and favors growth
of proximal-like tubules. Distal buds typical of controls (arrows) branch from the proxirnalized tubules after RA
is withdrawn. Whole mounts magnification: A, B, and C, x 25.
scription reaction consisted of 300 ng of linearized plasmid template, 2 pl of 10 x transcription buffer (BRL),
2 p1 of 100 mM DTT, 0.8 U of RNAase inhibitor (RNAsin, Promega, Madison, WI),80 pCi t35Sl-UTP (40
mCi/ml stock, Amersham), 1 pl each of 10 mM stocks of
ATP, CTP, GTP, in a total volume of 19 p1. Reaction
was started by adding 1 p1 (20 U) of the appropriate
RNA polymerase (BRL)that allowed synthesis of sense
and antisense riboprobes. Samples were incubated at
37°C for 2 hr. Plasmid DNA template was digested by
adding 1 ~1 of RNAase-free DNAase (Promega) and
incubated a t 37°C for 15 min. The labeled probe was
submitted to alkaline hydrolysis for 60 min and purified on G-50 Sephadex columns. Sections were then
prehybridized and hybridized with sense and antisense
RNA probes, being subsequently washed and treated
with RNAase (Sassoon et al., 1988; Rishi et al., 1995).
The slides were finally dehydrated, dried, and coated
with Kodak NTB2 emulsion. Slides were developed in
Kodak D-19 developer a t 1 and 3 weeks and photographed in a Leitz Orthoplan microscope. Each experimental condition was tested 2-3 times.
of the epithelial tubules; however higher RA concentrations
to 10-'M) suppress formation of distal
tubules and favor growth of proximal-like tubules (Fig.
1B and Cardoso et al., 1995). In the present work we
studied the lungs treated with RA lO-'M, in which the
effects on patterning were most evident. These lungs,
although showing proximal features of differentiation
such as ciliated cells in the epithelium, appeared less
mature than equivalent controls (compare proximal epithelia of day 13.5, control, and RA at day 9 in Fig. 2).
The 10-5M RA-treated lung phenotype promptly reverted to the control phenotype by culturing the RAtreated lungs in control medium (Fig. 1C). Newly
formed distal buds arising from the proximalized tubules showed the same features as distal tubules of
controls (Fig. 1C and Fig. 9).
Hox mRNA Quantitation In Vivo
Densitometric measurements were performed on RT/
PCR Southern blots of rat lungs freshly isolated a t gestational days 13.5, 15.5, 18.5, and 21.5 and probed for
Hoxa-2 and Hoxb-6 (Fig. 3). Hoxu-2 mRNA levels remained unchanged throughout development. Hoxb -6
levels decreased significantly and were almost undetectable at late gestation (Figs. 3 and 41, as previously
shown by Northern analysis (Bogue et al., 1994).
RESULTS
Morphology of Control and RA-Treated Lungs
Control cultured lungs exhibited airway branching
and differentiation of both epithelium and mesenchyme, reproducing the overall proximal-to-distal pattern seen in lungs in vivo (Fig. 1A; see also Cardoso et Hox mRNA Quantitation In Vitro and Effects
al., 1995).Briefly, proximal tubules (airways) are lined of RA
The relative levels of Hoxa-2 and Hoxb-6 mRNA in
by a tall columnar epithelium with ciliated and secretory cells (Fig. 2B) and are surrounded by either a control lungs a t days 3 and 5 mirrored the changes in
smooth muscle layer or cartilage primordia. Distal ep- mRNA levels observed a t equivalent time points in
ithelial tubules (putative bronchioles and alveoli) are vivo (Fig. 4).At lO-'M RA Horn-2 mRNA levels are
acinus-shaped, with cuboidal cells and lack an under- significantly higher than controls at day 5. We could
lying smooth muscle layer (not shown). Treatment of not detect statistically significant differences in
H o d 8 mRNA levels between control and RA-treated
lung explants with low RA concentrations (
lO-'M) does not affect the overall pattern of branching lungs by quantitative PCR (Fig. 4).
50
CARDOSO ET AL.
Fig. 2. Histological features of the epithelia of a day 13.5 embryonic rat lung (A), proximal tubules of a
9-day control cultured lung (B), and a proximalired tubule of a 9-day IO-%l RA-treated lung (C).The
epithelium of the RA-treated lung, although showing proximal features of differentiation such as the presence
of ciliated cells (arrowheads), resembles the immature epithelium of the 13.5 day lung. Toluidine blue-stained
plastic sections; magnification: A and C, x 650; B, x 950.
In Situ Hybridization:Hox and Shh mRNA
Expression In Vivo and In Vitro and Effects
of RA
Hoxa-2. Hoxa-2 mRNA is expressed in mesenchyma1 cells of the day 13-14 rat lung along the main axis
of the respiratory tract; trachea and lung exhibited a
similar intensity of signal (Fig. 5A). At later gestational ages, such as days 18.5 (Fig. 5C,D) and 21.5 (Fig.
5E,F), while Hoxu-2 mRNA levels remain unaltered
(Fig. 4), its distribution changes, localizing mostly to
the mesenchyme associated with cartilage, smooth
muscle layer of airways, and vascular smooth muscle
(Fig. 5C-F). Control cultured lungs a t day 5, similarly
to day 18.5 fetal lungs, show Hoxu-2 mRNA signals in
cartilage primordia and smooth muscle of large proximal airways (Fig. 5G). Distal mesenchyme of the cultured lungs also expressed Hoxu-2 mRNA, however diffusely distributed and at much lower levels than in
proximal mesenchyme. Vascular expression was not
seen in vitro because blood vessels appear not to develop in vitro. We assessed Hoxa-2 mRNA expression
in lungs treated with lO-'M RA for 12 hr (see Fig.
and days (Fig*5H)*At both time points treated
lungs showed much higher H o ~ a - 2mRNA levels than
equivalent control lungs. At day 5, the lung proximalto-distal pattern was altered and, in contrastto controls, Hoxa-2 mRNA was homogeneously distributed in
the mesenchyme, with no preferential localization to
particular structures of the lung (Fig. 5H).
7ApB)
Fig. 3. Hox gene expression in embryonic rat lungs: representative
Southern blots of rat cDNA from rat lungs. After RT/PCR amplification
gels were blotted and hybridized to a Hoxa-2 and Hoxb-6 32P labelled
cDNA probe. Filters were reprobed with p-actin probe. Hoxa-2 mRNA
levels do not change throughout gestation, however Hoxb-6 mRNA significantly decrease in late
lungs.
51
RETINOIC ACID AND PATTERNING OF THE LUNG
--T
i n vivo-
W
T
BJ
HOXZI-2
Hoxb-6
100
h
s
v
9
0
50
0
13.5
15.5
18.5
21.5
gestational d a y
Hoxb-6
Hoxa-2
200
-^__
100
**
1
T
z
Y
n
s
Y
0
0
100
ti
rT-
0
50
0
0
13.5 C 3 C5
13.5 C 3
C5
R5
R5
Fig. 4. Hox gene expression in embryonic rat lungs in vivo and in
vitro, and effects of RA. Histograms show quantitation of RT/PCR-Southern blots from freshly isolated embryonic rat lungs (gestational day 13.521.5) and cultured lungs in control medium (C3, C5) and in the presence
of RA 10-5M (R5) for 5 days. Filters were probed with Hoxa-2 and
Hoxb-6;the densitometric values obtained from Southern blots were normalized by respective p-actin values as shown in blots in Figure 3. Values
(bars and lines representing mean and standard deviation) were expressed as percentage from the gestation day 13.5 lung value in both in
vivo and in vitro studies. Number of samples "n" equals 3 (gestation day
15.5,C3,C5, and R5),4 (gestation days 13.5and 18.5), and 5 (gestation
day 21.5). Hoxa-2 mANA levels do not change throughout gestation in
vivo or with time in culture. Hoxb-6 levels significantly decrease with
gestation age and time in culture. Five-day RA-treated lungs show significantly higher levels of Hoxa-2 mRNA than respective controls and day
13.5 lungs. Hoxb-6 mRNA levels in RA-treated lungs at day 5 are not
significantly higher than respective controls. 'Significantly different (P <
0.05) from day 13.5 lung but not different from respective control; ""significantly different (P i0.05) from day 13.5 lung and respective control.
Hoxb-6. Lungs of gestation day 13-14 show a gradient of Hoxb-6 mRNA expression in the respiratory
tract. Background levels of grains are seen in trachea
and proximal lung; high levels of expression are found
in mesenchymal cells associated with the distal epithelial tubules (Fig. 6A). At late stages of development,
days 18.5 (Fig. 6C,D) and 21.5 (not shown) fetal lung
expresses progressively lower levels of Hoxb-6 mRNA.
Signals are almost undetectable in the 18.5-day lung
(Fig. 6C,D). Similarly Hoxb-6 signals are barely detectable in the mesenchyme of 5-day control cultured
lungs, and the proximal-distal mRNA gradient previously observed is no longer seen (Fig. 6E).Short exposures to 10m6MRA (12 hr) appears to expand slightly
the domain of Hoxb-6 expression to a more proximal
location (Fig. 7D).In contrast to the results obtained by
quantitative PCR (Fig. 41, in situ hybridization of
5-day RA-treated lungs appear to display much stronger Hoxb-6 mRNA signals than 5-day controls (Fig.
6E,F). Signals are no longer localized to distal lung as
in the 13.5 day lung, but are seen throughout the mesenchyme of the entire RA-treated explant.
Shh. High levels of Shh mRNA expression are observed in the epithelia of the 13-14 day rat esophagus
and the respiratory tract (arrows in Fig. 8A). Shh transcripts are not detected a t sites other than the epithelium, and signals are similar in trachea and lung; thus
no proximal-distal gradient was identified (Fig. 8A).
Days 18.5 (Fig. 8B,C) and 21.5 (Fig. 8D,E) fetal lungs
and trachea had progressively less Shh expression in
52
CARDOSO ET AL.
Fig. 5.
RETINOIC ACID AND PATTERNING OF THE LUNG
their epithelia. Low levels of Shh transcripts, comparable to day 18.5 lungs, are also found in 5-day control
cultured lungs (Fig. 8F).Exposure to 10m5MRA for 12
hours did not seem to alter the preexisting high levels
of expression of Shh mRNA of the early cultured lung
(data not shown). In contrast, RA exposure for 5 days
strongly upregulated Shh in the lung epithelium and
prevented the normal decrease in Shh seen in controls
(Fig. 8G). When RA is washed out, distal buds form
(Fig. lC, and also indicated by arrowheads in Fig. 9)
and appear similar to the distal lung buds of control
lungs, expressing low levels of epithelial Shh mRNA
(arrowheads in Fig. 9B). Areas that retain the morphologic features of the proximalized lung (large arrows in
Fig. 9) continue to express high levels of Shh (large
arrow in Fig. 9B).
SP-C. We have previously shown by in situ hybridization that SP-C mRNA is localized to the distal tubules of control lungs and that its expression is suppressed by 10p5MRA treatment (Cardoso et al., 1995).
In lungs where RA effects have been reversed by washing out RA, the newly formed buds express high levels
of SP-C mRNA (arrowheads in Fig. 9A).
DISCUSSION
We have previously shown that RA affects the pattern of lung formation during embryonic development,
tending to proximalize the developing lung and to prevent formation of distal lung tubules (Cardoso et al.,
1995). The demonstration of RARs and retinoid binding proteins in the developing lung (Dolle et al., 1990)
suggests that RA-responsive signalling pathways may
participate in the structural and biochemical alterations we have observed. The potential role of RA in
lung development has been demonstrated by double
null mutations of RARs that result in the absence or
hypoplasia of the lung (Mendelsohn et al., 1994).These
observations lead us to propose that RA and associated
molecules may play important roles in normal lung
development.
A number of studies of developing systems suggest
that RA acts in part via its ability to influence the site
and time of expression of Hox genes. Hox genes are
present in the lung a t early stages of development (reviewed in Cardoso, 1995), although it has not been
shown that lung development depends on any single
~~
Fig. 5. In situ hybridization of Hoxa-2 mRNA expression in the developing rat lung at gestational days 13.5 (A, anti-sense; B, sense strand
negative control) and 18.5 (C, antisense; D, phase contrast), trachea at
gestational day 21 (E, antisense; F, phase contrast), control 5-day cultured lung (0)and 5-day 10-5MRA-treated lungs (H).Hoxa-2 mRNA
(arrows) is diffusely expressed in the mesenchyme of the 13.5 day lung.
Later in gestation and in culture Hoxa-2 is abundant in the mesenchyme
associated with smooth muscle layer of vessels (v), airways (a), and
cartilage (ct) in proximal airways; low Hoxa-2 signal is also seen diffusely
in distal mesenchyme of control cultured lungs. Under RA-treatment
Hoxa-2signal is strongly upregulated in the mesenchyme along the entire
proximal-distal axis of the lung. Magnification: A and 8, x 70; C and D,
x95: E and F, x50; G, x55; H, x60.
53
homeobox gene, as in the case of the spleen (Roberts et
al., 1994) or on a Hox code established by overlapping
domains of Hox gene expression, as in the case of the
limb (Hunt et al., 1991, Izpisua-Belmonte et al., 1990).
The main purpose of our study was to see if changes
in lung patterning were associated with changes in expression of pattern-related genes. Thus, we designed
the study to investigate Hox and Shh gene expression
a t the RA concentration that generated an easily recognizable patterning phenotype, without evidence of
cellular toxicity (i0-5M).
Although we expected to find an overall increase in
Hox gene expression in our system upon RA administration, we chose to study the RA effects on the expression of one anterior (Hoxa-2) and a relatively posterior
(Hoxb-6)Hox gene in the lung because they would be
expected to respond differently to RA (Simeone et al.,
1990) and because they have different patterns of expression along the respiratory tract; Hoxa-2 is present
in the embryonic trachea and lung, while Hoxb-6 is
confined to the lung itself (reviewed in Cardoso, 1995).
The in vivo and in vitro patterns of Hox gene expression seen in our study suggest that these genes play
distinct roles in lung development. Hoxa-2 appears to
participate in the formation of proximal mesenchymal
derivatives and vasculogenesis because its expression
is progressively restricted to the mesenchyme associated with differentiating cartilage primordia, airway
smooth muscle, and vessels. Hoxb-6 may be part of a
complex network that regulates lung distal epithelial
branching during development because its expression
appears to be spatially and temporally associated with
the process of formation of distal lung tubules. We speculate that such a network, like that described in other
systems (Rancourt et al., 1995), may involve cooperativity with other homeobox genes, such as H o x b d ,
which share the same pattern of expression with
Hoxb-6 (Holland and Hogan, 1988; Volpe and Nielsen,
1994), and the thyroid transcription factor-1 (TTF-1)
(Lazzaro et al., 19911,as well as several growth factors.
One of the problems of implicating Hox genes individually in lung development is that there is probably
functional overlap of these genes. Perhaps because of
such redundancy, targeted knockout of either Hoxa-2
or Hoxb-6 results in no abnormal lung phenotype (Rijli
et al., 1993; Gendron-Maguire et al., 1993; Rancourt et
al., 1995).
Shh is a signalling molecule in limb bud morphogenesis, present in the limb mesenchyme. Shh mRNA is
activated by RA and induces Hoxd gene expression
(Riddle et al., 1993; reviewed in Johnson and Tabin,
1995). Shh mRNA and protein are expressed at many
sites of epithelial-mesenchymal interactions, such as
the branchial arches, gut, respiratory and urinary tract
in the mouse embryo, where its role is still unclear
(Echelard et al., 1993; Bitgood and McMahon, 1995;
Marti et al., 1995). We found that Shh mRNA is expressed at high levels in the epithelium of the day
13-14 rat lung and trachea, with no proximal-distal
54
CARDOSO ET AL.
Fig. 6. In situ hybridization of Hoxb-6 mRNA expression in gestational
day 13.5 rat lung (A, antisense; 6,sense), gestational day 18.5 lung (C,
antisense; D, phase contrast), control 5-day cultured lung (E) and 5day
10M6MRA-treated lungs (F).Hoxbd signals (large arrows) are restricted
to the distal mesenchyme of the 13.5 day lung. Hoxb-6 mRNA levels are
almost undetectable later in gestation and in culture. Hoxb-6 is upregu-
lated in RA-treated lungs; no proximal-distal gradient of expression is
observed. The coarse grains of variable size (small double arrows in C
and E) are not true signal, and result from tissue birefringency. tr, trachea;
a, airways in the lung in vivo and in vitro. Magnification: A-D, x 80; E and
F, x 60.
RETINOIC ACID AND PATTERNING OF THE LUNG
55
Fig. 7. Effect of 12-hr exposures of the lung to RA 10-5M on Hoxa-2 (A, 8 ) and Hoxb-6 (C, D) mRNA
expression. Control (A, C). RA-treated (6,D) lungs. Short exposures to RA do not alter patterning of the lung;
however, they markedly upregulate Hoxa-2 mRNA signals (arrows in A and B) along its entire proximal-distal
axis. RA appears to extend ff~xb-6expression (arrows in C and D)to a slightly more proximal site. Magnification: A-D, x 50.
mRNA gradient. Lung maturation in vivo and in vitro
was associated with a decline in the Shh levels in the
epithelium; however in the presence of exogenous RA,
lung explants maintain Shh expression a t high levels
with a distribution similar to that seen in day 13-14
lungs (Fig. 8A and G). RA therefore prevents the normal decline in Shh mRNA seen a t late time points in
controls, and appears to hold the lung in a proximal
immature pattern. This hypothesis is further supported by histologic features of immaturity seen in the
proximalized epithelium (Fig. 2). Interestingly, at late
times in culture both control and RA-treated lungs express Shh and SP-C mRNA in distal epithelium in an
inverse fashion; when Shh is upregulated, SP-C is
downregulated and vice versa. In contrast, distal buds
of uncultured day 13.5 lungs simultaneously express
Shh (Fig. 8A) and SP-C (data not shown, Wert et al.,
1993)mRNAs, and likely both genes are present in the
same cells in the early lung bud. When RA is removed
from the culture media, newly formed epithelial buds
56
CARDOSO ET AL.
Fig. 8. In situ hybridization of Shh mRNA expression in the developing rat lung at gestational days 13.5 (A, inset shows isolated lung) and
18.5 (8, antisense; C, phase contrast), gestational day 21.5 trachea (D,
antisense; E, phase contrast), control 5-day cultured lung (F), and 5-day
l 0 - W RA-treated lungs (G, antisense; H, sense strand negative control).
Shh signals (arrows) are very strong in the epithelium of the day 13.5
esophagus (es), trachea (tr), and lung (lu). Shh mRNA is uniformly ex-
pressed along the proximal-distal axis of the respiratory tract. Shh signals
decrease in the airway epithelium of the lung and trachea later in gestation (8, D) and after 5 days in culture (F). RA markedly increases Shh
mRNA signals in the lung epithelium (G), and does not alter the proximaldistal mRNA expression pattern seen in controls. Magnification: A, F. G,
and H, x60; inset, x80; B and C, x70; D and E, x50.
express high levels of SP-C and low levels of Shh
mRNAs (Fig. 9) in a fashion that is comparable to controls of the same age. Thus it seems that RA withdrawal releases the lung from the proximalized pattern
and allows newly formed distal buds to express Shh
and SP-CmRNAs in a fashion that is typical of mature
lungs.
We have shown that RA rapidly alters Hox gene expression, prior to changes in lung morphology and
SP-C mRNA suppression. We hypothesize that such
early changes in Hox expression are a major determinant of subsequent alterations in developmental
events. Longer exposures to RA showed more marked
changes in both lung patterning and Hox gene expression. It is likely that in our model, RA acts by maintaining prolonged high levels of Hoxu-2, Hozb-6, and
other RA-responsive patterning genes from the Ho3t
family as well as Shh. The proximalizing phenomenon
may involve retinoid signalling molecules such as
RAR-p and RAR-y which are characteristically expressed in the proximal respiratory tract a t early
stages of the development (Dolle et al., 1990).
Difference in Hoxb-6 expression between day 5 control and RA-treated samples appeared greater by in
situ hybridization than by PCR analysis. Such difference may be ascribed to several factors and includes:
(1)variability in the response of the explants to RA in
the PCR analysis, as shown by standard deviations in
Figure 4; the mean value of Hoxb-6 expression in RA
treated samples appears twice that of controls and,
likely, an increase in the number of samples could demonstrate a statistically significant difference between
RETINOIC ACID AND PATTERNING OF THE LUNG
57
Fig. 9. In situ hybridizationof Sf-C(A)and Sbh(B) mRNA expression
in lungs treated with RA lO-’M for 3 days, washed out and cultured in
control medium for additional 2 days. Newly formed distal buds (arrowheads in A, B, and also shown in Fig. 1C) express Sf-C, a marker of
distal lung epithelium and Shh mRNA in the same fashion of control 5-day
cultured lungs (see Shh expression in distal lung shown in Fig. 8F and
Fig. 9s). The remaining proximalized tubules (large arrow in A and 6)do
not express SP-C (Fig. 9A), but express high levels of Shh mRNA (Fig.
9B) characteristic of the RA-treated lung epithelium (shown in Fig. 8G).
Magnification: A and B, x 65.
these groups; (2) Apparent non-linearity of in situ hybridization method on detection of signals, so that increases in Hoxb-6 expression appear more noticeable
when the baseline signal is low (as in controls); (3)
changes in the proportion of epithelium vs. mesenchyme; there is less mesenchyme in controls than in
RA-treated explants, thus, signals by in situ hybridization appear more prominent; (4) Lower sensitivity of
the PCR analysis, as compared to in situ hybridization
on detection of changes in Hoxb-6, which is less inducible by RA than other 3’ genes (such as Hoxu-2) (Simeone et al., 1990).
We have not yet characterized the molecular sequence of events and roles that Hox and Shh genes play
in lung patterning, but it is clear that exogenous RA
profoundly affects this process. Good candidates for
participating in this network are members of the fibroblast growth factor (FGF) family and their receptors.
Limb bud formation appears to be regulated by a network involving endogenous RA, Shh, Hoxb, and Hoxd
genes, as well as FGF-4 and FGF-8 (reviewed in Tabin,
1995). FGFs and FGF receptors are expressed in the
developing lung (Stark et al., 1991; Peters et al., 1992;
Han et al., 1992;Mason et al., 1994; Finch et al., 1995,)
and appear to have a crucial role in lung epithelial
branching (Peters et al., 1994; Nogawa and Ito, 1995).
Our studies suggest that molecular paradigms developed for skeletal structures, limb bud, and brain development apply in general to the development of branch-
ing visceral organs such as the lung. The proximalized
lung is intended to be a model where pattern formation
is altered; it does not reproduce a physiological situation, However, this model allows us to infer that high
concentrations of RA at early stages of development are
incompatible with distal lung formation, fostering a
more immature proximal phenotype.
ACKNOWLEDGMENTS
We thank Dr. Andrew P. McMahon (Harvard Biolabs) for the gift of the Shh cDNA clone. We thank
Barbara Miranda and Anne Hinds for technical assistance. This work was supported by grants from the
National Institute of HealtWNHLBI (PO1 HL47049
and R01 HL34768) and the Parker B. Francis Foundation.
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