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DEVELOPMENTAL DYNAMICS 217:62–74 (2000)
Regulation of the Hoxa4 and Hoxa5 Genes in the
Embryonic Mouse Lung by Retinoic Acid and TGF␤1:
Implications for Lung Development and Patterning
ALAN I. PACKER,1 KARIMI G. MAILUTHA,1 LORETTE A. AMBROZEWICZ,4 AND
DEBRA J. WOLGEMUTH1–5*
1
Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York
2
Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, New York
3
Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, New York, New York
4
Institute of Human Nutrition, Columbia University College of Physicians and Surgeons, New York, New York
5
Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York
ABSTRACT
We have previously described a
5‘ cis-acting retinoic acid response element that
is required for a subset of Hoxa4 expression, including the midgestation mouse lung. As both
retinoids and Hox genes have been implicated in
lung development and patterning, we have examined Hoxa4 expression in the developing mouse
lung and extended our work on its regulation. At
E12.5, a Hoxa4/lacZ transgene is expressed in the
mesenchymal compartment of the lung. Later in
development expression is restricted to the proximal mesenchyme and is also observed in smooth
muscle cells, subepithelial fibroblasts, and alveolar cells. We show that both Hoxa4 and Hoxa5 are
upregulated when cultured in the presence of
all-trans retinoic acid. In addition, retinoic acid
extends the domain of Hoxa4 and Hoxa5 expression to the periphery of the explants where the
distal epithelia are developing. Interestingly, the
effect of retinoic acid on Hoxa5 expression was
not observed in a Hoxa4 mutant background.
In contrast, TGF␤1 was found to downregulate
both Hoxa4 and Hoxa5 expression in cultured
lung explants. We also establish that retinoic
acid has the effect of proximalizing the mouse
lung when cultured in a serum-free medium,
as evidenced by reduced expression of the
distal marker surfactant protein-C. Lungs from
Hoxa4 mutant embryos exhibited a similar response to retinoic acid, suggesting that Hoxa4
alone is not required for the proximalizing
effect. Based on their retinoid-dependent expression, we conclude that members of the group
4 and/or group 5 Hox genes are likely to be involved in patterning of the mouse lung. Dev Dyn
2000;217:62–74. © 2000 Wiley-Liss, Inc.
of inductive interactions. These interactions, usually
involving reciprocal signals between contiguous epithelial and mesenchymal cell layers, are mediated by soluble and membrane-bound polypeptide growth factors,
adhesion molecules, and components of the extracellular matrix. In recent years a great deal of progress has
been made in identifying such factors through tissue
explant studies, in vivo gene targeting, and the analysis of gene expression patterns. In particular, a number
of the proteins and other molecules that are required
for the development of the mammalian lung have been
identified (reviewed in Hogan and Yingling, 1998). In
the mouse embryo, lung development begins at embryonic day 9.5 (E9.5) as the ventral foregut is induced to
grow caudally to form an epithelial outgrowth (the
pulmonary endoderm) surrounded by mesodermallyderived splanchnic mesenchyme. Thereafter, lung morphogenesis is divided into the pseudoglandular (E9.5E16.2), canalicular (E16.6-E17.4) and terminal sac
stages (E17.4-P5), during which branching morphogenesis and septation occur (Ten Have-Opbroek, 1991).
Among the secreted factors that have been found to
play critical roles in mouse lung development are EGF
(Miettinen et al., 1997); TGF␤1 (Serra et al., 1994;
Zhao et al., 1996; Zhao et al., 1998); FGF-1, 7, and 10
(Nogawa and Ito, 1995; Cardoso et al., 1997; Bellusci et
al., 1997b; Park et al., 1998); HGF (Ohmichi et al.,
1998); BMP-4 (Bellusci et al., 1996); and sonic hedgehog (Bellusci et al., 1997a; Pepicelli et al., 1998, Litingtung et al., 1998). Transcription factors such as N-myc
(Moens et al., 1992); TTF-1 (Minoo et al., 1995); Gli2
and 3 (Motoyama et al., 1998); Hoxa5 (Aubin et al.,
1997); and the retinoid receptors RAR␣ and ␤2 (Men-
Key words: Hoxa4; Hoxa5; retinoic acid; TGF␤1;
embryonic lung patterning
Grant sponsor: National Institutes of Health; Grant number: R01
HD18122; Grant sponsor: United States Department of Agriculture;
Grant number: 97–35200 – 4291.
*Correspondence to: Debra J. Wolgemuth, Ph.D., Department of
Genetics and Development, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032.
E-mail: djw3@columbia.edu
Received 16 July 1999; Accepted 6 October 1999
INTRODUCTION
The growth, patterning, and differentiation of tissues in metazoans is orchestrated by a complex series
© 2000 WILEY-LISS, INC.
Hox REGULATION AND LUNG PATTERNING
delsohn et al., 1994b) are also required for normal lung
development (reviewed in Cardoso, 1995).
The retinoid signaling pathway is of particular interest in lung biology. The RAR␣/␤2 double knockout results in agenesis of the left lung and hypoplasia of the
right lung (Mendelsohn et al., 1994). In addition, there
is evidence to suggest that maternal-fetal transmission
of retinol and/or retinoic acid is involved in lung maturation in the human fetus (reviewed in Chytil, 1992;
Shenai et al., 1992). Attempts to elucidate the mechanisms of action of retinoic acid in lung development
have focused on organ culture of the rat lung as a
model. The addition of all-trans retinoic acid to embryonic rat lung explants promotes the development of
proximal airways while suppressing the development
of distal epithelia (Cardoso et al., 1995). Proximal epithelial cells are relatively slowly proliferating cells surrounded by a layer of smooth muscle cells or cartilage.
These epithelia give rise to the bronchial portion of the
adult lung. Distal epithelial cells, by contrast, are relatively rapidly proliferating cells that lack surrounding
smooth muscle or cartilage, and they are fated to give
rise to the gas exchange surface of the lung—the respiratory acini.
It has been known for some time that the fate of the
various lung epithelia is completely dependent on signals from the adjacent mesenchyme. Tracheal, proximal, and distal mesenchymal cells specifically promote
the differentiation of their own cognate epithelium in
tissue recombination experiments where they are
grafted to a denuded endodermal layer (Wessells, 1970;
Shannon et al., 1998). That retinoic acid might be affecting lung epithelia via the mesenchyme was suggested by tissue and organ culture experiments employing the mouse embryonic lung as a model. Retinoic
acid was found to stimulate proliferation of both lung
mesenchymal and epithelial cells when co-cultured
(Schuger et al., 1993). However, when cultured separately, increased proliferation was observed only in the
mesenchymal cells, suggesting that the effect of retinoic acid on the epithelial cells was an indirect one
(Schuger et al., 1993). Interestingly, retinoic acid promoted increased branching of mouse embryonic lungs
in organ culture, but had no apparent effect on proximal/distal patterning of the kind that had been observed in retinoic acid-treated rat lung explants
(Schuger et al., 1993; Cardoso et al., 1995). Whether
the observed effects of retinoic acid are due to the direct
activation of the synthesis of mesenchymal-derived
growth factors, such as the EGFs or FGFs, or whether
there are intermediary transcription factors that carry
out this program, is not yet clear.
Among the genes whose expression has been examined in retinoic acid-treated rat lung explants are the
Hox genes, some of which had been shown previously to
be retinoid-responsive in midgestation mouse embryos
(Kessel and Gruss, 1991). Hox genes encode a family of
39 highly conserved transcription factors involved in
the patterning of axial and appendicular structures
63
(reviewed in Krumlauf, 1994) and particular organs
(Hsieh-Li et al., 1995; Manley and Capecchi, 1995).
In the cultured rat lung, Hoxa2 (Cardoso et al., 1996),
Hoxa5 (Bogue et al., 1994), and Hoxb6 (Cardoso
et al., 1996) were found to be upregulated by retinoic
acid. These Hox genes are expressed in the lung mesenchyme and it was proposed that they mediate the
effects of retinoic acid on lung patterning (Cardoso et
al., 1996). Recently, it was reported that mice
lacking the Hoxa5 gene die perinatally due to respiratory distress that arises from specific defects in lung
development (Aubin et al., 1997). The mid-late gestation lung in the Hoxa5 null embryo was found to have
a disorganized mesenchymal compartment, reduced
branching, thickened alveolar walls, and disorganization of the proximal bronchi and distal respiratory pathways (Aubin et al., 1997). These observations confirm that at least one Hox gene is essential for
lung development, and suggest that retinoid-dependent Hox gene expression may be involved in lung
patterning.
We and others have been studying the regulation
and expression of the Hoxa4 gene in mouse embryos.
An initial examination of Hoxa4 expression in the embryonic lung by in situ hybridization showed expression in the mesenchymal compartment at E12.5 and
E15.5 (Galliot et al., 1989). To begin a characterization
of Hoxa4 cis-acting elements, a line of transgenic mice
was generated in which it was demonstrated that 3.8
kb of 5' flanking sequence in addition to intronic sequences were necessary and sufficient to drive appropriate lacZ reporter gene expression in the embryo
(Behringer et al., 1993; Keegan et al., 1997). In the
lung, expression was reported at E11.5, specifically in
the lower half of the lung (Behringer et al., 1993).
Subsequently, it was found that a conserved retinoic
acid response element (RARE) in the 5' flanking region
(at ⫺2.9 kb) was specifically required for Hoxa4/lacZ
transgene expression in the gut, kidney, and lung when
expression was examined at E14.5 (Packer et al., 1998).
Given the retinoid-dependent expression of Hoxa4 in
the developing lung, the reported effects of retinoic acid
on the patterning of the rat lung in organ culture, and
the requirement for a neighboring gene, Hoxa5, in
mouse lung development, we have undertaken an analysis of Hoxa4 expression and regulation in the embryonic lung. We show that Hoxa4 is expressed at a uniformly high level in the lung in mid-late gestation, and
that the specification of its expression may be determined by the opposing effects of all-trans retinoic acid
and TGF␤1. In addition, we find that Hoxa5, expressed
in the lung in a pattern similar to that of Hoxa4, also
responds to all-trans retinoic acid and TGF␤1, but that
its response to retinoic acid is abolished in the Hoxa4
mutant background. Finally, we report that all-trans
retinoic acid can alter the patterning of cultured embryonic mouse lungs from both wild-type and Hoxa4
mutant backgrounds.
64
PACKER ET AL.
RESULTS
Expression of the Hoxa4/Lacz Transgene
in the Embryonic Mouse Lung
We have initiated a survey of Hoxa4 expression in
the embryonic mouse lung through the use of our
Hoxa4/lacZ transgenic line (Behringer et al., 1993). At
E9.5 and E10.5, we observed no transgene expression
in the developing lung (not shown), suggesting that
expression is initiated at or around E11.5. At E12.5,
transgene expression was restricted to the mesenchymal compartment in the lower half of the lobes (Fig.
1A). Of particular interest is the modestly elevated
expression apparent in the mesenchymal cells that are
directly apposed to the developing epithelia (Fig. 1A).
After E12.5 it is no longer possible to distinguish reasonably well between epithelial and mesenchymal compartments in the intact lung, but it is clear at E14.5
that expression of the Hoxa4/lacZ transgene persists at
a high level and in a somewhat polarized pattern, with
expression specifically absent in the distal, peripheral
regions of the lung (Fig. 1B). High level expression
continued at E16.5 (Fig. 1C) and E18.5 (Fig. 1D) as the
domain of transgene expression was expanded.
Sections of the stained lungs revealed the particular
cell types expressing Hoxa4. As was evident in the
whole lung, expression of the transgene was first seen
in the mesenchymal cells at E12.5 (Fig. 2A). At E14.5,
in addition to the expression in the mesenchyme, staining was also observed in the smooth muscle cells surrounding the proximal epithelial cells of the bronchi
(Fig. 2B). At E16.5 expression was also occasionally
seen in subepithelial fibroblasts (Fig. 2C). Finally, at
E18.5 expression was apparent in cells of the maturing
alveoli (Fig. 2D). This dynamic pattern of expression
suggests roles both in patterning of the lung via mesenchymal-epithelial signaling, and in the differentiation of particular cell types.
Retinoid-Responsiveness of the Endogenous
Hoxa4 and Hoxa5 Genes in the Embryonic Lung
As indicated in the Introduction, exogenous retinoids
have been found to promote the expression of particular Hox genes in rat lung explants (Cardoso et al.,
1996). In addition, our previous results indicate that
expression of the murine Hoxa4 gene in the lung requires an intact 5' retinoic acid response element
(Packer et al., 1998). To extend these results we examined the expression of the endogenous Hoxa4 gene in
response to exogenous all-trans-retinoic acid in an organ culture setting. In these experiments we cultured
E14.5 lung explants on 0.8 ␮m Millipore filters in serum-free DMEM/F12 medium (see Experimental Procedures for details of the culture protocol). Initially we
cultured the lung explants for 4 hr with or without 10⫺5
M all-trans retinoic acid (Cardoso et al., 1996). This
was followed by whole-mount in situ hybridization
with a digoxigenin-labeled Hoxa4 riboprobe. The pattern of expression of the endogenous Hoxa4 gene in an
untreated explant was quite similar to the pattern of
Hoxa4/lacZ transgene expression in that there was a
distal, peripheral region where expression was not detected (compare Fig. 1B (transgene) and Fig. 3A (endogenous)). In addition, sections of untreated explants
revealed that expression was restricted to the mesenchymal compartment, as observed for expression of the
transgene, again suggesting that the transgene expression faithfully recapitulates Hoxa4 expression in the
lung (compare Fig. 2A (transgene) and Fig. 3C, D (endogenous)). No specific staining was observed with a
sense Hoxa4 riboprobe (not shown).
Retinoic acid treatment (10⫺5 M) resulted in an expansion of the domain of expression of Hoxa4 in the
lung (Fig. 3B). This expansion was also seen in sections
of retinoic acid-treated explants where mesenchymal
expression was found to extend to peripheral regions
surrounding the distal epithelia (Fig. 3D, F). Since
Hoxa5 expression had been shown to be responsive to
retinoic acid in the rat lung (Bogue et al., 1994), and
since mice lacking Hoxa5 exhibit significant lung defects (Aubin et al., 1997), we also examined the effect of
retinoic acid on Hoxa5 in our culture system. E14.5
lung explants were cultured for 4 hr as before either in
medium alone or in medium containing 10⫺5 M alltrans retinoic acid. Whole-mount in situ hybridization
with a digoxgenin-labeled Hoxa5 riboprobe revealed
that the effect of retinoic acid on Hoxa5 expression was
at least as robust as that observed on Hoxa4. Hoxa5
expression in the untreated lung was found to be restricted to the proximal mesenchyme and was essentially identical to that of Hoxa4 (Fig. 4A). This pattern
is similar to that reported by Aubin and colleagues
(1997) in that it is restricted to the mesenchyme, although they did not describe the polarized expression
that we observe here. The retinoic acid-treated lung
clearly exhibited an increased intensity of Hoxa5 expression that extended to the entire region of the explant, including the most distal regions (Fig. 4B). We
also examined the response of each gene to a lower
concentration of retinoic acid (10⫺6 M). This concentration also resulted in an expansion of Hox gene expression, but it appeared to be less dramatic than that
observed in response to 10⫺5 M (data not shown).
Given the demonstrated importance of Hoxa5 in lung
development (Aubin et al., 1997), the overlap of Hoxa4
and Hoxa5 expression in the lung at E12.5–E14.5, and
the similar response of each gene to exogenous retinoic
acid, we were interested in determining if Hoxa4 is in
fact required for the response of Hoxa5 to retinoic acid.
It has already been demonstrated that Hoxa4 is required for Hoxa5 expression in the anterior portion of
the prevertebral column (Aubin et al., 1998). When we
examined retinoic acid-treated E14.5 lung explants
from embryos lacking Hoxa4 we found that there was
no increase in Hoxa5 expression by whole-mount in
situ hybridization (Fig. 4C, D). We suggest that the
effect of retinoic acid on Hoxa5 is mediated by a shared
Hoxa4/Hoxa5 cis-acting enhancer whose activity is af-
Hox REGULATION AND LUNG PATTERNING
fected by the neo insertion used to generate the Hoxa4
mutation (see Discussion).
Effect of TGF␤1 on Hoxa4 and Hoxa5
Expression in the Embryonic Lung
A recent study examining cultured murine fetal
lungs presented evidence that TGF␤1 (2 ng/ml) could
reduce the levels of Hoxb5 protein after three days of
culture (Chinoy et al., 1998). This prompted us to examine Hoxa4 and Hoxa5 expression in cultured E14.5
murine lungs after 18 hr of exposure to TGF␤1 at the
same concentration. Overall, the effects of TGF␤1 that
we observed were the opposite of those observed in
response to all-trans retinoic acid. By whole-mount in
situ hybridization, Hoxa4 (Fig. 5A, B) and Hoxa5 (Fig.
5C, D) expression was modestly reduced. A direct comparison of the effects of retinoic acid and TGF␤1 on
Hoxa4 expression is shown in Fig. 5E, highlighting the
opposing effects of these two factors in determining the
distribution of Hoxa4 mRNA in the lung.
Effect of All-Trans Retinoic Acid on Lung
Morphology
Previous reports have established that all-trans retinoic acid can affect the patterning of the developing rat
lung by promoting the formation of proximal airways
and suppressing the formation of distal airways (Cardoso et al., 1995, 1996). However, the addition of exogenous retinoic acid to mouse embryonic lung explants
was reported to promote branching morphogenesis
with no apparent effect on lung patterning (Schuger et
al., 1993). The difference in the responses of rat and
mouse tissue to retinoic acid might be attributed to
differences in the culture protocols. For example, the
medium used by Schuger and colleagues contained 2%
fetal calf serum to arrest branching and a maximum
concentration of 10⫺6 M retinoic acid. In contrast, Cardoso and colleagues used a serum-free medium and up
to 10⫺5 M retinoic acid to establish its effect on patterning. To determine if we could demonstrate an effect
of retinoic acid on lung patterning in the mouse, we
cultured E12.5 explants in our standard, serum-free
culture medium for 48 hr. Examples of such cultures
are shown in Figures 6A and B. The explant cultured in
the presence of 10⫺5 M retinoic acid (Fig. 6B) clearly
differed in appearance from the explant cultured in the
absence of retinoic acid (Fig. 6A). In particular, the
morphology of the epithelia appeared to have been
affected by the retinoic acid treatment in that large
airways now extended to the periphery of the lung.
To determine if the effect of retinoic acid on the
mouse lung was similar to that reported for the rat, we
carried out the same culture experiment followed by
whole-mount in situ hybridization with a digoxigeninlabeled riboprobe for surfactant protein-C (SP-C). Expression of SP-C has been shown to be restricted to the
distal epithelia of the lung (Cardoso et al., 1995). Figures 6C and D show that there is a dramatic decrease
in SP-C expression in the mouse lung explant treated
65
with retinoic acid, indicating that retinoic acid has
affected the differentiation pathway of the epithelia in
the cultured mouse explant. Figures 6E and F show
sections of these lungs, again demonstrating the loss of
SP-C expression. The normal pattern of SP-C expression in untreated lungs also corresponded with the
degree of epithelial cell proliferation, as evaluated by
bromodeoxyuridine uptake in E14.5 lung explants over
a 24-hour period (Fig. 5G). Most of the characteristically rapidly proliferating cells of the distal airways
were located at the periphery of the explant, roughly
overlapping with the pattern of SP-C expression. The
columnar, proximal epithelial cells were located in the
interior of the explant and exhibited significantly less
proliferation.
Effect of Retinoic Acid on Lung Morphology
in the Absence of Hoxa4
It is likely that the dramatic effects of retinoic acid on
lung patterning are mediated by a group of factors
rather than a single downstream target. Such factors
must meet several criteria, including i) expression in
the mesenchyme, since the development of the epithelia—including the effects of retinoids on epithelia—
requires the presence of mesenchymal cells; ii) sensitivity to exogenous retinoids and/or direct activation by
retinoids through a cis-acting retinoic acid response
element; and iii) a demonstration that the effect of
retinoic acid on lung patterning is in some way altered
by the absence of the factor. Previous studies in the rat
have suggested that Hox genes are good candidates,
particularly since particular genes like Hoxa2 and
Hoxb6 are expressed in the lung mesenchyme in a
polarized manner and are responsive to retinoic acid
(Cardoso et al., 1996). However, the rat is not a convenient genetic model wherein the effect of retinoic acid
Fig. 1. (Figure on following page.) Expression of the Hoxa4/lacZ transgene in the mouse lung from E12.5 to E18.5. In all figures, “proximal” refers
to the central/rostral regions of the lung, i.e., the bronchial region of the lung,
while “distal” refers to the more peripheral and caudal regions of the lung. A:
Expression at E12.5 is restricted to the mesenchymal compartment in the
lower half of the lobes (arrow). Arrowheads indicate slightly elevated expression in mesenchymal cells directly apposed to the developing epithelia. B:
Expression at E14.5 is observed in proximal regions of the lung (P), with the
most distal, peripheral region of the lung exhibiting no expression of the
transgene (D). Rings of expression surrounding developing proximal epithelia are apparent (arrowhead). C: Expression at E16.5 is again observed in
more proximal regions of the lung (arrow), now slightly expanded when
compared to the expression at E14.5. D: Expression at E18.5 is observed
throughout the lung.
Fig. 2. (Figure on following page.) Sections of embryonic lungs expressing the Hoxa4/lacZ transgene. A: Sections reveal that expression at
E12.5 is restricted to the mesenchymal compartment (M) and excluded
from the epithelial cells (E). B: In sections at E14.5, expression is also
observed in the smooth muscle cells (arrowheads) surrounding the epithelial cells of a proximal bronchus. Note the tall columnar epithelium
characteristic of proximal airways (arrow). C: In sections at E16.5, expression is observed in subepithelial fibroblasts (arrowheads). D: In
sections at E18.5, expression is seen in squamous epithelial cells of
maturing alveoli (arrowheads).
Figure 1.
(Legend on preceding page.)
Figure 2.
(Legend on preceding page.)
Hox REGULATION AND LUNG PATTERNING
Fig. 3. Effects of all-trans retinoic acid on expression of the endogenous Hoxa4 gene in the embryonic mouse lung. E14.5 lung explants
were cultured for 4 hr (see Experimental Procedures) and Hoxa4 expression determined by whole-mount in situ hybridization. A: In a whole lung
explant cultured in the absence of retinoic acid, expression of Hoxa4 is
observed in proximal regions (P), but not in distal regions (D). B: A whole
lung explant cultured in the presence of retinoic acid exhibiting a large
expansion of Hoxa4 expression to more distal, peripheral regions (compare with A). C: 10⫻ magnification of a section of a lung explant cultured
in the absence of retinoic acid shows mesenchymal Hoxa4 expression in
proximal regions of the lung (P) and lack of expression in epithelia. The
67
bracketed regions include the most distal regions (D) containing epithelia
that are not in contact with Hoxa4-expressing mesenchyme. D: 10⫻
magnification of a section of a lung explant cultured in the presence of
retinoic acid. The area of Hoxa4-expressing mesenchyme now extends
to the periphery of the lung. The bracketed areas indicate that the distal
(D), non-expressing regions are only one epithelial layer wide. E: 25⫻
magnification of the section shown in C, with bracketed regions again
identifying distal epithelia (D) surrounded by Hoxa4-free mesenchyme. F:
25⫻ magnification of the section shown in D, with bracketed regions
enclosing the distal-most epithelia (D) in the explant that are nonetheless
in contact with Hoxa4-expressing mesenchyme.
68
PACKER ET AL.
Fig. 4. Effects of all-trans retinoic acid on expression of the endogenous Hoxa5 gene in the embryonic mouse lung. A: In a whole lung
explant at E14.5 cultured in the absence of retinoic acid, expression of
Hoxa5 is restricted to proximal regions (P) and excluded from more distal
regions (D). B: A whole lung explant cultured in the presence of 10⫺5 M
retinoic acid exhibits elevated Hoxa5 expression and an expansion of that
expression to include more distal regions of the explant (compare with A).
C: Hoxa5 expression in an E14.5 lung explant from a Hoxa4 mutant
embryo cultured for 4 hr in the absence of retinoic acid. D: Hoxa5
expression in an E14.5 lung explant from a Hoxa4 mutant embryo cultured for 4 hr in the presence of 10⫺5 M retinoic acid. There is no
significant effect of retinoic acid on Hoxa5 expression in this mutant
background (compare with C).
on lung patterning can be tested on gene-targeted embryos.
In order to determine if the absence of Hoxa4 alone
can alter the proximalizing effects of retinoic acid on
the mouse lung in culture, we carried out an additional
explant experiment in which E12.5 lungs from a Hoxa4
mutant background were cultured with or without
10⫺5 M retinoic acid. The 48-hour culture period was
followed by whole-mount in situ hybridization with an
SP-C riboprobe to examine the patterning of the lung
epithelia. As shown in Figure 7, the effect of retinoic
acid on SP-C expression is similar to that observed in
lungs from a wild-type background (compare to Fig.
6C,D).
DISCUSSION
Hox Gene Expression in the Embryonic
Mouse Lung
We have described the expression of a Hoxa4/lacZ
transgene in the embryonic and fetal mouse lung
from E9.5 to E18.5. At E12.5 expression was restricted to the mesenchymal compartment of the
lung, with strongest expression in mesenchymal cells
apposed to the epithelia. At E14.5 expression in the
mesenchyme persists, particularly in regions surrounding the proximal epithelia, and was also observed in smooth muscle cells. Expression in subepithelial fibroblasts and alveolar cells was seen at
E16.5–E18.5. We had demonstrated previously that
Hox REGULATION AND LUNG PATTERNING
69
Fig. 5. Effects of TGF␤1 on Hoxa4 and Hoxa5 expression in the
embryonic mouse lung. E14.5 lung explants were cultured for 18 hr
followed by whole-mount in situ hybridization. A: Hoxa4 expression in an
explant cultured in the absence of TGF␤1. B: Hoxa4 expression in an
explant cultured in the presence of TGF␤1 (2 ng/ml). Note the reduced
level of expression when compared to the untreated explant. C: Hoxa5
expression in an explant cultured in the absence of TGF␤1. D: Hoxa5
expression in an explant cultured for in the presence of TGF␤1 (2 ng/ml).
Note the reduction in Hox gene expression in response to TGF␤1 treatment. E: Comparison of Hoxa4 expression in E14.5 lung explants cultured in retinoic acid (10⫺5 M) for 4 hr (left) and cultured in TGF␤1 (2
ng/ml) for 18 hr (right) and processed in parallel. Again, note the differences in the level of expression, particularly in the distal regions of the
lung where Hoxa4 transcription has been promoted by retinoic acid.
this transgene faithfully recapitulates the pattern of
expression of the endogenous Hoxa4 gene in the embryo, although the lung was not examined in detail
(Behringer et al., 1993; Packer et al., 1998). This is
supported in the present study by whole-mount in
situ hybridization experiments at E14.5 showing
70
PACKER ET AL.
that, as for the Hoxa4/lacZ transgene, Hoxa4 is expressed in the proximal mesenchyme.
Most of the initial reports of Hox function in vertebrate embryos emphasized their role in the specification of the axial and appendicular skeletons, with functions similar to those suggested for the Drosophila
homeotic genes in patterning metameric units of the
embryo (reviewed in Krumlauf, 1994). More recent
studies have established that Hox genes can also function in the patterning of embryonic structures that are
polar but not metameric, such as the urogenital tract
(Hsieh-Li et al., 1995) and possibly the hindgut (Roberts et al., 1995) and heart (Searcy and Yutzey, 1998).
A similar role has been proposed for Hox genes in lung
development (Kappen, 1996; Mollard and Dziadek,
1997). Of the 39 Hox genes, 20 are expressed either in
the fetal lung, the neonatal lung, or both (reviewed in
Cardoso, 1995; Kappen, 1996). Of note is Hoxa5, whose
expression in the proximal mesenchyme (Aubin et al.,
1997; this study) is required for normal lung development (Aubin et al., 1997). Most similar to the pattern of
Hoxa4 expression, Hoxa2 is restricted to the proximal
mesenchyme and mesenchymal derivatives (Cardoso et
al., 1996), and Hoxb5 is found in mesenchymal cells at
E13.5 and later in subepithelial fibroblasts and late
fetal epithelial cells (Volpe et al., 1997). Some Hox
genes, like Hoxb3 and Hoxb4, are uniformly expressed
throughout the lung mesenchyme (Bogue et al., 1996).
Others, like Hoxb6, exhibit a different pattern of expression, being restricted to mesenchyme surrounding
the distal epithelia (Cardoso et al., 1996). Although
Hoxa5 is the only Hox gene for which there is definitive
evidence from gene targeting for a role in lung development, the varying patterns of expression of many
other Hox genes along the proximal-distal axis suggest
that together these genes have significant roles in lung
development and patterning. The elevated expression
of Hoxa4 at E12.5 in cells directly apposed to the epi-
Fig. 6. Effect of all-trans retinoic acid on lung morphology. A: E12.5
lung explant cultured for 48 hr in the absence of retinoic acid. Note the
characteristic T-shaped morphology of the distal epithelia at the periphery
of the explant (arrowheads). B: E12.5 lung explant cultured for 48 hr in
the presence of 10⫺5 M retinoic acid. Note the large airways extending to
the periphery of the explant (arrowheads). C E12.5 lung explant cultured
for 48 hr in the absence of retinoic acid and processed for whole-mount
in situ hybridization with an SP-C riboprobe. Expression is confined to the
distal epithelia. D: E12.5 lung explant cultured for 48 hr in the presence
of 10⫺5 M retinoic acid and processed for whole-mount in situ hybridization with an SP-C probe. SP-C expression is greatly reduced, suggesting
that retinoic acid has altered the differentiation pathway of the epithelial
cells to a more proximal fate. Large airways can be seen extending to the
periphery of the explant (arrowheads). E: Section of the untreated explant
showing SP-C expression in the distal epithelial cells. F: Section of the
retinoic acid-treated explant showing reduced SP-C expression. G: Bromodeoxyuridine incorporation into E14.5 explants cultured for 24 hr. Most
of the proliferating cells are in the cuboidal epithelia at the edge of the
tissue (arrowheads), corresponding to the region of highest SP-C expression (compare with E). Columnar epithelial cells lining the more proximal
airways in the interior of the explant exhibited much less proliferation (P).
thelia, followed by its expression at E14.5 in smooth
muscle cells surrounding developing proximal airways,
suggests that Hoxa4 might have a role in the development of the smooth muscle cell lineage. Interestingly,
overexpression of Hoxa4 in the developing gut appears
to affect the differentiation of smooth muscle cells from
Hox REGULATION AND LUNG PATTERNING
71
Fig. 7. Effect of all-trans retinoic acid on lung morphology in the
absence of Hoxa4. A: E12.5 lung explant from a Hoxa4 mutant embryo
cultured for 48 hr and processed for SP-C whole-mount in situ hybridization. B: E12.5 lung explant from a Hoxa4 mutant embryo cultured for 48
hr in the presence of 10⫺5 M retinoic acid and processed in parallel with
the wild-type explant. The reduction in SP-C expression observed upon
retinoic acid treatment is comparable to that observed in wild-type explants (see Fig. 6C, D).
the enteric mesenchyme (Tennyson et al., 1993), suggesting a possible parallel function.
have been identified (Hoxa1, Hoxb1, Hoxa4, Hoxb4,
Hoxd4), only the three group 4 paralogs have been
identified in the embryonic lung, with Hoxd4 expression lost after E12.5 (Gaunt et al., 1989). We suggest
that Hoxa4 might be a central transcription factor in
the mesenchymal response to endogenous retinoids
during lung patterning and branching morphogenesis.
That mice lacking Hoxa4 have no readily apparent
lung defects (Horan et al., 1994) might be due to functional redundancy with other Hox genes, particularly
the group 4 paralogs Hoxb4 and Hoxd4, both of which
are expressed in the embryonic lung (see below). In
addition, the dramatic lung phenotype observed in the
RAR␣/␤2 knockout mice (Mendelsohn et al., 1994) suggests that other genes expressed very early in lung
development must be essential downstream targets for
retinoids.
The inability of retinoic acid to promote expression of
Hoxa5 in the Hoxa4 mutant background is also of interest. Although Hoxa4 is required for expression of
Hoxa5 in the anterior prevertebrae, its expression elsewhere in the embryo is apparently Hoxa4-independent
(Aubin et al., 1998), suggesting that it is unlikely that
Hoxa4 protein is required in trans to promote Hoxa5
expression in the lung. A more attractive possibility is
that a retinoid-dependent, lung-specific enhancer is
shared by Hoxa4 and Hoxa5. Interestingly, an 11.1-kb
genomic fragment of the mouse Hoxa5 locus fails to
promote reporter gene expression in the lung, suggesting that a lung-specific element might be shared with a
Retinoid-Dependent Hox Gene Expression
in the Embryonic Mouse Lung
In addition to our previous work showing the dependence of Hoxa4 lung expression on a conserved retinoic
acid response element (Packer et al., 1998), we show
here that exogenous retinoic acid can promote the expression of both Hoxa4 and Hoxa5 expression in the
proximal mesenchyme at E14.5. In addition to upregulating their expression, retinoic acid also expands the
domain of expression of both genes into mesenchymal
cells that surround distal epithelia at the periphery of
the lung at E14.5. This is similar to the effect of retinoic
acid on the expression of the Hoxa2 gene in the rat lung
(Cardoso et al., 1996). It has been proposed that the
proximalizing effect of retinoids on the lung might be
due to their effect on downstream target genes in the
mesenchyme whose polarized distribution would be altered by elevated levels of retinoic acid (Cardoso et al.,
1996). Both the ubiquitously expressed RAR␣ and
RAR␤2 and the mesenchyme-specific RAR␥ are the
candidate retinoid receptors to mediate retinoid function in the lung mesenchyme (Dollé et al., 1990). Interestingly, of the three Hox genes whose polarized expression in the proximal mesenchyme has been
established (Hoxa2, Hoxa4, Hoxa5), a cis-acting retinoic acid response element has been identified only for
Hoxa4. Of the five Hox genes for which such elements
72
PACKER ET AL.
more 3' Hox gene such as Hoxa4 (Larochelle et al.,
1999). Transcriptional interference in cis by the inserted neo cassette in the Hoxa4 mutant might then
interfere with the response of Hoxa5 to exogenous retinoic acid.
Opposing Effects of Retinoic Acid and TGF␤1
on Hoxa4 and Hoxa5 Expression
Since the retinoid receptors and retinoid binding proteins expressed in the embryonic lung mesenchyme do
not appear to be restricted along the proximal-distal
axis (Dollé et al., 1990), the specification of Hoxa4
expression in the proximal mesenchyme may depend
on an inhibitory factor in the distal mesenchyme.
There is very little known about tissue-specific inhibition of Hox gene transcription, but, as stated earlier,
there was a report of downregulation of Hoxb5 protein
expression in the lung in response to TGF␤1 after three
days in culture (Chinoy et al., 1998). Our results indicate that TGF␤1 can inhibit Hoxa4 and Hoxa5 expression in E14.5 explants after only 18 hr.
TGF␤-dependent signal transduction appears to depend on the Smad family of proteins. A recent study
suggests that the Smad3 and Smad4 proteins exhibit
sequence-specific DNA binding to TGF␤-inducible elements in the promoter of the human plasminogen activator inhibitor-type 1 gene (Dennler et al., 1998). Two
of these “CAGACA” elements can be found in the 5'
Hoxa4 regulatory region, at ⫺2.1 kb (AAACAGACA)
and ⫺1.8 kb (GCACAGACA), although it is not known
whether these are actually functional elements mediating TGF␤1-dependent inhibition of Hoxa4 expression. Mutagenesis of these sequences may determine
their roles, if any, in vivo. Perhaps most relevant to this
study, the essential Smad signaling protein, Smad4, is
expressed most highly in the distal mesenchyme in the
lung (Zhao et al., 1998). This leads to a possible model
of Hoxa4 regulation in the embryonic lung, whereby
retinoids activate Hoxa4 throughout the mesenchyme,
followed by TGF␤1/Smad4-dependent inhibition in the
distal mesenchyme. Alternatively, other members of
the TGF␤ gene superfamily may play a role in inhibiting Hox gene expression in the lung, including BMP4,
which is highly expressed in the distal endoderm (Bellusci et al., 1996).
The Role of Retinoid-Dependent Hoxa4
Expression in Lung Development and
Patterning
We have shown that exogenous all-trans retinoic acid
has the effect of proximalizing E12.5 mouse lung explants as evidenced by loss of expression of the distal
epithelial marker SP-C. The establishment of this effect in the mouse model should allow for the identification of molecules downstream of the retinoids that
are required in the mesenchyme for patterning of the
lung epithelium. To this end we have tested the effects
of retinoic acid on lung development in the absence of
Hoxa4. We find that the absence of Hoxa4 by itself does
not affect the ability of exogenous retinoic acid to proximalize the lung as measured by SP-C expression. As
discussed earlier, given the partial redundancy of Hox
gene function observed in many experimental settings,
(Horan et al., 1995) it is likely that two or more Hox
genes are required to mediate the patterning effects of
retinoic acid in the lung. The Hoxb4 gene, expressed in
the embryonic and fetal lung, was recently shown to
regulated by a retinoic acid response element (Gould et
al., 1998), raising the possibility that it has an essential retinoid-dependent function in the lung. Alternatively, retinoids may be affecting lung patterning
through the regulation of another class of molecules, as
yet unidentified.
Further experiments such as these, carried out in
varying mutant backgrounds, may allow for the identification of developmental pathways and networks
that operate in the embryonic and fetal lung. Moreover,
Hox genes are expressed in the mesenchymal compartments of other tissues such as the kidney, gut, and
reproductive tract, where retinoid receptors have been
identified. This approach may therefore be useful in
identifying signaling pathways in those tissues where
retinoids and mesenchymal-epithelial interactions are
known to be essential for proper development.
EXPERIMENTAL PROCEDURES
Mice
The generation of the Hoxa4/lacZ transgenic and
Hoxa4 mutant lines has been described (Behringer et
al., 1993; Horan et al., 1994). For timed matings, the
day of the detection of the vaginal plug was considered
embryonic day (E) 0.5, and the developmental stage
was determined according to Theiler (1972). Genotyping of Hoxa4 mutant embryos was carried out by
Southern analysis using a Hoxa4 digoxigenin-labeled
probe (Horan et al., 1994).
␤-Galactosidase Detection
Embryonic tissues carrying a lacZ reporter gene
were fixed, processed, stained with X-gal (Bachem, Torrance, CA) and processed for histology as described
previously (Behringer et al., 1993).
Embryonic Lung Organ Culture
Embryonic tissues were dissected, rinsed briefly in
phosphate-buffered saline, pH 7.4, and placed on floating 0.8 ␮m nitrocellulose filters (Millipore, Bedford,
MA) in a Falcon 24-well plate. Care was taken to place
the tissues at the liquid/air interface. The culture medium (1 ml/well) consisted of DMEM/F12 (Gibco, Grand
Island, NY) containing 1X insulin/transferrin/selenium
(ITS) supplement (Gibco) and 1% penicillin/streptomycin. All cultures were maintained in a humidified 37°C
incubator at 5% CO2. All-trans-retinoic acid-treated
cultures (Sigma Chemical Company, St. Louis, MO)
contained 10⫺5 M all-trans-retinoic acid diluted from
the original stock in dimethyl sulfoxide (DMSO) while
control cultures contained DMSO alone at an equiva-
73
Hox REGULATION AND LUNG PATTERNING
lent concentration. Cultures treated with human
TGF␤1 (Genzyme) contained 2 ng/ml diluted in
DMEM/F12 culture medium from the original stock. In
cultures maintained for 48 hr, the medium was replenished after 24 hr.
Whole-Mount In Situ Hybridization
At the end of the culture period, tissues were fixed
overnight in freshly prepared 4% paraformaldehyde
and then processed for whole-mount in situ hybridization according to Wilkinson (1993). Riboprobes were
labeled with digoxigenin using the Boehringer Mannheim RNA labeling kit, according to the manufacturer’s
instructions. The template for synthesis of the Hoxa4
antisense riboprobe was a 0.8 kb XbaI-HindIII fragment corresponding to the 3' untranslated region. The
template for synthesis of the Hoxa5 antisense riboprobe was a 1.2 kb EcoRI-HindIII insert (Watrin and
Wolgemuth, 1993). This template was linearized at an
internal BglII site to allow synthesis of a 0.8 kb probe
that excludes the homeobox. The template for synthesis of the SP-C probe was the 571 base pair rat cDNA
described in Cardoso et al. (1995). Whole-mount in situ
hybridization was carried out exactly according to
Wilkinson (1993), except that the prehybridization and
hybridization steps were performed at 70°C. Occasionally, fixed tissues were stored at ⫺20°C in 100% methanol for up to 10 days before in situ hybridization. No
significant differences in staining were observed in
stored tissues. The alkaline phospatase-coupled antidigoxigenin antibody and NBT/BCIP detection reagents were from Boehringer Mannheim. Following
staining, tissues were post-fixed overnight in 4% paraformaldehyde, placed in methanol for 10 min and isopropanol for 15 min, and embedded in paraffin. Sections were cut at 15 ␮m, deparaffinized in Histoclear
for 2 min, and mounted with the aqueous mounting
medium Crystal/Mount (Biomeda). All photomicrographs were taken using Fujicolor 100 film. Sections
were viewed and photographed on a Leitz photomicroscope under bright field optics. Whole tissues were
viewed and photographed on a Wild Heerbrugg photomicroscope under dark field optics.
Bromodeoxyuridine Labeling
and Immunohistochemistry
To examine cell proliferation, lung explants were
cultured in the presence of 10 ␮M bromodeoxyuridine
(Sigma) for 24 hr. Explants were fixed overnight in 4%
paraformaldehyde, dehydrated, embedded in paraffin,
sectioned at 6 ␮M, and immunohistochemistry was
carried out with a monoclonal antibody specific for
bromodeoxyuridine (Sigma; 1:1000 dilution) and Vectastain ABC reagents (Vector Labs, Burlingame, CA)
as described previously (Packer et al., 1997).
ACKNOWLEDGMENTS
We are grateful to Chris Marshall and Xiangyuan
Wang for excellent technical assistance and to Dr.
Wellington Cardoso (Boston University) for providing
the SP-C probe. We also thank Dr. Jeanine D’Armiento
for a critical review of the manuscript. This work was
supported by NIH grant R01 HD18122 to D.J.W., a
minority supplement to NIH R01 HD18122 to K.G.M.,
and USDA grant 97–35200 – 4291 to A.I.P.
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