1 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA
2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA
* Author for correspondence (e-mail: b.hogan{at}cellbio.duke.edu)
Accepted 5 January 2005
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SUMMARY |
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Key words: Lung development, Nmyc, Conditional inactivation, Progenitor cells, Sox proteins, Growth, Differentiation
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Introduction |
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Nmyc is a member of a small family of proto-oncogenes (Myc,
Lmyc and Nmyc) encoding basic helix-loop-helix-leucine zipper
(bHLHZ) proteins. Myc proteins can either activate or repress transcription by
interacting with specific binding partners, and by recruiting cofactors,
including histone and chromatin protein modifying enzymes, to the vicinity of
a very large number of target genes (for reviews, see
Eisenman, 2001;
Levens, 2003
;
Patel et al., 2004
;
Zeller et al., 2003
) (see also
http://www.myccancergene.org/site/mycTargetDB.asp).
Myc proteins appear to coordinate many interdependent processes, including
cell growth (increase in cell mass), cell proliferation (DNA replication and
cell cycle progression), differentiation and apoptosis. Recent gain- and
loss-of-function studies in the embryonic nervous system have highlighted
roles for Nmyc both in promoting cell cycle progression in
undifferentiated progenitor cells and in inhibiting their differentiation in
response to specific signaling pathways. In mouse cerebellar granule neuron
progenitors, for example, Nmyc is directly upregulated by sonic
hedgehog acting as a mitogen, and Nmyc overexpression in the same
cells stimulates cyclin D1 accumulation and cell cycle progression
(Kenney et al., 2003
).
Moreover, recent studies have shown that Nmyc protein is partially stabilized
in neuronal cells by activity of the PI3K pathway
(Kenney et al., 2004
). By
contrast, conditional deletion of Nmyc in the embryonic nervous
system results in a decrease in the pool of granule cell precursors, largely
due to an increase in their expression of cell cycle inhibitors and premature
differentiation (Knoepfler et al.,
2002
).
As indicated earlier, it is well established that Nmyc is required
for lung development (Moens et al.,
1992; Moens et al.,
1993
; Sawai et al.,
1993
; Stanton et al.,
1992
). Embryos homozygous for a hypomorphic mutation
(Nmyc9a/9a) die at birth with lungs that are about half
the normal size, and that contain fewer branches and highly enlarged air
spaces. Compound mutants between Nmyc9a and a null allele
(NmycBRP) have an even more severe reduction in lung
development. To further explore how Nmyc functions in the lung, we
have exploited transgenic and conditional gene deletion techniques to
overexpress Nmyc, or, conversely, to remove one or both copies of the
gene specifically in the epithelium of the developing lung.
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Materials and methods |
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The Sftpc-cre transgene was constructed by cloning the same
SFTPC promoter upstream of the rabbit ß-globin second intron,
followed by a modified cre recombinase gene containing a Kozak
consensus sequence, an N-terminal nuclear localization signal, and the
ß-globin polyadenylation signal. The modified 5' sequences of the
cre recombinase gene are:
ACCATGGCTCCCAAGAAGAAGAGGAAGgtg, with the Kozak sequence
underlined, the start ATG in bold and the first cre recombinase codon
shown in lower case. A line was established from a male giving specific
temporal and spatial expression of cre in the lung epithelium from E10.5, as
judged by crossing with a Rosa26R female and analyzing lacZ
expression. It was maintained by crossing with ICR mice.
Nmycflox mice were generated as described
(Knoepfler et al., 2002), and
maintained as homozygotes on the 129xC57BL/6 background.
129S4-Gt(ROSA)26Sortm1Sor mice from The Jackson Laboratory on the
C57BL/6 background were maintained by interbreeding.
ß-Gal staining
Lungs were fixed in 4% paraformaldehyde in PBS (pH 7.4), permeabilized in 2
mM MgCl2, 0.01% NaDeoxycholate, 0.02% NP-40, stained with X-gal
overnight, embedded in paraffin wax, sectioned at 7 µm, and counterstained
with Eosin.
BrdU incorporation
BrdU (Amersham Bioscience, UK) was injected intraperitoneally into pregnant
females at a dose of 10 µl per gram bodyweight. After 1 hour, embryonic
lungs were fixed in 4% paraformaldehyde in PBS (pH 7.4). For
immunohistochemistry, BrdU monoclonal antibody (Sigma, St Louis, MO, USA) was
used, in combination with with MOM-blocking solution (Vector Laboratories,
Burlingame, CA, USA).
RT-PCR
Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, USA). The
cDNA was synthesized from 1 µg total RNA using SuperScriptTM
First-strand Synthesis Kit (Invitrogen). Primer sets were:
claudin 6, 5'-ATGGCCTCTACTGGTCTGCAAATC-3' and 5'-GCATCACACATAATTCTTGGTGGG-3';
cyclin D2, 5'-TGAGCACATCCTTCGCAAGC-3' and 5'-CTCACAGGTCAACATCCCG-3';
Nmyc, 5'-CGAATTGGGCTACGGAGATGCT-3' and 5'-TTGTGCTGCTGATGGATGGG-3';
Nol5a, 5'-TGAAGGAAGCTGTGGTTCAGG-3' and 5'-CTAATCCTCCTGTGCTTTCTG-3';
Ppan, 5'-ATGGGGCAGTCCGGGCGGTC-3' and 5'-GCTCACTGATGTCCTGCAGTC-3';
Rog, 5'-CCCCACTCCAGGATCTTTTCC-3' and 5'-AGGTGGCAGCAGANGAGGTAG-3';
Scgb1a1, 5'-TGAAGATCGCCATCACAATC-3' and 5'-ATCTTGCTTACACAGAGGAC-3'; and
Sox9, 5'-ACGTGTGGATGTCGAAGCAG-3' and 5'-ACTGGTTGTTCCCAGTGCTG-3'.
The primers for Sftpa, Sftpb, Sftpc, Aquaporin 5 (Aqp5)
and ßactin were as described
(Okubo and Hogan, 2004).
In situ hybridization
Mouse Sftpc, rat Scgb1a1 and mouse Foxj1 cDNA
have been described previously (Weaver et
al., 1999). Digoxigenin (DIG) or fluorescein-labeled antisense
cRNA probes were made using T7 or SP6 RNA polymerase. Briefly, paraffin
wax-embedded sections were dewaxed, pretreated with proteinase K and
hybridized with one DIG-labeled and one fluorescein-labeled probe at 60°C
overnight. Slides were then washed sequentially in 5xSSC, 2xSSC
(50% formamide), 0.1xSSC and blocking solution (supplied in TSA Plus
Biotin System, Perkin Elmer). The DIG-labeled probe was detected using
POD-coupled anti-DIG (Roche), followed by standard biotin-tyramide and
streptavadin-HRP amplification, and visualized using Cy3-tyramide (Perkin
Elmer). HRP activity was quenched by treatment with 1%
H2O2 in methanol (15 minutes). This was followed by
detection of the fluorescein-labeled RNA probe with POD-coupled
anti-fluorescein (Roche), and similar amplification and visualization with
fluorescein-tyramide. Nuclei were counterstained with DAPI.
Immunohistochemistry
The following reagents were used: rabbit polyclonal antibody to Nmyc
(SantaCruz); phosphohistone H3 (Upstate Biotechnology); cyclin D1 (clone AM29,
Zymed); cleaved caspase-3 (Cell Signaling Technology, Beverly MA); Scgb1a1
(kindly provided by Barry Stripp, University of Pittsburgh); Sox2 (kindly
provided by Larysa Pevny, UNC Chapel Hill); and hamster monoclonal anti-Gp38
(University of Iowa Hybridoma Bank). The rabbit Sox9 antibody was kindly
provided by Dr Francis Poulat, CNRS, Montpellier, and was used at a dilution
of 1:3000. It was raised against amino acids 408-504 of the human protein
(Gasca et al., 2002), a region
showing significant similarity only with sequences in mouse Sox10. However,
RT-PCR analysis indicates that Sox10 is not expressed in either
wild-type or transgenic embryonic mouse lung (data not shown). The specificity
of the rabbit antibody for Sox9 in the chick was described previously
(Moniot et al., 2004
), and was
confirmed in this study by specific staining of Sertoli cells in the mouse
testis (E18.5; data not shown). The Nmyc antibody showed no staining of
neurones in which Nmyc was deleted
(Knoepfler et al., 2002
), and
only low-level staining of epithelial cells in conditional mutant lungs
(Fig. 7F).
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DNA Affymetrix analysis
Total RNA (10 µg) was extracted from three different transgenic and two
wild-type lungs (right caudal lobe) by using RNeasy kit. All subsequent
reactions using Affymetrix GeneChip Mouse 430A arrays and statistical analyses
were carried out in the Duke Center for Genome Technology Microarray core
facility as detailed previously (Okubo and
Hogan, 2004). All primary data files are freely available (see
Tables S1 and S2 in supplementary material).
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Results |
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The genes downregulated in Sftpc-NmycEGFP lungs also fell into
several categories (see Table S2 in supplementary material). One of the
largest (12% of total) encodes specialized products characteristic of type I
and II alveolar cells, numerous genes encoding transmembrane proteins involved
in the directed movement of ions and molecules into and out of cells, and
proteins involved in cellular immunity (complement, immunoglobulin). Another
category includes proteins involved in cell adhesion, extracellular matrix
production and cell structure (26%). This grouping may reflect the simpler
cuboidal morphology of the distal epithelium in transgenic lungs compared with
the more complex three-dimensional organization of the primitive alveoli in
the non-transgenic lungs. However, a number of the genes in this category are
similar to those downregulated in other epithelial cells, such as
keratinocytes (Arnold and Watt,
2001), and/or are direct targets of Myc repression
(http://www.myccancergene.org/site/mycTargetDB.asp).
In the transgenic lungs, we saw no evidence for epithelial cells detaching
from the basal lamina, although branching morphogenesis was severely
disrupted. RT-PCR confirmed the up- and downregulation of a number of the
genes detected by microarray analysis (Fig.
4E).
Conditional deletion of Nmyc leads to abnormal lung development
To study the effect of conditional disruption of Nmyc, we used a
Sftpc-cre transgene, driving cre recombinase specifically in lung
endoderm from about E10.5 (Okubo and
Hogan, 2004), in combination with a previously described floxed
allele of Nmyc (Knoepfler et al.,
2002
).
To generate cre(+);Nmycflox/flox mice, we initially
crossed Sftpc-cre and homozygous Nmycflox/flox
mice (Fig. 6A). As expected,
about 50% of the F1 offspring were cre(+);Nmycflox/+. It
was verified repeatedly that the Nmycflox allele was
deleted in the lung epithelium of embryos inheriting Sftpc-cre
(Fig. 6B and data not shown).
Previous studies had shown that mice heterozygous for both a germline null
allele of Nmyc, and a conditional null allele in the nervous system
(Knoepfler et al., 2002), have
only a mild phenotype of reduced body or brain mass. It was therefore
surprising to observe that about half of the
cre(+);Nmycflox/+ pups died at or shortly after birth with
a very severe lung phenotype (Fig.
7A,B; Table 1).
This result was obtained whether the cre transgene was inherited from the
mother or father. The remaining pups (both males and females, in normal ratio)
developed normally and had no obvious defects in lung morphology when analyzed
as adults.
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To confirm that the cre transgene is active, we crossed cre(+);Nmycflox/+ mice with mice compound heterozygous for Nmycflox/+ and the Rosa26R reporter allele. These compound heterozygotes were generated by crossing Nmycflox/flox mice (129xC57BL/6) with Rosa26R mice (C57BL/6) (see Materials and methods). This showed that recombination had indeed taken place throughout the epithelium in both cre(+);Nmycflox/flox and cre(+);Nmycflox/+ lungs at E16.5 and E18.5 (see Fig. S2 in supplementary material). In this set of experiments, 25% (4/16) of the cre;Nmycflox/+ lungs were abnormal (Table 1).
Increased apoptosis, reduced proliferation, and evidence for depletion of progenitor pool in conditional mutant lungs
At E18.5, the lobulation pattern and tracheal morphology of
cre(+);Nmycflox/flox and affected
cre(+);Nmycflox/+ lungs were normal. However, they were
composed of numerous large fluid-filled sacs, containing cellular debris,
lined by highly attenuated epithelial cells, and separated by a thin layer of
mesoderm (Fig. 7A,B;
Fig. 8F; and data not
shown).
When examined earlier (E14.5-E16.5), the external dimensions of the lungs
of cre(+);Nmycflox/flox and affected
cre(+);Nmycflox/+ embryos were approximately normal
(Fig. 7C,D). This was in
contrast to the report that Nmyc9a/9a or
Nmyc9a/BRP mutant lungs were about half the size of wild
type (Moens et al., 1993).
However, internally, the conditional mutant lungs showed very reduced
branching, with a few expanded tubes separated by abundant mesoderm. RT-PCR
analysis showed that both the levels of Nmyc RNA and protein were
significantly reduced at these stages (Fig.
7E,F). Epithelial cell size was irregular, and dead cells were
frequently observed in the lumen, suggesting a high level of apoptosis
(Fig. 8D-F). This was confirmed
by staining sections with an antibody to cleaved caspase 3 (a marker for
apoptosis), which revealed many positive cells in both the epithelium and
mesenchyme (Fig. 8G-L). BrdU
labeling at E15.5 and E16.5 (Fig.
8M-O) showed a striking reduction in the proportion of
proliferating cells in the mutant lung endoderm (17% compared with 65% for
wild type at E15.5). These results indicate that Nmyc deletion
severely inhibits both cell proliferation and survival.
Finally, we explored the differentiation of epithelial cells in the
conditional mutant lungs. There was a clear reduction of the number of
Sox9-positive cells in cre(+);Nmycflox/flox lungs at E14.5
and E16.5 (Fig. 5B,D),
suggesting that the pool of progenitor cells is not maintained but is lost
through apoptosis or differentiation. The presence in E18.5 conditional mutant
lungs of both Sftpc-positive type II cells and Gp38
(T1)-positive, attenuated type I cells shows that differentiation does
occur (Fig. 9B,D). Evidence
that some differentiation may be premature comes from the analysis of lung
differentiation markers at E16.5 by RT-PCR. As shown earlier in
Fig. 1A, expression of
Aqp5 (a marker for type I cells) is normally not upregulated until
E17.5-E18.5. However, Aqp5 was detected in
cre(+);Nmycflox/flox lungs at E16.5, although it was
absent from cre(-);Nmycflox/+ lungs at the same age
(Fig. 9E).
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Discussion |
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How do high levels of Nmyc perform this function in the lung? Our
array analysis provides evidence that overexpression of Nmyc leads to
the upregulation of many well-documented Myc target genes that promote cell
growth (increase in cell mass), RNA processing and nucleolar structure, DNA
replication, and transit of the cell cycle. It is therefore tempting to
speculate that Nmyc functions not only by controlling positive and negative
cell cycle regulators but also by promoting a particular functional
organization of the nucleus, including changes associated with S-phase, and/or
elevated levels of nucleolar proteins such as fibrillarin and nucleostemin,
which are characteristic of embryonic, stem and cancer cells
(Newton et al., 2003;
Tsai and McKay, 2002
). Our
results also suggest Nmyc functions in the lung through maintaining high
levels the expression of the HMGbox protein Sox9. At present, we do not know
the significance of this finding, as the effect of overexpressing or deleting
Sox9 in the lung has not been reported. However, recent studies show
that Sox9 protein is also localized in the proliferative compartment of the
adult intestine and its overexpression in cell lines inhibits the expression
of genes involved in epithelial cell differentiation
(Blache et al., 2004
).
The increased proliferation seen in Sftpc-Nmyc transgenic lungs
was also accompanied by apoptosis. The induction of both apoptosis and
hyperproliferation in response to Myc overexpression has been well documented
in other systems, particularly under conditions in which the availability of
cell survival factors becomes rate limiting (for reviews, see
Hipfner and Cohen, 2004;
Hueber and Evan, 1998
). Recent
studies have suggested a mechanism by which cells undergoing apoptosis are
extruded from an epithelial layer
(Rosenblatt et al., 2001
), and
this might account for the dead cells seen in the lumen of both transgenic and
conditional mutant lungs.
Nmyc is essential for the proliferation and survival of embryonic lung epithelial cells and for normal epithelial-mesenchymal interactions
Previous studies on embryos carrying germline mutations in Nmyc
had suggested that lung development is particularly sensitive to reductions in
Nmyc levels. Our results here support this conclusion and show that
conditional deletion of Nmyc leads to inhibition of cell
proliferation and to extensive cell death. Dying cells drop out of the
epithelial layer and the survivors that fill the vacated space may possibly
undergo premature differentiation, so that, by E18.5, only attenuated type I
cells and a few type II cells line the large sacs. The extensive apoptosis
seen in the conditional mutant lungs is in marked contrast to the effect of
deleting Nmycflox in neuronal progenitors in the
developing brain. In this organ, there is little cell death of the neuroblasts
in which Nmyc is deleted, and they appear to undergo cell cycle
arrest and precocious differentiation
(Knoepfler et al., 2002).
There are at least two possible reasons for this discrepancy between lung and
brain. First, in the lung, other members of the myc gene family
apparently cannot compensate for the absence of an essential function normally
provided by Nmyc. By contrast, in the brain Myc can
presumably compensate for the absence of Nmyc
(Knoepfler et al., 2002
).
Another reason may relate to the different organization of the cell layers in
the two organs, and to the recent proposal that absence of Myc places cells at
a disadvantage when competing with their neighbors for limited access to
growth factors or necessary substrates (de
la Cova et al., 2004
; Moreno
and Basler, 2004
). In the case of the developing cerebellum, the
progenitor cells in the germinal layer that undergo neurogenic (asymmetric)
cell divisions rather than proliferative (symmetric) division generate
daughter cells that move into the developing brain and differentiate. There
may therefore be relatively little selective pressure in the germinal
epithelium against cells with reduced or absent Nmyc expression. By
contrast, in the lung, all the daughters of epithelial cells normally remain
attached to a common, continuous basal lamina. Therefore, since recombination
takes place asynchronously, the cells that are the first to lose Nmyc
will be at a growth disadvantage compared with their neighbors, and may be
competed or forced off the substrate on which they depend for their survival.
Restriction in access to essential factors for cell growth and survival may
also account for the high level of apoptosis seen in the mesenchyme of
conditional mutant lungs at E15.5 and E16.5, even though Nmyc is not
expressed in this cell population. We and others have shown that, in the
developing lung, the endoderm produces factors, such as sonic hedgehog, that
are necessary for the survival and proliferation of the mesoderm
(Gebb and Shannon, 2000
;
Weaver et al., 2003
).
A general conclusion from our gain- and loss-of-function studies is that
Nmyc plays a key role in controlling the flow of cells into and out of a
distal progenitor pool during lung development. It will therefore be important
to determine how factors such as Wnts, Tgfßs and Igfs regulate the
expression of Nmyc RNA, and the phosphorylation and turnover of the
protein, at different stages of embryogenesis
(Frederick et al., 2004;
Kenney et al., 2004
) (our
unpublished observations suggesting that Nmyc is a direct target of
Wnt signaling in the embryonic lung).
Effects of acute Nmyc hemizygosity on lung development
A striking finding of this study was the failure in lung development in
half (40/81) of the embryos in which only one copy of Nmyc is deleted
by cre recombination. The precise proportion of affected embryos varied from
53% (28/52) in the F1 generation from crossing
Sftpc-cre;Nmyc+/+ with Nmycflox/flox
mice, to 62% (8/13) in the F2 generation
(Fig. 6), and 25% (4/16) when
Sftpc-cre(+); Nmycflox/+ mice were crossed with
Nmycflox/+;Rosa26R mice
(Table 1). There are two
possible explanations for these results, neither of which can be excluded
without further extensive experimentation. The first possibility is that the
(129xC57BL/6) background on which the Nmycflox
allele is maintained is segregating for a modifier gene that decreases the
probability that a lung epithelial cell in which only one Nmyc allele
is active will continue to proliferate. The second, more speculative, model
proposes that the level of Nmyc transcription is normally tightly
controlled and involves an autoregulatory negative-feedback loop acting early
in development (Penn et al.,
1990). According to this model, in the developing embryo the
probability that Nmyc transcription is initiated is low and, once one
allele is active in a cell, the transcription of the other allele is inhibited
by the feedback loop. This essentially monoallelic expression is then
maintained, possibly by epigenetic modification. Consequently, the epithelium
of the primary buds of heterozygous cre(+);Nmycflox/+
embryos will be a mosaic of cells in which either one or the other of the
Nmyc alleles is active. Those cells in which the
Nmycflox allele is active will become functionally null
after recombination. Depending on the proportion of cells in the early lung
primordium with each allele active, and on the level of competition between
normal and mutant cells, the conditional mutant lungs will be either abnormal,
a mosaic of normal and abnormal tissue, or completely normal. To further test
this model will require analysis of the transcription or epigenetic
modification of distinguishable Nmyc alleles at the single-cell level
in the embryonic lung. Meanwhile, whichever model is correct, our findings
raise the interesting possibility that, prior to amplification of Nmyc or
aberrant activation of Myc, lung tumor cells should be particularly sensitive
to agents downregulating Nmyc expression.
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Supplementary material |
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ACKNOWLEDGMENTS |
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