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First published online April 12, 2006
doi: 10.1242/10.1242/dev.02310
Review |
Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, USA
* Author for correspondence (e-mail: wcardoso{at}lung.bumc.bu.edu)
SUMMARY
During early respiratory system development, the foregut endoderm gives rise to the tracheal and lung cell progenitors. Through branching morphogenesis, and in coordination with vascular development, a tree-like structure of epithelial tubules forms and differentiates to produce the airways and alveoli. Recent studies have implicated the fibroblast growth factor, sonic hedgehog, bone morphogenetic protein, retinoic acid and Wnt signaling pathways, and various transcription factors in regulating the initial stages of lung development. However, the precise roles of these molecules and how they interact in the developing lung is subject to debate. Here, we review early stages in lung development and highlight questions and controversies regarding their molecular regulation.
Introduction
The basic design of the mammalian respiratory system, referred to here as the trachea and lung, is that of a tree of epithelial tubules in which air is cleaned, humidified and delivered to numerous alveolar units closely apposed to blood vessels, where the circulating blood is oxygenated.
The respiratory system arises from the ventral foregut endoderm. The
process initiates with the establishment of respiratory cell fate in the
primitive foregut. This is followed by the development of a tree-like system
of epithelial tubules and vascular structures (see
Fig. 1), which ultimately gives
rise to the mature airways and alveoli. The foregut endoderm differentiates
into various epithelial cell types, which line the inner surface of the
developing lung and trachea. The lung mesenchyme originates from the lateral
plate mesoderm and gives rise to multiple components of the lung, including
its connective tissue, endothelial cell precursors, the smooth muscle that
surrounds airways and blood vessels, the cartilage of the trachea, the
lymphatics, and the mesothelial cells that cover the outer surface of the
lung, the pleura. The lung vasculature forms, in part, by migration of blood
vessels from the aortic arches and from the left atrium to the lung
(angiogenesis). Blood vessels also develop by vasculogenesis in the lung
mesenchyme near developing epithelial buds; a rudimentary capillary network
initially forms and expands, and later connects to the larger vessels to give
rise to the lung vasculature (Wood et
al., 1997
; Demello et al.,
1997; Gebb and Shannon,
2000). There is evidence that, during organogenesis, blood vessels
serve as a source of inductive signals to the epithelium
(Lammert et al., 2001;
Matsumoto et al., 2001).
However, the role of the vasculature in epithelial patterning has still to be
clearly demonstrated in the developing lung.
The mechanisms that control respiratory system formation have been the
subjects of an increasing number of studies. This review focuses on our
current knowledge of the molecular regulation of early lung development.
Topics discussed here include lung endodermal specification, lung primordium
formation, and the regulation of the initial stages of branching morphogenesis
and differentiation in the embryonic lung. We address questions such as `when
and how is respiratory cell fate established?', `how do lung buds form?', `how
are stereotypical patterns of airway branching and cellular diversity
generated in the developing lung?' and `which pathways and targets are key to
these processes?'. Most of what is described refers to mouse lung development
because of the genetic data available
(Table 1). Lung vascular
development and later events, such as sacculation and alveoli formation, are
not discussed in this review (for reviews, see
Pauling and Vu, 2004;
Williams, 2003;
Bourbon et al., 2005).
|
Foregut morphogenesis and establishment of endodermal cell fate
Following gastrulation (embryonic day E7.5 in mice), the definitive
endoderm undergoes complex morphogenetic movements that ultimately lead to the
formation of the primitive gut tube. The foregut represents the most anterior
(cranial) region of this tube, while the midgut and hindgut are located at
progressively more posterior regions, towards the caudal end of the embryo
(Wells and Melton, 1999).
Transcription factor genes such as Foxa1, Foxa2, Gata4 and
Gata6, which are expressed early in the endoderm, are crucial for the
survival, differentiation and morphogenesis of the foregut
(Kuo et al., 1997;
Morrisey et al., 1998;
Ang and Rossant, 1994;
Wan et al., 2005). By
E8.0-9.5, the local expression of transcription factors along the
anteroposterior (AP) axis of the gut endoderm marks organ-specific domains (or
fields; see Fig. 1A). For
example, the homeodomain protein gene Nkx2.1 [also known as thyroid
transcription factor 1 (Titf1) or T/EBP] is expressed in the
thyroid and respiratory fields (Kimura et
al., 1996), Hex (hematopoietically expressed homeobox) is
expressed in the thyroid and liver fields
(Martinez Barbera et al.,
2000), and the Pdx1 (pancreas-duodenal-associated
homeobox gene) is expressed in the pancreatic and duodenal fields
(Offield et al., 1996). In
addition, morphogenetic movements foster dynamic interactions between the
endoderm and neighboring structures, such as the heart, notochord or the
septum transversum (the mesodermal cells that give rise to the diaphragm).
Exposure of the endoderm to diffusible signals from these structures at
crucial developmental windows is essential for endodermal cell fate
specification (Kumar and Melton,
2003; Bort et al.,
2004).
Fibroblast growth factor 4 (Fgf4), bone morphogenetic protein 2 (Bmp2) and
retinoic acid (RA) are among the signals that confer AP identity to the early
endoderm. They render the endoderm competent to respond to signals from the
adjacent mesoderm or from nearby structures to initiate morphogenesis
(Tiso et al., 2002;
Stafford and Prince, 2002;
Wells and Melton, 2000). In
zebrafish, disrupted RA signaling during gastrulation results in the loss of
liver and pancreatic (posterior) fates, while thyroid and pharynx (anterior)
fates remain unaltered. Conversely, excess RA induces hepatic and pancreatic
cell fates at more anterior domains
(Stafford and Prince, 2002).
In mice and rats, RA signaling initiates soon after gastrulation
(Rossant et al., 1991), but
does not seem to be as crucial for foregut AP identity as it is in the
zebrafish.
|
; Kumar and Melton,
2003). By contrast, relatively fewer studies have focused on the
lung. Lineage analysis suggests that the progenitor cells of the trachea and
proximal lung differ in origin from those that will form the distal region of
the lung (Perl et al., 2002).
Precisely when respiratory cell fate is established in the foregut endoderm is
still unclear. Respiratory progenitors are first visualized by in situ
hybridization as a group of Nkx2.1-expressing endodermal cells in the
prospective lung/tracheal region of the foregut, at 
E9
(Minoo et al., 1999).
Nkx2.1 transcripts have been reported by RT-PCR to be present in the
foregut as early as E8-8.5 (eight-somite stage)
(Serls et al., 2005). These
signals, however, probably originate from the thyroid primordium, as the
thyroid starts to develop at least 1 day before the lung
(Lazzaro et al., 1991). The
surfactant-associated protein C (Sftpc) gene is the most specific
marker of lung epithelial cell lineage, but its expression is consistently
detected only by E10-10.5, after the primary buds form
(Wert et al., 1993).
Studies in mouse foregut cultures strongly indicate that Fgfs emanating
from the adjacent heart influence the AP fate of the ventral foregut endoderm
in a concentration-dependent manner. A model of foregut specification has been
proposed, in which different thresholds of Fgfs pattern the endoderm into
different foregut derivatives, including the liver and lung
(Fig. 2A). If cultured alone,
the endoderm adopts a `default' pancreatic fate; adding increasing amounts of
cardiac mesoderm or Fgf2 to the endoderm results first in induction of liver,
and then of lung or thyroid fates (Rossi
et al., 2001; Serls et al.,
2005; Jung et al.,
1999). In this system, the induction of lung cell fate appears to
involve Fgfr4 signaling (Serls et al.,
2005). Lung development, however, is apparently normal in
Fgfr4-null mice (Weinstein et
al., 1998). Currently, there is no evidence that sonic hedgehog
(Shh) or Bmps, which are present in the foregut, are involved in lung endoderm
specification. In summary, lung specification is likely to depend on signaling
molecules, such as Fgfs from the heart, and local transcription factors that
have yet to be identified.
Nkx2.1 and distal lung development
Although Nkx2.1 is the earliest known marker of the presumptive
respiratory region, Nkx2.1-null mutant mice do have lungs
(Minoo et al., 1999). These
lungs, however, are highly abnormal and consist of two main bronchi, which
give rise to cystic structures, lined by columnar cells with scattered cilia -
features that are reminiscent of those found in proximal airways. Strikingly,
marker analysis shows that the epithelium fails to express any of the
surfactant-protein genes typically found in the normal distal lung. Whether
distal lung progenitors are specified but not maintained in these mice cannot
be determined without the identification of additional early markers of lung
cell fate. Besides Nkx2.1, no other early marker is currently
available. The presence of relatively preserved features of proximal
differentiation in Nkx2.1-null mutants suggests that Nkx2.1
may not be crucial for the developmental program of progenitor cells of the
proximal lung (Minoo et al.,
1999).
|
).
These studies suggest that Nkx2.1 is essential for the developmental
program of epithelial cells of the distal lung and that Nkx2.1 is required for
expression of several lung markers, such as Sftpc
(Kelly et al., 1996). Although
the promoter region of Nkx2.1 has been studied, little is known about
the cis-active regulatory sequences that direct Nkx2.1 expression to
the lung (Pan et al.,
2004).
Primary lung bud formation
Lung bud morphogenesis: the role of Fgf10 and Fgfr2b
In mice, lung primordial buds form at E9.5 (25-somite stage;
Fig. 1). As determined by
studies in Drosophila, the budding of the developing tracheal system
is initiated by the expression of a Fgf ligand (branchless) at
prospective sites of budding; this is followed by the local activation of a
Fgf receptor (breathless) in the endoderm to induce budding
(Sutherland et al., 1996). In
mammals, signaling by Fgf10 and Fgfr2b is crucial for lung bud formation.
Fgf10 is a chemotactic and proliferation factor for the endoderm
(Bellusci et al., 1997b;
Park et al., 1998). Deletion
of either Fgf10 or Fgfr2b in mice results in lung agenesis
and multiple abnormalities (De Moerlooze
et al., 2000; Min et al.,
1998; Sekine et al.,
1999). The overlapping features of Fgf10- and
Fgfr2b-null mutants confirm Fgfr2b as the major receptor for Fgf10.
Interestingly, unlike Fgf10-null mutants, Fgfr2b-null mice
form an underdeveloped lung bud that soon undergoes apoptosis
(De Moerlooze et al., 2000).
This has been attributed to Fgf10-mediated activation of Fgfr1b, a receptor
that also binds to Fgf10, but with much lower affinity
(Lu et al., 1999). It is thus
not able to maintain lung epithelial survival and the lung morphogenetic
program that is normally carried out by Fgf10 and Fgfr2b signaling. Although
tracheal morphogenesis has been reported to be normal in Fgf10-null
mice at birth, a recent analysis of Fgf10 heterozygous mice has
revealed that the size and number of tracheal submucosal glands are
significantly reduced (Rawlins and Hogan,
2005). These structures develop postnatally from the tracheal
epithelium and probably recapitulate the Fgf-dependent program of budding and
branching seen in the embryonic lung.
What controls lung primordium positioning in the foregut tube?
Little is known about the genes that control the positioning of the lung
primordium in the foregut or the boundaries of the Fgf10 domain in
the foregut (Fig. 2B). There is
evidence, however, that Fgf10 expression and bud formation in the
lung field are crucially dependent on RA
(Desai et al., 2004). RA
synthesis and use are prominent throughout the E8.5-9.5 mouse foregut
(Malpel et al., 2000). Yet,
disruption of RA signaling in the foregut affects the lung most dramatically
and leads to several abnormalities, including lung agenesis
(Wilson et al., 1953;
Mollard et al., 2000).
Culturing E8.5 foregut explants in the presence of a RA receptor antagonist
prevents lung buds from forming. In this model, RA selectively regulates
Fgf10 where the lung and neighboring stomach form
(Desai et al., 2004). This
seems to involve signaling by RA receptor ß in the mesoderm
(Desai et al., 2006).
Gli and T-box (Tbx) transcription factors have been also implicated in the
formation of the lung primordium. Gli1, Gli2 and Gli3 are transcriptional
effectors of the Shh signaling pathway that are present in the foregut
mesoderm and later in the lung mesenchyme
(Hui et al., 1994). In
Gli2/Gli3 double-null mice, lung and tracheal primordium never form;
other foregut derivatives develop but are smaller than normal, and most
embryos die by E10.5 (Motoyama et al.,
1998). This phenotype is intriguing, as it is more severe than
that of Shh-null mutants
(Pepicelli et al., 1998), and
also because there is no evidence that during development these Gli proteins
are preferentially expressed in the lung field. Presumably Gli2 and Gli3
induce a currently unidentified mesodermal diffusible signal that is required
for bud formation.
In chick embryos, Tbx4 and Fgf10 are co-expressed in the
foregut mesoderm in the lung field in a domain that coincides with that of
Nkx2.1 in the endoderm, except in its most anterior portion. Studies
in ovo show that misexpression of Tbx4 induces ectopic Fgf10
expression and ectopic buds that express Nkx 2.1 mRNA. Tbx4 and Fgf10
are not required to initiate Nkx.2.1 expression, but appear to play a
role in defining the posterior boundary of Nkx2.1 and the lung
primordium (Sakiyama et al.,
2003). However, the genetic inactivation of Tbx4 in mice
does not prevent lung bud formation
(Naiche and Papaioannou,
2003). The overlapping expression of Tbx2, Tbx3, Tbx4 and
Tbx5 in the developing foregut and lung mesoderm suggests that these
genes may have a redundant role in foregut and lung morphogenesis
(Chapman et al., 1996).
RA, Fgf10, Gli2, Gli3, Tbx2, Tbx3 and Tbx4, discussed here, all have in common expression in the foregut mesoderm at the onset of lung development and a potential, or demonstrated, involvement in primary lung bud induction.
Making the respiratory tree
From E10.5-E17.0, the lung epithelium undergoes branching morphogenesis to form the respiratory (or bronchial) tree. This process has been extensively studied in the lung (see Table 1), but many questions and controversies about its molecular regulation remain unresolved.
Fgf10 acts as the signal that triggers secondary and subsequent budding, as
it does during primary budding (Fig.
3A). This, however, remains to be rigorously tested by conditional
inactivation of Fgf10. At least during the initial generation of
branches, lung buds arise in a highly stereotypical fashion. It has been
proposed that the expression pattern of branchless in the developing
Drosophila trachea or Fgf10 in the lung is invariant within
a three-dimensional grid and could be set by global regulators of axis
formation, such as the Hox genes (Metzger
and Krasnow, 1999). Indeed, several Hox family members are
expressed in partially overlapping domains along the AP axis of the mouse
developing lung (Bogue et al.,
1996; Volpe et al.,
1997; Aubin et al.,
1997). However, the lack of dramatic changes in the AP axis of the
lung in single or double Hox-null mutants suggests that the role of these
genes in lung patterning is still unclear.
An intriguing, dynamic pattern of expression of the Sry-like HMG box
transcription factor Sox2 in the developing lung and thyroid
epithelium has led to the hypothesis that local downregulation of
Sox2 may be required for commencement of bud morphogenesis. During
lung branching morphogenesis, Sox2 is associated with the epithelium
that is less morphogenetically active, and expression is lost at sites where
nascent buds arise (Ishii et al.,
1998). The role of Sox2 in the lung remains to be demonstrated;
mice deficient in Sox2 die prior to organogenesis because of the inability of
the stem cells to proliferate (Avilion et
al., 2003).
Left-right asymmetry and branching
Left and right lungs are asymmetric, as is apparent by their distinct
patterns of branching and by the different number of lobes on each side (lobes
are morphological units of the lung that are covered by the visceral pleura).
The number of lobes varies in different species. Murine lungs
characteristically have one lobe on the left and four lobes on the right (see
Fig. 1D). Asymmetry of the lung
is dependent on left-right (LR) determinants. The process is part of an early
global program of axis specification that is regulated by several Tgfb-related
molecules, such as activin receptor 2, Lefty1, Lefty2 and growth
differentiation factor 1, and by the bicoid type homeobox gene Pitx2
(Oh and Li, 1997;
Meno et al., 1998;
Kitamura et al., 1999;
Rankin et al., 2000;
Lin et al., 1999). Disruption
of these genes results in laterality defects in multiple organs. In the lung,
these defects commonly manifest as isomerism, the presence of equal numbers of
lobes (with either right or left pattern) on both sides
(Table 1). Interestingly, most
of these genes are expressed in the mouse foregut mesoderm only transiently
(E8-8.5). Except for Pitx2, expression of LR determinants ceases by
the time the lung primordium forms (E9.5). The relatively late appearance of
Pitx2 has led to the hypothesis that this gene acts as an executor of
early genetic programs that control asymmetry in different structures of the
embryo (Kitamura et al.,
1999). Among the three isoforms (a, b, c), only Pitx2c is
asymmetrically expressed in the left lung. Pitx2-null mice show right
pulmonary isomerism (four-lobed lungs bilaterally)
(Kitamura et al., 1999). Thus,
during normal development, Pitx2 could presumably influence gene expression in
the left lung, allowing budding only at specific sites. By doing so, Pitx2
would generate a simpler pattern of branching (and lobation), characteristic
of the left lung. How Pitx2 exerts its functions, and whether Pitx2
and Fgf10 interact in the lung mesenchyme, are unknown. Only a
limited number of Pitx2 targets have been reported, and they shed
little light on these issues (Ganga et
al., 2003).
Control of budding by sprouty and Shh
The exchange of signals between the growing bud and the surrounding
mesenchyme establishes feedback responses that control the size and shape of
the bud during branching. This is illustrated by the mechanisms that control
Fgfr2b activity or Fgf10 expression by the sprouty (Spry) or the Shh
pathways, respectively (Fig.
3B-D).
Spry genes encode a family of cysteine-rich proteins (there are four
members in mice) that interact with crucial elements of the receptor tyrosine
kinase Rtk-Ras-Erk/Mapk cascade and interfere with the intensity or timing of
Rtk signaling by ligands such as Fgf and Egf
(Kim and Bar-Sagi, 2004). In
the mouse lung epithelial cell line MLE15, Spry2 inhibits Fgf10-Fgfr2
signaling by binding to Frs2 (fibroblast growth factor receptor substrate 2),
Grb2 (growth factor receptor bound protein 2) and Raf (v-raf-leukemia viral
oncogene 1), and by disassociating from Gap1 (GTPase-activating protein 1) and
Shp2 (Src homology 2-containing phosphotyrosine phosphatase)
(Tefft et al., 2002).
In the developing E11-12 mouse lung, Spry2 is expressed at the
tips of the growing epithelial buds, while Spry4 is present in the
surrounding distal lung mesenchyme. Spry2 is one of the earliest
targets to be induced in the lung epithelium in response to Fgf10
(Mailleux et al., 2001). By
acting as a Fgf10-dependent inhibitor of Fgfr2b activity, Spry2 limits the
proliferation or migration of the lung epithelium when buds are forming. This
could be part of a mechanism to control the size of the bud or to stop bud
formation and, ultimately, to inhibit branching morphogenesis. Consistent with
this role, reducing Spry2 activity in lung organ cultures results in
increased branching (Tefft et al.,
1999), as also reported for the tracheal system of
Drosophila in Spry mutants
(Hacohen et al., 1998).
Conversely, overexpression of Spry2 or misexpression of
Spry4 in the distal lung epithelium of transgenic mice severely
impairs branching (Mailleux et al.,
2001; Perl et al.,
2003).
|
). Data from lung organ culture and in vivo studies support
the idea that Shh negatively regulates Fgf10 expression in the lung
(Bellusci et al., 1997a;
Lebeche et al., 1999). These
findings have led to the proposal that Shh at the bud tips progressively
downregulates Fgf10 expression as a bud grows towards the
Fgf10-expressing mesenchyme, thus limiting further bud outgrowth. By
doing so, Shh would contribute to controlling bud size. Interestingly, Shh may
also control the shape of the bud by preventing the widespread expression of
Fgf10 in the mesenchyme and the generalized activation of Fgfr2b in
the distal epithelium. In lungs from Shh-null mice, Fgf10
transcripts are found diffusely expressed in the lung mesenchyme. Instead of
forming typical buds, the epithelium develops as large cystic structures and
branching morphogenesis is severely disrupted
(Pepicelli et al., 1998).
Precisely how Shh helps to control the spatial pattern of Fgf10
expression is unknown. One potential way involves Shh induction of the
hedgehog interacting protein Hhip1 in the distal mesenchyme. Hhip1
inhibits Shh signaling by ligand sequestration and, thus, releases
Shh-mediated repression of Fgf10 locally (see
Fig. 3D). In the lungs of
Hhip1-null mice, branching is inhibited because of increased Shh
activity and the nearly complete repression of Fgf10 expression in
the developing lung (Chuang et al.,
2003).
Bmp and Wnt signaling: positive or negative regulators of branching?
There have been apparently conflicting reports about how Bmp and Wnt
signaling influence lung branching morphogenesis. Among the Bmp ligands
present in the embryonic lung, Bmp4 is the best studied. Bmp4 is
transcribed in the lung mesenchyme from its earliest stages, but it is not
present in the epithelium until branching initiates. A
Bmp4lacZ reporter mouse reveals a striking distribution of
Bmp4 in the ventral foregut mesoderm at the prospective lung region
as early as E9 (Weaver et al.,
1999). The biological significance of this finding remains to be
determined. By E11-12, Bmp4 transcripts are found in the distal lung
epithelium and in the proximal mesenchyme
(Weaver et al., 1999;
Weaver et al., 2003). The
Bmp4 receptor (type I, or Alk3), and the Bmp transducing Smad1 protein are
present both in the epithelium and mesenchyme of the embryonic lung
(Bellusci et al., 1996;
Chen et al., 2005). A detailed
analysis of Bmpr2 distribution in the early embryonic lung is not currently
available. The patterns above suggest that Bmp4 signaling can be activated
both in an autocrine fashion (in the epithelium) and in a paracrine fashion
(in the mesenchyme).
The precise role of Bmp4 in the developing lung in vivo remains unclear, in
part because of the early embryonic death of Bmp4-null mice
(Winnier et al., 1995). It
has been previously proposed that, during branching, Bmp4 is induced and
activated in the epithelium of distal buds to limit Fgf10-mediated bud
outgrowth. This model is supported by the following observations
(Lebeche et al., 1999;
Weaver et al., 2000).
Analyses of E11.5 lungs undergoing branching morphogenesis show that
Bmp4 expression in the lung epithelium is highest in distal buds,
near Fgf10-expressing cells. Bmp4 is not induced in the
epithelium of E11.5 lungs during bud initiation but appears later, once the
bud is elongating. In mesenchyme-free lung epithelial cultures, recombinant
Fgf10 induces budding and Bmp4 expression, while recombinant Bmp4
inhibits the Fgf10-mediated budding in these cultures.
Paradoxically, when recombinant Bmp4 is administered to intact lung
explants in which the epithelium and mesenchyme are present, branching is
enhanced (Bragg et al., 2001).
An alternative model has been proposed to explain how Bmp4 can have both
positive and negative effects in the lung. The model predicts that the
mesenchyme influences the ability of the epithelium to respond to Bmp4. When
Bmp4 signaling is activated in the epithelium in an autocrine fashion,
proliferation is inhibited (but see
Eblaghie et al., 2006). In the
intact lung, however, Bmp4 present in the distal epithelium may also activate
Bmp signaling in the adjacent mesenchyme (paracrine fashion). Bmp4, then,
induces a currently unidentified distal mesenchymal signal that enhances
proliferation of distal epithelial buds
(Bragg et al., 2001). In this
way, negative or positive effects on branching would depend on whether Bmp
signaling is activated via an autocrine or a paracrine mechanism
(Fig. 3B). Regulation of
Bmp4 in the epithelium is complex and dependent on signals such as
Wnt (see below), Fgf10 and Pod1 (Tcf21), a transcriptional factor present in
the mesenchyme (Quaggin et al.,
1999).
The role of Wnt signaling in lung branching morphogenesis has also been
debated. Several Wnt ligands, frizzled receptors and components of the Wnt
canonical pathway, such as ß-catenin, and Tcf/Lef transcription factors
(see
http://www.stanford.edu/~rnusse/wntwindow.html)
are present in the developing lung
(Bellusci et al., 1996;
Lako et al., 1998;
Zakin et al., 1998;
Tebar et al., 2001).
Activation of canonical Wnt signaling can be monitored by detection of nuclear
translocated ß-catenin, and by analysis of a Wnt responsive reporter
mouse (TOPGAL), in which lacZ is expressed where the
ß-catenin-Lef1/Tcf complex activates the transcription of Wnt targets
(Nelson and Nusse, 2004;
DasGupta and Fuchs, 1999). In
the E11-13 lung, ß-catenin is expressed throughout the entire lung
epithelium. However, nuclear-localized ß-catenin, Tcf/Lef transcripts and
lacZ-TOPGAL expression are increased in the distal lung epithelium,
the sites that are actively branching
(Okubo and Hogan, 2004;
De Langhe et al., 2005).
Disruption of canonical Wnt signaling at these sites by targeted deletion of
ß-catenin, or by targeted expression of the Wnt inhibitor dickkopf 1 in
vivo, prevents distal lung buds from forming and markedly interferes with
branching morphogenesis. The defect appears to result, at least in part, from
failure to induce proper levels of Fgfr2b in the distal lung
epithelium where Wnt/ß-catenin signaling is inhibited
(Mucenski et al., 2003;
Shu et al., 2005). It has
been pointed out, however, that although ß-catenin deletion is a good
method for disrupting all Wnt canonical signaling, other ß-catenin
functions, not necessarily related to Wnt signaling, may be also affected
(Dean et al., 2005). Indeed,
ß-catenin is also found in cell membranes in a cadherin-bound form that
regulates cell adhesion. Thus, it is possible that the branching defect
reported in the models in which ß-catenin was deleted from the epithelium
could have resulted from changes to both its Wnt and non-Wnt functions
(Dean et al., 2005).
Interestingly, the results above conflict with that of two other models
that show increased branching morphogenesis as a consequence of disrupted Wnt
signaling. In one model, lung explants were treated with morpholino
oligonucleotides against ß-catenin; the other is a genetic model in which
mice lack the Wnt5a gene (a non-canonical Wnt, normally present in
the lung epithelium and mesenchyme) (Li et
al., 2002; Dean et al.,
2005). In both cases, Fgf10 expression was locally
increased in these lungs. It is possible that this discrepancy in results is
due to the fact that in the morpholino and Wnt5a models, Wnt
signaling was inactivated in both the epithelium and mesenchyme. In addition,
canonical and non-canonical Wnts (and even different Wnt family members) may
have distinct functions (Fig.
3C,E,F). Further studies are required to clarify these issues.
The discussion in this section underscores the complexity of the Bmp4 and Wnt signaling during branching, and the importance of taking into account the overall balance of these signals in the epithelium and mesenchyme. In addition, both Bmp4 and canonical Wnt are required for the establishment of distal epithelial cell fate in the lung (see below).
Tgfb signaling as a negative regulator of branching morphogenesis
Tgfb1, Tgfb2 and Tgfb3, members of the Tgfb subfamily, have also been
implicated in the control of lung branching morphogenesis. These Tgfb ligands,
their receptors (Tgfbr1 and Tgfbr2) and transducing proteins Smad2 and Smad3,
are expressed in the developing mouse lung. Many of the biological activities
of these Tgfb proteins differ only in the intensity of their effects (reviewed
by Massague, 2000). During
lung branching morphogenesis, Tgfb1 is transcribed in the mesenchyme
adjacent to the epithelium, without an obvious proximodistal gradient
(Lebeche et al., 1999).
However Tgfb1 protein accumulates in stalks and in regions in between buds,
where extracellular matrix components collagen I, collagen III and fibronectin
are also present (Heine et al.,
1990). Tgfb2 is expressed in the distal lung epithelium,
while Tgfb3 is present in the proximal lung epithelium, mesenchyme
and pleura (Pelton et al.,
1991). Analysis of Tgfb2- or Tgfb3-null mice
reveals that lung branching morphogenesis is affected by the lack of
Tgfb3, but not by Tgfb2 deficiency
(Sanford et al., 1997;
Kaartinen et al., 1995).
Exogenous Tgfb1 inhibits branching morphogenesis, growth and
differentiation in cultured mouse embryonic lungs
(Serra et al., 1994;
Zhao et al., 1996). This
effect has been also reported in transgenic mice misexpressing Tgfb1
in the distal epithelium (Zhou et al.,
1996). Interestingly, Tgfb1-null mice show no obvious
structural lung defect and die perinatally of a diffuse inflammatory syndrome
(Letterio et al., 1994).
Whether Tgfb1 is dispensable for lung morphogenesis has been debated, as there
is evidence that the phenotype may have been rescued by maternal transfer of
Tgfb1. Studies in NIH3T3 fibroblasts, lung and prostate organ cultures
indicate that activation of Tgfb1 signaling in mesenchymal cells markedly
inhibits Fgf10 expression (Beer et
al., 1997; Lebeche et al.,
1999; Tomlinson et al.,
2004). In the developing lung, Tgfb1 may be part of a mechanism
that prevents Fgf10 from being expressed in the mesenchyme of bud
stalks or in more proximal regions of the lung. At these sites, Tgfb1 could
also induce synthesis of extracellular matrix components and prevent budding
locally (Fig. 3B).
Refining patterning and preventing ectopic budding
Branching patterns can be further refined by mechanisms that regulate bud
formation at specific locations in the developing lung. For example, the
transcription factor Foxf1 is expressed throughout the mesenchyme of
both lungs from the earliest developmental stages. However, analysis of
Foxf1 heterozygous mice at E10-11 suggests that Foxf1 is required for
proper gene expression and budding selectively in the cranial, middle and
accessory lobes of the right lung. These mice show low levels of
Fgf10 and Gli3 mRNAs, and display altered lung bud
orientation and ectopic budding in these lobes, among other abnormalities
(Lim et al., 2002;
Mahlapuu et al., 2001). The
presence of lung defects that resemble those from Gli3-null mutants
suggest a genetic interaction between Foxf1 and Shh signaling via
Gli3 (Fig. 3C). Foxf1
expression is in part regulated by Shh signaling through Shh-dependent
processing of Gli3 (Li et al.,
2004).
Other genes are dynamically expressed at the stalks of branching tubules in
a pattern that suggests a role in preventing local budding
(Fig. 3B). This is the case of
the axon guidance molecules netrin 1 and netrin 4, and the extracellular
matrix protein Tgfb induced (Tgfbi or ßig-h3) in the
E11-13 mouse lung. There is evidence that netrins and their receptors Unc5b
and Dcc (deleted in colorectal cancer) are present in the lung epithelium at
these stages. Moreover, exogenous netrin inhibits Fgf-mediated Erk
phosphorylation and bud formation in mesenchyme-free lung epithelial explants
(Liu et al., 2004). These
observations favor the hypothesis that, during branching morphogenesis, netrin
expression in stalks prevents buds from forming in these region by interfering
with local activation of Fgf signaling
(Liu et al., 2004). This
hypothesis, however, has not been confirmed in vivo, as genetic inactivation
of netrins or other axon guidance molecules, such as semaphorins (or Slit and
Robo proteins) in mice has not resulted in obvious defects in lung branching
morphogenesis (Hinck, 2004).
Recent evidence demonstrating that Slit2 and netrin 1 act synergistically in
mammary gland ductal morphogenesis suggests that deciphering the role of axon
guidance molecules in the lung will require simultaneous inactivation of more
than one member of these functionally related families of genes
(Strickland et al.,
2006).
Tgfbi, another molecule dynamically expressed in stalks and in
interbud regions of the developing lung, is present in the mesenchyme, at
sites traditionally associated with Tgfb-induced deposition of extracellular
matrix and decreased epithelial cell proliferation
(Serra et al., 1994;
Zhao et al., 1996;
Heine et al., 1990;
Lu et al., 2004).
Tgfbi is known to be induced by Tgfb1 and to mediate Tgfb responses
in some cell systems by a mechanism that is still unclear
(Kim et al., 2003). Tgfbi
binds to a variety of matrix molecules, such as fibronectin and collagen
(Billings et al., 2002).
Whether Tgfbi restricts bud formation in stalks during lung branching
morphogenesis is currently unknown.
What is downstream of Fgf signaling during lung bud formation?
There is still little understanding of the cellular mechanisms of lung bud
formation and of the targets of Fgf10 in this process. In Drosophila,
Fgf signaling activation causes dynamic actin-based filopodial extensions at
the tip of primary branches, leading to epithelial migration. This is in part
promoted by the induction of pointed (Spdef - Mouse Genome
Informatics) an Ets transcription factor
(Samakovlis et al., 1996;
Sutherland et al., 1996). In
the E11.5 mouse lung, two Ets family members, Pea3 and Erm (Etv4 and Etv5,
respectively), have been identified in the distal epithelium. Although both
are downstream of the Fgfr2b pathway, they do not seem to be required for
branching, as shown by expression of a dominant-negative Erm targeted
to the lung in vivo (Liu et al.,
2003).
A microarray-based screen to identify Fgf10 targets in mesenchyme-free lung
epithelial buds has shown that the initial stages of budding are characterized
by upregulation of genes traditionally associated with: cell rearrangement and
cell migration [Tm4sf3, transmembrane 4 superfamily member 3
(Tspan8); the Notch signaling antagonist Numb; and
Lmo7, LIM domain only 7]; inflammatory processes (annexins); lipid
metabolism [HSL, hormone-sensitive lipase (Lipe)];
proteolysis (cathepsin H, Timp3); and metastatic behavior
(Tacstd2, tumor-associated calcium signal transducer 2), but not cell
proliferation (Lu et al.,
2005a). This
observation is consistent with previous studies that show local changes in
proliferation are not the triggering event that initiates lung budding
(Nogawa et al., 1998).
|
| Box 1. Myogenesis and bud morphogenesis
Airway smooth muscle starts to form relatively early in the developing
mouse lung. A smooth muscle layer adjacent to the epithelium of the trachea
and primary lung buds is present at E11 and continues to form around proximal,
but not distal, epithelial tubules as branching proceeds
(Tollet et al., 2001
|
Patterning the mesenchyme
Integrating epithelial and pleural signals
Lung mesenchyme development is crucially influenced by signals from the
epithelium and the pleura (Fig.
3E, Box 1,
Table 1) that, in concert,
appear to maintain a balance of differentiated and proliferating multipotent
progenitors while the lung grows (Weaver
et al., 2003). Epithelial buds are a source of trophic or
differentiation signals for the mesenchyme, such as Shh, Vegf and
Wnt7b, among others. Lungs from Shh-null mice show inhibited
mesenchymal cell proliferation and smooth muscle differentiation, and severe
hypoplasia, a condition characterized by abnormal small lungs
(Pepicelli et al., 1998).
Wnt7b-null mouse mutants also have defective vascular smooth muscle
differentiation and are also severely hypoplastic
(Shu et al., 2002). Vegf is
crucial for several aspects of vascular development, and for
epithelial-endothelial interactions during branching morphogenesis
(Pauling and Vu, 2004;
Del Moral et al., 2006).
The transcription factors Foxa1 and Foxa2 are present in
the lung epithelium and have been shown to influence both epithelial and
mesenchymal programs by regulating the levels of expression of Shh
and Wnt7b. The conditional deletion of Foxa1 and
Foxa2 in the distal lung epithelium of transgenic mice inhibits
Shh and Wnt7b expression and results in disrupted branching
morphogenesis and smooth muscle differentiation
(Fig. 3E)
(Wan et al., 2005). It has
been proposed that the pleura controls proliferation and the differentiation
status of the distal mesenchyme (Weaver
et al., 2003). Indeed, mesothelial cells of the pleura express a
number of signaling molecules such as Fgf9, Tgfb3 and RA, which can
mediate these activities (Colvin et al.,
2001; Bragg et al.,
2001; Malpel et al.,
2000). Mice in which these pathways have been inactivated have
pulmonary hypoplasia. Fgf9 (also present in the distal epithelium), probably
signaling via Fgfr1c, acts as a trophic factor for the distal mesenchyme.
Fgf9 mutants have a markedly reduced number of mesenchymal cells,
which results in less Fgf10 being available overall to induce normal
epithelial branching (Colvin et al.,
2001). Fgf9 prevents Shh-induced differentiation of the lung
mesenchyme into smooth muscle in vitro
(Weaver et al., 2003).
The RA synthesizing enzyme Raldh2
(Niederreither et al., 1999)
is co-expressed with Fgf9 in the pleura, and it is possible that
Fgf9 expression is regulated by RA in mesothelial cells
(Malpel et al., 2000;
Colvin et al., 1999). In the
developing heart, RA from mesothelial cells of the epicardium induces
Fgf9, which then activates an Fgf pathway that is essential for
cardiomyoblast expansion (Lavine et al.,
2005). As in Fgf9-null mice, lung hypoplasia has been
also associated with disruption of RA signaling
(Wilson et al., 1953).
Generating cell diversity
Mechanisms influencing proximal-distal cell fate and epithelial differentiation
The mature respiratory epithelium consists of multiple cell types,
including ciliated, neuroendocrine and secretory cells present in proximal
regions of the respiratory system, and type I and type II cells that are
typical of the distal alveolar region of the lung. Cell fate is established
along the proximodistal axis of the respiratory epithelium as lung buds form
and branch, and seems to depend on a distal signaling center in which Bmp4 and
Wnt canonical signaling are crucial. When Bmp signaling is inhibited in
transgenic mice by targeting a dominant-negative Bmp receptor
(dnAlk6) or Bmp antagonists (noggin, gremlin) to the distal lung
epithelium, development of the distal epithelium is severely impaired and the
lung becomes `proximalized' (Weaver et
al., 1999; Lu et al.,
2001). In the model proposed by the authors of these studies,
disrupted Bmp signaling makes lung epithelial cells from distal buds acquire a
proximal phenotype and stop branching. Proximalization also results from the
targeted deletion of ß-catenin in the distal lung epithelium
(Weaver et al., 1999;
Mucenski et al., 2003;
Lu et al., 2001). Thus, a
gradient of Bmp4 and Wnt signaling, with the highest levels of activation in
distal epithelial buds, is thought to prevent distal cells from assuming a
proximal phenotype (Fig. 3F).
At subsequent stages, other molecules contribute to the differentiation of the
epithelium into specific cell types. For example, in the proximal lung, the
forkhead box transcription factor Foxj1 is required to form ciliated
cells (Chen et al., 1998).
Disrupted distal lung development is also seen in transgenic mice in which
RA signaling is constitutively activated in the distal lung epithelium
throughout branching morphogenesis
(Wongtrakool et al., 2003).
Endogenous RA signaling is active during primary lung bud formation, but is
downregulated in the epithelium once secondary budding and branching initiates
(Malpel et al., 2000).
Interestingly, in the transgenic model above, RA signaling persists in the
distal epithelium, as during early developmental stages. As a result, distal
lung progenitors are present but do not undergo further differentiation and
remain immature (Wongtrakool et al.,
2003). Thus, in the developing lung, RA seems to act as a
developmental switch. RA is initially `on' to activate an early developmental
program, but later it has to be turned `off' to allow subsequent stages of
this program to take place in the distal lung.
Maintaining progenitor cells while differentiating
As epithelial cells in branching airways continue to differentiate, it is
crucial to maintain and expand a pool of uncommitted progenitor cells for
continued growth. It has been proposed that this pool resides in the distal
lung, as a population of proliferating immature epithelial cells that
expresses high levels of the proto-oncogene Mycn. Targeted disruption
of Mycn expression in distal lung epithelial cells of mutant mice
inhibits distal lung proliferation and induces premature differentiation
(Okubo et al., 2005).
Inhibition of Wnt canonical signaling in mutant mice causes a similar lung
phenotype and shows that Wnt controls levels of Mycn and
Bmp4 expression in the distal lung
(Shu et al., 2005)
(Fig. 3F). There is also
evidence that activation of Fgf10 and Fgfr2b signaling in the developing
pancreas, tooth, skin and the lung may be required to expand or maintain a
pool of epithelial progenitor cells during organogenesis
(Harada et al., 2002;
Bhushan et al., 2001;
Norgaard et al., 2003).
Cell plasticity: reprogramming cell fates
There is accumulated evidence in vitro and in vivo that developmental
programs can be altered in cells that have initially embarked on a specific
lineage pathway, simply by changing the type or amount of signals in the local
environment. This has been documented in tissue recombination experiments in
vitro. Reprogramming of tracheal epithelium or ureteric bud by lung mesenchyme
has been demonstrated by the induction of a lung-specific pattern of branching
and differentiation that is not normally present in tracheal or ureteric
progenitor cells (Lin et al.,
2003; Shannon,
1994). These observations are consistent with the idea that local
inductive signals from the mesenchyme confer novel position-specific
information that radically changes epithelial cell fate. Fgf proteins,
collagen XVIII, Wnt2, Shh and transferrin are some of the molecules that have
been implicated in the in vitro reprogramming of the lung epithelium
(Lin et al., 2003;
Hyatt et al., 2004;
Ohtsuka et al., 2001). Lung
epithelial reprogramming has also been shown in vivo in transgenic mice
harboring a Sfptc-driven constitutively active ß-catenin/Lef
fusion protein construct (Okubo and
Hogan, 2004). Remarkably, these lungs lack differentiated lung
cell types and show a hyperproliferative epithelium that expresses Cdx1,
Atoh1 and other genes involved in the establishment of intestinal cell
lineages. Although it is not clear exactly when the transgene starts to act,
the successful targeting of the transgene to Sftpc-expressing cells
suggests that increased Wnt signaling leads lung progenitor cells to change
their fate into an intestinal secretory cell fate. Whether Wnt signaling
influences the initial specification of the lung field in the primitive
foregut, remains to be investigated.
Conclusion
Overall, the studies so far suggest that the major events in early lung
morphogenesis are controlled by a relatively limited group of molecules (Fgfs,
Tgfb, Shh, Wnt proteins). Novel insights will be gained by exploring the
different ways by which expression or activation of these molecules is
controlled; these include gene methylation, endogenous microRNAs and
proteolysis, among other mechanisms (Lu et
al., 2005b; Harris et al.,
2006; Li et al.,
2004). A crucial role for heparan and chondroitin sulfate
proteoglycans as modulators of growth factor distribution and signaling in
organogenesis has been well documented in several developing systems. In
Drosophila, integrity and proper sulfation of heparan are essential
for Fgf signaling and tracheal morphogenesis
(Kamimura et al., 2001). There
is evidence that this is also true for the mammalian lung, but the mechanisms
remain to be understood (Izvolsky et al.,
2003; Shannon et al.,
2003).
The lack of early markers of lung progenitor cells represents a clear gap of knowledge in the field. Because Sftpc expression cannot be identified prior to the emergence of primary buds, Nkx2.1 is the only early marker currently available for these cells. A confounding issue is that this gene is also expressed by the thyroid. Laser capture microdissection approaches and detailed gene profiling analysis of the developing foregut will be useful to find other markers of lung progenitor cells. Also crucial will be the development of tools for targeting genes to these early progenitors in the foregut in future functional studies.
Still relatively little is known about the changes that are induced in the milieu around the nascent lung bud and in the bud itself when Fgf10 activates Fgfr2b. How do cells rearrange to form new buds? These questions require powerful image analysis systems and an array of markers, which may be already available.
A rather more complex problem is the understanding of how the coordinates that set up the three-dimensional pattern of morphogens, such as Fgf10, are established in the lung. Finally, there is the much debated issue of stem cells. What are these cells? Where are their niches in the developing lung? How can they be identified? Tackling these issues will provide insights into the molecular and cellular mechanisms by which the lung develops.
Note added in proof
A recent report by Eblaghie et al.
(Eblaghie et al., 2006)
provides genetic evidence that Bmpr1a mediates an autocrine signaling required
for distal lung epithelial cell proliferation and survival.
ACKNOWLEDGMENTS
We thank Mark Krasnow, Jerry Brody and Mary Williams for helpful discussions. We also thank present and former members of the laboratory (Felicia Chen, Konstantin Izvolsky, Jeff Sedita, Tushar Desai, Cherry Wongtrakool and Sarah Malpel) for valuable insights and for some of the work cited here. We are grateful to Brigid Hogan for critical reading of the manuscript. The authors research is, in part, supported by NIH/NHLBI.
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