1 Developmental Biology Program
and Department of Surgery, Complementary molecular and genetic approaches
are yielding information about gain- versus loss-of-function phenotypes
of specific genes and gene families in the embryonic, fetal, neonatal, and adult lungs. New insights are being derived from the conservation of function between genes regulating branching morphogenesis of the
respiratory organs in Drosophila and
in the mammalian lung. The function of specific morphogenetic genes in
the lung are now placed in context with pattern-forming functions in
other, better understood morphogenetic fields such as the limb bud.
Initiation of lung morphogenesis from the floor of the primitive
foregut requires coordinated transcriptional activation and repression involving hepatocyte nuclear factor-3
sprouty; branchless; breathless; vascular endothelial growth
factor; hepatocyte forkhead homologue; surfactant protein A; pulmonary
neuroendocrine cells; Notch; laminin
A remarkable feat of tissue
engineering provides a vast and extremely thin surface for transfer of
gasses between air and blood...how little we know for sure and how much
remains to be learned. Functional homologies between the genes regulating branching
morphogenesis in the tracheal system of
Drosophila and genes regulating
mammalian lung morphogenesis are providing new insights into the
conserved mechanisms of respiratory organogenesis (9, 13, 46).
Drosophila tracheae arise from a
series of lateral, segmentally distributed respiratory placodes on the
larval surface. The primary tracheal branch is a multicellular
structure. The secondary branches arise as unicellular tubes derived
from extensions of the most distal cell on the tip of the primary
branch. Tertiary cytoplasmic extensions then arise from the secondary
branch cells and conduct gas to individual cells in the larva.
Functional analysis of null mutant phenotypes (Table
1) has revealed that, in
Drosophila, the
branchless gene activates the
breathless gene in tracheal cells to
induce primary branching (36, 37). Critical positive regulators of
tracheal branching morphogenesis in
Drosophila are now known to be highly
conserved with mammalian genes that induce respiratory branching
morphogenesis: branchless is
homologous to fibroblast growth factor (FGF)-10, whereas
breathless is homologous to the
FGF-receptor (FGFR) family. Functional homology is also suggested by
the finding that transgenic expression of a dominant negative FGFR from
the surfctant protein (SP) C promoter in mice results in profound
inhibition of lung morphogenesis distal to the primary bronchi (34).
FGF-10 is expressed in murine embryonic lung mesenchyme and exerts a
chemotactic effect on embryonic lung epithelium (32). Null mutation of
FGF-10 in mice results in a lack of respiratory organogenesis distal to
the carina as well as a complete absence of limb buds (27). The
Drosophila
sprouty gene product is a negative
modulator of FGF signaling. Null mutation of
sprouty results in a strong
gain-of-function phenotype in tracheal branching in
Drosophila. Several human sprouty
homologues have recently been discovered (9). Lessons from the studies
of airway morphogenesis in Drosophila
include the following: 1)
morphogenesis involves both positive and negative regulators,
2) the signaling genes are conserved
through evolution, and 3) the
signaling molecules function throughout development so that studies of
early development will be likely to increase our understanding of later
physiological processes.
ABSTRACT
, Sonic hedgehog,
patched, Gli2, and Gli3 as well as Nkx2.1. Subsequent
inductive events require epithelial-mesenchymal interaction mediated by
specific fibroblast growth factor ligand-receptor signaling as well as modulation by other peptide growth factors including epidermal growth
factor, platelet-derived growth factor-A and transforming growth
factor-
and by extracellular matrix components. A scientific rationale for developing new therapeutic approaches to urgent questions
of human pulmonary health such as bronchopulmonary dysplasia is
beginning to emerge from work in this field.
INTRODUCTION
Julius H. Comroe,
1965 MOLECULAR EMBRYOLOGY of the lung
is still a young field: the term was coined in 1992 (49). However,
significant progress has been made in identifying molecular determinants of embryonic lung morphogenesis and cell lineage differentiation during the past six years. The following report of a
recent National Heart, Lung, and Blood Institute Workshop, compiled
from individual presenter's remarks, briefly highlights recent
scientific advances in the molecular embryology of the lung, places
them in the context of likely future advances, and suggests new
experimental approaches. Advances in the field are rapidly improving
our understanding of the molecular pathobiology of human pulmonary
disease. Work in this field is not only increasing our comprehension of
interactive and parallel pathways mediated by known genes but is also
discovering novel targets. Thus new rational therapeutic approaches to
lung disease, especially in the developing lung, are likely to emerge.
GENETICS OF TRACHEAL BRANCHING MORPHOGENESIS IN
DROSOPHILA: CONSERVATION OF GENE
FUNCTION1
Table 1.
Selected Drosophila null mutant phenotypes that give rise to defects of
respiratory tracheal organogenesis
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PATTERN FORMATION IN THE ANTERIOR FOREGUT AND THE MORPHOGENETIC FUNCTION OF NKX2.12 |
---|
Pattern formation in the early embryonic anterior foregut involves Nkx2.1, which is also known as either thyroid transcription factor-1 or thyroid-specific enhancer-binding protein (T/ebp). Nkx2.1 is a homeobox gene involved in the positive and negative regulation of lung-specific genes (28). Nkx2.1 expression has been detected in the primitive anterior foregut and the two main bronchi of the primitive lung bud. It is expressed only in the epithelium. However, at later stages, Nkx2.1 expression is extinguished in some specific respiratory epithelial lineages while continuing to be expressed at high levels in others. The Nkx2.1 null mutation in mice results in a severe loss of lung morphogenesis (18). The Nkx2.1 null mutants fail to separate the trachea from the esophagus. These mice have a common tube leading from the pharynx to the stomach, suggesting that Nkx2.1 must play a significant role in the early ventralization of the pharynx. The lungs arise as a pair of epithelial bags bulging from the sides of the tracheoesophageal common lumen but do not undergo significant further morphogenesis. Alterations in gene expression of vascular endothelial growth factor isoforms, SPs, and 10-kDa Clara cell secretory protein (CC10) demonstrate that the absence of Nkx2.1 arrests lung development at an early stage. The study of regulatory regions of the Nkx2.1 gene reveals that Nkx2.1 has multiple promoters containing binding sites for both ubiquitous and specific transcription factors including GATA, hepatocyte nuclear factor (HNF)-3, and Nkx2.1 itself, the latter finding suggesting a possible positive regulatory mechanism to sustain expression of Nkx2.1. Thus initiation of Nkx2.1 expression may require morphogenetic signaling, but expression may then be maintained by autoregulation.
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SONIC HEDGEHOG AND GLI GENES FUNCTION IN EPITHELIAL-MESENCHYMAL INTERACTION IN EMBRYONIC LUNG MORPHOGENESIS3 |
---|
Lung pattern formation, both branching and differentiation, depends on epithelial-mesenchymal interactions. Hedgehog was first discovered in a genetic screen in the fruit fly by Nusslein-Volhard and Weischaus (31). Subsequently, a hedgehog protein family comprising Sonic, Indian, Desert, and Tiggy-Winkle hedgehog proteins has become widely studied in mammals. Sonic hedgehog (Shh) null mutant mice exhibit grossly abnormal foregut and lung development (6). Transgenic mice overexpressing Shh in the lung epithelium from the SP-C promoter die shortly after birth, have an abundance of mesenchyme, and lack functional alveoli. Abrogation of Shh function with either antisense oligodeoxynucleotides or neutralizing antibodies reduces embryonic rat lung branching in culture. Shh has been identified in the developing lung epithelium and the early embryonic lung: its expression pattern coincides with Nkx2.1. Shh proprotein is cleaved into 19-kDa NH2-terminal and 26- to 28-kDa COOH-terminal peptides. The NH2-terminal peptide is modified by the addition of a cholesterol moiety that acts as a cell membrane anchor. The COOH-terminal peptide is freely diffusible and exerts distant effects in a concentration-dependent manner. Soluble, secreted Shh acts on adjacent fibroblasts by binding to the patched (Ptc) transmembrane receptor (10). Antisense inhibition of Ptc also resulted in diminished rat lung branching in culture. On Shh binding, Ptc releases the segment polarity gene smoothened (Smo). Once Smo is released, zinc finger (Gli) proteins are activated to function as transcriptional factors.
There are three separate Gli proteins (14). Gli3 null
[Gli3(/
)] mice show a mild lung phenotype,
whereas Gli2/Gli3 double-null mutants do not develop lungs and have
tracheoesophageal fistulas, a phenotype that is strikingly similar to
the Nkx2.1 and Shh null mutations (8, 29). Gli2(
/
) mice
have unilobar lungs bilaterally (in contrast to the normal phenotype of
four lobes on the right and one on the left). Together, these studies
suggest that the Shh-Ptc-Gli pathway is involved in early pattern
formation in the lung, although the downstream targets of Gli remain to
be elucidated. The similarity of the double-null mutant phenotype of
Gli2/Gli3 to Nkx2.1 and Shh suggests functional interactions between
these gene families during lung morphogenesis.
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TRANSCRIPTION FACTORS INTERACT IN REGULATORY NETWORKS TO DETERMINE LUNG EPITHELIAL CELL FATE4 |
---|
Regulatory networks determining cell fate and proliferation are
controlled at the transcriptional and signaling levels by interplay
among numerous genes and gene families (51). Epithelial differentiation
of foregut endoderm is directed, at least in part, by the interactions
among HNF-3, various other forkhead homologue family members, and
the homeodomain protein Nkx2.1 as well as by GATA family members. These
genes interact at the level of target gene transcription, mediating
expression of SP genes as well as of transcription factors. For
example, GATA-6 regulates SP-A and SP-C; Nkx2.1 regulates SP-A, SP-B,
SP-C, SP-D, and CC10; and HNF-3
regulates SP-B and CC10, whereas
Nkx2.1 also regulates itself (52). Understanding the role of these
transcription factors has been advanced by studies in transgenic mice
and gene deletion. New results show that hepatocyte forkhead
homologue-4, HNF-3
, and Nkx2.1 play critical roles in determining
epithelial cell differentiation in transgenic and null mutant mice in
vivo. This supports a model in which combinations of transcription
factors interact in regulatory pathways that determine lung epithelial cell fate and differentiation.
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SP-A: FUNCTION IN VIVO AND ITS TRANSCRIPTIONAL REGULATION5 |
---|
SP-A is initially expressed during the pseudoglandular stage of lung
development in distal tubular epithelia. SP-A levels accumulate during
development, reaching maximal levels in the adult lung. Cellular sites
of SP-A production in the adult are tracheal and bronchial gland cells,
Clara cells, and alveolar epithelial type 2 cells (AEC2). SP-A null
[SP-A(/
)] mice have normal appearing lungs
and normal surfactant metabolism and survive and breed normally in a
pathogen-free environment. However, surfactant from
SP-A(
/
) mice is inhibited by plasma protein and has
reduced surface tension-lowering properties (20). SP-A(
/
)
mice also clear group B streptococci from the lung less efficiently
than wild-type mice (21). Macrophages from SP-A(
/
) mice
phagocytose group B streptococci less efficiently and produce reduced
levels of oxygen radicals in response to group B streptococcal
infection. These in vivo studies support a postnatal role for SP-A in
lung function and protection from bacterial injury.
To determine the mechanisms that upregulate SP-A levels after injury, SP-A transcription has been studied in MLE-15 cells. Nkx2.1, B-Myb, and nuclear factor (NF)-1 have been identified as critical regulators. Nkx2.1 binds four cis-acting elements in the SP-A promoter (4). Site 3 and 4 interactions appear to be involved in the upregulation of transcription. Site 1 is closely juxtaposed to an NF-1 consensus element, and mutation of the NF-1 site affects SP-A transcription and Nkx2.1 binding. A consensus Myb site 1 required for SP-A expression and phosphorylation of B-Myb markedly enhances its activity as a trans-activator of SP-A transcription. Phosphorylated B-Myb is principally detected in proliferating cells, suggesting that B-Myb may be an important regulator of SP-A expression in the regenerating epithelium. SP-A, like many cell-specific genes including SP-B, SP-C, and CC10, is transcriptionally regulated by combinatorial interactions of ubiquitously expressed transcriptional factors. Future challenges include determining the unique combinations and timing of control of transcriptional regulators that lead to normal levels of SP-A after lung injury.
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MESENCHYMAL SIGNALS AS INDUCERS OF LUNG EPITHELIAL PHENOTYPE6 |
---|
Studies in avian and mammalian species have consistently demonstrated that branching of the presumptive pulmonary epithelium requires interactions with the underlying mesenchyme. This interaction is highly specific because only pulmonary mesenchyme can induce pulmonary development.
Because commitment to distal lung differentiation has already occurred by the time that the lung rudiment is identifiable, i.e., cells expressing SP-C are already present in the initial lung buds, studies of the interaction between the pulmonary mesenchyme and epithelium are complicated. However, grafting lung mesenchyme (LgM) onto tracheal epithelium (TrE) results in morphogenesis of supernumary branches identical to normal peripheral lung buds: SP-C expression is induced within 24 h after grafting. However, TrE can respond to LgM only during a restricted window in development. Also, LgM can only induce epithelia that are competent to respond to substances it produces; neither esophageal nor intestinal epithelium will respond. Mesenchyme from the trachea or bronchus proximal to the distal tips are noninductive: reciprocal combinations of tracheal mesenchyme with lung epithelium resulted in cyst formation and no branching. However, TrE recombination with tracheal mesenchyme did induce mucin expression, a proximal epithelial cell marker. Transfilter tissue recombinations reproduced these findings, indicating that the inductive events seen were mediated by soluble factors and that tissue-tissue contact was not necessary. Deleting individual components of the defined medium revealed that FGF-7 is necessary but not sufficient to induce SP-C expression in TrE (41-43).
In summary, LgM is a potent inducer of both branching morphogenesis and SP-C expression. Production of this inductive activity is both temporally and spatially restricted in the embryonic lung and is diffusible across a filter. The inductive effects of LgM on LgE and TrE can be replaced in part by a defined medium containing growth factors and using an extracellular matrix to support epithelial cell differentiation.
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FGF SIGNALING IN LUNG EPITHELIAL BRANCHING AND DIFFERENTIATION7 |
---|
FGF signaling activates a number of intracellular pathways fundamental for cell proliferation, differentiation, and pattern formation in several developing systems. A restricted number of FGF family ligands and all FGFRs are present in the embryonic lung, and their expression is regulated in time and space. Perturbation of the FGF signaling pathway during lung development results in dramatic abnormalities of epithelial branching and differentiation. Some aspects of differentiation appear to depend on a specific ligand: in AEC cultures, FGF-7 can induce an AEC2-like phenotype, whereas FGF-1 cannot, even though FGF-1 and FGF-7 can both bind the same FGFR-2IIIb subtype (5). However, some effects of FGF ligands appear to be determined by the temporospatial distribution of FGFRs and heparan sulfate proteoglycans, which also influence ligand-receptor interactions: FGF-1 induces budding in epithelial cultures at sites with the highest concentration of FGFRs. FGF-10 is expressed at high levels during lung development in the distal mesenchyme at prospective sites of bud formation (2). In cultured embryonic lungs, FGF-10-soaked beads attract distal epithelial buds that eventually surround the bead, suggesting that FGF-10 may be acting as a guidance signal for the distal epithelium (32). But FGF-10 did not interfere with epithelial differentiation and had a weak effect on proliferation. The FGF-10 null mutation resulted in the absence of lung distal to the carina as well as the absence of limbs (27), establishing that FGF-10 signaling plays a role in organizing both limb and lung development.
In summary, FGF signaling regulates embryonic lung epithelial cell patterning, differentiation, and proliferation. Further studies of the interactions between FGF signaling and other signaling pathways will be fundamental for understanding the specific roles of FGF signaling in lung morphogenesis.
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MECHANISTIC INSIGHTS FROM LIMB BUD DETERMINATION8 |
---|
Pattern formation of both the wing and the leg in the chick is determined by the apical ectodermal ridge and zone of polarizing activity (ZPA), which are located on the anterior and posterior sides, respectively, of the limb rudiment (47, 48). Transplantation or ectopic expression of ZPA activity results in limb duplication. The key signaling processes in limb morphogenesis involve FGF-8 and-10, Shh, bone morphogenetic protein-2, and retinoic acid (RA) signaling (11). RA is required for the initial inductive event but is then not required for subsequent limb growth (12, 47): an RA-degrading enzyme is present once the limb bud has formed. Retinoid X receptors and RA-receptor antagonists result in limb pattern defects: antiretinoids block establishment of the ZPA and block the expression of Shh and bone morphogenetic protein-2 but not of FGF-8 and homeobox gene (Hox) D-13 (24). Recently, it has emerged that the morphogenetic effects of RA may be mediated by an RA-activated cysteine kinase-1. FGF signaling induces the expression of RA-activated cysteine kinase-1, which, in turn, activates and stabilizes protein kinase C.
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THE PULMONARY NEUROENDOCRINE SYSTEM AND LUNG MORPHOGENESIS9 |
---|
Pulmonary neuroendocrine (PNE) cells are among the first cells to differentiate from the primitive lung epithelium. PNE cells are surrounded by a greater density of proliferating cells than elsewhere in the epithelium. PNE cells express a number of proliferative cytokines, including calcitonin gene-related peptide and bombesin. Bombesin-like peptides (BLPs) promote branching morphogenesis and stimulate both epithelial and mesenchymal cell proliferation, AEC2 differentiation, surfactant phospholipid synthesis, and secretion as well as Clara cell and PNE cell differentiation (19). Stimulation of surfactant phospholipid synthesis by gastrin-releasing peptide in embryonic day 20 rat AECs depends on coculture with fetal lung fibroblasts, suggesting a mesenchymal-epithelial interaction. The effect of BLP on lung morphogenesis may also depend on mesenchymal cells. A working hypothesis for the role of PNE cells in lung development is that PNE cells release BLP and calcitonin gene-related peptide that stimulate lipofibroblasts in the lung mesenchyme to interact with airway epithelium, thereby regulating AEC2 differentiation.
Recent data indicate that the murine homologue of the Drosophila gene Notch is expressed in developing lung. Abrogation of Notch gene expression with antisense oligodeoxynucleotides induces PNE cell differentiation in embryonic day 12 rat lung buds.
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CELL ADHESION AND PULMONARY ARCHITECTURE10 |
---|
Laminins (LNs), major protein components of basement membranes, are
hetereotrimeric glycoproteins consisting of ,
, and
polypeptide chains linked together by disulfide bonds. Whereas LN1 and
LN2 are constitutively expressed by embryonic lung epithelial and
mesenchymal cells, synthesis of
LN-
1 chain (and thus LN1) requires epithelial-mesenchymal contact. Immunohistochemical studies on
cocultures treated with brefeldin A, an inhibitor of protein secretion,
indicated that both epithelial and mesenchymal cells synthesize the
LN-
1 chain on heterotypic
cell-cell contact. Lung explants exposed to monoclonal
anti-LN-
1 chain antibodies exhibited alterations in
peribronchial cell shape and decreased smooth muscle development, as
indicated by low levels of
-actin and desmin.
Lung embryonic mesenchymal cells can differentiate into smooth muscle
cells in culture. Smooth muscle differentiation is stimulated by cell
spreading, takes place in <24 h, and is independent of cell
proliferation and cell-cell contact. Cell spreading or elongation appears to be critical because prevention of cell spreading prevents smooth muscle differentiation. In organotypic cocultures of embryonic mesenchymal and epithelial cells, epithelial cells aggregate into cysts
that form the basement membrane and become surrounded by mesenchymal
cells. The mesenchymal cells in contact with this basement membrane
spread and differentiate into smooth muscle, whereas the remainder of
the mesenchymal cells remain round in shape and devoid of smooth muscle
markers. Inhibition of LN polymerization by exposure of the organotypic
cultures to antibodies to the globular region of the
LN-1 or
LN-
1 chain blocks assembly of
the basement membrane, mesenchymal cell spreading, and smooth muscle
differentiation. Thus it appears that LN synthesis is regulated by
epithelial-mesenchymal cell interaction and may regulate smooth muscle
development by facilitating mesenchymal cell spreading along the airway
basement membrane (38-40).
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RECOMBINANT ADENOVIRAL VECTORS ARE USEFUL TOOLS TO DETERMINE CYTOKINE FUNCTION IN LUNG MORPHOGENESIS AND INFLAMMATION11 |
---|
Recombinant replication-deficient adenovirus 5 vectors engineered to express specific cytokines and receptors efficiently transfer and express these genes in lung tissue (7, 44, 45). The trachea is the most accessible route of administration of adenoviral vectors in vivo and results in infection primarily of the respiratory epithelium. Expression of a gene(s) begins within a few hours and extends over several days. No genomic integration occurs. Adenoviral vector load can be titrated, and multiple vectors can be administered simultaneously. For receptor and metabolic inhibitor studies, only the infected cells are affected. An acute inflammatory response can occur with high viral loads, but the immune reactions generated in mature animals may not be a consideration in embryonic and neonatal lungs. Intrauterine administration delivers the vector to the fetal lung. Intratracheal administration to embryonic lungs in culture results in efficient infection and gene expression in the primitive airway epithelium.
Current first-generation vectors have been effective in elucidating
cytokine function in adult, fetal, and embryonic lungs. New
developments include construction of replication-deficient vectors with
inducible promoters to investigate the introduction of lethal genes and
helper-dependent "empty" adenoviral vectors that exhibit
prolonged expression and can incorporate many genes. Proinflammatory
cytokines such as tumor necrosis factor- and immune-regulating
cytokines such as interleukin (IL)-4, IL-6, and IL-12 have been tested.
Chemokines such as IL-8 and lymphotactin cause accumulation of
inflammatory cells, whereas inhibitory cytokines such as IL-10
interfere with cytokine function. In general, although these factors
cause marked temporary changes in lung morphology, there is a
remarkable lack of sustained changes when they are expressed at low to
intermediate levels. Growth and differentiating cytokines such as
granulocyte-macrophage colony-stimulating factor and transforming
growth factor (TGF)-
, when expressed at high levels, cause matrix
accumulation and fibrogenesis, whereas in the embryonic lung explant,
dominant negative TGF-
type II receptor (TGF-
IIR) causes a
similar gain-of-function phenotype for branching morphogenesis as does
abrogation of the TGF-
ligand function. The application of
adenoviral vectors can be made on any genetic background and can be
used to replace specific cytokine function in the lung epithelium of
null mutant or transgenic mice.
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INTEGRATION OF SIGNALING SYSTEMS IN LUNG MORPHOGENESIS12 |
---|
It is clear from the foregoing sections that initiation of pulmonary
morphogenesis from the epithelium lining the floor of the embryonic
foregut requires integrated signaling and coordinated transcriptional
activation and repression involving HNF-3, Shh, Ptc, Gli2, and Gli3 as
well as Nkx2.1 (Table 2). Subsequent early inductive events mediating not only primary tracheal branching but also
the organization of secondary and tertiary branching events as well as
lung angiogenesis involve reciprocal mesenchymal-epithelial and
epithelial-mesenchymal signaling mediated by diffusible FGF family
peptides that signal through cognate FGFRs.
|
Signaling by other classes of peptide growth factors also plays pivotal
roles in lung morphogenesis, determining the rate of cell proliferation
and differentiation, activation or repression of key transcripitional
factors such as Nkx2.1, and synthesis of key extracellular matrix
elements such as LN. In general, peptide growth factor receptors with
intracellular tyrosine kinase signaling domains, such as epidermal
growth factor (EGF) receptor, platelet-derived growth factor (PDGF)
receptor, FGFR, insulin-like growth factor receptor-1, and c-Met, are
inductive and/or permissive for embryonic lung morphogenesis, whereas
activation of TGF- type I and II receptors with intracellular
serine/threonine kinase domains is inhibitory for embryonic lung
morphogenesis (16, 26, 54-57). But how these positive and negative
pathways are integrated at the molecular level to determine embryonic
lung morphogenesis is almost completely unknown.
In early murine embryonic lung in culture, abrogation of TGF-
signaling, either by antisense oligodeoxynucleotide abrogation of
TGF-
IIR expression, by immunoperturbation of TGF-
IIR
ligand-receptor interactions, or by intratracheal microinjection of
recombinant adenoviruses expressing a dominant negative TGF-
IIR, all
result in a strong gain-of-function phenotype for early murine
embryonic lung branching morphogenesis. The positive effects of
exogenous EGF or PDGF-A ligands are potentiated by abrogation of
TGF-
IIR signaling, suggesting that endogenous TGF-
signaling
negatively regulates EGF and PDGF signaling. Similarly, abrogation of
betaglycan (TGF-
IIIR) expression with antisense
oligodeoxynucleotides stimulates lung morphogenesis in culture and
strongly inhibits the effectiveness of exogenous TGF-
ligands,
particularly TGF-
2, to inhibit lung morphogenesis in culture (31).
Recently, Smad family proteins have emerged as key downstream mediators
of the TGF-
-receptor signaling pathway. Briefly, Smads 2 and 3 bind
to the TGF-
IR and TGF-
IIR heteromeric complexes and are
phosphorylated by the receptor serine/threonine kinases. Smads 2 and/or
3 can then activate Smad 4, which transduces the TGF-
signal to the
nucleus. Smads 6 and 7 are inhibitors of activation of Smads 2 and/or
3. Abrogation of Smads 2 and 3 or 4 with antisense
oligodeoxynucleotides results in a strong gain-of-function phenotype
for branching morphogenesis of early murine embryonic lung in culture,
similar to that obtained after abrogation of TGF-
II or TGF-
IIIR
signaling. It will be important to determine whether Smads are key
focal regulators of the inductive tyrosine kinase cognate receptor
pathways and the inhibitory serine/threonine kinase cognate receptor pathways.
The concept was introduced above that striking cDNA sequence homologies suggest conservation of function between the breathless, branchless, and sprouty genes that control tracheal branching in Drosophila respiratory organogenesis and their respective murine homologues FGF-10, FGFR2, and msprouty. Antisense oligodeoxynucleotides against murine sprouty2 (mspry2) expression result in a strong gain-of-function phenotype for branching morphogenesis of early murine embryonic lungs in culture. Thus growth factor antagonists and binding proteins such as mspry2 and betaglycan may modulate and thus integrate peptide growth factor signaling during lung branching morphogenesis.
In summary, induction and modulation of embryonic lung morphogenesis by transcriptional factor and by peptide growth factor signaling mechanisms can occur at a number of levels of integration including temporospatial and stoichiometric regulation of ligand, cognate receptor, and ligand binding proteins and intracellular effector protein expression and function as well as of transcriptional factor activation and suppression of key developmental gene promoters.
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WHAT REMAINS TO COMPLETE A MOLECULAR PROFILE OF LUNG DEVELOPMENT, AND WHAT IS THE POTENTIAL FOR APPLICATION OF THIS INFORMATION TO QUESTIONS OF HUMAN HEALTH? |
---|
Major advances have been made over the past six years, arising from
investigator-initiated, candidate gene approaches, in determining the
function of individual molecules during the embryogenesis of the lung.
For example, the transcriptional factor Nkx2.1 has been shown to
function as a "master gene" that induces and maintains lung
morphogenesis and differentiation of lung epithelial cell lineages.
Expression of Nkx2.1 is downregulated in lung tissue obtained from both
premature human and premature baboon infants with severe
bronchopulmonary dysplasia. TGF-1 activity is also markedly
increased in tracheal effluent fluid obtained from premature human
infants at risk for developing bronchopulmonary dysplasia, and TGF-
signaling is known to negatively regulate lung morphogenesis as well as
expression of the Nkx2.1 and SP genes. Thus new molecular therapeutic
targets are being identified from candidate gene studies on the
molecular embryology of the lung. These targets are quite likely to be
amenable to rational therapeutic intervention to prevent or ameliorate
the effects of abnormal signaling that culminate in human lung disease.
Future challenges include not only comprehending individual gene
functions as well as interactions between known candidate genes but
also the discovery of novel pathways, interactions, entry points, and
targets that are emerging from as yet incompletely characterized
genomic information. This will require significant investments in new
biotechnologies as well as in bioinformatics (Table
3).
|
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ACKNOWLEDGEMENTS |
---|
This is a report on a National Heart, Lung, and Blood Institute-sponsored workshop held at the Natcher Conference Center, National Institutes of Health, Bethesda, MD, on June 1, 1998. The chairs were Merton Bernfield, David Warburton, and Mary Williams.
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FOOTNOTES |
---|
1 Presented by Mark Krasnow.
2 Presented by Parviz Minoo.
3 Presented by Martin Post.
4 Presented by Jeffrey Whitsett.
5 Presented by Thomas Korfhagen.
6 Presented by John Shannon.
7 Presented by Wellington Cardoso.
8 Presented by Gregor Eichele.
9 Presented by Mary Sunday.
10 Presented by Lucia Shuger.
11 Presented by Jack Gauldie.
12 Presented by David Warburton.
Address for reprint requests and other correspondence: D. Warburton, Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, 4650 Sunset Blvd., MS 35, Los Angeles, CA 90027 (E-mail: dwarburton{at}chla.usc.edu).
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