INVITED REVIEW
Molecular embryology of the lung: then, now, and in the future

David Warburton1,2, Jingsong Zhao2, Mary Anne Berberich3, and Merton Bernfield4

1 Developmental Biology Program and Department of Surgery, Childrens Hospital Los Angeles Research Institute, and 2 Center for Craniofacial Molecular Biology, University of Southern California Schools of Medicine and Dentistry, Los Angeles, California 90027; 3 Developmental Biology and Pediatrics Group, Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and 4 Division of Developmental and Newborn Biology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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-3beta , 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-beta 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.

sprouty; branchless; breathless; vascular endothelial growth factor; hepatocyte forkhead homologue; surfactant protein A; pulmonary neuroendocrine cells; Notch; laminin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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.
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
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INTRODUCTION
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INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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.

                              
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Table 1.   Selected Drosophila null mutant phenotypes that give rise to defects of respiratory tracheal organogenesis


    PATTERN FORMATION IN THE ANTERIOR FOREGUT AND THE MORPHOGENETIC FUNCTION OF NKX2.12
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INTRODUCTION
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SONIC HEDGEHOG AND GLI...
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MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
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WHAT REMAINS TO COMPLETE...
REFERENCES

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.


    SONIC HEDGEHOG AND GLI GENES FUNCTION IN EPITHELIAL-MESENCHYMAL INTERACTION IN EMBRYONIC LUNG MORPHOGENESIS3
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INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
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SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
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WHAT REMAINS TO COMPLETE...
REFERENCES

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.


    TRANSCRIPTION FACTORS INTERACT IN REGULATORY NETWORKS TO DETERMINE LUNG EPITHELIAL CELL FATE4
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ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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-3beta , 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-3beta 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-3beta , 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.


    SP-A: FUNCTION IN VIVO AND ITS TRANSCRIPTIONAL REGULATION5
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ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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.


    MESENCHYMAL SIGNALS AS INDUCERS OF LUNG EPITHELIAL PHENOTYPE6
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ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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.


    FGF SIGNALING IN LUNG EPITHELIAL BRANCHING AND DIFFERENTIATION7
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INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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.


    MECHANISTIC INSIGHTS FROM LIMB BUD DETERMINATION8
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ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
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SP-A: FUNCTION IN VIVO...
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WHAT REMAINS TO COMPLETE...
REFERENCES

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.


    THE PULMONARY NEUROENDOCRINE SYSTEM AND LUNG MORPHOGENESIS9
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INTRODUCTION
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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.


    CELL ADHESION AND PULMONARY ARCHITECTURE10
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Laminins (LNs), major protein components of basement membranes, are hetereotrimeric glycoproteins consisting of alpha , beta , and gamma  polypeptide chains linked together by disulfide bonds. Whereas LN1 and LN2 are constitutively expressed by embryonic lung epithelial and mesenchymal cells, synthesis of LN-alpha 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-alpha 1 chain on heterotypic cell-cell contact. Lung explants exposed to monoclonal anti-LN-alpha 1 chain antibodies exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of alpha -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-beta 1 or LN-gamma 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).


    RECOMBINANT ADENOVIRAL VECTORS ARE USEFUL TOOLS TO DETERMINE CYTOKINE FUNCTION IN LUNG MORPHOGENESIS AND INFLAMMATION11
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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-alpha 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)-beta , when expressed at high levels, cause matrix accumulation and fibrogenesis, whereas in the embryonic lung explant, dominant negative TGF-beta type II receptor (TGF-beta IIR) causes a similar gain-of-function phenotype for branching morphogenesis as does abrogation of the TGF-beta 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.


    INTEGRATION OF SIGNALING SYSTEMS IN LUNG MORPHOGENESIS12
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INTRODUCTION
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PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
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WHAT REMAINS TO COMPLETE...
REFERENCES

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.

                              
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Table 2.   Selected murine null mutation and transgenic models that result in informative defects of respiratory organogenesis

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-beta 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-beta signaling, either by antisense oligodeoxynucleotide abrogation of TGF-beta IIR expression, by immunoperturbation of TGF-beta IIR ligand-receptor interactions, or by intratracheal microinjection of recombinant adenoviruses expressing a dominant negative TGF-beta 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-beta IIR signaling, suggesting that endogenous TGF-beta signaling negatively regulates EGF and PDGF signaling. Similarly, abrogation of betaglycan (TGF-beta IIIR) expression with antisense oligodeoxynucleotides stimulates lung morphogenesis in culture and strongly inhibits the effectiveness of exogenous TGF-beta ligands, particularly TGF-beta 2, to inhibit lung morphogenesis in culture (31). Recently, Smad family proteins have emerged as key downstream mediators of the TGF-beta -receptor signaling pathway. Briefly, Smads 2 and 3 bind to the TGF-beta IR and TGF-beta 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-beta 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-beta II or TGF-beta 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.


    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?
TOP
ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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-beta 1 activity is also markedly increased in tracheal effluent fluid obtained from premature human infants at risk for developing bronchopulmonary dysplasia, and TGF-beta 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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Table 3.   Rapid new biotechnologies and bioinformatic-based approaches to accelerate post-genomic era progress in molecular embryology of lung


    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.


    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).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
GENETICS OF TRACHEAL BRANCHING...
PATTERN FORMATION IN THE...
SONIC HEDGEHOG AND GLI...
TRANSCRIPTION FACTORS INTERACT...
SP-A: FUNCTION IN VIVO...
MESENCHYMAL SIGNALS AS INDUCERS...
FGF SIGNALING IN LUNG...
MECHANISTIC INSIGHTS FROM LIMB...
THE PULMONARY NEUROENDOCRINE...
CELL ADHESION AND PULMONARY...
RECOMBINANT ADENOVIRAL VECTORS...
INTEGRATION OF SIGNALING...
WHAT REMAINS TO COMPLETE...
REFERENCES

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