Wnt signaling in lung organogenesis

[Organogenesis 4:2, 100-108; April/May/June 2008]; ©2008 Landes Bioscience

Review

Wnt signaling in lung organogenesis Stijn P. De Langhe and Susan D. Reynolds* Department of Pediatrics; National Jewish Medical Research Center; Denver, Colorado USA

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Although the proximal respiratory system plays a critical role in lung diseases that are a direct consequence of environmental exposures as well as those that are exacerbated by environmental triggers, the relative lack of information regarding roles for Wnt/β-catenin in specification and specialization of these prevents further consideration in this review. The distal respiratory system is divided into the conducting airway and alveolar regions. The airway is comprised of a series of dichotomously branched tubes that decrease in caliber from proximal (trachea), through the central airways (bronchi, bronchial and bronchioles) to the distal region (terminal and respiratory bronchioles). In contrast, alveoli are established through post-natal septation of primary sacules. Epithelial structures lining the arborized system are derived from the foregut endoderm while adjacent mesenchymal structures are derived primarily from the splanchic mesenchyme and first appear on the dorsal aspect of the trachea at the level of the first bifurcation.5 Nervous tissue is present in the form of two large nerve trunks that parallel the airway tree6 and are likely to be derived from the neural crest. The airway and alveolar epithelium exhibit specialized structural features that are specific to regulation of the epithelial barrier, host defense, immunomodulation, water/ ion balance, metabolism, detoxification and protection from physical stress. Epithelial specialization along the proximal to distal axis is paralleled by adaptation of mesenchymally-derived structures which support the external tubular structures and regulate the tone of the internal airways. Regional distinctions in airway structure and function are governed by epithelial mesenchymal interactions that involve the main signaling cascades including the Wnt/β-catenin pathway.

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Reporter transgene, knockout, and misexpression studies support the notion that Wnt/β-catenin signaling regulates aspects of branching morphogenesis, regional specialization of the epithelium and mesenchyme, and establishment of progenitor cell pools. As demonstrated for other foregut endoderm-derived organs, β-catenin and the Wnt/β-catenin signaling pathway contribute to control of cellular proliferation, differentiation and migration. However, the contribution of Wnt/β-catenin signaling to these processes is shaped by other signals impinging on target tissues. In this review, we will concentrate on roles for Wnt/β-catenin in respiratory system development, including segregation of the conducting airway and alveolar compartments, specialization of the mesenchyme, and establishment of tracheal asymmetries and tracheal glands.

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Key words: morphogenesis, respiratory, airway, alveolar, mesenchyme, endoderm

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The Respiratory System

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The respiratory system is divided into sub-regions that perform specialized functions important for the ultimate goal of delivering oxygen to and removing carbon dioxide from the blood stream. The proximal respiratory system is divided into the nasal and oral cavities and the larynx. The nose and mouth (except in rodents) serve as the primary interface between the respiratory system and the environment, function in humidification of inspired air, and are the first line of defense for protection from injurious agents including pathogens, particles, and chemical or gaseous pollutants. These regions exhibit complex structural and functional characteristics that have been extensively reviewed elsewhere.1 Little is known of roles for Wnt/ β-catenin signaling in patterning or functional specialization of these tissues. The third component of the proximal respiratory system is the larynx. This region is composed of complex cartilaginous support structures, an epithelial lining, and an extensive muscular and neural network. Although evidence for Wnt/β-catenin signaling during development of this region has been reported2 high levels of endogenous β-gal activity limit interpretation of studies relying exclusively on LacZ reporter constructs. The larynx is a common target for reconstructive surgery and reparative and signaling mechanisms have been reviewed in the regenerative medicine literature.3,4 *Correspondence to: Susan D. Reynolds; Department of Pediatrics; National Jewish Medical Research Center; Goodman Building; K1026; 1400 Jackson Street; Denver, Colorado 80206 USA; Tel.: 303.270.2920; Email: [email protected] Submitted: 01/31/08; Accepted: 03/06/08 Previously published online as an Organogenesis E-publication: http://www.landesbioscience.com/journals/organogenesis/article/5856 100

Axial Coordinates of the Respiratory System The respiratory system exhibits structural variation along three axes: rostral-caudal, lateral (left-right), and dorsal-ventral. As indicated above, the nasal cavity anchors the rostral end of the primary axis and the lung lobes the caudal region. In humans the left lung is trilobed and the right lung is bilobed. In contrast, in the mouse the right lung is divided into four lobes that branch from the main stem bronchus (cranial, medial, caudal and accessory lobes), whereas the left lung consists of only one lobe. The dorsal-ventral axis is most easily visualized according to the relative positions of the trachea (ventral) and the esophagus (dorsal). While the basic mechanisms governing establishment of functional and structural diversity within the respiratory system are likely to be conserved among mammals, anatomical distinctions are likely to influence susceptibility to injurious agents and the consequent

Organogenesis

2008; Vol. 4 Issue 2

Wnt and lung development

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Patterning of the endoderm. The distal respiratory system including the trachea and lung is derived from the definitive endoderm. This germ layer is established during gastrulation starting on embryonic day 6 (E6) in the mouse. The definitive endoderm is patterned through interactions with the primitive endoderm, particularly the anterior visceral endoderm.17 Thus, anterior-posterior patterning of the visceral endoderm, which is dependent on Wnt/βcatenin signaling, is superimposed on the definitive endoderm as the primitive streak is formed and migrates along the anterior-posterior axis of the preimplantation embryo (reviewed by Marikawa).18 These signals collaborate with the node, a specialized structure at the anterior end of the primitive streak, to further establish the anterior-posterior and dorsal-ventral axes.19 Regio-specific deletion of β-catenin, mosaic analysis and reporter gene analysis indicate that β-catenin-mediated signaling in the visceral endoderm and node are necessary for elaboration of paracrine factors that act on the migrating endoderm.20-22 Patterning of the hindgut. Genetic modification of the β-catenin locus in numerous organs supports the notion that β-catenindependent signaling is tightly regulated and that “just-right” levels are needed to foster appropriate cell fate decisions. An example of this type of analysis is that of Okubo and Hogan,23 which demonstrated that transgenic expression of a β-catenin/Lef1 fusion resulted in transdetermination of the lung endoderm. Since interpretation of these data require knowledge of hindgut differentiation, this subject is briefly reviewed herein. Evidence for Wnt/β-catenin signaling in the endoderm is first evidenced by expression of the TCF/Lef1 reporter transgenes TOPGAL24 and BATGAL21 during gastrulation. These reporter genes are highly expressed first in the presumptive hindgut region and then upregulated in foregut regions. Deletion of β-catenin in the definitive endoderm about E6.5 resulted in formation of multiple hearts and suggested that β-catenin signaling is necessary to maintain endodermal cell fate during gastrulation.25,26 Wnt ligands are detected in various compartments of the endoderm between E12.5 and E 16.5.27 During this period, β-catenin-dependent gene expression in the hindgut is restricted to post-mitotic differentiated villus cells and is dependent on TCF3.28 In contrast, Wnt/β-catenin signaling in the adult is limited to mitotic cells of the crypt of Leiberkuhn and is dependent on TCF4. Mosaic intestines composed of wild type cells and those in which β-catenin dependent gene expression was potentated through expression of a Lef1-N-terminally truncated β-catenin fusion were cleared of modified cells by adulthood.29 These data suggest that both the level of signaling and the transcriptional cofactors vary as a function of time and that both quality and quantity of β-catenin signaling impact hindgut tissue morphogenesis and epithelial maintenance in the adult. Patterning of the foregut. Organ anlagen within the foregut are established as a consequence of inductive processes mediated by heartderived FGF’s reviewed in ref.30 By E9.0 transcriptional domains

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Lung development is divided into four stages that are defined by changes in the structure of the airway tubes and morphological modifications of epithelial cells. This staging method has been extensively reviewed elsewhere12,13 and is summarized here. In th emouse, the pseudoglandular stage initiates with formation of the lung buds on embryonic day 9.5 and lasts through embryonic day 16.5. During this period, the primordial airway tubules are established and the endoderm-derived epithelium exhibits a columnar morphology. Between embryonic days 14.5 and 16.5, distinct bronchial and respiratory systems are established and bronchial and acinar tubules are recognized. Bronchial epithelial cells continue to be cuboidal in shape while the epithelium lining the acinar tubules is low columnar to cubiodal. At the end of the pseudoglandular stage the branching pattern of the conducting airway is established. After this stage, structural modifications are limited to changes in the length and diameter of individual airway segments. The canalicular period runs between embryonic days 16.5 and 17.5. During this time, the airway epithelium downregulates expression of surfactant protein C,14 and begins to exhibit differentiated characteristics such as expression of the secretory cell marker, Clara cell secretory protein (CCSP, also referred to as CC10, CC16, uteroglobin).15 These primordial structures have smooth walls and lack alveolar sacs. The prospective pulmonary acinus is lined by cuboidal type 2 cells and a small number of squamous type 1 cells. Surfactant protein C mRNA and protein are restricted to the alveolar region at this stage. The saccular period runs between embryonic day 17.5 and postnatal day 5 in mice. During this period, the alveolar ducts and alveolar sacs are formed. The latter structures are lined by a flattened epithelium and the mesenchymal tissue thins. The alveolar stage occurs during the postnatal period and can last as long as three months in mice.16 During this time alveolar septation initiates and leads to a tremendous increase in the surface area of the lung. As discussed below, Wnt/β-catenin signaling is not necessary for the establishment of the primary branching pattern of the lung but is necessary for appropriate branching morphogenesis. Stabilization strategies indicate that the magnitude, location and duration of

Changing Roles for Wnt/β-catenin Signaling in Airway Growth and Differentiation

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Stages of Lung Development

β-catenin signaling alter differentiation of the epithelium and the mesenchyme. However, a specific impact of Wnt/β-catenin on later stages of lung morphogenesis will require development of regulatable and cell type specific strategies to modify gene expression during this time frame.

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reparative mechanisms within these species. Thus, analysis of chronic lung disease must assess initiation and progression in structurally and functionally analogous regions of the respiratory tree. The mouse trachea and external lobar bronchi are supported by cartilage and the epithelial barrier is composed of a basal cell containing, pseudostratified, mucociliary epithelium. This region is similar to the first six generations of the human lung with the exception that glandular structures in the mouse are restricted to the first three-four tracheal rings of the trachea7 whereas they are uniformly distributed along the proximal human airway.8 Species-specific variation in the ultrastructural and molecular characteristics of epithelial secretory cells has also been recognized.9,10 Although this analysis suggests that human and rodent terminal bronchioles are similar, the presence of respiratory bronchioles in the human complicates analysis of lung mechanics and reparative mechanisms. Recognition of species-specific variation in respiratory structure and function should serve as a cautionary note but should not prohibit modeling of human lung disease in rodents. Importantly, similarities in the processes rather than the specific players should be sought.11

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TOPGAL and BATGAL studies. Analysis of β-catenin-dependent gene expression in respiratory tissues has relied heavily on the TOPGAL and BATGAL reporter transgenes. Both transgenes utilize an optimized and multimerized TCF promoter and as such are reporters for interactions between β-catenin and TCF factors. This is an important consideration as these transgenes may report only TCF-dependent β-catenin-dependent gene activity and be insensitive to variation in the types of TCF factors present. Furthermore, these transgenes will not detect gene expression in response to interaction of β-catenin with other transcriptional cofactors such as the SOX and PITX transcription factors. Dynamic expression of Lef1, the Tcf ’s35,36 and various Sox37 factors have been reported in lung development and variation in the types of DNA-binding transcription factors will likely influence β-catenin-dependent gene expression. A final concern is that sensitivity may vary between the TOPGAL (cytoplasmic β-gal reporter) and BATGAL (nuclear localized β-gal reporter) transgenes and that the relatively long half-life of the β-gal protein may obscure the cellular-specificity and pulsatile nature of Wnt/β-catenin signaling. With these caveats in mind, expression of the TOPGAL and BATGAL transgenes has been used to draw generalized conclusions regarding the pattern of β-catenin stabilization in the developing lung. Within the respiratory region, the TOPGAL reporter is expressed in the undivided proximal endodermal tube and lung buds as early as E9.5.23 This pattern is maintained as the tracheal and esophagus separate and the lung buds grow between E10 and E11.5.23,35,38,39 Between E12.5 and E18.5 analysis of TOPGAL and BATGAL transgene activity suggests a dynamic pattern of TCF/βcatenin-dependent gene expression. Reporter gene activity is found in the tracheal epithelium and cartilaginous condensations at E12.5 but is restricted to the bronchial epithelium at E13.5.35,38 The distal lung epithelium expresses both reporters by E9.5. The pattern of TCF/β-catenin-dependent gene activity in the distal lung at later time points is somewhat variable and dependent on the reporter transgene analyzed. In general, transgene activity clears from central airways between E13.5 and post-natal day 14.23,38 Colocalization of β-gal and the airway secretory cell marker CCSP suggests that residual transgene positive cells are relatively undifferentiated.38 At

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Painting with Broad Strokes: Analysis of Reporter Gene Activity, Lineage Tracing and β-catenin Deletion/Stabilization Studies

E14.5, expression in the distal tip epithelium is either extinguished (TOPGAL)35 or restricted to a subset of early alveolar type 2 cells (BATGAL).38 In the post-natal period, the TOPGAL transgene is expressed in the surface epithelium and in gland buds.40 In the adult, the TOPGAL transgene is highly expressed in the distal trachea, and sporadically in both airway secretory and ciliated cells, parabronchial smooth muscle, and rarely in the alveolar space (Reynolds SD, unpublished data). Lineage tracing in the lung epithelium. Analysis of epithelial specification and differentiation has relied heavily on two “workhorse” promoters, the surfactant protein C promoter from human (Sfptc, abbreviated Spc) and the Clara cell secretory protein (Scgb1A1, abbreviated CCSP in this manuscript but also referred to as CC10) from rat15 or mouse.41 The strengths and weaknesses of transgenic approaches relying on these promoters have been recognized and appropriate cautions to interpretation of data indicated.42-45 Importantly, both the SPC and the CCSP genes are developmentally regulated. As a consequence, Cre recombinase mediated modification of various loci is limited to developmental period in which these genes are expressed and to epithelial cell layer. Use of these two promoters has yielded a great deal knowledge regarding roles for Wnt/β-catenin signaling during branching morphogenesis but little is known of roles for this signaling pathway in later lung development or in maintenance of adult lung tissues. Since the SPC and CCSP promoters have been utilized to conditionally knockout β-catenin46 and to express constitutively active forms of this protein23,47 the patterns of CRE-mediated recombination of the Z/AP (ROSA26 promoter driving expression of a floxed LacZ cassette and a downstream alkakine phosphatase reporter48) and Z/EG (ROSA26 promoter driving expression of a floxed LacZ cassette and a downstream enhanced green fluorescent protein reporter49) recombination substrates will be reviewed. Lineage tracing studies using a human SPC promoter-regulated transgene indicate that distal lung lineages (intrapulmonary airways and alveolar sacs) are established between E4.5–6.5.42 However, endogenous surfactant protein C gene activity is not detected until E10-10.5 and is localized to the distal tip epithelium.14 Consistent with this pattern, distal progenitor cells labeled at E11.5 were restricted to a small number of cells positioned along the bronchial tubes and clusters of cells at the bronchial tips and lateral buds. Further analysis using the Spc promoter regulated rtTA (rtTA, reverse Tet transactivator) and Tet(O)-CRE (Tet(O), Tet responsive element, sometimes abbreviated TRE) approach demonstrated that extrapulmonary airway lineages (trachea and bronchi) are distinct from distal lineages. A subset of proximal lineage cells was established between E8.5 and E10.5 and was restricted to the distal portion of the trachea and to the lateral margins of this structure. Interestingly, tracheal glands (submucosal glands) were not tagged, even when recombination was induced from E0.5 through postnatal day 7. These data may indicate that submucosal glands are derived from a distinct set of progenitor cells. However, caution is warranted considering that submucosal glands are formed in the post-natal period (a time when the Spc promoter is inactive in airways), the fact that submucosal glands are limited to the first 3–4 tracheal rings (a region in which very low levels of transgene activity was detected), and the fact that lineage tracing studies strongly support a lineage relationship between bronchial and glandular lineages.7,40,50 These studies demonstrate that the

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that define the pancreas, liver, lung/trachea and thymus are evident. The homeodomain transcription factor Nkx2.1 marks the thymic anlagen on E8-8.531 and the respiratory region on E9.32 Although Nkx2.1 is expressed in both the trachea and lung primordium it is necessary only for generation of distal lung lineages. Nkx2.1 null lungs fail to undergo branching morphogenesis (see below), do not express the distal epithelial marker surfactant protein C, and have defects in expression of extracellular matrix components (reviewed by Cardoso).30 An emerging view of Nkx2.1 regulated gene expression suggests that this transcription factor interacts with GATA6 and that this heterodimeric transcription factor complex regulates HDAC and Hop, factors involved in feedback regulation.33,34 Direct or indirect interactions of Nkx2.1/GATA6 with the Wnt/β-catenin pathway have not been reported but are suggested by overlap in gene expression patterns and reporter gene activity (see below).

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these mice was decreased secondary and tertiary branching which lead to elongated bronchiolar tubules that were poorly branched and enlarged at the tips. Expression of airway epithelial markers (CCSP, FoxJ1 and β-tubulin) was unaltered. However, expression of the alveolar type 2 cell marker SPC was severely decreased. PECAM, a vascular endothelial marker, was diminished and alterations in expression of α-smooth muscle actin suggested that β-catenin deletion altered epithelial-mesenchymal interactions necessary for normal lung development. Interestingly, transcription of factors necessary for primary branching morphogenesis (Nkx2.1, Fgf10, Bmp4 and Shh) was unaltered and suggested that β-catenin dependent processes were downstream of these pathways and that processes regulated by β-catenin were dispensable for establishment of the main-stem bronchi and the intralobular main axial pathway and for differentiation of cells of the conducting airway epithelium. Thus, β-catenin dependent processes appear to refine the signals established by the other major signaling pathways during branching morphogenesis of the lung. β-catenin stabilization studies. Cytoplasmic concentrations of β-catenin are maintained at relatively low levels through GSK3βmediated phosphorylation of amino acids encoded within exon 3 of the Catnb gene.59 Strategies for stabilization of β-catenin include transgenic methods such as expression of an N-terminal truncation (dN89-β-catenin)60 or a fusion of a truncated β-catenin to transcriptional co-factors (Lef1-dN89-β-catenin).23 Additionally, CRE recombinase-mediated deletion of exon 3 has been used to generate cell type-specific stabilization of β-catenin. Studies utilizing these approaches have further supported the importance of β-catenin dependent gene expression in various aspects of organogenesis and tissue maintenance but have added the further concept that timing, cellular location and level of β-catenin dependent signaling are tightly regulated. Importantly, stabilization of β-catenin leads to apoptosis of intestinal progenitors,29 loss of hematopoietic stem cells,61,62 cell fate transitions,53 and time-dependent impact on organ size63 that may be a direct or an indirect consequence of alterations in the status of β-catenin signaling or regulation of cellular interactions.24,60,64 Two methods have been used to stabilize β-catenin in the developing lung. Okubo and Hogan used a Spc promoter-regulated Lef1-dN89 β-catenin to alter β-catenin abundance starting approximately at E10.5.23 Importantly, Lef1 is expressed throughout the presumptive lung epithelium at this time.35 Expression of the stabilized Lef1/β-catenin fusion did not impact morphogenesis of the trachea or bronchi. However, primary bronchial tubes were wider than normal and opened directly into sacules lined with a simple cuboidal or columnar epithelium. Analysis of epithelial differentiation markers indicated decreased differentiation of airway progenitor cells to secretory and ciliated cell types and an absence of mature alveolar type 2 and type 1 cells. Thus, constitutive β-catenin signaling in the developing foregut endoderm partially inhibited branching morphogenesis and blocked expression of lung specific differentiation genes. Unexpectedly, cells with secretory morphology were found to express markers of intestinal secretory cells including both the paneth and goblet cell types. These data were strictly interpreted as an indication of a role for β-catenin in cell fate transition with very high levels of signaling leading to suppression of foregut fates. An alternative method for stabilization of β-catenin utilized the rCCSP-rtTA/Tet(O)-Cre system in combination with the

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Spc-rtTA/Tet(O)-Cre system can be used to effectively modify gene expression within specific temporal and spatial windows of lung development and that this method particularly well suited to analysis of the distal lung. Lineage tracing studies using a ratCCSP promoter-regulated rtTA allow tagging of bronchial and bronchiolar epithelial cells starting at E14.5.43 Although mouse alveolar cells do not express detectable levels of CCSP,51 a subset of alveolar type 2 cells was tagged using this system starting on E18.5. However, the tag was not transferred to alveolar type 1 cells. This is in contrast with results using the Spc system42 which demonstrated a clear and previously documented lineage relationship between alveolar type 2 and type 1 cells.52 Extensive/complete tagging of epithelial cells that are enriched in regions overlying cartilaginous rings on the ventral side of the trachea and scattered cells on the membranous dorsal region was achieved in animals exposed to Dox between E6.5 and post-natal day 7. These data indicate that the ratCCSP-rtTA/Tet(O)-Cre system can be used to effectively modify gene expression during the later stage of lung development and that modifications are biased but not restricted to the airway epithelium. β-catenin knockout studies. Conditional modification of the β-catenin locus is commonly used to determine roles for Wnt/ β-catenin in tissue morphogenesis and cell fate specification. With the exception of the gut where β-catenin is a proximal regulator of cell proliferation and differentiation, a common trend among these studies is the finding that β-catenin is necessary early in organogenesis and may function in suppression of alternative cell fates.53-55 This activity may be through regulation of gene expression but may also be a function of alterations in cell adhesion.55 In derivatives of the foregut endoderm, relatively late deletion of β-catenin exons 2–656 has little impact on differentiated functions in the otherwise wild type liver or in endocrine function in the pancreas.57 However, β-catenin deletion has an impact on liver regeneration58 and on acinar cell specification in the pancreas.57 In the lung two approaches have been used to investigate roles for β-catenin in lineage specification. Morpholino technology has been used in lung organ cultures to knockdown β-catenin in both the epithelial and mesenchymal compartments.39 This treatment resulted in an effective reduction in β-catenin protein levels and an increase in the number of branches. However, this strategy would be expected to alter β-catenin mediated processes in both the epithelium and mesenchyme and suggests that the balance between β-catenin dependent signaling in these two compartments is critical for establishment of the correct number of secondary branches. The β-catenin locus has also been modified in vivo using the Spc-rtTA/Tet(O)-Cre and CCSP-rtTA/Tet(O)-Cre approaches46 in combination with the CatnbfloxedE2-6 allele.56 Interestingly, β-catenin deletion using the CCSP system resulted in no perceptible alteration to lung structure. These data were interpreted in light of previous studies42,43 and supported the conclusion that the airway and alveolar lineages were distinct by the pseudoglandular to canalicular transition and that β-catenin was unnecessary for airway epithelial differentiation, for establishment and differentiation of the parabronchial mesenchyme, or for generation of the vasculature. In contrast, deletion of β-catenin in the presumptive epithelium using the Spc promoter resulted in profound perturbation of normal epithelial, mesenchymal and vascular development. The primary defect in www.landesbioscience.com

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Painting in the Pointillist Style: Analysis of Roles for Wnt/β-catenin in Patterning of the Mesenchyme

tooth and skin.71-73 Using a hypomorphic Fgf10 allele Ramasamy and colleagues,74 showed that FGFR2b signaling via FGF10 controls the proliferation of the pulmonary epithelial progenitors74 in part by autoregulation of β-catenin signaling in the epithelium. This correlation of a reduction in epithelial FGF signaling and epithelial TOPGAL activity has also been demonstrated in lungs of a mouse Apert disease model.75 Intriguingly, regulation of epithelial β-catenin signaling by FGF10 and concomitant upregulation of Fgfr2b receptor expression, results in a strengthening of this signaling cascade locally, maintaining the distal epithelial progenitor state. β-catenin’s promiscuity between cell adhesion and Wnt signaling. Extensively reviewed in reference 76. The β-catenin protein was initially discovered for its role in cell adhesion.77 As a component of adherens junctions, it promotes cell adhesion by binding to the intracellular domain of the transmembrane protein cadherin, a Ca2+dependent homotypic adhesion molecule. This adhesion function is based on a subcellular pool of β-catenin that is membraneassociated and stable.78,79 The structural and functional integrity of the cadherin-catenin complex is regulated by phosphorylation.80 Serine/threonine phosphorylation of β-catenin81 or E-cadherin25 results in increased stabilization of the cadherin-catenin complex. However, tyrosine phosphorylation of β-catenin Tyr residue Y654 by Src or the epidermal growth factor (EGF) receptor82 disrupts binding of β-catenin to cadherin. In general, activation of tyrosine kinases results in a loss of cadherin-mediated cell-cell adhesion and an increase in the level of cytoplasmic β-catenin and subsequent β-catenin signaling in the nucleus,82-86 either by direct release of β-catenin into the cytoplasm or by activating cadherin endocytosis.87 In contrast, activation of PTPases or inactivation of tyrosine kinase receptors, stabilizes the cadherin-catenin complex and results in increased cadherin-mediated cell-cell adhesion and decreased TCF/β-catenin mediated gene transcription.88-91 In addition to these kinase/phosphatase pathways, the protein phosphatase 2A is known to regulate Wnt/β-catenin signaling and over expression of the B56γ subunit under regulation of the SPC promoter has profound effects on branching morphogenesis potentially through down regulation of β-catenin.92 Therefore, phosphorylation-dependent release of β-catenin from the cadherin complex not only regulates the integrity and function of the adhesion complex, but may also be an alternative mechanism for activating β-catenin signaling. Mesenchymal β-catenin signaling relies on the Pitx family of transcription factors. During development and in adult tissues, mesenchymal cells serve as precursors to diverse cell lineages, including smooth muscle cells (SMCs), endothelial cells, pericytes, lipocytes and stromal fibroblasts. The proper generation of these cell types likely relies on the controlled amplification of lineage-restricted and non-restricted mesenchymal precursors followed by their timely differentiation into the appropriate progeny. The distal lung contains two distinct mesenchymal cell populations: sub-epithelial and sub-mesothelial. Sub-epithelial cells express Ptch and respond to epithelially-derived SHH and develop in the lung capillary plexus,93,94 while transient fate analysis studies using an Fgf10-LacZ reporter, show that the sub-mesothelial cells express high levels of Fgf10 and serve as progenitors to parabronchial smooth muscle cells (PSMCs).95 The PSMC progenitor status is maintained by mesothelial-derived FGF9.93 With time, the PSMC progenitors relocate around the bronchi, and under the influence of an

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CatnbfloxedExon3 allele.47,65 Other studies using this transgenic system, suggest that recombination occurred ~E14.5 in airways and ~E18.5 in the alveolar space.43,47 Recombination in both compartments was indicated by increased levels of β-catenin in airway epithelial cells and in cells with the morphological characteristics of alveolar type 2 cells. Key observations from this study were that branching morphogenesis was not impacted by “late” stabilization of β-catenin. However, airway epithelial cell phenotype was impacted by β-catenin stabilization. Subsets of airway epithelial cells exhibited a mucus-producing phenotype while other cells were squamous in morphology. Overall levels of CCSP immunopositive cells in the airway epithelium were low while increased expression of SPC was noted. Future studies are needed to correlate these phenotypic changes with expression of the stabilized β-catenin. Late stabilization of β-catenin also resulted in airspace enlargement that could be a consequence of β-catenin stabilization or expression of the rCCSP-rtTA transgene.44,45 Many of the phenotypic consequences of β-catenin stabilization noted in the rCCSP-rtTA/Tet(O)-Cre model may be related to the proposed lineage relationship between airway and alveolar cells.66 However, airway and alveolar dysplasia and high frequency of adenocarcinomas in this model could be accounted for by a chronic injury/repair process suggested by morphological and phenotypic changes to the airway and alveolar epithelium. Future studies allowing stabilization of β-catenin in either the airway or alveolar compartment are needed to resolve this interesting issue.

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Branching morphogenesis of the lung. This subject has been extensively reviewed in recent publications and roles for Wnt/βcatenin in this process have been well documented.30,67-69 In this section we will review new data that has been presented since publication of these reviews. FGF10 and β-catenin regulates epithelial progenitor state in the lung. The developing lung provides a good system for studying the regulators of epithelial and mesenchymal cell lineage formation70 and thus regulators of epithelial progenitor fates have been elucidated. As with other organ systems, lung development involves a balance between expansion of the number of undifferentiated progenitor cells and withdrawal of cells from this pool through differentiation. Understanding how this balance is achieved is relevant to a number of clinical problems; for example, the pulmonary dysplasia, lung repair after injury, and the progression of lung cancer. 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 FGF and canonical Wnt signaling are crucial. For example, hyperactive β-catenin signaling leads to aberrant amplification of distal lung progenitor cells, partly through the regulation of N-myc expression23,37,38 and prevents distal cells from assuming a proximal phenotype. On the other hand targeted disruption of β-catenin or N-myc in the distal lung epithelium23,46 results in premature differentiation and reduced epithelial cell proliferation. β-catenin signaling also regulates the levels of Bmp4 and Fgfr2b expression in distal lung epithelium.38 FGFR2b signaling in turn, is critical for maintenance and expansion of the pool of epithelial progenitor cells, not only in lungs, but also in developing pancreas,

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and in the lung in particular.100 This could be accounted for by the possible existence of a feedback loop involving vascular endothelial growth factor (VEGF), which is known to act via FLK1. Supporting this possibility, Vegf was found to be upregulated in experiments aiming to analyze changes in global gene expression using Affimetrix micro-arrays (De Langhe S, unpublished). Our results also raise the possibility that a distinction can be drawn between the amplification of the angioblast, involving VEGF and subsequent differentiation via the activation of β-catenin signaling in these cells.

Morphogenesis of Proximal Respiratory Structures

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Patterning of the trachea. Respiratory structures derived from the anterior end of the foregut endoderm exhibit exquisite asymmetries along the three major axes. Specifically, the trachea is a tubular structure that is supported by cartilaginous rings on the ventral surface and contracted by smooth muscle located on the dorsal surface. Glands, termed submucosal glands, are located in the proximal 3–4 segments of the mouse trachea7,105 and the distribution of secretory cells (enriched in cartilaginous zones) and ciliated cells (enriched in intercartilagenous zones) varies with changes in the mesenchymally-derived support structures.106 Lateral asymmetries are typified by variation in the height of the epithelium and the number and phenotype of basal cells. Signaling mechanisms leading to establishment of these asymmetries involve many of the molecular players instrumental in branching morphogenesis of the lung. However, interactions with the Wnt/β-catenin pathway have not been addressed extensively. Ventral to dorsal patterning of the trachea is likely to involve gradients of SHH, BMP’s and FGF’s which impose both positive and negative interactions on each other.107 These pathways converge to upregulate Nkx2.1 in the trachea and differentially regulate Sox2 and p63 in the trachea (Sox2-low, p63-low) and esophagus (Sox2-high, p63-high).108 SHH109 working directly or indirectly through SOX92 may also be important for patterning of the tracheal cartilage and potentially regulate expression of Lef1 in this tissue.8 Alternatively, PITX2, β-catenin and Lef1 may interact within the tracheal epithelium to foster gland formation as shown in the distal airway100 and in vitro.110 Analysis of the TOPGAL and BATGAL transgenes indicates that β-catenin is stabilized in the anterior foregut prior to separation of the trachea and esophagus and that transgene activity is maintained in the trachea through the post-natal period into adult hood.23,40 Although β-catenin mediated transcription is evident at the right time and place interactions between this and other signaling pathways have not been determined. Furthermore tissue recombination and bead experiments indicate that the tracheal epithelium exhibits a time limited capacity to undergo branching morphogenesis and to express distal airway markers.111 However, the specific combination of signals and events that regulate this window of competence are yet to be determined. Branching morphogenesis of the submucosal glands. Submucosal glands are an important source of antimicrobial factors and mucus needed for clearance of pathogens. They are formed during the post-natal period well after the period when distal lung mesenchyme can induce a lung-like pattern of branching reviewed in ref.112 Pathological alterations to submucosal glands include hypertrophy and hyperplasia and these changes are a significant contributor

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epithelially-derived signal, BMP4, differentiate into mature PSMCs.95 The myogenic program is then completed along the proximal airways, where the progenitors encounter Laminin-2 and Fibronectin in the epithelial basement membrane.96-98 For a long time, the role of mesenchymal canonical Wnt signaling has been elusive. The lack of significant activity of well established Wnt reporter lines in the mesenchyme including the TOPGAL and BATGAL mice certainly did not support an important role for Wnt signaling in the mesenchyme during organogenesis. However, the expression of several Wnt receptors in the lung mesenchyme had been reported.97 Furthermore, over expression of Wnt5a has been shown to either directly or indirectly regulate Fgf10 expression in the mesenchyme99 while Wnt7b has been demonstrated to act on lung vascular SMCs through Frizzled 1 and LRP5.52 Using Dermo1Cre/+mediated conditional inactivation (CKO) of β-catenin to study the role of β-catenin signaling in mouse embryonic mesodermal lineages we recently shed light on this enigma.100 Besides Lef1/TCF mediated β-catenin signaling, β-catenin can also act through the PITX family of transcription factors,101 which are abundantly expressed in developing mesenchymal tissues.102 Dermo1-cre/β-catenin CKO embryos have multiple mesenchymal-related defects that are remarkably reminiscent of a double knock out of Pitx1 and Pitx2 genes.103 Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by β-catenin signaling. By focusing on the lungs of the conditional mutant embryos and combining fate analysis and global gene expression pattern studies, it was shown that mesenchymal β-catenin signaling has a dual, lineagedependant function. It regulates the formation and amplification of Fgf10-expressing PSMC progenitors but does not affect their differentiation. Yet, it is required for proper differentiation of endothelial cells. These findings reveal a critical requirement for β-catenin signaling in the development of multiple mesenchymal lineages.100 Mesenchymal canonical Wnt signaling controls the amplification of PSMC progenitors. Interestingly mesenchymal β-catenin signaling via the PITX family of transcription factors has similar functions as epithelial β-catenin signaling in controlling differentiation and/or proliferation of embryonic progenitor cells through a the tight regulation of FGF and β-catenin signaling. Fgfr2b and N-Myc are downstream targets of canonical Wnt signaling in the epithelium38 while mesenchymal β-catenin signaling, regulates the expression of Fgfr2c and c-Myc.100 The response of the CKO mesenchyme to FGF9, produced mostly by the mesothelium and acting on the sub-mesothelial mesenchyme, is severely impaired in Dermo1-cre/β-catenin CKO lungs, hampering the amplification of Fgf10-expressing PSMC progenitors. Interestingly, β-catenin abrogation in the mesenchyme specifically interferes with the amplification of the PSMC progenitors but does not affect, at least in vitro, their differentiation into smooth muscle cells.100 Ablation of mesenchymal canonical Wnt signaling impairs angioblast differentiation. Inactivation of β-catenin in angioblasts throughout the embryo and in the lung inhibits their differentiation into mature endothelial cells.100 Interestingly ablation of β-catenin in mature endothelial cells using a Tie2-cre driver line does not affect vasculogenesis and angiogenesis, nor did it affect PECAM expression 104. Thus, β-catenin signaling plays a role very early in endothelial cell differentiation. Remarkably, mesenchymal deletion of β-catenin resulted in increased Flk1/LacZ expression throughout the embryo www.landesbioscience.com

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disease. Further analysis of Wnt/β-catenin signaling may elucidate mechanisms through which primary injury to one compartment compromises functionality of adjacent regions in diseases such as chronic obstructive lung disease and asthma. Acknowledgements

This work was supported by a N.T.H. R.O. grant (HL075585-03) and a Cystic Fibrosis Foundation grant to S.D.R. References

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1. Harkema JR, Carey SA, Wagner JG. The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicol Pathol 2006; 34:252-69. 2. Elluru RG, Whitsett JA. Potential role of Sox9 in patterning tracheal cartilage ring formation in an embryonic mouse model. Arch Otolaryngol Head Neck Surg 2004; 130:732-6. 3. Rice MA, Dodson BT, Arthur JA, Anseth KS. Cell-based therapies and tissue engineering. Otolaryngol Clin North Am 2005; 38:199-214. 4. Bucheler M, Haisch A. Tissue engineering in otorhinolaryngology. DNA Cell Biol 2003; 22:549-64. 5. Yamada T, Suzuki E, Gejyo F, Ushiki T. Developmental changes in the structure of the rat fetal lung, with special reference to the airway smooth muscle and vasculature. Arch Histol Cytol 2002; 65:55-69. 6. Tollet J, Everett AW, Sparrow MP. Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development. Dev Dyn 2001; 221:48-60. 7. Borthwick DW, West JD, Keighren MA, Flockhart JH, Innes BA, Dorin JR. Murine submucosal glands are clonally derived and show a cystic fibrosis gene-dependent distribution pattern. Am J Respir Cell Mol Biol 1999; 20:1181-9. 8. Duan D, Sehgal A, Yao J, Engelhardt JF. Lef1 transcription factor expression defines airway progenitor cell targets for in utero gene therapy of submucosal gland in cystic fibrosis. Am J Respir Cell Mol Biol 1998; 18:750-8. 9. Massaro GD, Singh G, Mason R, Plopper CG, Malkinson AM, Gail DB. Biology of the Clara cell. Am J Physiol 1994; 266:101-6. 10. Plopper CG. Comparative morphologic features of bronchiolar epithelial cells. The Clara cell. Am Rev Respir Dis 1983; 128:37-41. 11. Shapiro SD. Evolving concepts in the pathogenesis of chronic obstructive pulmonary disease. Clin Chest Med 2000; 21:621-32. 12. Ten Have-Opbroek AA. Lung development in the mouse embryo. Exp Lung Res 1991; 17:111-30. 13. Ten Have-Opbroek AA. The development of the lung in mammals: an analysis of concepts and findings. Am J Anat 1981; 162:201-19. 14. Wert SE, Glasser SW, Korfhagen TR, Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 1993; 156:426-43. 15. Stripp BR, Sawaya PL, Luse DS, Wikenheiser KA, Wert SE, Huffman JA, Lattier DL, Singh G, Katyal SL, Whitsett JA. cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem 1992; 267:14703-12. 16. Shi W, Bellusci S, Warburton D. Lung development and adult lung diseases. Chest 2007; 132:651-6. 17. Thomas P, Beddington R. Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr Biol 1996; 6:1487-96. 18. Marikawa Y. Wnt/beta-catenin signaling and body plan formation in mouse embryos. Semin Cell Dev Biol 2006; 17:175-84. 19. Tam PP, Steiner KA. Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo. Development 1999; 126:5171-9. 20. Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W. Requirement for beta-catenin in anterior-posterior axis formation in mice. J Cell Biol 2000; 148:567-78. 21. Maretto S, Cordenonsi M, Dupont S, Braghetta P, Broccoli V, Hassan AB, Volpin D, Bressan GM, Piccolo S. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci USA 2003; 100:3299-304. 22. Merrill BJ, Pasolli HA, Polak L, Rendl M, Garcia-Garcia MJ, Anderson KV, Fuchs E. Tcf3: a transcriptional regulator of axis induction in the early embryo. Development 2004; 131:263-74. 23. Okubo T, Hogan BL. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm. J Biol 2004; 3:11. 24. DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 1999; 126:4557-68. 25. Lickert H, Domon C, Huls G, Wehrle C, Duluc I, Clevers H, Meyer BI, Freund JN, Kemler R. Wnt/(beta)-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine. Development 2000; 127:3805-13. 26. Lickert H, Kutsch S, Kanzler B, Tamai Y, Taketo MM, Kemler R. Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev Cell 2002; 3:171-81.

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to morbidity in mucosecretory diseases including cystic fibrosis, asthma and COPD.8,113,114 Extensive analysis of Lef1 expression in three species has characterized epithelial processes leading to submucosal gland formation.8,40,115-117 Gland formation is initiated when cytokeratin 14/LEF1 dual positive cells within the surface epithelium coalesce to form the gland bud. These cells then invade the interstitium and cells at the distal tips of the elongating tubules maintain high level expression of Lef1. Analysis of Wnt3a and Lef1 knockout mice in combination with immunostaining for TCF4 led to the conclusion that Wnt3a (or a functionally redundant Wnt pathway ligand) acts on keratin 14-expressing cells within the surface epithelium to stabilize β-catenin.40 This protein interacts with TCF4 to activate Lef1 gene expression. These studies refined the model of branching morphogenesis in the upper airway and demonstrated that the budding process occurred independently of LEF1 but that LEF1 was necessary for tubulogenesis. In this context, LEF1 was a critical mediator of proliferation and gland progenitor cell survival. Mesenchymal signals necessary for gland formation were investigated by Rawlings and Hogan.112 These studies indicate that Bmp4 is expressed in the peritracheal mesenchyme and that its distribution is restricted to regions surrounding the presumptive gland bud. Gland formation was also shown to be dependent on FGF10. This study also identified the TNF superfamily member ectodysplesin as a critical epithelial factor for gland formation.

Future Directions

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Analysis of Wnt/β-catenin signaling in lung organogenesis, branching morphogenesis of the airway, and differentiation of the alveolar compartment suggests that this pathway functions primarily in refinement of morphogenic processes initiated by other signaling cascades. Thus, the relative position of the Wnt/β-catenin pathway within the hierarchy of signals that shape respiratory system is distinct from that found in hindgut-derived tissues (intestine and colon) and varies as a function of the morphogenic process under consideration. When existing data are interpreted in this context, time- and tissue-dependent variation in cellular interpretation of endogenous or engineered Wnt/β-catenin signals is more readily understood. The permissive function of the Wnt/β-catenin pathway within the developing respiratory system is distinct from the instructive activity in the gut and has important implications regarding signals necessary for maintenance of slowly-renewing tissues (Stripp and Reynolds 2008, In press) and mechanisms leading to dysplasia118,119 and oncogenesis.120 Future analysis of roles for the Wnt/β-catenin pathway in maintenance and repair of the adult lung will require development of methods for conditional modulation of Wnt-β-catenin pathway gene expression in the late prenatal, early postnatal and adult periods as well as development of sophisticated methods for in vitro expansion and analysis of differentiated cells. These tools will undoubtedly allow identification of developmental processes that are recapitulated during repair and remodeling as well as detection of novel interactions between Wnt/β-catenin and other signaling pathways that are specific to long-lived epithelia such as that of the adult respiratory system. Finally the structural and functional diversity of the respiratory system predicts regionally-specific activities for the Wnt/β-catenin pathway that may contribute to the distinct pathophysiology of the proximal and distal lung in chronic lung 106

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55. Smith AN, Miller LA, Song N, Taketo MM, Lang RA. The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev Biol 2005; 285:477-89. 56. Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 2001; 128:1253-64. 57. Murtaugh LC, Law AC, Dor Y, Melton DA. Beta-catenin is essential for pancreatic acinar but not islet development. Development 2005; 132:4663-74. 58. Sekine S, Gutierrez PJ, Lan BY, Feng S, Hebrok M. Liver-specific loss of beta-catenin results in delayed hepatocyte proliferation after partial hepatectomy. Hepatology 2007; 45:361-8. 59. Gordon MD, Nusse R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 2006; 281:22429-33. 60. DasGupta R, Rhee H, Fuchs E. A developmental conundrum: a stabilized form of betacatenin lacking the transcriptional activation domain triggers features of hair cell fate in epidermal cells and epidermal cell fate in hair follicle cells. J Cell Biol 2002; 158:331-44. 61. Kirstetter P, Anderson K, Porse BT, Jacobsen SE, Nerlov C. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nat Immunol 2006. 62. Scheller M, Huelsken J, Rosenbauer F, Taketo MM, Birchmeier W, Tenen DG, Leutz A. Hematopoietic stem cell and multilineage defects generated by constitutive beta-catenin activation. Nat Immunol 2006. 63. Heiser PW, Lau J, Taketo MM, Herrera PL, Hebrok M. Stabilization of beta-catenin impacts pancreas growth. Development 2006; 133:2023-32. 64. Vasioukhin V, Degenstein L, Wise B, Fuchs E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc Natl Acad Sci USA 1999; 96:8551-6. 65. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M, Taketo MM. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. Embo J 1999; 18:5931-42. 66. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005; 121:823-35. 67. Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, Cardoso WV. The molecular basis of lung morphogenesis. Mech Dev 2000; 92:55-81. 68. Maeda Y, Dave V, Whitsett JA. Transcriptional control of lung morphogenesis. Physiol Rev 2007; 87:219-44. 69. Pongracz JE, Stockley RA. Wnt signalling in lung development and diseases. Respir Res 2006; 7:15. 70. Cardoso WV, Lu J. Regulation of early lung morphogenesis: questions, facts and controversies. Development 2006; 133:1611-24. 71. Harada H, Toyono T, Toyoshima K, Yamasaki M, Itoh N, Kato S, Sekine K, Ohuchi H. FGF10 maintains stem cell compartment in developing mouse incisors. Development 2002; 129:1533-41. 72. Bhushan A, Itoh N, Kato S, Thiery JP, Czernichow P, Bellusci S, Scharfmann R. Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 2001; 128:5109-17. 73. Norgaard GA, Jensen JN, Jensen J. FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development. Dev Biol 2003; 264:323-38. 74. Ramasamy SK, Mailleux AA, Gupte VV, Mata F, Sala FG, Veltmaat JM, Del Moral PM, De Langhe S, Parsa S, Kelly LK, Kelly R, Shia W, Keshet E, Minoo P, Warburton D, Bellusci S. Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development. Dev Biol 2007; 307:237-47. 75. De Langhe SP, Carraro G, Warburton D, Hajihosseini MK, Bellusci S. Levels of mesenchymal FGFR2 signaling modulate smooth muscle progenitor cell commitment in the lung. Dev Biol 2006; 299:52-62. 76. Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science 2004; 303:1483-7. 77. Kemler R. From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 1993; 9:317-21. 78. Jamora C, Fuchs E. Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol 2002; 4:101-8. 79. Gumbiner BM. Regulation of cadherin adhesive activity. J Cell Biol 2000; 148:399-404. 80. Lilien J, Balsamo J, Arregui C, Xu G. Turn-off, drop-out: functional state switching of cadherins. Dev Dyn 2002; 224:18-29. 81. Bek S, Kemler R. Protein kinase CKII regulates the interaction of beta-catenin with alphacatenin and its protein stability. J Cell Sci 2002; 115:4743-53. 82. Roura S, Miravet S, Piedra J, Garcia de Herreros A, Dunach M. Regulation of E-cadherin/ Catenin association by tyrosine phosphorylation. J Biol Chem 1999; 274:36734-40. 83. Danilkovitch-Miagkova A, Miagkov A, Skeel A, Nakaigawa N, Zbar B, Leonard EJ. Oncogenic mutants of RON and MET receptor tyrosine kinases cause activation of the beta-catenin pathway. Mol Cell Biol 2001; 21:5857-68. 84. Morali OG, Delmas V, Moore R, Jeanney C, Thiery JP, Larue L. IGF-II induces rapid betacatenin relocation to the nucleus during epithelium to mesenchyme transition. Oncogene 2001; 20:4942-50. 85. Piedra J, Miravet S, Castano J, Palmer HG, Heisterkamp N, Garcia de Herreros A, Dunach M. p120 Catenin-associated Fer and Fyn tyrosine kinases regulate beta-catenin Tyr-142 phosphorylation and beta-catenin-alpha-catenin Interaction. Mol Cell Biol 2003; 23:2287-97.

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

27. Lickert H, Kispert A, Kutsch S, Kemler R. Expression patterns of Wnt genes in mouse gut development. Mech Dev 2001; 105:181-4. 28. Kim BM, Mao J, Taketo MM, Shivdasani RA. Phases of canonical Wnt signaling during the development of mouse intestinal epithelium. Gastroenterology 2007; 133:529-38. 29. Wong MH, Huelsken J, Birchmeier W, Gordon JI. Selection of multipotent stem cells during morphogenesis of small intestinal crypts of Lieberkuhn is perturbed by stimulation of Lef-1/beta-catenin signaling. J Biol Chem 2002; 277:15843-50. 30. Cardoso WV. Molecular regulation of lung development. Annu Rev Physiol 2001; 63:471-94. 31. Serls AE, Doherty S, Parvatiyar P, Wells JM, Deutsch GH. Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Development 2005; 132:35-47. 32. Minoo P, Su G, Drum H, Bringas P, Kimura S. Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(-/-) mouse embryos. Dev Biol 1999; 209:60-71. 33. Yin Z, Gonzales L, Kolla V, Rath N, Zhang Y, Lu MM, Kimura S, Ballard PL, Beers MF, Epstein JA, Morrisey EE. Hop functions downstream of Nkx2.1 and GATA6 to mediate HDAC-dependent negative regulation of pulmonary gene expression. Am J Physiol Lung Cell Mol Physiol 2006; 291:191-9. 34. Zhang Y, Rath N, Hannenhalli S, Wang Z, Cappola T, Kimura S, Atochina-Vasserman E, Lu MM, Beers MF, Morrisey EE. GATA and Nkx factors synergistically regulate tissuespecific gene expression and development in vivo. Development 2007; 134:189-98. 35. De Langhe SP, Sala FG, Del Moral PM, Fairbanks TJ, Yamada KM, Warburton D, Burns RC, Bellusci S. Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev Biol 2005; 277:316-31. 36. Tebar M, Destree O, de Vree WJ, Ten Have-Opbroek AA. Expression of Tcf/Lef and sFrp and localization of beta-catenin in the developing mouse lung. Mech Dev 2001; 109:437-40. 37. Okubo T, Knoepfler PS, Eisenman RN, Hogan BL. Nmyc plays an essential role during lung development as a dosage-sensitive regulator of progenitor cell proliferation and differentiation. Development 2005; 132:1363-74. 38. Shu W, Guttentag S, Wang Z, Andl T, Ballard P, Lu MM, Piccolo S, Birchmeier W, Whitsett JA, Millar SE, Morrisey EE. Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev Biol 2005; 283:226-39. 39. Dean CH, Miller LA, Smith AN, Dufort D, Lang RA, Niswander LA. Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland. Dev Biol 2005; 286:270-86. 40. Driskell RR, Goodheart M, Neff T, Liu X, Luo M, Moothart C, Sigmund CD, Hosokawa R, Chai Y, Engelhardt JF. Wnt3a regulates Lef-1 expression during airway submucosal gland morphogenesis. Dev Biol 2007; 305:90-102. 41. Reynolds SD, Hong KU, Giangreco A, Mango GW, Guron C, Morimoto Y, Stripp BR. Conditional clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells. Am J Physiol Lung Cell Mol Physiol 2000; 278:1256-63. 42. Perl AK, Tichelaar JW, Whitsett JA. Conditional gene expression in the respiratory epithelium of the mouse. Transgenic Res 2002; 11:21-9. 43. Perl AK, Wert SE, Loudy DE, Shan Z, Blair PA, Whitsett JA. Conditional recombination reveals distinct subsets of epithelial cells in trachea, bronchi, and alveoli. Am J Respir Cell Mol Biol 2005; 33:455-62. 44. Whitsett JA, Perl AK. Conditional control of gene expression in the respiratory epithelium: A cautionary note. Am J Respir Cell Mol Biol 2006; 34:519-20. 45. Sisson TH, Hansen JM, Shah M, Hanson KE, Du M, Ling T, Simon RH, Christensen PJ. Expression of the reverse tetracycline-transactivator gene causes emphysema-like changes in mice. Am J Respir Cell Mol Biol 2006; 34:552-60. 46. Mucenski ML, Wert SE, Nation JM, Loudy DE, Huelsken J, Birchmeier W, Morrisey EE, Whitsett JA. beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis. J Biol Chem 2003; 278:40231-8. 47. Mucenski ML, Nation JM, Thitoff AR, Besnard V, Xu Y, Wert SE, Harada N, Taketo MM, Stahlman MT, Whitsett JA. Beta-catenin regulates differentiation of respiratory epithelial cells in vivo. Am J Physiol Lung Cell Mol Physiol 2005; 289:971-9. 48. Lobe CG, Koop KE, Kreppner W, Lomeli H, Gertsenstein M, Nagy A. Z/AP, a double reporter for cre-mediated recombination. Dev Biol 1999; 208:281-92. 49. Novak A, Guo C, Yang W, Nagy A, Lobe CG. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 2000; 28:147-55. 50. Engelhardt JF, Schlossberg H, Yankaskas JR, Dudus L. Progenitor cells of the adult human airway involved in submucosal gland development. Development 1995; 121:2031-46. 51. Singh G, Katyal SL. Clara cells and Clara cell 10 kD protein (CC10). Am J Respir Cell Mol Biol 1997; 17:141-3. 52. Warburton D, Bellusci S, De Langhe S, Del Moral PM, Fleury V, Mailleux A, Tefft D, Unbekandt M, Wang K, Shi W. Molecular mechanisms of early lung specification and branching morphogenesis. Pediatr Res 2005; 57:26-37. 53. Miller LA, Smith AN, Taketo MM, Lang RA. Optic cup and facial patterning defects in ocular ectoderm beta-catenin gain-of-function mice. BMC Dev Biol 2006; 6:14. 54. Kreslova J, Machon O, Ruzickova J, Lachova J, Wawrousek EF, Kemler R, Krauss S, Piatigorsky J, Kozmik Z. Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function. Genesis 2007; 45:157-68.

www.landesbioscience.com

Organogenesis

107


OT D

IST RIB

UT E

.

112. Rawlins EL, Hogan BL. Intercellular growth factor signaling and the development of mouse tracheal submucosal glands. Dev Dyn 2005; 233:1378-85. 113. Jeffery PK. Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 2001; 164:28-38. 114. Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003; 167:1360-8. 115. Duan D, Yue Y, Zhou W, Labed B, Ritchie TC, Grosschedl R, Engelhardt JF. Submucosal gland development in the airway is controlled by lymphoid enhancer binding factor 1 (LEF1). Development 1999; 126:4441-53. 116. Driskell RR, Liu X, Luo M, Filali M, Zhou W, Abbott D, Cheng N, Moothart C, Sigmund CD, Engelhardt JF. Wnt-responsive element controls Lef-1 promoter expression during submucosal gland morphogenesis. Am J Physiol Lung Cell Mol Physiol 2004; 287:752-63. 117. Liu X, Driskell RR, Engelhardt JF. Airway glandular development and stem cells. Curr Top Dev Biol 2004; 64:33-56. 118. Chilosi M, Zamo A, Doglioni C, Reghellin D, Lestani M, Montagna L, Pedron S, Ennas MG, Cancellieri A, Murer B, Poletti V. Migratory marker expression in fibroblast foci of idiopathic pulmonary fibrosis. Respir Res 2006; 7:95. 119. Chilosi M, Poletti V, Zamo A, Lestani M, Montagna L, Piccoli P, Pedron S, Bertaso M, Scarpa A, Murer B, Cancellieri A, Maestro R, Semenzato G, Doglioni C. Aberrant Wnt/ beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol 2003; 162:1495-502. 120. Tennis M, Van Scoyk M, Winn RA. Role of the wnt signaling pathway and lung cancer. J Thorac Oncol 2007; 2:889-92.

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

86. Playford MP, Bicknell D, Bodmer WF, Macaulay VM. Insulin-like growth factor 1 regulates the location, stability, and transcriptional activity of beta-catenin. Proc Natl Acad Sci USA 2000; 97:12103-8. 87. Le TL, Yap AS, Stow JL. Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J Cell Biol 1999; 146:219-32. 88. Balsamo J, Arregui C, Leung T, Lilien J. The nonreceptor protein tyrosine phosphatase PTP1B binds to the cytoplasmic domain of N-cadherin and regulates the cadherin-actin linkage. J Cell Biol 1998; 143:523-32. 89. Bonvini P, An WG, Rosolen A, Nguyen P, Trepel J, Garcia de Herreros A, Dunach M, Neckers LM. Geldanamycin abrogates ErbB2 association with proteasome-resistant betacatenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription. Cancer Res 2001; 61:1671-7. 90. Hellberg CB, Burden Gulley SM, Pietz GE, Brady-Kalnay SM. Expression of the receptor protein-tyrosine phosphatase, PTPmu, restores E-cadherin-dependent adhesion in human prostate carcinoma cells. J Biol Chem 2002; 277:11165-73. 91. Nawroth R, Poell G, Ranft A, Kloep S, Samulowitz U, Fachinger G, Golding M, Shima DT, Deutsch U, Vestweber D. VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts. EMBO J 2002; 21:4885-95. 92. Everett AD, Kamibayashi C, Brautigan DL. Transgenic expression of protein phosphatase 2A regulatory subunit B56gamma disrupts distal lung differentiation. Am J Physiol Lung Cell Mol Physiol 2002; 282:1266-71. 93. del Moral PM, De Langhe SP, Sala FG, Veltmaat JM, Tefft D, Wang K, Warburton D, Bellusci S. Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung. Dev Biol 2006; 293:77-89. 94. White AC, Xu J, Yin Y, Smith C, Schmid G, Ornitz DM. FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains. Development 2006; 133:1507-17. 95. Mailleux AA, Kelly R, Veltmaat JM, De Langhe SP, Zaffran S, Thiery JP, Bellusci S. Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage. Development 2005; 132:2157. 96. Yang Y, Beqaj S, Kemp P, Ariel I, Schuger L. Stretch-induced alternative splicing of serum response factor promotes bronchial myogenesis and is defective in lung hypoplasia. J Clin Invest 2000; 106:1321-30. 97. De Langhe SP, Sala FG, Del Moral PM, Fairbanks TJ, Yamada KM, Warburton D, Burns RC, Bellusci S. Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev Biol 2005; 277:316. 98. Jakkaraju S, Zhe X, Pan D, Choudhury R, Schuger L. TIPs are tension-responsive proteins involved in myogenic versus adipogenic differentiation. Dev Cell 2005; 9:39-49. 99. Li C, Hu L, Xiao J, Chen H, Li JT, Bellusci S, Delanghe S, Minoo P. Wnt5a regulates Shh and Fgf10 signaling during lung development. Dev Biol 2005; 287:86-97. 100. De Langhe SP, Carraro G, Tefft D, Li C, Xu X, Chai Y, Minoo P, Hajihosseini MK, Drouin J, Kaartinen V, Bellusci S. Formation and Differentiation of Multiple Mesenchymal Lineages during Lung Development Is Regulated by beta-catenin Signaling. PLoS ONE 2008; 3:1516. 101. Kioussi C, Briata P, Baek SH, Rose DW, Hamblet NS, Herman T, Ohgi KA, Lin C, Gleiberman A, Wang J, Brault V, Ruiz Lozano P, Nguyen HD, Kemler R, Glass CK, Wynshaw Boris A, Rosenfeld MG. Identification of a Wnt/Dvl/beta-Catenin→Pitx2 pathway mediating cell-type-specific proliferation during development. Cell 2002; 111:673-85. 102. Kitamura K, Miura H, Miyagawa Tomita S, Yanazawa M, Katoh Fukui Y, Suzuki R, Ohuchi H, Suehiro A, Motegi Y, Nakahara Y, Kondo S, Yokoyama M. Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 1999; 126:5749-58. 103. Marcil A, Dumontier E, Chamberland M, Camper SA, Drouin J. Pitx1 and Pitx2 are required for development of hindlimb buds. Development 2003; 130:45-55. 104. Cattelino A, Liebner S, Gallini R, Zanetti A, Balconi G, Corsi A, Bianco P, Wolburg H, Moore R, Oreda B, Kemler R, Dejana E. The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility. J Cell Biol 2003; 162:1111-22. 105. Innes BA, Dorin JR. Submucosal gland distribution in the mouse has a genetic determination localized on chromosome 9. Mamm Genome 2001; 12:124-8. 106. Plopper CG, Mariassy AT, Wilson DW, Alley JL, Nishio SJ, Nettesheim P. Comparison of nonciliated tracheal epithelial cells in six mammalian species: ultrastructure and population densities. Exp Lung Res 1983; 5:281-94. 107. Que J, Choi M, Ziel JW, Klingensmith J, Hogan BL. Morphogenesis of the trachea and esophagus: current players and new roles for noggin and Bmps. Differentiation 2006; 74:422-37. 108. Que J, Okubo T, Goldenring JR, Nam KT, Kurotani R, Morrisey EE, Taranova O, Pevny LH, Hogan BL. Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm. Development 2007; 134:2521-31. 109. Miller LA, Wert SE, Clark JC, Xu Y, Perl AK, Whitsett JA. Role of Sonic hedgehog in patterning of tracheal-bronchial cartilage and the peripheral lung. Dev Dyn 2004; 231:57-71. 110. Vadlamudi U, Espinoza HM, Ganga M, Martin DM, Liu X, Engelhardt JF, Amendt BA. PITX2, beta-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter. J Cell Sci 2005; 118:1129-37. 111. Shannon JM, Nielsen LD, Gebb SA, Randell SH. Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev Dyn 1998; 212:482-94.

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