Submucosal Gland Development Research Articles

Cell competition drives bronchiolization and pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive respiratory scarring disease arising from the maladaptive differentiation of lung stem cells into bronchial epithelial cells rather than into alveolar type 1 (AT1) cells, which are responsible for gas exchange. Here, we report that healthy lungs maintain their stem cells through tonic Hippo and β-catenin signaling, which promote Yap/Taz degradation and allow for low-level expression of the Wnt target gene Myc. Inactivation of upstream activators of the Hippo pathway in lung stem cells inhibits this tonic β-catenin signaling and Myc expression and promotes their Taz-mediated differentiation into AT1 cells. Vice versa, increased Myc in collaboration with Yap promotes the differentiation of lung stem cells along the basal and myoepithelial-like lineages allowing them to invade and bronchiolize the lung parenchyma in a process reminiscent of submucosal gland development. Our findings indicate that stem cells exhibiting the highest Myc levels become supercompetitors that drive remodeling, whereas loser cells with lower Myc levels terminally differentiate into AT1 cells.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

FGF10 is an essential regulator of tracheal submucosal gland morphogenesis

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. In the trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with an additional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins and antimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals that regulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development of murine tracheal SMGs, with gland development arresting at the early bud stage in the absence of FGF10 signalling. As Fgf10 knockout mice are lethal at birth, inducible knockdown of Fgf10 at late embryonic stages was used to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygous Fgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. The reduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction in overall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both the number of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMG-dependent respiratory health.

Stem Cell Biology of Airway Submucosal Glands during Development and Disease

Lineage‐tracing studies in the mouse lung suggest that multiple region‐specific progenitors contribute to regenerative plasticity of the airway epithelium. Airway submucosal glands (SMGs) are thought to house stem cell niches for the surface airway epithelium; however, the phenotype of the SMG‐derived progenitor cells remains unclear. SMGs are tubuloacinar structures embedded within the mesenchyme beneath the surface of all cartilaginous airways in humans and the proximal trachea of mice. These structures secrete important factors that regulate airway clearance and maintain sterility at homeostasis and in response to injury and infection. Secretory functions of SMGs are severely affected in cystic fibrosis, a genetic disease caused by mutation in the cystic fibrosis transmembrane conductance regulator, and disease‐associated alterations in neuropeptide (CGRP) expression alter the slowly cycling stem cell niches of SMGs. In this context, CGRP acts as both a secretagogue to stimulate gland secretion following injury—a response defected in cystic fibrosis—and a mitogen of glandular progenitor cells. To better define the contribution of SMG stem cells in airway regeneration and abnormalities to these processes in cystic fibrosis, we have dissected Wnt‐regulated mechanism that control both the formation of glandular stem cell niches during development and lineage‐commitment of glandular stem cells during airway regenerative responses following injury. Using lineage tracing in mice, we demonstrate that multipotent glandular myoepithelial cells form very early during SMG development in a Wnt/TCF‐dependent manner. In the adult mouse trachea, SMG myoepithelial cells can give rise to seven cell types in the proximal airway following severe injury. Similar to the stem cell program adopted during SMG development, Wnt/TCF signals appear to control self‐renewal and lineage commitment of glandular myoepithelial stem cells following injury. These studies clarify important stem cell targets for therapy in the cystic fibrosis lung.Support or Funding InformationNIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multipotent Myoepithelial Progenitor Cells Are Born Early during Airway Submucosal Gland Development.

Airway submucosal glands (SMGs) are facultative stem cell niches for the surface epithelium, but the phenotype of the SMG-derived progenitor cells remains unclear. In other organs, glandular myoepithelial cells (MECs) have been proposed to be multipotent progenitors for luminal cells. We sought to determine the developmental phase during which mouse tracheal glandular MECs are born and whether these MECs are progenitors for other cell phenotypes during SMG morphogenesis. To approach this question, we localized two MEC protein markers (α-smooth muscle actin [αSMA/ACTA2] and smooth muscle myosin heavy chain 11 [SMMHC/MYH11]) during various stages of SMG development (placode, elongation, branching, and differentiation) and used ACTA2-CreERT2 and MYH11-CreERT2 transgenic mice to fate map MEC-derived lineages during SMG morphogenesis. Both αSMA- and SMMHC-expressing cells emerged early after placode formation and during the elongation phase of SMG development. Lineage tracing in newborn mice demonstrated that lineage-positive MECs are born at the tips of invading tubules during the elongation phase of gland development. Lineage-positive MECs born within the first 7 days after birth gave rise to the largest percentage of multipotent progenitors capable of contributing to myoepithelial, serous, mucous, and ductal cell lineages. Serial tamoxifen-induction of both Cre-driver lines demonstrated that lineage-positive multipotent MECs contribute to ∼ 60% of glandular cells by 21 days after birth. In contrast, lineage-traced MECs did not contribute to cell types in the surface airway epithelium. These findings demonstrate that MECs born early during SMG morphogenesis are multipotent progenitors with the capacity to differentiate into other glandular cell types.

Postnatal morphological and lectin histochemical observation of the submucosal glands of rat nasopharynx

The development of submucosal glands of rat nasopharynx was studied with respect to their morphological maturation and glycoprotein alterations during the postnatal period. This study examined the histological morphology with hematoxylin–eosin and the binding pattern of lectins, soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin-I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and succinylated WGA (sucWGA) on frozen sections from newborn into adulthood. At birth, nasopharyngeal glands consisted of rudimentary secretory units which by postnatal day 3 (PN3) showed the characteristic features of salivary glands comprised of mixed mucous and serous cells. With maturation, serous cells increased in number and were arranged in clusters. Lectin reactivity at birth was detected at the acinar cell basal membranes for DBA, SBA, VVA, UEA-1 and PNA. At PN3, lectins labeled the apical cytoplasm and basolateral membranes of mucous cells and progressively with maturation, extended from the apical to basal portions of the cytoplasm with variable reactivity of VVA, PNA and sucWGA. Serous cells were labeled by UEA-1 starting from PN10 and also by PNA in adults. Ducts showed variable lectin reaction on the luminal membrane with strong reactivity of DBA and UEA-1 at PN21. Taken together, lectin histochemistry indicated the transitional occurrence of glycoproteins depending on the stage of maturation of the glands. Moreover, these results emphasize the difference in the morphology and lectin histochemistry between the nasopharyngeal and palatine glands.

FGF and EDA pathways control initiation and branching of distinct subsets of developing nasal glands

Hypertrophy, hyperplasia and altered mucus secretion from the respiratory submucosal glands (SMG) are characteristics of airway diseases such as cystic fibrosis, asthma and chronic bronchitis. More commonly, hyper-secretion of the nasal SMGs contributes to allergic rhinitis and upper airway infection. Considering the role of these glands in disease states, there is a significant dearth in understanding the molecular signals that regulate SMG development and patterning. Due to the imperative role of FGF signalling during the development of other branched structures, we investigated the role of Fgf10 during initiation and branching morphogenesis of murine nasal SMGs. Fgf10 is expressed in the mesenchyme around developing SMGs while expression of its receptor Fgfr2 is seen within glandular epithelial cells. In the Fgf10 null embryo, Steno's gland and the maxillary sinus gland were completely absent while other neighbouring nasal glands showed normal duct elongation but defective branching. Interestingly, the medial nasal glands were present in Fgf10 homozygotes but missing in Fgfr2b mutants, with expression of Fgf7 specifically expressed around these developing glands, indicating that Fgf7 might compensate for loss of Fgf10 in this group of glands. Intriguingly the lateral nasal glands were only mildly affected by loss of FGF signalling, while these glands were missing in Eda mutant mice, where the Steno's and maxillary sinus gland developed as normal. This analysis reveals that regulation of nasal gland development is complex with different subsets of glands being regulated by different signalling pathways. This analysis helps shed light on the nasal gland defects observed in patients with hypohidrotic ectodermal dysplasia (HED) (defect EDA pathway) and LADD syndrome (defect FGFR2b pathway).

Understanding the development of the respiratory glands.

The submucosal glands (SMGs) of the respiratory system are specialized structures essential for maintaining airway homeostasis. The significance of SMGs is highlighted by their involvement in respiratory diseases such as cystic fibrosis, asthma and chronic bronchitis, where their phenotype and function are severely altered. Uncovering the normal development of the airway SMGs is essential to elucidate their role in these disorders, however, very little is known about the cellular mechanisms and intracellular signals involved in their morphogenesis. This review describes in detail the embryonic developmental journey of the nasal SMGs and the postnatal development of the tracheal SMGs in the mouse. Current knowledge of the genes and signalling molecules involved in SMG organogenesis is also explored. Here we review the temporal localisation and development of the murine respiratory glands in the hope of stimulating further research into the mechanisms required for successful SMG patterning and function.

Sox2 modulatesLef-1expression during airway submucosal gland development

Tracheobronchial submucosal glands (SMGs) are derived from one or more multipotent glandular stem cells that coalesce to form a placode in surface airway epithelium (SAE). Wnt/β-catenin-dependent induction of lymphoid enhancer factor (Lef-1) gene expression during placode formation is an early event required for SMG morphogenesis. We discovered that Sox2 expression is repressed as Lef-1 is induced within airway SMG placodes. Deletion of Lef-1 did not activate Sox2 expression in SMG placodes, demonstrating that Lef-1 activation does not directly inhibit Sox2 expression. Repression of Sox2 protein in SMG placodes occurred posttranscriptionally, since the activity of its endogenous promoter remained unchanged in SMG placodes. Thus we hypothesized that Sox2 transcriptionally represses Lef-1 expression in the SAE and that suppression of Sox2 in SMG placodes activates Wnt/β-catenin-dependent induction of Lef-1 during SMG morphogenesis. Consistent with this hypothesis, transcriptional reporter assays, ChIP analyses, and DNA-protein binding studies revealed a functional Sox2 DNA binding site in the Lef-1 promoter that is required for suppressing β-catenin-dependent transcription. In polarized primary airway epithelium, Wnt induction enhanced Lef-1 expression while also inhibiting Sox2 expression. Conditional deletion of Sox2 also enhanced Lef-1 expression in polarized primary airway epithelium, but this induction was significantly augmented by Wnt stimulation. Our findings provide the first evidence that Sox2 acts as a repressor to directly modulate Wnt-responsive transcription of the Lef-1 gene promoter. These studies support a model whereby Wnt signals and Sox2 dynamically regulate the expression of Lef-1 in airway epithelia and potentially also during SMG development.

Submucosal gland development in the human fetal trachea xenograft model: implications for fetal gene therapy

Background/Purpose Our previous work in a human-fetal trachea xenograft model suggests potential benefits of treating cystic fibrosis in utero. The target for postnatal gene therapy in cystic fibrosis is tracheal submucosal glands (SMGs). The aim of this study was to determine if SMG development in our model recapitulates normal trachea development and its validity for studying fetal gene transfer. Methods Fetal tracheas were divided into developmental phases: early, mid, and late. Fetal tracheas were xenografted onto immunocompromised mice and analyzed for SMG developmental staging and mucopolysaccharide production. Results There were no significant differences in gland number, size, or density from early through late phase between groups. Xenografted tracheas demonstrated a similar progression through the stages of SMG development as controls after an initial phase shift. Control and xenografted tracheas demonstrated characteristic patterns of acidic mucin production at the base of the SMGs. Conclusions Fetal trachea xenograft SMG recapitulates normal development and is a valid model for studying human fetal gene transfer. The accessibility of SMG stem cells in early tracheal development may afford a unique window of opportunity for gene transfer. This model has the benefit of providing access to human fetal tracheas in vivo and permits the study of novel fetal gene therapy strategies.

Wnt3a regulates Lef-1 expression during airway submucosal gland morphogenesis

Regulation of the lymphoid enhancer factor 1 (Lef-1) transcription factor is important for the inductive formation of many epithelial-derived appendages including airway submucosal glands (SMGs). Although Wnts have been linked to developmental processes involving transcriptional activation of the Lef-1 protein, there is little in vivo information directly linking Wnts with the transcriptional regulation of the Lef-1 promoter. In the present study, we hypothesized that Wnt3a directly regulates Lef-1 gene expression required for SMG morphogenesis in mice. In support of this hypothesis, TOPGAL reporter mice demonstrated activation of β-catenin/Tcf complexes during early phases of SMG development and immunolocalization studies confirmed abundant expression of Tcf4, but not Tcf1 or Tcf3, at this stage. ChIP analysis in primary airway epithelial cells revealed that Tcf4 associates with a known Wnt Responsive Region in the Lef-1 promoter and transfection of Cos-1 cells with dominant active β-catenin and Tcf4 synergistically activated the Lef-1 promoter. Using Wnt3a deficient and Lef-1 promoter-GFP reporter mice, we also demonstrate that Wnt3a induces Lef-1 gene expression in newly forming SMG buds of mice and is required for the maintenance of gland bud growth. These findings provide the first in vivo evidence that Wnt3a can transcriptionally regulate the Lef-1 gene.

Intercellular growth factor signaling and the development of mouse tracheal submucosal glands

To provide a genetic framework for investigating changes in airway submucosal gland function in human respiratory disease, we have investigated their counterparts in normal and mutant mice. We describe their morphogenesis in relation to the expression of genes encoding conserved intercellular signaling pathways. Submucosal glands are severely reduced in number and size in mice heterozygous for Fgf10. Glands are completely absent in mice lacking Ectodysplasin (Eda) and Edaradd (Eda receptor adaptor protein), members of the tumor necrosis (TNF) superfamily of signaling factors. Furthermore, components of the Eda and closely related pathways are transcribed throughout the respiratory system in the adult mouse. Finally, the temporal and spatial pattern of Bmp4 expression suggests that it may control submucosal gland development and homeostasis. Taken together, our observations have important implications for the better understanding of the submucosal gland remodeling that occurs in human respiratory disease.

DEVELOPMENTAL EXPRESSION OF CATENINS AND ASSOCIATED PROTEINS DURING SUBMUCOSAL GLAND MORPHOGENESIS IN THE AIRWAY

Although lymphoid enhancer binding factor-1 (Lef-1) plays an obligatory role in airway submucosal gland (SMG) development, its expression alone is not an adequate signal for initiating gland morphogenesis. Because Lef-1 forms a bipartite transcription factor with beta-catenin to mediate wnt pathway signaling, we investigated the expression of beta-catenin and associated proteins during SMG development with both in situ hybridization and immunocytochemistry. Unexpectedly, high levels of E-cadherin mRNA were expressed by cells in developing gland buds from the earliest stages through subsequent differentiation into mature glands. In contrast, a decreased level of E-cadherin immunoreactivity in stage I gland bud cells suggested that post-translational modulation of E-cadherin protein levels may play a critical role in early stages of gland morphogenesis. Adenomatous polyposis coli (APC) mRNA was expressed relatively weakly in the developing ferret trachea, but higher levels of protein staining were observed throughout the cytoplasm of gland buds and surface epithelial cells. B-Catenin mRNA was abundantly expressed throughout the tracheal epithelium and at the highest levels in primordial gland buds. B-Catenin protein localized to the basolateral membranes of all airway epithelial cell types. However, no detectable increases in nuclear or cytoplasmic staining were associated with gland buds, as would be expected if beta-catenin served as a transcriptional cofactor for Lef-1 in gland morphogenesis. Additional studies demonstrated the gamma-catenin distribution to be remarkably similar to that of beta-catenin, whereas alpha-catenin staining was more diffuse in the cytoplasm of airway epithelial and gland bud cells. These descriptive results do not rule out a role for wnt signaling in SMG development , but provide no evidence that beta-catenin, or gamma-catenin, is a cofactor in Lef-1 regulation of SMG development.

Submucosal gland development in the airway is controlled by lymphoid enhancer binding factor 1 (LEF1).

Previous studies have demonstrated that transcription of the lymphoid enhancer binding factor 1 (Lef1) gene is upregulated in submucosal gland progenitor cells just prior to gland bud formation in the developing ferret trachea. In the current report, several animal models were utilized to functionally investigate the role of LEF1 in initiating and supporting gland development in the airway. Studies on Lef1-deficient mice and antisense oligonucleotides in a ferret xenograft model demonstrate that LEF1 is functionally required for submucosal gland formation in the nasal and tracheal mucosa. To determine whether LEF1 expression was sufficient for the induction of airway submucosal glands, two additional model systems were utilized. In the first, recombinant adeno-associated virus was used to overexpress the human LEF1 gene in a human bronchial xenograft model of regenerative gland development in the adult airway. In a second model, the LEF1 gene was ectopically overexpressed under the direction of the proximal airway-specific CC10 promoter in transgenic mice. In both of these models, morphometric analyses revealed no increase in the number or size of airway submucosal glands, indicating that ectopic LEF1 expression alone is insufficient to induce submucosal gland development. In summary, these studies demonstrate that LEF1 expression is required, but in and of itself is insufficient, for the initiation and continued morphogenesis of submucosal glands in the airway.

Murine Submucosal Glands Are Clonally Derived and Show a Cystic Fibrosis Gene–Dependent Distribution Pattern

Submucosal glands (SMGs) are the major site of expression of the cystic fibrosis (CF) transmembrane conductance regulator gene (CFTR) in the human lung. As such, SMGs may be a critical component of CF lung disease pathogenesis and an important target for gene therapy. Gene-targeted mouse models exist for CF and these are used to validate gene therapy or other interventions and to dissect CF phenotypes. It is important, therefore, to compare human and mouse SMGs. We show that SMGs in the mouse are similar in structure, cell types, and Cftr expression to those in the human. Murine SMGs were found to be present in the proximal regions of the trachea at the same density as in humans but, unlike in humans, did not extend below the trachea. Upon investigation of homozygous Cftr tm1HGU and Cftr tm1G551D mutant mice, SMGs were found to extend more distally than those in wild-type control mice (P < 0.05). To investigate the development of SMGs we generated aggregation chimeric mice. Chimeric offspring contained a contribution of transgenic cells that were detectable either by DNA in situ hybridization (reiterated beta-globin transgene TgN[Hbb-bl]83Clo) or beta-galactosidase histochemistry (Lac Z reporter gene TgR[ROSA26]- 26Sor). Analysis of the distribution of transgenic cells in chimeric SMGs suggests that SMGs are clonally derived.

Differentially regulated epithelial expression of an Eph family tyrosine kinase (fHek2) during tracheal surface airway and submucosal gland development.

A ferret model was used to evaluate the potential role of an Eph family tyrosine kinase (fHek2) in tracheal development of surface airway epithelium and submucosal glands. A partial 2.6-kb cDNA fragment of fHek2 was isolated from a ferret tracheal/lung cDNA library. Sequence analysis demonstrated that this gene is the ortholog to the previously cloned human Hek2 gene. In situ hybridization analysis of fHek2 mRNA expression on ferret tracheal developmental time points revealed an expression pattern within a subset of surface airway epithelial cells which remained relatively constant throughout tracheal development (from -2 d in utero to adult). In contrast, developing tracheal submucosal glands at 3-day postnatal time points demonstrated little fHek2 mRNA expression. However, expression of fHek2 significantly increased more than 4-fold over the course of gland development to adulthood. These findings, which demonstrate a uniquely regulated pattern of fHek2 mRNA expression between surface airway epithelium and submucosal glands, have implications on regulatory processes which control differentiation and/or maturation of secretory structures in the lung. Such findings may be useful in further delineating the mechanisms which control cellular differentiation in the lung and how these processes are abnormally regulated in hypersecretory diseases such as chronic bronchitis, asthma, and cystic fibrosis.

Developmental expression patterns of CFTR in ferret tracheal surface airway and submucosal gland epithelia.

Submucosal glands are a major site of cystic fibrosis transmembrane conductance regulator (CFTR) expression in the human airway and may play an important role in the pathogenesis of cystic fibrosis. In humans, strategies for gene targeting to submucosal glands will likely be dependent on in utero gene transfer to submucosal gland progenitors because of the inaccessibility of these regions from the fully developed airway. However, little is known about the ontogeny of CFTR gene expression in developing submucosal glands and the potential functional role(s) CFTR may have during gland development. To this end, we describe the partial cloning of the ferret CFTR cDNA which was used for in situ mRNA localization studies in developing ferret trachea. The ferret animal model is attractive for studies pertaining to the development of tracheal submucosal glands because postnatal tracheal development in this species mirrors in utero gland development in humans. Sequence analysis of the first nucleotide binding domain (NBD1) from ferret CFTR revealed a striking homology to the human gene at both the DNA (94.4%) and amino acid level (97%). Interestingly, this high level of amino acid homology extends to a group of mammalian species (ferret, human, sheep, and bovine) which have similar lung morphologies with respect to the presence of surface airway goblet cells and submucosal glands. In contrast, mouse and rat airways which are quite divergent from those of ferret, human, sheep, and bovine species with respect to secretory cell types in the airway and abundance of submucosal glands, demonstrate significantly less homology (80%) with respect to CFTR amino acid sequence in the NBD1 domain. Given that traditional phylogenetic classification of these species does not mirror the evolutionary conservation of CFTR, such findings would suggest that certain aspects of lung morphology may be reflected in the evolutionary conservation of CFTR NBD1 amino acid sequences. CFTR in situ hybridization studies in ferret trachea demonstrate a developmental increase (3-4 fold) in CFTR mRNA expression within the surface airway epithelium between -2 day to 5 wk which mirrors an increase in ciliogenesis over this time frame. Additionally, all stages of gland development including the most primordial gland-forming buds could be seen to contain infrequent cells which highly express CFTR at a level that remained constant throughout development. Such findings suggest that differentiation of gland progenitor cells to CFTR-expressing submucosal gland cells occurs very early within submucosal gland development and morphogenesis.

Progenitor cells of the adult human airway involved in submucosal gland development.

A bronchial xenograft model of the human airway was used to identify submucosal gland progenitor cells within the surface airway epithelium. Lineage analysis using recombinant retroviruses has demonstrated considerable diversity in the cellular composition of expanded clones within reconstituted xenograft airway epithelium. These findings provide evidence for the existence of multiple progenitors in the airway with either limited or pluripotent capacity for differentiation. Furthermore, the development of transgene-expressing submucosal glands was associated with a single subset of surface airway epithelial clones. This gland progenitor cell demonstrated two discernible characteristics consistent with the identification of an airway stem cell including: (1) pluripotent capacity for airway differentiation and (2) a two-fold higher proliferative rate than other observed clone types. The number of progenitor cells involved in gland development was also assessed by clonal analysis using alkaline phosphatase and beta-galactosidase transgenes. These studies demonstrated that more than one airway progenitor cell is involved in the initial stages of gland development. A second explanation for the high prevalence of non-clonality in developing glands was suggested from three-dimensional reconstruction of transgene marked glands. These reconstruction experiments demonstrated that 27% of glands contained more than one duct to the surface airway epithelium. This observation suggests a novel mechanism of gland morphogenesis by which independently formed glands interact to join glandular lumens. Such a mechanism of glandular development and morphogenesis may play an important role in normal submucosal gland development and/or the progression of hypersecretory diseases of the adult human airway as seen in cystic fibrosis, chronic bronchitis and asthma. The identification of progenitor cells with the capacity to form submucosal glands has implications on the targets for gene therapy in cystic fibrosis.

Progenitor cells of the adult human airway involved in submucosal gland development

Progenitor cells of the adult human airway involved in submucosal gland development

Human Fetal Lung Fibroblasts Promote Invasion of Extracellular Matrix by Normal Human Tracheobronchial Epithelial Cells In Vitro: A Model of Early Airway Gland Development

Epithelial invasion of extracellular matrix (ECM) is important during lung development and the pathogenesis of bronchogenic neoplasms. Airway submucosal gland development begins when clusters of surface epithelial cells invade the lamina propria between the tenth and thirtieth weeks of fetal life. The factors regulating this transient normal invasive behavior are unknown. We observed that normal human tracheobronchial epithelial (HTBE) cells from adult necropsy specimens penetrate collagen matrices when co-cultured with human fetal lung fibroblasts (HFLF). Invading clusters of epithelial cells resembled primordial glands, forming tubular structures and undergoing dichotomous branching. Using 48 different tracheobronchial specimens, we compared paired cultures of HTBE cells without (control) and with HFLF co-culture. Fixed, vertically sectioned culture substrates were examined, and invaginated epithelial cell clusters as well as total invading HTBE cells were counted. The co-cultured condition resulted in significantly more epithelial invaginations per two sections (4 +/- 1 versus 22 +/- 3, P < 0.0005) and more invading HTBE cells per two sections (31 +/- 9 versus 194 +/- 27, P < 0.0005) than controls. Epithelial invasion was noted by 36 h in culture and was greatest at HTBE cell/HFLF ratios near 1 and HFLF passages between 10 and 16. Epithelial cells co-cultured with a fibroblast cell line derived from the adult bronchiole showed no increase in ECM invasion compared with controls. These results demonstrate that fetal mesenchymal cells are capable of promoting invasive behavior in mature epithelial cells in vitro. Given the fibroblast type and passage specificity, this model should prove useful for investigating the cellular and molecular regulation of epithelial ECM invasion.