Canalicular Stage Research Articles

Knockout of Insulin-Like Growth Factor-1 Receptor Impairs Distal Lung Morphogenesis

BackgroundInsulin-like growth factors (IGF-I and -II) are pleiotropic regulators of somatic growth and development in vertebrate species. Endocrine and paracrine effects of both hormones are mediated by a common IGF type 1 receptor (IGF-1R). Lethal respiratory failure in neonatal IGF-1R knockout mice suggested a particular role for this receptor in pulmonary development, and we therefore investigated the consequences of IGF-1R inactivation in lung tissue.Methods and FindingsWe first generated compound heterozygous mutant mice harboring a hypomorphic (Igf1rneo) and a null (Igf1r−) allele. These IGF-1Rneo/− mice express only 22% of normal IGF-1R levels and are viable. In adult IGF-1Rneo/− mice, we assessed lung morphology and respiratory physiology and found normal histomorphometric characteristics and normal breathing response to hypercapnia. We then generated homozygous IGF-1R knockout mutants (IGF-1R−/−) and analyzed their lung development during late gestation using histomorphometric and immunohistochemical methods. IGF-1R−/− embryos displayed severe lung hypoplasia and markedly underdeveloped diaphragms, leading to lethal neonatal respiratory distress. Importantly, IGF-1R−/− lungs from late gestation embryos were four times smaller than control lungs and showed markedly thickened intersaccular mesenchyme, indicating strongly delayed lung maturation. Cell proliferation and apoptosis were significantly increased in IGF-1R−/− lung tissue as compared with IGF-1R+/+ controls. Immunohistochemistry using pro-SP-C, NKX2-1, CD31 and vWF as markers revealed a delay in cell differentiation and arrest in the canalicular stage of prenatal respiratory organ development in IGF-1R−/− mutant mice.Conclusions/SignificanceWe found that low levels of IGF-1R were sufficient to ensure normal lung development in mice. In contrast, complete absence of IGF-1R significantly delayed end-gestational lung maturation. Results indicate that IGF-1R plays essential roles in cell proliferation and timing of cell differentiation during fetal lung development.

Oxygen dose responsiveness of human fetal airway smooth muscle cells

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18-20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca(2+) ([Ca(2+)](i)) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca(2+) regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca(2+) imaging showed robust [Ca(2+)](i) responses to 1 μM acetylcholine (ACh) and 10 μM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca(2+). Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca(2+)](i) responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

Lung Injury in Preterm Neonates: The Role and Therapeutic Potential of Stem Cells

Continuous improvements in perinatal care have allowed the survival of ever more premature infants, making the task of protecting the extremely immature lung from injury increasingly challenging. Premature infants at risk of developing chronic lung disease or bronchopulmonary dysplasia (BPD) are now born at the late canalicular stage of lung development, just when the airways become juxtaposed to the lung vasculature and when gas-exchange becomes possible. Readily available strategies, including improved antenatal management (education, regionalization, steroids, and antibiotics), together with exogenous surfactant and exclusive/early noninvasive ventilatory support, will likely decrease the incidence/severity of BPD over the next few years. Nonetheless, because of the extreme immaturity of the developing lung, the extent to which disruption of lung growth after prematurity and neonatal management lead to an earlier or more aggravated decline in respiratory function in later life is a matter of concern. Consequently, much more needs to be learned about the mechanisms of lung development, injury, and repair. Recent insight into stem cell biology has sparked interest for stem cells to repair damaged organs. This review summarizes the exciting potential of stem cell-based therapies for lung diseases in general and BPD in particular.

Sex-specific perinatal expression of glutathione peroxidases during mouse lung development

Reports indicate that antioxidant enzymes like the glutathione peroxidases (GPx) can be regulated by sex steroids. The GPx, a major class of antioxidants involved in H2O2 and lipid hydroperoxides neutralization, showed an age- and sex-specific expression in many adult organs including the lung. High levels of androgens in the male lung are known to delay the surge of surfactant synthesis during gestation in several species. However, the impact of male androgens on antioxidant GPx early in life remains to be determined. The objective was to study the lung sex-specific expression of GPx during BALB/c mouse perinatal development. The mRNA expression of four seleno-dependent Gpx (Gpx1 to 4) in the lung of both sexes was characterized by real-time PCR from gestational day 15 to postnatal day 30, covering the entire canalicular, saccular and alveolar stages. Immunohistochemistry of GPx-1, -3 and -4, and seleno-dependent GPx enzymatic assays were also performed in the lung. We found a transient lower Gpx1 mRNA level in male than in female lungs during the first 5 days after birth, corresponding to the saccular phase. This dimorphic expression was concomitant to a sex difference in GPx enzymatic activity corrected for blood. It is, to our knowledge, the first report of a sex dimorphism for murine lung enzymatic antioxidant defenses during the perinatal period.

Learning how Mechanical Forces Regulate Lung Development:Opportunities for Translational Research

Lung growth and development during fetal life are critical for extrauterine survival. Pulmonary hypoplasia secondary to congenital diaphragmatic hernia, oligohydramnios, etc, is an important cause of neonatal morbidity and mortality. In fact, pulmonary hypoplasia is the most common finding in neonatal autopsies. In addition, more than 20,000 babies are born every year in the United States before 27 weeks of gestation (canalicular stage of lung development). These disorders have in common an incomplete development of the lungs. Despite the improvement in neonatal care, these conditions can cause serious short-term and long-term morbidities. Currently, the management is primarily supportive and there is not specific treatment to stimulate the growth and development of the lungs.

Expression analysis of asthma candidate genes during human and murine lung development

BackgroundLittle is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.ObjectiveTo investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.MethodsAsthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.ResultsIn total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.ConclusionsOur data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.

Expression of E-Cadherin and Beta-Catenin in the Developing Lung after Prenatal Administration of Vitamin A

Cadherin-catenin complexes stabilize adhesive contacts between the neighboring cells, direct actin cytoskeleton reorganization, and are tightly linked to several key signal transduction networks, which makes them crucial players during lung morphogenesis. Vitamin A ant its derivatives (retinoids) are potent regulators of cell proliferation and differentiation essential for normal lung development. In order to investigate vitamin A influence on E-cadherin and beta-catenin during lung development we conducted an in vivo study in which pregnant mice were subjected to subcutaneous administration of vitamin A on the 12th gestational day. The lungs from control and treated animals were collected daily from the 15th gestational day till the 3th day of life and processed for routine immunohistochemistry. According to our results, beta-catenin expression was not changed by vitamin A administration, while the expression of E-cadherin increased. E-cadherin expression was significantly higher in the lungs of vitamin A-treated animals at the 15th and 18th gestational days, at the day of birth and at the first and second day of life. Our previous works showed that prenatal administration of vitamin A modified branching morphogenesis resulting in a more mature lung at the canalicular and subsequent stages of lung development, a feature that can be explained by its influence on the regulation of the correct size and shape of the epithelial growing buds in which the E-cadherin spatial and temporal expression is of pivotal importance.

Stem Cell Repair of the Preterm Lung: A Breakthrough in the Making?

Continuous improvements in perinatal care have allowed the survival of ever more premature infants, making the task of protecting the more premature lung from injury increasingly challenging. Premature infants at risk of developing chronic lung disease of prematurity or bronchopulmonary dysplasia (BPD) are now born at the late canalicular stage of lung development, just when the airways become juxtaposed to the lung vasculature and when gas-exchange becomes possible. Readily available strategies including improved antenatal management (education, regionalization, steroids, antibiotics) together with prophylactic surfactant and early non-invasive ventilatory support targeting lower oxygen saturations will likely decrease the incidence/severity of BPD over the next few years. Nonetheless, the long-term consequences of early interference with lung development may reach into adulthood. Consequently, much more needs to be learned about the mechanisms of lung development, injury and repair. Recent insight into stem cell biology has sparked interest for stem cells to repair damaged organs. This lecture will summarize the exciting potential of stem cell-based therapies for the prevention or treatment of BPD.

Structural and Functional Development of the Respiratory System in a Newborn Marsupial with Cutaneous Gas Exchange

Marsupials are born with structurally immature lungs and rely, to varying degrees, on cutaneous gas exchange. With a gestation of 13 d and a birth weight of 13 mg, the fat-tailed dunnart (Sminthopsis crassicaudata) is one of the smallest and most immature marsupial newborns. We determined that the skin is almost solely responsible for gas exchange in the early neonatal period. Indeed, fewer than 35% of newborn dunnarts were observed to make any respiratory effort on the day of birth, with pulmonary ventilation alone not meeting the demand for oxygen until approximately 35 d postpartum. Despite the lack of pulmonary ventilation, the phrenic nerve had made contact with the diaphragm, and the respiratory epithelium was sufficiently developed to support gas exchange on the day of birth. Both type I and type II (surfactant-producing) alveolar epithelial cells were present, with fewer than 7% of the cells resembling undifferentiated alveolar epithelial precursor cells. The type I epithelial cells did, however, display thickened cytoplasmic extensions, leading to a high diffusion distance for oxygen. In addition, the architecture of the lung was immature, resembling the early canalicular stage, with alveolarization not commencing until 45 d postpartum. The pulmonary vasculature was also immature, with a centrally positioned single-capillary layer not evident until 100 d postbirth. These structural limitations may impede efficient pulmonary gas exchange, forcing the neonatal fat-tailed dunnart to rely predominately on its skin, a phenomenon supported by a low metabolic rate and small size.

Expression of Hypoxia-Inducible Factors, Regulators, and Target Genes in Congenital Diaphragmatic Hernia Patients

Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension and has high morbidity and mortality rates. The cause and pathophysiology of CDH are not fully understood. However, impaired angiogenesis appears to play an important role in the pathophysiology of CDH. Therefore, we examined different components of an important pathway in angiogenesis: hypoxia-inducible factors (HIFs); HIF regulators von Hippel-Lindau (VHL) and prolyl 3-hydroxylase (PHD3); and HIF target genes vascular endothelial growth factor A ( VEGF-A ) and vascular endothelial growth factor receptor 2 ( VEGFR-2 ). Quantitative polymerase chain reaction of lung tissue showed a significantly decreased expression of VEGF-A mRNA in the alveolar stage of lung development in CDH patients compared with matched control patients. In the canalicular stage, no differences for VEGF-A were seen between the lungs of CDH patients and those of control patients. Other components of angiogenesis (VHL, HIF-1α, HIF-2α, HIF-3α, VEGFR-2 mRNA, PHD3 protein) that were analyzed showed no differences in expression between CDH and control patients, independent of the developmental stage. A lower expression of VEGF mRNA in CDH patients in the alveolar stage, possibly as a result of downregulation of HIF-2α might indicate a role for these factors in the pathophysiology of CDH.

Aquaporin 5 distribution pattern during development of the mouse sublingual salivary gland.

Aquaporin 5 (AQP5) is important in salivary fluid secretion, and has been found in acinar cells of salivary glands in several species. Recently, studies have shown the AQP5 transcript and protein expression patterns as well as the temporal-spatial protein distribution during development of the mouse submandibular salivary gland. The AQP5 distribution pattern of the closely located sublingual gland (SLG) is, however, not well known. Thus, in this study, the Aqp5 RNA expression pattern and the temporal-spatial distribution of AQP5 protein in prenatal development and in adult mouse SLG was investigated. SLGs from embryonic day 14.5 (E14.5) to 18.5 and postnatal days 0 (P0), 25, and 60 were examined using real time PCR and immunohistochemistry. The Aqp5 transcript was detected from E13.5 and was found to increase towards birth and in young adults. The protein was first detected in a scattered pattern in the canalicular stage and became more organized in the luminal membrane of the acinar cells towards birth. During all postnatal developmental stages studied, AQP5 was localized in the luminal and lateral membrane of acinar cells. AQP5 was also detected in the intercalated duct and additional apical membrane staining in the entire intralobular duct was found in the terminal bud stage. These results indicate that AQP5 plays a role during embryonic salivary gland development.

Apolipoprotein A-I, A-II, and H mRNA and protein accumulation sites in the developing lung in late gestation

BackgroundExpression of apolipoprotein A-I (apoA-I), A-II, and H was previously observed at 16 to 50-fold higher levels in the fetal than the adult mouse lung. Here, sites of apoA-I, A-II, and H mRNA and protein accumulation were determined in mouse fetal lungs by in situ hybridization and immunohistochemistry in late gestation.ResultsExpression sites vary for the three genes and change for the distal epithelium before the end of the canalicular stage, thus where and when the surge of surfactant synthesis occurs. Messenger of apoH, but not those of apoA-I and A-II, was also observed in the proximal epithelium and smooth muscles surrounding arteries. In contrast to apoC-II protein, none of the three studied apolipoproteins accumulated within secretory granule-like structures. Immunohistochemistry revealed that apoA-I and apoH accumulated mainly in capillaries. Three different positive signals with the anti-apoA-II antibody were found: one transient signal in the nucleus of a portion of mesenchymal cells, a second at lower levels throughout the mesenchyme, and another in capillaries with a specific increase from gestation day 17.5/18.5.ConclusionTemporal and geographic co-expression of apoAI, AII, and H genes with surfactant production site suggests that the three apolipoproteins are secreted to play roles supporting the lung-specific surfactant lipid-related metabolism.

Embryonic over‐expression of RAGE in mouse lung causes an imbalance between apoptosis and proliferation leading to severe lung hypoplasia

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors prominently detected in lung epithelium. Previous experiments demonstrate that over-expression of RAGE in murine alveolar type (AT) II cells during development causes lung hypoplasia and neonatal lethality. In order to characterize lung hypoplasticity, proliferation and apoptosis were evaluated in RAGE over-expressing mice. Histology spanning developmental milestones revealed marked loss of lung tissue beginning in the canalicular stage of lung development and continuing throughout the saccular period (embryonic day (E) 15.5–18.5). In situ cell death detection using TUNEL assay revealed a significant elevation in apoptotic cells in RAGE over-expressing pulmonary tissue at E17.5 compared to age-matched controls. The balance between cell death and proliferation was also characterized by PCNA staining. These findings were further supported by electron micrographic analysis of E18.5 lungs that demonstrated extracellular matrix degradation and ATI and ATII cell death in RAGE over-expressed mice. The data suggest that elevated RAGE expression alters the normal balance between proliferation and apoptosis in the developing lung leading to significant loss in respiratory surface area. Supported by the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.

Selective Toll-Like Receptor Expression in Human Fetal Lung

Toll-like receptors (TLRs) are critical components of the innate immune system, acting as pattern recognition molecules and triggering an inflammatory response. TLR associated gene products are of interest in modulating inflammatory-related pulmonary diseases of the neonate. The ontogeny of TLR-related genes in human fetal lung has not been previously described and could elucidate additional functions and identify strategies for attenuating the effects of fetal inflammation. We examined the expression of 84 TLR-related genes on 23 human fetal lung samples from three groups with estimated ages of 60 (57-59 d), 90 (89-91 d), and 130 (117-154 d) d. By using a false detection rate algorithm, we identified 32 genes displaying developmental regulation with TLR2 having the greatest up-regulation of TLR genes (9.2-fold increase) and TLR4 unchanged. We confirmed the TLR2 up-regulation by examining an additional 133 fetal lung tissue samples with a fluorogenic polymerase chain reaction assay (TaqMan) and found an exponential best-fit curve during the study time. The best-fit curve predicts a 6.1-fold increase from 60 to 130 d. We conclude that TLR2 is developmentally expressed from the early pseudoglandular stage of lung development to the canalicular stage.

Expression of SHH signaling pathway components in the developing human lung

The Sonic hedgehog (Shh) cascade is crucial for the patterning of the early lung morphogenesis in mice, but its role in the developing human lung remains to be determined. In the present study, the expression patterns of SHH signaling pathway components, including SHH, PTCH1, SMO, GLI1, GLI2 and GLI3 were examined by in situ hybridization and immunohistochemistry, and compared with the equivalent patterns in mice. Our results showed that, as in mice, SHH was expressed in the epithelium of the developing human lung. However, SHH receptors (PTCH1 and SMO) and SHH signaling effectors (GLI1-3) were strongly detected in the human lung epithelium, but weakly in the mesenchyme, slightly different from their expressions in mice. Furthermore, the expression levels of SHH signaling pathway genes in human lung, but not that of GLI1, were subsequently downregulated at the canalicular stage evaluated by real-time PCR, coincident with a decline in the developing murine lung. In conclusion, in spite of slight differences, the considerable similarities of gene expression in human and mice suggest that conserved molecular networks regulate mammalian lung development.

ErbB4 regulates the timely progression of late fetal lung development

The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development.

Levels of Dihydrotestosterone, Testosterone, Androstenedione, and Estradiol in Canalicular, Saccular, and Alveolar Mouse Lungs

Androgens and estrogens are known regulators of fetal and postnatal lung development, but their levels in the developing lung have never been determined. We present here, for the first time, a gas chromatography-mass spectrometry (GC/MS) quantification of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar stage lungs of both sexes. Testosterone, androstenedione, and estradiol were observed in all the analyzed lung samples from gestation day (GD) 16.5 to postnatal day (PN) 30, totalizing 383 individual mice. Levels of these three steroids decreased between birth and PN 5. In contrast, dihydrotestosterone was detected only in male samples on GD 19.5, PN 0, and PN 30. A significant sex difference was observed for testosterone and androstenedione but not for estradiol. Steroid levels were also determined in skinned hind legs for comparison. Three-way analysis of variance revealed that tissue (lung or leg) had a significant effect on testosterone levels for both sexes, but not on androstenedione and estradiol levels. Low but significant testosterone and androstenedione levels were observed in all the females and in prepubertal male samples. These levels must be sufficient to induce androgen receptor activation, as suggested by our recent report showing the presence of androgen receptor in the nucleus of several lung cells in corresponding developmental ages and sexes.

A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in Asthma

Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes. We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma. Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV(1), FVC, and FEV(1)/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study. Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7-16 wk) and the canalicular (gestational age: 17-26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV(1) (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV(1)/FVC (combined P = 0.003). Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.

The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells

The conducting airways (bronchi and bronchioles) and peripheral gas exchange (alveolar) regions of the mammalian lung are generated by a process of branching morphogenesis. Evidence suggests that during embryonic development, the undifferentiated epithelial progenitors are located at the distal tips of the branching epithelium. To test this hypothesis, we used an Id2-CreER(T2) knock-in mouse strain to lineage trace the distal epithelial tip cells during either the pseudoglandular or canalicular phases of development. During the pseudoglandular stage, the tip cells both self-renew and contribute descendents to all epithelial cell lineages, including neuroendocrine cells. In addition, individual Id2(+) tip cells can self-renew and contribute descendents to both the bronchiolar and alveolar compartments. By contrast, during the later canalicular stage, the distal epithelial tip cells only contribute descendents to the alveoli. Taken together, this evidence supports a model in which the distal tip of the developing lung contains a multipotent epithelial population, the fate of which changes during development.