Volumetric analysis of the terminal ductal lobular unit architecture and cell phenotypes in the human breast.

The major lactiferous ducts of the human breast branch out and end at terminal ductal lobular units (TDLUs). Despite their functional and clinical importance, the three-dimensional (3D) architecture of TDLUs has remained undetermined. Our quantitative and volumetric imaging of healthy human breast tissue demonstrates that highly branched TDLUs, which exhibit increased proliferation, are uncommon in the resting tissue regardless of donor age, parity, or hormonal contraception. Overall, TDLUs have a consistent shape and branch parameters, and they contain a main subtree that dominates in bifurcation events and exhibits a more duct-like keratin expression pattern. Simulation of TDLU branching morphogenesis in three dimensions suggests that evolutionarily conserved mechanisms regulate mammary gland branching in humans and mice despite their anatomical differences. In all, our data provide structural insight into 3D anatomy and branching of the human breast and exemplify the power of volumetric imaging in gaining a deeper understanding of breast biology.

Neuropeptides regulate embryonic salivary gland branching through the FGF/FGFR pathway in aging klotho-deficient mice.

Salivary gland branching morphogenesis is regulated by the functional integration of neuronal signaling, but the underlying mechanisms are not fully understood in aging accelerated klotho-deficient (Kl-/-) mice. Here, we investigated whether the neuropeptides substance P (SP) and neuropeptide Y (NPY) affect the branching morphogenesis of embryonic salivary glands in aging Kl-/- mice. In the salivary glands of embryonic Kl-/- mice, morphological analysis and immunostaining revealed that epithelial bud formation, neuronal cell proliferation/differentiation, and the expression of the salivary gland functional marker ZO-1 were decreased in embryonic ductal cells. Incubation with SP/NPY at E12-E13d promoted branching morphogenesis, parasympathetic innervation, and epithelial proliferation in salivary glands of embryonic Kl-/- mice. The ERK inhibitor U0126 specifically inhibited neuronal substance-induced epithelial bud formation in the embryonic salivary gland. RNA-seq profiling analysis revealed that the expression of fibroblast growth factors/fibroblast growth factors (FGFs/FGFRs) and their receptors was significantly regulated by SP/NPY treatment in the embryonic salivary gland (E15). The FGFR inhibitor BGJ389 inhibited new branching formation induced by SP and NPY treatment and ERK1/2 expression. These results showed that aging may affect virtually the development of salivary gland by neuronal dysfunction. The neuropeptides SP/NPY induced embryonic salivary gland development through FGF/FGFR/ERK1/2-mediated signaling.

Protocol for Imaging the Same Class IV Neurons at Different Stages of Development.

In this protocol, we focused on analyzing internal branches of Drosophila class IV neurons. These neurons are characterized by their highly branched axons and dendrites and intricately tile the larval body. As Drosophila larvae progress through developmental stages, the dendritic arbors of Class IV neurons undergo notable transformations. As Drosophila larvae develop, their Class IV dendritic arbors grow. In the initial 24 h after egg laying (AEL), the dendrites are smaller than segments. During the subsequent 24 h of the first instar larval stage, dendritic arbors outpace segment growth, achieving tiling. After 48 h, arbors and segments grow concurrently. Epidermal cells near Class IV dendrites expand in proportion to segment growth. This observation suggested that Class IV cells might grow via branch dilation-uniformly elongating branches, akin to Class I cells [1,2]. To understand whether the class IV complex arbor structure is formed by dilation or simply from growing tips, we developed this protocol to introduce a systematic approach for quantitatively assessing the growth dynamics of internal branches. Key features • This protocol employs imaging the same neuron over different development times • Drosophila embryo and larvae genotype is ;;ppkCD4-tdGFP, which explicitly tags class IV neurons • This protocol for the preparation of agar pads to mount and image Drosophila larvae is adapted from Monica Driscoll's method • Neurons are imaged without the use of anesthetics and for a short duration of time • This technique involves the use of a spinning disk confocal microscope.

Salivary gland developmental mechanics.

The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

Tight Junction Component Occludin Binds to FIP5 to Regulate Endosome Trafficking and Mitotic Spindle Function.

The genetic basis of vertebrate emergence during metazoan evolution has remained largely unknown. Understanding vertebrate-specific genes, such as the tight junction protein Occludin (Ocln), may help answer this question. Here, it is shown that mammary glands lacking Ocln exhibit retarded epithelial branching, owing to reduced cell proliferation and surface expansion. Interestingly, Ocln regulates mitotic spindle orientation and function, and its loss leads to a range of defects, including prolonged prophase and failed nuclear and/or cytoplasmic division. Mechanistically, Ocln binds to the RabGTPase-11 adaptor FIP5 and recruits recycling endosomes to the centrosome to participate in spindle assembly and function. FIP5 loss recapitulates Ocln null, leading to prolonged prophase, reduced cell proliferation, and retarded epithelial branching. These results identify a novel role in OCLN-mediated endosomal trafficking and potentially highlight its involvement in mediating membranous vesicle trafficking and function, which is evolutionarily conserved and essential.

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Spatially distinct epithelial and mesenchymal cell subsets along progressive lineage restriction in the branching embryonic mammary gland.

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis.

Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland.

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs.

A Decision Tree to Guide Human and Mouse Mammary Organoid Model Selection.

Over the past 50 years, researchers from the mammary gland field have launched a collection of distinctive 3D cell culture systems to study multiple aspects of mammary gland physiology and disease. As our knowledge about the mammary gland evolves, more sophisticated 3D cell culture systems are required to answer more and more complex questions. Nowadays, morphologically complex mammary organoids can be generated in distinct 3D settings, along with reproduction of multiple aspects of the gland microenvironment. Yet, each 3D culture protocol comes with its advantages and limitations, where some culture systems are best suited to study stemness potential, whereas others are tailored towards the study of mammary gland morphogenesis. Therefore, prior to starting a 3D mammary culture experiment, it is important to consider and select the ideal culture model to address the biological question of interest. The number and technical requirements of novel 3D cell culture methods vastly increased over the past decades, making it currently challenging and time consuming to identify the best experimental testing. In this chapter, we provide a summary of the most promising murine and human 3D organoid models that are currently used in mammary gland biology research. For each model, we will provide a brief description of the protocol and an overview of the expected morphological outcome, the advantages of the model, and the potential pitfalls, to guide the reader to the best model of choice for specific applications.

Branching morphogenesis of the mouse mammary gland after exposure to benzophenone-3.

Pubertal mammary branching morphogenesis is a hormone-regulated process susceptible to exposure to chemicals with endocrine disruptive capacity, such as the UV-filter benzophenone-3 (BP3). Our aim was to assess whether intrauterine or in vitro exposure to BP3 modified the branching morphogenesis of the female mouse mammary gland. For this, pregnant mice were dermally exposed to BP3 (0.15 or 50 mg/kg/day) from gestation day (GD) 8.5 to GD18.5. Sesame oil treatment served as control. Changes of the mammary glands of the offspring were studied on postnatal day 45. Further, mammary organoids from untreated mice were cultured under branching induction conditions and exposed for 9 days to BP3 (1 × 10-6 M, 1 × 10-9 M, or 1 × 10-12 M with 0.01% ethanol as control) to evaluate the branching progression. Mice that were exposed to BP3 in utero showed decreased mRNA levels of progesterone receptor (PR) and WNT4. However, estradiol and progesterone serum levels, mammary histomorphology, proliferation, and protein expression of estrogen receptor alpha (ESR1) and PR were not significantly altered. Interestingly, direct exposure to BP3 in vitro also decreased the mRNA levels of PR, RANKL, and amphiregulin without affecting the branching progression. Most effects were found after exposure to 50 mg/kg/day or 1 × 10-6 M of BP3, both related to sunscreen application in humans. In conclusion, exposure to BP3 does not impair mammary branching morphogenesis in our models. However, BP3 affects PR transcriptional expression and its downstream mediators, suggesting that exposure to BP3 might affect other developmental stages of the mammary gland.

Mechanism of branching morphogenesis inspired by diatom silica formation.

The silica-based cell walls of diatoms are prime examples of genetically controlled, species-specific mineral architectures. The physical principles underlying morphogenesis of their hierarchically structured silica patterns are not understood, yet such insight could indicate novel routes toward synthesizing functional inorganic materials. Recent advances in imaging nascent diatom silica allow rationalizing possible mechanisms of their pattern formation. Here, we combine theory and experiments on the model diatom Thalassiosira pseudonana to put forward a minimal model of branched rib patterns-a fundamental feature of the silica cell wall. We quantitatively recapitulate the time course of rib pattern morphogenesis by accounting for silica biochemistry with autocatalytic formation of diffusible silica precursors followed by conversion into solid silica. We propose that silica deposition releases an inhibitor that slows down up-stream precursor conversion, thereby implementing a self-replicating reaction-diffusion system different from a classical Turing mechanism. The proposed mechanism highlights the role of geometrical cues for guided self-organization, rationalizing the instructive role for the single initial pattern seed known as the primary silicification site. The mechanism of branching morphogenesis that we characterize here is possibly generic and may apply also in other biological systems.

Acinar-ductal cell rearrangement drives branching morphogenesis of the murine pancreas in an IGF/PI3K-dependent manner.

During organ formation, progenitor cells need to acquire different cell identities and organize themselves into distinct structural units. How these processes are coordinated and how tissue architecture(s) is preserved despite the dramatic cell rearrangements occurring in developing organs remain unclear. Here, we identified cellular rearrangements between acinar and ductal progenitors as a mechanism to drive branching morphogenesis in the pancreas while preserving the integrity of the acinar-ductal functional unit. Using ex vivo and in vivo mouse models, we found that pancreatic ductal cells form clefts by protruding and pulling on the acinar basement membrane, which leads to acini splitting. Newly formed acini remain connected to the bifurcated branches generated by ductal cell rearrangement. Insulin growth factor (IGF)/phosphatidylinositol 3-kinase (PI3K) pathway finely regulates this process by controlling pancreatic ductal tissue fluidity, with a simultaneous impact on branching and cell fate acquisition. Together, our results explain how acinar structure multiplication and branch bifurcation are synchronized during pancreas organogenesis.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study, we dissected the roles of BMP receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Mesenchymal Vangl facilitates airway elongation and widening independently of the planar cell polarity complex.

A hallmark of mammalian lungs is the fractal nature of the bronchial tree. In the adult, each successive generation of airways is a fraction of the size of the parental branch. This fractal structure is physiologically beneficial, as it minimizes the energy needed for breathing. Achieving this pattern likely requires precise control of airway length and diameter, as the branches of the embryonic airways initially lack the fractal scaling observed in those of the adult lung. In epithelial monolayers and tubes, directional growth can be regulated by the planar cell polarity (PCP) complex. Here, we comprehensively characterized the roles of PCP-complex components in airway initiation, elongation, and widening during branching morphogenesis of the murine lung. Using tissue-specific knockout mice, we surprisingly found that branching morphogenesis proceeds independently of PCP-component expression in the developing airway epithelium. Instead, we found a novel, Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme. Specifically, mesenchymal loss of Vangl1/2 leads to defects in branch initiation, elongation, and widening. At the cellular level, we observe changes in the shape of smooth muscle cells that indicate a potential defect in collective mesenchymal rearrangements, which we hypothesize are necessary for lung morphogenesis. Our data thus reveal an explicit function for Vangl that is independent of the core PCP complex, suggesting a functional diversification of PCP components in vertebrate development. These data also reveal an essential role for the embryonic mesenchyme in generating the fractal structure of airways of the mature lung.

Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development.

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

ß-Catenin in the kidney stroma modulates pathways and genes to regulate kidney development.

BACKGROUND: Kidney development is regulated by cellular interactions between the ureteric epithelium, mesenchyme, and stroma. Previous studies demonstrate essential roles for stromal ß-catenin in kidney development. However, how stromal ß-catenin regulates kidney development is not known. We hypothesize that stromal ß-catenin modulates pathways and genes that facilitate communications with neighboring cell populations to regulate kidney development. RESULTS: We isolated purified stromal cells with wild type, deficient, and overexpressed ß-catenin by fluorescence-activated cell sorting and conducted RNA Sequencing. A Gene Ontology network analysis demonstrated that stromal ß-catenin modulates key kidney developmental processes, including branching morphogenesis, nephrogenesis and vascular formation. Specific stromal ß-catenin candidate target genes that may mediate these effects included secreted, cell-surface and transcriptional factors that regulate branching morphogenesis and nephrogenesis (Wnts, Bmp, Fgfr, Tcf/Lef) and secreted vascular guidance cues (Angpt1, VEGF, Sema3a). We validated established ß-catenin targets including Lef1 and novel candidate ß-catenin targets including Sema3e which have unknown roles in kidney development. CONCLUSIONS: These studies advance our understanding of gene and biological pathway dysregulation in the context of stromal ß-catenin misexpression during kidney development. Our findings suggest that during normal kidney development, stromal ß-catenin may regulate secreted and cell-surface proteins to communicate with adjacent cell populations.

The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Prenatal triclosan exposure impairs mammalian lung branching morphogenesis through activating Bmp4 signaling.

Triclosan (TCS) is a commonly used antibacterial agent present in personal care and household products. Recently, there have been increasing concerns about the association between children's health and TCS exposure during gestation, but the toxicological effects of TCS exposure on embryonic lung development remain undetermined. In this study, through using an ex vivo lung explant culture system, we found that prenatal exposure to TCS resulted in impaired lung branching morphogenesis and altered proximal-distal airway patterning. These TCS-induced dysplasias are accompanied by significantly reduced proliferation and increased apoptosis within the developing lung, as a consequence of activated Bmp4 signaling. Inhibition of Bmp4 signaling by Noggin partially rescues the lung branching morphogenesis and cellular defects in TCS-exposed lung explants. In addition, we provided in vivo evidence that administration of TCS during gestation leads to compromised branching formation and enlarged airspace in the lung of offspring. Thus, this study provides novel toxicological information on TCS and indicated a strong/possible association between TCS exposure during pregnancy and lung dysplasia in offspring.

FGF signaling regulates salivary gland branching morphogenesis by modulating cell adhesion.

Loss of FGF signaling leads to defects in salivary gland branching, but the mechanisms underlying this phenotype remain largely unknown. We disrupted expression of Fgfr1 and Fgfr2 in salivary gland epithelial cells and found that both receptors function coordinately in regulating branching. Strikingly, branching morphogenesis in double knockouts is restored by Fgfr1 and Fgfr2 (Fgfr1/2) knock-in alleles incapable of engaging canonical RTK signaling, suggesting that additional FGF-dependent mechanisms play a role in salivary gland branching. Fgfr1/2 conditional null mutants showed defective cell-cell and cell-matrix adhesion, both of which have been shown to play instructive roles in salivary gland branching. Loss of FGF signaling led to disordered cell-basement membrane interactions in vivo as well as in organ culture. This was partially restored upon introducing Fgfr1/2 wild-type or signaling alleles that are incapable of eliciting canonical intracellular signaling. Together, our results identify non-canonical FGF signaling mechanisms that regulate branching morphogenesis through cell-adhesion processes.

FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.