Biophysics of morphogenesis in the vertebrate lung.

Morphogenesis is a physical process that sculpts the final functional forms of tissues and organs. Remarkably, the lungs of terrestrial vertebrates vary dramatically in form across species, despite providing the same function of transporting oxygen and carbon dioxide. These divergent forms arise from distinct physical processes through which the epithelium of the embryonic lung responds to the mechanical properties of its surrounding mesenchymal microenvironment. Here we compare the physical processes that guide folding of the lung epithelium in mammals, birds, and reptiles, and suggest a conceptual framework that reconciles how conserved molecular signaling generates divergent mechanical forces across these species.

Correlation of fetal lung area with MRI derived pulmonary volume.

BACKGROUND: Neonatal chest-Xray (CXR)s are commonly performed as a first line investigation for the evaluation of respiratory complications. Although lung area derived from CXRs correlates well with functional assessments of the neonatal lung, it is not currently utilised in clinical practice, partly due to the lack of reference ranges for CXR-derived lung area in healthy neonates. Advanced MR techniques now enable direct evaluation of both fetal pulmonary volume and area. This study therefore aims to generate reference ranges for pulmonary volume and area in uncomplicated pregnancies, evaluate the correlation between prenatal pulmonary volume and area, as well as to assess the agreement between antenatal MRI-derived and neonatal CXR-derived pulmonary area in a cohort of fetuses that delivered shortly after the antenatal MRI investigation. METHODS: Fetal MRI datasets were retrospectively analysed from uncomplicated term pregnancies and a preterm cohort that delivered within 72 h of the fetal MRI. All examinations included T2 weighted single-shot turbo spin echo images in multiple planes. In-house pipelines were applied to correct for fetal motion using deformable slice-to-volume reconstruction. An MRI-derived lung area was manually segmented from the average intensity projection (AIP) images generated. Postnatal lung area in the preterm cohort was measured from neonatal CXRs within 24 h of delivery. Pearson correlation coefficient was used to correlate MRI-derived lung volume and area. A two-way absolute agreement was performed between the MRI-derived AIP lung area and CXR-derived lung area. RESULTS: Datasets from 180 controls and 10 preterm fetuses were suitable for analysis. Mean gestational age at MRI was 28.6 ± 4.2 weeks for controls and 28.7 ± 2.7 weeks for preterm neonates. MRI-derived lung area correlated strongly with lung volumes (p < 0.001). MRI-derived lung area had good agreement with the neonatal CXR-derived lung area in the preterm cohort [both lungs = 0.982]. CONCLUSION: MRI-derived pulmonary area correlates well with absolute pulmonary volume and there is good correlation between MRI-derived pulmonary area and postnatal CXR-derived lung area when delivery occurs within a few days of the MRI examination. This may indicate that fetal MRI derived lung area may prove to be useful reference ranges for pulmonary areas derived from CXRs obtained in the perinatal period.

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 using genetic mouse models, we dissected the roles of bone morphogenetic protein (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.

Congenital disorders are medical conditions that are present from birth. Although many congenital disorders are rare, they can have a severe impact on the quality of life of those affected. For example, congenital pulmonary airway malformation (CPAM) is a rare congenital disorder that occurs in around 1 out of every 25,000 pregnancies. In CPAM, abnormal, fluid-filled sac-like pockets of tissue, known as cysts, form within the lungs of unborn babies. After birth, these cysts become air-filled and do not behave like normal lung tissue and stop a baby's lungs from working properly. In severe cases, babies with CPAM need surgery immediately after birth. We still do not understand exactly what the underlying causes of CPAM might be. CPAM is not considered to be hereditary ­ that is, it does not appear to be passed down in families ­ nor is it obviously linked to any environmental factors. CPAM is also very difficult to study, because researchers cannot access tissue samples during the critical early stages of the disease. To overcome these difficulties, Luo et al. wanted to find a way to study CPAM in the laboratory. First, they developed a non-human animal 'model' that naturally forms CPAM-like lung cysts, using genetically modified mice where the gene for the signaling molecule Bmpr1a had been deleted in lung cells. Normally, Bmpr1a is part of a set of the molecular instructions, collectively termed BMP signaling, which guide healthy lung development early in life. However, mouse embryos lacking Bmpr1a developed abnormal lung cysts that were similar to those found in CPAM patients, suggesting that problems with BMP signalling might also trigger CPAM in humans. Luo et al. also identified several other genes in the Bmpr1a-deficient mouse lungs that had abnormal patterns of activity. All these genes were known to be controlled by BMP signaling, and to play a role in the development and organisation of lung tissue. This suggests that when these genes are not controlled properly, they could drive formation of CPAM cysts when BMP signaling is compromised. This work is a significant advance in the tools available to study CPAM. Luo et al.'s results also shed new light on the molecular mechanisms underpinning this rare disorder. In the future, Luo et al. hope this knowledge will help us develop better treatments for CPAM, or even help to prevent it altogether.

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

The people behind the papers - Yongjun Yin and David Ornitz.

During alveologenesis, multiple mesenchymal cell types play crucial roles in maximising the lung surface area. In their study, David Ornitz and colleagues define the repertoire of lung fibroblasts, with a particular focus on alveolar myofibroblasts. To know more about their work, we spoke to the first author, Yongjun Yin, and the corresponding author, David Ornitz, Alumni Endowed Professor at the Department of Developmental Biology, Washington University School of Medicine, St. Louis.

Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFBs) and a stable but poorly described population of lipid-rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFBs). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single-cell RNA sequencing and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a MyoFB differentiation program that is distinct from other mesenchymal cell types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

Vangl-dependent mesenchymal thinning shapes the distal lung during murine sacculation.

The planar cell polarity (PCP) complex is speculated to function in murine lung development, where branching morphogenesis generates an epithelial tree whose distal tips expand dramatically during sacculation. Here, we show that PCP is dispensable in the airway epithelium for sacculation. Rather, we find a Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme: loss of Vangl1/2 inhibits mesenchymal thinning and expansion of the saccular epithelium. Further, loss of mesenchymal Wnt5a mimics sacculation defects observed in Vangl2-mutant lungs, implicating mesenchymal Wnt5a/Vangl signaling as a key regulator of late lung morphogenesis. A computational model predicts that sacculation requires a fluid mesenchymal compartment. Lineage-tracing and cell-shape analyses are consistent with the mesenchyme acting as a fluid tissue, suggesting that loss of Vangl1/2 impacts the ability of mesenchymal cells to exchange neighbors. Our data thus identify an explicit function for Vangl and the pulmonary mesenchyme in actively shaping the saccular epithelium.

Bias in the prenatal lung measurements in fetal congenital diaphragmatic hernia with intrauterine growth restriction.

OBJECTIVES: The failure of a fetus to develop to its full potential due to maternal or placental factors is known as intrauterine growth restriction (IUGR). Fetal head growth is usually preserved in that situation producing a potential discordance between head and body size. Our goal is to discover if IUGR has an impact on the prenatal ultrasound measurements taken to assess pulmonary development in congenital diaphragmatic hernia (CDH). METHODS: A retrospective chart review (IRB#2017-6361) was performed on all prenatally diagnosed CDH patients from 2007 to 2016. Patient demographics, fetal and neonatal anthropometric measurements, and fetal lung parameters were the main subjects of the data that were gathered. Fetal growth was assessed by the curves based on US data by Olsen et al. and by Peleg et al. Of 147 CDH patients, 19 (12.9 %) patients were diagnosed with IUGR before the 30th gestational week while there were 20 (13.6 %) patients after the 30th gestational week. RESULTS: Patients with IUGR and the observed-to-expected lung-to-head ratio (O/E LHR) less than 25 % had better survival rates both to discharge and date compared to non IUGR group (p=0.226, OR 2.25 95 % CI 0.60-1.08 and p=0.175, OR 2.40 95 % CI 0.66-1.17, respectively). Moreover, the ECMO need of the patients who had IUGR and O/E LHR less than 25 % was significantly less than the patients without IUGR (38.5 vs. 80.0 %, p=0.005). CONCLUSIONS: This study confirms that the intrauterine measurements to predict pulmonary hypoplasia in CDH patients are misleading in the presence of IUGR and cause an overestimation.

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

Assessment of mediastinal shift angles in congenital pulmonary airway malformation: a new fetal magnetic resonance imaging indicator of congenital lung disease.

BACKGROUND: The mediastinal shift angle is a new fetal magnetic resonance imaging (MRI) index that is reportedly correlated with postnatal survival in fetuses with congenital diaphragmatic hernia. However, its correlation in patients with congenital pulmonary airway malformation (CPAM) has not been assessed. OBJECTIVE: This study aimed to establish a normal range for the right/left mediastinal shift angles, to evaluate the mediastinal shift angle in fetuses with CPAM, to compare the mediastinal shift angle with the CPAM volume ratio, and to evaluate the predictive value of the mediastinal shift angle measurements. MATERIALS AND METHODS: To establish the normal range, we measured the mediastinal shift angle bilaterally in 124 fetuses without any lung abnormality (the control group). Subsequently, the mediastinal shift angle was measured in 32 fetuses pathologically diagnosed with CPAM. Moreover, the mediastinal shift angle and CPAM volume ratio were compared using fetal MRI. RESULTS: The mean values for the right/left mediastinal shift angles were 18.6°/26.3° and 39.2°/35.9° for control fetuses and fetuses with CPAM, respectively. The mediastinal shift angle and the CPAM volume ratio showed a positive statistical correlation. The area under the curve demonstrated high discriminatory accuracy for the mediastinal shift angle (0.76). CONCLUSION: The mediastinal shift angle has potential to replace the CPAM volume ratio for evaluating the severity of CPAM in fetal MRI.

Leptin deficiency, a potential mechanism for impaired fetal lung development in uteroplacental insufficiency?

Uteroplacental insufficiency (UPI) is a major cause of fetal growth restriction (FGR). Leptin, an adipokine, has been shown to play a vital role in fetal organogenesis. There is evidence reporting leptin deficiency in preterm and growth-restricted fetuses. In this issue of Pediatric Research, Yuliana et al. report leptin expression and lung development in UPI-induced FGR rats. UPI-induced FGR rats expressed decreased lung leptin and had impaired lung development, as shown by decreased surface area and lung volume. They also found a significant association between lung radial alveolar count, serum leptin, von Willebrand factor, and specific metabolites on metabolomic analyses. Previous studies on leptin supplementation in vivo have been associated with improvement in lung maturation; supporting the evidence, that leptin improves lung growth and development in FGR and may have future therapeutic potential in the improvement of respiratory outcomes in these infants. Future studies to support evidence of this association in humans are warranted.

Bronchial tree of the human embryo: Examination based on a mammalian model.

The symmetry of the right and left bronchi, proposed in a previous comparative anatomical study as the basic model of the mammalian bronchial tree, was examined to determine if it applied to the embryonic human bronchial tree. Imaging data of 41 human embryo specimens at Carnegie stages (CS) 16-23 (equivalent to 6-8 weeks after fertilization) belonging to the Kyoto collection were obtained using phase-contrast X-ray computed tomography. Three-dimensional bronchial trees were then reconstructed from these images. Bronchi branching from both main bronchi were labeled as dorsal, ventral, medial, or lateral systems based on the branching position with numbering starting cranially. The length from the tracheal bifurcation to the branching point of the labeled bronchus was measured, and the right-to-left ratio of the same labeled bronchus in both lungs was calculated. In both lungs, the human embryonic bronchial tree showed symmetry with an alternating pattern of dorsal and lateral systems up to segmental bronchus B9 as the basic shape, with a more peripheral variation. This pattern is similar to that described in adult human lungs. Bronchial length increased with the CS in all labeled bronchi, whereas the right-to-left ratio was constant at approximately 1.0. The data demonstrated that the prototype of the human adult bronchial branching structure is formed and maintained in the embryonic stage. The morphology and branching position of all lobar bronchi and B6, B8, B9, and the subsegmental bronchus of B10 may be genetically determined. On the other hand, no common structures between individual embryos were found in the peripheral branches after the subsegmental bronchus of B10, suggesting that branch formation in this region is influenced more by environmental factors than by genetic factors.

Distribution of calcitonin gene-related peptide and vascular endothelial growth factor in the mouse lung at the embryonic and postnatal stages / Distribución del péptido relacionado con el gen de la calcitonina y el factor de crecimiento endotelial vascular en el pulmón de ratón en las etapas embrionaria y posnatal

SUMMARY: Neuropeptide calcitonin gene-related peptide (CGRP) is a neurotransmitter related to vasculogenesis during organ development. The vascular endothelial growth factor A (VEGF-A) is also required for vascular patterning during lung morphogenesis. CGRP is primarily found in organs and initially appears in pulmonary neuroendocrine cells during the early embryonic stage of lung development. However, the relationship between CGRP and VEGF-A during lung formation remains unclear. This study investigates CGRP and VEGF-A mRNA expressions in the embryonic, pseudoglandular, canalicular, saccular, and alveolar stages of lung development from embryonic day 12.5 (E12.5) to postnatal day 5 (P5) through quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Further, we analyzed the expression of CGRP via immunohistochemistry. The VEGF-A mRNA was mainly scattered across the whole lung body from E12.5. CGRP was found to be expressed in a few epithelial cells of the canalicular and the respiratory bronchiole of the lung from E12.5 to P5. An antisense probe for CGRP mRNA was strongly detected in the lung from E14.5 to E17.5. Endogenous CGRP may regulate the development of the embryonic alveoli from E14.5 to E17.5 in a temporal manner.

El péptido relacionado con el gen de la calcitonina (CGRP) es un neurotransmisor vinculado con la vasculogénesis durante el desarrollo de órganos. El factor de crecimiento endotelial vascular A (VEGF-A) también se requiere para el patrón vascular durante la morfogénesis pulmonar. El CGRP se encuentra principalmente en los órganos y aparece inicialmente en las células neuroendocrinas pulmonares durante la etapa embrionaria temprana del desarrollo pulmonar. Sin embargo, la relación entre CGRP y VEGF-A durante la formación de los pulmones sigue sin estar clara. Este estudio investiga las expresiones de ARNm de CGRP y VEGF-A en las etapas embrionaria, pseudoglandular, canalicular, sacular y alveolar del desarrollo pulmonar desde el día embrionario 12,5 (E12,5) hasta el día postnatal 5 (P5) a través de la reacción en cadena de la polimerasa cuantitativa en tiempo real. (qRT-PCR) e hibridación in situ. Además, analizamos la expresión de CGRP mediante inmunohistoquímica. El ARNm de VEGF-A se dispersó principalmente por todo parénquima pulmonar desde E12,5. Se encontró que CGRP se expresaba en unas pocas células epiteliales de los bronquiolos canaliculares y respiratorios del pulmón desde E12,5 a P5. Se detectó fuertemente una sonda antisentido para ARNm de CGRP en el pulmón de E14,5 a E17,5. El CGRP endógeno puede regular el desarrollo de los alvéolos embrionarios de E14,5 a E17,5 de manera temporal.

Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

Evaluation of the cytotoxic effect of titanium dioxide nanoparticles in human embryonic lung cells.

Background/aim: Titanium dioxide nanoparticles are widely used in a variety of products, including sunscreens, paints, and ceramics. However, their increasing use has raised concerns about their potential health risks. Titanium dioxide nanoparticles have been shown to have the ability to enter the bloodstream and accumulate in various tissues, reaching the fetus via the placenta. The aim of this study was to investigate the cytotoxic effects of titanium dioxide nanoparticles on a human embryonic lung cell line (HEL 299/An1) and the formation of oxidative DNA damage. Materials and methods: The cytotoxic effects of brookite-based titanium dioxide nanoparticles (<100 nm) were assessed using the 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay for 24 and 48 h. Cell titanium levels were determined using inductively coupled plasma mass spectrometry. Oxidative DNA damage was assessed by measuring the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) as a biomarker. Results: Titanium dioxide nanoparticles caused dose-dependent cytotoxicity in HEL 299/An1 cells. The IC50 values were 25.93 µM and 0.054 µM after 24 h and 48 h of exposure, respectively. Cell titanium levels were found to be 25,967 ppb after 24 h and 210,353 ppb after 48 h (p < 0.01). 8-OHdG was detected at 32.96 ng/mL after 24 h of exposure and 17.89 ng/mL after 48 h of exposure. Conclusion: In our study, it was shown that titanium nanoparticles caused dose-dependent cytotoxicity and oxidative DNA damage in human embryonic lung cells. The nanoparticles also accumulated in cells and were taken up in higher amounts after 48 h of exposure. These findings suggest that titanium dioxide nanoparticles may pose a health risk, especially for pregnant women who may not be aware of their pregnancy. Therefore, it is important to take preventive measures to reduce exposure to these nanoparticles.

FGFR2b signalling restricts lineage-flexible alveolar progenitors during mouse lung development and converges in mature alveolar type 2 cells.

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and AT2) progenitors during embryonic lung development are poorly defined. Current models of distal epithelial lineage formation fail to capture the heterogeneity and dynamic contribution of progenitor pools present during early development. Furthermore, few studies explore the pathways involved in alveolar progenitor specification and fate. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (FGFR2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular stage lung development. Our results suggest that a significant proportion of AT2 and AT1 progenitors are lineage-flexible during late pseudoglandular stage development, and that lineage commitment is regulated in part by FGFR2b signalling. We have characterized a set of direct FGFR2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes converge on a subpopulation of AT2 cells later in development and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of pneumocytes by elucidating the role of FGFR2b signalling in these cells during early airway epithelial lineage formation, as well as during repair after injury.

SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling.

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

Adaptation of the Oxygen Sensing System during Lung Development.

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia.

Transmural pressure signals through retinoic acid to regulate lung branching.

During development, the mammalian lung undergoes several rounds of branching, the rate of which is tuned by the relative pressure of the fluid within the lumen of the lung. We carried out bioinformatics analysis of RNA-sequencing of embryonic mouse lungs cultured under physiologic or sub-physiologic transmural pressure and identified transcription factor-binding motifs near genes whose expression changes in response to pressure. Surprisingly, we found retinoic acid (RA) receptor binding sites significantly overrepresented in the promoters and enhancers of pressure-responsive genes. Consistently, increasing transmural pressure activates RA signaling, and pharmacologically inhibiting RA signaling decreases airway epithelial branching and smooth muscle wrapping. We found that pressure activates RA signaling through the mechanosensor Yap. A computational model predicts that mechanical signaling through Yap and RA affects lung branching by altering the balance between epithelial proliferation and smooth muscle wrapping, which we test experimentally. Our results reveal that transmural pressure signals through RA to balance the relative rates of epithelial growth and smooth muscle differentiation in the developing mouse lung and identify RA as a previously unreported component in the mechanotransduction machinery of embryonic tissues.