Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

A local translation program regulates centriole amplification in the airway epithelium.

Biogenesis of organelles requires targeting of a subset of proteins to specific subcellular domains by signal peptides or mechanisms controlling mRNA localization and local translation. How local distribution and translation of specific mRNAs for organelle biogenesis is achieved remains elusive and likely to be dependent on the cellular context. Here we identify Trinucleotide repeat containing-6a (Tnrc6a), a component of the miRNA pathway, distinctively localized to apical granules of differentiating airway multiciliated cells (MCCs) adjacent to centrioles. In spite of being enriched in TNRC6A and the miRNA-binding protein AGO2, they lack enzymes for mRNA degradation. Instead, we found these apical granules enriched in components of the mRNA translation machinery and newly synthesized proteins suggesting that they are specific hubs for target mRNA localization and local translation in MCCs. Consistent with this, Tnrc6a loss of function prevented formation of these granules and led to a broad reduction, rather than stabilization of miRNA targets. These included downregulation of key genes involved in ciliogenesis and was associated with defective multicilia formation both in vivo and in primary airway epithelial cultures. Similar analysis of Tnrc6a disruption in yolk sac showed stabilization of miRNA targets, highlighting the potential diversity of these mechanisms across organs.

Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don't proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63CreERT2 and KRT5-CreERT2-labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63CreERT2-labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3-/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.

Airway basal cells show regionally distinct potential to undergo metaplastic differentiation.

Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

Prominin 1 and Notch regulate ciliary length and dynamics in multiciliated cells of the airway epithelium.

Differences in ciliary morphology and dynamics among multiciliated cells of the respiratory tract contribute to efficient mucociliary clearance. Nevertheless, little is known about how these phenotypic differences are established. We show that Prominin 1 (Prom1), a transmembrane protein widely used as stem cell marker, is crucial to this process. During airway differentiation, Prom1 becomes restricted to multiciliated cells, where it is expressed at distinct levels along the proximal-distal axis of the airways. Prom1 is induced by Notch in multiciliated cells, and Notch inactivation abolishes this gradient of expression. Prom1 was not required for multicilia formation, but when inactivated resulted in longer cilia that beat at a lower frequency. Disruption of Notch resulted in opposite effects and suggested that Notch fine-tunes Prom1 levels to regulate the multiciliated cell phenotype and generate diversity among these cells. This mechanism could contribute to the innate defense of the lung and help prevent pulmonary disease.

Intravitreal Bevacizumab Is Associated With Prolonged Ventilatory Support in Preterm Infants With Bronchopulmonary Dysplasia.

BACKGROUND: Intravitreal bevacizumab (IVB), an anti-vascular endothelial growth factor (VEGF) antibody, is a widely adopted treatment for retinopathy of prematurity (ROP). Although animal studies have demonstrated that IVB inhibits alveologenesis in neonatal rat lung, the clinical influence of IVB on respiratory outcomes has not been studied. RESEARCH QUESTION: Does IVB affect the respiratory outcome in preterm infants with bronchopulmonary dysplasia? STUDY DESIGN AND METHODS: We retrospectively assessed very low birth weight (VLBW) preterm infants admitted to our neonatal ICU between January 2016 and June 2021. Furthermore, we evaluated the short-term respiratory outcomes after IVB therapy in VLBW preterm infants requiring ventilatory support at 36 weeks' postmenstrual age (PMA). RESULTS: One hundred seventy-four VLBW preterm infants with bronchopulmonary dysplasia were recruited. Eighty-eight infants showed ROP onset before being ventilator free, and 78 infants received a diagnosis of the most severe ROP before being ventilator free. Among them, 32 received a diagnosis with type 1 ROP and received IVB treatment. After adjusting for gestational age, birth body weight, and baseline respiratory status, we discovered that IVB is associated significantly with prolonged ventilatory support and a lower likelihood of becoming ventilator free (hazard ratio, 0.53; P = .03). INTERPRETATION: IVB may have a short-term respiratory adverse effect in patients requiring ventilatory support at 36 weeks' PMA. Therefore, long-term follow-up for respiratory outcomes may be considered in VLBW infants who receive IVB treatment.

Maturation for regeneration.

Alveolar type 2 cells are recognized as epithelial progenitors of the lung gas-exchange region. In this issue of Cell Stem Cell, Penkala et al. (2021) provide evidence that this is not so during neonatal life, and that alveolar type I cell reprograming is a key event during regeneration post-hyperoxia injury.

E2F4's cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6.

Multiciliated cells play critical roles in the airway, reproductive organs, and brain. Generation of multiple cilia requires both activation of a specialized transcriptional program and subsequent massive amplification of centrioles within the cytoplasm. The E2F4 transcription factor is required for both roles and consequently for multiciliogenesis. Here we establish that E2F4 associates with two distinct components of the centriole replication machinery, Deup1 and SAS6, targeting nonhomologous domains in these proteins. We map Deup1 and SAS6 binding to E2F4's N-terminus and show that this domain is sufficient to mediate E2F4's cytoplasmic role in multiciliogenesis. This sequence is highly conserved across the E2F family, but the ability to bind Deup1 and SAS6 is specific to E2F4 and E2F5, consistent with their shared roles in multiciliogenesis. By generating E2F4/E2F1 chimeras, we identify a six-residue motif that is critical for Deup1 and SAS6 binding. We propose that the ability of E2F4 and E2F5 to recruit Deup1 and/or SAS6, and enable centriole replication, contributes to their cytoplasmic roles in multiciliogenesis.

Disproportionate Vitamin A Deficiency in Women of Specific Ethnicities Linked to Differences in Allele Frequencies of Vitamin A-Related Polymorphisms.

Background: While the current national prevalence rate of vitamin A deficiency (VAD) is estimated to be less than 1%, it is suggested that it varies between different ethnic groups and races within the U.S. We assessed the prevalence of VAD in pregnant women of different ethnic groups and tested these prevalence rates for associations with the vitamin A-related single nucleotide polymorphism (SNP) allele frequencies in each ethnic group. Methods: We analyzed two independent datasets of serum retinol levels with self-reported ethnicities and the differences of allele frequencies of the SNPs associated with vitamin A metabolism between groups in publicly available datasets. Results: Non-Hispanic Black and Hispanic pregnant women showed high VAD prevalence in both datasets. Interestingly, the VAD prevalence for Hispanic pregnant women significantly differed between datasets (p = 1.973 × 10-10, 95%CI 0.04-0.22). Alleles known to confer the risk of low serum retinol (rs10882272 C and rs738409 G) showed higher frequencies in the race/ethnicity groups with more VAD. Moreover, minor allele frequencies of a set of 39 previously reported SNPs associated with vitamin A metabolism were significantly different between the populations of different ancestries than those of randomly selected SNPs (p = 0.030). Conclusions: Our analysis confirmed that VAD prevalence varies between different ethnic groups/races and may be causally associated with genetic variants conferring risk for low retinol levels. Assessing genetic variant information prior to performing an effective nutrient supplementation program will help us plan more effective food-based interventions.

Prematurity alters the progenitor cell program of the upper respiratory tract of neonates.

The impact of prematurity on human development and neonatal diseases, such as bronchopulmonary dysplasia, has been widely reported. However, little is known about the effects of prematurity on the programs of stem cell self-renewal and differentiation of the upper respiratory epithelium, which is key for adaptation to neonatal life. We developed a minimally invasive methodology for isolation of neonatal basal cells from nasopharyngeal (NP) aspirates and performed functional analysis in organotypic cultures to address this issue. We show that preterm NP progenitors have a markedly distinct molecular signature of abnormal proliferation and mitochondria quality control compared to term progenitors. Preterm progenitors had lower oxygen consumption at baseline and were unable to ramp up consumption to the levels of term cells when challenged. Although they formed a mucociliary epithelium, ciliary function tended to decline in premature cells as they differentiated, compared to term cells. Together, these differences suggested increased sensitivity of preterm progenitors to environmental stressors under non-homeostatic conditions.

Airway basal stem cells generate distinct subpopulations of PNECs.

Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Hippo-Yap/Taz signaling: Complex network interactions and impact in epithelial cell behavior.

The Hippo pathway has emerged as a crucial integrator of signals in biological events from development to adulthood and in diseases. Although extensively studied in Drosophila and in cell cultures, major gaps of knowledge still remain on how this pathway functions in mammalian systems. The pathway consists of a growing number of components, including core kinases and adaptor proteins, which control the subcellular localization of the transcriptional co-activators Yap and Taz through phosphorylation of serines at key sites. When localized to the nucleus, Yap/Taz interact with TEAD transcription factors to induce transcriptional programs of proliferation, stemness, and growth. In the cytoplasm, Yap/Taz interact with multiple pathways to regulate a variety of cellular functions or are targeted for degradation. The Hippo pathway receives cues from diverse intracellular and extracellular inputs, including growth factor and integrin signaling, polarity complexes, and cell-cell junctions. This review highlights the mechanisms of regulation of Yap/Taz nucleocytoplasmic shuttling and their implications for epithelial cell behavior using the lung as an intriguing example of this paradigm. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Signaling Pathways > Cell Fate Signaling Establishment of Spatial and Temporal Patterns > Cytoplasmic Localization.

Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells.

Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.

Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus.

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.

Jagged and Delta-like ligands control distinct events during airway progenitor cell differentiation.

Notch signaling regulates cell fate selection during development in multiple organs including the lung. Previous studies on the role of Notch in the lung focused mostly on Notch pathway core components or receptor-specific functions. It is unclear, however, how Jagged or Delta-like ligands collectively or individually (Jag1, Jag2, Dll1, Dll4) influence differentiation of airway epithelial progenitors. Using mouse genetic models we show major differences in Jag and Dll in regulation and establishment of cell fate. Jag ligands had a major impact in balancing distinct cell populations in conducting airways, but had no role in the establishment of domains and cellular abundance in the neuroendocrine (NE) microenvironment. Surprisingly, Dll ligands were crucial in restricting cell fate and size of NE bodies and showed an overlapping role with Jag in differentiation of NE-associated secretory (club) cells. These mechanisms may potentially play a role in human conditions that result in aberrant NE differentiation, including NE hyperplasias and cancer.

CCN1-Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis.

Cellular communication network factor 1 (CCN1) is a dynamically expressed, matricellular protein required for vascular development and tissue repair. The CCN1 gene is a presumed target of Yes-associated protein (YAP), a transcriptional coactivator that regulates cell growth and organ size. Herein, we demonstrate that the CCN1 promoter is indeed a direct genomic target of YAP in endothelial cells (ECs) of new blood vessel sprouts and that YAP deficiency in mice downregulates CCN1 and alters cytoskeletal and mitogenic gene expression. Interestingly, CCN1 overexpression in cultured ECs inactivates YAP in a negative feedback and causes its nuclear exclusion. Accordingly, EC-specific deletion of the CCN1 gene in mice mimics a YAP gain-of-function phenotype, characterized by EC hyperproliferation and blood vessel enlargement. CCN1 brings about its effect by providing cells with a soft compliant matrix that creates YAP-repressive cytoskeletal states. Concordantly, pharmacological inhibition of cell stiffness recapitulates the CCN1 deletion vascular phenotype. Furthermore, adeno-associated virus-mediated expression of CCN1 reversed the pathology of YAP hyperactivation and the subsequent aberrant growth of blood vessels in mice with ischemic retinopathy. Our studies unravel a new paradigm of functional interaction between CCN1 and YAP and underscore the significance of their interplay in the pathogenesis of neovascular diseases.

Yap and its subcellular localization have distinct compartment-specific roles in the developing lung.

Although the Hippo-yes-associated protein (Yap) pathway has been implicated in lung development, the specific roles for Yap and its nucleocytoplasmic shuttling in the developing airway and alveolar compartments remain elusive. Moreover, conflicting results from expression studies and differences in the lung phenotypes of Yap and Hippo kinase null mutants caused controversy over the dynamics and significance of Yap subcellular localization in the developing lung. Here, we show that the aberrant morphogenesis of Yap-deficient lungs results from the disruption of developmental events specifically in distal epithelial progenitors. We also show that activation of nuclear Yap is enough to fulfill the Yap requirements to rescue abnormalities in these lungs. Remarkably, we found that Yap nucleocytoplasmic shuttling is largely dispensable in epithelial progenitors for both branching morphogenesis and sacculation. However, if maintained transcriptionally active in airways, nuclear Yap profoundly alters proximal-distal identity and halts epithelial differentiation. Taken together, these observations provide novel insights into the crucial importance of Hippo-Yap signaling in the lung prenatally.

Author Correction: Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis.

This corrects the article DOI: 10.1038/ncomms15857.