Lung patterning: Is a distal-to-proximal gradient of cell allocation and fate decision a general paradigm?: A gradient of distal-to-proximal distribution and differentiation of tip progenitors produces distinct compartments in the lung.

Recent studies support a model in which the progeny of SOX9+ epithelial progenitors at the distal tip of lung branches undergo cell allocation and differentiation sequentially along the distal-to-proximal axis. Concomitant with the elongation and ramification of lung branches, the descendants of the distal SOX9+ progenitors are distributed proximally, express SOX2, and differentiate into cell types in the conducting airways. Amid subsequent sacculation, the distal SOX9+ progenitors generate alveolar epithelial cells to form alveoli. Sequential cell allocation and differentiation are integrated with the branching process to generate a functional branching organ. This review focuses on the roles of SOX9+ cells as precursors for new branches, as the source of various cell types in the conducting airways, and as progenitors of the alveolar epithelium. All of these processes are controlled by multiple signaling pathways. Many mouse mutants with defective lung branching contain underlying defects in one or more steps of cell allocation and differentiation of SOX9+ progenitors. This model provides a framework to understand the molecular basis of lung phenotypes and to elucidate the molecular mechanisms of lung patterning. It builds a foundation on which comparing and contrasting the mechanisms employed by different branching organs in diverse species can be made.

Wnt5a-Vangl1/2 signaling regulates the position and direction of lung branching through the cytoskeleton and focal adhesions.

Lung branching morphogenesis requires reciprocal interactions between the epithelium and mesenchyme. How the lung branches are generated at a defined location and projected toward a specific direction remains a major unresolved issue. In this study, we investigated the function of Wnt signaling in lung branching in mice. We discovered that Wnt5a in both the epithelium and the mesenchyme plays an essential role in controlling the position and direction of lung branching. The Wnt5a signal is mediated by Vangl1/2 to trigger a cascade of noncanonical or planar cell polarity (PCP) signaling. In response to noncanonical Wnt signaling, lung cells undergo cytoskeletal reorganization and change focal adhesions. Perturbed focal adhesions in lung explants are associated with defective branching. Moreover, we observed changes in the shape and orientation of the epithelial sheet and the underlying mesenchymal layer in regions of defective branching in the mutant lungs. Thus, PCP signaling helps define the position and orientation of the lung branches. We propose that mechanical force induced by noncanonical Wnt signaling mediates a coordinated alteration in the shape and orientation of a group of epithelial and mesenchymal cells. These results provide a new framework for understanding the molecular mechanisms by which a stereotypic branching pattern is generated.

Regulation of Sufu activity by p66ß and Mycbp provides new insight into vertebrate Hedgehog signaling.

Control of Gli function by Suppressor of Fused (Sufu), a major negative regulator, is a key step in mammalian Hedgehog (Hh) signaling, but how this is achieved in the nucleus is unknown. We found that Hh signaling results in reduced Sufu protein levels and Sufu dissociation from Gli proteins in the nucleus, highlighting critical functions of Sufu in the nucleus. Through a proteomic approach, we identified several Sufu-interacting proteins, including p66ß (a member of the NuRD [nucleosome remodeling and histone deacetylase] repressor complex) and Mycbp (a Myc-binding protein). p66ß negatively and Mycbp positively regulate Hh signaling in cell-based assays and zebrafish. They function downstream from the membrane receptors, Patched and Smoothened, and the primary cilium. Sufu, p66ß, Mycbp, and Gli are also detected on the promoters of Hh targets in a dynamic manner. Our results support a new model of Hh signaling in the nucleus. Sufu recruits p66ß to block Gli-mediated Hh target gene expression. Meanwhile, Mycbp forms a complex with Gli and Sufu without Hh stimulation but remains inactive. Hh pathway activation leads to dissociation of Sufu/p66ß from Gli, enabling Mycbp to promote Gli protein activity and Hh target gene expression. These studies provide novel insight into how Sufu controls Hh signaling in the nucleus.

Notch Signaling Controls Transdifferentiation of Pulmonary Neuroendocrine Cells in Response to Lung Injury.

Production of an appropriate number of distinct cell types in precise locations during embryonic development is critical for proper tissue function. Homeostatic renewal or repair of damaged tissues in adults also requires cell expansion and transdifferentiation to replenish lost cells. However, the responses of diverse cell types to tissue injury are not fully elucidated. Moreover, the molecular mechanisms underlying transdifferentiation remain poorly understood. This knowledge is essential for harnessing the regenerative potential of individual cell types. This study investigated the fate of pulmonary neuroendocrine cells (PNECs) following lung damage to understand their plasticity and potential. PNECs are proposed to carry out diverse physiological functions in the lung and can also be the cells of origin of human small cell lung cancer. We found that Notch signaling is activated in proliferating PNECs in response to epithelial injury. Forced induction of high levels of Notch signaling in PNECs in conjunction with lung injury results in extensive proliferation and transdifferentiation of PNECs toward the fate of club cells, ciliated cells and goblet cells. Conversely, inactivating Notch signaling in PNECs abolishes their ability to switch cell fate following lung insult. We also established a connection between PNEC transdifferentiation and epigenetic modification mediated by the polycomb repressive complex 2 and inflammatory responses that involve the IL6-STAT3 pathway. These studies not only reveal a major pathway that controls PNEC fate change following lung injury but also provide tools to uncover the molecular basis of cell proliferation and fate determination in response to lung injury. Stem Cells 2018;36:377-391.

Selective Ablation of Tumor Suppressors in Parafollicular C Cells Elicits Medullary Thyroid Carcinoma.

Among the four different types of thyroid cancer, treatment of medullary thyroid carcinoma poses a major challenge because of its propensity of early metastasis. To further investigate the molecular mechanisms of medullary thyroid carcinoma and discover candidates for targeted therapies, we developed a new mouse model of medullary thyroid carcinoma based on our CGRPCreER mouse line. This system enables gene manipulation in parafollicular C cells in the thyroid, the purported cells of origin of medullary thyroid carcinoma. Selective inactivation of tumor suppressors, such as p53, Rb, and Pten, in mature parafollicular C cells via an inducible Cre recombinase from CGRPCreER led to development of murine medullary thyroid carcinoma. Loss of Pten accelerated p53/Rb-induced medullary thyroid carcinoma, indicating interactions between pathways controlled by tumor suppressors. Moreover, labeling differentiated parafollicular C cells by CGRPCreER allows us to follow their fate during malignant transformation to medullary thyroid tumor. Our findings support a model in which mutational events in differentiated parafollicular C cells result in medullary thyroid carcinoma. Through expression analysis including RNA-Seq, we uncovered major signaling pathways and networks that are perturbed following the removal of tumor suppressors. Taken together, these studies not only increase our molecular understanding of medullary thyroid carcinoma but also offer new candidates for designing targeted therapies or other treatment modalities.

A conserved MST1/2-YAP axis mediates Hippo signaling during lung growth.

Hippo signaling is a critical player in controlling the growth of several tissues and organs in diverse species. The current model of Hippo signaling postulates a cascade of kinase activity initiated by the MST1/2 kinases in response to external stimuli. This leads to inactivation of the transcriptional coactivators, YAP/TAZ, due to their cytoplasmic retention and degradation that is correlated with YAP/TAZ phosphorylation. In most tissues examined, YAP plays a more dominant role than TAZ. Whether a conserved Hippo pathway is utilized during lung growth and development is unclear. In particular, the regulatory relationship between MST1/2 and YAP/TAZ in the lung remains controversial. By employing the Shh-Cre mouse line to efficiently inactivate genes in the lung epithelium, we show that loss of MST1/2 kinases in the epithelium can lead to neonatal lethality caused by lung defects. This is manifested by perturbation of lung epithelial cell proliferation and differentiation. These phenotypes are more severe than those produced by Nkx2.1-Cre, highlighting the effects of differential Cre activity on phenotypic outcomes. Importantly, expression of YAP targets is upregulated and the ratio of phospho-YAP to total YAP protein levels is reduced in Mst1/2-deficient lungs, all of which are consistent with a negative role of MST1/2 in controlling YAP function. This model gains further support from both in vivo and in vitro studies. Genetic removal of one allele of Yap or one copy of both Yap and Taz rescues neonatal lethality and lung phenotypes due to loss of Mst1/2. Moreover, knockdown of Yap in lung epithelial cell lines restores diminished alveolar marker expression caused by Mst1/2 inactivation. These results demonstrate that MST1/2 inhibit YAP/TAZ activity and establish a conserved MST1/2-YAP axis in coordinating lung growth during development.

Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis.

During mammalian spermatogenesis, the diploid spermatogonia mature into haploid spermatozoa through a highly controlled process of mitosis, meiosis and post-meiotic morphological remodeling (spermiogenesis). Despite important progress made in this area, the molecular mechanisms underpinning this transformation are poorly understood. Our analysis of the expression and function of the putative serine-threonine kinase Fused (Fu) provides critical insight into key steps in spermatogenesis. In this report, we demonstrate that conditional inactivation of Fu in male germ cells results in infertility due to diminished sperm count, abnormal head shaping, decapitation and motility defects of the sperm. Interestingly, mutant flagellar axonemes are intact but exhibit altered periaxonemal structures that affect motility. These data suggest that Fu plays a central role in shaping the sperm head and controlling the organization of the periaxonemal structures in the flagellum. We show that Fu localizes to multiple tubulin-containing or microtubule-organizing structures, including the manchette and the acrosome-acroplaxome complex that are involved in spermatid head shaping. In addition, Fu interacts with the outer dense fiber protein Odf1, a major component of the periaxonemal structures in the sperm flagellum, and Kif27, which is detected in the manchette. We propose that disrupted Fu function in these structures underlies the head and flagellar defects in Fu-deficient sperm. Since a majority of human male infertility syndromes stem from reduced sperm motility and structural defects, uncovering Fu׳s role in spermiogenesis provides new insight into the causes of sterility and the biology of reproduction.

Differential regulation of Gli proteins by Sufu in the lung affects PDGF signaling and myofibroblast development.

Mammalian Hedgehog (Hh) signaling relies on three Gli transcription factors to mediate Hh responses. This process is controlled in part by a major negative regulator, Sufu, through its effects on Gli protein level, distribution and activity. In this report, we showed that Sufu regulates Gli1 protein levels by antagonizing Numb/Itch. Otherwise, Numb/Itch would induce Gli1 protein degradation. This is in contrast to inhibition of Spop-mediated degradation of Gli2/3 by Sufu. Thus, controlling protein levels of all three Gli genes by Sufu is a conserved mechanism to modulate Hh responses albeit via distinct pathways. These findings in cell-based assays were further validated in vivo. In analyzing how Sufu controls Gli proteins in different tissues, we discovered that loss of Sufu in the lung exerts different effects on Hh target genes. Hh targets Ptch1/Hhip are upregulated in Sufu-deficient lungs, consistent with Hh pathway activation. Surprisingly, protein levels of Hh target Gli1 are reduced. We also found that myofibroblasts are absent from many prospective alveoli of Sufu-deficient lungs. Myofibroblast development is dependent on PDGF signaling. Interestingly, analysis of the Pdgfra promoter revealed a canonical Gli-binding site where Gli1 resides. These studies support a model in which loss of Sufu contributes to compromised Pdgfra activation and disrupts myofibroblast development in the lung. Our work illustrates the unappreciated complexity of Hh responses where distinct Hh targets could respond differently depending on the availability of Gli proteins that control their expression.

Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis.

Pulmonary neuroendocrine cells (PNECs) are proposed to be the first specialized cell type to appear in the lung, but their ontogeny remains obscure. Although studies of PNECs have suggested their involvement in a number of lung functions, neither their in vivo significance nor the molecular mechanisms underlying them have been elucidated. Importantly, PNECs have long been speculated to constitute the cells of origin of human small-cell lung cancer (SCLC) and recent mouse models support this hypothesis. However, a genetic system that permits tracing the early events of PNEC transformation has not been available. To address these key issues, we developed a genetic tool in mice by introducing a fusion protein of Cre recombinase and estrogen receptor (CreER) into the calcitonin gene-related peptide (CGRP) locus that encodes a major peptide in PNECs. The CGRP(CreER) mouse line has enabled us to manipulate gene activity in PNECs. Lineage tracing using this tool revealed the plasticity of PNECs. PNECs can be colabeled with alveolar cells during lung development, and following lung injury, PNECs can contribute to Clara cells and ciliated cells. Contrary to the current model, we observed that elimination of PNECs has no apparent consequence on Clara cell recovery. We also created mouse models of SCLC in which CGRP(CreER) was used to ablate multiple tumor suppressors in PNECs that were simultaneously labeled for following their fate. Our findings suggest that SCLC can originate from differentiated PNECs. Together, these studies provide unique insight into PNEC lineage and function and establish the foundation of investigating how PNECs contribute to lung homeostasis, injury/repair, and tumorigenesis.

Hedgehog signaling: From basic research to clinical applications.

Studies of the major signaling pathways have revealed a connection between development, regeneration, and cancer, highlighting common signaling networks in these processes. The Hedgehog (Hh) pathway plays a central role in the development of most tissues and organs in mammals. Hh signaling is also required for tissue homeostasis and regeneration in adults, while perturbed Hh signaling is associated with human cancers. A fundamental understanding of Hh signaling will not only enhance our knowledge of how the embryos are patterned but also provide tools to treat diseases related to aberrant Hh signaling. Studies have yielded a basic framework of Hh signaling, which establishes the foundation for addressing unresolved issues of Hh signaling. A detailed characterization of the biochemical interactions between Hh components will help explain the production of graded Hh responses required for tissue patterning. Additional cell biological and genetic studies will offer new insight into the role of Hh signaling in homeostasis and regeneration. Finally, drugs that are capable of manipulating the Hh pathway can be used to treat human diseases caused by disrupted Hh signaling. These investigations will serve as a paradigm for studying signal transduction/integration in homeostasis and disease, and for translating discovery from bench to bedside.

Fused (Stk36) is a ciliary protein required for central pair assembly and motile cilia orientation in the mammalian oviduct.

BACKGROUND: Motile cilia on the inner lining of the oviductal epithelium play a central role in ovum transport toward the uterus and subsequent fertilization by sperm. While the basic ultrastructure of 9+2 motile cilia (nine peripheral microtubule doublets surrounding a central pair) has been characterized, many important steps of ciliogenesis remain poorly understood. RESULTS: Our previous studies on mammalian Fused (Fu) (Stk36), a putative serine-threonine kinase, reveal a critical function of Fu in central pair construction and cilia orientation of motile cilia that line the tracheal and ependymal epithelia. These findings identify a novel regulatory component for these processes. In this study, we show that Fu is expressed in the multi-ciliated oviductal epithelium in several vertebrates, suggesting a conserved function of Fu in the oviduct. In support of this, analysis of Fu-deficient mouse oviducts uncovers a similar role of Fu in central pair construction and cilia orientation. We also demonstrate that Fu localizes to motile cilia and physically associates with kinesin Kif27 located at the cilium base and known central pair components Spag16 and Pcdp1. CONCLUSIONS: Our results delineate a novel pathway for central pair apparatus assembly and add important insight to the biogenesis and function of oviductal motile cilia.

The alveolus: Our current knowledge of how the gas exchange unit of the lung is constructed and repaired.

The mammalian lung completes its last step of development, alveologenesis, to generate sufficient surface area for gas exchange. In this process, multiple cell types that include alveolar epithelial cells, endothelial cells, and fibroblasts undergo coordinated cell proliferation, cell migration and/or contraction, cell shape changes, and cell-cell and cell-matrix interactions to produce the gas exchange unit: the alveolus. Full functioning of alveoli also involves immune cells and the lymphatic and autonomic nervous system. With the advent of lineage tracing, conditional gene inactivation, transcriptome analysis, live imaging, and lung organoids, our molecular understanding of alveologenesis has advanced significantly. In this review, we summarize the current knowledge of the constituents of the alveolus and the molecular pathways that control alveolar formation. We also discuss how insight into alveolar formation may inform us of alveolar repair/regeneration mechanisms following lung injury and the pathogenic processes that lead to loss of alveoli or tissue fibrosis.

mTORC1 signaling facilitates differential stem cell differentiation to shape the developing murine lung and is associated with mitochondrial capacity.

Formation of branched organs requires sequential differentiation of stem cells. In this work, we find that the conducting airways derived from SOX2+ progenitors in the murine lungs fail to form without mTOR complex 1 (mTORC1) signaling and are replaced by lung cysts. Proximal-distal patterning through transitioning of distal SOX9+ progenitors to proximal SOX2+ cells is disrupted. Mitochondria number and ATP production are reduced. Compromised mitochondrial capacity results in a similar defect as that in mTORC1-deficient lungs. This suggests that mTORC1 promotes differentiation of SOX9+ progenitors to form the conducting airways by modulating mitochondrial capacity. Surprisingly, in all mutants, saccules are produced from lung cysts at the proper developmental time despite defective branching. SOX9+ progenitors also differentiate into alveolar epithelial type I and type II cells within saccules. These findings highlight selective utilization of energy and regulatory programs during stem cell differentiation to produce distinct structures of the mammalian lungs.

A functional circuit formed by the autonomic nerves and myofibroblasts controls mammalian alveolar formation for gas exchange.

Alveolar formation increases the surface area for gas exchange. A molecular understanding of alveologenesis remains incomplete. Here, we show that the autonomic nerve and alveolar myofibroblast form a functional unit in mice. Myofibroblasts secrete neurotrophins to promote neurite extension/survival, whereas neurotransmitters released from autonomic terminals are necessary for myofibroblast proliferation and migration, a key step in alveologenesis. This establishes a functional link between autonomic innervation and alveolar formation. We also discover that planar cell polarity (PCP) signaling employs a Wnt-Fz/Ror-Vangl cascade to regulate the cytoskeleton and neurotransmitter trafficking/release from the terminals of autonomic nerves. This represents a new aspect of PCP signaling in conferring cellular properties. Together, these studies offer molecular insight into how autonomic activity controls alveolar formation. Our work also illustrates the fundamental principle of how two tissues (e.g., nerves and lungs) interact to build alveoli at the organismal level.

Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria.

Alveolar formation requires coordinated movement and interaction between alveolar epithelial cells, mesenchymal myofibroblasts, and endothelial cells/pericytes to produce secondary septa. These processes rely on the acquisition of distinct cellular properties to enable ligand secretion for cell-cell signaling and initiate morphogenesis through cellular contraction, cell migration, and cell shape change. In this study, we showed that mitochondrial activity and distribution play a key role in bestowing cellular functions on both alveolar epithelial cells and mesenchymal myofibroblasts for generating secondary septa to form alveoli in mice. These results suggest that mitochondrial function is tightly regulated to empower cellular machineries in a spatially specific manner. Indeed, such regulation via mitochondria is required for secretion of ligands, such as platelet-derived growth factor, from alveolar epithelial cells to influence myofibroblast proliferation and contraction/migration. Moreover, mitochondrial function enables myofibroblast contraction/migration during alveolar formation. Together, these findings yield novel mechanistic insights into how mitochondria regulate pivotal steps of alveologenesis. They highlight selective utilization of energy in cells and diverse energy demands in different cellular processes during development. Our work serves as a paradigm for studying how mitochondria control tissue patterning.

The lungs display an intricate, tree-shaped structure which enables the complex gas exchanges required for life. The end of each tiny 'branch' hosts delicate air sacs, or alveoli, which are further divided by internal walls called septa. In mammals, this final structure is acquired during the last stage of lung development. Then, many different types of cells in the immature alveoli multiply and reach the right location to start constructing additional septa. While the structural changes underlining alveoli maturation are well-studied, the energy requirements for that process remain poorly understood. In particular, the exact role of the mitochondria, the cellular compartments that power most life processes, is still unclear. Zhang et al. therefore set out to map, in detail, the role of mitochondria in alveolar development. Microscope imaging revealed how mitochondria were unevenly distributed within the lung cells of newborn mice. Mitochondria accumulated around the machinery that controls protein secretion in the epithelial cells that line the air sacs, and around the contractile apparatus in the underlying cells (the 'myofibroblasts'). Genetically altering the mice to reduce mitochondrial activity or perturb mitochondrial location in these two cell types produced defective alveoli with fewer septa, but it had no effect on lung development before alveoli formation. This suggests that the formation of alveoli requires more energy than other steps of lung development. Disrupting mitochondrial activity or location also compromised how epithelial cells produced chemical signals necessary for the contraction or migration of the myofibroblasts. Together, these results highlight the importance of tightly regulating mitochondrial activity and location during lung patterning. In the future, this insight could lay the groundwork to determine how energy requirements in various tissues shape other biological processes in health and disease.

A mammalian Wnt5a-Ror2-Vangl2 axis controls the cytoskeleton and confers cellular properties required for alveologenesis.

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a-Ror2-Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior.

The lungs enable the exchange of gases between inhaled air and the bloodstream. This exchange happens in structures called alveoli, which have a large surface area that aids in efficient gas exchange. Shortly after birth in mice, or during the last few months before birth in humans, alveoli develop folds called secondary septa that increase their surface area and improve the efficiency of gas exchange. Several types of cells work together to form secondary septa. Surface cells called epithelia and underlying "myofibroblast" cells and small blood vessels must both communicate and move together to build the septa. The processes that control the formation of septa have not been fully studied. In other cases, a cell signaling pathway known as the planar cell polarity (PCP) pathway has been shown to help coordinate cell movements. The PCP pathway works by changing the cytoskeleton of cells, which is the series of protein fibers that give cells their shape and structure and the ability to move. Zhang et al. have now studied septa in mouse lungs and revealed how three genes ­ Wnt5a, Ror2 and Vangl2 ­ in the PCP pathway control this process. This pathway oversees changes to the cytoskeleton in both epithelial cells and myofibroblasts, helping the cells to change shape and move together to form septa. Unusually, the PCP pathway has different effects in different cells, rather than affecting all cells similarly. This is partly due to so-called PDGF signals from the epithelial cells that help to guide the growth and movement of myofibroblasts. This process is helped by the epithelial cells changing their shape to accommodate myofibroblasts during septa formation. Further analysis also showed reduced PCP signaling in patients with chronic obstructive pulmonary disease, also known as COPD. This could be a factor in the extensive lung damage seen in these patients. These findings help to explain a key lung development process and may provide new insights to understand lung diseases such as COPD.

YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis.

Branching morphogenesis is a fundamental program for tissue patterning. We show that active YAP, a key mediator of Hippo signaling, is distributed throughout the murine lung epithelium and loss of epithelial YAP severely disrupts branching. Failure to branch is restricted to regions where YAP activity is removed. This suggests that YAP controls local epithelial cell properties. In support of this model, mechanical force production is compromised and cell proliferation is reduced in Yap mutant lungs. We propose that defective force generation and insufficient epithelial cell number underlie the branching defects. Through genomic analysis, we also uncovered a feedback control of pMLC levels, which is critical for mechanical force production, likely through the direct induction of multiple regulators by YAP. Our work provides a molecular pathway that could control epithelial cell properties required for proper morphogenetic movement and pattern formation.

Alveolar type II cells possess the capability of initiating lung tumor development.

Identifying cells of tumor origin is a fundamental question in tumor biology. Answers to this central question will not only advance our understanding of tumor initiation and progression but also have important therapeutic implications. In this study, we aimed to uncover the cells of origin of lung adenocarcinoma, a major subtype of non-small cell lung cancer. To this end, we developed new mouse models of lung adenocarcinoma that enabled selective manipulation of gene activity in surfactant associated protein C (SPC)-expressing cells, including alveolar type II cells and bronchioalveolar stem cells (BASCs) that reside at the bronchioalveolar duct junction (BADJ). Our findings showed that activation of oncogenic Kras alone or in combination with the removal of the tumor suppressor p53 in SPC⁺ cells resulted in development of alveolar tumors. Similarly, sustained EGF signaling in SPC⁺ cells led to alveolar tumors. By contrast, BASCs failed to proliferate or produce tumors under these conditions. Importantly, in a mouse strain in which Kras/p53 activity was selectively altered in type II cells but not BASCs, alveolar tumors developed while BADJs retained normal architecture. These results confirm and extend previous findings and support a model in which lung adenocarcinoma can initiate in alveolar type II cells. Our results establish the foundation for elucidating the molecular mechanisms by which lung cancer initiates and progresses in a specific lung cell type.

Evaluation of hydrate-screening methods.

The purpose of this work is to evaluate the effectiveness and reliability of several common hydrate-screening techniques, and to provide guidelines for designing hydrate-screening programs for new drug candidates. Ten hydrate-forming compounds were selected as model compounds and six hydrate-screening approaches were applied to these compounds in an effort to generate their hydrate forms. The results prove that no screening approach is universally effective in finding hydrates for small organic compounds. Rather, a combination of different methods should be used to improve screening reliability. Among the approaches tested, the dynamic water vapor sorption/desorption isotherm (DVI) method and storage under high humidity (HH) yielded 60-70% success ratios, the lowest among all techniques studied. The risk of false negatives arises in particular for nonhygroscopic compounds. On the other hand, both slurry in water (Slurry) and temperature cycling of aqueous suspension (TCS) showed high success rates (90%) with some exceptions. The mixed solvent systems (MSS) procedure also achieved high success rates (90%), and was found to be more suitable for water-insoluble compounds. For water-soluble compounds, MSS may not be the best approach because recrystallization is difficult in solutions with high water activity. Finally, vapor diffusion (VD) yielded a reasonably high success ratio in finding hydrates (80%). However, this method suffers from experimental difficulty and unreliable results for either highly water-soluble or water-insoluble compounds. This study indicates that a reliable hydrate-screening strategy should take into consideration the solubility and hygroscopicity of the compounds studied. A combination of the Slurry or TCS method with the MSS procedure could provide a screening strategy with reasonable reliability.