Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization

To identify pathways involved in adult lung regeneration, we employ a unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact lung. We show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce angiocrine growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. Endothelial cells trigger expansion of cocultured epithelial cells, forming three-dimensional angiospheres reminiscent of alveolar-capillary sacs. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibits production of MMP14, impairing alveolarization. MMP14 promotes expansion of epithelial progenitor cells by unmasking cryptic EGF-like ectodomains that activate the EGF receptor (EGFR). Consistent with this, neutralization of MMP14 impairs EGFR-mediated alveolar regeneration, whereas administration of EGF or intravascular transplantation of MMP14(+) PCECs into pneumonectomized Vegfr2/Fgfr1-deficient mice restores alveologenesis and lung inspiratory volume and compliance function. VEGFR2 and FGFR1 activation in PCECs therefore increases MMP14-dependent bioavailability of EGFR ligands to initiate and sustain alveologenesis.     

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.     

Testis development requires the repression of Wnt4 by Fgf signaling

The bipotential gonad expresses genes associated with both the male and female pathways. Adoption of the male testicular fate is associated with the repression of many female genes including Wnt4. However, the importance of repression of Wnt4 to the establishment of male development was not previously determined. Deletion of either Fgf9 or Fgfr2 in an XY gonad resulted in up-regulation of Wnt4 and male-to-female sex reversal. We investigated whether the deletion if Wnt4 could rescue sex reversal in Fgf9 and Fgfr2 mutants. XY Fgf9/Wnt4 and Fgfr2/Wnt4 double mutants developed testes with male somatic and germ cells present, suggesting that the primary role of Fgf signaling is the repression of female-promoting genes. Thus, the decision to adopt the male fate is based not only on whether male genes, such as Sox9, are expressed, but also on the active repression of female genes, such as Wnt4. Because loss of Wnt4 results in the up-regulation of Fgf9, we also tested the possibility that derepression of Fgf9 was responsible for the aspects of male development observed in XX Wnt4 mutants. However, we found that the relationship between these two signaling factors is not symmetric: loss of Fgf9 in XX Wnt4(-/-) gonads does not rescue their partial female-to-male sex-reversal.     

A Novel Five Gene Signature Derived from Stem-Like Side Population Cells Predicts Overall and Recurrence-Free Survival in NSCLC

Gene expression profiling has been used to characterize prognosis in various cancers. Earlier studies had shown that side population cells isolated from Non-Small Cell Lung Cancer (NSCLC) cell lines exhibit cancer stem cell properties. In this study we apply a systems biology approach to gene expression profiling data from cancer stem like cells isolated from lung cancer cell lines to identify novel gene signatures that could predict prognosis. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stem like properties. Differentially expressed genes that were over or under-expressed at least two fold commonly in all 4 cell lines were identified. We found 354 were upregulated and 126 were downregulated in SP cells compared to MP cells; of these, 89 up and 62 downregulated genes (average 2 fold changes) were used for Principle Component Analysis (PCA) and MetaCore™ pathway analysis. The pathway analysis demonstrated representation of 4 up regulated genes (TOP2A, AURKB, BRRN1, CDK1) in chromosome condensation pathway and 1 down regulated gene FUS in chromosomal translocation. Microarray data was validated using qRT-PCR on the 5 selected genes and all showed robust correlation between microarray and qRT-PCR. Further, we analyzed two independent gene expression datasets that included 360 lung adenocarcinoma patients from NCI Director''s Challenge Set for overall survival and 63 samples from Sungkyunkwan University (SKKU) for recurrence free survival. Kaplan-Meier and log-rank test analysis predicted poor survival of patients in both data sets. Our results suggest that genes involved in chromosome condensation are likely related with stem-like properties and might predict survival in lung adenocarcinoma. Our findings highlight a gene signature for effective identification of lung adenocarcinoma patients with poor prognosis and designing more aggressive therapies for such patients.     

Loss of PECAM-1 function impairs alveolarization

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.     

Chick pulmonary Wnt5a directs airway and vascular tubulogenesis

Wnt5a is an important factor patterning many aspects of early development, including the lung. We find pulmonary non-canonical Wnt5a uses Ror2 to control patterning of both distal air and vascular tubulogenesis (alveolarization). Lungs with mis/overexpressed Wnt5a develop with severe pulmonary hypoplasia associated with altered expression patterns of Shh, L-CAM, fibronectin, VEGF and Flk1. This hypoplastic phenotype is rescued by either replacement of the Shh protein or inhibition of fibronectin function. We find that the effect of Wnt5a on vascular patterning is likely to be through fibronectin-mediated VEGF signaling. These results demonstrate the pivotal role of Wnt5a in directing the essential coordinated development of pulmonary airway and vasculature, by affecting fibronectin levels directly, and by affecting the fibronectin pattern of expression through its regulation of Shh. Data herein suggest that Wnt5a functions in mid-pulmonary patterning (during alveolarization), and is distinct from the Wnt canonical pathway which is more important in earlier lung development.     

Identification of dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes

The development of epithelial appendages, including hairs, glands and teeth starts from ectodermal placodes, and is regulated by interplay of stimulatory and inhibitory signals. Ectodysplasin-A1 (Eda-A1) and Wnts are high in hierarchy of placode activators. To identify direct targets of ectodysplasin pathway, we performed microarray profiling of genes differentially regulated by short exposure to recombinant Eda-A1 in embryonic eda^−/− skin explants. Surprisingly, there were only two genes with obvious involvement in Wnt pathway: dkk4 (most highly induced gene in the screen), and lrp4. Both genes colocalized with Eda-A1 receptor Edar in placodes of ectodermal organs. They were upregulated upon Edar activation while several other Wnt associated genes previously suggested as Edar targets were unaffected. However, low dkk4 and lrp4 expression was retained in the absence of NF-κB signalling in eda^−/− hair placodes. We provide evidence that this expression was dependent on Wnt activity present prior to Eda-A1/Edar signalling. Dkk4 was recently suggested as a key Wnt antagonist regulating lateral inhibition essential for correct patterning of hair follicles. Several pieces of evidence suggest Lrp4 as a Wnt inhibitor, as well. The finding that Eda-A1 induces placode inhibitors was unexpected, and underlines the importance of delicate fine-tuning of signalling during placode formation.     

Wnt11/Fgfr1b cross-talk modulates the fate of cells in palate development

Various cellular and molecular events underlie the elevation and fusion of the developing palate that occurs during embryonic development. This includes convergent extension, where the medial edge epithelium is intercalated into the midline epithelial seam. We examined the expression patterns of Wnt11 and Fgfr1b - which are believed to be key factors in convergent extension - in mouse palate development. Wnt-11 overexpression and beads soaked in SU5402 (an Fgfr1 inhibitor) were employed in in vitro organ cultures. The results suggested that interactions between Wnt11 and Fgfr1b are important in modulating cellular events such as cell proliferation for growth and apoptosis for fusion. Moreover, the Wnt11 siRNA results showed that Wnt11-induced apoptosis was necessary for palatal fusion. In summary, Fgfr1b induces cell proliferation in the developing palate mesenchyme so that the palate grows and contacts each palatal shelf, with negative feedback of Fgfs triggered by excessive cell proliferation then inhibiting the expression of Fgfr1b and activating the expression of Wnt11 to fuse each palate by activating apoptosis.     

A model of postnatal formation of alveoli in rat lung.

In rats, and many other species, most lung alveoli are formed after birth. Septation of the large air saccules existing at birth has been considered as the main mechanism for alveoli formation. However, other undefined means of alveolarization have also been postulated to account for the large increase in gas-exchange surface area that takes place in the lung as the rat grows larger. Moreover, recent results show that the majority of alveoli in rat lung are formed by means other than septation of saccules existing at birth, but these mechanisms have not been identified up to the present. In this study, a mathematical model of alveolarization in rat lung is presented. The model is based on three postulates: (a) new saccules continue to be formed up to adulthood according to certain rules; (b) all these saccules subsequently septate generating a certain number of alveoli; (c) once formed, the saccules (and alveoli) do not change in volume, but newly-formed saccules are larger than the preceding ones according to a given law. The model accurately predicts the experimentally-known values at different ages of total alveolar volume, alveolar number, volume of the average alveolus, gas-exchange surface area, and alveolar volume distribution for normal rats and for rats in which septation is inhibited by treatment with dexamethasone or hypoxia during the early postnatal weeks of life.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa‐B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKβ, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKβ demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKβ induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKβ regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKβ regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKβ as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKβ activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation

Implantation of mammalian embryos depends on differentiation of the blastocyst to a competent state and of the uterine endometrium to a receptive state. Communication between the blastocyst and uterus ensures that these changes are temporally coordinated. Although considerable evidence indicates that the blastocyst induces expression of numerous genes in uterine tissue, potential signaling mechanisms have yet to be identified. Moreover, whereas a surge of maternal estradiol occurring on Day 4 of pregnancy in the mouse is critically required for many of the peri-implantation uterine changes, whether this surge also affects blastocyst gene expression has not been established. We show here that mouse morulae express genes encoding several members of the Wnt family of signaling molecules. Additional Wnt genes are newly expressed following development to blastocyst. Unexpectedly, Wnt5a and Wnt11 are expressed in embryos that undergo the morula-to-blastocyst transition in vivo, but only weakly or not at all in embryos that do so in vitro. Upregulation of Wnt11 is temporally coordinated with the surge of maternal estradiol on Day 4. Wnt11 fails to be upregulated in blastocysts obtained from mice ovariectomized early on Day 4 or from mice treated with the estradiol antagonist, ICI 182,780. Administration of estradiol-17beta or its metabolite, 4-OH-estradiol, to ovariectomized mice restores Wnt11 expression. Moreover, Wnt11 expression is not upregulated when blastocysts are trapped in the oviduct following ligation of the utero-tubal junction, nor when estradiol-17beta or 4-OH-estradiol are administered to blastocysts in vitro. These results establish a comprehensive profile of Wnt gene expression during late preimplantation development, demonstrate that estradiol regulates gene expression in the blastocyst via uterine factors, and identify Wnts as potential mediators of embryo-uterine communication during implantation.     

Inhibited Wnt Signaling Causes Age-Dependent Abnormalities in the Bone Matrix Mineralization in the Apert Syndrome FGFR2S252W/+ Mice

Apert syndrome (AS) is a type of autosomal dominant disease characterized by premature fusion of the cranial sutures, severe syndactyly, and other abnormalities in internal organs. Approximately 70% of AS cases are caused by a single mutation, S252W, in fibroblast growth factor receptor 2 (FGFR2). Two groups have generated FGFR2 knock-in mice Fgfr2^S252W/+ that exhibit features of AS. During the present study of AS using the Fgfr2^S252W/+ mouse model, an age-related phenotype of bone homeostasis was discovered. The long bone mass was lower in 2 month old mutant mice than in age-matched controls but higher in 5 month old mutant mice. This unusual phenotype suggested that bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to maintain bone homeostasis, might be involved. BMSCs were isolated from Fgfr2^S252W/+ mice and found that S252W mutation could impair osteogenic differentiation BMSCs but enhance mineralization of more mature osteoblasts. A microarray analysis revealed that Wnt pathway inhibitors SRFP1/2/4 were up-regulated in mutant BMSCs. This work provides evidence to show that the Wnt/β-catenin pathway is inhibited in both mutant BMSCs and osteoblasts, and differentiation defects of these cells can be ameliorated by Wnt3a treatment. The present study suggested that the bone abnormalities caused by deregulation of Wnt pathway may underlie the symptoms of AS.     

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.     

Integration of FGF and TWIST in calvarial bone and suture development

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.     

CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma

We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC) samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET) to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC) and adenocarcinoma (AC) subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP) pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.