Lung Bud Research Articles

Foxf1 and SP-C contains a Cell-Type-Specific Transcriptional Activation Domain and is Expressed in Mouse Lungs

Lung development in the mammal carries series of steps starting from simple lung buds to the elaborately branched organ that fulfil the vital function of gaseous exchange. Strict transcriptional control is essential for lung development. To screen the genes responsible for the regional growth and patterning we used RNA Insitu hybridization to identify genes that show restricted expression patterns in the embryonic lung. During organogenesis, the expression of Foxf1 becomes restricted to the splanchnic mesoderm, which provides the mesenchymal cells of the intestinal tract and gut derivates such as lungs and liver. Surfactant is a lipoprotein substance that is synthesized and secreted by alveolar type II epithelial cells and acts to reduce surface tension at the air-alveolar interface. SP-C a 5 kDa, hydro-phobic, surfactant-associated protein. We used the technique of in situ hybridization to localize SP-C mRNA in mice lung tissue. SP-C mRNA was present in all of the epithelial cells of the prealveolar region of day 13 gestational age mice lung tissue. Our data are suggestive that SP-C is present in fetal lung prealveolar epithelial cells much earlier in gestation than are differentiated, surfactant-producing alveolar type II cells.

Development of the cardiorespiratory system in dogs from days 16 to 46 of pregnancy.

Dogs have been studied for several reasons, such as the genetic improvement, their use as experimental models, in zoonotic research, cell therapy and as a model for human diseases. However, many features relating to the embryonic development of dogs remain unknown because of the absence of embryological studies. Considering the importance of the cardiorespiratory system in the development of embryos, the aim of this study was to investigate the development of the main cardiorespiratory organs of dog embryos and foetuses with estimated gestational ages from 16 to 46days using macro- and microscopic descriptions. On day 16 of development, the neural tube and crest were formed, the anterior and posterior neuropore closure had begun and the somites had developed. Between days 22 and 27 of gestation, the lung buds and the initial formation of the primary bronchi and heart chambers were observed. The heart chambers exhibited the endo-, myo- and epicardial layers but did not have obvious differences in thickness among each other. Between days 41 and 46 of gestation, the nasal conchae and septa and trachea were formed, which exhibited characteristic epithelia. The lung formation and lobation were complete. The heart and major vessels exhibited mature histological architecture when their anatomical development was complete. The results of this study contribute to a more accurate definition of the embryonic and foetal developmental stages in dogs.

A Retinoic Acid-Hedgehog Cascade Coordinates Mesoderm-Inducing Signals and Endoderm Competence during Lung Specification

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA), and hedgehog (Hh), but how these pathways interact ina gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures, we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then itpromotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.

Congenital Lesions of the Lung

1. Clinicians can provide better care for patients with congenital lung abnormalities when they understand the developmental sequence and different types of abnormalities. 2. Knowing the appropriate antenatal and postnatal imaging strategies for congenital lung abnormalities will optimize diagnosis and guide treatment. After completing this article, readers should be able to: 1. Understand the developmental sequence of congenital lung abnormalities (CLA). 2. Distinguish the different types of CLA. 3. Identify the antenatal and postnatal imaging to optimize the diagnosis of CLA. 4. Know the treatment strategies for CLA. Congenital lung abnormalities (CLAs), also termed bronchopulmonary malformation (BPM), are increasingly identified in the perinatal setting. The use of ultrasonography with improved resolution for antenatal screening, along with supplementary fetal magnetic resonance imaging (MRI) has increased the detection of pulmonary lesions. The estimated incidence is 1 in 2,000 to 1 in 2,500 live births. A large percentage of these will remain asymptomatic in the neonatal period and later. An understanding of the pathophysiology and timing of the development of these lesions is essential in the diagnosis and treatment of these infants. CLAs are likely to represent a spectrum of developmental miscues, along with tracheobronchial obstruction, that result in the downstream formation of these lesions. (1) Lung development occurs through 5 distinct stages as follows: Embryonic (4–7 weeks): Lung buds develop off the primitive foregut. Lobar branching occurs along with vascular inflow and outflow development. Pseudoglandular (7–17 weeks): The tracheobronchial tree grows and branches, ultimately forming 20 generations of airways. Vascular ingrowth matches the tracheobronchial tree to the preacinar level and achieves a mature configuration. Arteries follow airway branching with the veins located in the adjacent connective tissue. Cannicular (16–23 weeks): Terminal branching of the bronchial tree occurs, forming respiratory bronchioles and initiating the formation of air-blood interface along with the differentiation of type 1 and 2 pneumocytes. Type …

Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.

Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD.

Thyroid bud morphogenesis requires CDC42- and SHROOM3-dependent apical constriction.

ABSTRACTEarly development of the gut endoderm and its subsequent remodeling for the formation of organ buds are accompanied by changes to epithelial cell shape and polarity. Members of the Rho-related family of small GTPases and their interacting proteins play multiple roles in regulating epithelial morphogenesis. In this study we examined the role of Cdc42 in foregut development and organ bud formation. Ablation of Cdc42 in post-gastrulation mouse embryos resulted in a loss of apical-basal cell polarity and columnar epithelial morphology in the ventral pharyngeal endoderm, in conjunction with a loss of apical localization of the known CDC42 effector protein PARD6B. Cell viability but not proliferation in the foregut endoderm was impaired. Outgrowth of the liver, lung and thyroid buds was severely curtailed in Cdc42-deficient embryos. In particular, the thyroid bud epithelium did not display the apical constriction that normally occurs concurrently with the outgrowth of the bud into the underlying mesenchyme. SHROOM3, a protein that interacts with Rho GTPases and promotes apical constriction, was strongly expressed in the thyroid bud and its sub-cellular localization was disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutant embryos, the thyroid bud epithelium showed no apical constriction, while the bud continued to grow and protruded into the foregut lumen. Our findings indicate that Cdc42 is required for epithelial polarity and organization in the endoderm and for apical constriction in the thyroid bud. It is possible that the function of CDC42 is partly mediated by SHROOM3.

Neuropilin-2 genomic elements drive cre recombinase expression in primitive blood, vascular and neuronal lineages.

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2-Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue-specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709-717, 2015. © 2015 Wiley Periodicals, Inc.

A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice.

BackgroundThe GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs.ResultsWe have generated a Gata6H2B-Venus knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution.ConclusionsExpression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6H2B-Venus/H2B-Venus homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0086-5) contains supplementary material, which is available to authorized users.

Wnt4 is essential to normal mammalian lung development

Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4−/− embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4−/− lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development.

Switching of the Laryngeal Cavity From the Respiratory Diverticulum to the Vestibular Recess: A Study Using Serial Sagittal Sections of Human Embryos and Fetuses

A cecum-like protrusion of the pharynx (the laryngeal cecum or vestibular recess [VR]) develops immediately anterior to the laryngeal part of the respiratory diverticulum. An expansion of the VR has been well described, whereas the fate of the diverticulum is still obscure, although its pharyngeal opening corresponds to the glottis. We observed sagittal sections of 10 embryos (five specimens at 5-6weeks and another five at 7-8weeks) and eight fetuses at 25-30weeks. At 5-6weeks, a lumen of the laryngeal part of the respiratory diverticulum appeared, and subsequently, the VR opened into the epithelial lamina. Because of this discrete separation, it seemed unlikely that the pharyngeal pouches contributed to the laryngeal epithelium. At 6-7weeks, the VR exhibited a high boot-shaped lumen with canalization to the diverticular lumen at the level of the cricoid cartilage. Thus, in a midline area between the bilateral arytenoid cartilages, double laryngeal lumina were evident, separated by the thick midline epithelial lamina. At 25-30weeks, the inferior part of the VR lumen had become enlarged because of the destruction of the epithelial lamina along the arytenoid and corniculate cartilages. In contrast, candidates for the initial diverticular lumen remained as epithelial slits in the anterosuperior side of the transverse arytenoid muscle. Therefore, the final anterior and lateral laryngeal walls seemed to originate from the VR with canalization, in contrast to the part of the posterior wall derived from the initial diverticular wall.

Heterochrony and early left-right asymmetry in the development of the cardiorespiratory system of snakes.

Snake lungs show a remarkable diversity of organ asymmetries. The right lung is always fully developed, while the left lung is either absent, vestigial, or well-developed (but smaller than the right). A ‘tracheal lung’ is present in some taxa. These asymmetries are reflected in the pulmonary arteries. Lung asymmetry is known to appear at early stages of development in Thamnophis radix and Natrix natrix. Unfortunately, there is no developmental data on snakes with a well-developed or absent left lung. We examine the adult and developmental morphology of the lung and pulmonary arteries in the snakes Python curtus breitensteini, Pantherophis guttata guttata, Elaphe obsoleta spiloides, Calloselasma rhodostoma and Causus rhombeatus using gross dissection, MicroCT scanning and 3D reconstruction. We find that the right and tracheal lung develop similarly in these species. By contrast, the left lung either: (1) fails to develop; (2) elongates more slowly and aborts early without (2a) or with (2b) subsequent development of faveoli; (3) or develops normally. A right pulmonary artery always develops, but the left develops only if the left lung develops. No pulmonary artery develops in relation to the tracheal lung. We conclude that heterochrony in lung bud development contributes to lung asymmetry in several snake taxa. Secondly, the development of the pulmonary arteries is asymmetric at early stages, possibly because the splanchnic plexus fails to develop when the left lung is reduced. Finally, some changes in the topography of the pulmonary arteries are consequent on ontogenetic displacement of the heart down the body. Our findings show that the left-right asymmetry in the cardiorespiratory system of snakes is expressed early in development and may become phenotypically expressed through heterochronic shifts in growth, and changes in axial relations of organs and vessels. We propose a step-wise model for reduction of the left lung during snake evolution.

Left Lung Agenesis with Bicuspid Aortic Valve

Agenesis of the lung is an extremely rare congenital anomaly representing failure of development of the primitive lung bud. Here we are presenting a case of left lung agenesis with bicuspid aortic valve in a 17 year old girl who came with the complaints of backache only. The diagnosis was made on the basis of CECT and 2D Echocardiography. DOI: http://dx.doi.org/10.3126/jnps.v34i2.8792 J Nepal Paediatr Soc 2014;34(2):147-149

An interplay of geometry and signaling enables robust lung branching morphogenesis.

Early branching events during lung development are stereotyped. Although key regulatory components have been defined, the branching mechanism remains elusive. We have now used a developmental series of 3D geometric datasets of mouse embryonic lungs as well as time-lapse movies of cultured lungs to obtain physiological geometries and displacement fields. We find that only a ligand-receptor-based Turing model in combination with a particular geometry effect that arises from the distinct expression domains of ligands and receptors successfully predicts the embryonic areas of outgrowth and supports robust branch outgrowth. The geometry effect alone does not support bifurcating outgrowth, while the Turing mechanism alone is not robust to noisy initial conditions. The negative feedback between the individual Turing modules formed by fibroblast growth factor 10 (FGF10) and sonic hedgehog (SHH) enlarges the parameter space for which the embryonic growth field is reproduced. We therefore propose that a signaling mechanism based on FGF10 and SHH directs outgrowth of the lung bud via a ligand-receptor-based Turing mechanism and a geometry effect.

Kyste bronchogénique diaphragmatique : une localisation exceptionnelle

A 64-year-old man complained of persistent dyspnea and bilateral basi-thoracic pain with shoulder irradiation. Chest computed tomography revealed a heterogeneous left diaphragmatic mass, while magnetic resonance imaging showed hypo-T1 and hyper-T2 signal. Positron-emission tomography did not show any hypermetabolism. Video-assisted thoracic surgery was decided. At inspection, tumour appeared within the posterior costal part of the diaphragmatic muscle. Tumour resection was extended to a 8-cm-long portion of the lumbar part of diaphragm. Diaphragm was repaired with non-absorbable interrupted sutures. Postoperative course was uneventful. Final pathology revealed an intra-diaphragmatic bronchogenic cyst, which is an exceptional condition. Primary diaphragmatic tumours are very rare and preoperative diagnosis cannot be affirmed. Embryologic hypotheses (migration along the oesophagus or envelopment within diaphragmatic precursors of an abnormal supernumerary lung bud) including recent molecular findings of deregulated pathways (fibroblast growth factor-10 and NOTCH) are discussed.

Bronchogenic Cyst

Introduction: Bronchogenic cysts are congenital anomalies caused by abnormal bronchial development from the primitive ventral foregut, which arises from cells isolated from the main pulmonary branching when lung bud separates from the primitive gut.Materials & Methods: We reviewed all pediatric patients with bronchogenic cyst who underwent surgery in our thoracic surgical unit- III during 2007-2009. They comprised 1 male and 5 female patients, with an age range of 2 to 17 years (mean age 5.66 years).Results: Symptoms were present in 5 patients (83%), cough was the most common symptom. Other symptoms included purulent sputum, chest pain, fever, dyspnea, anorexia and/or weight loss, and haemoptysis. All patients underwent chest x-ray posteroanterior and lateral view and CT scan of chest for diagnostic purpose. Two of the cysts were mediastinal and rests were intraparenchymal. Surgery performed were resection (2), lobectomy (4). Complications of bronchogenic cysts occurred in 4 patients (67%). In 1 patient (Patient 2), a ruptured, infected, parenchymal bronchogenic cyst caused pleuritis and empyemaThere was no operative deaths & no in-hospital mortality.Conclusions: We conclude that one should go for surgical resection of all suspected bronchogenic cysts in operable candidates.DOI: http://dx.doi.org/10.3329/jpsb.v1i2.19524

Abnormal Sonic hedgehog signaling in the lung of rats with esophageal atresia induced by adriamycin.

Abnormal lung development was recently described in the rat model of esophageal atresia and tracheoesophageal fistula (EA-TEF). Since in this condition the ventral-to-dorsal switch of Shh expression in the foregut is disturbed, the present study tested the hypothesis that this abnormal expression at the emergence of the tracheobronchial bud might be translated into the developing lung. Pregnant rats received either 1.75 mg/kg i.p. adriamycin or vehicle from E7 to E9. Three groups were studied: control and adriamycin-exposed with and without EA-TEF. Embryos were recovered and the lungs were harvested and processed for reverse transcription polymerase chain reaction and immunofluorescence analysis of the Shh signaling cascade. Shh signaling was downregulated at the late embryonic stage of lung development (E13) in embryos with EA-TEF. Throughout the subsequent stages of development, the expression of both Shh and its downstream components increased significantly and remained upregulated throughout gestation. Immunofluorescent localization was consistent with these findings. Defective Shh signaling environment in the foregut is present beyond the emergence of lung buds and probably impairs lung development. Later in gestation, lungs exhibited a remarkable ability to upregulate the Shh cascade, suggesting a compensatory response. These findings may be relevant to understand pulmonary disease suffered by children with EA-TEF.

Cardiovascular ATIP (Angiotensin receptor type 2 interacting protein) expression in mouse development

ATIPs (Angiotensin receptor type 2 [AT2] interacting proteins) are described as AT2 interacting protein variants, whereas their expression and functions during development are not known yet. Here, we provide a detailed expression pattern of ATIP variants during mouse development by visualizing Mtus1 promotor activity, Mtus1 RNA, and subsequent ATIP protein expression. ATIPs are strongly expressed in the developing cardiovascular system, including the vascular plexus of the yolk sac and the fetal vascular part of the placenta. Moreover, ATIP is expressed spatially distinct during eye and limb bud development, and in later stages in lung and nervous system. The three murine ATIP isoforms are expressed in a distinct manner, whereupon isoform 1 and 4 are correlated to cardiovascular, lung, and limb bud development and isoform 3 is restricted to brain and eye development. Interestingly, Mtus1 expression is not necessarily correlated to Agtr2 expression, suggesting novel but yet unknown functions for ATIP, independent of AT2 signaling. ATIPs seem to be mainly involved in the developmental regulation of the cardiovascular system and may act in different AT2-dependent and -independent manners. Hence, these results deliver valuable information to further elucidate the different functions of ATIPs in the processes of mammalian development.

Epithelial-mesenchymal interactions: a fundamental Developmental Biology mechanism.

Interactions between epithelium and mesenchyme are common features of early stages of morphogenesis in different organs. In this historical review article, we retrospectively analyze the most important contribution to the definition and characterization of these interactions in three different organogenetic systems, including kidney, lung and limb bud. Tubule formation in the kidney is an example of an organogenetic event which involves interaction between the ureteric epithelium and the underlying mesenchyme that, in turn, induces the branching of the ureteric epithelium. In contrast, in lung organogenesis, interactive signaling occurs between the endodermal epithelium and the mesenchyme, leading to an alveolar structure. Finally, limb bud development results from a series of epithelial-mesenchymal interactions between the mesenchymal cells of the lateral plate mesoderm and the overlying ectodermal cells.

Unilateral Hypoplasia of Lung in Association with Dilated Stomach

ABSTRACTHuman lung growth starts as a primitive lung bud in early embryonic life and its growth is determined by various factors which occur during development. Pressure effects during intrauterine life from neighboring organs can impede the growth and lead to a hypoplastic lung. These could be due to associated congenital abnormalities of diaphragm, kidney, heart, GIT, etc. Dilated stomach is a rare association with a hypoplastic lung. We describe a case of congenital unilateral pulmonary hypoplasia in association with dilated stomach in a 25-year-old man who presented with exacerbation of bronchial asthma.How to cite this articleKasilingam SK, Jindal S. Unilateral Hypoplasia of Lung in Association with Dilated Stomach. J Postgrad Med Edu Res 2014;48(1):40-42.

Heparin binding VEGF isoforms attenuate hyperoxic embryonic lung growth retardation via a FLK1-neuropilin-1-PKC dependent pathway

BackgroundPrevious work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to determine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to identify the target cell for VEGF action.MethodsTimed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were treated ± Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody or GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence studies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies of lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF.ResultsHeparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced epithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required NRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated that the VEGF-mediated anti-apoptotic effects were dependent on PKC activation.ConclusionsVegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine signal that in turn inhibits epithelial cell apoptosis.