Transforming growth factor beta 1 is an epithelial-derived signal peptide that influences otic capsule formation.

Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work indicates that many growth factors are distributed in the early embryo in vivo in a temporal-spatial pattern that correlates with sites of ongoing morphogenetic events. We report here that the localization of transforming growth factor beta 1 (TGF-beta 1) in both epithelial and mesenchymal tissues of the mouse inner ear between 10 and 16 days of embryonic development (E10-E16). In addition, utilizing a high-density culture system as an in vitro model of otic capsule chondrogenesis, we show that modulation of chondrogenesis by TGF-beta 1 in cultured mouse periotic mesenchyme mimics the in vitro effects of otic epithelium on the expression of chondrogenic potential. We provide evidence of a causal relationship of this growth factor to otic capsule formation in situ by demonstrating that the actual sequence of chondrogenic events that occur in the developing embryo is reproduced in culture by the addition of exogenous TGF-beta 1 peptide. Furthermore, in cultures of mesenchyme containing otic epithelium, we demonstrate the localization of endogenous TGF-beta 1, first within the epithelial tissue and later within both the epithelium and its surrounding periotic mesenchyme, contrasted to an absence of endogenous TGF-beta 1 in cultures of mesenchyme alone. Our results suggest that TGF-beta 1 is one of the signal molecules that mediate the effects of otic epithelium in influencing the formation of the cartilaginous otic capsule.     

Mesodermal deletion of transforming growth factor-beta receptor II disrupts lung epithelial morphogenesis: cross-talk between TGF-beta and Sonic hedgehog pathways

In vertebrates, Sonic hedgehog (Shh) and transforming growth factor-beta (TGF-beta) signaling pathways occur in an overlapping manner in many morphogenetic processes. In vitro data indicate that the two pathways may interact. Whether such interactions occur during embryonic development remains unknown. Using embryonic lung morphogenesis as a model, we generated transgenic mice in which exon 2 of the TbetaRII gene, which encodes the type II TGF-beta receptor, was deleted via a mesodermal-specific Cre. Mesodermal-specific deletion of TbetaRII (TbetaRII(Delta/Delta)) resulted in embryonic lethality. The lungs showed abnormalities in both number and shape of cartilage in trachea and bronchi. In the lung parenchyma, where epithelial-mesenchymal interactions are critical for normal development, deletion of mesenchymal TbetaRII caused abnormalities in epithelial morphogenesis. Failure in normal epithelial branching morphogenesis in the TbetaRII(Delta/Delta) lungs caused cystic airway malformations. Interruption of the TbetaRII locus in the lung mesenchyme increased mRNA for Patched and Gli-1, two downstream targets of Shh signaling, without alterations in Shh ligand levels produced in the epithelium. Therefore, we conclude that TbetaRII-mediated signaling in the lung mesenchyme modulates transduction of Shh signaling that originates from the epithelium. To our knowledge, this is the first in vivo evidence for a reciprocal and novel mode of cross-communication between Shh and TGF-beta pathways during embryonic development.     

Androgen sensitivity and gene expression in ras + myc-induced mouse prostate carcinomas.

We established an androgen-sensitive cell line (BR31-5) from a ras + myc-induced mouse prostate carcinoma and used this cell line together with a previously reported transplantable androgen-independent mouse prostate carcinoma to investigate patterns of expression for apoptosis-related genes in an androgen-deprived environment. Single cell suspensions derived from the BR31-5 cell line were inoculated into the flank of intact or castrated adult male C57BL/6 mice and tumors were harvested 12 days post-inoculation for Northern blotting. A transplantable androgen-independent prostate cancer was also inoculated into intact or castrated mice and tumors harvested 21 days later. Tumor volume analyses showed that BR31-5 carcinomas were androgen-sensitive. Northern blotting showed that mRNA levels for two apoptosis-related genes, transforming growth factor-beta 1 and c-myc, were significantly elevated to a similar extent in carcinomas grown in castrated hosts compared to intact hosts for both the androgen-sensitive BR31-5 and androgen-independent carcinomas. Levels of mRNA for tissue type plasminogen activator, shown previously to be elevated in androgen-independent carcinomas following growth in castrates, were also increased in BR31-5 carcinomas under similar androgen-deprived conditions but to a lesser extent. Interestingly, testosterone repressed prostate mRNA No. 2 levels shown previously to be similar in both the intact and castrated groups for androgen-independent carcinomas were significantly increased in the castrated group compared to the intact group for BR31-5 carcinomas. Therefore, specific patterns of expression for apoptosis-related genes may be able to discriminate androgen-sensitive and androgen-independent prostate cancer under androgen-deprived conditions.     

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.     

Alpha v beta 6 integrin down-regulates the MMP-13 expression in oral squamous cell carcinoma cells

The integrin alphavbeta6, a receptor for fibronectin, vitronectin, tenascin and TGF-beta latency-associated peptide (LAP), is not detectable on normal oral epithelium but is neo-expressed in oral squamous cell carcinomas (OSCC) and epithelial dysplasia. Previously it has been shown that alphavbeta6 integrin can up-regulate MMP-3 and -9 expression in OSCC cells. Using beta6-transfected and control OSCC cells we demonstrate that alphavbeta6 integrin down-regulates MMP-13 expression at both mRNA and protein level. Although expressing less MMP-13, beta6-transfected cells were found to have similar collagenolytic activity as control cells and invade at similar levels through type I collagen. Growth of the tumour cells in organotypic culture and confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6 expression. Furthermore, human squamous cell carcinomas of the tongue with high expression of alphavbeta6 showed lower MMP-13 levels than carcinomas with low levels of alphavbeta6. Our results suggest that alphavbeta6 down-regulates MMP-13 expression in OSCC cells and that MMP-13 is not essential for the degradation of type I collagen by OSCC cells.     

v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis

Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis.     

Retinoic acid-induced alterations in the expression of growth factors in embryonic mouse palatal shelves

Retinoic acid (RA) is teratogenic in many species, producing multiple malformations, including cleft palate. The effects of RA which lead to cleft palate vary depending on the stage of development exposed. After exposure of embryonic mice to RA on gestation day (GD) 10, abnormally small palatal shelves form. After exposure on GD 12 shelves of normal size form, but fail to fuse, as the medial cells proliferate and differentiate into a nasal-like epithelium. Growth factors and their receptors play an important role in regulating development, and the expression of EGF receptors, EGF, TGF-alpha, TFG-beta 1, and TGF-beta 2 has been reported in the mouse embryo. In a variety of cell types in culture, these growth factors are capable of regulating proliferation, differentiation, expression of matrix proteins, and other cellular events including epithelial-mesenchymal transformations. The present study examines immunohistochemically the expression of EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 in the control embryonic palatal shelves from GD 12 to 15 and the effects of RA treatment on GD 10 or 12 on their expression on GD 14 and 16. These growth factors were shown to have specific temporal and spatial expression in the palatal shelf. With advancing development the levels of TGF-alpha decreased while the expression of EGF increased. TGF-beta 2 localization became regional by GD 14-15, with higher levels found in epithelial cells and chondrogenic mesenchyme. TGF-beta 1 occurred in epithelial and mesenchymal cells and distribution did not change substantially with advancing development. RA exposure altered the expression of TFG-alpha, TGF-beta 1, and TGF-beta 2, but significant effects on EGF were not found. The effects on TGF-alpha and TGF-beta 1 expression were dependent on the gestational age exposed. Levels of TGF-alpha on GD 14 decreased after RA exposure on GD 10, but increased after GD 12 exposure. TGF-beta 1 expression in the mesenchyme was increased after exposure on GD 12, but was unaffected by RA on GD 10. After exposure on either day, the levels of TGF-beta 2 increased in GD 14 nasal epithelial cells. Acting in concert, growth factors could regulate events critical to formation of the secondary palate, including cessation of medial epithelial cell proliferation, synthesis of extracellular matrix proteins in the mesenchyme, programmed cell death of medial epithelial peridermal cells, and transformation of basal epithelial medial cells to mesenchymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming growth factor beta 1 in liver carcinogenesis: messenger RNA expression and growth effects

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of hepatocyte proliferation. Since loss of sensitivity to growth inhibition is thought to contribute to the development of neoplasia, we analyzed the expression of TGF-beta 1 mRNA during hepatocarcinogenesis in vivo and in cultured liver epithelial cells (oval cells) obtained from carcinogen-treated animals. We found that TGF-beta 1 mRNA increases in the liver during carcinogenesis and that, at the early stages of the process, oval cells but not hepatocytes contain the growth factor mRNA. Moreover, immortalized, nontumorigenic oval cells (LE/6 cell line) continued to produce TGF-beta 1 mRNA in culture. TGF-beta 1 message markedly decreased upon cell transformation, but message levels, although generally low, were variable in various tumor cell clones. A consistent feature of the tumorigenic cell lines was a loss of sensitivity to TGF-beta 1 growth inhibition. Tumor cells could bind TGF-beta 1 with similar capacity as normal cells and had the same type of receptors (Mr 280,000, 85,000, and 65,000) capable of binding iodinated TGF-beta 1, suggesting that the loss of sensitivity to TGF-beta 1 in transformed liver epithelial cells involves postreceptor mechanisms. Further studies showed that c-myc is not a target for TGF-beta 1 in liver epithelial cells and that TGF-beta 1 no longer induces fibronectin mRNA in transformed cells. The data presented are consistent with the hypothesis that TGF-beta 1 secreted during liver carcinogenesis may inhibit the proliferation of normal cells while providing a selective advantage for the growth of cells that are "partially transformed" and are unresponsive to the factor.     

Reciprocal interactions between epithelium, mesenchyme, and epidermal growth factor (EGF) in the regulation of mandibular mitotic activity in the embryonic chick

Mandibular epithelia and mesenchyme from chick embryos of Hamburger and Hamilton (H.H.) stage 18-25 were cultured intact, in isolation, or in recombinations in the presence or absence of 5-40 ng/ml epidermal growth factor (EGF). 3H-thymidine labelling demonstrated that mesenchyme influenced epithelial mitotic activity and vice versa. EGF can substitute for the epithelial effect. The stimulation of mesenchymal proliferation by H.H. 18 and 22 epithelia correlated with high levels of epithelial proliferation. Epithelial proliferation was low at H.H. 25 and unaffected by mesenchyme or by EGF. Epithelial stimulation of mesenchymal proliferation began earlier (H.H. 18) than did mesenchymal stimulation of epithelial proliferation (H.H. 22); i.e., within the ages tested, the epithelium initiated these reciprocal mitogenic interactions. That epithelial dependence on mesenchyme coincided with epithelial bone-evoking properties, suggested a) that mesenchyme promotes or maintains epithelial bone-promoting activity and b) that the critical differentiative influence of epithelium on mesenchyme is a mitogenic one. The temporal correlation between a sharp decline in mesenchymal proliferation and termination of the osteogenic epithelial-mesenchymal interaction at H.H. 25 further supports a connection between epithelial maintenance of mesenchymal proliferation and epithelial evocation of osteogenesis.     

Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation of myc-transfected cells by epidermal growth factor.

To identify functional relationships between oncogenes and growth factors, we compared the effects of transfected myc and ras oncogenes on the responsiveness of Fischer rat 3T3 cells to three growth factors: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta). Control cells did not grow in soft agar under any conditions. ras-Transfected cells grew in soft agar under all conditions tested and were insensitive to the stimulatory effects of exogenous growth factors. These cells secreted elevated levels of both EGF-like factors and TGF-beta, suggesting that the lack of responsiveness of these cells to exogenous growth factors arose from autocrine stimulation. myc-Transfected cells displayed conditional anchorage-independent growth: they formed numerous colonies in soft agar in the presence of EGF but relatively few colonies in the presence of PDGF or TGF-beta. Secretion of EGF-like factors and TGF-beta by these cells was not elevated above that of control cells. These results suggest a model for the mechanism of cooperation between myc and ras oncogenes in which ras-like genes induce growth factor production, while myc-like genes increase the responsiveness of cells to these factors.     

Snail family members and cell survival in physiological and pathological cleft palates

Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor beta gene (TGF-beta) superfamily members, and TGF-beta(3) null mutant mice (TGF-beta(3)-/-) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-beta(3)-/-mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-beta(1), in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-beta(3) mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-beta(3), TGF-beta(1) is upregulated in the mesenchyme, and that in both physiological (avian) and pathological (TGF-beta(3)-/-mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium.     

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.     

Treatment with all-trans-retinoic acid decreases levels of endogenous TGF-beta(1) in the mesenchyme of the developing mouse inner ear

Background: Previous studies have shown that in utero exposure of the mouse embryo to high doses of all-trans-retinoic acid (atRA) produces defects of the developing inner ear and its surrounding cartilaginous capsule, while exposure of cultured periotic mesenchyme plus otic epithelium to high doses of exogenous atRA results in an inhibition of otic capsule chondrogenesis. Methods: In this study, we examine the effects of atRA exposure on the endogenous expression of transforming growth factor-beta(1) (TGF-beta(1)), a signaling molecule that mediates the epithelial-mesenchymal interactions that guide the development of the capsule of the inner ear. Results: Our results demonstrate a marked reduction in immunostaining for TGF-beta(1) in the periotic mesenchyme of atRA-exposed embryos of age E10.5 and E12 days in comparison with control specimens. Consistent with these in vivo findings, high-density cultures of E10.5 periotic mesenchyme plus otic epithelium, treated with doses of atRA that suppress chondrogenesis, showed significantly decreased levels of TGF-beta(1), as compared with TGF-beta(1) levels in untreated control cultures. Furthermore, we demonstrate a rescue of cultured periotic mesenchyme plus otic epithelium from atRA-induced chondrogenic suppression by supplementation of cultures with excess TGF-beta(1). Conclusions: Our results support the hypothesis that TGF-beta(1) plays a role in mechanisms of atRA teratogenicity during inner ear development.     

Three-dimensional epithelial and mesenchymal cell co-cultures form early tooth epithelium invagination-like structures: expression patterns of relevant molecules

Epithelium invagination is the key feature of early tooth development. In this study, we built a three-dimensional (3D) model to represent epithelium invagination-like structure by tissue engineering. Human normal oral epithelial cells (OECs) and dental pulp stem cells (DPSCs) were co-cultivated for 2-7 weeks on matrigel or collagen gel to form epithelial and mesenchymal tissues. The histological change and gene expression were analyzed by HE staining, immunostaining, and quantitative real-time RT-PCR (qRT-PCR). After 4 weeks of cultivation, OECs-formed epithelium invaginated into DPSCs-derived mesenchyme on both matrigel and collagen gel. OEC-DPSC co-cultures on matrigel showed typical invagination of epithelial cells and condensation of the underlying mesenchymal cells. Epithelial invagination-related molecules, CD44 and E-cadherin, and mesenchymal condensation involved molecules, N-cadherin and Msx1 expressed at a high level in the tissue model, suggesting the epithelial invagination is functional. However, when OECs and DPSCs were co-cultivated on collagen gel; the invaginated epithelium was transformed to several epithelial colonies inside the mesenchyme after long culture period. When DPSCs were co-cultivated with immortalized human OECs NDUSD-1, all of the above-mentioned features were not presented. Immunohistological staining and qRT-PCR analysis showed that p75, BMP2, Shh, Wnt10b, E-cadherin, N-cadherin, Msx1, and Pax9 are involved in initiating epithelium invagination and epithelial-mesenchymal interaction in the 3D OEC-DPSC co-cultures. Our results suggest that co-cultivated OECs and DPSCs on matrigel under certain conditions can build an epithelium invagination-like model. This model might be explored as a potential research tool for epithelial-mesenchymal interaction and tooth regeneration.     

Analysis of cadherin/catenin complexes in transformed thyroid epithelial cells: modulation by beta 1 integrin subunit

We have analysed the expression of cadherin/catenin complex molecules in PC C13 rat thyroid cells transformed in vitro with different oncogenes. No significant downregulation of either E-cadherin, alpha-, beta- and gamma-catenin was detected following the introduction of activated forms of myc, adenovirus E1A, ras, raf, myc + ras, E1A + raf. However, ras- and raf-transformed PC C13 cells showed altered adherens junctions. An altered distribution of cadherin/catenin complexes characterized by radially oriented membrane spikes perpendicular to cell edges was the most prominent feature evidenced by immunofluorescence. No beta1 integrin localization was observed in areas where this altered pattern of E-cadherin expression was detected. However, beta1 integrin subunit expression was detected at areas of cell-cell contact where E-cadherin showed a normal pattern of expression. Furthermore, ras- and raf-transformed PC C13 cells showed the ability to migrate in collagen gels, in contrast to their normal untransformed counterpart. Overexpression of beta1 integrin was found to restore normal E-cadherin localization at cell-cell contacts and to partially inhibit the ability to migrate in collagen gels. Finally, two cell lines obtained by ras transformation in vivo, and derived from a rat primary thyroid carcinoma (TK6) and its lung metastasis (MPTK6), were found to have lost gamma-catenin expression. TK6 lost also E-cadherin expression and membrane localization of alpha-catenin. These results suggest that: i) in vitro thyroid cell transformation is associated to a change in cadherin/catenin complexes distribution rather than to a decrease in expression; ii) in vivo transformation is associated to the loss of expression of some of these molecules likely due to tumor progression; iii) alterations in beta1 integrin subunit expression can result in changes in cadherin/catenin function thus implying that an integrin-cadherin synergy may exist in thyroid cells.     

Lipid signalling pathways in normal and ras-transfected NIH/3T3 cells

The role of ras oncogenes in cellular signalling pathways involving phospholipid breakdown was studied in untransfected and proto-H-ras and mutated H-, K- and N-ras transfected NIH/3T3 cells. When the cells were grown at low cell densities, all of the ras transfected cells had 2-4 fold higher diacylglycerol (DAG) levels compared to growing NIH/3T3 cells. At high cell densities, DAG levels decreased in the former and increased in contact inhibited NIH/3T3 cells. In this regard, only cells transformed by mutated cellular and viral H-ras oncogenes (but not by the H-ras proto-oncogene) had elevated DAG levels compared to contact inhibited NIH/3T3 cells. The basal levels of inositol phosphates in ras transfected cells were not significantly different from NIH/3T3 cells and did not vary with cell density. Thus, the elevated DAG levels are not a consequence of increased phosphoinositide hydrolysis. The latter was stimulated by serum and bombesin only in normal and proto-H-ras transfected cells. In contrast, stimulation by bradykinin was observed only in cells transformed by mutated cellular ras oncogenes. Furthermore, aluminum fluoride stimulated phosphoinositide breakdown in the latter cells indicating that there was no uncoupling of the G protein from phospholipase C. Treatment of ras transfected cells with dibutyryl cyclic AMP (DB-cAMP), which causes an inhibition of growth and a reversal of the transformed morphology, did not alter the basal levels of inositol phosphates, DB-cAMP, however, did lower DAG levels in some of the transformed cell lines, but elevated DAG levels in low density NIH/3T3 cells. These findings indicate that the ras gene product p21 is not involved in phosphoinositide hydrolysis and that DAG levels do not correlate with cell growth in either normal or ras transfected NIH/3T3 cells. Thus, p21 appears to alter cell growth through mechanism(s) independent of lipid signalling pathways.     

FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains

Morphogenesis of the lung is regulated by reciprocal signaling between epithelium and mesenchyme. In previous studies, we have shown that FGF9 signals are essential for lung mesenchyme development. Using Fgf9 loss-of-function and inducible gain-of-function mouse models, we show that lung mesenchyme can be divided into two distinct regions: the sub-mesothelial and sub-epithelial compartments, which proliferate in response to unique growth factor signals. Fibroblast growth factor (FGF) 9 signals from the mesothelium (the future pleura) to sub-mesothelial mesenchyme through both FGF receptor (FGFR) 1 and FGFR2 to induce proliferation. FGF9 also signals from the epithelium to the sub-epithelial mesenchyme to maintain SHH signaling, which regulates cell proliferation, survival and the expression of mesenchymal to epithelial signals. We further show that FGF9 represses peribronchiolar smooth muscle differentiation and stimulates vascular development in vivo. We propose a model in which FGF9 and SHH signals cooperate to regulate mesenchymal proliferation in distinct submesothelial and subepithelial regions. These data provide a molecular mechanism by which mesothelial and epithelial FGF9 directs lung development by regulating mesenchymal growth, and the pattern and expression levels of mesenchymal growth factors that signal back to the epithelium.