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The effects of fibroblast growth factor 10 on colonic epithelium development, using hypomorphic mice studies, are presented.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.07.001 Byline: Stijn P. De Langhe (a)(b), Gianni Carraro (a), David Warburton (a), Mohammad K. Hajihosseini (c), Saverio Bellusci (a) Keywords: FGF10; FGFR2b; Smooth muscle cells; Lung; Apert syndrome Abstract: Fibroblast growth factor (FGF) signaling has been shown to regulate lung epithelial development but its influence on mesenchymal differentiation has been poorly investigated. To study the role of mesenchymal FGF signaling in the differentiation of the mesenchyme and its impact on epithelial morphogenesis, we took advantage of Fgfr2c.sup.+/[DELTA] mice, which due to a splicing switch express Fgfr2b in mesenchymal tissues and manifest Apert syndrome-like phenotypes. Using a set of in vivo and in vitro studies, we show that an autocrine FGF10-FGFR2b signaling loop is established in the mutant lung mesenchyme, which has several consequences. It prevents the entry of the smooth muscle progenitors into the smooth muscle cell (SMC) lineage and results in reduced fibronectin and elastin deposition. Levels of Fgf10 expression are raised within the mutant mesenchyme itself. Epithelial branching as well as epithelial levels of FGF and canonical Wnt signaling is dramatically reduced. These defects result in arrested development of terminal airways and an 'emphysema like' phenotype in postnatal lungs. Our work unravels part of the complex interactions that govern normal lung development and may be pertinent to understanding the basis of respiratory defects in Apert syndrome. Author Affiliation: (a) Developmental Biology Program, Department of Surgery, Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA (b) Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB)-Ghent University, Technologiepark 927, B-9052 Ghent, Zwijnaarde, Belgium (c) School of Biological Sciences, University of East Anglia (UEA), Norwich, Norfolk, UK Article History: Received 2 March 2006; Revised 20 June 2006; Accepted 5 July 2006

The role of fibroblast growth factor 9 on lung mesenchymal and epithelial proliferation and differentiation, using mouse inactivation studies, is presented.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.022 Byline: Pierre-Marie Del Moral (a)(b), Frederic G. Sala (a), Denise Tefft (a)(b), Wei Shi (a)(b), Eli Keshet (c), Saverio Bellusci (a), David Warburton (a)(b) Keywords: Vascular endothelial growth factor-A (VEGF-A); Fetal liver kinase-1 (Flk-1); Bone morphogenetic protein-4 (BMP-4); Murine Sprouty-2 (mSpry-2); Murine Sprouty-4 (mSpry-4) Abstract: Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis. Author Affiliation: (a) Developmental Biology Program, Saban Research Institute, Children's Hospital Los Angeles, Department of Pediatric Surgery, USC Keck School of Medicine, 4650 Sunset Blvd., Los Angeles, CA 90027, USA (b) Center for Craniofacial Molecular Biology, Keck School of Medicine and School of Dentistry, University of Southern California, CA, USA (c) Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Article History: Received 20 November 2004; Revised 10 October 2005; Accepted 14 November 2005

The role of vascular endothelial growth factor-A in activating mouse lung morphogenesis is presented.

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must he tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis. Keywords: Lung; Shp2; Branching morphogenesis; Erk phosphorylation; Smooth muscle cell

Members of the Dickkopf (Dkk) family of secreted proteins are potent inhibitors of Wnt/[beta]-catenin signaling. In this study we show that Dkk1, -2, and -3 are expressed distally in the epithelium, while Kremen1, the needed co-receptor, is expressed throughout the epithelium of the developing lung. Using TOPGAL mice [DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557-4568] to monitor the Wnt pathway, we show that canonical Wnt signaling is dynamic in the developing lung and is active throughout the epithelium and in the proximal smooth muscle cells (SMC) until E12.5. However, from E13.5 onwards, TOPGAL activity is absent in the SMC and is markedly reduced in the distal epithelium coinciding with the onset of Dkk-1 expression in the distal epithelium. To determine the role of Wnt signaling in early lung development, E11.5 organ cultures were treated with recombinant DKK1. Treated lungs display impaired branching, characterized by failed cleft formation and enlarged terminal buds, and show decreased [alpha]-smooth muscle actin ([alpha]-SMA) expression as well as detects in the formation of the pulmonary vasculature. These defects coincide with a pattern of decreased fibronectin (FN) deposition. DKK1-induced morphogenetic defects can be mimicked by inhibition of FN and overcome by addition of exogenous FN, suggesting an involvement of FN in Wnt-regulated morphogenetic processes. Keywords: Lung; Dickkopf-1; Wnt signaling; Fibronectin; Branching morphogenesis; Smooth muscle; Vascular development

Tumor necrosis factor-[alpha] converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNF[alpha] , TGF[alpha], several other cytokines, as well as the receptors for TNF[alpha], and neuregulin (ErbB4). Mice with [TACE.sup.[delta]Zn/[delta]Zn] null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that [TACE.sup.[delta]Zn/[delta]Zn] null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of [TACE.sup.[delta]Zn/[delta]Zn] knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in [TACE.sup.[delta]Zn/[delta]Zn] knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling. Keywords: TACE; Heart development; Cardiomyocyte

The mammalian lung develops through branching morphogenesis which is controlled by growth factors, hormones, and extracellular matrix proteins. We have evaluated the role of EGF-receptor signaling in lung morphogenesis by analyzing the developmental phenotype of lungs in mice with an inactivated the EGF-receptor gene both in vivo and in organ culture. Neonatal EGF-receptor-deficient mice often show evidence of lung immaturity which can result in visible respiratory distress. The lungs of these mutant mice had impaired branching and deficient alveolization and septation, resulting in a 50% reduction in alveolar volume and, thus, a markedly reduced surface for gas exchange. The EGF-receptor inactivation also resulted in type II pneumocyte immaturity, which was apparent from their increased glycogen content and a reduced number of lamellar bodies. The defective branching was already evident at Day 12 of embryonic development. When explants of embryonic lungs from Day 12 embryos were cultured under defined conditions, the branching defect in EGF-receptor-deficient lungs was even more pronounced, with only half as many terminal buds as normal lungs. EGF treatment stimulated the expression of surfactant protein C and thyroid transcription factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor signaling regulates the expression of these marker genes during type II pneumocyte maturation. Taken together, our data indicate that signal transduction through the EGF receptor plays a major role in lung development and that its inactivation leads to a respiratory distress-like syndrome.

TGF-[Beta]1 is a known inhibitor of branching morphogenesis when added exogenously to mouse embryonic lungs in culture. However, the issue of whether endogenous TGF-[Beta] signaling has a function in the process of lung organogenesis is not completely resolved. We utilized immunoperturbation and antisense oligodeoxynucleotide inhibitory strategies to abrogate TGF-[Beta] type II receptor function in embryonic mouse lungs undergoing branching morphogenesis in serumless explant culture. Antisera directed against a TGF-[Beta] type II receptor N-terminal peptide that perturbs TGF-[Beta] ligand-receptor binding increased branching by 70%. Similarly, antisense TGF-[Beta] type II receptor oligodeoxynucleotides (40/[[micro]molar]) resulted in a 58% increase in branching, compared to scrambled and mismatched sequence controls, while TGF-[Beta] type II receptor mRNA and its protein expression levels were suppressed by 95 and 84%, respectively. Addition of exogenous TGF-[Beta]1 did not overcome the stimulatory effects either of TGF-[Beta] type II receptor immunoperturbation or of antisense oligodeoxynucleotide treatment on lung branching morphogenesis. Using in situ hybridization and immunohistochemistry, both TGF-[Beta] type II receptor mRNA and protein were localized to the epithelium lining the developing airways, and to the surrounding mesenchyme, indicating that TGF-[Beta] type II receptor is an important regulator of epithelial-mesenchymal interaction. Exogenous TGF-[Beta]1 decreased cyclin A mRNA levels in control embryonic lung explants, while TGF-[Beta] type II receptor antisense oligodeoxynucleotides prevented the downregulation of cyclin A mRNA expression by exogenous TGF-[Beta]1. In addition, PCNA immunostaining of the primitive bronchial epithelium was increased in the presence of TGF-[Beta] type II receptor antisense oligodeoxynucleotides either alone or together with exogenous TGF-[Beta]1, whereas TGF-[Beta]1 alone decreased PCNA staining. Thus, abrogation of TGF-[Beta] type II receptor expression prevented TGF-[Beta]1-induced epithelial cell [G.sub.1] arrest. These results demonstrate, for the first time, that abrogation of the TGF-[Beta] type II receptor stimulates embryonic lung organogenesis in culture and reverses the negative influence of endogenous TGF-[Beta] signaling upon epithelial cell cycle progression.

TTF-1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. In vitro, TTF-1 can activate the promoter of thyroid- and pulmonary-specific genes. We postulated that TTF-1 not only is essential for the activation of tissue-specific genes, but also may directly participate in epithelial cell morphogenesis. To test this postulate, we used an antisense oligonucleotide inhibitory strategy in an in vitro model of embryonic mouse lung branching morphogenesis. This strategy suppressed TTF-1 translation and inhibited lung branching morphogenesis. The resulting abnormal phenotype was characterized by hyperplastic and unorganized proliferation of epithelial cells in the airways. The mesenchymal compartment of the lung appeared to be unaffected. These results demonstrate, for the first time, that the expression of a homeoprotein transcriptional regulator is necessary for lung epithelial morphogenesis.

A 5' 15-mer antisense oligodeoxynucleotide (ODN) directed against epidermal growth factor (EGF) precursor mRNA in embryonic mouse lung in culture under chemically established, serumless settings was used to impede EGF expression and branching morphogenesis. A 73% fall in protein synthesis, a 64% fall in RNA content, a 73% fall in DNA content, a 75% decline in branching morphogenesis and a 90% impairment of EGF immunoreactive peptide synthesis were caused by EGF ODN. Antisense influences were partly reversed by supplementing antisense ODN with EGF. Pulmonary organogenesis is influenced by EGF.

Experimental evidence is rapidly emerging that the coupling of positive regulatory signals with the induction of negative feedback modulators is a mechanism of fine regulation in development. Studies in Drosophila and chick have shown that members of the SPROUTY family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. We and others have shown that fibroblast growth factor 10 is a key positive regulator of lung branching morphogenesis. Herein, we provide direct evidence that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is down-regulated in the clefts between new branches at E12.5. We found that mSprouty2 was expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression was restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 was most highly expressed in the subjacent epithelial terminal buds. FGF10 beads up-regulated the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 showed greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduced branching and decreased mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 did not change Shh and FgfR2 mRNA levels in the lungs. We investigated Sprouty2 function during lung development by two different but complementary approaches. The targeted overexpression of mSprouty2 in the peripheral lung epithelium in vivo, using the surfactant protein C promoter, resulted in a low level of branching, lung lobe edges abnormal in appearance, and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intratracheal adenovirus microinjection also resulted in a low level of branching. These results indicate for the first time that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth.

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor- [Alpha] converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-[Alpha] (TNF-[Alpha]). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a 'sheddase' during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-[Alpha] or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development. [C] 2001 Academic Press Key Words: TACE; TNF-[Alpha]; EGF; ectodomain shedding; lung branching morphogenesis; lung explant culture; SP-C; vasculogenesis.