Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a receptor-mediated mechanism.

Pulmonary neuroendocrine cells are localized predominantly at airway branchpoints. Previous work showed that gastrin-releasing peptide (GRP), a major pulmonary bombesin-like peptide, occurred in neuroendocrine cells exclusively in branching human fetal airways. We now demonstrate that GRP and GRP receptor genes are expressed in fetal mouse lung as early as embryonic day 12 (E12), when lung buds are beginning to branch. By in situ hybridization, GRP receptor transcripts were at highest levels in mesenchymal cells at cleft regions of branching airways and blood vessels. To explore the possibility that bombesin-like peptides might play a role in branching morphogenesis, E12 lung buds were cultured for 48 hr in serum-free medium. In the presence of 0.10-10 microM bombesin, branching was significantly augmented as compared with control cultures, with a peak of 94% above control values at 1 microM (P < 0.005). The bombesin receptor antagonist [Leu13- psi(CH2NH)Leu14]bombesin alone (100 nM) had no effect on baseline branching but completely abolished bombesin-induced branching. A bombesin-related peptide, [Leu8]phyllolitorin also increased branching (65% above control values at 10 nM, P < 0.005). [Leu8]Phyllolitorin also significantly augmented thymidine incorporation in cultured lung buds. Fibronectin, which is abundant at branchpoints, induces GRP gene expression in undifferentiated cell lines. These observations suggest that BLPs secreted by pulmonary neuroendocrine cells may contribute to lung branching morphogenesis. Furthermore, components of branchpoints may induce pulmonary neuroendocrine cell differentiation as part of a positive feedback loop, which could account in part for the high prevalence of these cells at branchpoints.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

Overexpression of a Kinase-deficient Transforming Growth Factor-β Type II Receptor in Mouse Mammary Stroma Results in Increased Epithelial Branching

Members of the transforming growth factor-β (TGF-β) superfamily signal through heteromeric type I and type II serine/threonine kinase receptors. Transgenic mice that overexpress a dominant-negative mutation of the TGF-β type II receptor (DNIIR) under the control of a metallothionein-derived promoter (MT-DNIIR) were used to determine the role of endogenous TGF-βs in the developing mammary gland. The expression of the dominant-negative receptor was induced with zinc and was primarily localized to the stroma underlying the ductal epithelium in the mammary glands of virgin transgenic mice from two separate mouse lines. In MT-DNIIR virgin females treated with zinc, there was an increase in lateral branching of the ductal epithelium. We tested the hypothesis that expression of the dominant-negative receptor may alter expression of genes that are expressed in the stroma and regulated by TGF-βs, potentially resulting in the increased lateral branching seen in the MT-DNIIR mammary glands. The expression of hepatocyte growth factor mRNA was increased in mammary glands from transgenic animals relative to the wild-type controls, suggesting that this factor may play a role in TGF-β-mediated regulation of lateral branching. Loss of responsiveness to TGF-βs in the mammary stroma resulted in increased branching in mammary epithelium, suggesting that TGF-βs play an important role in the stromal–epithelial interactions required for branching morphogenesis.

A role for Timeless in epithelial morphogenesis during kidney development

Central to the process of epithelial organogenesis is branching morphogenesis into tubules and ducts. In the kidney, this can be modeled by a very simple system consisting of isolated ureteric bud (UB) cells, which undergo branching morphogenesis in response to soluble factors present in the conditioned medium of a metanephric mesenchyme cell line. By employing a targeted screen to identify transcription factors involved early in the morphogenetic program leading to UB branching, we identified the mammalian ortholog of Timeless (mTim) as a potential immediate early gene (IEG) important in this process. In the embryo, mTim was found to be expressed in patterns very suggestive of a role in epithelial organogenesis with high levels of expression in the developing lung, liver, and kidney, as well as neuroepithelium. In the embryonic kidney, the expression of mTim was maximal in regions of active UB branching, and a shift from the large isoform of mTim to a smaller isoform occurred as the kidney developed. Selective down-regulation of mTim resulted in profound inhibition of embryonic kidney growth and UB morphogenesis in organ culture. A direct effect on the branching UB was supported by the observation that down-regulation of mTim in the isolated UB (cultured in the absence of mesenchyme) resulted in marked inhibition of morphogenesis, suggesting a key role for Tim in the epithelial cell morphogenetic pathway leading to the formation of branching tubules.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes

Intercellular signaling by fibroblast growth factors plays vital roles during embryogenesis. Mice deficient for fibroblast growth factor receptors (FgfRs) show abnormalities in early gastrulation and implantation, disruptions in epithelial–mesenchymal interactions, as well as profound defects in membranous and endochondrial bone formation. Activating FGFR mutations are the underlying cause of several craniosynostoses and dwarfism syndromes in humans. Here we show that a heterozygotic abrogation of FgfR2-exon 9 (IIIc) in mice causes a splicing switch, resulting in a gain-of-function mutation. The consequences are neonatal growth retardation and death, coronal synostosis, ocular proptosis, precocious sternal fusion, and abnormalities in secondary branching in several organs that undergo branching morphogenesis. This phenotype has strong parallels to some Apert's and Pfeiffer's syndrome patients.

Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development.

Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.

Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis

SMAD2 is a member of the transforming growth factor β and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior–posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis.

Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: Implications for morphogenesis and organization of encapsidated RNA

The capsid protein of hepatitis B virus, consisting of an “assembly” domain (residues 1–149) and an RNA-binding “protamine” domain (residues 150–183), assembles from dimers into icosahedral capsids of two different sizes. The C terminus of the assembly domain (residues 140–149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell. We now have defined the location of this peptide in capsids assembled in vitro by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy. The labeled protein is unimpaired in its ability to form capsids. Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers. Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds. Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds. Capsids assembled by expressing the full-length protein in Escherichia coli package bacterial RNAs in amounts equivalent to the viral pregenome. Our density map of these capsids reveals a distinct inner shell of density—the RNA. The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.