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PubMed1997

The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae.

Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved kinase cascades that are required for the response of eukaryotic cells to a wide variety of environmental stimuli. MAP kinase pathways are also required for the execution of developmental and differentiative programs in a variety of cell and tissue types. SMK1 encodes a developmentally regulated MAP kinase in yeast that is required for spore wall morphogenesis. Cyclin-dependent kinase-activating kinases (CAKs) phosphorylate a conserved threonine residue in the activating loop of cyclin-dependent kinases. CAK1 encodes the major CAK activity in yeast and is required for cell cycle progression. The work presented here demonstrates that CAK1 functions positively in the spore wall morphogenesis pathway. First, CAK1 has been isolated as a dosage suppressor of a conditional smk1 mutant that is defective for spore wall morphogenesis. Second, CAK1 mRNA accumulates during spore development contemporaneously with SMK1 mRNA. Third, cak1 mutant strains have been isolated that are able to complete meiosis I and II but are specifically defective in assembly of the spore wall. These results show that cell cycle progression and morphogenetic pathways can be regulated by a single gene product and suggest mechanisms for coordinating these processes during development.

Alleles↗
PubMed1997

Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi.

We identified a gene of the fungal pathogen Candida albicans, designated EFG1, whose high-level expression stimulates pseudohyphal morphogenesis in the yeast Saccharomyces cerevisiae. In a central region the deduced Efg1 protein is highly homologous to the StuA and Phd1/Sok2 proteins that regulate morphogenesis of Aspergillus nidulans and S. cerevisiae, respectively. The core of the conserved region is homologous to the basic helix-loop-helix (bHLH) motif of eukaryotic transcription factors, specifically to the human Myc and Max proteins. Fungal-specific residues in the bHLH domain include the substitution of an invariant glutamate, responsible for target (E-box) specificity, by a threonine residue. During hyphal induction EFG1 transcript levels decline to low levels; downregulation is effected at the level of transcriptional initiation as shown by a EFG1 promoter-LAC4 fusion. A strain carrying one disrupted EFG1 allele and one EFG1 allele under the control of the glucose-repressible PCK1 promoter forms rod-like, pseudohyphal cells, but is unable to form true hyphae on glucose-containing media. Overexpression of EFG1 in C. albicans leads to enhanced filamentous growth in the form of extended pseudohyphae in liquid and on solid media. The results suggest that Efg1p has a dual role as a transcriptional activator and repressor, whose balanced activity is essential for yeast, pseudohyphal and hyphal morphogenesis of C. albicans. Functional analogies between Efg1p and Myc are discussed.

Amino Acid Sequence↗
PubMed2004

BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana.

The BLADE-ON-PETIOLE1 (BOP1) gene of Arabidopsis thaliana is required for proper leaf morphogenesis. BOP1 regulates leaf differentiation in a proximal-distal manner, and represses the expression of three class I knotted-like homeobox (knox) genes during leaf formation. Utilizing a map-based approach, we identified the molecular nature of the BOP1 gene, which encodes a BTB/POZ domain protein with ankyrin repeats. BOP1 is a member of a small gene family in Arabidopsis that includes the disease resistance regulatory protein NPR1. Insertions in and around BOP1 cause distinct lesions in leaf morphogenesis, revealing complex regulation of the locus. BOP1 transcripts are initially detectable in embryos, where they specifically localize to the base of the developing cotyledons near the SAM. During vegetative development, BOP1 is expressed in young leaf primordia and at the base of the rosette leaves on the adaxial side. During reproductive development, BOP1 transcripts are detected in young floral buds, and at the base of the sepals and petals. Our results indicate that BOP1 encodes a putative regulatory protein that modulates meristematic activity at discrete locations in developing lateral organs. This is the first report on a plant protein that plays a key role in morphogenesis with the distinctive combinatorial architecture of the BTB/POZ and ankyrin repeat domains.

Amino Acid Sequence↗
PubMed2006

Dynamics of vascular branching morphogenesis: the effect of blood and tissue flow.

Vascularization of embryonic organs or tumors starts from a primitive lattice of capillaries. Upon perfusion, this lattice is remodeled into branched arteries and veins. Adaptation to mechanical forces is implied to play a major role in arterial patterning. However, numerical simulations of vessel adaptation to haemodynamics has so far failed to predict any realistic vascular pattern. We present in this article a theoretical modeling of vascular development in the yolk sac based on three features of vascular morphogenesis: the disconnection of side branches from main branches, the reconnection of dangling sprouts ("dead ends"), and the plastic extension of interstitial tissue, which we have observed in vascular morphogenesis. We show that the effect of Poiseuille flow in the vessels can be modeled by aggregation of random walkers. Solid tissue expansion can be modeled by a Poiseuille (parabolic) deformation, hence by deformation under hits of random walkers. Incorporation of these features, which are of a mechanical nature, leads to realistic modeling of vessels, with important biological consequences. The model also predicts the outcome of simple mechanical actions, such as clamping of vessels or deformation of tissue by the presence of obstacles. This study offers an explanation for flow-driven control of vascular branching morphogenesis.

Animals↗
PubMedDate not supplied

Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components.

Epithelial-mesenchymal interactions play an important role in controlling epidermal morphogenesis and homeostasis but little is known about the mechanisms of these interactions. To examine whether diffusible factors produced by fibroblasts and/or keratinocytes support epidermal morphogenesis and basement membrane formation, organotypic keratinocyte monocultures were established in media collected either from organotypic fibroblast or keratinocyte-monocultures or from keratinocyte-fibroblast cocultures, and the expression of keratin 10, 16, and 17 and basement membrane components (types IV and VII collagen, laminin 5, nidogen, BP 180, LAD-1) were examined. We found that diffusible factors released by keratinocytes were not sufficient to support the establishment of normalized epidermal phenotype and deposition of basement membrane components in contrast to fibroblast- or keratinocyte/fibroblast-derived factors. Keratinocytes appear to affect the spectrum of secreted soluble factors, as keratinocyte/fibroblast-derived factors were more effective to accomplish continuous linear deposition of laminin 5 and of nidogen. The finding that released amounts of keratinocyte growth factor and granulocyte macrophage colony stimulating factor were not sufficient to fully support epidermal morphogenesis and deposition of basement membrane components is suggestive for the involvement of other released diffusible factors. Generation of organotypic keratinocyte monocultures in the presence of fibroblast- or keratinocyte/fibroblast-derived soluble factors resulted in enhanced expression of keratins K16 and K17 and the absence of type IV collagen. This observation indicates that next to paracrine acting factors, epidermal homeostasis is controlled by mutual keratinocyte-fibroblast interaction.

Basement Membrane↗
PubMed2005

A role for microfilament-based contraction in branching morphogenesis of the ureteric bud.

BACKGROUND: Branching morphogenesis of the ureteric bud/collecting duct epithelium is an important feature of kidney development. Recent work has identified many transcription factors and paracrine signaling molecules that regulate branching, but the physical mechanisms by which these signals act remain largely unknown. The actin cytoskeleton is a common component of mechanisms of morphogenesis. We have therefore studied the expression of, and requirement for actin filaments in the ureteric bud, a branching epithelium of the mammalian kidney. METHODS: Embryonic kidney rudiments were grown in organ culture. Actin expression in kidneys growing normally and those in which branching was inhibited was examined using labeled phalloidin. The morphogenetic effects of inhibiting actin organization and tension using cytochalasin D, butanedione monoxime, and Rho kinase ROCK inhibitors were assessed using immunofluorescence. RESULTS: F-actin is expressed particularly strongly in the apical domains of cells at the tips of branching ureteric bud, but this expression depends on the bud actively growing and branching. Blocking the polymerization of actin using cytochalasin D inhibits ureteric bud branching reversibly, as does blocking myosin function using butadiene monoxime. Inhibiting the activation of ROCK, a known activator of myosin, with the drugs Y27632 or with H1152 inhibits the expression of strong actin bundles in the ureteric bud tips and inhibits ureteric bud branching without inhibiting other aspects of renal development. CONCLUSION: The formation of tension-bearing actin-myosin complexes is essential for branching morphogenesis in the developing kidney.

Actin Cytoskeleton↗
PubMed1998

Repression of the A8L gene, encoding the early transcription factor 82-kilodalton subunit, inhibits morphogenesis of vaccinia virions.

The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and A8L genes, respectively. A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particles. The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle. To address these and other questions, we constructed and characterized a conditionally lethal recombinant vaccinia virus in which the A8L gene is stringently repressed by the Escherichia coli lac operator system. The amount of 82-kDa protein synthesized could be regulated by the amount of inducer: from undetectable to higher than normal levels. Virus replication, as determined by plaque formation or virus yield upon synchronous infection, was dependent on inducer. Nevertheless, de novo synthesis of the 82-kDa subunit was not required for viral early, intermediate, and late gene expression or DNA replication. Overexpression of the A8L gene alone, produced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect. Laser confocal fluorescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated in the absence of inducer, indicating that the A8L protein has a role in morphogenesis of the core and subsequent events.

Animals↗
PubMed1999

The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells.

Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.

Carcinoma, Renal Cell↗
PubMed2000

Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis.

The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Delta, and end3Delta mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

Actins↗
PubMed2002

Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity.

The actin cytoskeleton plays a major role in morphological development of neurons and in structural changes of adult neurons. This article reviews the myriad functions of actin and myosin in axon initiation, growth, guidance and branching, in morphogenesis of dendrites and dendritic spines, in synapse formation and stability, and in axon and dendrite retraction. Evidence is presented that signaling pathways involving the Rho family of small GTPases are key regulators of actin polymerization and myosin function in the context of different aspects of neuronal morphogenesis. These studies support an emerging theme: Different aspects of neuronal morphogenesis may involve regulation of common core signaling pathways, in particular the Rho GTPases.

Actins↗
PubMed2003

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity.

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 microg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair.

Animals↗
PubMed2004

Matrix GLA protein modulates branching morphogenesis in fetal rat lung.

The regulation of matrix gamma-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.

Animals↗
PubMed2003

Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro.

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene (SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein (CC10), sonic hedgehog (Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.

Animals↗
PubMed2005

Iroquois genes influence proximo-distal morphogenesis during rat lung development.

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.

Animals↗
PubMed1999

Adenovirus-mediated decorin gene transfer prevents TGF-beta-induced inhibition of lung morphogenesis.

Excessive transforming growth factor (TGF)-beta signaling has been implicated in pulmonary hypoplasia associated with bronchopulmonary dysplasia, a chronic lung disease of human prematurity featuring pulmonary fibrosis. This implies that inhibitors of TGF-beta could be useful therapeutic agents. Because exogenous TGF-beta ligands are known to inhibit lung branching morphogenesis and cytodifferentiation in mouse embryonic lungs in ex vivo culture, we examined the capacity of a naturally occurring inhibitor of TGF-beta activity, the proteoglycan decorin, to overcome the inhibitory effects of exogenous TGF-beta. Intratracheal microinjection of a recombinant adenovirus containing decorin cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. Although exogenous TGF-beta efficiently decreased epithelial lung branching morphogenesis in control cultures, TGF-beta-induced inhibition of lung growth was abolished after epithelial transfer of the decorin gene. Additionally, exogenous TGF-beta-induced antiproliferative effects as well as the downregulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs. Moreover, lung branching inhibition by TGF-beta could be restored by the addition of decorin antisense oligodeoxynucleotides in culture, indicating that decorin is both specifically and directly involved in suppressing TGF-beta-mediated negative regulation of lung morphogenesis. Our findings suggest that decorin can antagonize bioactive TGF-beta during lung growth and differentiation, establishing the rationale for decorin as a candidate therapeutic approach to ameliorate excessive levels of TGF-beta signaling in the developing lung.

Adenoviridae↗
PubMed2000

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model.

Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P < 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself.

Animals↗
PubMed1999

Expression of c-ret promotes morphogenesis and cell survival in mIMCD-3 cells.

c-Ret, a protein tyrosine kinase receptor, and its ligand glial-derived neurotropic factor (GDNF) are critical for early regulation of ureteric bud development and nephrogenesis. To address whether c-ret directly initiates epithelial cell morphogenesis, the c-ret receptor was expressed in murine inner medullary collecting duct cells (mIMCD-3, a cell line of ureteric bud origin, which has no detectable endogenous c-ret expression). Stable expression of wild-type c-ret was found to yield a constitutively tyrosine-phosphorylated receptor, with no change after the addition of GDNF. Examination of mRNA from these cells demonstrated the message for endogenous GDNF, suggesting that c-ret was potentially being constitutively activated by an autocrine mechanism. When mIMCD-3 cells stably expressing the phosphorylated c-ret receptor were cultured in a type I collagen matrix, they exhibited little GDNF-independent or -dependent branching process formation at early time points compared with the known morphogen hepatocyte growth factor (HGF) (48 h; control, 0.33 +/- 0.33; GDNF, 1.0 +/- 0.58, P = nonsignificant; and HGF, 6.33 +/- 0.33 processes/20 cell clusters, P < 0.001), whereas extended culture (7 days) under serum-free conditions revealed a marked increase in cell survival and the spontaneous development of rudimentary branching process formation. Extended culture (7 days) of c-ret-expressing clones in type I collagen with the epithelial morphogens HGF and/or epidermal growth factor (EGF) resulted in the development of complex three-dimensional spiny cysts, whereas parental mIMCD-3 cells died under these conditions. We conclude that activated c-ret appears to mediate epithelial morphogenesis by prolonging cell survival and, in conjunction with activation of the morphogenic receptors c-met and the EGF receptor, initiates the events required for very early branching morphogenesis.

Animals↗
PubMed2005

NO mediates mural cell recruitment and vessel morphogenesis in murine melanomas and tissue-engineered blood vessels.

NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, alpha-smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell-derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial-mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell-derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.

Animals↗