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PubMed1993

Ecdysteroid induction of embryonic morphogenesis in a parasitic wasp.

Development of the parasitic wasp Copidosoma floridanum (Hymenoptera: Encyrtidae) is unusual in two ways. As many as 3000 embryos are formed from a single egg and embryonic morphogenesis is closely synchronized with the onset of the metamorphosis of its host. Given this extreme synchrony between parasite and host development, we undertook a series of experiments to determine whether host endocrine factors regulate C. floridanum embryonic morphogenesis. Here we report that C. floridanum embryos must develop for 9 days before acquiring the competence to undergo morphogenesis. Furthermore, several pieces of evidence suggest that ecdysteroids of host origin regulate induction of C. floridanum morphogenesis. First, competent embryos initiated morphogenesis when transplanted into host larvae and pupae, host stages possessing elevated ecdysteroid titers, but not when transplanted into adult moths. Second, morphogenesis was arrested by ablation of the host's source of ecdysone, but could be rescued by injection of 20-hydroxyecdysone in a dose-dependent manner. Finally, a segment of DNA encoding a zinc finger nearly identical in sequence to a portion of the ecdysone receptor of Drosophila melanogaster was isolated and characterized from C. floridanum. This putative ecdysone receptor probe indicated that expression of this gene was correlated with the initiation of C. floridanum embryonic morphogenesis. The temporal pattern of putative receptor RNA accumulation increased in association with the onset of morphogenesis, while the spatial pattern of expression was associated with the invagination of cells forming the gastrula. Together, these data suggest that ecdysone of host origin is directly involved in the induction of C. floridanum embryonic morphogenesis.

Amino Acid Sequence↗
PubMed2000

Role of hyaluronan and CD44 in in vitro branching morphogenesis of ureteric bud cells.

Mutual interaction between the metanephric mesenchyme (MM) and the ureteric bud (UB) in the developing kidney leads to branching morphogenesis and the formation of the ureteric tree. A UB-derived cell line, stimulated by conditioned medium derived from an embryonic MM cell line (or, similarly, by 10% fetal calf serum), forms branching tubules under three-dimensional culture conditions (H. Sakurai et al., 1997, Proc. Natl. Acad. Sci. USA 94, 6279-6284). The formation of branching tubules in this simple in vitro system for early nephrogenesis is highly sensitive to the matrix environment, a key component of which is the glycosaminoglycan hyaluronan (HA). Consistent with this, we found that HA in the extracellular environment markedly stimulated the formation of cellular processes and multicellular cords (early steps in branching morphogenesis) and also acted as a cell survival factor. Inhibition of HA binding to the cells by addition of blocking antibodies to CD44, the principal cell surface receptor for HA, or degradation of HA by the addition of Streptomyces hyaluronidase resulted in decreased cell survival and diminished morphogenesis, indicating that the HA-CD44 axis plays a central role in in vitro branching morphogenesis. Analysis of the expression of a large number of genes displayed on a cDNA array revealed that significant changes in gene expression in cells undergoing morphogenesis in the presence of HA were limited to a small subset of genes regulating apoptosis, proliferation, and morphogenesis. This included upregulation by HA of its receptor, CD44, which was found to largely localize to the tips of branching cellular processes. In the embryonic kidney, HA was found near the developing ureteric tree and CD44 was expressed basolaterally in UB-derived structures. In addition, both UB and MM appear to express HA synthase, suggesting their ability to secrete HA. We propose that HA promotes branching morphogenesis by creating a positive feedback loop that results in (1) enhanced interaction of HA-CD44 at branching tips (possibly leading to localization of HA binding morphoregulatory factors at the tips) and (2) an activated transcriptional program favoring cell survival/proliferation and migration/morphogenesis of cells through matrix by the expression of key morphoregulatory molecules. Furthermore, since HA, hyaluronidase, and CD44 have been functionally implicated in branching morphogenesis in this model, and since HA, CD44, and HA synthase are all expressed in an appropriate spatiotemporal fashion in the developing kidney, we propose that these molecules may, together, constitute a morphoregulatory pathway that plays a key role in sequential cycles of branching morphogenesis in the UB.

Animals↗
PubMed1997

Oxidized LDL inhibits vascular endothelial cell morphogenesis in culture.

Human umbilical cord vein endothelial cells can be induced to undergo morphogenesis (tube formation) by phorbol ester (TPA) when cultured on or in three-dimensional collagen gels. Induction of morphogenesis by TPA is accompanied by increased activity of the collagenase gene transcription factors, ETS1 and API, and the elaboration of collagenase by the endothelial cells. In the present study, we used endothelial cell elongation as a measure of morphogenesis and showed that oxidized low density lipoprotein (oxLDL) inhibited endothelial cell migration in monolayer cultures and TPA-induced morphogenesis in collagen gels in a dose-dependent manner. Moreover, the inhibition was positively correlated with the extent of LDL oxidation. In contrast, native LDL stimulated cell migration and TPA-induced morphogenesis under the same culture conditions. However, in the absence of TPA, LDL showed no effect on EC morphogenesis. Further studies showed that inhibition of TPA-induced endothelial cell morphogenesis by oxLDL is correlated with suppression of the protein kinase C (PKC) and ETS1/AP1 activities. The results indicated that the inhibition of endothelial cell morphogenesis by oxLDL is probably mediated through inhibition of the TPA-activated PKC pathway and its subsequent suppression of the ETS1/AP1 activity. The results also indicated that EC migration can be mediated through PKC-dependent and independent pathways and only the former pathway can induce EC morphogenesis as well.

Cell Division↗
PubMed2000

The molecular basis of lung morphogenesis.

To form a diffusible interface large enough to conduct respiratory gas exchange with the circulation, the lung endoderm undergoes extensive branching morphogenesis and alveolization, coupled with angiogenesis and vasculogenesis. It is becoming clear that many of the key factors determining the process of branching morphogenesis, particularly of the respiratory organs, are highly conserved through evolution. Synthesis of information from null mutations in Drosophila and mouse indicates that members of the sonic hedgehog/patched/smoothened/Gli/FGF/FGFR/sprouty pathway are functionally conserved and extremely important in determining respiratory organogenesis through mesenchymal-epithelial inductive signaling, which induces epithelial proliferation, chemotaxis and organ-specific gene expression. Transcriptional factors including Nkx2.1, HNF family forkhead homologues, GATA family zinc finger factors, pou and hox, helix-loop-helix (HLH) factors, Id factors, glucocorticoid and retinoic acid receptors mediate and integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Signaling by the IGF, EGF and TGF-beta/BMP pathways, extracellular matrix components and integrin signaling pathways also directs lung morphogenesis as well as proximo-distal lung epithelial cell lineage differentiation. Soluble factors secreted by lung mesenchyme comprise a 'compleat' inducer of lung morphogenesis. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as FGFR, EGFR, IGFR, PDGFR and c-met stimulate lung morphogenesis. On the other hand, cognate receptors with serine/threonine kinase intracellular signaling domains, such as the TGF-beta receptor family are inhibitory, although BMP4 and BMPR also play key inductive roles. Pulmonary neuroendocrine cells differentiate earliest in gestation from among multipotential lung epithelial cells. MASH1 null mutant mice do not develop PNE cells. Proximal and distal airway epithelial phenotypes differentiate under distinct transcriptional control mechanisms. It is becoming clear that angiogenesis and vasculogenesis of the pulmonary circulation and capillary network are closely linked with and may be necessary for lung epithelial morphogenesis. Like epithelial morphogenesis, pulmonary vascularization is subject to a fine balance between positive and negative factors. Angiogenic and vasculogenic factors include VEGF, which signals through cognate receptors flk and flt, while novel anti-angiogenic factors include EMAP II.

Animals↗
PubMed1993

AFR1 acts in conjunction with the alpha-factor receptor to promote morphogenesis and adaptation.

Mating pheromone receptors activate a G-protein signaling pathway that induces changes in transcription, cell division, and morphogenesis needed for the conjunction of Saccharomyces cerevisiae. The C terminus of the alpha-factor pheromone receptor functions in two complex processes, adaptation and morphogenesis. Adaptation to alpha-factor may occur through receptor desensitization, and alpha-factor-induced morphogenesis forms the conjugation bridge between mating cells. A plasmid overexpression strategy was used to isolate a new gene, AFR1, which acts together with the receptor C terminus to promote adaptation. The expression of AFR1 was highly induced by alpha-factor. Unexpectedly, cells lacking AFR1 showed a defect in alpha-factor-stimulated morphogenesis that was similar to the morphogenesis defect observed in cells producing C-terminally truncated alpha-factor receptors. In contrast, AFR1 overexpression resulted in longer projections of morphogenesis, which suggests that this gene may directly stimulate morphogenesis. These results indicate that AFR1 encodes a developmentally regulated function that coordinates both the regulation of receptor signaling and the induction of morphogenesis during conjugation.

Acclimatization↗
PubMed1998

TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.

Islets of Langerhans are microorgans scattered throughout the pancreas, and are responsible for synthesizing and secreting pancreatic hormones. While progress has recently been made concerning cell differentiation of the islets of Langerhans, the mechanism controlling islet morphogenesis is not known. It is thought that these islets are formed by mature cell association, first differentiating in the primitive pancreatic epithelium, then migrating in the extracellular matrix, and finally associating into islets of Langerhans. This mechanism suggests that the extracellular matrix has to be degraded for proper islet morphogenesis. We demonstrated in the present study that during rat pancreatic development, matrix metalloproteinase 2 (MMP-2) is activated in vivo between E17 and E19 when islet morphogenesis occurs. We next demonstrated that when E12.5 pancreatic epithelia develop in vitro, MMP-2 is activated in an in vitro model that recapitulates endocrine pancreas development (Miralles, F., P. Czernichow, and R. Scharfmann. 1998. Development. 125: 1017-1024). On the other hand, islet morphogenesis was impaired when MMP-2 activity was inhibited. We next demonstrated that exogenous TGF-beta1 positively controls both islet morphogenesis and MMP-2 activity. Finally, we demonstrated that both islet morphogenesis and MMP-2 activation were abolished in the presence of a pan-specific TGF-beta neutralizing antibody. Taken together, these observations demonstrate that in vitro, TGF-beta is a key activator of pancreatic MMP-2, and that MMP-2 activity is necessary for islet morphogenesis.

Animals↗
PubMed1984

Transferrin is required for early tooth morphogenesis.

The role of circulating molecules during early tooth morphogenesis was studied in organ cultures of mouse embryonic molar-tooth germs. Special attention was focused on the effect of transferrin and insulin, which are necessary for the growth of most cells in culture. The requirement of serum factors for tooth morphogenesis was shown to diminish as the developmental stage advances from the bud stage in day-13 embryos to the cap stage at day 15. The day-15 teeth underwent morphogenesis and cell differentiation in unsupplemented basal culture medium, but the addition of transferrin (50 micrograms/ml) was necessary for the morphogenesis of day-14 tooth germs. We demonstrated, by using transferrin-depleted serum, that transferrin is also necessary for the morphogenesis of day-13 tooth germs. However, some still-unidentified serum components are also required for the morphogenesis of the bud-stage day-13 teeth. These factors apparently do not include insulin, since it was shown to inhibit tooth development. Analysis of the DNA content of tooth germs cultured in various culture media showed that the ability of transferrin to support tooth morphogenesis correlated with a stimulation of growth. The results support our earlier suggestions that transferrin functions as a fetal growth factor. The availability of the transferrin-containing chemically defined medium facilitates studies on the roles of other growth factors during tooth development.

Animals↗
PubMed1999

The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. We show here that hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in discs morphogenesis, especially in the fusion of the two lateral wing discs. We show that these defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role of puc in discs morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role of puc in disc morphogenesis. Furthermore, we show that the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence for a role of the peripodial membrane in disc morphogenesis. We discuss a general model whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges.

Animals↗
PubMed1982

Mandibular morphogenesis and craniofacial malformations.

Four questions have been addressed in this review. What is morphogenesis? I conclude that morphogenesis is simply development or change of shape and distinguish morphogenesis both from differentiation and from growth, although predominant growth along one axis can alter shape. Morphogenesis can be considered at all levels from the molecule to the population. I concentrated on cells, tissues, and organs. How does the mandible develop? A brief overview of the origin, migration, and differentiation of the neural crest and other cells which constitute the mandible has been provided. How is mandibular morphogenesis controlled? Several lines of evidence are presented to show that basic elements of morphogenesis, eg number and shape of skeletal elements in the mandible, are a property possessed by the mesenchymal cells which will form those tissues, before they differentiate into cartilage or bone. Reaggregated mesenchymal cells form site-specific-shaped cartilages. First arch neural crest, transplanted to sites of presumptive second and third arch neural crest, migrates to the second arch region but forms first branchial arch skeletal and muscle elements in that ectopic site. Morphogenesis is an intrinsic property of the neural crest and of the mesenchymal cells which arise from the crest. What is the developmental basis of craniofacial malformations? Evidence is presented to show that defects at any stage of mandibular development can lead to craniofacial malformations. Absence of the neural crest, abnormal migration of neural crest-derived cells, abnormal extracellular environments, defective interactions between or differentiation of mesenchymal cells, altered inductive tissue interactions, and deficiencies in epigenetic interactions between components of the mandible can all lead to craniofacial malformations. The challenge for the clinical and basic craniofacial biologist is to identify the defective cellular process which has produced the malformation and to devise preventative or corrective procedures which restore that process to normality.

Animals↗
PubMedDate not supplied

Glucocorticoids, TGF-beta, and embryonic mouse salivary gland morphogenesis.

Branching morphogenesis is a fundamental embryologic process in many developing organs: salivary gland, lung, pancreas, mammary gland, and kidney. Glucocorticoids (CORT) are known to regulate morphogenesis in several branching organs (e.g., lung); we hypothesize that CORT is also important to embryonic mouse salivary gland morphogenesis. We studied the CORT-glucocorticoid receptor (GR) signal transduction pathway during embryonic mouse submandibular gland development. Western analysis demonstrates that the 96-kDa GR is present in E14 to E18 submandibular glands. The embryonic GR is functional, as defined by its ability to bind a DNA CORT response element, for all gestational ages evaluated (E14 to E18); increasing GR levels are observed with progressive development. The level of endogenous corticosterone in embryonic submandibular glands was also determined. Using radioimmunoassays corticosterone is first detected on day 15 of gestation; there is a progressive increase in steroid levels from day 15 to 18 of gestation. In addition, we investigated the effect of exogenous CORT on submandibular gland morphogenesis in vivo and in vitro. In vivo experiments were performed using maternal injection of CORT or sham injection on day 12 of gestation with sacrifice 72 hr post-injection (E15). CORT treatment significantly increases embryonic submandibular gland growth in vivo. To further analyze the effect of exogenous CORT on embryonic submandibular gland morphogenesis, E13 mouse submandibular glands were cultured under serum-free, chemically-defined conditions. A significant (P < 0.05) enhancement of mean branching ratios (72 hr/0 hr) is detected in CORT-supplemented explants (10(-5) M to 10(-8) M) compared to control explants; doses more dilute than 10(-8) M CORT do not significantly (P > 0.10) increase the induction of branching. In addition, CORT administration to embryonic mice in utero enhances the expression of acinar-cell-specific mucin protein. These data indicate that the CORT-GR signal transduction pathway plays an important role in salivary gland morphogenesis. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo confirms the presence of TGF-beta 1 and TGF-beta 2 and identifies TGF-beta 3 in embryonic submandibular glands. To begin to identify a molecular mechanism of CORT-GR mediated submandibular gland morphodifferentiation, we studied the effect of CORT on growth factor gene expression, specifically TGF-beta. Northern analysis suggests that the CORT-GR signal transduction pathway modulates the rate of morphogenesis by regulating TGF-beta 2 and TGF-beta 3 mRNA expression.

Analysis of Variance↗
PubMed1995

Hydrocortisone and progesterone regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1-10 microgram/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01-1 microgram/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 micrograms/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells.

Animals↗
PubMed1997

Transforming growth factor-beta selectively inhibits branching morphogenesis but not tubulogenesis.

When cultured in type I collagen gels, two kidney-derived cell lines, Madin-Darby canine kidney (MDCK) cells and murine inner medullary collecting duct (mIMCD3) cells, from branching tubular structures in the presence of Swiss 3T3 conditioned medium, in which hepatocyte growth factor (HGF) is the major branching tubule inducing factor. However, upon incubation with transforming growth factor-beta (TGF-beta) in the presence of 3T3 conditioned medium, MDCK tubulogenesis and branching was markedly inhibited. In contrast, mIMCD3 cells, which are much less susceptible to growth and tubulogenesis inhibition by TGF-beta, formed long straight tubulelike structures in presence of TGF-beta, suggesting a dissociation between tubulogenesis and branching morphogenesis. Interestingly, those long tubules that did branch often superficially resembled the early branching ureteric bud in embryonic kidneys. Quantitation of branching events revealed a selective branch-inhibiting effect of TGF-beta on mIMCD3 cells at concentrations between 0.02 and 2 ng/ml. There was no qualitative or quantitative difference among TGF-beta 1, -beta 2, and -beta 3 on inhibition of branching events, suggesting existence of potentially redundant mechanisms for modulating branching morphogenesis. Concentrations of TGF-beta that resulted in long nonbranching tubules also altered the profile of extracellular matrix-degrading proteases and their inhibitors expressed by developing tubules. Ratios of urokinase type plasminogen activator (u-PA) to plasminogen activator inhibitor (PAI-l) and matrix metalloprotease (MMP)-1 to tissue inhibitor of metalloprotease (TIMP)-1 were both markedly decreased. In addition, apart from a direct effect on epithelial cell branching morphogenesis, TGF-beta downregulated the expression of HGF mRNA in Swiss 3T3 cells. Thus TGF-beta exerts at least three distinct effects relevant to tubulogenesis and branching morphogenesis inhibition of branching morphogenesis alone (mIMCD3 cells), inhibition of both tubulogenesis and branching morphogenesis (MDCK cells), and inhibition of the expression of growth factor which induce tubulogenesis and branching morphogenesis (3T3 cells). In the context of epithelial tissue development, which requires tightly regulated branching tubulogenesis of epithelial cells, the data suggest a model where branching patterns are regulated by a precise temporal and spatial balance between branching morphogens such as HGF and inhibitory morphogens such as members of the TGF-beta superfamily [e.g., TGF-beta isoforms, certain bone morphogenetic proteins].

Animals↗
PubMed2000

Inhibition of distal lung morphogenesis in Nkx2.1(-/-) embryos.

In vitro and in vivo results are consistent with a critical role for NKX2.1, an epithelial homeodomain transcription factor in lung morphogenesis. Nkx2.1 null mutant embryos die at birth due to respiratory insufficiency caused by profoundly abnormal lungs. However, the precise role of NKX2.1 in the multistep process of lung structural morphogenesis and differentiation of various pulmonary cell types remains unknown. In the current study, we tested the hypothesis that the mutant lungs do not undergo branching morphogenesis beyond the formation of the mainstem bronchi and therefore consist solely of dilated tracheobronchial structures. To test this hypothesis, we determined the spatial and temporal expression pattern of a number of extracellular matrix (ECM) proteins and their cellular receptors, including alpha-integrins, laminin, and collagen type IV. Although laminin is expressed in the mutant Nkx2.1(-/-) lungs, expression of alpha-integrins and collagen type IV is significantly reduced or absent. In addition, examination of regionally specific expression of differentially spliced Vegf (vascular endothelial growth factor) transcripts, clearly indicates that the epithelial phenotype of the Nkx2.1(-/-) lungs is similar to the tracheobronchial epithelium. In contrast to wild-type lungs in which both Vegf1 and Vegf3 are developmentally expressed, Nkx2.1(-/-) lungs are characterized by predominant expression of Vegf1 and reduced or absent Vegf3. A similar pattern of Vegf expression is also observed in isolated tracheo-bronchial tissue. The sum of these findings suggest that at least two separate pathways may exist in embryonic lung morphogenesis: proximal lung morphogenesis is Nkx2.1 independent, while distal lung morphogenesis appears to be strictly dependent on the wild-type activity of Nkx2.1.

Actins↗
PubMed1993

Morphogenesis of the avian trunk neural crest: use of morphological techniques in elucidating the process.

Morphological data generated from light and electron microscopy form the basis of our understanding of avian morphogenesis. Because chicken embryos are readily and cheaply obtained and are easily accessible for experimental manipulation, morphogenetic processes have been studied extensively in this species. Such studies have allowed us to identify the cells involved during morphogenesis, observe the shape changes or cellular translocations that accompany a morphogenetic process, and determine the timing of these events. Elucidation of the molecular basis of morphogenesis has awaited the integration of several additional approaches. Among these are experimental embryology, which has allowed us to understand cellular behavior associated with morphogenesis; immunocytochemistry, which has identified the macromolecular cues that regulate cell movements and the environmental factors that control them; and molecular techniques, which will permit us eventually to clarify the genetic regulation of morphogenesis. Although current research in development is heavily biased towards molecular biology, morphological studies continue to frame the questions that are now being addressed using molecular techniques. This review focuses on the cells of the neural crest as a model system where questions of avian morphogenesis have been profitably addressed.

Animals↗
PubMed1998

Abrogation of Smad3 and Smad2 or of Smad4 gene expression positively regulates murine embryonic lung branching morphogenesis in culture.

Smad genes are recently identified intracellular effectors for receptor signaling in the BMP/activin/TGF-beta pathway. Since TGF-beta ligands are known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad genes negatively regulate lung organogenesis. Antisense oligodeoxynucleotides were designed to attenuate Smad3 and Smad2 gene expression in embryonic (E11) mouse lungs over 4 days in culture. Endogenous Smad3 and Smad2 mRNA levels were suppressed by 97 and 91%, respectively, in cultured embryonic lungs when antisense oligodeoxynucleotide (40 microM) to Smad was added, compared to scrambled and sense sequence controls. The corresponding Smad3 and Smad2 protein amounts were also decreased respectively by 86 and 90% in lungs treated with Smad3 and Smad2 antisense oligodeoxynucleotide. Phenotypically, Smad antisense oligodeoxynucleotides resulted in a concentration-dependent increase in lung branching: embryonic lung branching was stimulated by up to 53% in culture with 40 microM antisense oligodeoxynucleotide, whereas both scrambled and sense controls showed no stimulatory effect. Thus, inhibition of endogenous Smad3 and Smad2 gene expression resulted in stimulation of embryonic lung branching similar to that caused by inhibition of TGF-beta type II receptor expression and signaling (J. Zhao et al., 1996, Dev. Biol. 180, 242-257). Abrogation of Smad4 (DPC4), the downstream mediator of Smad3 and Smad2 proteins, with antisense oligodeoxynucleotide, also resulted in increased branching morphogenesis. Furthermore, while TGF-beta alone inhibited lung branching morphogenesis in culture, addition of exogenous TGF-beta 1 could not overcome the stimulatory effect on lung branching of Smad antisense oligodeoxynucleotide treatment. By immunohistochemistry, Smad proteins were localized mainly to the epithelial cells lining the branching distal airways, indicating that Smad genes could regulate lung morphogenesis through mesoderm-endoderm interaction. Our results demonstrate, for the first time, that abrogation of Smad2 and Smad3 or of Smad4 gene expression stimulated early mouse embryonic lung branching morphogenesis in culture, possibly through reversing the negative influence of endogenous TGF-beta signaling upon lung branching morphogenesis.

Animals↗
PubMed1998

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER.

Animals↗
PubMed1992

Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis.

A major unsolved problem in developmental biology is to determine when and how time- and position-restricted instructions are signaled and received during secondary embryonic inductions such as branching morphogenesis. The mouse embryonic lung rudiment was used to test the hypothesis that endogenous peptide growth factors, specifically epidermal growth factor (EGF), serve as instructive epigenetic signals for morphogenesis. The presence of EGF precursor mRNA transcripts was detected using the reverse-transcriptase-coupled polymerase chain reaction both in E11-E17-day mouse embryo lung tissues in vivo and in E11-day lung cultured for up to 7 days in vitro under chemically defined, serum-free conditions. Immunolocalization identified a position-restricted distribution of EGF in and around the primitive airways both during in vivo lung morphogenesis and in culture. EGF receptors (EGFR) coimmunolocalized with EGF in the primitive airways. Addition of exogenous EGF to lungs in culture resulted in significant concentration-dependent stimulation of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA. Conversely, the addition of tyrphostin (specific EGF receptor kinase antagonist) to lungs in culture resulted in concentration-dependent inhibition of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA without apparent cytotoxicity. The inhibition of the EGF signal by tyrphostin was confirmed by immunoprecipitation of tyrosine phosphoproteins. We conclude that early mouse embryo lungs express EGF transcripts and corresponding EGF peptides in a specific position-restricted distribution which coimmunolocalizes with EGFR in the primitive airways, while stimulatory and inhibitory studies indicate a functional role for the transduced EGF signal in the epigenetic regulation of lung branching morphogenesis. We speculate that the peptide growth factor EGF serves a function in secondary embryonic morphogenetic inductions, which may be modulated by interaction with other growth factors.

Animals↗
PubMed1998

Roles of hepatocyte growth factor/scatter factor and transforming growth factor-beta1 in mammary gland ductal morphogenesis.

Epithelial-mesenchymal interactions are responsible for the unique pattern of ductal branching morphogenesis characteristic of the mammary gland. To investigate the factors which control the elongation and branching of lactiferous ducts, we developed an in vitro model of ductal morphogenesis in which clonal mouse mammary epithelial cells (TAC-2 cells) are grown in collagen gels. In this experimental system, fibroblast conditioned medium (CM)3 stimulates the formation of extensively arborized tubules. The molecule responsible for this tubulogenic effect was identified as hepatocyte growth factor/scatter factor (HGF/SF). To determine whether HGF/SF plays a role in mammary gland morphogenesis in vivo, the expression of HGF/SF and its receptor, c-Met, were analyzed in the rat mammary gland during pregnancy, lactation, and involution. Levels of HGF/SF and c-Met transcripts were progressively reduced during pregnancy, were virtually undetectable during lactation, and increased again during involution. Collectively, these in vitro and in vivo findings suggest that HGF/SF is a paracrine mediator of mammary gland ductal morphogenesis. We subsequently investigated the effect of another multifunctional cytokine, namely TGF-beta1, on branching morphogenesis of TAC-2 cells. TGF-beta1 had a striking biphasic effect: whereas relatively high concentrations of this cytokine inhibited colony formation, lower concentrations stimulated extensive elongation and branching of epithelial cords. Taken together, these studies indicate that HGF/SF is a stromal-derived paracrine mediator of mammary ductal morphogenesis, and that when present at low concentrations, TGF-beta1 can contribute to this process.

Animals↗