Biomedical subjects

Pablo Bringas

Publications and source records attributed to Pablo Bringas.

At least 19 recordsLinked to original sources

PubMed2007

Establishment and characterization of an immortomouse-derived odontoblast-like cell line to evaluate the effect of insulin-like growth factors on odontoblast differentiation.

Insulin-like growth factors (IGF-I and IGF-II) play important roles in regulating growth and differentiation of many different organs including teeth. The presence of these factors in the developing tooth has been demonstrated. In vitro studies using tooth explants grown in the presence of IGFs suggest that they promote differentiation of ameloblast and odontoblasts cells. This is achieved by inducing or repressing gene expression associated with these cells. Since some of the genes involved in tooth differentiation are expressed by both cells, to determine the effect of IGF on odontoblast cell differentiation we first need a cell line in which a controlled environment can be created. In this study, we report the establishment and characterization of an Immortomouse-derived odontoblast-like cell line. This conditional cell line can grow indefinitely under permissive conditions in the presence of INF-gamma at 33 degrees C, differentiate into odontoblast-like cells and produce a mineralized extracellular matrix when the INF-gamma is removed and cell maintained at 39 degrees C. Addition of exogenous IGFs to the media results in an accelerated production of a mineralized matrix. This is the result of increased transcription of genes associated with bone mineralization while down regulating genes associated with dentin formation like DSPP. This data suggest that IGFs induce dental papillae mesenchyme cells to produce a bone-like mineralized extracellular matrix.

Animals↗

PubMed2006

The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development.

During craniofacial development, Meckel's cartilage and the mandible bone derive from the first branchial arch, and their development depends upon the contribution of cranial neural crest (CNC) cells. We previously demonstrated that conditional inactivation of Tgfbr2 in the neural crest of mice (Tgfbr2(fl/fl);Wnt1-Cre) results in severe defects in mandibular development, although the specific cellular and molecular mechanisms by which TGF-beta signaling regulates the fate of CNC cells during mandibular development remain unknown. We show here that loss of Tgfbr2 does not affect the migration of CNC cells during mandibular development. TGF-beta signaling is specifically required for cell proliferation in Meckel's cartilage and the mandibular anlagen and for the formation of the coronoid, condyle and angular processes. TGF-beta-mediated connective tissue growth factor (CTGF) signaling is critical for CNC cell proliferation. Exogenous CTGF rescues the cell proliferation defect in Meckel's cartilage of Tgfbr2(fl/fl);Wnt1-Cre mutants, demonstrating the biological significance of this signaling cascade in chondrogenesis during mandibular development. Furthermore, TGF-beta signaling controls Msx1 expression to regulate mandibular osteogenesis as Msx1 expression is significantly reduced in Tgfbr2(fl/fl);Wnt1-Cre mutants. Collectively, our data suggest that there are differential signal cascades in response to TGF-beta to control chondrogenesis and osteogenesis during mandibular development.

Animals↗

PubMed2006

Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion.

Palatal fusion is a complex, multi-step developmental process; the consequence of failure in this process is cleft palate, one of the most common birth defects in humans. Previous studies have shown that regression of the medial edge epithelium (MEE) upon palatal fusion is required for this process, and TGF-beta signaling plays an important role in regulating palatal fusion. However, the fate of the MEE and the mechanisms underlying its disappearance are still unclear. By using the Cre/lox system, we are able to label the MEE genetically and to ablate Tgfbr2 specifically in the palatal epithelial cells. Our results indicate that epithelial-mesenchymal transformation does not occur in the regression of MEE cells. Ablation of Tgfbr2 in the palatal epithelial cells causes soft palate cleft, submucosal cleft and failure of the primary palate to fuse with the secondary palate. Whereas wild-type MEE cells disappear, the mutant MEE cells continue to proliferate and form cysts and epithelial bridges in the midline of the palate. Our study provides for the first time an animal model for soft palate cleft and submucous cleft. At the molecular level, Tgfb3 and Irf6 have similar expression patterns in the MEE. Mutations in IRF6 disrupt orofacial development and cause cleft palate in humans. We show here that Irf6 expression is downregulated in the MEE of the Tgfbr2 mutant. As a recent study shows that heterozygous mutations in TGFBR1 or TGFBR2 cause multiple human congenital malformations, including soft palate cleft, we propose that TGF-beta mediated Irf6 expression plays an important, cell-autonomous role in regulating the fate of MEE cells during palatogenesis in both mice and humans.

Animals↗

PubMed2006

Altering biomineralization by protein design.

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.

Ameloblasts↗

PubMed2006

An in vitro model for characterizing the post-migratory cranial neural crest cells of the first branchial arch.

The cranial neural crest (CNC) is a transient cell population that originates at the crest of the neural fold and gives rise to multiple cell types during craniofacial development. Traditionally, researchers have used tissue explants, such as the neural tube, to obtain primary neural crest cells for their studies. However, this approach has inevitably resulted in simultaneous isolation of neural and non-neural crest cells as both of these cells migrate away from tissue explants. Using the Wnt1-Cre/R26R mouse model, we have obtained a pure population of neural crest cells and established a primary CNC cell culture system in which the cell culture medium best supports the proliferation of E10.5 first branchial arch CNC cells and maintains these cells in their undifferentiated state. Differentiation of CNC cells can be initiated by switching to a differentiation medium. In this model, cultured CNC cells can give rise to neurons, glial cells, osteoblasts, and other cell types, faithfully mimicking the differentiation process of the post-migratory CNC cells in vivo. Taken together, our study shows that the Wnt1-Cre/R26R mouse first branchial arch provides an excellent model for obtaining post-migratory neural crest cells free of any mesodermal contaminants. The cultured neural crest cells are under sustained proliferative, undifferentiated, or lineage-enhanced conditions, hence, serving as a tool for the investigation of the regulatory mechanism of CNC cell fate determination in normal and abnormal craniofacial development.

Animals↗

PubMed2006

Amelogenin and ameloblastin show growth-factor like activity in periodontal ligament cells.

Enamel proteins, particularly amelogenin, have been associated with other functions in addition to regulating enamel biomineralization. Extracts of enamel proteins are currently being used to regenerate periodontal tissues, and new studies suggest that enamel proteins might have chondrogenic and osteogenic properties. In this study, we wanted to determine the effect, if any, of purified recombinant amelogenin and ameloblastin on the adhesion, proliferation, and differentiation of periodontal ligament cells in vitro. Immortomouse-derived periodontal ligament (PDL) cells were grown under permissive and differentiation conditions in the presence of different concentrations of mouse recombinant amelogenin, recombinant ameloblastin, or both. Cells were collected after 4 h to determine attachment, after 24 h to determine proliferation, and after 7, 14, 21 and 28 d to determine differentiation using reverse transcription-polymerase chain reaction (RT-PCR). Both amelogenin and ameloblastin had a small, but statistically significant, effect on increasing the cell attachment and proliferation of PDL cells. Both amelogenin and ameloblastin modulated bone morphogenetic protein (BMP) expression, down-regulated the expression of collagen type I, and induced the de novo expression of osteocalcin. Amelogenin also induced the expression of bone sialoprotein. These results suggest that amelogenin, as well as ameloblastin, might have some 'growth factor' activity during periodontium development and regeneration.

Amelogenin↗

PubMed2006

TGFbeta-mediated FGF signaling is crucial for regulating cranial neural crest cell proliferation during frontal bone development.

The murine frontal bone derives entirely from the cranial neural crest (CNC) and consists of the calvarial (lateral) aspect that covers the frontal lobe of brain and the orbital aspect that forms the roof of bony orbit. TGFbeta and FGF signaling have important regulatory roles in postnatal calvarial development. Our previous study has demonstrated that conditional inactivation of Tgfbr2 in the neural crest results in severe defects in calvarial development, although the cellular and molecular mechanisms by which TGFbeta signaling regulates the fate of CNC cells during frontal bone development remain unknown. Here, we show that TGFbeta IIR is required for proliferation of osteoprogenitor cells in the CNC-derived frontal bone anlagen. FGF acts downstream of TGFbeta signaling in regulating CNC cell proliferation, and exogenous FGF2 rescues the cell proliferation defect in the frontal primordium of Tgfbr2 mutant. Furthermore, the CNC-derived frontal primordium requires TGFbeta IIR to undergo terminal differentiation. However, this requirement is restricted to the developing calvarial aspect of the frontal bone, whereas the orbital aspect forms despite the ablation of Tgfbr2 gene, implying a differential requirement for TGFbeta signaling during the development of various regions of the frontal bone. This study demonstrates the biological significance of TGFbeta-mediated FGF signaling cascade in regulating frontal bone development, suggests that TGFbeta functions as a morphogen in regulating the fate of the CNC-derived osteoblast and provides a model for investigating abnormal craniofacial development.

Animals↗

PubMed2005

Dentin sialoprotein and dentin phosphoprotein overexpression during amelogenesis.

The gene for dentin sialophosphoprotein produces a single protein that is post-translationally modified to generate two distinct extracellular proteins: dentin sialoprotein and dentin phosphoprotein. In teeth, dentin sialophosphoprotein is expressed primarily by odontoblast cells, but is also transiently expressed by presecretory ameloblasts. Because of this expression profile it appears that dentin sialophosphoprotein contributes to the early events of amelogenesis, and in particular to those events that result in the formation of the dentino-enamel junction and the adjacent "aprismatic" enamel. Using a transgenic animal approach we have extended dentin sialoprotein or dentin phosphoprotein expression throughout the developmental stages of amelogenesis. Overexpression of dentin sialoprotein results in an increased rate of enamel mineralization, however, the enamel morphology is not significantly altered. In wild-type animals, the inclusion of dentin sialoprotein in the forming aprismatic enamel may account for its increased hardness properties, when compared with bulk enamel. In contrast, the overexpression of dentin phosphoprotein creates "pitted" and "chalky" enamel of non-uniform thickness that is more prone to wear. Disruptions to the prismatic enamel structure are also a characteristic of the dentin phosphoprotein overexpressing animals. These data support the previous suggestion that dentin sialoprotein and dentin phosphoprotein have distinct functions related to tooth formation, and that the dentino-enamel junction should be viewed as a unique transition zone between enamel and the underlying dentin. These results support the notion that the dentin proteins expressed by presecretory ameloblasts contribute to the unique properties of the dentino-enamel junction.

Amelogenesis↗

PubMed2005

Effect of N'-nitrosonornicotine (NNN) on murine palatal fusion in vitro.

Maternal smoking has been linked to an increased risk for orofacial clefts. N'-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines that has been shown to be linked to the deleterious effects of tobacco and could be linked to the formation of cleft palate birth defects. The effect of NNN on palatal fusion was examined using an in vitro organ culture model of palatal development. The organ cultures were exposed to NNN (0.01, 0.1, 1, 10 and 100 mM) and the effects on palatal development characterized at defined points. Palatal fusion was evaluated at embryonic day 13 (E13)+72 h by characterizing the remaining medial edge epithelium (MEE) and determining the extent of fusion compared to controls. The NNN-treated group (1 mM) had more MEE remaining in the palatal midline than the untreated group at E13+72 h (P<0.05). Changes in cell proliferation in the MEE resulting from NNN exposure were examined by BrdU incorporation in replicating DNA. Changes in the pattern of MEE cell death were examined by TUNEL. BrdU incorporation and TUNEL staining showed that the NNN (1 mM)-treated palates had more MEE cell proliferation and less apoptosis than the untreated-palates at E13+24 h (P<0.05). The mechanism altered by NNN was further evaluated by characterizations of extracellular signal-regulated kinase (ERK) 1/2, p38 and c-jun amino-terminal kinase (JNK). NNN at 1 mM induced ERK1/2 phosphorylation, but reduced p38 phosphorylation (P<0.05, P<0.01, respectively) in the MEE. The results suggest that NNN inhibited palatal fusion by effects on cell proliferation and MEE cell death.

Animals↗

PubMed2005

LEF1 is a critical epithelial survival factor during tooth morphogenesis.

LEF1 is a cell-type-specific transcription factor and mediates Wnt signaling pathway by association with its co-activator beta-catenin. Wnt signaling is known to be critical for the specification of cranial neural crest (CNC) cells and may regulate the fate diversity of the CNC during craniofacial morphogenesis. Loss of Lef1 results in arrested tooth development at the late bud stage and LEF1 is required for a relay of a Wnt signaling to a cascade of FGF signaling activities to mediate the epithelial-mesenchymal interaction during tooth morphogenesis. It remains unclear, however, what is the cellular mechanism of LEF1 signaling in regulating tooth morphogenesis. To test the hypothesis that LEF1 signaling regulates the fate of the dental epithelial and the CNC-derived mesenchymal cells during tooth morphogenesis, we investigated and compared the cellular migration, proliferation, and apoptotic activity within the tooth germ between the wild-type and Lef1 null mutant mice. Using the Wnt1-Cre/R26R transgenic system for indelibly marking the progenies of CNC cells, we show that there is no CNC migration defect in the Lef1 null mutant mice, indicating that the arrest in tooth development is not the result of shortage of the CNC contribution into the first branchial arch in the Lef1 mutant. Furthermore, there is no alteration in cell proliferation or condensation of the CNC-derived dental mesenchyme in the Lef1 null mutant, suggesting that LEF1 may not affect the cell cycle progression of the multipotential CNC cells during tooth morphogenesis. Importantly, apoptotic activity is significantly increased within the dental epithelium in the Lef1 null mutant mice. As the result of this increased cell death, the bud stage tooth germ fails to advance to the cap stage in the absence of Lef1. Inhibition of apoptotic activity by FGF4 rescues the tooth development in the Lef1 null mutant. Our studies suggest that LEF1 is a critical survival factor for the dental epithelial cells during tooth morphogenesis.

Animals↗

PubMed2005

Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate.

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.

Animals↗

PubMed2005

Smad1 expression and function during mouse embryonic lung branching morphogenesis.

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.

Animals↗

PubMed2005

IGFs increase enamel formation by inducing expression of enamel mineralizing specific genes.

Insulin-like growth factors (IGF-I and IGF-II) have been shown to play an important role in growth and differentiation in a number of tissues including mineralizing bone. Little is known about their role in tooth mineralization. Previous work in our laboratory has shown the presence of IGFs ligands, their receptors, and their binding proteins during mouse tooth morphogenesis. The expression of IGF I coincides with the expression of amelogenin, ameloblastin and enamelin at the late bell and secretory stage. The objective of this study is to determine the mechanisms by which IGFs modulate enamel and dentin formation. Mouse first mandibular molars were dissected from E16 and E17 mouse embryos and placed in organ culture in the presence of IGF-I or IGF-II. The molars were harvested after 12 days for histological examination or 1 day for mRNA expression analysis by real-time RT-PCR. Our results show an increase in enamel deposition, and an induction of enamelin, amelogenin and collagen type I mRNA expression, while expression of DSPP was down-regulated. These results suggest that IGFs increase enamel formation by the induction of gene expression of enamel related genes. Studies are underway to determine a possible mechanism for these factors.

Amelogenin↗

PubMed2005

PDGFR-alpha signaling is critical for tooth cusp and palate morphogenesis.

Platelet-derived growth factor receptor alpha (PDGFR-alpha) and PDGF ligands are key regulators for embryonic development. Although Pdgfralpha is spatially expressed in the cranial neural crest (CNC)-derived odontogenic mesenchyme, mice deficient for Pdgfralpha are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfralpha and Pdgfa are specifically and consistently expressed in the CNC-derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfralpha and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfralpha gene does not affect proper odontoblasts proliferation and differentiation in the CNC-derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfralpha-/- mice have complete cleft palate. We show that the cleft palate in Pdgfralpha mutant mice results from an extracellular matrix defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFRalpha and PDGFA are critical regulators for the continued epithelial-mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFRalpha signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development.

Animals↗

PubMed2005

Functional significance of Smad2 in regulating basal keratinocyte migration during wound healing.

Members of the transforming growth factor-beta (TGF-beta) superfamily are critical regulators for wound healing. Transduction of TGF-beta signaling depends on activation of Smad2 and Smad3 by heteromeric complexes of ligand-specific receptors. Mice lacking Smad3 show accelerated wound healing, whereas the biological significance of Smad2-mediated TGF-beta signaling in wound healing remains unknown. To understand the function of Smad2 in regulating wound healing, we investigated the effect of Smad2 overexpression on epithelialization of incision wounds. Cutaneous wounds made in K14-Smad2 mice showed delayed healing. This delay in wound healing resulted from a defect in basal keratinocyte migration in K14-Smad2 mice. Instead of basal keratinocytes, the suprabasal layer of keratinocytes migrated into the wound region. Furthermore, overexpression of Smad2 activated the Smad2/Smad4 complex in keratinocytes and inhibited keratin 16 (K16) expression. As K16 functions as a critical mediator for reorganization of keratin filaments following skin injury, we propose that altered K16 expression affects the migration of basal keratinocytes in the K14-Smad2 mice. Taken together, these findings demonstrate a crucial role of TGF-beta signaling mediator Smad2 in regulating keratinocyte migration and re-epithelialization during wound healing. The K14-Smad2 transgenic mice can serve as an animal model for the investigation of TGF-beta signaling mechanism in regulating wound healing.

Animals↗

PubMed2005

Meckel's cartilage differentiation is dependent on hedgehog signaling.

The hedgehog (Hh) signaling pathway has been shown to be essential for craniofacial development. Although mandibular arch derivatives are largely absent in Shh null mice, little is known about the role of Hh signaling during Meckel's cartilage development per se. Mandible development is dependent on the morphogenesis of Meckel's cartilage, which then serves as a template for subsequent skeletal differentiation. In this study, we examine the biological function of Hh signaling during Meckel's cartilage development in vivo and in vitro. E13.5 Shh null mice present a small mesenchymal condensation in the region of a presumptive Meckel's cartilage in the hypoplastic mandibular arch. By E15.5, the Shh mutant exhibits a mere remnant of the mandibular arch, without evidence of Meckel's cartilage differentiation. Further, wild-type embryonic (E11 or E12) mandibular explants cultured for up to 5 days in the presence of cyclopamine, a steroidal alkaloid that specifically disrupts the Hh signaling pathway, exhibit a stage-dependent inhibition of Meckel's cartilage chondroblast differentiation to mature chondrocytes. This phenotype can be rescued by exogenous FGF8, a downstream effector of Hh signaling. Taken together, our results indicate that the Hh signaling pathway is critical to Meckel's cartilage ontogenesis and the rate of chondrogenesis, but not to initial primordium formation. The reliance on Hh signaling is stage dependent.

Animals↗

PubMed2004

FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis.

FGF8 has been shown to play important morphoregulatory roles during embryonic development. The observation that craniofacial, cardiovascular, pharyngeal, and neural phenotypes vary with Fgf8 gene dosage suggests that FGF8 signaling induces differences in downstream responses in a dose-dependent manner. In this study, we investigated if FGF8 plays a dose-dependent regulatory role during embryonic submandibular salivary gland (SMG) morphogenesis. We evaluated SMG phenotypes of Fgf8 hypomorphic mice, which have decreased Fgf8 gene function throughout embryogenesis. We also evaluated SMG phenotypes of Fgf8 conditional mutants in which Fgf8 function has been completely ablated in its expression domain in the first pharyngeal arch ectoderm from the time of arch formation. Fgf8 hypomorphs have hypoplastic SMGs, whereas conditional mutant SMGs exhibit ontogenic arrest followed by involution and are absent by E18.5. SMG aplasia in Fgf8 ectoderm conditional mutants indicates that FGF8 signaling is essential for the morphogenesis and survival of Pseudoglandular Stage and older SMGs. Equally important, the presence of an initial SMG bud in Fgf8 conditional mutants indicates that initial bud formation is FGF8 independent. Mice heterozygous for either the Fgf8 null allele (Fgf8(+/N)) or the hypomorphic allele (Fgf8(+/H)) have SMGs that are indistinguishable from wild-type (Fgf8(+/+)) mice which suggest that there is not only an FGF8 dose-dependent phenotypic response, but a nonlinear, threshold-like, epistatic response as well. We also found that enhanced FGF8 signaling induced, and abrogated FGF8 signaling decreased, SMG branching morphogenesis in vitro. Furthermore, since FGF10 and Shh expression is modulated by Fgf8 levels, we postulated that exogenous FGF10, Shh, or FGF10 + Shh peptide supplementation in vitro would largely "rescue" the abnormal SMG phenotype associated with decreased FGF8 signaling. This is as expected, though there is no synergistic effect with FGF10 + Shh peptide supplementation. These in vitro experiments model the principle that mutations have different effects in the context of different epigenotypes.

Animals↗

PubMed2004

Sonic hedgehog signaling plays an essential role during embryonic salivary gland epithelial branching morphogenesis.

Gene targeting studies indicate that sonic hedgehog (Shh) signaling plays an essential role during craniofacial development. Because numerous mandibular derivatives (e.g., teeth, tongue, Meckel's cartilage) are absent in Shh null mice and the embryonic submandibular salivary gland (SMG) develops from the mandibular arch, we postulated that Shh signaling is important for embryonic SMG development. To address this question, we first determined the spatiotemporal distribution of Shh; two transmembrane proteins, patched 1 (Ptc) and Smoothened (Smo), which act as a negative or a positive regulator of the Shh signal, respectively; and the Gli 3 transcription factor, which is downstream of the Shh signal. The epithelial localization of Shh, Ptc, Smo, and Gli 3 suggests that Shh signaling may act within the epithelium in a juxtacrine manner. The SMG phenotype in our embryonic day (E) 18.5 Shh null mice can be characterized as "paedomorphic," that is, it fails to progress to ontogenic stages beyond the Early Pseudoglandular ( approximately E14). In a complementary set of experiments, we used organ culture to evaluate the effect of enhanced or abrogated Shh signaling on embryonic SMG development in vitro. Paired E13 (Late Initial Bud stage) or E14 (Pseudoglandular stage) SMGs were cultured in the presence or absence of exogenous Shh peptide supplementation; Shh-supplemented explants exhibit a significant stage-dependent increase in branching morphogenesis compared with control explants. Furthermore, by using cyclopamine, a steroidal alkaloid that specifically disrupts the Shh pathway, to abrogate endogenous Shh signaling in vitro, we found a significant decrease in branching in cyclopamine-treated explants compared with controls, as well as a significant decrease in epithelial cell proliferation. Our results indicate that Shh signaling plays an essential role during embryonic SMG branching morphogenesis. Exogenous FGF8 peptide supplementation in vitro rescues the abnormal SMG phenotype seen in cyclopamine-treated explants, demonstrating that overexpression of a parallel, but related, downstream signaling pathway can compensate for diminished Shh signaling and restore embryonic SMG branching morphogenesis.

Animals↗