Biomedical subjects

T Jaskoll

Publications and source records attributed to T Jaskoll.

At least 19 recordsLinked to original sources

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.

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PubMed2001

The functional genomic response of developing embryonic submandibular glands to NF-kappa B inhibition.

BACKGROUND: The proper balance between epithelial cell proliferation, quiescence, and apoptosis during development is mediated by the specific temporal and spatial appearance of transcription factors, growth factors, cytokines, caspases, etc. Since our prior studies suggest the importance of transcription factor NF-kappaB during embryonic submandibular salivary gland (SMG) development, we attempted to delineate the emergent dynamics of a cognate signaling network by studying the molecular patterns and phenotypic outcomes of interrupted NF-kappaB signaling in embryonic SMG explants. RESULTS: SN50-mediated inhibition of NF-kappaB nuclear translocation in E15 SMG explants cultured for 2 days results in a highly significant increase in apoptosis and decrease in cell proliferation. Probabilistic Neural Network (PNN) analyses of transcriptomic and proteomic assays identify specific transcripts and proteins with altered expression that best discriminate control from SN50-treated SMGs. These include PCNA, GR, BMP1, BMP3b, Chk1, Caspase 6, E2F1, c-Raf, ERK1/2 and JNK-1, as well as several others of lesser importance. Increased expression of signaling pathway components is not necessarily probative of pathway activity; however, as confirmation we found a significant increase in activated (phosphorylated/cleaved) ERK 1/2, Caspase 3, and PARP in SN50-treated explants. This increased activity of proapoptotic (caspase3/PARP) and compensatory antiapoptotic (ERK1/2) pathways is consistent with the dramatic cell death seen in SN50-treated SMGs. CONCLUSIONS: Our morphological and functional genomic analyses indicate that the primary and secondary effects of NF-kappaB-mediated transcription are critical to embryonic SMG developmental homeostasis. Relative to understanding complex genetic networks and organogenesis, our results illustrate the importance of evaluating the gene, protein, and activated protein expression of multiple components from multiple pathways within broad functional categories.

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PubMed2001

Developmental expression of survivin during embryonic submandibular salivary gland development.

BACKGROUND: The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival. RESULTS: We investigated the developmental expression of survivin in Pseudoglandular (approximately E14), Canalicular (approximately E15) and Terminal Bud (approximately E17) Stage SMGs. We report a significant 26% increase in transcript levels between the Canalicular and Terminal Bud Stages. Immunohistochemical studies demonstrate nuclear-localized survivin protein in epithelial cells bounding forming lumina in Canalicular and Terminal Bud Stage SMGs. CONCLUSIONS: Survivin is known to be a pro-survival and anti-apoptotic factor. Given that survivin translocation into the nucleus is required for the induction of entry into the cell cycle and the inhibition of apoptosis, our demonstration of nuclear-localized survivin protein in presumptive ductal and proacinar lumen-bounding cells suggests that survivin may be a key mediator of embryonic SMG epithelial cell survival.

Amino Acid Sequence↗

PubMed2001

Embryonic mouse submandibular salivary gland morphogenesis and the TNF/TNF-R1 signal transduction pathway.

TNF is a pleiotropic cytokine that modulates cell proliferation and apoptosis. The objective of the present study was to investigate the possible function(s) of the TNF/TNF-R1 signaling pathway in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro experiments to investigate the phenotypic outcomes of enhanced and deficient ligand. The results of these experiments indicate that the TNF/TNF-R1 signal transduction pathway plays an important role in balancing cell proliferation and apoptosis during SMG duct and presumptive acini formation.

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PubMed2001

An alternatively spliced Muc10 glycoprotein ligand for putative L-selectin binding during mouse embryonic submandibular gland morphogenesis.

Late-gestation (embryonic day 18; E18) mouse submandibular glands (SMG) comprise a network of large and small ducts that terminate in lumen-containing, presumptive acini (terminal buds) expressing unique, cell membrane-associated embryonic mucin. The objective here was to clone and sequence embryonic low molecular-weight SMG mucin, predict its secondary structure, and begin to investigate its possible role in SMG development. Evidence was found that: (1) embryonic low molecular-weight mucin is an alternatively spliced Muc10 gene product, 220 amino acids in size (approximately 25 kDa), rich in potential O-glycosylation sites, and variably glycosylated (approximately 40 and 68 kDa); (2) consensus secondary-structure prediction for embryonic low molecular-weight mucin is consistent with a molecule that is anchored to the plasma membrane, directly or indirectly (via a glycolipid), and has a protein core that serves as a scaffold for carbohydrate presentation; (3) embryonic L-selectin is immunolocalized to the plasma membrane region of terminal-bud epithelial cells in a pattern similar to that seen for embryonic mucin; (4) embryonic, but not adult, mucin is able to bind L-selectin and does so endogenously in E18 SMG. As the primary role of L-selectin is to mediate cell adhesion and its ligands are mucin-like glycoproteins, it is suggested that this embryonic low molecular-weight mucin be termed MucCAM.

Age Factors↗

PubMed2001

Interleukin-6 signaling and embryonic mouse submandibular salivary gland morphogenesis.

Interleukin-6 (IL-6) is a multifunctional cytokine that mediates cell growth, differentiation, and survival. It was the objective of the present study to investigate the possible function(s) of IL-6 signaling in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro strategies to investigate the phenotypic outcomes of enhanced IL-6-induced signaling and immunoperturbation of IL-6 binding to cognate receptors. These experiments demonstrate: (1) there is a significant increase of IL-6 mRNA with progressive SMG development, and that this is highly correlated with TNF transcript levels; (2) IL-6 and its cognate receptors are immunolocalized in SMG branching epithelia from the canalicular stage to the late terminal bud stage, as are other constituents of the IL-6 pathway; (3) as compared to controls, IL-6-supplemented explants exhibit a substantial increase in overall size and in the number of ductal branches and terminal buds, as well as a highly significant increase in epithelial cell proliferation; (4) SMG explants cultured in the presence of anti-IL-6 neutralizing antibodies exhibit a marked decrease in epithelial ducts and terminal buds, concomitant with a significant decline in cell proliferation and a highly significant increase in apoptosis. Taken together, our experimental results indicate that IL-6 signaling is important to SMG developmental homeostasis.

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PubMed2000

Thrombospondin-2 gene expression and protein localization during embryonic mouse palate development.

The mammalian palate develops from projections of the paired maxillary processes termed palatal shelves. Shelf growth is an essential in normal palatal morphogenesis. Mesenchymal proliferation in the palatal shelves is modulated by transforming growth factor-1 (TGF-1), among other growth factors. Several pathways effect TGF-beta activation, including one which utilizes thrombospondin (TSP). TSP-1 is a major activator of TGF-beta in vivo and has been localized in head mesenchyme, including palates. TSP-2 appears to inhibit TSP-1 activation of latent TGF-beta by competitively binding the latent TGF-beta. Here the TSP-2 mRNA transcript and the immunolocalization of TSP-2 protein with progressive palatogenesis were quantified. There was a significant (p < 0.05) decline of TSP-2 transcript with palatal maturation; there was no evidence correlating the TSP-2 transcription with the amount of activated TGF-beta. At the vertical shelf stage of palatogenesis, TSP-2 protein was found throughout the extracellular matrix of shelf mesenchyme. By the horizontal shelf stage, TSP-2 protein was principally localized to the ossification centres of the developing maxilla, both in extracellular matrix and bone; far less was seen in palatal shelves proper. These results suggest that TSP-2 is multifunctional during embryonic palate formation.

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PubMed2000

Mouse submandibular gland morphogenesis: a paradigm for embryonic signal processing.

Signal processing is the sine qua non of embryogenesis. At its core, any single signal transduction pathway may be understood as classic Information Theory, adapted as an open system such that, because of networking, the "receiver" is presented with more information than was initially signaled by the "source". Over 40 years ago, Waddington presented his "Epigenetic Landscape" as a metaphor for the hierarchical nature of embryogenesis. Mathematically, Waddington's landscape may be modeled as a neural net. The "black box" of the neural net is an interacting network of signal transduction pathways (using hormones, growth factors, cytokines, neurotransmitters, and others) which inform the Boolean logic gates. An emerging theme in developmental biology is that defined sets of epigenetic circuits are used in multiple places, at multiple times, for similar and sometimes different purposes during organogenesis. As we show here, submandibular gland embryonic and fetal development is a splendid paradigm of these epigenetic circuits and their phenotypic outcomes, such as branching and lumen formation.

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PubMed1999

Submandibular gland morphogenesis: stage-specific expression of TGF-alpha/EGF, IGF, TGF-beta, TNF, and IL-6 signal transduction in normal embryonic mice and the phenotypic effects of TGF-beta2, TGF-beta3, and EGF-r null mutations.

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell-cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF-alpha/EGF/EGF-R; IGF-II/IGF-IR/IGF-IIR; TGF-betas and cognate receptors; TNF, IL-6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF-beta2, TGF-beta(3), EGF-R). Among many observations, the following seem of particular importance: (1) TGF-alpha and EGF-R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF-R1 signalling; (3) TGF-beta2 and TGF-beta3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF-R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies.

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PubMed1998

Insulin-like growth factor II receptor, transforming growth factor-beta, and Cdk4 expression and the developmental epigenetics of mouse palate morphogenesis and dysmorphogenesis.

The B10/B10.A congenic mouse pair serves as a model for identifying specific genes related to morphogenesis and dysmorphogenesis of the embryonic palate and other organs. The present report describes our initial investigation of the Fraser-Juriloff paradigm, which proposes that susceptibility to malformation results from genetically determined differences in normal developmental patterns. Specifically, we evaluated the relationship between Igf2r gene expression, transforming growth factor-beta (TGF-beta) activation, and cdk4 gene expression. By using in situ hybridization, RNase protection assays, indirect immunofluorescence, Western blots, and bioassays, we show 1) the presence of insulin-like growth factor II (IGF-II), IGF-II receptor (IGF-IIR), IGF-IR, TGF-beta, plasminogen, plasminogen activators [urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA)], and Cdk4 in developing palates; 2) on embryonic day 14 (E14), which is a critical day for palatal growth, B10.A embryos have 82% greater IGF-IIR mRNA than B10; 3) on E14, B10.A embryonic palates have a 57% greater level of active TGF-beta2 than B10, although the total TGF-beta2 is nearly identical; and 4) on E14, B10 embryonic palates have a 52% greater level of Cdk4 mRNA than B10.A palates, a measure of cell cycle progression. Because cellular activation of latent TGF-beta appears to require binding to the mannose-6-phosphate (M6P) binding site of the IGF-IIR and is plasmin and plasminogen activator dependent, the positive correlation of IGF-IIR levels and active TGF-beta2 levels seems to be key. Thus, the strain variation of TGF-beta2/IGF-IIR-mediated growth inhibition in late G1 phase would appear to account for the slower growth and development of B10.A palates relative to B10. Elevated corticosteroid (CORT) exposure in E14 B10.A embryos significantly increases TGF-beta levels, 87% of which is TGF-beta2, as well as the levels of active TGF-beta, 64% of which is TGF-beta2. Without exogenous CORT, B10.A embryos do not have clefts; hence, we present an outline of pathogenesis: slower growing B10.A embryos have an up-regulation of IGF-IIR, which serves to sequester IGF-II from the growth-promoting IGF-IR and to bind more CORT-up-regulated, latent TGF-beta2 for subsequent plasmin-dependent activation; higher levels of TGF-beta2 signaling down-regulate Cdk4 and result in greater palatal growth inhibition at a critical stage of palatogenesis and, thus, cleft palate. We present an epigenetic model of information processing related to cell proliferation. The model is a dynamical network that uses continuous logic to learn its rules from changing conditions.

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PubMed1998

Mouse submandibular gland mucin: embryo-specific mRNA and protein species.

Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein.

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PubMed1996

Developmental expression and CORT-regulation of TGF-beta and EGF receptor mRNA during mouse palatal morphogenesis: correlation between CORT-induced cleft palate and TGF-beta 2 mRNA expression.

Glucocorticoids (CORT) have been shown to induce cleft palate in mice. Although the pathogenetic pathway of CORT-induced cleft palate has been investigated for several decades, the molecular details remain to be elucidated. Since growth factors have been shown to regulate palate morphogenesis, and the expression of several growth factors or their receptors, e.g. TGF-beta, EGF receptor (EGF-R), are known to be modulated by CORT, we postulate that CORT modulation of growth factor (or receptor) gene expression is a key mechanism involved in CORT-induced cleft palate. To test this hypothesis, we analyzed the steady-state levels (Northern and RNase protection) and developmental expression (in situ hybridization) of four CORT-responsive genes--TGF-Beta 1, TGF- beta 2, TGF-beta 3, and EGF receptor (EGF-R)--in developing mouse palates in the presence or absence of exogenous CORT. Pregnant B10.A dams were injected on day 12 of gestation with CORT or sham-injected and embryonic palates were collected at 1, 2, and 3 days postinjection (E13-E15). During mouse palate development, significant increases in TGF-beta 1 and TGF-beta 3 mRNA levels, as well as significant decrease in TGF-beta 2 mRNA levels, are detected; no significant difference in EGF-R transcript level is observed with progressive development. In CORT-exposed palates, we demonstrate no significant differences in the direction or magnitude of change with time in TGF-beta 1, TGF-beta 3, and EGF-R mRNA levels compared to controls. However, CORT delays by 1 day the down-regulation of palatal TGF-beta 2 transcript normally seen on day 14 of gestation. TGF-beta 2 is known to inhibit cell proliferation. The level of TGF-beta 2 mRNA, the only isoform primarily expressed in the palatal mesenchyme, significantly decreases with progressive palatal development; this down-regulation of TGF-beta 2 expression is associated with increased mesenchymal cell proliferation and palatal shelf growth. CORT, at a critical stage of palatogenesis, induces a delay in the normal down-regulation of TGF-beta 2 gene expression. Given that CORT is known to inhibit mesenchymal cell proliferation and palatal shelf growth, we conclude that the CORT-induced delay in the normal down-regulation of TGF-beta 2 gene expression is probably key event in the pathogenesis of CORT-induced cleft palate.

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PubMed1996

Glucocorticoids, tumor necrosis factor-alpha, and epidermal growth factor regulation of pulmonary morphogenesis: a multivariate in vitro analysis of their related actions.

The mouse lung commences development on embryonic day 11 as an epithelial evagination from the posterior pharyngeal wall into undifferentiated mesenchyme, this epithelium bifurcating to form the lung primordium. Branching morphogenesis, as well as terminal differentiation, requires epithelial-mesenchymal interactions utilizing precise regulatory controls. Not surprisingly, specific hormones and growth factors appear to play a key role in this regulation. We report here a series of experiments designed to investigate morphodifferentiation (epithelial branching number, generation number, and fractal dimension) and histodifferentiation (cell morphology and SP-A immunolocalization), as they relate to glucocorticoid (CORT)-regulation of growth factor function and expression (Northern analysis). These experiments were conducted in embryonic lung primordia (E11.5-E12) cultured under defined conditions in the presence of single or combined CORT, TNF-alpha, and EGF supplementation. EGF supplementation enhances branching morphogenesis, but not immunodetectable SP-A expression, in embryonic lung primordia cultured for 4 or 7 days. TNF-alpha supplementation also enhances branching morphogenesis on days 4 or 7 in vitro; on day 7, SP-A expression is also enhanced. By contrast, the introduction of exogenous CORT to embryonic explants cultured 4 or 7 days markedly alters morphodifferentiation and histodifferentiation. Early on it would appear to enhance morphodifferentiation by changing the process of branching, while contemporaneously initiating precocious SP-A expression; later on, it alters morphogenesis by continued terminal differentiation of normal lung epithelium and a singular transdifferentiation of lung mesenchyme into an epithelioid morphotype expressing SP-A. This is correlated with a CORT-induced, highly significant, down-regulation of TGF-beta 2 and TGF-beta 3 transcripts. Explants supplemented with CORT + TNF-alpha or CORT + EGF.demonstrate a microanatomy and SP-A expression pattern identical to that seen with CORT supplementation alone. EGF inhibits the accelerated lung maturation normally seen in the presence of exogenous TNF-alpha alone, suggesting a relationship between these two seemingly disparate regulatory pathways.

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PubMed1996

Developmental expression of insulin-like growth factor II receptor (IGF-IIR) in congenic mouse embryonic lungs: correlation between IGF-IIR mRNA and protein levels and heterochronic lung development.

Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2a) and B10 (H-2b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF-IIR levels with progressive development in both strains. Further, slower-developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory Distress Syndrome in prenatal newborns.

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PubMed1996

The glucocorticoid-glucocorticoid receptor signal transduction pathway, transforming growth factor-beta, and embryonic mouse lung development in vivo.

Lung morphogenesis has been shown to be regulated by glucocorticoids (CORT). Because CORT has been primarily thought to affect fetal lung development, previous studies have focused on the role of CORT receptor (GR)-mediated regulation of fetal lung development. Although endogenous CORT increases during embryonic and fetal stages and exogenous CORT treatment in vivo and in vitro clearly accelerates embryonic lung development, little is known about the morphoregulatory role of the embryonic CORT-GR signal transduction pathway during lung development. In this study, we characterize the embryonic mouse CORT-GR pathway and demonstrate: stage-specific in situ patterns of GR immunolocalization; similarity in GR relative mobility with progressive (E13 --> E17) development; that embryonic GR can be activated to bind a GR response element (GRE); significantly increasing levels of functional GR with increasing lung maturation; and the presence of heat shock protein (hsp) 70 and hsp90 from early (E13) to late (E17) developmental stages. These results support the purported importance of the embryonic CORT-GR signal transduction pathway in progressive lung differentiation. To demonstrate that the embryonic CORT-GR directed pathway plays a role in lung development, early embryonic (E12) lungs were exposed to CORT in utero and surfactant-associated protein A (SP-A) expression was analyzed; CORT treatment up-regulates SP-A mRNA expression and spatiotemporal protein distribution. Finally, to determine whether CORT-GR-directed pulmonary morphogenesis in vivo involves the modulation of growth factors, we studied the effect of CORT on TGF-beta gene expression. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo indicates that CORT regulates the rate of lung morpho- and histodifferentiation by down-regulating TGF-beta 3 gene expression.

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PubMed1994

Effect of in ovo retinoic acid exposure on forebrain neural crest: in vitro analysis reveals up-regulation of N-CAM and loss of mesenchymal phenotype.

In a prior study of in ovo exogenous retinoic acid (RA) exposure, we observed a prolonged expression of cell surface N-CAM in cranial neural crest (NC) cells exhibiting migratory failure. In the present studies, we employed an experimental strategy in which embryos were first exposed to exogenous RA in ovo and incubated for 45-60 hr; this was followed by extirpation and in vitro culturing of these same RA-exposed cranial neural tubes. NC cell outgrowth from the explant was assayed, as was the immunohistochemical localization of HNK-1 and N-CAM antigens. In RA-exposed explants, the size of the NC cell outgrowths were comparable to controls. However, almost all NC cells lost their mesenchymal phenotype and were arranged in an "epithelioid" pattern of tightly packed polygonal cells that expressed N-CAM at adjacent cell boundaries. By contrast, control NC cells were flattened and multipolar in shape and expressed HNK-1, rarely co-expressing N-CAM. These observations indicate that RA modulates NC cell N-CAM expression and microanatomical phenotype, a finding consistent with prior in ovo studies of RA-exposure. Several possible explanations are considered.

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PubMed1994

Tumor necrosis factor-alpha and embryonic mouse lung morphogenesis.

The ontogeny of the embryonic and fetal lung involves complex interactions between epithelial and mesenchymal primordia which require a specific program of gene regulation and signal transduction. Past studies in our laboratory using congenic mouse strains indicate that one or more genes which map to the H-2 region of chromosome 17 regulate the rate of lung morphogenesis, defined in this context as differentiative heterochrony among strains. Since hormones and growth factors are the messengers of morphogenesis, it was logical to propose that tumor necrosis factor-alpha (TNF-alpha), a well-characterized cytokine whose gene maps to the D-region of the H-2 complex, is a putative mediator of lung morphogenesis. We investigated this proposition using immunochemical methods and a serumless, chemically defined in vitro model system. Our results demonstrate that: (1) TNF-alpha has a specific spatiotemporal localization, in vivo and in vitro; (2) TNF-alpha receptor, in vivo and in vitro, is localized throughout the embryonic lung; (3) TNF-alpha supplementation in vitro of embryonic lung primordia has a marked dose-dependent, stimulatory effect on branching morphogenesis and surfactant-associated protein (SP-A) expression; (4) multiple immunoreactive proteins, including 17, 26, and 68 kDa species, are expressed during development in vivo, and a subset of these are expressed in vitro; and (5) both time- and glucocorticoid-dependent changes occur in the in vivo expression pattern of TNF-alpha immunoreactive proteins after 4 and 7 days in vitro, including the up-regulation of a novel 40 kDa protein. Given that glucocorticoids (CORT) regulate TNF-alpha expression and TNF-alpha's ability to stimulate pulmonary morphodifferentiation and histodifferentiation, we conclude that TNF-alpha is an autocrine/paracrine pulmonary cytokine, probably a component of the lung morphogenesis pathway regulated by CORT.

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PubMed1993

Raphe of the posterior neural tube in the chick embryo: its closure and reopening as studied in living embryos with a high definition light microscope.

Chick embryos cultured on a curved substratum show a transient enlargement of the posterior neuropore (PN), mimicking the temporary delay of PN closure as seen in the curly tail (ct) mouse mutant (van Straaten et al. [1993] Development 117:1163-1172). In the present study the PN enlargement in the chick embryo was investigated further with a high definition light microscope (HDmic), allowing high resolution viewing of living embryos in vitro. The temporary PN enlargement appeared due to considerable reopening of the raphe of the posterior neural tube, which was followed by reclosure after several hours. The raphe was subsequently studied in detail. It appeared very irregular, with small zones of apposed, open and fused neural folds. During closure, these raphe features shifted posteriorly. A distinct fusion sequence between surface epithelium and neuroepithelium was not seen. During experimental reopening of the raphe in vitro, small bridges temporarily arose, broke and disappeared quickly; they likely represented the first adhesion sites between the neural folds. More prominent adhesion sites partly detached, resulting in bridging filopodia-like connections; they probably represented the first anteroposterior locations of neural fold fusion. Our observations in the living chick embryo in vitro thus show that the caudal neural tube has an irregular raphe with few adhesion sites, which can be readily reopened. As a result of the irregularity, the PN does not close zipper-like, but button-like by forming multiple closure sites.

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