Search PubMed⌕ Search

Biomedical subjects

C M Chuong

Publications and source records attributed to C M Chuong.

At least 55 records · Page 3Linked to original sources
PubMed1996

Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance.

Early molecular events during the development and regeneration of skin appendages were studied using cultured chicken skin explants with epithelial-mesenchymal recombination. The explant epithelium was separated from the mesenchyme, rotated 90 degrees or 180 degrees, recombined with the mesenchyme, and cultured. After this procedure, existing feather buds disappeared and new buds were regenerated. The location of the new buds is determined by the original dermal condensations, whereas the orientation is dictated by the original epithelium. The temporal expression of key morphogenetic molecules was examined 3, 6, and 20 h after recombination by whole-mount in situ hybridization and immunostaining. The results showed the following. (i) Placode formation and the expression of wingless-int (Wnt) 7a and Msx-1 in the placode epithelium are mesenchyme dependent. (ii) Hox C6 and neural cell adhesion molecule (NCAM) expression in the anterior mesenchyme is placode epithelium dependent. (iii) Bone morphogenetic protein (BMP)-2, BMP-4, and fibroblast growth factor (FGF)-4 expression in the original dermal condensations was unaffected by recombination. (iv) Old dermal condensations can induce new placodes with new Wnt 7a, sonic hedgehog (Shh), and Msx-1 and -2 expression. (v) The new placode epithelium can then induce new Hox C6 and NCAM microgradients in the feather bud mesenchyme. (vi) The order of appearance can be classified into four groups in the following order: BMP-2, BMP-4, and FGF-4 (peptide growth factors); Wnt 7a and Shh (Drosophila segment polarity gene homologs); Msx-1 and Msx-2 (Msx class homeobox genes); and then Hox C6 (Hox class homeobox genes) and NCAM (adhesion molecules). These results suggest an order for the molecular cascade during the inductive phase of skin appendage development.

Animals↗
PubMed1995

Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis.

The molecular signaling of secondary induction is a fundamental process in organogenesis during embryonic development. To study the signal transduction pathways involved, we used developing chicken skin as a model and focused on the roles of intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of forming dermis around H and H stage 30. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. The presence and activity of PKC, PKA, and P-CREB in developing chicken skin are further characterized by immunoblot, kinase activity, and gel shift assays. To explore their physiological significance, embryonic chicken dorsal skin explants were treated with different modulators in medium or in beads for localized effects. The results showed that PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. We observed that activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented.

Animals↗
PubMed1995

Homeobox genes Msx-1 and Msx-2 are associated with induction and growth of skin appendages.

The mechanism involved in the morphogenesis of skin appendages is a fundamental issue underlying the development and healing of skin. To identify molecules involved in the induction and growth of skin appendages, we studied the expression of two homeobox genes, Msx-1 and Msx-2, during embryonic chicken skin development. We found that i) both Msx-1 and Msx-2 are early markers of epithelial placodes for skin appendages; ii) both Msx-1 and Msx-2 are expressed in the growing feather bud epithelia but not in the interbud epithelia; iii) although mostly overlapping, there are differences between the expression of the two Msx genes, Msx-1 being expressed more toward the anterior whereas Msx-2 is expressed more toward the distal feather bud; iv) there is no body-position-specific expression pattern as was observed for members of the Hox A-D clusters; v) in the feather follicle, Msx-1 and 2 are expressed in the collar and barb ridge epithelia, both regions of continuous cell proliferation; vi) when feather-bud growth was inhibited by forskolin, an activator of adenylyl cyclase, the expression of both genes was reduced. These results showed that Msx genes are specifically expressed in epithelial domains destined to become skin appendages. Its function in skin-appendage morphogenesis may be twofold, first in making epithelial cells competent to become skin appendages and, second, in making epithelial cells maintain their potential for continuous growth.

Animals↗
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.

Animals↗
PubMed1994

cDCC (chicken homologue to a gene deleted in colorectal carcinoma) is an epithelial adhesion molecule expressed in the basal cells and involved in epithelial-mesenchymal interaction.

Cloning of human DCC (deleted in colorectal carcinoma, Fearon et al., 1990) showed that it is an immunoglobulin superfamily member homologous to neural cell adhesion molecules (N-CAM). To explore the normal function of this molecule, we have cloned a chicken homologue to DCC (cDCC) and raised an antibody to DCC. cDCC is a protein of 160 kDa with an expression pattern distinct from those of other immunoglobulin family members including N-CAM and Ng-CAM. Transgene expression of cDCC in fibroblasts led to increased cell-cell adhesion. Localization studies in chicken and mouse embryos showed that DCC is expressed in the epithelia of skin, gut, lung, and bladder. In adult, the expression of DCC is limited to the basal layer of stratified epithelium in skin, crypt regions of intestinal villi, and stem cells in mammary duct. Cell aggregation assay using embryonic chicken skin epithelial cells and antibody to DCC showed it is a Ca2+ independent cell adhesion molecule. In epithelial-mesenchymal interactions during feather morphogenesis, antibody to DCC suppressed the formation of dermal condensations and the polarized localization of N-CAM and fibronectin. These results implied that DCC is an epithelial cell adhesion molecule required for mediating critical functions in epithelial-epithelial and epithelial-mesenchymal interactions.

Animals↗
PubMed1993

Tenascin is associated with articular cartilage development.

The roles of tenascin in cartilage development and function remain unclear. Based on the observation that tenascin is particularly abundant at the epiphyseal extremities of developing cartilaginous models of long bones in chick and mouse embryo, we tested the hypothesis that tenascin is involved in articular cartilage development. Immunofluorescence analysis revealed that tenascin was first localized in the cell condensation region of Day 4 chick embryo limb buds, where the cartilaginous models form. With further development, tenascin gene expression became indeed restricted to the articular cap of the models. Tenascin persisted in the articular cartilage of postnatal chickens but appeared to decrease with age. The protein was also abundant in embryonic and adult tracheal cartilage rings which, like articular cartilage, persist throughout postnatal life. Similar patterns of tenascin expression were seen in mouse. Using monoclonal antibodies to avian tenascin variants, we found that the bulk of articular cartilage contained the shortest tenascin variant (Tn190), whereas the largest variant (Tn230) was present in tissues associated or interacting with articular cartilage (ligaments and meniscus). The protein and its mRNA, however, were undetectable in growth plate cartilage undergoing maturation and endochondral ossification. This inverse correlation between chondrocyte maturation and tenascin production was corroborated by the finding that tenascin gene expression decreased markedly during maturation of chondrocytes in culture and during formation of a secondary ossification center within the articular cap in vivo. Thus, tenascin is intimately associated with the development of articular cartilage and other permanent cartilages whereas absence or reduced amounts of this matrix protein characterize transient cartilages which undergo maturation and are replaced by bone.

Animals↗
PubMed1993

The making of a feather: homeoproteins, retinoids and adhesion molecules.

We have been using feather development as a model for understanding the molecular basis of pattern formation and to explore the roles of homeoproteins, retinoids and adhesion molecules in this process. Two kinds of homeobox (Hox) protein gradients in the skin have been identified: a 'microgradient' within a single feather bud and a 'macrogradient' across the feather tract. The asynchronous alignment of different Hox macrogradients establishes a unique repertoire of Hox expression patterns in skin appendages within the integument, designated here as the 'Hox codes of skin appendages'. It is hypothesized that these Hox codes contribute to the phenotypic determination of skin appendages. High doses of retinoic acid cause a morphological transformation between feather and scale, while low doses of retinoic acid cause an alteration of the axial orientation of skin appendages. We have tested the ability of molecules directly involved in the feather formation process to mediate the action of the Hox codes, and surmise that adhesion molecules are potential candidates. Using specific Fabs to suppress the activity of adhesion molecules, we have found that L-CAM is involved in the formation of the hexagonal pattern, N-CAM is involved in mediating dermal condensations, tenascin is involved in feather bud growth and elongation, and integrin beta-1 is essential for epithelial-mesenchymal interactions. More work is in progress to fully understand the molecular pathways regulating the feather formation process.

Animals↗
PubMed1993

Adhesion molecules in skeletogenesis: II. Neural cell adhesion molecules mediate precartilaginous mesenchymal condensations and enhance chondrogenesis.

Neural cell adhesion molecules (NCAM) was expressed transiently by mesenchymal cells in precartilaginous condensations of the embryonic chicken limb but was lost upon differentiation into cartilage. Consequently, NCAM was present in the periphery of the limb anlagen but was absent in the cartilaginous center of the growing limb. To determine NCAM function in limb bud chondrogenesis we incubated dissociated stage 22/23 distal mesenchymal limb bud cells with Fab' fragments of antibodies to NCAM. Cell aggregation was inhibited by incubating the cells with anti-NCAM Fab'. These results suggest that NCAM may mediate the formation of precartilaginous condensations. This hypothesis was further tested using micromass cultures. NCAM expression in micromass cultures in vitro recapitulated that in vivo. NCAM was enriched in condensations of 2 day cultures, but was diminished and concentrically distributed around cartilage nodules in 4 day cultures. Anti-NCAM Fab' fragments reduced the area occupied by precartilaginous condensations and the degree of chondrogenic differentiation. Control antibody against chicken embryo fibroblasts had no effect. The effect of overexpressing NCAM was analyzed by electroporating expression vectors directing the synthesis of chicken NCAM. Limb bud cells cultured after electroporation with an NCAM expression vector displayed larger cartilage nodules and greater chondrogenic differentiation than cells electroporated with vector alone. The expression of NCAM in electroporated cells also increased. Control experiments using plasmids encoding beta-galactosidase indicated that approximately 10% of the limb bud cells were transfected under these conditions. The results suggest that NCAM is involved in the chondrogenesis pathway by mediating the formation of precartilaginous condensations.

Animals↗
PubMed1993

Activin enhances chondrogenesis of limb bud cells: stimulation of precartilaginous mesenchymal condensations and expression of NCAM.

The roles of activin action in chicken limb development were analyzed. Activin enhanced chondrogenesis up to five-fold in a concentration-dependent manner in limb bud micromass cultures, with a half-maximal dose around 30 ng/ml (1.25 nM). The response of limb bud cells (stage 22/23) to activin appeared higher within 24 hr after culture. Activin increased the size of precartilaginous condensations. No corresponding increase in cell number was observed with total DNA or [3H]thymidine incorporation. Activin treatment resulted in increased expression of NCAM in precartilaginous condensations and tenascin in cartilage nodules, suggesting that the mechanism of activin is mediated by increased cell adhesion and recruitment of mesenchymal cells into condensations. Our results demonstrate a novel function of activin and imply that activin, together with other TGF beta superfamily members, is involved in the induction of limb chondrogenesis.

Activins↗
PubMed1993

Adhesion molecules and homeoproteins in the phenotypic determination of skin appendages.

We examined the roles of adhesion molecules and homeoproteins in the morphogenesis of skin appendages using feather as a model. The expression pattern of these molecules in different stages of feather development were very dynamic. For example, neural cell adhesion molecules are present first in the dermal condensations, then in distal bud epithelium, then in the dermal papilla, and finally in the marginal and axial plates. Tenascin is present first in the placode, then in the anterior bud epithelium and mesoderm, and then in the dermal papilla. The expression patterns suggest that the adhesion molecules are involved in forming the boundary of cell groups that interact to form skin appendages. Antibody perturbation of embryonic skin-explant cultures showed that liver cell adhesion molecules are involved in establishing the hexagonal pattern, neural cell adhesion molecules are involved in the formation of dermal condensations, tenascin appears to be involved in the growth of feather buds, and integrin is essential for epithelial-mesenchymal interactions. Using antibodies to XlHbox 1 (similar to Hox 3.3 or C6) and Hox 4.2 (or D4), we showed that there is a homeoprotein gradient within the feather buds, and that the expression pattern is position-specific. It is hypothesized that Hox codes, derived from the combined expression pattern of homeoproteins, determine the phenotypes and orientation of skin appendages. Experiments using retinoids in the media or retinoid-soaked beads to create a local retinoid gradient are consistent with this hypothesis. As demonstrated here, feather development provides an excellent opportunity to analyze the molecular cascade of skin-appendage morphogenesis.

Animals↗
PubMed1993

Asymmetric patterns of gap junctional communication in developing chicken skin.

To study the pattern of gap junctional communication in chicken skin and feather development, we injected Lucifer Yellow into single cells and monitored the transfer of the fluorescent dye through gap junctions. Dye coupling is present between cells of the epithelium as well as between cells of the mesoderm. However, dye transfer did not occur equally in all directions and showed several consistent patterns and asymmetries, including: (1) no dye coupling between mesoderm and epithelium, (2) partial restriction of dye coupling at the feather bud/interbud boundary during early feather bud development, (3) preferential distribution of Lucifer Yellow along the anteroposterior axis of the feather placode and (4) absence of dye coupling in some epithelial cells. These results suggest the presence of preferential pathways of communication that may play a role in the patterning of chicken skin.

Animals↗
PubMed1992

Replication-defective virus infection of feather buds produces a localized region of beta-galactosidase activity.

We are interested in using retroviral vectors to trace cell lineage and to introduce exogenous genes in chicken skin explant cultures. Here the LZ10 virus carrying the gene encoding beta-galactosidase was introduced to the skin explants by two different means: a) the virus was added to the media or b) the virus was microinjected into regions of the developing feather buds. Infection by microinjection led to localized expression of beta-galactosidase in the developing feather bud, while, surprisingly, infection by adding the virus to the culture media led to localized band of beta-galactosidase expression in the middle of the feather filament. The significance of this finding in skin morphogenesis and as a tool for experimental embryology is discussed.

Animals↗
PubMed1992

Adhesion molecules in skeletogenesis: I. Transient expression of neural cell adhesion molecules (NCAM) in osteoblasts during endochondral and intramembranous ossification.

We report that neural cell adhesion molecules (NCAM) are expressed transiently in developing chicken osteoblasts during osteogenesis using immunostaining on cryostat sections. NCAM is strongly expressed in most osteoblasts along bone trabeculae that coincide with the presence of collagen I and alkaline phosphatase activity. In endochondral ossification, NCAM is highly expressed in osteogenic buds as seen in the epiphysis and diaphysis of tibia and vertebrae. In intramembranous ossification, NCAM is seen in osteogenic condensation of calvaria and in the periosteum of tibial diaphysis. The expression is transient because NCAM is not expressed in mesenchymal cells before osteogenic condensation and NCAM expression is lost in osteocytes in later stages. The staining pattern suggests that NCAM is present on the cell membrane of osteoblasts. Using a specific monoclonal antibody, the osteoblast NCAM is shown to contain polysialic acid, which is enriched in embryonic brain. Northern blot analysis using chicken brain NCAM cDNA as probes showed two major sizes of mRNA at 6.4 and 4.2 kb in calvarial mRNA as opposed to bands at 7.2, 6.4, and 4.2 kb in the brain. An immunoblot showed major proteins at Mr 165 and 110 kd, unlike brain NCAM, which are 180, 140, and 120 kD. That NCAM is involved in bone morphogenesis is consistent with the general hypothesis that NCAM plays pivotal roles in mesenchymal condensation, as shown in the formation of muscle, kidney, skin, and cartilage. The results establish NCAM as a cell surface molecule expressed transiently during osteoblast lineage. The implication that NCAM may mediate osteoblast interaction and regulate skeletal morphogenesis is discussed.

Animals↗
PubMed1992

Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin.

To understand cell interactions during induction of skin appendages, we studied the roles of adhesion molecules N-CAM, tenascin, integrin, and fibronectin during feather development. Tenascin appeared in a periodic pattern on epithelia and was so far the earliest molecule detected in placodes. Three placode domains were identified: the anterior was positive for tenascin, the distal positive for N-CAM, and the posterior lacking both. Integrin appeared in dermal-epidermal junctions of placodes. In feather buds, sagittal sections revealed a transient anterior-posterior asymmetry with tenascin and N-CAM enriched in the anterior mesoderm. Tangential sections revealed a lateral-medial asymmetry with tenascin distributed in a ring shape and N-CAM in an "X" shape. Integrin was diffusely distributed within buds. Later tenascin and N-CAM were enriched in dermal papilla, the inducer of skin appendages. Perturbation of embryonic skin explant cultures with antibodies showed that anti-integrin beta 1 and anti-fibronectin blocked epithelial-mesenchymal interaction, anti-N-CAM caused uneven segregation of mesenchymal condensation, and anti-tenascin inhibited feather bud elongation. Dose-response curves showed gradual effects by these antibodies. The results indicated that these adhesion molecules are independently regulated and each contributes in different phases during morphogenesis of skin appendages.

Animals↗
PubMed1992

Mechanism of skin morphogenesis. II. Retinoic acid modulates axis orientation and phenotypes of skin appendages.

The factors that determine the axial orientation and phenotypes of skin appendages were analyzed by studying the effect of retinoic acid (RA) on embryonic chicken skin explant cultures. With RA uniformly distributed in the culture media, the feather buds became smaller, were disoriented or were transformed into scale-like structures in a concentration-dependent manner (from 0.05-2.5 microM). With RA distributed as a gradient created by a RA-soaked anion exchange bead, a radial zone of inhibition with a rim of disoriented buds was observed. The new axis of the disoriented buds appeared to be determined by a combination of the original feather axis determining force and a new axial force pointing centrifugally away from the RA source. This observed result can be simulated with a computer model using a vectorial sum of different feather axial determination forces. The size of the inhibited zone is linearly correlated to the RA concentration and may be used to quantify the morphogenetic activity of retinoids. These effects are specific to developmental stages (Hamburg and Hamilton stage 31-34). Both all-trans and 13-cis RA have morphogenetic activity. Retinol has no effect and retinal has a small inhibitory effect but neither phenotypic transformation nor axial disorientation were observed. The antero-posterior gradient of homeoprotein XlHbox 1 in feather buds became diffusive after RA treatment. RA dissolves dermal condensations and the distribution of N-CAM is altered from an anterior localized pattern to a diffusive presence in the bud cores. Endogenous retinoids in developing skins show developmental stage-dependent changes both quantitatively and qualitatively. The results suggest that RA either is or can modulate the endogenous morphogen(s) that determine the orientation and phenotype of skin appendages, and that this morphogenetic pathway involves Hox genes and adhesion molecules.

Animals↗
PubMed1991

Adhesion molecules in skin development: morphogenesis of feather and hair.

Figure 9 summarizes the morphogenetic process of feather and hair. Hair of feathers are formed from a layer of homogeneously distributed mesenchymal cells. The mesenchymal cells start to condense to form foci in response to some unidentified induction signal (Fig. 9B). Several adhesion molecules, including L-CAM, N-CAM, integrin, tenascin, as well as proteoglycan, are involved. These adhesion molecules appear to have different roles in this process, because perturbation with specific antibodies leads to different aborted patterns. Hair or feather follicles then form following cell proliferation and epithelial invagination (Fig. 9C). The dermal papilla is enriched with N-CAM and tenascin, whereas the feather collar (equivalent of hair matrix) is enriched with L-CAM and PDGF receptor. Epithelial cells in the feather collar receive a signal from the dermal papilla and are able to continue to divide. Several growth factors, such as PDGF and EGF, may be involved. As epithelial cells are pushed upwards, they differentiate and keratinize in a cylindrical structure into hair. In feather, another morphogenetic event takes place to form the branched structure. The epithelial cylinder of the feather shaft invaginates to form rows of cells that die to become space and create the secondary branch or barbs (Fig. 9D). N-CAM is enriched in the cells destined to die and appears to form the border of cell groups within which the "death signal" is transmitted. In some, but not all, feathers the same process is repeated, in a way analogous to fractal formation, to form the tertiary branches or the barbules (Fig. 9E). Thus, in each step of the morphogenesis of feather and hair, different adhesion molecules are expressed and are involved in different functions: induction, mesenchymal condensation, epithelial folding, and cell death, depending on different scenarios. We have just begun to elucidate these molecular events.

Animals↗