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Biomedical subjects

C M Chuong

Publications and source records attributed to C M Chuong.

At least 37 records · Page 2Linked to original sources
PubMed1999

Avian integument provides multiple possibilities to analyse different phases of skin appendage morphogenesis.

To analyse the morphogenic events during skin appendage formation, it is important to have an animal model that offers distinct patterns at various stages of development and is accessible to analysis using state of the art technology. The avian integument is such a model. Combining experimental embryologic approaches, organ cultures, and gene transduction technology, we are now able to begin to address the molecular basis of pattern formation, primordium initiation, anterior-posterior axis formation, proximo-distal axis formation, phenotypic determination, and others. Parallel mechanisms are usually found in feathers and hairs, and the avian integument model has matured to be a major source of new findings in the study of skin appendage morphogenesis. More information on the avian integument model can be found at website http://www.hsc.usc.educmchuong.

Animals↗
PubMed1999

Epidermal dysplasia and abnormal hair follicles in transgenic mice overexpressing homeobox gene MSX-2.

The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin beta 1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages.

Abnormalities, Multiple↗
PubMed1999

Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model.

How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity revealed a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiated all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression was induced later as a dot within the primordia. RCAS was used to overexpress Wnt-7a in reconstituted feather explants derived from stage 29 dorsal skin to further test its function in feather formation. Control skin formed normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression led to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also had inhibited elongation of the P-D axes. This was not due to a lack of cell proliferation, which actually was increased although randomly distributed. While morphogenesis was perturbed, differentiation proceeded as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 were diffusely expressed. The results showed that ectopic Wnt-7a expression enhanced properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds.

Animals↗
PubMed1999

Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia.

Periodic patterning is a fundamental organizing process in biology. Using a feather reconstitution assay, we traced back to the initial stage of the patterning process. Cells started from an equivalent state and self-organized into a periodic pattern without previous cues or sequential propagation. When different numbers of dissociated mesenchymal cells were confronted with a piece of same-sized epithelium, the size of feather primordia remained constant, not the number or interbud spacing, suggesting size determination is intrinsic to dissociated cells. Increasing bone morphogenetic protein (BMP) receptor expression in mesenchymal cells decreased the size of primordia while antagonizing the BMP pathway with Noggin increased the size of primordia. A threshold number of mesenchymal cells with a basal level of adhesion molecules such as NCAM were sufficient to trigger the patterning process. The process is best visualized by the progressive restriction of beta-catenin transcripts in the epidermis. Therefore, feather size, number and spacing are modulated through the available morphogen ligands and receptors in the system.

Animals↗
PubMed1998

Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning; however, no molecular candidates have been identified. Here we show that the feather tract is initiated by a continuous stripe of Shh, Fgf-4, and Ptc expression in the epithelium, which then segregates into discrete feather primordia that are more strongly Shh and Fgf-4 positive. The primordia also become Bmp-2 and Bmp-4 positive. Bead-mediated delivery of BMPs inhibits local feather formation in contrast with the activators, SHH and FGF-4, which induce feather formation. Both FGF-4 and SHH induce local expression of Bmp-4, while BMP-4 suppresses local expression of both. FGF-4 also induces Shh. Based on these findings, we propose a model that involves (1) homogeneously distributed global activators that define the field, (2) a position-dependent activator of competence that propagates across the field, and (3) local activators and inhibitors triggered in sites of individual primordia that act in a reaction-diffusion mechanism. A computer simulation model for feather pattern formation is also presented.

Animals↗
PubMed1998

Lineage and pluripotentiality of epithelial precursor cells in developing chicken skin.

How do epithelial cells in developing skin accommodate the constantly growing embryo? Where do cells in skin appendages come from? Are they derivatives of a single appendage stem cell, or are they polyclonal? Here we analyze these issues in developing chicken skin using a replication-defective virus carrying beta-galactosidase and DiI microinjections. The results demonstrate that in early skin, epithelial cells labelled near the spine show a parallel linear stripe distribution pattern that is perpendicular to the midline of the trunk. This is similar to the human lines of Blaschko, a linear pattern on the skin, which many skin nevoid or acquired disorders follow. In later skin, feather buds form and contain a mixture of labeled and unlabeled cells, attesting to their polyclonal origin. When cells are traced for shorter time intervals, the labeled progeny appear to follow certain rules. The degree of cell dispersion and mixing increases with a longer incubation period between the time of labeling and detection. The spatial maturation sequence of skin appendages is not regulated by the order in which epithelial cells are generated. Epithelial cells at this developmental stage are pluripotent and competent to respond to new signals to assume appropriate fates according to their micro-environment. The results suggest that local interactions act upon the originally linearly deposited pluripotential epithelial cells to form skin appendages.

Animals↗
PubMed1998

Retroviral gene transfer in chondrogenic limb bud micromass cultures.

We report development of a model of retroviral gene transduction in high-density limb bud cell micromass culture. The replication competent avian retrovirus RCAS BP (A) carrying the human placental alkaline phosphatase gene (RCAS AP) was used as a marker for retroviral infection and spread. The final protocol balances the need to allow time for retroviral integration and gene transduction against loss of chondrogenic potential when limb bud cells are plated at low density. It includes: (i) incubation of the dissociated limb bud cells with RCAS virus for 2 h followed by low-density culture for 48 h to allow retroviral gene expression; and (ii) secondary replating as high-density micromass culture to initiate chondrogenesis. The pattern and level of chondrogenesis in the retrovirus-transduced micromass cultures is similar to regular micromass cultures. At least 40%-50% of cells express the retroviral-transduced genes 24 h after high-density plating. This new approach facilitates ectopic gene expression in micromass culture, enabling molecular dissection of chondrogenesis and serves as a model for gene transduction in other organotypic cultures.

Alkaline Phosphatase↗
PubMed1997

Isolation and characterization of chicken beta-catenin.

beta-catenin interacts with a number of proteins in different important biological processes, including cell adhesion through cadherins, actin organization through fascin, body axis determination through Wnt signaling, tumor suppression through APC, and transcriptional activation through LEF-1. To examine its function in chicken embryogenesis, we isolated the chicken homolog of beta-catenin from a chicken embryo cDNA library. The sequence is highly conserved at the amino acid level between chicken, mouse (99%), human (99%) and Xenopus (97%). In-situ hybridization and immunostaining showed that in the developing limb, it is specifically expressed in the apical ectodermal ridge, suggesting a role in epithelial-mesenchymal interactions.

Amino Acid Sequence↗
PubMed1997

Molecular histology in skin appendage morphogenesis.

Classical histological studies have demonstrated the cellular organization of skin appendages and helped us appreciate the intricate structures and function of skin appendages. At this juncture, questions can be directed to determine how these cellular organizations are achieved. How do cells rearrange themselves to form the complex cyto-architecture of skin appendages? What are the molecular bases of the morphogenesis and histogenesis of skin appendages? Recently, many new molecules expressed in a spatial and temporal specific manner during the formation of skin appendages were identified by molecular biological approaches. In this review, novel molecular techniques that are useful in skin appendage research are discussed. The distribution of exemplary molecules from different categories including growth factors, intracellular signaling molecules, homeobox genes, adhesion molecules, and extracellular matrix molecules are summarized in a diagram using feather and hair as models. We hope that these results will serve as the ground work for completing the molecular mapping of skin appendages which will refine and re-define our understanding of the developmental process beyond relying on morphological criteria. We also hope that the listed protocols will help those who are interested in this venture. This new molecular histology of skin appendages is the foundation for forming new hypotheses on how molecules are mechanistically involved in skin appendage development and for designing experiments to test them. This may also lead to the modulation of healing and regeneration processes in future treatment modalities.

Animals↗
PubMed1997

Asymmetric expression of Notch/Delta/Serrate is associated with the anterior-posterior axis of feather buds.

We studied the roles of Notch, Delta, and Serrate in vertebrate epithelial appendage morphogenesis using feather as a model and found the following. (1) C-Notch-1, C-Delta-1, and C-Serrate-1 are not expressed at the early placode stage and are therefore not involved in the determination of bud versus interbud compartments. (2) From symmetric short buds to asymmetric long buds, C-Delta-1 and C-Serrate-1 are expressed in the posterior bud mesenchyme in a nested fashion, while C-Notch-1 is expressed as a stripe perpendicular to the anterior-posterior (A-P) axis and positioned posterior to the midpoint. (3) Epithelial-mesenchymal recombination with rotation led to the disappearance of these genes followed by their reappearance with new positions appearing to predict their new morphological orientation. (4) Conditions leading to branched buds (e.g., recombination of later buds) show polarized staining patterns before branching occurs. (5) Conditions leading to symmetrical round buds (e.g., treated with the protein kinase A agonist forskolin) suppress expression of all three genes. These results lead us to hypothesize that Notch, Delta, and Serrate are involved in establishing the A-P asymmetry of feather buds.

Animals↗
PubMed1997

Activation of protein kinase A is a pivotal step involved in both BMP-2- and cyclic AMP-induced chondrogenesis.

We studied the roles of protein kinase A (PKA) activation and cyclic AMP response element binding protein (CREB) phosphorylation in chondrogenesis using serum-free chicken limb bud micromass cultures as a model system. We showed the following points: 1) in micromass cultures, activation of PKA enhances chondrogenesis and increases the phosphorylation of CREB; 2) BMP-2, a chondrogenic stimulator, increases PKA activity and the level of phosphorylated CREB (P-CREB); 3) H8, a PKA inhibitor, inhibits chondrogenesis; 4) the chondrogenic activities of BMP-2 and cAMP are suppressed by H8; and 5) long-term TPA treatment (a protein kinase C (PKC) modulator) inhibits chondrogenesis and decreases the levels of CREB and P-CREB. These results suggest that activation of PKA is a physiological event during chondrogenesis that is involved in the chondrogenic effects of both BMP-2 and cyclic AMP (cAMP)-dependent pathways.

Animals↗
PubMed1997

Expression of two Ig family adhesion molecules in the murine hair cycle: DCC in the bulge epithelia and NCAM in the follicular papilla.

The hair cycle involves remodeling of cells and of cell groups into a complex follicular structure. During skin appendage development, adhesion molecules such as neural cell adhesion molecule (NCAM) and deleted in colon carcinoma (DC) participate in the formation of cell groups. NCAM has been found to be expressed in the mesenchyme during mouse hair follicle induction. DCC expression has been observed in the epithelial cells of the developing feather. We postulate that these two molecules may also define cell groups in the cycling hair follicle. Here we report their spatio-temporal expression patterns during the depilation-induced murine hair cycle. NCAM expression was also examined in positive and negative hair-inductive follicular papilla cell lines. Throughout the hair cycle, DCC expression was confined to the basal keratinocytes of the epidermis and the epithelial portion of the hair follicle. During mid-anagen, two types of deleted in colon carcinoma staining were observed. One was a cell surface pattern seen in the epithelial cells in the bulge region where the follicular stem cells reside. The other was a diffuse cytoplasmic staining pattern in the transient hair follicle epithelia located below the bulge region. Prominent NCAM staining was observed in the follicular papilla throughout the hair cycle and was accompanied by weak staining of the matrix epithelia. NCAM expression correlated with hair induction by a follicular papilla cell line. The results suggest that DCC and NCAM define the permanent cell groups of the hair follicle and that NCAM is important for hair induction.

Animals↗
PubMed1997

Dual action of sonic hedgehog on chondrocyte hypertrophy: retrovirus mediated ectopic sonic hedgehog expression in limb bud micromass culture induces novel cartilage nodules that are positive for alkaline phosphatase and type X collagen.

Members of the vertebrate hedgehog gene family (HH) are involved in patterning and modulation of differentiation. Recently it has been shown that ectopic expression of HH gene family members in vivo blocks chondrocyte maturation through activation of a parathyroid hormone related peptide (PTHrP) dependent negative regulatory loop in the perichondrium. However, the direct effect of HH on chondrocyte maturation has not been tested. Here, we studied the effect of retroviral overexpression of the chicken sonic hedgehog gene (Shh) on the growth and maturation of limb bud cells in micromass cultures. Shh is neither expressed nor required for the initiation of cellular condensation in normal micromass cultures. With Shh over-expression, micromass cultures developed novel tightly whorled nodules in addition to the normal Alcian Blue positive cartilage nodules. We characterized the new nodules and showed that they are strongly positive for alkaline phosphatase, enriched in type X collagen and weakly positive for Alcian Blue staining. Shh overexpression also increased cell proliferation, but this cannot account for the formation of the new nodules. This current study shows that misexpression of Shh in in vitro chondrogenic cultures promotes characteristics of hypertrophic chondrocytes. Thus HH has two complementary functions; a direct positive effect on chondrocyte hypertrophy in the absence of PTHrP pathway, and an indirect negative feedback loop through PTHrP to prevent other less differentiated chondrocytes from becoming hypertrophic. These two complementary actions of HH coordinate the progression of cartilage maturation.

Alkaline Phosphatase↗
PubMed1996

Local delivery of TGF beta2 can substitute for placode epithelium to induce mesenchymal condensation during skin appendage morphogenesis.

Development of skin appendages requires interactions between the epithelium and mesenchyme. Without the epithelium, dermal condensations cannot develop, and those already formed will disintegrate. Here we explored the molecular basis of this epithelial requirement and tried to identify the molecule(s) responsible by using the chick feather bud development as a model. TGF beta2 is a likely candidate because its message is predominantly expressed in the feather bud epithelium, and the protein is enriched in the dermal-epidermal junction within the bud. We tested this hypothesis by placing TGF beta-soaked beads on skin explants. We found that TGF beta2, but not TGF beta1, beads placed on top of epithelially stripped mesenchymes can induce dermal condensations. NCAM and tenascin-C (Tn-C) are expressed and protein kinase C is suppressed in the normal feather bud domain. This molecular organization is lost in denuded mesenchyme but can be restored by TGF beta2-coated beads. Subsequently, the TGF beta2-induced dermal condensations can induce nascent epithelium to form skin appendages. Together with our recent findings that ectopic Sonic hedgehog (Shh) expression causes wider TGF beta expression and larger dermal condensation, these results strongly suggest that TGF beta2 produced by epithelial placode is downstream to Shh and plays a key role in the induction of dermal condensation by activating the expression of NCAM and Tn-C, and by suppressing PKC expression.

Animals↗
PubMed1996

cAMP, an activator of protein kinase A, suppresses the expression of sonic hedgehog.

In Drosophila, it has been shown that protein kinase A and hedgehog have antagonistic actions during the formation of imaginal disks. In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. using an in vitro explant culture model we showed that dibutyryl cAMP, a protein kinase A (PKA) activator, suppresses the expression of Sonic hedgehog, (Shh) and continuous feather growth. The results suggest that Shh and PKA also have antagonistic action during vertebrate skin morphogenesis.

Animals↗
PubMed1996

Sonic Hedgehog in feather morphogenesis: induction of mesenchymal condensation and association with cell death.

Sonic hedgehog is involved in vertebrate tissue interactions during development. During early feather development, Sonic hedgehog appears very early in epithelial placodes. During late feather development, Sonic hedgehog expression precedes the development of the marginal plates and is specifically localized in the marginal plate epithelium, which will later undergo cell death. By using retroviral vectors, exogenous Sonic hedgehog overexpression in developing feathers induced enlarged feather buds that have either lost their anterior-posterior polarity or exhibited reverse orientation. The enlarged dermal condensations may be mediated through broader TGF-beta 2 expression and reduced protein kinase C (PKC) expression. Reciprocal mesenchymal interaction is required for the induction and maintenance of Sonic hedgehog in the epithelial placodes. In scaleless mutant, Sonic hedgehog is absent in the apteric region and aberrantly expressed in the mesenchyme of the abnormal feather ridge. These findings suggest that Sonic hedgehog mediates key interactions between the epithelium and mesenchyme during feather morphogenesis.

Animals↗
PubMed1996

FGF induces new feather buds from developing avian skin.

Induction of skin appendages involves a cascade of molecular events. The fibroblast growth factor (FGF) family of peptide growth factors is involved in cell proliferation and morphogenesis. We explored the role of the FGFs during skin appendage induction using developing chicken feather buds as a model. FGF-1, FGF-2, or FGF-4 was added directly to the culture medium or was released from pre-soaked Affigel blue beads. Near the midline, FGFs led to fusion of developing feather buds, representing FGFs' ability to expand feather bud domains in developing skin. In lateral regions of the explant where feather placodes have not formed, FGF treatment produces a zone of condensation and a region with an increased number of feather buds. In ventral epidermis that is normally apteric (without feathers), FGFs can also induce new feather buds. Like normal feather buds, the newly induced buds express Shh. The expression of Grb, Ras, Raf, and Erk, intracellular signaling molecules known to be downstream to tyrosine kinase receptors such as the FGF receptor, was enriched in feather bud domains. Genistein, an inhibitor of tyrosine kinase, suppressed feather bud formation and the effect of FGF. These results indicate that there are varied responses to FGFs depending on epithelial competence. All the phenotypic responses, however, show that FGFs facilitate the formation of skin appendage domains.

Animals↗