The Molecular Circuit Regulating Tooth Development in Crocodilians.

Alligators have robust regenerative potential for tooth renewal. In contrast, extant mammals can either renew their teeth once (diphyodont dentition, as found in humans) or not at all (monophyodont dentition, present in mice). Previously, the authors used multiple mitotic labeling to map putative stem cells in alligator dental laminae, which contain quiescent odontogenic progenitors. The authors demonstrated that alligator tooth cycle initiation is related to ß-catenin/Wnt pathway activity in the dental lamina bulge. However, the molecular circuitry underlying the developmental progression of polyphyodont teeth remains elusive. Here, the authors used transcriptomic analyses to examine the additional molecular pathways related to the process of alligator tooth development. The authors collected juvenile alligator dental laminae at different developmental stages and performed RNA-seq. This data shows that Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) pathways are activated at the transition from pre-initiation stage (bud) to initiation stage (cap). Intriguingly, the activation of Wnt ligands, receptors and co-activators accompanies the inactivation of Wnt antagonists. In addition, the authors identified the molecular circuitry at different stages of tooth development. The authors conclude that multiple pathways are associated with specific stages of tooth development in the alligator. This data shows that Wnt pathway activation may play the most important role in the initiation of tooth development. This result may offer insight into ways to modulate the genetic controls involved in mammalian tooth renewal.

Immunohistochemical localization of deleted-in-colon-cancer (DCC) protein in human epithelial, neural, and neuro-endocrine tissues in paraffin sections with antigen retrieval.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins, and it is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is know to occur, resulting in different isoforms of the protein. Using a polyclonal antibody against the DCC gene product, we have demonstrated, by antigen retrieval immunostaining, the presence of a DCC protein isoform on the cell surface of goblet cells in the G-I tract, cytoplasm of squamous epithelium in the skin & esophagus and transitional epithelium in the urinary bladder, ductal glandular epithelium of endometrium, endocervix, prostate, gall bladder and breast, cytoplasm of neuron in the cerebral cortex and Purkinje cells in the cerebellum. In addition, we also demonstrated DCC protein expression in neuroendocrine cells including argentaffin cells of the stomach, small intestine, appendix and colon, C cells of thyroid gland, chromaffin cells of the adrenal gland, islets of Langerhans in the pancreas and neurons of the sympathetic ganglion. This restrictive distribution suggests the DCC gene products may be abundant expression in neuroendocrine cells of human tissue.

A Novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells.

The templates required for inducing posttranscriptional gene silencing (PTGS) effects have been investigated in human prostate cancer LNCaP cells. Transfection of a mRNA-cDNA hybrid construct was found to result in a relatively long-term interference of specific gene expression. Androgen-stimulated expression of bcl-2 has been reported to increase the tumorigenic and metastatic potentials of human prostate cancer LNCaP cells, as well as their resistance to many apoptotic stimuli. The addition of bcl-2 antisense oligonucleotides, however, restored apoptosis. Our studies demonstrate gene silencing effects of the mRNA-cDNA transfection that is similar to those of PTGS/RNAi in this in vitro prostate cancer cell model. A potential RNA-directed RNA polymerase activity was also detected which is alpha-amanitin-sensitive. These findings indicate that a novel gene silencing system may exist in mammalian cells.

D-RNAi (messenger RNA-antisense DNA interference) as a novel defense system against cancer and viral infections.

D-RNAi (Messenger RNA-antisense DNA interference), a novel posttranscriptional phenomenon of silencing gene expression by transfection of mRNA-aDNA hybrids, was originally observed in the effects of bcl-2 on phorbol ester-induced apoptosis in human prostate cancer LNCaP cells. This phenomenon was also demonstrated in chicken embryos and a human CD4(+) T cell line, H9. The in vivo transduction of beta-catenin D-RNAi was shown to knock out more than 99% endogenous beta-catenin gene expression, while the in cell transfection of HIV-1 D-RNAi homolog rejected viral gene replication completely. D-RNAi was found to have long-term gene knockout effects resulting from a posttranscriptional gene silencing mechanism that may involve the homologous recombination between intracellular mRNA and the mRNA components of a D-RNAi construct. These findings provide a potential intracellular defense system against cancer and viral infections.

beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin.

We explored the role of beta-catenin in chicken skin morphogenesis. Initially beta-catenin mRNA was expressed at homogeneous levels in the epithelia over a skin appendage tract field which became transformed into a periodic pattern corresponding to individual primordia. The importance of periodic patterning was shown in scaleless mutants, in which beta-catenin was initially expressed normally, but failed to make a punctuated pattern. To test beta-catenin function, a truncated armadillo fragment was expressed in developing chicken skin from the RCAS retrovirus. This produced a variety of phenotypic changes during epithelial appendage morphogenesis. In apteric and scale-producing regions, new feather buds with normal-appearing follicle sheaths, dermal papillae, and barb ridges were induced. In feather tracts, short, wide, and curled feather buds with abnormal morphology and random orientation formed. Epidermal invaginations and placode-like structures formed in the scale epidermis. PCNA staining and the distribution of molecular markers (SHH, NCAM, Tenascin-C) were characteristic of feather buds. These results suggest that the beta-catenin pathway is involved in modulating epithelial morphogenesis and that increased beta-catenin pathway activity can increase the activity of skin appendage phenotypes. Analogies between regulated and deregulated new growths are discussed.

Suppression of activin-induced apoptosis by novel antisense strategy in human prostate cancer cells.

Apoptosin, a novel gene encoding a mitotic kinase-motif protein, is stimulated by activin, a member of TGF-beta family, in human LNCaP prostate cancer cells and in patient tissues. We employed a gene knockout methodology based on the covalent bonding of chemically modified antisense probes to apoptosin mRNAs in LNCaP cells. The mRNA-antisense hybrid duplexes were neither translated nor post-transcriptionally modified, resulting in no protein synthesis. Introducing antisense apoptosin into activin-induced apoptotic LNCaP cells prevented apoptosis, interfered with genomic DNA fragmentation and released cell cycle checkpoint. These findings suggest that the apoptosin, in addition to p53, is important in apoptotic regulation of human prostate cancers.

In vivo analysis of cancerous gene expression by RNA-polymerase chain reaction.

An easy and routine procedure to amplify messenger RNA (mRNA) libraries from a few tissue cells can provide molecular gene expression profiles at high resolution. A novel PCR-like method, the RNA-PCR, was developed to generate high quality and quantity mRNAs from as few as 20 cells (2 pg mRNAs). The principle relies upon the cycling steps of promoter-linked double-stranded cDNA synthesis and promoter-driven transcription to amplify mRNAs up to 250-fold/cycle with good representation of high and low copy mRNAs. The amplified mRNA libraries were shown to possess high fidelity, purity, specificity and reproducibility for in vivo analyses of cancerous gene expression in human prostate cancers.

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.

Successive formative stages of precartilaginous mesenchymal condensations in vitro: modulation of cell adhesion by Wnt-7A and BMP-2.

High-density chick limb bud cell culture is a useful model to study mesenchymal condensatifons and chondrogenesis. Most previous studies have focused on the effects of soluble reagents on terminal chondrogenic differentiation and have not defined the early cellular processes and signaling events. In this study, we defined five successive stages in the differentiation process: 1) dissociated cells, 2) small aggregates, 3) formation of cell clusters, 4) precartilaginous condensations, and 5) cartilage nodule. We used RCAS retrovirus-mediated Wnt-7a gene transduction to test the effect of Wnt-7a on the differentiation process. We found that Wnt-7a suppressed chondrogenic differentiation. Wnt-7a did not inhibit the initiation of condensation formation but blocked the progression of precartilaginous condensations to cartilage nodules. The Wnt-7a-transduced cultures showed characteristics of a less mature culture with persistent expression of NCAM, N-cadherin, wider distribution of integrin beta1 and fibronectin, and suppression of tenascin-C. BMP-2 is known to enhance chondrogenic differentiation in these cultures by promoting cell clusters to form continuous sheet-like precartilaginous condensations. However, cultures exposed to both BMP-2 and Wnt-7a showed inhibition of chondrogenic differentiation. Different signaling molecules such as Wnt-7a and BMP-2 may have antagonistic effects on cartilage differentiation and the gradient of the two molecules may be involved in defining the boundaries of the initial precartilaginous condensation. We propose that the shape of the precartilaginous condensations may be modulated by local concentrations of signaling molecules, such as Wnt-7a and BMP-2, which act to alter cell-substrate and cell-cell adhesions.

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.

Phenotypic determination of epithelial appendages: genes, developmental pathways, and evolution.

Epithelial appendages are derivatives of epithelia that elaborate to form specialized structures and functions. The appendage can protrude out, such as in teeth and feathers, or invaginate in, such as in glands. The epithelia can be ectodermal, such as in hairs, or endodermal, such as in livers. Using feather as a prototype of epithelial appendage, we study the molecular signals involved in the successive stages of epithelial-mesenchymal interactions during morphogenesis. We propose that these form the basics of gene networks, which can be integrated to gene supernetwork and totinetwork. Because the unit of development is molecular pathway rather than single molecule, and the unit of morphogenesis is cell group rather than single cell, we make the analogy between genes/developmental pathways and words/sentences. The study of developmental pathways in epithelial appendage organogenesis will help us to understand the grammar of genes and the basic rules in constructing regulated new growth. This knowledge may contribute to the study of cancer biology (deregulated new growth) and organ regeneration.

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.

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.

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.

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.

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.

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.

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.