Avian scale development. IX. Scale formation by scaleless (sc/sc) epidermis under the influence of normal scale dermis

Unlike normal scutate scales whose outer and inner epidermal surfaces elaborate β (β-keratins) and α (α-keratins) strata, respectively, the scaleless mutant's anterior metatarsal epidermis remains flat and elaborates only an α stratum. Reciprocal epidermal-dermal recombinations of presumptive scale tissues from normal and mutant embryos have demonstrated that the scaleless defect is expressed only by the epidermis. In fact, the scaleless anterior metatarsal epidermis is unable to undergo placode formation. More recently, it has been determined that the absence of epidermal placode morphogenesis into a definitive scale ridge actually results in the establishment of a scale dermis which is incapable of inducing the outer and inner epidermal surfaces of scutate scales. Can the initial genetic defect in the scaleless anterior metatarsal epidermis be overcome by replacing the defective dermis with a normal scutate scale dermis, i.e., a dermis with scale ridges already present? Or, are the genes involved in the production of a β stratum regulated by events directly associated with morphogenesis of the epidermal placode? In the present study, we combined scaleless anterior metatarsal epidermis (stages 36 to 42) with normal scutate scale dermis (stage 40, 41, or 42) old enough to have acquired its scutate scale-inducing ability. After 7 days of growth as chorioallantoic membrane grafts, we observed grossly and histologically, typical scutate scales in these recombinant grafts. Electron microscopic and electrophoretic analyses have verified that these recombinant scales are true scutate scales. The scaleless mutation, known to be expressed initially by the anterior metatarsal epidermis, can be overcome by exposing this epidermis to appropriate inductive cues, i.e., cues that direct the differentiation of the outer and inner epidermal surfaces of the scutate scales and the production of specific structural proteins. We have determined that the time between stages 38 and 39 is the critical period during which the normal scutate scale dermis acquires these inductive abilities.     

Differences between normal and scaleless (sc/sc) chicken epidermal cell surfaces detected with wheat germ agglutinin

Dissociated epidermal cells derived from the backskin of scaleless chick embryos (stage 34 or 35) form larger agglutinates with wheat germ agglutinin (WGA) than epidermal cells from normal embryonic skin. [³H]Acetyl WGA binding to the scaleless cells is twice as great as to normal epidermal cells. Treatment of these cells with concanavalin A (conA) results in equivalent agglutination of both mutant and normal epidermal cells, whereas neither scaleless nor normal epidermal cells are agglutinated by Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) or Ulex europeus agglutinin (UEA). This alteration in cell surface carbohydrates may be related to the failure of the scaleless mutant embryonic epidermis to undergo normal morphogenesis.     

Avian scale development. XI. Initial appearance of the dermal defect in scaleless skin

During erythropoiesis, the decrease of complexity of a RNA population is an important process as is globin mRNA accumulation. To determine the sequential control process of gene expression, many genomic clones which express in mouse reticulocytes were obtained and used for the titration of each mRNA level in the different stages of erythroid cells. The level of mRNAs of rt-clones decreases depending on the maturation of erythroid cells, and the coordinated and sequential control of this level is likely to be one of the factors affecting this process.     

Origin of feathers: Feather beta (beta) keratins are expressed in discrete epidermal cell populations of embryonic scutate scales

The feathers of birds develop from embryonic epidermal lineages that differentiate during outgrowth of the feather germ. Independent cell populations also form an embryonic epidermis on scutate scales, which consists of peridermal layers, a subperiderm, and an alpha stratum. Using an antiserum (anti-FbetaK) developed to react specifically with the beta (beta) keratins of feathers, we find that the feather-type beta keratins are expressed in the subperiderm cells of embryonic scutate scales, as well as the barb ridge lineages of the feather. However, unlike the subperiderm of scales, which is lost at hatching, the cells of barb ridges, in conjunction with adjacent cell populations, give rise to the structural elements of the feather. The observation that an embryonic epidermis, consisting of peridermal and subperidermal layers, also characterizes alligator scales (Thompson, 2001. J Anat 198:265-282) suggests that the epidermal populations of the scales and feathers of avian embryos are homologous with those forming the embryonic epidermis of alligators. While the embryonic epidermal populations of archosaurian scales are discarded at hatching, those of the feather germ differentiate into the periderm, sheath, barb ridges, axial plates, barbules, and marginal plates of the embryonic feather filament. We propose that the development of the embryonic feather filament provides a model for the evolution of the first protofeather. Furthermore, we hypothesize that invagination of the epidermal lineages of the feather filament, namely the barb ridges, initiated the formation of the follicle, which then allowed continuous renewal of the feather epidermal lineages, and the evolution of diverse feather forms.     

Region-specific expression of scutate scale type beta keratins in the developing chick beak.

This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed.     

The expression of tenascin by neural crest cells and glia.

The extracellular matrix glycoprotein tenascin is concentrated in both the embryo and adult in regions where cell motility is taking place. For example, during avian neural crest morphogenesis tenascin is concentrated in the rostral half of the sclerotome, precisely where the neural crest cells themselves are found. Previous in vitro studies indicated that somite cells were the source of this tenascin, implying a role for tenascin in directing the ventral migration of neural crest cells and thus the establishment of the periodic arrangement of the PNS. In this study, we have used a cDNA probe to identify the source of tenascin found along the pathways of the neural crest using in situ hybridization. In tissue sections, individual cells found along the neural crest migratory pathways, both before entering the somites and within the somites, are strongly labelled by the tenascin cDNA. In vitro neural crest cells are more strongly labelled with the tenascin probe than somite cells. Finally, western blotting has been used to identify tenascin in culture medium conditioned by neural crest cells. This indicates that neural crest cells themselves are the source of much of the tenascin found lining their migratory pathways, and that interactions with somite cells may not be needed to induce the expression of tenascin. We have also studied the distribution of tenascin mRNA in the developing spinal cord and spinal ganglia. At embryonic days 7 and 10, tenascin cDNA hybridizes within cells that appear to be migrating from the ependymal layer to the white matter, as well as within cells in the dorsal roots.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered tenascin expression during spontaneous endometrial carcinogenesis in the BDII/Han rat.

The BDII/Han rat develops spontaneous endometrial adenocarcinoma, which appears virtually identical histologically to human endometrial adenocarcinoma. The incidence rate of cancer formation in the rat is 90% and the mean lifetime of the animals is 22 months. This animal model therefore, is useful in the study of molecular aspects of spontaneous transformation as well as mammalian neoplastic progression. In this study we address the in-situ expression of tenascin, an extracellular matrix glycoprotein, during normal cyclic growth, during development of proliferative states, and during malignant transformation of the endometrium. Trace amounts of immunocytochemically detectable tenascin were found in 10% of young BDII/Han rats with a normal estrus cycle. In these inbred animals no tenascin was detectable in uteri without neoplastic progressive alterations of the endometrium. Tenascin immunoreactivity first appeared during proliferation in one of three uteri with cystic glandular hyperplasia. Prominent tenascin expression was detectable in all adenomatous hyperplasia, but restricted to the stromal mesenchyme, that surrounded the glands. In all endometrial adenocarcinomas tested, essentially the entire extracellular space of the stromal mesenchyme was immunoreactive with anti-tenascin antibodies while the epithelial glands themselves were negative. This staining pattern was observed independent of the degree of tumor differentiation or extent of myometrial invasion. The tenascin staining pattern was not significantly altered in tumors transplanted into the soft tissues of the neck of female BDII/Han rats. From our studies we conclude that tenascin may be a marker for the early detection of proliferative endometrial states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue distribution and appearance in ontogeny of alpha/beta T cell receptor (TCR2) in chicken

We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens. We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens. TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens. Some TCR2+ cells were found in the cortex of bursal follicles and in liver. The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.     

How and when the regional competence of chick epidermis is established: feathers vs. scutate and reticulate scales, a problem en route to a solution

Most of the chick body is covered with feathers, while the tarsometatarsus and the dorsal face of the digits form oblong overlapping scales (scuta) and the plantar face rounded nonoverlapping scales (reticula). Feathers and scuta are made of beta-keratins, while the epidermis of reticula and inter-appendage or apteria (nude regions) express a-keratins. These regional characteristics are determined in skin precursors and require an epidermal FGF-like signal to be expressed. Both the initiation of appendages, their outline and pattern depend on signals from the dermis, while their asymmetry and outgrowth depend on epidermal competence. For example, the plantar dermis of the central foot pad induces reticula in a plantar or feathers in an apteric epidermis, in a hexagonal pattern starting from the medial point. By manipulating Shh levels in the epidermis, the regional appendage type can be changed from scuta or reticula to feather, whereas the inhibition of Wnt7a, together with a downregulation of Shh gives rise to reticula and in extreme cases, apteria. During morphogenesis of plantar skin, the epidermal expression of En-1, acting as a repressor both of Wnt7a and Shh, is linked to the formation of reticula. Finally, in birds, the complex formation of feathers, which can be easily triggered, even in the extra-embryonic somatopleure, may result from a basic genetic program, whereas the simple formation of scales appears secondarily derived, as requiring a partial (scuta) or total (reticula) inhibition of epidermal outgrowth and beta-keratin gene expression, an inhibition lost for the scuta in the case of feathered feet breeds.     

Nerve-dependent and -independent tenascin expression in the developing chick limb bud.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.     

The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans

Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations. Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes). The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila). The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi). Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA. By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences-- was found whose genomic DNA contained this sequence in detectable amounts. A computer search also failed to find the TG element in human mitochondrial DNA.      

Phylogeny and biogeography of the scaleless scale worm Pisione (Sigalionidae,Annelida)

Pisione is a scaleless group of small scale worms inhabiting sandy bottoms in shallow marine waters. This group was once considered rare, but now 45 described species can be characterized, among others, by their paired, segmental copulatory organs (one to multiple external pairs), which display a complexity of various accessory structures. The evolutionary significance of these unique organs was suggested in the late 1960s, but has been heavily debated since the late 1990s and remains controversial. In the present paper, we study the internal relationships within Pisione, employing combined phylogenetic analyses of both molecular and morphological data from 16 terminals of Pisione, as well as two terminals of Pisionidens, and eight additional scale worms as outgroups. Our taxon sampling covers all geographical areas where the genus has been reported, as well as most of their morphological and copulatory variability, including representatives of the “africana,” “remota,” “crassa,” and “papuensis” groups, established previously by Yamanishi. We hereby provide a first insight into the relationships of the genus, testing previously proposed hypotheses on the evolutionary significance of male copulatory structures within Pisione, while attempting to understand patterns of distribution. The phylogenetic analyses using maximum likelihood and Bayesian methods consistently recovered two large clades spanning the East Atlantic (including the Mediterranean) and the Indo‐Pacific–West Atlantic, respectively. Character optimization on our trees revealed a high degree of homoplasy in both non‐reproductive and sexual characters of Pisione, with buccal acicula found to be the sole apomorphy among the morphological features assessed herein, with none defining the biogeographical subclades within. Overall, our comparative analyses highlight the high degree of morphological variation in this widely distributed genus, rejecting previous assertions of an increasing number and complexity of copulatory structures across the genus.     

The chicken alpha 1 (XI) collagen gene is widely expressed in embryonic tissues.

Complementary DNA and genomic DNA clones corresponding to the chicken alpha 1 (XI) collagen gene were isolated and characterized. These recombinant DNA clones covered 2667 base pairs of the mRNA and encode 624 amino acids of the triple helical region plus the entire carboxyl-terminal propeptide. Northern blot analysis showed a major band of approximately 6.5 kilobases and a minor band of approximately 7.5 kilobases. A combination of Northern blot and in situ hybridization analyses showed that, in addition to its presence in cartilage, this mRNA also is present in a wide variety of chicken noncartilaginous embryonic tissues including brain, heart, skeletal muscle, calvaria, and skin, but was not detected in liver. Type II collagen mRNA has also been detected at low levels in these same tissues. Also, similar to the mRNA for the alpha 1 chain for type II collagen, the alpha 1 (XI) collagen mRNA is detected in limb mesenchyme prior to condensation and differentiation of the core mesenchyme into cartilage.     

The chicken stathmin gene and its expression in the embryo.

Stathmin, which functions as an intracellular relay in signal transduction pathways, has been suggested as a potential indicator of pluripotent cells in the early mouse embryo. In this study, chicken stathmin cDNA and genomic DNA were analyzed. In mammals stathmin consists of five exons and four introns; exons 3, 4, and 5 in the mammalian stathmin gene are equivalent to one relatively large exon in the chicken stathmin gene. Introns equivalent to introns 3 and 4 in the mammalian stathmin gene are not present in the counterpart gene in chickens and, although intron 2 was shown to be present in both mammals and birds, it is smaller in the chicken stathmin gene. Despite differences in the genomic organization of the gene and its smaller size in chickens compared with that in humans and mice, similarities in the coding sequences and in the expression of the chicken and mouse stathmin genes at certain stages of embryo development, as determined by whole-mount in situ hybridization experiments, suggest that their products are functional homologues. The argument is thus substantiated for further investigations into the use of regulatory regions of the stathmin gene in a system for the establishment of long-term cultures of germline competent chicken embryonic stem (ES) cells by the selective ablation of differentiated cells in culture using drug selection.     

The vesicular monoamine transporter 2 (VMAT2) is expressed by normal and tumor cutaneous mast cells and Langerhans cells of the skin but is absent from Langerhans cell histiocytosis.

Monoamine storage in secretory granules is mediated by the vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2). The aim of our study was to identify monoamine-handling normal and neoplastic inflammatory cells in the skin by their expression of VMAT1 and VMAT2. Normal skin from various parts of the body, as well as 21 cases of cutaneous mastocytosis and 10 cases of cutaneous Langerhans cell histiocytosis were analyzed by immunohistochemistry, radioactive in situ hybridization, and double-fluorescence confocal microscopy. VMAT2-positive cells in the subepidermal layer were identified as mast cells by their expression of tryptase. Neoplastic mast cells in all cases of cutaneous mastocytosis retained their VMAT2 positivity. The intraepidermal VMAT2-expressing cells were identified as Langerhans cells by their CD1a positivity. VMAT2 was absent from Langerhans cell histiocytosis. VMAT2 is an excellent marker for normal and neoplastic mast cells. The expression of VMAT2 demonstrates the capacity of mast cells for monoamine storage and handling. The presence of VMAT2 in epidermal Langerhans cells revealed a previously unrecognized monoamine-handling phenotype of these cells and indicates possible involvement of amine storage and release associated with antigen presentation. Absence of VMAT2 in neoplastic Langerhans cells indicates a loss of monoamine handling capacity of these cells during tumorigenesis.     

The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts.

Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl-terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that recognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor-like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phenotypic changes.     

Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis.

The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage-specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum.