Effects of retinoic acid and Gbx1 on feather-bud formation and epidermal transdifferentiation in chick embryonic cultured dorsal skin.

BACKGROUND: Retinoic acid, an active metabolite of retinol, is known to regulate cell proliferation, differentiation, and morphogenesis during normal development of many tissues. Using chick embryonic tarsometatarsal skin, we showed previously that the expression of Gbx1, a divergent homeobox gene, is increased in the epidermis through interaction with retinol-pretreated dermal fibroblasts followed by epidermal transdifferentiation to mucous epithelium. This present study was performed to elucidate the effects of retinoic acid and Gbx1 on feather-bud formation and epidermal transdifferentiation. RESULTS: We showed that Gbx1 was expressed in the chick embryonic dorsal epidermis as early as at placode stage (Hamburger and Hamilton stage 31) and increased in amount during feather-bud formation. Treatment with 1 µM retinoic acid for 24 hr inhibited feather-bud formation and induced the transdifferentiation of the epidermis to a mucosal epithelium with a concomitant increase in Gbx1 mRNA expression in the epithelium. Furthermore, transient transfection of the epidermis with Gbx1 cDNA by electroporation induced elongation of the feather bud, but did not result in transdifferentiation. CONCLUSIONS: These results indicate that Gbx1 was involved in the feather-bud formation and was one of target genes of retinoic acid and that other signals in addition to Gbx1 were required for epidermal mucous transdifferentiation.

Overexpression of hematopoietically expressed homeoprotein induces nonapoptotic cell death in mouse prechondrogenic ATDC5 cells.

Physiological cell death is an essential event in normal development and maintenance of homeostasis. Recently, the morphological and pharmacological characteristics of programmed cell death, which are distinct from those of apoptosis under physiological and pathological conditions, have been reported. However, the molecular mechanism and executioner of this type of cell death are unknown. We show that overexpression of hematopoietically expressed homeoprotein (Hex), a homeoprotein of divergent type, and enhanced green fluorescent protein (EGFP) fusion protein (Hex-EGFP) induces cell death in mouse chondrogenic cell line ATDC5. The expression rate of Hex-EGFP decreased more rapidly than that of EGFP 96 h after transfection. The time-lapse image of living cells revealed the Hex-EGFP-positive cells rapidly died in a necrosis-like fashion. The nuclei of Hex-EGFP-expressing cells were rarely fragmented; however, these cells were negative for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The expression rate of Hex-EGFP clearly increased by treatment with radical scavengers, propyl gallate and butylated hydroxyanisole, slightly increased with a caspase inhibitor, zVAD-fmk, and was not affected by N-acetyl cysteine in ATDC5 cells. A fluorescent probe indicated that reactive oxygen species (ROS) were localized near the nuclei in Hex-EGFP-positive cells. In differentiated ATDC5 cells, as hypertrophic chondrocyte-like cells, the expression rate of Hex-EGFP increased above that in uninduced ATDC5 cells. These results suggest that Hex induces nonapoptotic cell death through local accumulation of reactive oxygen species, and mature chondrocytes, which express Hex, might be able to escape cell death induced by Hex in cartilage.

Transdifferentiation of epidermis to mucous epithelium by retinol accompanies increase in transglutaminase 2/Gh and decrease in transglutaminase 3.

We showed previously that transdifferentiation of skin epidermis to mucous epithelium can be induced by treatment with 20 µM retinol for 1 d followed by culture for 4 d without retinol in chick embryonic tarsometatarsal skin. In mouse epidermal cells, 3 µM retinoic acid (an active metabolite of retinol) inhibits epidermal keratinization in consistent with an increase in transglutaminase (TG)2/Gh, while its physiological role in the skin is still unresolved. TG1, TG3 and TG5 are also found in mammalian keratinocytes and play an important role in the formation of the stratum corneum in the skin by the introduction of cross-links into proteins. The most characteristic enzyme function of TG family is calcium-dependent transamidation activity (transamidase) that introduces inter or intramolecular ε-(γ-glutamyl)lysine cross-links into the protein. TG2/Gh is a multifunctional protein and ubiquitously expressed member of transglutaminase family that has been implicated in a variety of biological processes. By in situ hybridization analysis, we showed that TG2/Gh mRNA expression started to increase throughout the skin during the culture for 1 d with retinol, while it was weak in the control skin. On the other hand, an expression of TG3 mRNA was increased in the keratinized epidermis of control skin but was decreased by retinol. In situ transamidase activity of transglutaminase was weak in retinol-pretreated skin. Therefore, it was indicated that functions other than transamidase of TG2/Gh protein might be important in retinol-induced epidermal mucous transdifferentiation.

Homeoprotein Hex is expressed in mouse developing chondrocytes.

Endochondral ossification is a complex process involving the formation of cartilage and the subsequent replacement by mineralized bone. Although the proliferation and differentiation of chondrocytes are strictly regulated, the molecular mechanisms involved are not completely understood. Here, we show that a divergent-type homeobox gene, hematopoietically expressed homeobox gene (HEX), is expressed in mouse chondrogenic cell line ATDC5. The expression of Hex protein drastically increased during differentiation. The chondrogenic differentiation-enhanced expression of Hex protein was also observed in chondrocytes in the tibia of embryonic day 15.5 (E15.5) mouse embryos. The localization of Hex protein in the chondrocytes of the tibia changed in association with maturation; namely, there was Hex protein in the cytoplasm near the endoplasmic reticulum (ER) in resting chondrocytes, which moved to the nucleus in prehypertrophic chondrocytes, and thereafter entered the ER in hypertrophic chondrocytes. These results suggest Hex expression and subcellular localization are associated with chondrocyte maturation.

Tgm2/Gh, Gbx1 and TGF-beta are involved in retinoic acid-induced transdifferentiation from epidermis to mucosal epithelium.

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.

Involvement of Hex in the initiation of feather morphogenesis.

In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.

Expression of Hex during feather bud development.

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.

Localization of HB9 homeodomain protein and characterization of its nuclear localization signal during chick embryonic skin development.

We detected HB9 protein during tarsometatarsal scale skin and late feather development. Immunofluorescent analyses with N-terminal 14 amino acids antiserum revealed that HB9 was strongly expressed in epidermal basal cells of the outer scale face in tarsometatarsal scale skin. Specific expression was also detected in dermal cells at the root region of the feather and around the feather follicle. Furthermore, we observed precise distribution of HB9 protein by immunoelectron microscopy. We detected HB9 protein not only in the nucleus, but also in the cytoplasm in tarsometatarsal scale skin. However, in feather skin HB9 protein was found in the nucleus but not in the cytoplasm. Cytoplasmic localization of HB9 protein in tarsometatarsal scale skin was observed especially in the endoplasmic reticulum and the Golgi apparatus. To address the mechanism of nuclear-cytoplasmic translocation, we determined the nuclear localization signal (NLS) sequences by using eukaryotic green fluorescent protein fusion protein in primary keratinocyte culture. Chick HB9 homeoprotein has two types of the NLS sequences in its homeodomain. One of them is a bipartite type as representatively found in Xenopus nucleoplasmin. The other is very similar to hexapeptide NLS sequences identified in pancreatic duodenum homeobox 1 (PDX1). These sequences functioned not only in keratinocytes but also in dermal fibroblasts. They are conserved in Xenopus, mouse, and human HB9 ortholog. These results indicate that HB9 protein might be involved in chick tarsometatarsal scale and feather development and that nuclear import of HB9 protein might be regulated by these NLS sequences in the homeodomain.

Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis.

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline-rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two-fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.

Acceleration of Retinol-Induced Epidermal Mucous Metaplasia by Stimulating the Dermal Adenylate Cyclase-cAMP System in Chick Embryonic Skin: Appearance of cAMP-Dependent Phosphorylated Proteins in Dermis of Retinol-Pretreated Skin after 2 h-Treatment with cAMP: (retinol/epidermal mucous metaplasia/dermis/adenylate cyclase/protein phosphorylation).

Epidermal mucous metaplasia of cultured 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing excess retinol (20 µM) for only 8-24 h and then in a chemically defined medium with Bt2 cAMP (0.2-2 mM) and without retinoids or serum for 2 days. In this work, stimulation of the adenylate cyclase-cAMP system in retinol-pretreated skin by forskolin, pertussis toxin, cholera toxin or AIF4 - was found to accelerate the synthesis of epidermal sulfated glycoprotein (mucin). In skin induced toward mucous metaplasia by retinol, treatment with forskolin for 1 day increased the cAMP content 10-fold in the dermis but only 2-fold in the epidermis over the control levels. The cAMP level of Bt2 cAMP (0.2 mM)-treated skin was 18 times higher in the dermis but rather lower in the epidermis than untreated skin. These results suggest the importance of an adenylate cyclase-cAMP system in the dermis of skin in stimulating mucous metaplasia induced by retinoids. In fact, cAMP-dependent protein phosphorylation was seen only in the dermis of retinol-pretreated skin after 2 h-treatment with cAMP. As no transfer of cAMP from the dermis to the epidermis of forskolin-treated skin was detected, there may be no gap junctional communication between the epidermis and the dermis, while the basement membrane becomes discontinuous during mucous metaplasia.

Induction of Mucous Metaplasia in Chick Embryonic Skin by Retinol-Pretreated Embryonic Chick or Quail Dermal Fibroblasts through Cell-Cell Interaction: Correlation of a Transient Increase in Retinoic Acid Receptor ß mRNA in Retinol-Treated Dermal Fibroblasts with Their Competence to Induce Epidermal Mucous Metaplasia: (epidermal mucous metaplasia/cell-cell interaction/retinol/RARß mRNA/dermal fibroblasts).

Epidermal mucous metaplasia of 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing 20 µM retinol for only 8 hr and then in a chemically defined medium without retinol for 2 days. Retinol primarily affects the dermal cells, which then transform the epithelial cells into mucus-secreting cells. In this study, we developed a system using a combination of retinol-pretreated chick or quail dermal fibroblasts and chick skin, and showed that retinol-pretreated quail embryonic dermal fibroblasts invaded the dermis of chick embryonic skin to beneath the epidermal basal cells within 1 day of culture and induced metaplasia, suggesting that epidermal mucous metaplasia of the skin was induced by the direct interaction of retinol-pretreated dermal fibroblasts with the epidermal cells or by low diffusible paracrine factor produced by the fibroblasts. Increase in retinoic acid receptor ß (RARß) mRNA in dermal fibroblasts was observed after 8 hr-treatment with retinol which preceded morphological changes induced by retinol and this increase was correlated with the competence of the dermal fibroblasts to induce epidermal mucous metaplasia. Thus some gene product(s) controlled by RARß in dermal fibroblasts may be an essential signal for induction of epidermal mucous metaplasia.

Short term retinol treatment in vitro induces stable transdifferentiation of chick epidermal cells into mucus-secreting cells.

Epidermal mucous metaplasia of cultured skin can be induced by treatment with excess retinol for several days (Fell 1957). In the induction of mucous metaplasia, retinol primarily affects the dermal cells and retinol-pretreated dermis can alter epidermal differentiation towards secretory epithelium (Obinata et al. 1987). In this work, we found that mucous metaplasia could be induced by culturing 13-day-old chick embryonic tarsometatarsal skin in medium containing retinol (20 µM) for only 8-24 h, followed by culture in a chemically defined medium (BGJb) without retinol or serum for 6 days. The application of cycloheximide together with retinol during the first 8 h of culture inhibited epidermal mucous metaplasia during subsequent culture for 6 days in BGJb, indicating that induction of a signal(s) in the dermis by excess retinol requires protein synthesis. However, the presence of 20 nM hydrocortisone (Takata et al. 1981) throughout the culture period did not inhibit retinol-induced epidermal mucous metaplasia of the epidermis. This indicates that a brief treatment of the skin with excess retinol determines the direction of epithelial differentiation toward secretory epithelium; this is a simpler in vitro system for the induction of epidermal mucous metaplasia than those established before.