Chick Embryonic Skin Research Articles

Electroporation of embryonic chick skin explant

Since the introduction of electroporation a decade ago, the chick embryo has evolved into a powerful system for studying gene regulation and function during development. Although this is a straightforward method for embryos, skin (especially lamellar tissue) present special challenges. Here we describe a protocol for electroporation of expression vectors into the chick skin explant, with precise spatial and temporal control. We have improved the design of the electroporation chamber, which can achieve precise regulation of the electroporation area by changing the parameters of the laser cutting of the acrylic plate. We give the electroporation parameters of chicken embryo skin, which can effectively improve the electroporation efficiency. This technique can be used for time-lapse imaging and to study gene regulation in chick embryo skin.

Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis

The Janus kinase (JAK)–signal transducer and activator of transcription (STAT) (JAK–STAT) pathway is shown to restrain the hair follicles in catagen and telogen and prevent anagen reentry in murine hair follicle cycling. The early roles of JAK-STAT pathway genes in skin development remain uncharacterized in mouse and chicken models. Here, we revealed the expression patterns of three JAK-STAT pathway genes (JAK1, JAK2, and TYK2) in chicken embryonic skin at E6–E10 stages which are key to feather follicle morphogenesis. Multiple sequence alignment of the three genes from chicken and other species all showed a closely related homology with birds like quail and goose. Whole mount in situ hybridization (WISH) revealed weak expression of JAK1, JAK2, and TYK2 in chicken skin at E6 and E7, and followed with the focally restricted signals in the feather follicles of neck and body skin located dorsally at E8 for JAK1, E9 for TYK2 and E10 for JAK2 gene. All three genes displayed stronger expression in feather follicles of neck skin than that of body skin. The expression levels of JAK1 and TYK2 were much stronger than those of JAK2. Quantitative real-time PCR (qRT-PCR) analysis revealed the increased expression tendency for JAK2 both in the neck and body skin from E6 to E10, and the much stronger expression in neck and body skin at later stages (E8-E10) than earlier stages (E6 and E7) for JAK1 and TYK2. Overall, these findings suggest that JAK1 and TYK2, not JAK2 are important to specify the feather follicle primordia, and to arrange the proximal–distal axis of feather follicles, respectively, during the morphogenesis of feather follicles in embryonic chicken skin.

Development Finance Takes ‘Private Turn’: Implications and Challenges Ahead

Changes in lectin activity during development of embryonic chick skin were studied. In the dorsal skin of the chick embryo in which feathers were formed, lectin activity first increased, during the period of dermal condensation, and then it decreased during the development of feathers. A similar change in lectin activity was also found in the anterior shank skin, the prospective scale region of the chick embryo. The embryonic cornea, in which no mesenchymal condensation took place, had lectin activity and did not show any developmental changes in lectin activity. Apteria regions of the dorsal skin, experimentally formed by treatment with hydrocortisone, gave low lectin activity. The lectin found in the embryonic skin showed specificity for lactose. The relationship found between lectin activity and dermal condensation in the embryonic chick skin is discussed.

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

BackgroundRegional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge.ResultsWe used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin.We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes.ConclusionWe demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1202-9) contains supplementary material, which is available to authorized users.

Retinoic Acid-Induced Epidermal Transdifferentiation in Skin

Retinoids function as important regulatory signaling molecules during development, acting in cellular growth and differentiation both during embryogenesis and in the adult animal. In 1953, Fell and Mellanby first found that excess vitamin A can induce transdifferentiation of chick embryonic epidermis to a mucous epithelium (Fell, H.B.; Mellanby, E. Metaplasia produced in cultures of chick ectoderm by high vitamin A. J. Physiol. 1953, 119, 470–488). However, the molecular mechanism of this transdifferentiation process was unknown for a long time. Recent studies demonstrated that Gbx1, a divergent homeobox gene, is one of the target genes of all-trans retinoic acid (ATRA) for this transdifferentiation. Furthermore, it was found that ATRA can induce the epidermal transdifferentiation into a mucosal epithelium in mammalian embryonic skin, as well as in chick embryonic skin. In the mammalian embryonic skin, the co-expression of Tgm2 and Gbx1 in the epidermis and an increase in TGF-β2 expression elicited by ATRA in the dermis are required for the mucosal transdifferentiation, which occurs through epithelial-mesenchymal interaction. Not only does retinoic acid (RA) play an important role in mucosal transdifferentiation, periderm desquamation, and barrier formation in the developing mammalian skin, but it is also involved in hair follicle downgrowth and bending by its effect on the Wnt/β-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families.

Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells

The ability of glutathione to affect melanocyte survival has fostered its use in a variety of applications related to epithelial cells. Our study focused on fibroblast migration and the effects of oxidative stress. We used scratch assays to measure cell migration: fibroblasts were harvested from embryonic chicks, grown to confluence in a monolayer, and the layer was scratched to initiate migration. Migration rates were measured over 8 h using photomicrographs, and vinculin expression as an indicator focal adhesion formation was measured using immunofluorescence. Addition of 200 μM glutathione to the culture media in which the cells grew resulted in a significantly increased rate of scratch closure. When the scratch assays were performed in the presence of 100 μM H2O2 (to simulate oxidative stress), the cells ceased to migrate. Addition of 200 μM glutathione to the H2O2-treated scratched layers resulted in a restoration of the scratch closure capabilities. At the subcellular level, addition of extracellular glutathione resulted in a redistribution of vinculin into fewer but larger aggregates. In cells at the edge of scratched monolayers that were treated with H2O2, vinculin particles were distributed throughout the cell in smaller aggregates; addition of glutathione resulted in vinculin aggregates that were larger and closer to the edges of the cell, indicating that these cells were more migratory. Our results suggest that glutathione promotes fibroblast migration, possibly via a mechanism that promotes the formation of focal adhesions.

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

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. 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. 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.

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.

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.

In search of the Golden Fleece: unraveling principles of morphogenesis by studying the integrative biology of skin appendages.

The mythological story of the Golden Fleece symbolizes the magical regenerative power of skin appendages. Similar to the adventurous pursuit of the Golden Fleece by the multi-talented Argonauts, today we also need an integrated multi-disciplined approach to understand the cellular and molecular processes during development, regeneration and evolution of skin appendages. To this end, we have explored several aspects of skin appendage biology that contribute to the Turing activator/inhibitor model in feather pattern formation, the topo-biological arrangement of stem cells in organ shape determination, the macro-environmental regulation of stem cells in regenerative hair waves, and potential novel molecular pathways in the morphological evolution of feathers. Here we show our current integrative biology efforts to unravel the complex cellular behavior in patterning stem cells and the control of regional specificity in skin appendages. We use feather/scale tissue recombination to demonstrate the timing control of competence and inducibility. Feathers from different body regions are used to study skin regional specificity. Bioinformatic analyses of transcriptome microarrays show the potential involvement of candidate molecular pathways. We further show Hox genes exhibit some region specific expression patterns. To visualize real time events, we applied time-lapse movies, confocal microscopy and multiphoton microscopy to analyze the morphogenesis of cultured embryonic chicken skin explants. These modern imaging technologies reveal unexpectedly complex cellular flow and organization of extracellular matrix molecules in three dimensions. While these approaches are in preliminary stages, this perspective highlights the challenges we face and new integrative tools we will use. Future work will follow these leads to develop a systems biology view and understanding in the morphogenetic principles that govern the development and regeneration of ectodermal organs.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Functional role of type VI collagen during early feather development of the chicken embryo in vitro

The regulatory function of type VI collagen during early feather development in embryonic chickens was investigated at the cellular and organ levels. Immunohistochemical studies of embryonic chicken skin showed that type VI collagen was distributed in spatial-specific and temporal-specific manners related to early feather development. To clarify the role of type VI collagen, we studied the feather development in intact, reconstituted and reconstituted gel skin cultures. When ethyl-3,4-dihydroxybenzoate (EDHB) was added to the medium of intact skin as an inhibitor of type VI collagen synthesis, the feather buds did not elongate and the number of neural cell adhesion molecule (NCAM)-positive cells was reduced. However, the magnitudes of both suppressive effects of EDHB were reduced by the addition of liquid type VI collagen. Similar improvement was also observed in the reconstituted skin with liquid type VI collagen and in the reconstituted gel skin with solid type VI collagen at a low concentration. Moreover, type VI collagen promoted feather bud development in the absence of EDHB. However, a high concentration of solid type VI collagen in the reconstituted gel skin arrested the feather bud elongation, and antitype VI collagen antibodies caused feather buds to become longer and smaller in the reconstituted skin. At the cellular level, type VI collagen affected the proliferation, migration and NCAM expression of mesenchymal cells. These results suggest that type VI collagen regulates early feather development by controlling mesenchymal cell behavior.

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.

Drm/Gremlin, a BMP antagonist, defines the interbud region during feather development.

The pattern of feather buds in a tract is thought to result from the relative ratios between activator and inhibitor signals through a lateral inhibition process. We analyse the role of Drm/Gremlin, a BMPs antagonist expressed during feather pattern formation, in the dermal precursor, the dense dermis, the interbud dermis and in the posterior dermal condensation. We have altered the activity of Drm in embryonic chick skin using retroviral vectors expressing drm/ gremlin and bmps. We show that expression of endogenous drm is under the control of a feedback loop induced by the BMP pathway, and that overexpression of drm results in fusion between adjacent feather buds. We propose that endogenous BMP proteins induce drm expression in the interbud dermis. In turn, the Drm/Gremlin protein limits the inhibitory effect of BMPs, allowing the adjacent row of feathers to form. Thus, the balance between BMPs and its antagonist Drm would regulate the size and spacing of the buds.

Dlx genes integrate positive and negative signals during feather bud development

In the embryonic chicken skin, feather buds and the intervening interbud tissue form in a reiterated and sequential pattern that is dependent on interactions between the epidermis and dermis. Feather promoting and inhibiting signals such as fibroblast growth factors (FGF) and bone morphogenetic proteins (BMP), respectively, direct the formation of this periodic pattern. However, the transcription factors that mediate the response to these signals and transmit this information to downstream effector genes are largely unknown. Here we have explored the DLX transcription factors as candidate transcriptional mediators downstream of the described feather patterning signals. We show that several Dlx members are expressed in the dermis and epidermis of the developing feather buds and their expression is induced in embryonic chick skin by the ectopic activation of BMP and FGF signaling. Misexpression of Dlx in the chick skin leads to both feather loss and feather bud fusions, suggesting that DLX proteins play a negative as well as a positive role in feather development. Moreover, DLX regulates the expression of NCAM and tenascin, molecules that are important for feather bud initiation as well as bud outgrowth and morphogenesis. Our results suggest that DLX transcription factors serve to integrate and transduce feather patterning signals to downstream effector molecules.

Expression and regulation of Groucho-related genes in the embryonic chicken feather bud.

The groucho-related gene (Grg) products modulate the transcriptional response to several extracellular signals, including the Wnts. In an effort to define the roles of Grgs in the morphogenesis of the feather bud, cDNAs encoding members of the Grg family were cloned from embryonic chick skin. In situ hybridization was used to localize transcripts for cGrg2, Grg3, Grg4, and Grg5 in embryos from day 6 through day 9. Expression of cGrg2, 3, and 5 is detected throughout the initial epidermal placode. As the buds mature, expression becomes limited to the posterior halves and eventually to the distal tip of the outgrowing bud. This pattern and the effects of forced activation of the bone morphogenetic protein and beta-catenin signal transduction pathways on Grg gene expression suggest that these genes act downstream of the early activation of the beta-catenin pathway that initiates placode formation. Induction of Grg genes by beta-catenin may serve as a negative feedback to modulate pathway activation while also altering the activity of other transduction pathways involved in bud patterning.

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.

FGF10 is a mesenchymally derived stimulator for epidermal development in the chick embryonic skin

The development of avian cutaneous appendages, feathers and scales, is known to arise from the epithelial–mesenchymal interaction. Here we show that FGF10 is associated with this developmental process as an early signal from mesenchymal cells underlying nascent cutaneous placodes. Expression of Fgf10 was detected in the mesenchymal cells underneath the developing placodes. Forced expression of Fgf10 in the femoral skin suppressed expression of Shh and a zinc finger gene snail-related ( cSnR), while induced expression of Bmp2 in the interbud region, resulting in thickening of the epidermal layer. Furthermore, forced expression of Fgf10 in the foot skin caused marked ingrowings of the epidermis. The cells in the epidermal ingrowings expressed β-catenin, proliferating cell nuclear antigen, and an epidermal stem cell marker p63. These results support the idea that FGF10 is a mesenchymally derived stimulator of epidermal development through crosstalk with bone morphogenetic protein (BMP), β-catenin, and other signaling pathways.

Colinearity and non-colinearity in the expression of Hox genes in developing chick skin.

Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. We found that these characteristics apply to several Hox genes expressed in developing chick skin (Hoxb-4, Hoxa-7 and Hoxc-8), and we classed this group of genes as regionally restricted. To our surprise, we found that most of the Hox genes we examined are regionally unrestricted in their expression in the embryonic chick skin. This second group includes the Hoxd genes, Hoxd-4 to Hoxd-13, Hoxa-11 and Hoxc-6. Temporally, the expression of the regionally restricted genes can be observed by E5 within the epidermis, whereas the spatially unrestricted genes are not expressed in the epidermis until E6.25. Unexpectedly, we found that all the unrestricted genes are expressed concomitantly and therefore do not conform to temporal colinearity. Moreover, the dermal expression for both groups occurs later, but maintains the same anteroposterior patterning to that seen previously in the epidermis. During embryonic day 7-8, expression for all genes is up-regulated within the dense dermis whilst being reduced within the inter-bud regions. Later expression within the bud mesenchyme is down-regulated whilst high levels of transcriptional activity are detectable within the epidermal sheath of each feather bud. These results indicate that the transcriptional activity of Hox genes in the developing chick skin could be important during embryonic skin patterning both by providing regionally restricted positional cues, and also by imparting generic signals necessary for feather morphology.

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin.

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271-283). During scale ridge elongation at 12-15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.