Involvement of selective epithelial cell death in the formation of feather buds on a bioengineered skin

Selective cell death by apoptosis plays important roles in organogenesis. Apoptotic cells are observed in the developmental and homeostatic processes of several ectodermal organs, such as hairs, feathers, and mammary glands. In chick feather development, apoptotic events have been observed during feather morphogenesis, but have not been investigated during early feather bud formation. Previously, we have reported a method for generating feather buds on a bioengineered skin from dissociated skin epithelial and mesenchymal cells in three-dimensional culture. During the development of the bioengineered skin, epithelial cavity formation by apoptosis was observed in the epithelial tissue. In this study, we examined the selective epithelial cell death during the bioengineered skin development. Histological analyses suggest that the selective epithelial cell death in the bioengineered skin was induced by caspase-3-related apoptosis. The formation of feather buds of the bioengineered skin was disturbed by the treatment with a pan-caspase inhibitor. The pan-caspase inhibitor treatment suppressed the rearrangement of the epithelial layer and the formation of dermal condensation, which are thought to be essential step to form feather buds. The suppression of the formation of feather buds on the pan-caspase inhibitor-treated skin was partially compensated by the addition of a GSK-3β inhibitor, which activates Wnt/β-catenin signaling. These results suggest that the epithelial cell death is involved in the formation of feather buds of the bioengineered skin. These observations also suggest that caspase activities and Wnt/β-catenin signaling may contribute to the formation of epithelial and mesenchymal components in the bioengineered skin.     

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.     

Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin.

To understand cell interactions during induction of skin appendages, we studied the roles of adhesion molecules N-CAM, tenascin, integrin, and fibronectin during feather development. Tenascin appeared in a periodic pattern on epithelia and was so far the earliest molecule detected in placodes. Three placode domains were identified: the anterior was positive for tenascin, the distal positive for N-CAM, and the posterior lacking both. Integrin appeared in dermal-epidermal junctions of placodes. In feather buds, sagittal sections revealed a transient anterior-posterior asymmetry with tenascin and N-CAM enriched in the anterior mesoderm. Tangential sections revealed a lateral-medial asymmetry with tenascin distributed in a ring shape and N-CAM in an "X" shape. Integrin was diffusely distributed within buds. Later tenascin and N-CAM were enriched in dermal papilla, the inducer of skin appendages. Perturbation of embryonic skin explant cultures with antibodies showed that anti-integrin beta 1 and anti-fibronectin blocked epithelial-mesenchymal interaction, anti-N-CAM caused uneven segregation of mesenchymal condensation, and anti-tenascin inhibited feather bud elongation. Dose-response curves showed gradual effects by these antibodies. The results indicated that these adhesion molecules are independently regulated and each contributes in different phases during morphogenesis of skin appendages.     

Formation of spacing pattern and morphogenesis of chick feather buds is regulated by cytoskeletal structures

Chick feather buds develop sequentially in a hexagonal array. Each feather bud develops with anterior posterior polarity, which is thought to develop in response to signals derived from specialized regions of mesenchymal condensation and epithelial thickening. These developmental processes are performed by cellular mechanisms, such as cell proliferation and migration, which occur during chick feather bud development. In order to understand the mechanisms regulating the formation of mesenchymal condensation and their role in feather bud development, we explanted chick dorsal skin at stage HH29+ with cytochalasin D, which inhibits cytoskeletal formation. We show that the aggregation of mesenchymal cells can be prevented by cytochalasin D treatment in a concentration-dependent manner. Subsequently, cytochalasin D disrupts the spacing pattern and inhibits feather bud axis formation as well. In addition, expression patterns of Bmp-4 and Msx-2, key molecules for early feather bud development, were disturbed by cytochalasin D treatment. Our results fully indicate that both the cytoskeletal structure and cell activity via gene regulation are of fundamental importance in mesenchymal condensation leading to proper morphogenesis of feather bud and spacing pattern formation.     

Transformation of amnion epithelium into skin and hair follicles

There is increasing interest into the extent to which epithelial differentiation can be altered by mesenchymal influence, and the molecular basis for these changes. In this study, we investigated whether amnion epithelium could be transformed into skin and hair follicles by associating E12.5 to E14.5 mouse amnion from the ROSA 26 strain, with mouse embryonic hair-forming dermis from a wild-type strain. These associations were able to produce fully formed hair follicles with associated sebaceous glands, and skin epidermis. Using beta-galactosidase staining we were able to demonstrate that the follicular epithelium and skin epidermis, but not the associated dermal cells, originated from the amnion. As Noggin and Sonic hedgehog (Shh) were recently shown to be required for early chick ventral skin formation, and able to trigger skin and feather formation from chick amnion, we associated cells engineered to produce those two factors with mouse amnion. In a few cases, we obtained hair buds connected to a pluristratified epithelium; however, the transformation of the amnion was impeded by uncontrolled fibroblastic proliferation. In contrast to an earlier report, none of our control amnion specimens autonomously transformed into skin and hair follicles, indicating that specific influences are necessary to elicit follicle formation from the mouse amnion. The ability to turn amnion into skin and its appendages has practical potential for the tissue engineering of replacement skin, and related biotechnological approaches.     

beta-catenin signaling can initiate feather bud development.

Intercellular signaling by a subset of Wnts is mediated by stabilization of cytoplasmic beta-catenin and its translocation to the nucleus. Immunolocalization of beta-catenin in developing chick skin reveals that this signaling pathway is active in a dynamic pattern from the earliest stages of feather bud development. Forced activation of this pathway by expression of a stabilized beta-catenin in the ectoderm results in the ectopic formation of feather buds. This construct is sufficient to induce bud formation since it does so both within presumptive feather tracts and in normally featherless regions where tract-specific signals are absent. It is also insensitive to the lateral inhibition that mediates the normal spacing of buds and can induce ectopic buds in interfollicular skin. However, additional patterning signals cooperate with this pathway to regulate gene expression within domains of stabilized beta-catenin expression. Localized activation of this pathway within the bud as it develops is required for normal morphogenesis and ectopic activation of the pathway leads to abnormally oriented buds and growths on the feather filaments. These results suggest that activation of the beta-catenin pathway initiates follicle development in embryonic skin and plays important roles in the subsequent morphogenesis of the bud.     

The making of a feather: Homeoproteins,retinoids and adhesion molecules

We have been using feather development as a model for understanding the molecular basis of pattern formation and to explore the roles of homeoproteins, retinoids and adhesion molecules in this process. Two kinds of homeobox (Hox) protein gradients in the skin have been identified: a ‘microgradient’ within a single feather bud and a ‘macrogradient’ across the feather tract. The asynchronous alignment of different Hox macrogradients establishes a unique repertoire of Hox expression patterns in skin appendages within the integument, designated here as the ‘Hox codes of skin appendages’. It is hypothesized that these Hox codes contribute to the phenotypic determination of skin appendages. High doses of retinoic acid cause a morphological transformation between feather and scale, while low doses of retinoic acid cause an alteration of the axial orientation of skin appendages. We have tested the ability of molecules directly involved in the feather formation process to mediate the action of the Hox codes, and surmise that adhesion molecules are potential candidates. Using specific Fabs to suppress the activity of adhesion molecules, we have found that L-CAM is involved in the formation of the hexagonal pattern, N-CAM is involved in mediating dermal condensations, tenascin is involved in feather bud growth and elongation, and integrin β-1 is essential for epithelial-mesenchymal interactions. More work is in progress to fully understand the molecular pathways regulating the feather formation process.     

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

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.     

Mechanism of skin morphogenesis. II. Retinoic acid modulates axis orientation and phenotypes of skin appendages.

The factors that determine the axial orientation and phenotypes of skin appendages were analyzed by studying the effect of retinoic acid (RA) on embryonic chicken skin explant cultures. With RA uniformly distributed in the culture media, the feather buds became smaller, were disoriented or were transformed into scale-like structures in a concentration-dependent manner (from 0.05-2.5 microM). With RA distributed as a gradient created by a RA-soaked anion exchange bead, a radial zone of inhibition with a rim of disoriented buds was observed. The new axis of the disoriented buds appeared to be determined by a combination of the original feather axis determining force and a new axial force pointing centrifugally away from the RA source. This observed result can be simulated with a computer model using a vectorial sum of different feather axial determination forces. The size of the inhibited zone is linearly correlated to the RA concentration and may be used to quantify the morphogenetic activity of retinoids. These effects are specific to developmental stages (Hamburg and Hamilton stage 31-34). Both all-trans and 13-cis RA have morphogenetic activity. Retinol has no effect and retinal has a small inhibitory effect but neither phenotypic transformation nor axial disorientation were observed. The antero-posterior gradient of homeoprotein XlHbox 1 in feather buds became diffusive after RA treatment. RA dissolves dermal condensations and the distribution of N-CAM is altered from an anterior localized pattern to a diffusive presence in the bud cores. Endogenous retinoids in developing skins show developmental stage-dependent changes both quantitatively and qualitatively. The results suggest that RA either is or can modulate the endogenous morphogen(s) that determine the orientation and phenotype of skin appendages, and that this morphogenetic pathway involves Hox genes and adhesion molecules.     

Induction of human keratinocytes into enamel-secreting ameloblasts

Mammalian tooth development relies heavily on the reciprocal and sequential interactions between cranial neural crest-derived mesenchymal cells and stomadial epithelium. During mouse tooth development, odontogenic potential, that is, the capability to direct an adjacent tissue to form a tooth, resides in dental epithelium initially, and shifts subsequently to dental mesenchyme. Recent studies have shown that mouse embryonic dental epithelium possessing odontogenic potential is able to induce the formation of a bioengineered tooth crown when confronted with postnatal mesenchymal stem cells of various sources. Despite many attempts, however, postnatal stem cells have not been used successfully as the epithelial component in the generation of a bioengineered tooth. We show here that epithelial sheets of cultured human keratinocytes, when recombined with mouse embryonic dental mesenchyme, are able to support tooth formation. Most significantly, human keratinocytes, recombined with mouse embryonic dental mesenchyme in the presence of exogenous FGF8, are induced to express the dental epithelial marker PITX2 and differentiate into enamel-secreting ameloblasts that develop a human-mouse chimeric whole tooth crown. We conclude that in the presence of appropriate odontogenic signals, human keratinocytes can be induced to become odontogenic competent; and that these are capable of participating in tooth crown morphogenesis and differentiating into ameloblasts. Our studies identify human keratinocytes as a potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.     

Trehalose 6‐phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.)

Trehalose 6‐phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2‐oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration‐dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.     

Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration

The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well‐known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1‐4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature‐dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage‐specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn‐fgfr1:EGFP)^pd1 completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn‐fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn‐fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin‐positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).     

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.     

Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development

P190-B RhoGAP (p190-B, also known as ARHGAP5) has been shown to play an essential role in invasion of the terminal end buds (TEBs) into the surrounding fat pad during mammary gland ductal morphogenesis. Here we report that embryos with a homozygous p190-B gene deletion exhibit major defects in embryonic mammary bud development. Overall, p190-B-deficient buds were smaller in size, contained fewer cells, and displayed characteristics of impaired mesenchymal proliferation and differentiation. Consistent with the reported effects of p190-B deletion on IGF-1R signaling, IGF-1R-deficient embryos also displayed a similar small mammary bud phenotype. However, unlike the p190-B-deficient embryos, the IGF-1R-deficient embryos exhibited decreased epithelial proliferation and did not display mesenchymal defects. Because both IGF and p190-B signaling affect IRS-1/2, we examined IRS-1/2 double knockout embryonic mammary buds. These embryos displayed major defects similar to the p190-B-deficient embryos including smaller bud size. Importantly, like the p190-B-deficient buds, proliferation of the IRS-1/2-deficient mesenchyme was impaired. These results indicate that IGF signaling through p190-B and IRS proteins is critical for mammary bud formation and ensuing epithelial-mesenchymal interactions necessary to sustain mammary bud morphogenesis.     

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.     

Expression of multiple delta-protocadherins during feather bud formation

The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.