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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY |
Laboratory of Animal Genetics and Breeding, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China
1 Corresponding author: rufusxu{at}yahoo.com.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: feather follicle • modified histological technique • goose embryo • development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The feather is a complex, highly organized epidermal derivative with hierarchical branches, and comprises a multilayered topological transformation of keratinocyte sheets (Chuong, 1993; Yu et al., 2004). The feathers of poultry can be divided into 3 major types: radially symmetrical downy feathers, bilaterally symmetrical contour feathers, and bilaterally asymmetrical flight feathers (Lucas and Stettenheim, 1972). The first generation of feathers, called the natal down (Yu et al., 2004), initially grows from the feather follicles that develop during the embryonic period. In fact, the various feathers (contour feathers, flight feathers, and downy feathers) belong to the third generation of feathers, which, in adults, replace the second-generation juvenile feathers that begin to form in the follicle late in embryonic life. To further elucidate the formation of feather follicles in geese, here the prenatal downy follicles are subdivided into 2 classes based on the diameter of the stemming follicles. Of them, the ones to develop contour feathers and flight feathers in postnatal life (called the primary feather follicles) have a larger diameter, and the other ones, which have a small diameter and emerge later than the primary follicles (called the secondary feather follicles), develop only downy feathers. A typical double-branched contour feather evolving in the closed pennaceous, pen pennaceous, and plumulaceous portions is composed of the calamus, which extends into the rachis, or central shaft of the feather (Dyck, 1985). The different types of feathers, including flight feathers, natal down, filoplumes, and afterfeathers have distinct microstructural features (Prum, 1999).
Although the structural features and detailed morphogenesis and growth of feather follicles in fowl have been described well (Haake et al., 1984; Jiang et al., 1999; Widelitz et al., 2003; Yu et al., 2004), only very limited information has been provided concerning the domesticated goose breeds (Luo, 1983; Wang et al., 1995). Even if a certain similarity exists in the general characteristics of feather morphogenesis, there are still various differences in the development of feather follicles among different species. Therefore, the objective of this study was mainly to focus on 1) the formation process of feather follicles and the developmental characteristics in the goose embryo; 2) changes of the densities of primary and secondary feather follicles on the thoracal, ventral, and dorsal tracts during varied stages of embryonic development; and 3) laying the groundwork for further studies of cellular and molecular mechanisms in the morphogenesis of feather follicles in geese.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; the goose embryos were randomly picked and sampled every 2 d. In total, 360 embryos of geese at different embryonic ages from embryonic days (E) 7, E8, E10, and up to E30 were used.
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Paraffin tissue sections were prepared for further histological processing as described by Edna (1992). The tissue was placed into the labeled plastic mold, dehydrated, cleared in xylene, and impregnated with wax by using a Shandon Pathcentre automated tissue processor (ThermoShandon, Pittsburgh, PA) according to modified methods, in which, after fixing the tissue in 10% formalin for 30 min, the following process was performed: 70% alcohol for 30 min, 80% alcohol for 6 h, 90% alcohol for 6 h, 95% alcohol for 6 h, and 100% alcohol 2 times for 6 h each. The formalin-fixed tissue was paraffin-embedded at 70° C by using a KD-BM tissue embedding processor (Jinhua Kedi Instrumental Equipment Co. Ltd., Zhejiang, China). Serial longitudinal and transverse sections of skin were cut at the desired thickness of 5 µm with a Leica RM 2135 microtome (Leica Microsystems, Wetzlar, Germany). The sections were transferred onto pure slides, which were then allowed to dry overnight and were stored at room temperature until ready for use.
After the sections were mounted on slides, a modification of the stain combination of hematoxylin and eosin described by Zheng (2005) was used to stain the sections; the method used to deparaffinize the paraffin sections was based on the following procedure: xylene, 2 times; 100% alcohol, 2 times; 90% alcohol, 1 time; 80% alcohol, 1 time for 10 to 20 s each; and tap water wash, 2 min. The sections were then rehydrated, the frozen section was air-dried; and the frozen section was finally fixed before staining.
Observation and Quantitative Measurements
With a JNOEC XS-213 biological microscope (Jiangnan Optics & Electronics Co. Ltd., Nanjing, China), we first observed the distribution of feather follicles from the skin sections at a magnification of 40 x . The follicle numbers of primary feather follicles (Pf) and secondary feather follicles (Sf) were counted from 10 fields of each transversal section at a magnification of 100 x and an actual field area of 11.25 mm2. For each feather tract, 2 sampling sections were observed. At the magnifications of 200 x , 400 x , and 800 x , respectively, the fine structures of the feather follicles at the different embryonic stages were observed and photographed from the tissue sections.
The following measurements were calculated on one transversal section from each embryo at each age: 1) density of Pf (follicles/cm2), and 2) density of the Sf (follicles/cm2). From these, the Sf:Pf ratio was obtained. Skin sections for measurement were selected at or above the (primary) sebaceous gland level, because those below the sebaceous gland were sometimes found to pass through the follicle bulb, particularly in the younger embryos. Shrinkage of skin samples during processing was corrected in the following way. The shrinkage ratios of skin samples were calculated based on the proportion between the area of skin section and that of the actual biopsy specimen on the feather tracts sampled on the different embryonic days.
Statistical Analyses
The following statistical model was used to analyze the relationship between the sampled and observed values for the factors of embryonic ages and feather tracts by using the GLM procedure of SPSS11.0 (Lin et al., 2002; Lu et al., 2002). The least squares mean, SE, and variance were estimated for the Pf and Sf densities in the geese:
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where Yij is the observed value of follicle density, µ is the population mean, 
i is the fixed effect of the ith factor of ages of embryonic geese (i = 14, 16, 18, ..., 30), ß j is the fixed effect of the jth factor of the feather tracts sampled (j = 1, 2, 3), and eij is the random error effect of each observation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The skin was still underdeveloped, and the epidermis layer was very thin and difficult to distinguish among structures, but a few dermal condensations had started to form (Figure 1B). By E11 to E12, the full-grown structure of the epidermis and dermis layer had developed and most feather primordia had become visible. At E12, the feather placode (short bud) was clearly distinguishable in the feather tracts, appearing as symmetrical structures and later with anterior-posterior and proximal-distal asymmetries (Figure 1C and 1D). The buds on the ventral tract elongated at E14 (Figure 1E), and they invaginated to form feather follicles that were the prototypes of Pf. The buds then developed into long buds at E16 (Figure 1F); meanwhile, the Sf, which were much smaller in size (diameter) than the Pf, gradually increased beginning on E18 (Figure 1G and 1H). The current results indicate that the formation of Sf occurred approximately 6 d later than that of the Pf, and that the Pf and Sf developed independently, with each of them generating from its own feather placode. This finding was also strongly supported by the photomicrographs taken from the transversal sections of skin tissues at the different embryonic stages (Figure 2).
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that both the Pf and Sf are arranged in a linear fashion. At E18 only a few Sf were formed, whereas the arrangement pattern of the Sf could be clearly observed on the thoracal tract at E26. One can also observe that both types of feather follicles developed independently, with no common organizational structure shared between the Pf and Sf, but that both follicles were connected by some muscles and nerves. From data on quantitative measurements of the thoracal, ventral, and dorsal feather tracts between E18 and E30, we found that the average diameter of the Pf (287.49 ± 39.79 µm) was approximately 7 times larger than that of the Sf (34.01 ± 8.39 µm; unpublished data).
Microstructures of the Feather Follicles
The photomicrographs in Figure 3 show the microstructures of feather follicles during the different embryonic stages. The results demonstrate that at E14, the outer epidermal wall of the underdeveloped cylindrical feather follicle presented an undifferentiated tubular collar that comprised 3 layers: the corneous (outermost) layer, the intermediate layer, and the germinative (inner basal) layer. The latter 2 would form the feather rachis and barbs. Obviously, the basal layer cells at the upper bud epidermal region initially started to form a series of blurred barb ridges. At that stage the blood vessels looked very thin and not very rich (Figure 3A). From the photomicrographs of the transversal sections of skin tissues at E16, approximately 12 to 16 radial barb ridges could clearly be seen, and with rich blood vessels in the central pulp (Figure 3B and 3C). The barb ridges differentiated into 3 longitudinal plates, these being the marginal plate, the barbule plate, and the axial plate, in sequence. The marginal plate cells showed a single layer of cells flanking each barb ridge. The longitudinal ridges would grow nonbranching keratin filaments with a basal calamus. Hence, with further development of the feather follicle, the new barb locus began to form and the borderlines of the barb ridges gradually became undistinguishable until they were differentiated into small subbridges at E20 (Figures 3D and 4A). This indicated that helical displacement of barb ridges within the follicle was taking place, and the new barb ridges were inferred to emerge at the locus later. During the same stage, most Sf were developing and some new Sf emerged (Figure 4).
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) showed that the feather follicle at E24 was richly connected with muscles in the dermis as well as with nerves and blood vessels. At this stage, the ensheathed filoplume and feather sheath were already highly developed. The muscles encircled each feather follicle, presenting a quadrangularly arranged network of muscles lying in the dermis (Figure 3F). These various types of muscles included erector muscles, depressor muscles, and retractor muscles arranged in antagonistic quadrilaterals for neighboring follicles to serve the purpose of pulling the feather in various directions (Yu et al., 2004). From E26 to E28, some downy follicles with the homologously branched barbs, which had a follicle microstructure similar to those represented in Figure 3D, were already mature (Figure 2F and Figure 4). Sebaceous gland tissues and sweat gland ducts were seen, which were distributed uniformly throughout the follicles (Figure 3G and 3H). The huge, very developed glands were associated with providing water resistance with oil in goose feathers. The number and secretory activity of sebaceous glands are usually considered one of the components having a significant effect on the quality of downy feathers.
Density Distribution of Feather Follicles
In the present study, quantitative measurements of the density of the Pf and Sf between E14 and E30 were conducted on the thoracal, ventral, and dorsal tracts, respectively. As shown in Table 2, the evolution of Pf densities in the 3 tracts showed a similar trend: the densities sharply increased from the lowest values at E14 to the highest values at E18, and then gradually declined. For example, the density of Pf on the thoracal tract was significantly (P < 0.01) enhanced from 4.19 follicles/cm2 at E14 to the highest density, 11.23 follicles/cm2 at E18, and then slightly declined from 10.91 follicles/cm2 at E20 to 8.92 follicles/cm2 at E30 (P < 0.05). There were no significant differences in the Pf densities between measurements taken on the thoracal, ventral, and dorsal tracts at the same embryonic ages. Coincidentally, the distinct increase in Sf density began from E18.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The arrangement of individual feathers within a feather tract on the skin is called micropatterning (Sengel, 1978). Pattern formation is a fundamental morphogenetic process that takes place during the morphogenesis stage (Jiang et al., 2004). In the chicken, the feather buds emerge sequentially across the tract from the middorsal line toward the lateral edges of the feather tract field and appear with a hexagonal feather pattern (Davidson, 1983a,b; Yu et al., 2004). Results of the current study were incongruous with this pattern in the chicken. The goose feather follicles within the dorsal tract were arranged in a linear fashion, and the same pattern was observed on the ventral and thoracal tracts. The pattern formation was determined by the interplay of signaling molecules with positive or negative roles on feather primordium formation, which functions by modulating the interactions between the epithelial and mesenchymal cells (Widelitz et al., 1999, 2003; Jiang et al., 2004). However, much of the molecular basis and many of the cellular mechanisms remain unknown (Yu et al., 2004). This work was expected to lay the foundation for further study of the cellular and molecular mechanisms in pattern formation of the feather follicles, which are arranged differently from chicken follicles.
To facilitate the description of the developmental characteristics of different feather follicles, we divided the feather follicles into Pf and Sf. The current results showed that the Sf in geese evolved later than the Pf, but the Sf did not derive from the Pf. There was not a dependent relationship between the 2 developmental processes. Hence, the distribution between the Pf and Sf was relatively independent. These findings differ from the representation of the skin follicles observed in sheep or goats, in which the secondary hair follicle was derived from the primary follicle, and the follicles appeared to be follicle groups that generally consisted of 3 primary follicles with a varying number of secondary follicles in wedge-shaped groups (Lyne, 1966; Parry et al., 1992).
Microstructure of Feather Follicles and Development
In this study, the microstructures of feather follicles in the goose embryo indicated that each of the bordered, coherent barb ridges appeared initially at E14 and then shot out toward the base of the feather bud to become the barbs. The remarkable observation at this stage was the formation of the feather follicle wall, which was understood to be the result of the epidermis surrounding the base, further invaginating into the dermis. These findings are consistent with studies on the follicle development of the chicken embryo at E10 to E11, in which the feather germ begins to grow faster and the basal layer of cells at the upper bud epidermal region begins to form a series of ridges, which are parallel to the long axis of the feather germ (Prum, 1999; Sawyer and Knapp, 2003). At this stage, the pulp (mesenchymal cells) is found at the center of the cylindrical follicle, which is composed of fibroblasts and extracellular matrix, as reviewed in Yu et al. (2004). By E16, the barb ridges appear to be radial and distinct in geese. This observation is in accordance with their presence in the chicken at E12 to E13, in which most feather germs of each barb ridge begin to differentiate into 3 longitudinal plates in sequence (the marginal plate, the barbule plates, and the axial plate); the 2 marginal plates of the 2 neighboring barb ridges constitute the barb septum (Yu et al., 2004). These findings demonstrate that during the embryonic stages of the goose, the epidermal layer cells at the bud region began to differentiate and generate into several types of cells, which separately constituted the feather sheath, barbs, pulp, and other parts of the cylindrical feather follicles.
The developmental mechanism of the Sf was less specialized than has been presented in previous literature. The current study on the Sf showed that the follicles evolved later than the Pf and developed independently of each other, as mentioned. Furthermore, similar microstructures between the Pf and Sf were found in the developmental stages before E16. Yu et al. (2002) showed that a radially symmetrical feather (downlike) is more primitive than a bilaterally symmetrical feather in terms of molecular and developmental mechanisms. The many varieties of feathers may result from modulation in the number, shape, and size of the rachis, barbs, and barbules (Lucas and Stettenheim, 1972; Prum and Williamson, 2001). The diversity of feathers is a consequence of microstructural variation in the rachis, barb rami, and barbules, of which the downy feathers typically have elongated barbules with nodal prongs that interact among barbs to form disorderly tangles that produce a large volume (Prum, 1999). The rachis has been considered as a special form of fused barb that appears later as an evolutionary novelty (Prum, 1999; Chuong et al., 2000). The fate of a feather follicle may be determined primarily by rachis formation and barb fusion or branching (Chuong and Edelman, 1985a,b; Yu et al., 2002). With the differentiation of the barbule plates, the diversity of feathers was determined based on the fusion of barb ridges on the anterior midline of the follicles (Prum, 1999). Hence, we concluded that the Pf with a rachis and a number of unbranched barbs can evolve into a contour feather, a flight feather, and the like. On the other hand, the Sf and a few Pf, having only homologously branched barbs with rami and barbules, generate the radially symmetrical downy feather. However, the regulatory mechanisms causing the morphogenetic differences between the Pf and Sf in the goose need to be studied.
Density of Feather Follicles
It is clear that the output of downy feathers is determined by the density of the feather follicles as well as the periodicities of feather growth. Meanwhile, the density also affects the down shape, which depends on the number, size, and length of the barbs or barbules, which play a very important role in the determination of feather quality. In the current study, the results of quantitative measurements of feather follicle densities demonstrated that the Pf density sharply reached the apex at E18, and then continuously descended until birth, and in the same stage, the Sf density gradually increased, whereas at E26 the Sf density became greater than that of the Pf. The decrease in Pf density can be explained by 1) an increase in body size, which caused a reduction in the number of Pf per unit of area, and 2) the lower formation ratio of Pf than of Sf, which could be clarified by the gradual increase in Sf:Pf values during the varied embryonic stages. These findings revealed a temporal and spatial diversity of feather development in the Pf and Sf of geese.
According to this presupposition, the density of feather follicles on the dorsal tract would be smaller than on the ventral tract or thoracal tract. In fact, no significant differences were observed among the densities of the 3 tracts, with the exception of the Sf densities observed on the tracts from E18 to E20 (P < 0.05). However, we observed the trend that the densities of feather follicles on the dorsal tract seemed to be smaller than those on the ventral tract or thoracal tract between E18 and E30. Certainly, the sexual dimorphism of feathers in adult geese was easily observed, but developmental differences of feather follicles between male and female animals during the embryonic ages were difficult to distinguish because no distinct sexual organs could be seen during the earlier developmental period. This was one of the main reasons that the formation of feather follicles was not described or discussed according to sexual differences. The relevant research is being carried out in postnatal geese.
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ACKNOWLEDGMENTS |
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Received for publication January 27, 2007. Accepted for publication May 25, 2007.
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