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Investigation of Feather Follicle Development in Embryonic Geese
MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Investigation of Feather Follicle Development in Embryonic Geese R. F. Xu,1 W. Wu, and H. Xu Laboratory of Animal Genetics and Breeding, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China feather follicles simply had radially symmetrical barb ridges, with much smaller diameters than the primary follicles, and that they developed only downy feathers. The primary and secondary follicles evolved independently of each other and formed ranks in a linear fashion. Moreover, quantitative measurements of the densities of both follicles confirmed that the density of the primary follicles sharply reached the maximum at E18, and then decreased gradually. Coincidentally, the secondary follicles started to increase from the age of E18, and up to E26 the density of the secondary follicles exceeded that of the primary follicles. Each of the primary feather follicles was richly encircled with muscles, which pointed to a quadrangularly arranged network in the dermis. The present work lays the foundation for further study of the cellular and molecular mechanisms of feather follicle morphogenesis in geese.
Key words: feather follicle, modified histological technique, goose embryo, development 2007 Poultry Science 86:2000–2007
INTRODUCTION The goose is one of the most widespread and important domesticated waterfowl in many countries. It is also the main resource for downy feathers (down), which are extensively used as high-grade insulation material in both clothing and bedding, because down is lighter and more compressible than other materials of similar insulation capacity. Intensive investigations of the characteristics of goose down quality relevant to morphology, biochemistry, and physics have been carried out (Bonser, 1995; Dawson et al., 2000; Taylor et al., 2004; Wilde et al., 2006). Furthermore, Lucas and Stettenheim (1972) previously studied the basics of feather morphogenesis, and Yu et al. (2004) also provided a comprehensive review of the developmental biology of feather follicles in the chicken. However, the development of feather follicles in the skin of the embryonic goose has not been studied widely. The feather is a complex, highly organized epidermal derivative with hierarchical branches, and comprises a ©2007 Poultry Science Association Inc. Received January 27, 2007. Accepted May 25, 2007. 1 Corresponding author: [email protected]
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).
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ABSTRACT In the present study, the process of feather follicle formation in the Zi goose, a Chinese indigenous breed, was investigated during various stages of embryonic development by using a modified histological processing method. The results showed that the feather placodes evolved initially at embryonic day (E) 12 on the spinal feather tract, emerging as symmetrical structures. Sequentially, the buds elongated from E14 to E16 with anterior-posterior and proximal-distal asymmetries, and invaginated to form the primary feather follicles, which were identified to develop the contour feathers or remiges. 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 and further invaginating into the dermis. With the differentiation of the barbule plates, the various types of feathers were determined. We proved that the secondary
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Table 1. The incubating procedure for goose eggs with an incubator Incubation phase
Temperature (°C)
RH (%)
Required conditions
1 to 9 d
38.0 to 38.5
55.0
10 to 13 d
37.8
60.0
14 to 20 d
37.5
55.5
21 to 28 d
37.2
68.0
29 to 31 d1
37.0
70.0
Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C
1
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.
MATERIALS AND METHODS Animals and Management The fertilized goose eggs used in this experiment for incubating embryos were collected from Zi geese originated in Jilin Province, northeastern China. The eggs were incubated in one incubator according to the procedures described in Table 1; 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.
the skin tissues were carefully clipped between 2 pieces of pressboard to flatten them. The specimens were then preserved in 10% buffered formalin for tissue sectioning and hematoxylin and eosin staining. After the initial sampling, additional skin specimens were taken from as close to the original sampling site as possible. 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 m 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.
Sampling and Histological Processing Skin samples were obtained from goose embryos from E7 to E30 after the embryos were preserved in 10% neutral buffered formalin for 20 to 24 h. Skin specimens of 1.5 cm2 were excised from the center-right side of the dorsal, ventral, and thoracal tracts of the sampled embryos, and
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
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Hatching time.
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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: Yij = + αi + βj + eij, where Yij is the observed value of follicle density, is the population mean, αi is the fixed effect of the ith factor of ages of embryonic geese (i = 14, 16, 18, ..., 30), βj 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.
Process of Feather Follicle Formation in the Goose Embryo
Figure 1. Formation of feather follicles. (Panel A) Photograph showing the developing skin of the goose embryo on embryonic day (E) 10, which appears as a smooth and transparent layer of mucous membrane. (Panel B) Longitudinal section (100×) of skin tissue indicating that the dense dermis is starting to form (marked with an arrow). (Panel C) Lateral view of the embryo showing the feather bud rows (indicated by an arrow) and the anterior-to-posterior (A to P) orientation of the spinal tract feather buds, which are parallel to the cephalic-caudal axis. (Panel D) Section of skin tissue from the spinal tract indicating the buds formed (A to P) and the proximal-to-distal (P to D) asymmetries on E12. (Panels E and F) Buds elongated on the ventral tract on E14 and E16 (indicated by arrows). (Panels G and H) Primary and secondary feather follicles on E18 and E20, respectively. Pr = primary feather follicles; Se = secondary feather follicles.
In this study, a series of photographs or photomicrographs of the skin sections at different embryonic stages were taken and observed during the 31 d of goose embryonic development under the aforementioned incubating and hatching conditions. These showed that, between approximately E7 and E8, the skin of the embryonic goose initially appeared as an intermediate layer, which consisted of the initiating structure of the surface periderm cells, intermediate cells (an intermediate ectodermal layer), and basal cells. By E10 the skin of the embryo had emerged as a smooth and transparent layer of mucous
membrane (Figure 1A). 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 anteriorposterior and proximal-distal asymmetries (Figure 1C
RESULTS
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sections at a magnification of 40×. 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× and an actual field area of 11.25 mm2. For each feather tract, 2 sampling sections were observed. At the magnifications of 200×, 400×, and 800×, 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.
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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). One can see in Figure 2 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
Figure 3. Photomicrographs showing the microstructures of feather follicles at the different embryonic stages. (Panel A) Showing the undeveloped barb ridges (Br) in the primary feather follicles (Pr) on the ventral tract on embryonic day (E) 14 (400×), and (panels B to C) showing the radial Br, and with rich blood vessels (BV) in the central pulp. The Br have differentiated into 3 longitudinal plates involving (panel B) the marginal plate (Mp), the barbule plate (Bp), and the axial plate (Ap) on the thoracal tract on E16 (400×), and (panel C) on the dorsal tract on E16 (400×). (Panel D) Showing the new barb locus forming on the thoracal tract on E20 (400×); (panel E) the completely developed feather muscles (Mu) on the thoracal tract on E22 (400×); (panel F) the exquisite Mu connections of mature feather follicles on the ventral tract on E24 (100×), which display a quadrangularly arranged network of muscles in the dermis; (panel G) the development of sebaceous gland (SG) tissues and BV in the skin on the ventral tract on E26 (100×); and (panel H) the development of sweat gland duct (SW) tissues and the mature secondary follicles (MSe) in the skin on the ventral tracts on E28 (100×).
the average diameter of the Pf (287.49 ± 39.79 m) was approximately 7 times larger than that of the Sf (34.01 ± 8.39 m; 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
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Figure 2. Photomicrographs showing the formation of the primary feather follicles (Pr) and secondary feather follicles (Se) taken from the transverse sections of skin tissues at different embryonic stages. (Panels A to C) Primary feather follicles formed with an asymmetrical pattern on the ventral and thoracal tracts on embryonic day (E) 14 to E16. No Se observed between the embryonic ages (panel A) on the ventral tract on E14 (100×), (panel B) on the thoracal tract on E16 (100×), and (panel C) on the dorsal tract on E16 (40×). (Panels D to F) Secondary follicles generated among the Pr. In the pictures, the Pr appear to be cavities because the feathers had been pulled out in the process of preparing the tissue sections (panel D) on the ventral tract on E20 (100×), (panel E) on the dorsal tracts on E22 (100×), and (panel F) on the thoracal tract on E26 (40×).
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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). During feather follicle development, the muscle connections of mature feather follicles, nerves, various glands, and blood vessels in the dermis gradually became rich and functional. The sections (Figure 3E and 3F) 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
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. The Sf density on the thoracal tract increased to 2.37 follicles/cm2 at E18, then increased significantly (P < 0.01) to 12.24 follicles/cm2 at E30. Similarly, the significant increases in Sf densities were also observed on the ventral and dorsal tracts, respectively. There were no significant differences in the Sf densities between the thoracal and ventral tracts at the same embryonic ages. Meanwhile, differences in the Sf densities were presented between the dorsal tract and the thoracal or ventral tracts from E18 to E20 (P < 0.05). Generally, the average Sf:Pf ratio increased in the 3 tracts from 0.18 at E18 to 1.37 at E30, showing the differences in the formation ratios between Pf and Sf.
DISCUSSION Feather Formation and Patterning Feathers are clustered in distinct feather tracts, which are bordered by apteria (Lucas and Stettenheim, 1972). In the chicken embryo, soon after the feather primordia generate from the flat epidermis in the tracts, they grow
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Figure 4. Photomicrographs showing the microstructures of the secondary feather follicles (Se) on the dorsal tract at different embryonic stages. (Panel A) An Se next to a primary one (Pr) on embryonic day (E) 20 (200×); (panel B) the amplified Se parallel to the one marked in the box of panel A (800×); (panel C) the amplified Se on E22 (800×); (panel D) the Se on E28 (200×).
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.
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FEATHER FOLLICLE DEVELOPMENT Table 2. Changes in the densities of primary and secondary feather follicles during the embryonic period of geese
Embryonic day 14 16 18 20 22 24 26 28 30
4.19 7.14 11.23 10.91 10.66 10.13 9.55 9.04 8.92
Thoracal tract (x ± SE)
Ventral tract (x ± SE)
Pf
Pf
± ± ± ± ± ± ± ± ±
Sf 0.56a 0.71 0.62 1.08 0.71 1.32 0.84 0.62 0.93b
— — 2.37 ± 4.38 ± 6.56 ± 9.01 ± 11.63 ± 11.88 ± 12.24 ±
0.24 0.32 0.57 1.36 1.92 2.11 2.27a
3.90 7.04 10.91 10.53 10.08 9.72 9.24 8.67 8.45
± ± ± ± ± ± ± ± ±
Dorsal tract (x ± SE)
Sf 0.68a 0.49 1.43 1.14 1.15 0.89 0.66 0.47 0.78b
— — 2.09 ± 4.04 ± 6.10 ± 8.40 ± 11.07 ± 11.48 ± 12.00 ±
1
Pf
0.12 0.23 0.59 1.82 1.94 2.00 1.69a
3.22 6.50 10.27 9.78 9.37 9.18 8.79 8.27 8.01
± ± ± ± ± ± ± ± ±
0.47a 0.50 1.08 0.70 0.41 0.71 0.60 0.27 0.42b
Sf
Sf:Pf
— — 1.19 ± 3.18 ± 5.04 ± 7.12 ± 9.36 ± 10.45 ± 10.51 ±
— — 0.18 0.37 0.59 0.85 1.16 1.30 1.37
0.20c 0.31c 0.62 0.88 0.91 1.27 1.12a
Values with different superscripts are different at P < 0.05 or P < 0.01. aValues compared with those in the same column of embryonic day (E) 18, P < 0.01. bValues compared with those in the same column at E18 or E20, P < 0.05. cValues compared with those on the thoracal or ventral tract on the same embryonic day, P < 0.05. 1 Pf indicates the density of mature primary feather follicles (follicles/cm2); Sf indicates the density of mature secondary feather follicles (follicles/ cm2); a dash (—) indicates values considered as not determined. At each age, 20 observations per bird of the 30 individuals were involved on different feather tracts. Here, the Pf:Sf ratio is the mean of the observed values of the 3 different tracts (thoracal, ventral, and dorsal) at the same age. a–c
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
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to form short feather buds at E10 (Widelitz et al., 2003). Compared with the chicken (as significantly less information is available on waterfowl), because of the much longer embryonic development period (31 d) of the goose than the chicken (21 d), the formation of feather follicles in the goose embryo was speculated to be later than that of the chicken. The results of this study confirm that the follicle developmental stage E12 to E18 of the goose embryo corresponds approximately to the stage E9 to E14 of the chicken. During this stage, the feather buds underwent active cell proliferation, migration, and differentiation to become the complex feather follicles that were the invaginated epidermis surrounding the feather filament cylinder with pulp inside (Yu et al., 2004). However, Hamburger and Hamilton (1951) also suggested that feather germs (feather follicles) formed at Hamburger and Hamilton stage 30 (E6.5 to E7.0) in the chicken. 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.
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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.
ACKNOWLEDGMENTS This work was supported by the Key Project of Science and Technology Plan of the Educational Department of Jilin Province (2006) and the Doctorial Foundation Project of Jilin Agricultural University of China (200615).
REFERENCES Bonser, R. H. C. 1995. Melanin and the abrasion resistance of feathers. Condor 97:590–591. Chuong, C. M. 1993. The making of a feather: Homeoproteins, retinoids and adhesion molecules. Bioessays 15:513–521. Chuong, C. M., and G. M. Edelman. 1985a. Expression of celladhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers. J. Cell Biol. 101:1009–1026. Chuong, C. M., and G. M. Edelman. 1985b. Expression of celladhesion molecules in embryonic induction. II. Morphogenesis of adult feathers. J. Cell Biol. 101:1027–1043. Chuong, C. M., R. Chodankar, R. B. Widelitz, and T. X. Jiang. 2000. Evo-devo of feathers and scales: Building complex epithelial appendages. Curr. Opin. Genet. Dev. 10:449–456. Davidson, D. 1983a. The mechanism of feather pattern development in the chick. 1. The time determination of feather position. J. Embryol. Exp. Morphol. 74:245–259. Davidson, D. 1983b. The mechanism of feather pattern development in the chick. II. Control of the sequence of pattern formation. J. Embryol. Exp. Morphol. 74:261–273. Dawson, A., S. A. Hinsley, P. N. Ferns, R. H. C. Bonser, and L. Eccleston. 2000. Rate of moult affects feather quality: A mechanism linking current reproductive effort to future survival. Proc. R. Soc. Lond. Ser. B 267:2093–2098. Dyck, J. 1985. The evolution of feathers. Zool. Scr. 14:137–153. Edna, B. 1992. AFIP Laboratory Methods in Histotechnology. Am. Regist. Pathol., Washington, DC. Haake, A. R., G. Konig, and R. H. Sawyer. 1984. Avian feather development: Relationships between morphogenesis and keratinization. Dev. Biol. 06:406–413.
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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.
FEATHER FOLLICLE DEVELOPMENT
Sawyer, R. H., and L. W. Knapp. 2003. Avian skin development and the evolutionary origin of feathers. J. Exp. Zool. 298:57–72. Sengel, P. 1978. Feather pattern development. Ciba Found. Symp. 28:51–70. Taylor, A. M., R. H. C. Bonser, and J. W. Farrent. 2004. The influence of hydration on the tensile and compressive properties of avian keratinous tissues. J. Mater. Sci. 39:939–942. Wang, J., G. M. Jing, M. Y. Zhu, and Y. R. Wang. 1995. Study of feather zone skin’s histological structure of Wanxi white geese and analysis on its velvet tegument. Anim. Husb. Vet. Med. 4:159–160. (in Chinese) Widelitz, R. B., T. X. Jiang, C. M. Chen, N. S. Stott, and C. M. Chuong. 1999. Wnt-7a in feather morphogenesis: Involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model. Development 126:2577–2587. Widelitz, R. B., T. X. Jiang, M. K. Yu, T. Shen, J. Y. Shen, P. Wu, Z. Yu, and C. M. Chuong. 2003. Molecular biology of feather morphogenesis: A testable model for evo-devo research. J. Exp. Zool. 298B:109–122. Wilde, T. P., D. L. McDowell, K. I. Jacob, and A. P. Aneja. 2006. A modified Mullins model for compressive behavior of goose down fiber assemblies. Mech. Adv. Mater. Struct 1:83–93. Yu, M. K., P. Wu, R. B. Widelitz, and C. M. Chuong. 2002. The morphogenesis of feathers. Nature 420:308–312. Yu, M., Z. Yue, P. Wu, D. Y. Wu, J. L. A. Mayer, M. Medina, R. B. Widelitz, T. X. Jiang, and C. M. Chuong. 2004. The developmental biology of feather follicles. Int. J. Dev. Biol. 48:181–191. Zheng, W. 2005. Experimental Protocols for Medical Molecular Biology in Chinese and English. Peking Union Med. Coll. Press, Beijing, China.
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Hamburger, V., and H. L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49–92. Jiang, T. X., H. S. Jung, R. B. Widelitz, and C. M. Chuong. 1999. Self-organization is the initial event in periodic feather patterning: Roles of signaling molecules and adhesion molecules. Development 126:4997–5009. Jiang, T. X., R. B. Widelitz, W. Shen, P. Will, D. Y. Wu, C. M. Lin, H. S. Jung, and C. M. Chuong. 2004. Integument pattern formation involves genetic and epigenetic controls: Feather arrays simulated by digital hormones. Int. J. Dev. Biol. 48:117–135. Lin, J. B., X. Chen, and M. D. Liu. 2002. SPSS11.0: Statistical Analysis Actual Practice Design. China Railway Press, Beijing, China. (in Chinese) Lu, W. D., Y. L. Zhu, J. Sha, and H. B. Zhu. 2002. SPSS for Windows Statistical Analysis. Publ. House of Electron. Ind., Beijing, China. (in Chinese) Lucas, A. M., and P. R. Stettenheim. 1972. Avian Anatomy— Integument. Agric. Handbook 362. Agric. Res. Serv., Washington, DC. Luo, K. 1983. Poultry Anatomy and Histology. Fujian Sci. Technol. Press, Fuzhou, China. (in Chinese) Lyne, A. G. 1966. The development of hair follicles. Aust. J. Sci. 28:370–377. Parry, A. L., B. W. Norton, and B. J. Restall. 1992. Skin follicle development in the Australian cashmere goat. Aust. J. Agric. Res. 43:857–870. Prum, R. O. 1999. Development and evolutionary origin of feathers. J. Exp. Zool. 285:291–306. Prum, R. O., and S. Williamson. 2001. Theory of the growth and evolution of feather shape. J. Exp. Zool. 291:30–57.
2007
Key words: feather follicle, modified histological technique, goose embryo, development 2007 Poultry Science 86:2000–2007
INTRODUCTION The goose is one of the most widespread and important domesticated waterfowl in many countries. It is also the main resource for downy feathers (down), which are extensively used as high-grade insulation material in both clothing and bedding, because down is lighter and more compressible than other materials of similar insulation capacity. Intensive investigations of the characteristics of goose down quality relevant to morphology, biochemistry, and physics have been carried out (Bonser, 1995; Dawson et al., 2000; Taylor et al., 2004; Wilde et al., 2006). Furthermore, Lucas and Stettenheim (1972) previously studied the basics of feather morphogenesis, and Yu et al. (2004) also provided a comprehensive review of the developmental biology of feather follicles in the chicken. However, the development of feather follicles in the skin of the embryonic goose has not been studied widely. The feather is a complex, highly organized epidermal derivative with hierarchical branches, and comprises a ©2007 Poultry Science Association Inc. Received January 27, 2007. Accepted May 25, 2007. 1 Corresponding author: [email protected]
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).
2000
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ABSTRACT In the present study, the process of feather follicle formation in the Zi goose, a Chinese indigenous breed, was investigated during various stages of embryonic development by using a modified histological processing method. The results showed that the feather placodes evolved initially at embryonic day (E) 12 on the spinal feather tract, emerging as symmetrical structures. Sequentially, the buds elongated from E14 to E16 with anterior-posterior and proximal-distal asymmetries, and invaginated to form the primary feather follicles, which were identified to develop the contour feathers or remiges. 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 and further invaginating into the dermis. With the differentiation of the barbule plates, the various types of feathers were determined. We proved that the secondary
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2001
Table 1. The incubating procedure for goose eggs with an incubator Incubation phase
Temperature (°C)
RH (%)
Required conditions
1 to 9 d
38.0 to 38.5
55.0
10 to 13 d
37.8
60.0
14 to 20 d
37.5
55.5
21 to 28 d
37.2
68.0
29 to 31 d1
37.0
70.0
Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C; turn eggs at least 2 or 3 times daily; proper ventilation Room temperature at 22 to 26°C
1
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.
MATERIALS AND METHODS Animals and Management The fertilized goose eggs used in this experiment for incubating embryos were collected from Zi geese originated in Jilin Province, northeastern China. The eggs were incubated in one incubator according to the procedures described in Table 1; 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.
the skin tissues were carefully clipped between 2 pieces of pressboard to flatten them. The specimens were then preserved in 10% buffered formalin for tissue sectioning and hematoxylin and eosin staining. After the initial sampling, additional skin specimens were taken from as close to the original sampling site as possible. 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 m 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.
Sampling and Histological Processing Skin samples were obtained from goose embryos from E7 to E30 after the embryos were preserved in 10% neutral buffered formalin for 20 to 24 h. Skin specimens of 1.5 cm2 were excised from the center-right side of the dorsal, ventral, and thoracal tracts of the sampled embryos, and
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
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Hatching time.
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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: Yij = + αi + βj + eij, where Yij is the observed value of follicle density, is the population mean, αi is the fixed effect of the ith factor of ages of embryonic geese (i = 14, 16, 18, ..., 30), βj 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.
Process of Feather Follicle Formation in the Goose Embryo
Figure 1. Formation of feather follicles. (Panel A) Photograph showing the developing skin of the goose embryo on embryonic day (E) 10, which appears as a smooth and transparent layer of mucous membrane. (Panel B) Longitudinal section (100×) of skin tissue indicating that the dense dermis is starting to form (marked with an arrow). (Panel C) Lateral view of the embryo showing the feather bud rows (indicated by an arrow) and the anterior-to-posterior (A to P) orientation of the spinal tract feather buds, which are parallel to the cephalic-caudal axis. (Panel D) Section of skin tissue from the spinal tract indicating the buds formed (A to P) and the proximal-to-distal (P to D) asymmetries on E12. (Panels E and F) Buds elongated on the ventral tract on E14 and E16 (indicated by arrows). (Panels G and H) Primary and secondary feather follicles on E18 and E20, respectively. Pr = primary feather follicles; Se = secondary feather follicles.
In this study, a series of photographs or photomicrographs of the skin sections at different embryonic stages were taken and observed during the 31 d of goose embryonic development under the aforementioned incubating and hatching conditions. These showed that, between approximately E7 and E8, the skin of the embryonic goose initially appeared as an intermediate layer, which consisted of the initiating structure of the surface periderm cells, intermediate cells (an intermediate ectodermal layer), and basal cells. By E10 the skin of the embryo had emerged as a smooth and transparent layer of mucous
membrane (Figure 1A). 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 anteriorposterior and proximal-distal asymmetries (Figure 1C
RESULTS
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sections at a magnification of 40×. 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× and an actual field area of 11.25 mm2. For each feather tract, 2 sampling sections were observed. At the magnifications of 200×, 400×, and 800×, 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.
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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). One can see in Figure 2 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
Figure 3. Photomicrographs showing the microstructures of feather follicles at the different embryonic stages. (Panel A) Showing the undeveloped barb ridges (Br) in the primary feather follicles (Pr) on the ventral tract on embryonic day (E) 14 (400×), and (panels B to C) showing the radial Br, and with rich blood vessels (BV) in the central pulp. The Br have differentiated into 3 longitudinal plates involving (panel B) the marginal plate (Mp), the barbule plate (Bp), and the axial plate (Ap) on the thoracal tract on E16 (400×), and (panel C) on the dorsal tract on E16 (400×). (Panel D) Showing the new barb locus forming on the thoracal tract on E20 (400×); (panel E) the completely developed feather muscles (Mu) on the thoracal tract on E22 (400×); (panel F) the exquisite Mu connections of mature feather follicles on the ventral tract on E24 (100×), which display a quadrangularly arranged network of muscles in the dermis; (panel G) the development of sebaceous gland (SG) tissues and BV in the skin on the ventral tract on E26 (100×); and (panel H) the development of sweat gland duct (SW) tissues and the mature secondary follicles (MSe) in the skin on the ventral tracts on E28 (100×).
the average diameter of the Pf (287.49 ± 39.79 m) was approximately 7 times larger than that of the Sf (34.01 ± 8.39 m; 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
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Figure 2. Photomicrographs showing the formation of the primary feather follicles (Pr) and secondary feather follicles (Se) taken from the transverse sections of skin tissues at different embryonic stages. (Panels A to C) Primary feather follicles formed with an asymmetrical pattern on the ventral and thoracal tracts on embryonic day (E) 14 to E16. No Se observed between the embryonic ages (panel A) on the ventral tract on E14 (100×), (panel B) on the thoracal tract on E16 (100×), and (panel C) on the dorsal tract on E16 (40×). (Panels D to F) Secondary follicles generated among the Pr. In the pictures, the Pr appear to be cavities because the feathers had been pulled out in the process of preparing the tissue sections (panel D) on the ventral tract on E20 (100×), (panel E) on the dorsal tracts on E22 (100×), and (panel F) on the thoracal tract on E26 (40×).
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XU ET AL.
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). During feather follicle development, the muscle connections of mature feather follicles, nerves, various glands, and blood vessels in the dermis gradually became rich and functional. The sections (Figure 3E and 3F) 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
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. The Sf density on the thoracal tract increased to 2.37 follicles/cm2 at E18, then increased significantly (P < 0.01) to 12.24 follicles/cm2 at E30. Similarly, the significant increases in Sf densities were also observed on the ventral and dorsal tracts, respectively. There were no significant differences in the Sf densities between the thoracal and ventral tracts at the same embryonic ages. Meanwhile, differences in the Sf densities were presented between the dorsal tract and the thoracal or ventral tracts from E18 to E20 (P < 0.05). Generally, the average Sf:Pf ratio increased in the 3 tracts from 0.18 at E18 to 1.37 at E30, showing the differences in the formation ratios between Pf and Sf.
DISCUSSION Feather Formation and Patterning Feathers are clustered in distinct feather tracts, which are bordered by apteria (Lucas and Stettenheim, 1972). In the chicken embryo, soon after the feather primordia generate from the flat epidermis in the tracts, they grow
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Figure 4. Photomicrographs showing the microstructures of the secondary feather follicles (Se) on the dorsal tract at different embryonic stages. (Panel A) An Se next to a primary one (Pr) on embryonic day (E) 20 (200×); (panel B) the amplified Se parallel to the one marked in the box of panel A (800×); (panel C) the amplified Se on E22 (800×); (panel D) the Se on E28 (200×).
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.
2005
FEATHER FOLLICLE DEVELOPMENT Table 2. Changes in the densities of primary and secondary feather follicles during the embryonic period of geese
Embryonic day 14 16 18 20 22 24 26 28 30
4.19 7.14 11.23 10.91 10.66 10.13 9.55 9.04 8.92
Thoracal tract (x ± SE)
Ventral tract (x ± SE)
Pf
Pf
± ± ± ± ± ± ± ± ±
Sf 0.56a 0.71 0.62 1.08 0.71 1.32 0.84 0.62 0.93b
— — 2.37 ± 4.38 ± 6.56 ± 9.01 ± 11.63 ± 11.88 ± 12.24 ±
0.24 0.32 0.57 1.36 1.92 2.11 2.27a
3.90 7.04 10.91 10.53 10.08 9.72 9.24 8.67 8.45
± ± ± ± ± ± ± ± ±
Dorsal tract (x ± SE)
Sf 0.68a 0.49 1.43 1.14 1.15 0.89 0.66 0.47 0.78b
— — 2.09 ± 4.04 ± 6.10 ± 8.40 ± 11.07 ± 11.48 ± 12.00 ±
1
Pf
0.12 0.23 0.59 1.82 1.94 2.00 1.69a
3.22 6.50 10.27 9.78 9.37 9.18 8.79 8.27 8.01
± ± ± ± ± ± ± ± ±
0.47a 0.50 1.08 0.70 0.41 0.71 0.60 0.27 0.42b
Sf
Sf:Pf
— — 1.19 ± 3.18 ± 5.04 ± 7.12 ± 9.36 ± 10.45 ± 10.51 ±
— — 0.18 0.37 0.59 0.85 1.16 1.30 1.37
0.20c 0.31c 0.62 0.88 0.91 1.27 1.12a
Values with different superscripts are different at P < 0.05 or P < 0.01. aValues compared with those in the same column of embryonic day (E) 18, P < 0.01. bValues compared with those in the same column at E18 or E20, P < 0.05. cValues compared with those on the thoracal or ventral tract on the same embryonic day, P < 0.05. 1 Pf indicates the density of mature primary feather follicles (follicles/cm2); Sf indicates the density of mature secondary feather follicles (follicles/ cm2); a dash (—) indicates values considered as not determined. At each age, 20 observations per bird of the 30 individuals were involved on different feather tracts. Here, the Pf:Sf ratio is the mean of the observed values of the 3 different tracts (thoracal, ventral, and dorsal) at the same age. a–c
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
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to form short feather buds at E10 (Widelitz et al., 2003). Compared with the chicken (as significantly less information is available on waterfowl), because of the much longer embryonic development period (31 d) of the goose than the chicken (21 d), the formation of feather follicles in the goose embryo was speculated to be later than that of the chicken. The results of this study confirm that the follicle developmental stage E12 to E18 of the goose embryo corresponds approximately to the stage E9 to E14 of the chicken. During this stage, the feather buds underwent active cell proliferation, migration, and differentiation to become the complex feather follicles that were the invaginated epidermis surrounding the feather filament cylinder with pulp inside (Yu et al., 2004). However, Hamburger and Hamilton (1951) also suggested that feather germs (feather follicles) formed at Hamburger and Hamilton stage 30 (E6.5 to E7.0) in the chicken. 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.
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XU ET AL.
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.
ACKNOWLEDGMENTS This work was supported by the Key Project of Science and Technology Plan of the Educational Department of Jilin Province (2006) and the Doctorial Foundation Project of Jilin Agricultural University of China (200615).
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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.
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