Feather Morphology of Four Different Mutations in the Japanese Quail

BREEDING AND GENETICS Feather Morphology of Four Different Mutations in the Japanese Quail K. M. CHENG Avian Genetics Laboratory, Department of Poultry Science, University of British Columbia, Vancouver, British Columbia, Canada V6T 2A2 A. H. BRUSH 06268

(Received for publication February 28, 1983) ABSTRACT Scanning electron microscopy and chemical analyses of feathers from four different Japanese quail mutants were carried out to correlate changes at the microscopic and molecular levels with the genetic analysis. All four mutants as well as the wild-type feathers contain similar polypeptides and share common filament-forming processes, a critical step in normal development. Thus, neither the structural genes nor the interaction of their products is abnormal. Both the gross and microscopic evidence demonstrates structural differences among mutants and between mutants and wild-type feathers. We suggest that the morphological abnormalities are the result of higher order processes, perhaps by mutation of some regulatory genes, which produces flaws in the function or organization of the follicle. (Key words: Japanese quail, feather, genetics, morphology, keratins, mutations) 1984 Poultry Science 6 3 : 3 9 1 - 4 0 0 INTRODUCTION

Feather formation is possibly the most complex developmental process occurring in the vertebrate skin (Spearman, 1966; Hodges, 1974; Sengel, 1976). On one hand, growth and morphogenesis involve a complex alignment of rows of epidermal cells to form the shaft, barbs, and barbules. Such processes have to be carefully regulated for the appropriate morphological units to form and interact. On the other hand, feathers are composed almost exclusively of keratins. These proteins are unique in several respects and consist of a relatively large number of similar, but not identical, subunits (Busch and Brush, 1979). Keratins from different parts of the feather have different electrophoretic patterns; this implies some difference in gene expression. The molecules that form feathers are more than simple homopolymers and obtain their unique chemical properties from both their composition and macromolecular organization (Brush, 1978). Although the shape and other functional properties of feathers are determined by an unknown number of regulatory genes, chemically the subunits of keratins are encoded by a family of closely related structural genes (Brush, 1975; Rogers, 1978; Molloy et al, 1982). The interactions between these two groups of genes and others (e.g., those affect-

ing pigmentation) result in the final product, the feather (Brush and Wyld, 1982). At the Avian Genetics Laboratory, we have discovered and maintained four different Japanese quail (Coturnix coturnix japonica) mutant strains involving structural abnormalities of both flight and contour feathers (Roberts and Fulton, 1979; Fulton et al., 1982a,c, 1983). In this study, we will describe the structural abnormalities of these feathers by scanning electron microscopy (SEM) and determine in each of the four mutants, whether the mutation involves one of the structural genes responsible for protein synthesis or the regulatory genes coordinating higher order organization during feather development. The combination of molecular and microscopic analyses permits the correlation of the changes at the two levels with the genetic analysis.

MATERIALS AND METHODS

Mutant Strains Feathers from four mutants of Japanese quail were analyzed. Rough-Textured (UBC-RT). Roberts and Fulton (1979) reported that this trait is caused by an autosomal recessive point mutation (rt). Progeny from rt/rt females also have

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The Biological Sciences Group, University of Connecticut, Storrs, Connecticut

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Scanning Electron

Microscopy

Natal down from the back of newly hatched chicks and adult flight and contour feathers were obtained from 3 to 5 individuals of each mutant strain. The adult feathers were first washed with a mild detergent, rinsed in distilled water, and air dried before being mounted for observation. Scanning electron micrographs of whole mounted feather parts were made with a 35 mm camera attached to Hitachi S-500 microscope. These micrographs were compared to those of feathers of wild-type individuals from the UBC-A strain, a randombred population.

trolled conditions on slab gels in an iso-lab apparatus. After the marker dye moved a predetermined distance, gels were removed and stained with Coomassie blue and destained in weak acetic acid solutions. The ability of the SH-keratin polypeptides to self-associate and form filaments was determined as described recently (Brush, 1983). This process is presumed to be a measure of the functional capacities of the proteins. RESULTS AND DISCUSSION Morph ological Characteristics of Natal Down (prepenna) Natal down of wild-type quail chicks consists of a bud-like calamus, a cluster of barbs with plumulaceous barbules (Fig. 1), and is very similar to the prepenna of chickens (Lucas and Stettenheim, 1972). Prepennae from UBC-DF chicks were smaller in appearance (Fig. 2) because their barbs were shorter than in the wild-type. Moreover, the barbules had smaller radial angles (the horizontal angle between the barb and the base of the barbule) compared to the wild-type prepennae. In some prepennae,

Chemical Analyses Feathers were washed, solubilized, and converted to the carboxymethyl (SCM) form for electrophoresis (O'Donnell, 1973). Soluble proteins were also maintained in the native thiol (SH-) form for use in the filamentation studies (Brush, 1983). Electrophoretic comparisons of SCM proteins were performed on polyacrylamide gels (PAGE) at pH 2.7 and 8.2 according to techniques described previously. Sodium dodecyl sulphate - PAGE (SDS-PAGE) was performed in 12.5% gels (Swank and Munkres, 1971) in a system modified to resolve smaller proteins. Molecular weights were estimated from Ferguson plots with a set of known weight standards. Both gel types were run in temperature con-

FIG. 1. Scanning electron type (UBC-A) prepenna. X 15.

micrograph

of

wild-

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reduced embryonic viability (maternal effect). Defective Feathers (UBC-DF). Breeding tests (Fulton et al., 1982b) showed that this trait is controlled by a dominant mutation (Df) at one locus whose manifestation is permitted by a recessive epistatic gene (mdf) at a second locus. The mutant thus has the genotype of (Df/df mdf/mdf) while individuals with genotype (Df/Df, mdf/mdf) appear to be embryonic lethals. Short Barb (UBC-SB). The short barb trait is controlled by a single autosomal recessive gene (sh) (Fulton etal, 1982c). Porcupine (UBC-PC). The porcupine trait is also controlled by a single autosomal recessive gene (pc). Porcupine quail have poor egg production, lower fertility, and higher embryonic and chick mortality compared to wild-type or heterozygotes (Fulton et al., 1982a).

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FIG. 3. Scanning electron micrograph of roughtextured (UBC-RT) prepenna, X 15.

the radial angles were so small that the barbules were clumped and fused together toward the distal portion of the barbs (Fig. 2). In other cases, the distal half of the barbs were completely bundled by and fused with the barbules. Prepennae from UBC-RT chicks were also smaller than wild-type (Fig. 3). In most of the prepennae that we observed, several barbs were totally bundled by and fused with their own barbules with very few irregularly spaced barbules joining them properly at an angle. These thick bundles probably resulted in the rough tactile sensation when the chicks were handled (Roberts and Fulton, 1979). Under these magnifications, we observed no difference between the prepennae from UBC-SB and UBC-PC chicks compared to the wild-type.

Defective Feathers. The feather from the UBC-DF mutant seemed to lack the interlocking web of the vane. In some individuals, only the outer vane of the remiges were affected (e.g., Fig. 4). In others, both the outer and the inner vanes were affected. Under magnification (Fig. 6), severe clumping of the barbules in some area of the barbs was observed. The barbs were also twisted in such a way that the hooklets of the distal barbules from one barb were not able to interlock with the proximal barbules of an adjacent barb. Higher magnification of the barbs and barbules in areas where the barbules appeared to be clumped (Fig. 8) revealed that the barbules were joined to the ramus at a vertical rather than a horizontal angle like that of the wild-type (Fig. 7). As a result, the distal and proximal barbules were close together over the dorsal ridge of the ramus. There also appeared to be some fusion of the bases of adjacent barbules. Whether this was caused by the same mechanism that resulted in similar phenomenon in the natal down of this mutant remains to be determined. Examinations under the same magnification of the areas of the feather where the barbs and barbules were twisted showed that many

Morphological Characteristics of Adult Feathers Figure 4 shows a secondary remex and a back contour feather from the wild-type strain and from each of the four mutants. The important features of a normal feather consist of the shaft (rachis), the barbs, and the barbules that interlock to form the vane (Fig. 5). (For detailed description of feather morphology, see Lucas and Stettenheim, 1972, p. 235.)

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FIG. 2. Scanning electron micrograph of defective feather (UBC-DF) prepenna. XI5.

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UBC

RT

UBC SB

FIG. 4. Secondary remiges and back contour feathers from wild-type (UBC-A) and the four mutants: defective feathers (UBC-DF), rough-textured (UBC-RT), short barb (UBC-SB), and porcupine (UBC-PC).

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UBC-A

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FIG. 6. Defective feathers (UBC-DF) secondary remex. X15, dorsal view.

FIG. 7. Wild-type (UBC-A) barbs and barbules of secondary remex. X 152, dorsal view.

FIG. 8. Defective feathers (UBC-DF) barbs and barbules (X152, dorsal view) showing clumping of distal and proximal barbules, and roughness of the rami.

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FIG. 5. Tip of wild-type (UBC-A) secondary remex. X 15, dorsal view.

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barbules were broken across the base (Fig. 9). The surface of the rami seemed to be rough and uneven compared to that of wild-type rami, and a definite dorsal ridge was difficult to observe. Similar roughness was also observed on many areas of the rachis. It is possible that the cortex in some parts of the mutant feather is particularly thin, thus exposing the contours of the pith cells. This is consistent with our observations that the rachis of mutant feathers was more translucent than normal ones. The outlines of the pith cells in the mutant rachis were clearly more visible compared to the wild-type when observed under a dissecting scope (40X). The cortex provides certain rigidity to the feather. In the area of the rami and barbules, the flange of the barbules stiffens the base against flexion, with the result that the barbules remain parallel even if they lack interlocking hooklets. Mutant feathers may not be as rigid in these and other parts because of a thinner cortical layer. This allows the barbs and barbules to be flexed out of alignment easily and expose these already more fragile parts to additional wear and tear. Consequently, there is more fraying and breaking of the barbs and barbules

FIG. 10. Rough-textured (UBC-RT) secondary remex. X 1 5, ventral view.

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FIG. 9. Defective feathers (UBC-DF) barbs and barbules (XI52, dorsal view) showing broken barbules.

of mutant feathers. The thickness of the cortex in different parts of the mutant feather can be examined and compared to that of the normal feather under the light microscope. Rough-Textured. The barbules of feathers from UBC-RT mutant were very different from those of the wild-type (Fig. 10). Only the plate of the base of these barbules was present; the flange and the pennulum were apparently missing. Unlike wild-type barbules, which were tapering, the plates of the mutant barbules were wider at the distal end. The radial angles were larger than 90 so that all the barbules were pointing towards the rachis rather than to the tip of the barb. Similar to the UBC-DF mutant, the barbules of UBC-RT were joined to the ramus at a vertical angle. As a result, the barbules were pointing "backwards" and "upwards", giving the phenotypic characteristics of "rough-textured". In some parts of the barbs, the plates of both distal and proximal barbules showed considerable curling along the long axis (Fig. 11). This led to the observation of "callus, button-like growth" by Roberts and Fulton (1979). It was also evident that on many barbs the distal barbules either overlapped tightly or

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Short Barb. Casual observation of the feathers from this mutant (Fig. 4) showed that the barbs on the leading edge of the remiges of some individuals were extremely short and had the appearance of having been broken. Many remiges also had an abnormally long and thick "barb" that originated from the plumulaceous portion of the vane and extended distally almost the whole length of the feather vane (Fig. 4, arrow). On some other remiges, although the barbs on the leading edge of the vane were short, their tips seemed to have fused together so that a thickened ridge was formed along the edge of the vane. This ridge could be teased apart easily from the rest of the vane with a pair of forceps. The result was a feather that looked exactly like the one shown in Figure 4. It is apparent that the abnormally long "barb" seen on some of the remiges was actually the ridge of fused tips separated from the barbs themselves. In some remiges the ridge was completely sheared from the vane leaving a row of barbs with broken ends. The length of the barbs seemed to be consistent on different remiges of the same individual but

FIG. 1 1 . R o u g h - t e x t u r e d (UBC-RT) barbules ( X 1 5 2 , dorsal view) showing barbule plates.

barbs ami curling of

FIG. 12. R o u g h - t e x t u r e d barbules ( X 1 5 2 , ventral view).

barbs

(UBC-RT)

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fused together at the base (Fig. 7). At higher magnification (Fig. 12), abnormal pennula in form of a row of hooklets along the ventral edge of the overlapping distal barbules could be seen. These were different from normal pennula, which also consist of dorsal and ventral cilia. These microscopic observations led us to conclude that the phenotype of UBC-RT is a result of an exchange of barbules between adjacent barbs during the development of the feather. Distal barbules joined the adjacent barb at the pennulum end of the barbule and thus becoming the abnormal proximal barbules of that barb. At the same time, proximal barbules joined adjacent barb in the same manner and became abnormal distal barbules. The pennula were therefore difficult to observe, either because they were not formed or because they were fused with the barb. The row of hooklets observed along the ventral edge of the distal barbules was actually the ventral teeth of what normally would be the proximal barbules. The wild-type proximal barbules have four very narrow ventral teeth, the tips of which are curved and pointed (Lucas and Stettenheim, 1972, p. 3 39). The barbule shown at the upper left hand corner of Figure 11, with part of pennulum still visible, should serve to demonstrate how a distal barbule joined the adjacent barb at the proximal position.

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FIG. 14. Magnified edge of the vane [X228, short barb (UBC-SB), back contour] showing fused barbules.

varied between individuals. For some mutants, the remiges were only slightly affected resulting in the appearance of notches on the leading edge (Fulton et al., 1982c). Examination of the contour feathers showed the same abnormality. In all of the contour feathers observed, barbs on both sides of the rachis were affected. The "ridge" of these feathers consists of bundles of barbulcs (Fig. 13). Higher magnification (Fig. 14) revealed the outlines of several barbs with their barbules overlapping and fused together. From these observations, we hypothesize that during the growth of the feather a flaw in coordination in either timing or geometry caused the developing barbs to fuse together and halted further development of the vane. Upon emergence from the feather sheath, the fused tips of the barbs were broken off through normal wear and tear and produced the short barb phenotype. Porcupine. After full emergence from the follicle, the feather vane of UBC-PC remained unfurled and was partly wrapped by the feather sheath at the proximal portion. However, we did not observe any abnormal changes in feather structure.

Chemical Analyses On the basis of comparisons at pH 2.7 and 8.2, all mutant feather types had PAGE patterns identical to normal, randombred individuals. Thus, the phenotypic differences were not the results of either new proteins or the absence of specific proteins in the mutant structures. A similar condition was reported in chicken frizzle feathers (Brush, 1972). Detectable PAGE differences appeared among feather parts (e.g., shaft, vane, plumulose, and pennaceous barbs) but not between the homologous parts of feathers of wild-type and mutants. Further, the molecular mass of the SCM-keratins (10.5 to 11 K daltons) was identical in all samples and like that in other avian species. On the basis of this comparison there were no detectable modifications in the nature or distribution of the protein structural elements between the wild-type and mutant feathers. The use of PAGE pattern and monomer size has become important in describing the organization and evolution of avian epidermal appendages (Brush and Wyld, 1980, 1982). The

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FIG. 13. Short barb (UBC-SB) back contour feather (X76, dorsal view) showing the thickened edge of the vane.

QUAIL FEATHER MORPHOLOGY

Implications: Relation and Morphology

of Molecular

Structure

The molecular organization of avian epidermal structures reflects a balance among the nature of constructional materials, the processes of fabrication and functional demands on the tissue. Differences that exist on a biochemical level among the tissues account for only some of these properties (Brush, 1980b; Brush and Wyld, 1982). All tissues share similar supramolecular features. One of these, a fibrous bundle network typical of keratinaceous structures, is produced by a uniform molecular process of polypeptide self-association (Brush, 1983). Much of the genetic heterogeniety detected on PAGE may be simple gene amplification to accommodate the rapid synthesis during growth. This does not exclude the alternative hypothesis that the heterogeniety

may play a role in the design or function of specific feather parts. In fact, both options are possible. The morphology of feathers, scale, and claw may also be influenced by other physical factors. For example, the early stages in morphogenesis of epithelial cell sheets are profoundly affected by physical interactions generated by the cells themselves (Oster and Alberch, 1982). Differences in the SCM-protein complement of feather parts and between feathers, down, scale, and claw, all suggest structural control not only through the distribution of specific protein subunits but also through mechanisms that determine the timing of production, the chemical composition, and the distribution of these polypeptides. Our findings are that all four feather mutants, as well as the wild-type feather, contain similar polypeptides and share common filament-forming processes, a critical step in normal development. Thus, neither the structural genes nor the interaction of their products is abnormal. This holds despite the fact that the traits are inherited in a simple Mendelian fashion. Microscopic observations further suggest that the differences in morphology are a result of a higher order process. The structural abnormalities might be caused by mutation of some, still unidentified, regulatory genes, which produce flaws in the function or organization of the follicle. Clearly there is a paradox. The morphological traits behave in a simple Mendelian fashion, yet there is no corresponding single, simple change at the molecular level. The role of central genetic information in the molecular biology and morphology of feathers remains a central problem to biology (Bonner, 1981). ACKNOWLEDGMENTS

We would like to thank J. E. Fulton and C. R. Nichols for technical assistance. Scanning electron microscopy was done at the Electron Microscopy Laboratory, Pests and Diseases Research Station, Agriculture Canada Research Branch, and we thank F. Skelton and B.Valentine for assistance. This study was supported in part by Natural Sciences and Engineering Research Council of Canada (NSERCC grant A-8062) and work at the University of Connecticut was supported by a grant from the National Science Foundation (DEB-80-16544). Quail genetic stocks at the Avian Genetics

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phylogenetic relationships among tissue types have also been estimated by amino-acid composition indices (Brush, 1980a). The important implications of the findings reported here are that there are no differences in the number, size, or distribution of the polypeptides in the parts of the mutant and wild-type feathers. On the basis of these data the causes of the morphological differences must be sought elsewhere. There were no differences between normal and mutant feather proteins in the ability to form filaments. No differences were detected in either the rate of filament formation or the efficiency (percent protein incorporation) among the mutants or between the mutants and wild-type. The process in quail was identical to that described for several other species (Brush, 1983). Thus, the most elementary organizational steps are the same in both mutants and wild-type. Differences in morphology must be caused by changes at other levels in organization or steps in the processing. Both the gross and microscopic evidence demonstrates structural differences among mutants and between mutants and wild-type feathers. We suggest that the morphological abnormalities are the result of higher order processes, perhaps a flaw in the function or organization of the follicle. This is compatible with the concept that much of the morphological variety of feathers is produced by higher order processes and not directly as the result of modification of structural genes.

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Laboratory are maintained with the. support of NSERCC grant A-8467.

REFERENCES

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