Abnormal Morphogenesis of Feather Structures and Pattern in the Chick Embryo Integument I I . HISTOLOGICAL DESCRIPTION HARRIS F. BROTMAN Department of Poultry Science, Cornell University, Ithaca, N e w York 14853

ABSTRACT The development of skin and feathers in highly feathered scaleless mutants and normal Single Comb White Leghorn chick embryos was analyzed histologically. In addition, the growth of mutant feathers in chorioallantoic membrane culture is reported as a verification of several inferences made from observations of serially staged fixed specimens. The most striking feature of scaleless high line feather development is the widespread appearance of condensed or nearly condensed dermis. The discrete arrangement of normal placodes with underlying condensed dermis is replaced in the mutant by a heterogeneously shaped group of extremely large islands of columnar (“placodized”) epithelium as long as 3,000 p. The shape and extent of the condensed areas of dermis reflect the shape and extent of the overlying “placodized” epithelium. The polarity of the epidermis in normal feather germs, i.e., thicker epidermis on the posterior surface, is absent in mutant feather germs. This absence of epidermal polarity is reflected in the aberrant outgrowth of the mutant feather primordia. In the mutant, the basal cell layer of the epidermis invades the dermal core of the aberrant feather germs and may form barb vane ridges or feather sheaths. This process has no counterpart in the development of normal down feathers.

Two main approaches to the study of the morphogenesis of down feather structure and distribution are the descriptive and the analytical. The latter approach, aimed at understanding the factors controlling morphogenesis of the down feather and its precisely organized distribution, relies upon an understanding of the anatomy of all stages in the development of the integument and one of its derivatives, the feathers. Thus, in studying the origins of inherited abnormalities of feathering, an understanding of the developmental anatomy in mutant as well as normal embryos is necessary for further analytical studies of the factors controlling feather morphogenesis. The early stages of normal feather development have been examined histologically by Holmes (‘35), Hosker (‘36), Watterson (‘42), Goff (‘49), Wessells (‘65), Sengel (‘71) and Stuart et al. (‘72). All have reported the origin of feather germs in the form of dermal condensations and epidermal placodes in various tracts between J.

EXP.

ZOOL.,200: 107-124

six and eight days of incubation. The epidermal placode and the dermal condensation beneath it are considered to constitute the germ or rudiment of a feather (Lucas and Stettenheim, ’72). The formation of dermal condensations in the dorsum takes place by means of focal migration of mesenchymal cells within the dermis (Sengel, ’71; Stuart et al., ’72) and localized mitotic activity within the prospective condensation site (Wessels, ’65). As the dermal cells migrate towards and proliferate within the site of the condensations, the overlying epidermis thickens and proliferates as well. The result of this activity is the elevation of the germ in the form of a hillock. Watterson (‘42) described in great detail the later stages, elongation and outgrowth, in the morphogenesis of the down feathers on the posterior surface of the wing, in particular the “developmental mechanics” of the epidermal rearrangements generating the complex form of the down feather.

107

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HARRIS F. BROTMAN

Several mutations are known which perturb the development of the precisely organized array of integumental derivatives in the chick embryo. Plumage mutations in the domestic fowl can be classified into the categories of (la) plumage deficiency due to abnormality of the structure and growth of feather germs, e.g., naked (n) (Hutt and Sturkie, '38); (lb) plumage deficiency due to absence of pterylae, e.g., congenital baldness (ba) (Hutt, '32; Sturkie, '42); naked neck (Na) (Hutt, '49) and apterylosis (Ap) (Sturkie, '42); (2) additional plumage, e.g., ptilopody (Serebrovski, '26; Danforth, '19). In addition to the mutations affecting distribution, there are a number that alter feather structure: frizzling (F) (Landauer and Dunn, '30; Hutt, '30); flightless (fl) (Warren, '32); fray (fr) (Warren, '38); and silkie (h) (Jones, '21). The original line of scaleless mutants (Abbott and Asmundson, '57) was fully viable during embryonic life and conditionally lethal during the brooding period and subsequently. Abbott ('65) successfully developed a more viable line of scaleless chicks by outcrossing and subsequently selecting for viability under cold stress. One correlated selection response was a severalfold increase in feathering. From this group, new lines were established by selection over seven generations for increased or decreased wing feathering. The lines are now called scaleless low and scaleless high. The effects of the scaleless mutation in the high line encompass all of the categories aforementioned. Scaleless low line embryos completely lack scales and most feathers. Sawyer and Abbott ('72) reported defective histogenesis and morphogenesis in the anterior shank skin of the scaleless low line embryo. Scale placodes did not form and subsequent morphogenetic changes of placodes into scale ridges were blocked. Goetinck and Sekellick ('70) studied the developmental events in the dorsal skin of this mutant during Stages 27-33. They found that the differentiation of the dorsal skin in the scaleless low line embryos mimics the early developmental events in the dorsal skin of genetically normal embryos. However, placodes and dermal condensations failed to form in the mutant. In pieces of scaleless low line backskin

cultured in vitro, Goetinck and Sekellick ('72) observed dermal condensations and feather development in contrast to the situation in vivo. They found that these feather germs differed from normal feather germs in arrangement and shape. A histological analysis indicated that shapes of the dermal condensations and the arrangement of the overlying epidermis were aberrant as well. The histology of the integument during development has not previously been examined in the scaleless high line. Macroscopic studies (Brotman, '76) suggested that feather development in scaleless high line embryos differed fundamentally from that of normal embryos. The purpose of the histological analysis reported here is to (1) identify the anatomical basis of the unusual morphogenesis of feathers in the scaleless high line at the tissue level, and (2) further our understanding of the action of the scaleless gene as it relates to the morphogenetic processes which engender feather structure and pattern. In addition, the growth of mutant feathers in chorioallantoic membrane (CAM) culture is reported as a verification of several inferences made from observation of serially staged fixed specimens. MATERIALS AND METHODS

Fertile eggs from (1) a commercial incrossbred Single Comb White Leghorn stock and (2) a line of scaleless birds selected for high feather number (Abbott, '65) were incubated in Jamesway 252 incubators at 37.5"C and 70% relative humidity prior to sacrifice for histological study. All embryos were staged according to the criteria established by Hamburger and Hamilton ('51). Stages 2 8 4 2 normal and scaleless high line embryos from 18 sire families were fixed for two days in Bouin's fixative and then stored in 70% ethanol. Whole embryos or excised tissue blocks from a representative sample of embryos at each stage were dehydrated in a graded series of ethanols and butanols, embedded in paraffin, serially sectioned at 7 p and stained with hematoxylin and eosin. The sections were photographed with a Zeiss photomicroscope. Cell density determinations were made at a magnification of X 640. Nuclei were

Stages 33-34 Asymmetrical Placode and Epidermal Pro1 iferation

0.

densation

Stage 33 Dermal Con-

C.

Stages 28-29 Dense D e n i s and Ptacode Formatlon

B.

A. Stage 27 Pre-Dense Dermis

3.6

3.6

2.4

2.0

1.1

Dermal Nuclear Density (NucIe I 1000, 3,

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-

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Dermal Nuclear Density

A

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16 II

H. Stage 33 Three MidDorsal Elevations; Condsn sed Dermis;

Stages 29-30 Dense and Condensed Dermis, Patches of Columnar €pi theliurn

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H (Figs. 6,7)

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A (Ftg. 1)

NORMAL

110

HARRIS F. BROTMAN TABLE 1

S u m m a r y of cell density c h a n g e s in the saddle s k i n of normal a n d scaleless h i g h line e m b r y o s Dermal cell density (nuclei/1,000 p 3 ) Stages Characteristic structures

27-28 28-29 30 33 34

Low cuboidal epithelium Placode-elevated epithelium Incipient dermal condensation Dermal condensation Feather germ or modified structures 50 p in height

counted and their distribution plotted with the aid of an ocular grid divided vertically and horizontally into ten equal columns and rows of ten squares. Each square measured 40 p on a side at X 640. Chlorioallantoic membrane preparations were incubated in a Lyon glass top incubator modified to maintain incubation conditions comparable to the Jamesway (Abbott and Craig, ’63). All embryos were staged according to the criteria established by Hamburger and Hamilton (‘51). Rectangular pieces of backskin (4-9 mmz) from the posterior saddle tract were excised from normal embryos of Stages 29-30 and from scaleless high line embryos Stages 32-33. Older stages of scaleless skin were used because feather primordia are evident macroscopically about a day later than in normal embryos (Brotman, ’76). Each piece of skin was grafted dermal side down on the CAM of a 10-day normal host. The site of the graft was a bifwcation of a major blood vessel. Before putting the graft in place, excess fluid was removed from the site with a Pasteur pipette. Grafts were observed through a Wild binocular dissecting scope (Model M-5) and camera lucida drawings made of the developing feathers at 6-hour intervals for 120 hours. After feather germs were obvious, three to six drops of a 15:l mixture of fresh albumin and Tyrode’s solution was added daily to the surface of each graft to avoid dessication of the developing structures (Rawles, ’63; Linsenmayer, ’72). RESULTS

Dense dermis formation The first histological indication of feather development in the normal dorsum (Stages 28-29) is a doubling in the density of the

Normal

sc Hi

1.1 2.0 2.4 3.6

0.8 1.8 2.3-3.0 2.5-3.4

3.6

3.5

superficial layer of mesenchyme beneath the bilayered cuboidal epithelium (table 1, text figs. 1A,B fig. 1). In the mutant, the mesenchymal density also approximately doubles (text figs. 1F,G and figs. 2, 4).

Placode formation The first indication of feather development in normal epidermis is the formation of a placode in Stages 28-29 (text fig. 1B). In normal embryos, the shape and dimensions of all placodes are the same. Each placode is a circular group of columnarized epithelial cells overlaid by a single layer of flattened peridermal cells. The diameter of the placode is about 300 p. Within a row, placodes are spaced 200-250 p apart. This is also the distance between the placodes of adjacent rows. In the mutant, the discrete placode arrangement is replaced by a heterogeneously shaped group of extremely large islands of columnar (“placodized) epithelium which appear during Stage 29 (text figs. lG,H). Placodes as long as 3,000 p and 250-350 p wide were measured. Dermal condensation formation The mesenchyme condenses beneath each normal placode as a result of focal migration of mesenchyme (Stuart et al., ’72) and mitotic activity (Wessells, ’65). The nuclear density of the condensation increases from 2.4 nuclei/l,OOO p 3 (text fig. 1C) to 3.6 nucleii1,OOO p 3 during Stages 33-34. In the mutant, the superficial dermis across the middorsum increases in density as a thick band to 2.6 nuclei/1,000 p3 (text fig. 1G). The large islands of columnar epithelium overlie similarly shaped areas of condensed dermis. There is a correla-

P

B ( F i g . 18)

a O.P.

Stage 38 Further subdivision of rapidly elongaling struc\ures.

M.N. Stages 37-38 Rapid outgrowrh and separation 01 lobes.

K , L . S a g e 37 El ongat ion and siibd 1\11 won 01 ridges and lobes by epidermal invasion. and barb vane ridge formation.

I.J. Stage 36 Rapid outgrowlh af ridges and hillocks: decrease in dermal density, barb vane ridge formalion.

H. Stage 35 Differenlial growth of ridge. elevation of short ridges and hillocks.

E.F.G. Stages 35-35 + Long dermal condensation as basis of ridge, overlaid by "placodized" epithelium. short ridges and hillocks variably arranped. -+

101

10:

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i M (~111.27)

]( N (Figs.

25,261

j( L (Figs. 23.24)

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F ( F i g . 12)

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I K (Fig. 19)

! I ( F i g . 20)

Loo?.%

E (Fig. 11)

Medial

Text fig. 2 Outgrowth and elongation. Sagittal sections showing the development of feathers in the normal (medial saddle) and scaleless high line (medial and lateral saddle) embryos, Stages 35-38. See text figure 1 for explanation of symbols.

[ D [ F i g . 22)

Circii lar Arrangemen1 ol 1 :-15 Barb Vane Ridges Arotind Vascular[zed Dermal Core

D. Stages 86-38

Vane Ridges

C. Slage 36 Dorsal and Ventral Barb

B. Stage 35 Dorsal Barb Vane Ridge

Proleciron of Feather Germ

Posterior

A . Stage 35

A ( F i g . 15)

NORMAL

25

E

1

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HARRIS F. BROTMAN

over the surface of the scaleless high line feather germs appear to be nearly constant. Thus, the polarity of the epidermis in normal feather germs, i.e., thicker epidermis on the posterior surface, is absent in scaleless high lines feather germs. This absence of epidermal polarity is reflected in the aberrant outgrowth of the mutant feather primordia (text figs. 2J-P).

tion between the presence of “placodized (i.e., columnar) epithelium and condensed dermis beneath it. The shape and extent of the condensed areas of dermis in normal and mutant embryos reflect the shape and extent of the overlying “placodized” epithelium.

Elongation and outgrowth The thicker posterior epithelial surface of normal feather germs foreshadows the direction of their elongation and outgrowth. During Stage 35 the apex of the normal germs projects about 100 p above the skin surface (text fig. 2A). The epithelium around the apex and the posterior surface has acquired an additional intermediate layer of cells and is 36 p thick. The mutant feather germs vary in shape and size from hillocks to domes to tableshapes with flat or undulating surfaces to long ridges (text figs. 2 6 1 ) . The feather germs project anteriorly or posteriorly, medially or laterally. As parts of the feather germs rise to heights of 150 p above the skin surface, the epidermis proliferates to thicknesses of 30-50 p with three to five cell layers. The feather germs may be classified on the basis oftheir shapes as (1) unilobed, normal in shape but with broader diameters; (2) multilobed, with large bases and distal sections extending in the same or different directions; or (3) ridged. An epidermis 30-50 p thick and a homgeneously dense dermal core are common to all the mutant germs at this stage.

Elevation of feather germ During Stages 33-34, the posterior epidermal surface of the normal placode becomes thicker with the addition of an intermediate cell layer (text fig. 1D). The apex of the feather germ is elevated from 50-70 p above the surface of the back skin with a corresponding increase in the thickness of the underlying dermal condensation. The nuclear density ofthe dermal condensations is 3.6 nuclei/1,000 p3. The feather germ has a hillock shape at the end of stage 34 (text fig. 1E). Although the epithelium of the mutant feather germs acquires an intermediate cell layer, the thicknesses of the epithelia

Barb uane ridge formation When the normal feather germ has elongated to approximately 300 p from its apex to its anterior base, its epidermis has also thickened by the addition of more intermediate cell layers (text fig. 2B). At this point an incipient barb vane ridge formed by the basal layers of the epidermis in the dorsal surface of the distal third appears. As the feather germs elongate, additional barb vane ridges appear around the dermal core as the basal layers of the epidermis throw themselves into columns of cells 70-85 p thick. The resultant configuration is a circular arrangement of 11 to 15 barb vane ridges

Text fig. 3 Camera lucida drawings viewed from above of pieces of scaleless high line saddle skin taken from different embryos and cultured on separate CAMS for 44 hours. The long feather germs have variable widths ranging from 0.2-0.5 mm. They are oriented to each other as they would be in situ. The lengths of the feather germs are: a, 1.9 mm; b, 1.7 mm; c, 1.2 mm; d, 2.3 mm; 4, 2.0 mm. Magnification X 20.

ABNORMAL MORPHOGENESIS IN CHICK EMBRYO INTEGUMENT A

A

5,

113

P

71

79 \36 86

92

Text fig. 4 Camera lucida drawings of the morphogenesis of a primordial ridge into abnormal feathers in a piece of Stage 34 scaleless high line saddle skin cultured on the CAM for 96 hours. A, anterior, P, posterior. Numbers refer to hours of development on the CAM. Magnification X 20.

around a vascularized dermal core (text fig. 2D). Each barb vane ridge represents a column of cells which is a prospective barb with two rows of attached barbules and will keratinize as such. In the mutant during Stages 36-37, the dermis in the apical regions of the germs appears less dense (text fig. 2L). Simultaneously, or soon after the uncondensed regions appear, barb vane ridge formation begins, with the epidermal border lining the dermis assuming a scalloped configuration (text figs. 21,K). At the same time, the lobes of the various long and short ridges are growing at different rates. In the mutant a process unlike any characterizing normal feather growth is then initiated. The epidermis appears to invade the feather germ in the form of barb vane ridges, sculpting or subdividing them into a further variety of configurations (text figs. 2K,M,N,O,P). The basal cell layers of these invasions seem to be proliferating as barb vane ridges extending into the dermal core of the feather (fig. 25).

The long ridges of the posterior saddle are subdivided by epidermal invasions that appear to be extensions of barb vane ridges (text fig. 2K). Figure 27 shows the results of differential growth and subdivision of a single ridge. The region on the right is being subdivided by two epidermal invasions which resemble barb vane ridges. Possibly, the two lobes on the right were separated at an earlier stage by a similar type of epidermal invasion. Presumably, the differentiated products of the invasion are the sheaths on their contiguous faces. The common boundary in the proximal region seems to be in the process of cornifying, so that the incipient separation will be completed as the whole germ grows out. Figure 26 shows two examples of the origin of two feather rudiments from each of two feather germs. Each doublet feather rudiment emerges from a single follicle. More advanced feather doublets, e.g., figure 29, are structures typical of the medial or lateral saddle of the scaleless high line embryo. Transverse sections of such doublets immediately above and be-

114

HARRIS F. BROTMAN

low the point of bifurcation (figs. 30, 31) show that (1) the remnant of the invading epidermis is a keratinized sheath separating the members of the doublet; and (2) each member of the doublet has about 30 barb vane ridges in contrast to normal feathers which have 11-15 (cf. text fig. 2D) Watterson, '42; Rawles, '72). Figure 28 illustrates a massive feather germ characteristic of the posterior medial saddle. Such feather germs contain 60 to 100 barb vane ridges. Figures 26 and 27 also show the growth of the epidermis downward into the loose dermal core. The epidermal barb vane ridges are evident. The invading epidermis seems to represent an amplification of one or more of the barb vane ridges. The morphogenetic origins of the unevenly contoured ridges characteristic of scaleless high line feather germ development seem to be differential growth of the various lobes and epidermal invasion. These putative processes sculpt, subdivide and compartmentalize these unusual structures which result in the shapes observed in macroand microphotographs of scaleless high line feather germs (Brotman, '76). The validity of these inferences would be supported by observation of such processes in situ through time. To circumvent the necessity of in ovo observation and recording of feather development, pieces of scaleless high line saddle skin were cultured on the chorioallantoic membrane (CAM) for 120 hours, and stages at 6-hour intervals were observed and drawn with the aid of a camera lucida. Growth of feathers on CAM Normal Chorioallantoic membrane grafting of saddle skin (Stages 29-30), which contains several rows of feather germs, lead to normal temporal and spatial development of these structures. The back skin curls slightly when cultured on the CAM but not enough to interfere with the development of the majority of the feathers as a hexagonal array. The appearance of feather germs in the cultured saddle skin of Stage 28 normal embryos is delayed for 48 hours from the time of CAM grafting. Due to either severe curling of the back skin on the CAM or the formation of cyst-like cavities between

the saddle skin and the CAM, the feather germs did not appear as a regular, hexagonal array. In all cases, the feather germs elongated normally, i.e., they went through the changes of shape characteristic of feather morphogenesis in vivo and ultimately appeared as long, slender, tapering rods. Scaleless high line In the eight pieces of Stage 34 scaleless high line saddle skin grown on the CAM, two or three long opaque areas, i.e., long feather primordia ranging in length from 1.2-2.3 mm, appeared after 18 to 36 hours of culture. Viewed from above after 40 to 50 hours of culture (text fig. 3), each opaque area appears like a tube with a wavy outline. The width of the opaque areas varies from 0.2-0.5 mm. The diameter of a normal feather germ is approximately 0.3 mm. Observations of the morphogenesis of a single, representative ridge from a CAM graft of a piece of Stage 34 scaleless high line skin through a 96-hour period are presented as a series of camera lucida drawings (text fig. 4). An evenly contoured, anteroposterior ridge that is 2.3 mm long appears at 23 hours of culture. Through the next 18 hours, the smoothly contoured ridge transforms into an undulating surface which then develops peaks of various shapes and sizes. Between 47 and 58 hours of culture, the posterior part of the ridge is divided into two regions by a cleft. The epidermal borders of the ridge are clearly distinguishable at 58 hours of culture and it is obvious that the deepest region of the cleft has an advancing epidermal border. The larger ridge anterior to the cleft is clearly subdivided by three tongues of epidermis at 86 hours of culture. Viewing this structure from above at 96 hours shows that the epidermis has partitioned the ridge into distinct units. Figure 32 is a transverse section near the base of two such structural complexes grown on the CAM for 96 hours. It is clear that a single, keratinizing epidermis serves as the sheath for each complex and as the partitioning agent. This figure shows the invading epidermal barb vane ridges, their fusion with other epidermal bar vine ridges and their incipient keratinization. Ultimately, epidermal invasion is responsible for the

ABNORMAL MORPHOGENESIS IN CHICK EMBRYO INTEGUMENT

115

that the loose mesenchyme of the dorsum becomes denser beginning about Stages 28-29. The epithelium of scaleless low line dorsal skin also elongates slightly. ScaleDISCUSSION less low line embryos, however, do not The most striking feature of scaleless develop discrete dermal condensations at high line feather development is the wide- Stages 30-31. This suggests that the process spread appearance of condensed or nearly of feather development is arrested during condensed dermis. Goetinck and Sekellick or just before Stages 30-31. The process of (‘70) observed the formation of dense dermal condensation is blocked in the dermis in Stage 35 scaleless low line back scaleless low line dorsal skin. Preceding skin and considered the cell density of this this block, however, is the absence of dermis to be close to the density of the cells placode or columnar epithelial formation. in dermal condensations of normal feather The appearance of condensed dermis in germs. In the scaleless high line embryo, the high line indicates that the result of it appears that the complete layer of selection for increased feathering in the dermis beneath the saddle epidermis has scaleless high line was to transcend the a cell density equivalent to a discrete, in- block to dermal condensation. The corcipient feather germ condensation in a related appearance of “placodized epithelial regions antecedent and above connormal embryo. An aberrant arrangement of epidermis densed dermis strongly suggests a causal was noted by Goetinck and Sekellick on relationship. The appearance of condensed the epidermal covering of feather germs dermis was accompanied by the lack of which developed in scaleless low line skin formation of normal dermal condensation cultured in vitro. They found a very thin patterns. Another degree of complexity is added region of epidermis over the center of the dermal condensation; the remainder of to the morphogenesis of the scaleless high the condensation was covered by thicker line feather germ when the epidermis inepidermis. Thus, the polarity of the epi- vades the dermal core. Except possibly for dermis in normal feather germs, i.e., the development of second-order barbs in thicker epidermis on the posterior surface, the normal down feathers by the formation is absent in scaleless high line feather of a new barb vane ridge between pairs of germs developing in vivo and in scaleless ventral barb vane ridges, as reported by low line feather germs in vitro. This ab- Watterson (’42), this phenomenon of episence of epidermal polarity is reflected in dermal invasion has no counterpart in the the seemingly random outgrowth of the development of normal down feathers. scaleless high line feather germs. Text figure 5 hypothesizes the morphoTable 1 summarizes and compares the genetic events in the subdivision of a changes in dermal cell densities in the scaleless high line feather germ by an episaddles of normal and scaleless high line dermal invasion. In stage A, one of the embryos. The changes in cell density are barb vane ridges is represented as having similar. In both, the dermal cell density a morphologically active basal cell layer. doubles before placode (normal) or elevated This basal cell layer proliferates into the epithelium (scaleless high line) formation dermal core of the feather germ (stage takes place. The cell densities of incipient B). The daughters of the basal cell layers and mature dermal condensations in the invading the dermal core arrive at posinormal embryo are similar to the cell tions where they keratinize in the form densities found in the condensed dermis of apposing feather sheaths (stages C, D). beneath the elevations in the saddle of In contrast, normal feather sheaths descaleless high line embryos. velop only from the keratinization of the Goetinck and Sekellick (‘70) described layer of epidermis that remains on the the histological events in the development outer surface of the feather rudiment. As of the scaleless low line saddle skin from the feather germ grows and the cohesive five and one-fourth to seven and three- forces between the prospective apposed fourths days (Stages 28-33). Although they feather sheaths weaken (stage E), the two did not present nuclear counts, they found lobes of the feather germ separate to form primary division of the monolith into substructures. Secondary substructures are generated by further epidermal invasions.

I

A

epidermis

7

separation of apposing keratinized sheaths (like separation of a zipper)

D

Text fig. 5 Diagram of the invasion of a short ridge by the epidermis, the differentiation of the invading epidermis into apposing keratinizing sheaths as part of the process which divides the ridge i n half.

t

-

morphologically active basal layer of an incipient epidermal invasion

*keratinization of t h e cell layers derived from the basal layer that i s morphologically active

ABNORMAL MORPHOGENESIS IN CHICK EMBRYO INTEGUMENT

two smaller rudiments, each with a smaller number of barbs than the parent structure. Thus, invations by the epidermis covering the dermal core of mutant feather germs appear capable of forming feather sheaths or barb vane ridges. ACKNOWLEDGMENTS

(1) Completed as part of a Ph.D. dissertation under the direction of Ursula K. Abbott, Department of Avian Sciences, Univerisity of California, Davis. (2) This work was partially supported by NIH Training Grant in Genetics 5TOlGM00701 and NSF Grant GB31879 to Ursula K. Abbott. ( 3 ) Sincere thanks to U. K. Abbott for advice in preparation of this manuscript, and to Rae Lester, Roger Sawyer, Nancy Brotman and Diane Bondioli for assistance with organization and illustrations. LITERATURE CITED Abbott, U. K. 1965 Selection for feather number in scaleless chickens. Poult. Sci., 44: 1347 (Abstract). Abbott, U. K., and V. S. Asmundson 1957 Scaleless, a n inherited ectodermal defect i n the domestic fowl. J. Hered., 48: 63-70. Abbott, U. K., and R. M. Craig 1963 The laboratory preparation of normal avian embryos. Poult. Sci., 42: 429437. Brotman, H. F. 1976 Abnormal morphogenesis of feather structure and pattern in the chick embryo integument. I. Macroscopic description. J. Exp. Zool., 196: 323-340. Danforth, C. H. 1919 The developmental relations of brachydactyly in the domestic fowl. Am. J. Anat., 25: 97-115. Goetinck, P. F., and M. J. Sekellick 1970 Early morphogenetic events in normal and mutant skin development i n the chick embryo and their relationship to alkaline phosphatase activity. Devel. Biol., 21: 349-363. 1972 Observations on collagen synthesis, lattice formation, and morphology of scaleless and normal embryonic skin. Devel. Biol., 28: 636-648. Goff, R. A. 1949 Development of the mesodermal constituents of feather germs of chick embryos. J. Morph., 85: 443-482. Hamburger, V., and H. Hamilton 1951 A series of normal stages i n the development of the chick embryo. J. Morph., 88: 49-92. Holmes, A. 1935 The pattern and symmetry of adult plumage units i n relation to the order and locus of origin of the embryonic feather papillae. Am. J. Anat., 56: 513537. Hosker, A. 1936 Studies on the epidermal

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structures of birds. Roy. SOC. London Phil. Trans. (Series B), 226: 143-188. Hutt, F. B. 1930 The genetics of the fowl. I. The inheritance of frizzled plumage. J. Genetics, 22: 109-127. 1932 Eight new mutations i n the domestic fowl. Proc. Intern. Congr. Genetic. 6th Congr., Ithaca, N. Y.,2: 96-97. 1949 Genetics of the Fowl. McGraw-Hill Book Co., Inc., New York. Hutt, F. B., and P. D. Sturkie 1938 Genetics of the fowl. IX. Naked, a new sex-linked mutati0n.J. Hered., 29: 370-379. Jones, S . V. H. 1921 Inheritance of silkiness i n fowls. J. Hered., 12: 117-128. Landauer, W., and L. C. Dunn 1930 The “frizzle” character of fowls. Its expression and inheritance. J. Hered., 21 :290-305. Linsenmayer, T. F. 1972 Control of integumentary patterns i n the chick. Devel. Biol., 27: 244-271. Lucas, A. F., and P. R. Stettenheim 1972 Avian Anatomy. Integument. Parts I & 11. Agriculture Handbook 362, Superintendent of Documents, U. S. Govt. Printing Off.,Washington, D. C. Rawles, M. E. 1963 Tissue interactions in scale and feather development as studied i n dermalepidermal recombinations. J. Embr. Exptl. Morph., I I : 765-789. 1972 Tract specificity in the structure of down feathers of the newly hatched chick. Proc. Nat. Acad. Sci.,69: 1136-1140. Sawyer, R. H., and U. K. Abbott 1972 Defective histogenesis and morphogenesis i n the anterior shank skin of the scaleless mutant. J. Exp. Zool., 181: 99-110. Sengel, P. 1971 The organogenesis and arrangement of cutaneous appendages i n birds. Advan. Mowhog., 9: 181-230. Serebrovsky, A. S . 1926 The genetics of the domestic fowl. 11. The genetics of leg feathering (trans. title). Mem. Anikowo Genetic. Sta., cited from abstract by L. C. Dunn (J. Hered., 20: 111-118) from translation by B. F. Glessing (29). Stuart, E. S., B. Garber and A. A. Moscona 1972 An analysis of feather germ formation i n the embryo and in vitro in normal development and in skin treated with hydrocortisone. J. Exp. Zool., 179: 97-118. Sturkie, P. D. 1942 A new type of autosomal nakedness in the domestic fowl. J. Hered., 33: 202-208. Warren, D. C. 1932 Flightlessheritable variation i n the domestic fowl. J Hered., 23: 449452. 1938 A heritable variation of feather structure in the fowl. J. Hered., 29: 91-93. Watterson, R. L. 1942 The morphogenesis of down feathers with special reference to the developmental history of melanophores. Physiol. Zool., 15: 234-259. Wessells, N. K. 1965 Morphology and proliferation during early feather development. Devel. Biol., 12: 131-153.

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PLATE 1 EXPLANATION O F FIGURES

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1

Transverse section i n the saddle of a Stage 27 normal embryo. The dermis is uncondensed and is covered by a uniformly low epithelium. X 32.

2

Transverse section in the saddle of a Stage 27 scaleless high line embryo. The dermis is uncondensed and is covered by a uniformly low epithelium. X 32.

3

Transverse section i n the saddle region of a Stage 29 normal embryo showing a single median placode overlying a band of dense dermis which stretches across the dorsum. X 32.

4

Transverse section of the saddle of a Stage 29 scaleless high line embryo showing a dense layer of superficial dermis (1.8 nuclei/1,000 p3) approximately 30 p thick. X 32.

5

Sagittal section in the saddle of a Stage 33 normal embryo showing a distinct dermal condensation beneath each placode. The condensations are 40-60 p deep. The length of a placode and associated dermal condensation is approximately 300 p. Note the regular, linear array of feather germs, each separated by a distance of approximately 200-250 p. X 16.

6

Transverse section of the saddle of a Stage 33 scaleless high line embryo showing three elevations in the middorsal regions. The dense dermis varies i n thickness from 40-60 p. The density of the dermis varies from 3.4 nuclei11,OOO p3 (medial elevation), to 2.8 nuclei/1,000 p3 (lateral elevations), to 2.5 nuclei/l,OOO ~3 laterally. x 32.

7

Transverse section through the medial and lateral elevations in the middorsum of the saddle region in a Stage 33 scaleless high line embryo. There is a n intermediate cell layer in each elevation that is absent i n the tall epidermis separating them. X 125.

8

Sagittal section in the saddle of a Stage 33+ normal embryo showing a regular linear array of asymmetrical placodes, each projecting approximately 50 p above the surface of the integument. X 16.

9

Sagittal section in the saddle of a Stage 34 normal embryo illustrating a n asymmetrical placode projecting approximately 50 p above the surface of the integument. The posterior epidermal surface of the placode which contains a n intermediate cell layer, is thicker than the anterior surface. The anterior surface of the placode is longer than the posterior surface. x 63.

10

Sagittal section i n the saddle of a Stage 34 normal embryo showing a feather germ i n the form of a symmetrically shaped hillock which is about 100 p tall. Note the polarization of the epidermis, i.e., the thicker epidermal covering of the posterior surface of the hillock. X 80.

ABNORMAL MORPHOGENESIS I N CHICK EMBRYO INTEGUMENT Harris F. Brotman

PLATE I

119

PLATE 2 EXPLANATION O F FIGURES

120

11

Sagittal section through one of the elevations of the middorsum in the saddle region of a Stage 35 scaleless high line embryo. Note the dermal condensation (nuclear density 3.0-3.6 nucleii1,OOO p3) which is 3 mm long. The condensation is the basis of one of the long middorsal ridges seen macroscopically. X 6.

12

Sagittal section of the lateral saddle of a Stage 36 scaleless high line embryo showing the elevation of three hillocks to a height of approximately 75 p. Note the relatively uniform epithelial thickness over each of the hillocks. X 32.

13

Sagittal section of feather germ i n the lateral saddle of Stage 35 scaleless high line embryo showing the various shapes and arrangements of hillocks and short ridges. Note the uniform thickness of the epidermis over the surfaces of the hillocks and ridges. X 32.

14

Sagittal section through the middorsum of the saddle of a Stage 35 scaleless h i i h line embryo showing the differential growth of one of-the long ridges. X 12.5.

15

Sagittal section of a Stage 35 normal embryo showing the incipient posterior projection of a feather germ. Note the vascularization of the feather germ, and the thicker epidermis on the posterior surface. X 100.

16, 17

Sagittal sections of the lateral saddle of Stage 36+ scaleless high line embryo showing a variety of hillocks and various orientations of outgrowth. These structures are approximately 125-150 p tall. X 12.5.

18

Normal feather germ elongating posteriorly over the integument of the Stage 35 dorsum. Note the incipient barb vane ridge formed by the basal region of the epidermis along the dorsal surface of the distal half of the feather. X 80.

19

Sagittal section of the elevated lobes of the ridges in the middorsum of the saddle of scaleless high line embryo. Note the epidermal invasions, which seem to be extensions of barb vane ridges, entering the dermal core and compartmentalizing the lobes. X 10.

20

Sagittal section of Stage 36 scaleless high line embryo showing the subdivision of one of the long ridges of the medial saddle by epidermal invasions near the the center of the ridge. X 8.

+

ABNORMAL MORPHOGENESIS I N CHICK EMBRYO INTEGUMENT Harris F . Brotman

PLATE 2

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PLATE 3 EXPLANATION OF FIGURES

21

Sagittal section of a feather of a Stage 36 normal embryo. The feather is approximately 500 p long. Dorsal and ventral barb vane ridges are evident. X 32.

22

Transverse and oblique section through three feather germs of the saddle of a Stage 37 normal embryo. T w o of the feather germs each contain 12 barb vane ridges, the other has 11. Each feather germ is surrounded by a keratinized sheath. X 63.

23,24 Sagittal sections i n the lateral saddle of Stage 37 scaleless high line embryo showing the development of a loose, mesenchymatous dermis i n the apices of the feather germs. X 32 and x 25.

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25

Epidermal invasion in a feather germ of the lateral saddle of a Stage 37 scaleless high line embryo. The invading epidermis seems to be a n amplification of one or more barb vane ridges. X 80.

26

Sagittal section of the saddle of a Stage 37 scaleless high line embryo showing the origin of a doublet fiom the same feather germ. The structure on the left is being subdivided by a n epidermal invasion. It is likely that a t least another doublet will originate from this feather germ. X 16.

27

Sagittal section of the saddle of a Stage 38 scaleless high line embryo showing the nearly complete separation of three lobes of a single ridge. Within the lobes, further subdivision is i n process as evidenced by the epidermal barb vane ridges advancing into the dermal core. X 12.5.

28

Sagittal section of the medial saddle of a Stage 38 scaleless high line embryo showing a n extremely large feather germ. Note the epidermal invasions seeming to subdivide this larger structure into quarters. Such structures may have 60-100 barb vane ridges. Large feather germs are typical of the lateral saddle as well. X 10.

29

Sagittal section of a feather germ which is nearly a doublet. The invading epidermis has keratinized and separated as two sheaths covering the apposing faces of this doublet. Lateral saddle, Stage 38 scaleless high line embryo. X 12.5.

30.31

Transverse sections of the regions immediately distal (30) and proximal (31) to the point at which a n incipient feather doublet bifurcates. The common boundary of the cylinders in (31)has a feather sheath-like character. Each member of the doublet has approximately 30 barb vane ridges. X 32.

ABNORMAL MORPHOGENESIS I N CHICK EMBRYO INTEGUMENT Harxis 8’. Brotman

PLATE 3

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ABNORMAL MORPHOGENESIS I N CHICK EMBRYO INTEGUMENT Harris F. Brotman

EXPLANATION OF FIGURE

32

124

Transverse section near the base of a complex of feathers i n a piece of Stage 34 scaleless high line back skin cultured on the CAM 96 hours. Note the subdivision of each complex by epidermal partitions which appear similar to the outer, keratinized sheath. Barb vane ridges extend from various areas into the dermal core and appear to fuse with each other to form partitions that eventually keratinize as feather sheaths. X 16.

PLATE 4

Abnormal morphogenesis of feather structures and pattern in the chick embryo integument. II. Histological description.

Abnormal Morphogenesis of Feather Structures and Pattern in the Chick Embryo Integument I I . HISTOLOGICAL DESCRIPTION HARRIS F. BROTMAN Department of...
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