J. Embryo!, exp. Morph. Vol. 30, 3, pp. 605-633, 1973
605
Printed in Great Britain
Feather pattern stability and reorganization
in cultured skin
By GENEVIEYE NOVEL 1
From the Laboratoire de Zoologie de VUniversite
scientifique et medicale de Grenoble
SUMMARY
1. The formation of the feather pattern has been studied in skin explants obtained from
the lumbar region of the spinal pteryla of 6\- to 7^-day chick embryos. Explants were cultured
in vivo on the chorioallantoic membrane (CAM) or in vitro, either on semi-solid natural
media (containing whole chick embryo extract (J£) or chick brain extract (EC)), or in a
liquid synthetic medium (199).
2. Feather pattern development was dependent on the culture method and also on the
way the explants were excised and treated before cultivation. When the mid-dorsal initial row
of rudiments was preserved at explantation, the feather pattern was stable on all media and
initial feather rudiments progressively gave rise to feather buds. When the mid-dorsal initial
row of rudiments was damaged at explantation, explants of younger stages (A, B or C)
cultured on media JE or EC underwent a complete reorganization of their initial feather
pattern; in other explants (of older stage D, or those of any stage cultured in medium 199 or
on the CAM) the initial feather pattern was stable. When the initial rudiments were destroyed
by dermo-epidermal dissociation and recombination, feather pattern reorganization was
observed in explants of any stage cultured //; vitro (on medium EC or in medium 199) (Table 1,
p. 625).
3. Feather pattern reorganization was characterized by the following events: during the
first 24-30 h of culture the initial feather rudiments gradually vanished; dermal condensations
and epidermal placodes disappeared; after which a new 'primary' row differentiated parallel
to the longitudinal edges of the explant's dermis, its distance from the medial dermal edge
was larger when the explant was obtained at a more advanced stage. The newly formed
feathers were arranged in longitudinal rows parallel to the cephalo-caudal axis of the dermis,
which was thus solely responsible for the polarity of the reorganized feather pattern.
4. The capacity of the feather pattern to reorganize shows that it is still labile at the time of
explantation and that the site of subsequent feathers is not yet determined in the unpatterned
dermis. The pattern is determined progressively from prospective row to prospective row
starting from the primary row. In normal development in situ, the role of the primary row is
supposedly played by the mid-dorsal row in the lumbar region of the spinal pteryla. Just
prior to the formation of feather condensations the dermal cells appear to be the transitory
seat of a passing peak of morphogenetic activity moving from the mid-dorsal line to the
lateral edges of the spinal tract.
INTRODUCTION
Repetitive anatomical structures are frequently found in living beings. The
regularly organized plumage of birds is a good example. Indeed feathers are
1
Author's address: Laboratoire de Zoologie, Universite scientifique et medicale de Grenoble, B.P. 53, 38041 Grenoble, France.
606
G. NOVEL
very regularly distributed and arranged on the body surface. How do such
periodic structures arise during embryonic development? It is well established
that the differentiation of the cutaneous appendages as individual organs
results from inductive interactions between dermis and epidermis (Sengel, 1958;
Rawles, 1963). However, little is known on the determination and the differentiation of the feather pattern as a whole. In a recent study of the differentiation
of the femoral pteryla, Linsenmayer (1972) concluded that the feather pattern is
capable of autonomous development, that it is determined late in development,
probably just prior to the formation of the feather rudiments, that the dermis
controls the spatial and temporal sequences of formation of the feather, and
that no one row serves as a specific 'initiator row' in whose absence the entire
area of skin would remain bare.
However, various experimental interventions on the 2-day embryo (spinalectomy: Sengel & Kieny, 1963; Mauger, 1972 c; X-irradiation or excision of the
somiticmesoderm: Sengel & Mauger, 1967; Mauger, 1970, 1972a, b; Mauger
& Sengel, 1970; injection of hydrocortisone: Ziist, 1971) result in malformations
of the spinal feather pattern, which suggest that the initial median (or paramedian) row(s) of rudiments play(s) a decisive role in the differentiation of the
subsequent lateral longitudinal rows. Bare patches of skin are produced
consisting either of a transverse belt of featherless skin stretching across the
whole width of the spinal pteryla or of two featherless notches indenting each
side of the feather tract. Under these experimental conditions, median featherless areas with feathers developing lateral to it were never obtained, indicating
that lateral feathers cannot develop in the absence of the median ones.
Furthermore, it is also possible with certain experimental procedures to
induce the differentiation of supernumerary feathers in normally apteric zones
such as the midventral apterium (Sengel & Kieny, 1967). These supernumerary
feathers are found to be regularly arranged in a hexagonal pattern as they are in
a normal pteryla. In addition, the extra feather field is frequently distributed
symmetrically on both sides of the plane of bilateral symmetry of the embryo.
Thus, even the non-feather-forming cutaneous tissues contain in a latent state
the morphogenetic factors necessary for the construction of an ordered plumage.
It was also shown that, in the midventral apterium, the incapacity of the skin to
produce feathers resides in the predermal mesenchyme (Sengel, Dhouailly &
Kieny, 1969).
When speaking of the feather distribution one should distinguish two kinds
of pattern: (1) the general outline of the feather fields or pterylae which are
separated from one another by apterous areas (apteria) and (2) the arrangement
of the individual feathers within each feather field. Indeed each pteryla has a
characteristic shape and size, whereas in all pterylae the feathers are arranged
according to a hexagonal array with minimal variations (such as interplumar
distances, diameter of feather buds, orientation of feather rows) from feather
tract to feather tract. Shape and size of pterylae, then, are region-dependent,
Feather pattern development
607
whereas the hexagonal feather pattern appears to be a general property of the
integument. According to Mauger (1972<z) the determination of the general
shape of the spinal pteryla occurs at a very early stage: it is already fixed in the
somitic cells of the embryos of the 2-day chick embryo, a long time before the
individualization of the dermis, where the feather condensations will ultimately
form.
The purpose of the present investigation is to analyse the mechanisms of the
formation of the hexagonal feather pattern. The use of organ culture techniques
appears to be a suitable method for the study of the differentiation of an isolated
piece of skin removed from the influence of its normal environment. It provides
the opportunity to modify several experimental factors such as the nature of the
substratum, the composition of the medium and the way in which the explant is
excised from the embryo and treated before explantation. By analysing the
behaviour of the skin in culture under these different experimental conditions,
it is possible to gain new knowledge on the developmental properties of skin as
a whole, of dermis and epidermis and their morphogenetic interactions. Indeed,
it was found that, under certain in vitro culture conditions, the feather pattern
present at the time of explantation would undergo a complete reorganization
during the first 24 h of culture (Sengel & Novel, 1970). This particular behaviour
of skin explants was then used in an attempt to answer following specific
questions: (1) Is there a morphogenetic interdependence between successive
rows of feather rudiments and, particularly, does the initial mid-dorsal row of
the lumbar region play an organizing role in the differentiation of the following
lateral rows? (2) Which of the constituents of embryonic skin, dermis or epidermis, contains the morphogenetic factors responsible for the construction of
the hexagonal pattern of the spinal pteryla and for the establishment of its
antero-posterior polarity ?
MATERIAL AND METHODS
I. Material
The experiments were performed on 6^- to 7^-day White Leghorn chick
embryos; four stages, A, B, C and D, were defined according to whether 0, 1, 2
or 3 lateral rows of feather rudiments were visible on each side of the middorsal row in the skin of the lumbosacral region at the time of explantation.
The following terminology will be used for embryonic feathers (Sengel, 1971):
(1) Feather rudiments are composed of a dermal condensation and an overlying
epidermal placode; they are flat or just starting to bulge out, their height being
less than 0-02 mm. (2) Feather buds bulge out above the surface of the skin; their
length is measurable (more than 0-02 mm); they consist of an epidermal sheath
and a dermal pulp. (3) Feather filaments are elongated buds characterized by the
differentiation of barb ridges. When no precise stage is meant, the word 'feather'
(for embryonic feather) will be used indifferently for any one of the three
mentioned structures.
608
G. NOVEL
Paramedian incision
0)
Median incision
(b)
Fig. 1. Types of excision of (stage C) dorsolumbar skin for the preparation of 3
types of explants: (a), (b) and (c). /, lateral; m, medial edges of explant; md, feather
rudiment of mid-dorsal initial row (stippled); \ md, halved feather rudiment of
mid-dorsal row.
II. Description of the lumbosacral portion of the spinal pteryla
In the lumbosacral region, from which all the explants used in this investigation were obtained, the first feather rudiments differentiate at 6|- days of incubation along the mid-dorsal line, in the plane of bilateral symmetry of the embryo.
Subsequent feathers appear in successive longitudinal and parallel rows on each
side of the initial feathers (Holmes, 1935). They are arranged in an alternate
fashion with respect to the feathers of the preceding row, so that each feather
rudiment is surrounded by six other approximatively equidistant rudiments.
The lumbosacral spinal pteryla is wider anteriorly than posteriorly: on each side
of the mid-dorsal row, there are from 10 to 12 lateral rows of feathers in the
lumbar region and from 6 to 7 lateral rows in the sacral region. Although any
hexagonal array admits three axes of symmetry, in the 'hexagonal' feather
pattern one of these axes is privileged by the fact that feathers arise in successive
longitudinal rows. In consequence, the hexagonal feather pattern is characterized
by an antero-posterior axis, parallel to or coincident with its plane of bilateral
symmetry. This antero-posterior axis is furthermore anatomically visualized by
the cephalo-caudal stretching of the embryo resulting in the successive feathers
of the longitudinal rows, being wider apart than those belonging to the other
Feather pattern development
609
two oblique axes, and is also polarized by the individual orientation of the
feather buds (later filaments) pointing their distal tips in the caudal direction.
III. Methods
1. Excision of explants
Rectangular blocks of skin (approximately 2-5 x 1-5 mm) were excised from
the lumbosacral region of the spinal pteryla. The length of the explants corresponded to the space prospectively or actually occupied by 7 or 8 median initial
feather rudiments. Three types of explants were obtained that differed in the way
they are excised:
(1) Type a. One of the longitudinal incisions was made along the first lateral
row of feathers in embryos of stages B, C, D or along its presumptive location
in embryos of stage A, so that the initial mid-dorsal feather primordia were left
intact within the explant and were aligned along its longitudinal medial edge
(Fig. la).
(2) Type b. This corresponded to the area of lateral skin left over after
excision of a type a explant: its medial longitudinal cut coincided with the first
lateral row of feathers or with its prospective site and eliminated the mid-dorsal
initial row entirely; thus, explants of this type did not contain any rudiments
when they were obtained at stage A; they comprised approximately half of the
rudiments of the first lateral row when excised at stages B, C or D (Fig. 1 b).
(3) Type c. One of the longitudinal incisions was made along the mid-dorsal
line and approximately halved the mid-dorsal rudiments, which were thus
damaged at explantation; the sectioned halves of the mid-dorsal initial row
were accordingly located along the median longitudinal edge of the explant
(Fig. lc).
The cephalo-caudal orientation of the explants was routinely marked by
carbon particles deposited at the anterior edge of each skin fragment.
2. Trypsinization and dissociation
For dissociation of dermis and epidermis, embryos were dissected in calciumand magnesium-free Earle's solution. Skin fragments were incubated at 4°C for
10-15 mi n (Rawles, 1963) in 0-5 %trypsin (lyophilized, Choay) and 1 % pangestin
(Difco) made up in Ca- and Mg-free Earle's saline, after which they were
mechanically split into dermis and epidermis. Both tissues were thoroughly
washed in two changes of Tyrode's solution, and thereafter placed in an inactivating 1:1 mixture of either ovalbumine or horse serum and Tyrode's solution.
3. Reassociation
In all cases the epidermis was reassociated to its own dermis. Three types of
recombinations were performed (Fig. 2). In the first one the epidermis was
redeposited on the dermis with its original cephalo-caudal orientation; in the
610
G. NOVEL
Dmd
\
Emd
Dmd
Dmd
Fig. 2. Dermo-epidermal recombinations of type a explants without (0°) or with
rotation of the epidermis by 180° or 90° with respect to the dermal polarity (arrow
pointing towards cranial edge of dermis). Dl, Lateral, Dm, medial edges of dermis;
Dmd, dermal condensation of mid-dorsal initial feather rudiment; El, lateral, Em,
medial edges of epidermis; Emd, epidermal placode of mid-dorsal initial feather
rudiment. Stages of examples shown are: A for 0°, B for 180°, and C for 90°
rotations. Dermis shown in stipple, epidermis in solid lines.
two other types the cephalo-caudal axis of the epidermis was turned respectively
by 180° or by 90° to the left or to the right with respect to the cephalo-caudal
orientation of the dermis.
4. Culture
(a) In vivo. Skin grafts were placed on a small blood vessel of the chorioallantoic membrane of a 10- to 11-day chick embryo host (Rawles, 1963). After
the first 24 h of culture they were humidified daily with a 1:1 mixture of ovalbumine and Tyrode's solution. Explants were observed regularly twice a day
for 2\ or 3^ days of culture before fixation.
(b) In vitro. Skin fragments were cultured in vitro at 38-5 °C according to two
organ culture methods. The duration of the culture was variable and will be
indicated in appropriate sections of the Results.
(a) Culture on natural media according to the method of Wolff & Haffen (1952).
Two semi-solid media were used: the standard medium (JE) composed of 6
parts of 1 % agar in Gey's saline, 3 parts of Tyrode's solution, 3 parts of 9-day
chick embryo extract and 400 i.u,/ml of penicillin, and a medium (EC) in
which the total embryo extract was replaced by an equal amount of brain
extract from 17- to 18-day chick embryos (Sengel, 1961). Medium EC with
brain extract is particularly favourable for the differentiation of feather rudiments and growth of feather filaments (Sengel, 1961). For this reason it was used
exclusively in the experiments where skin constituents were separated and reassociated after trypsin digestion.
(/?) Culture in a synthetic medium. The second method involved a liquid
medium used in Falcon dishes. Explants were placed on different types of substratum (black Millipore filters, non-incubated chick egg vitelline membrane,
611
Feather pattern development
Type c explants, on medium EC
iO-37
iO-47
0 58 i
Type c explants, on medium JE
iO-36
;o-46
;0-59
Type a 0'1 recombinants, on medium EC
1
:b-28
iO-44
::::0-53
iO-59
Type a 180 recombinants, on medium EC
Type a 90° recombinants, on medium EC
I?)
iO-29:
—i
(2)
©
:o-55
01
0-2
0-3
0-4
0-5
0-6
-Width of explant -
0-7
0-8
0-9
Fig. 3. Feather pattern reorganization. Variation of the mean position (expressed,
inside the bars,as a decimal fraction of theexplant's width: abscissa 0 corresponding
to the medial edge of dermis, and abscissa 1 to its lateral edge) of the newly formed
primary row of feather rudiments as a function of the stage (A, B, C or D) at which
the skin was explanted. Circled numbers give number of explants. Mean values are
given with their confidence interval forP < 005.
612
G. NOVEL
agar gel or lens paper) which was in turn placed on a stainless-steel grid. The
synthetic medium used was Difco TC medium 199 with phenol red, supplemented with 5 % glutamine and 400 i.u./ml of penicillin.
5. Measurements
An ocular micrometric scale with graduations of 0-025 mm was used for all
measures, at a magnification of x 40.
(a) Length of embryonic feathers. After 3% days of culture, explants were fixed
in Bouin-Hollande's solution and the length of outgrowing feather buds was
measured.
For the analysis of feather pattern formation, it is required that the first
formed row of feather rudiments be recognized, so that the sequence of appearance of the successive rows of feathers may be determined. In explants cultured
in the synthetic medium, it was easy to recognize the first formed row by direct
observation. This was not the case for the cultures on natural media, because of
the milky appearance of the substratum. Consequently, in experimental series
involving natural media, other criteria were used, together with direct periodic
observation, for the characterization of the first differentiating row (thereafter
called 'primary' row): it was found that the primary row either contained the
longest feather buds or displayed the best morphogenetic performance, the
latter being defined as the sum of the lengths of the feather buds in each of the
longitudinal rows. In 90 % of the cases both criteria characterized the same row;
in the 10 % remaining cases the longest feather bud was not located in the row
with maximal morphogenetic performance but in one of the adjacent rows, and
the identification of the primary row was left to subjective judgment. For
comparison, these quantitative criteria were also used in several series of in vivo
cultures on the CAM and in vitro cultures in medium 199.
In order to obtain clear differences in morphogenetic performances of newly
formed* rows and also to avoid overlapping of feather filaments that would
obscure their pattern, it was found convenient not to keep the cultures for more
than 4 days. Indeed, beyong 96 h of cultivation the growth of the first formed
feather buds slows down, and practically stops at 5 days. The differences in
length of the buds of the successive rows decrease and are finally no longer
measurable. Consequently, duration of the culture was voluntarily limited to 3^
days.
(b) Determination of the position of the primary row. As the primary row was
always oriented parallel to the cephalo-caudal axis of the dermal component
of the explant, its position within the width of the explant was defined by
measuring its distance from the medial edge of explant's dermis. This distance
was expressed as a decimal fraction of the explant's total width.
* For simplicity, the expression 'newly formed' will be applied only to those feathers or
rows of feathers that developed after the dedifferentiation of the initial feather pattern,
although, strictly speaking, newly formed feathers also appear in non-reorganizing explants.
613
Feather pattern development
Type c explants, on medium EC
0
6 12 I— 18H I 24
Type c explants, on medium JE.
30
1 1-36 H 42 48 54
hours of
culture
34)(34)(34
0
6
12 1—18-1
Type a 0 recombinants, on medium EC
100
H42-I 48 54
hours of
culture
W
90
80
70
60
5 50
40
30
20
10
0
6
12 18 I-24H I—30
hours of
culture
Fig. 4. Analysis of feather pattern reorganization. Vertical bars give percentage of
explants in which rudiments present at explantation were still visible (white bars),
had regressed and become undetectable (small dots), and in which newly formed
rudiments had differentiated (black bars) as a function of time. Circled numbers give
number of explants.
614
G. NOVEL
6. Histology
Explants were fixed in Bouin-Hollande's solution, every 6 h during the first
54 h of culture, and sectioned at 5 /«n. Sections were stained with haematoxylineosin.
RESULTS
The behaviour of skin explants varied according to several factors: the type
of excision, the nature of the medium, the stage at explantation and also
according to whether or not the explant had been dissociated into dermis and
epidermis and reassociated before explantation.
Under all experimental conditions that were tested, however, feather rudiments differentiated in the majority of the explants and finally occupied the
whole surface of the explants at the end of the cultivation period. It was also
observed that the shape of the explant became distorted in a quite constant
manner: although explants were always excised as rectangles, their lateral
(dermal) edge shrank more severely than did the medial one; as a consequence
the explant assumed a trapezoidal shape and the longitudinal rows of feathers
were more or less curved, anteriorly and more so posteriorly, in lateral directions.
Figs. 6-8, 10, 11, 15, 18, 22 and 23 are typical examples of this feature.
I. Behaviour of non-dissociated skin
1. Explants in which the mid-dorsal feather rudiments are preserved (excision
type a)
(a) In vitro culture in synthetic medium. Fifty-three explants of skin of stage
A, B, C or D were cultured on Millipore filter in the synthetic medium 199. At
explantation, they comprised, besides the mid-dorsal row, from 0 to 3 lateral
rows of feather rudiments (Fig. la). These were constituted by circular opaque
patches of 0-15 mm in diameter composed of a thickened epidermal placode and
an underlying lenticular dermal condensation.
Forty-five of the 53 explants were placed directly in the culture medium; the
8 remaining explants (trypsinized non-dissociated controls) were placed for 1015 min in the trypsin-pangestin solution, washed and then explanted without
dissociation. The 53 explants were regularly observed every 12 h for the first 2
days of culture and fixed after 3^ days of culture.
All explants developed according to the normal sequence. There was no
difference between trypsinized and non-trypsinized fragments: the feather
rudiments that were already present at explantation gradually gave rise to
feather buds, while new longitudinal rows differentiated successively in mediolateral direction.
After 3^ days of culture, the initial mid-dorsal row appeared to be the most
advanced in the development of its feathers; it contained the longest buds (0-25
mm of mean length) and was characterized by the best morphogenetic performance (Fig. 5,1-55 mm; Fig. 6,2-55 mm).
Feather pattern development
615
(b) In vitro culture on natural media (EC andJE). Fifty-eight explants of stage
A, B, C or D were cultured on medium EC and 27 on medium JE. At explantation they contained, besides the initial intact mid-dorsal row of feather rudiments,
from 0 to 3 lateral rows (Fig. 1 a).
Thirty-six of the 58 fragments were explanted directly on medium EC without
prior treatment; the 22 remaining explants were first placed in the trypsinpangestin solution (trypsinized non-dissociated controls) and then explanted on
medium EC. In this series the fragments were observed regularly every 6 h for
the first 2 days of culture and fixed after 3£ days.
On either medium, all explants, whether trypsinized or not, developed as in
the preceding series: the initial feather rudiments progressively formed feather
buds, while new longitudinal lateral rows of feather rudiments differentiated in
mediolateral sequence.
After 3 j days of culture the initial mid-dorsal row contained the longest
feather buds (0-25 mm of mean length) and displayed the best morphogenetic
performance (1-58 mm) (Fig. 7).
2. Explants in which the initial mid-dorsal feather rudiments were damaged or
eliminated at explanation (excision type b or <?)
(a) In vivo culture on the chorioallantoic membrane. Twenty-seven explants of
stage A, B, C or D were grafted on the chorioallantoic membrane and cultured
for 2>\ days. Explants were excised in pairs on either side of the mid-dorsal line
according to type c, so that each of the explants contained, along its mid-dorsal
edge, part of the damaged initial mid-dorsal feather rudiments and from 0 to 3
lateral rows of feather rudiments (Fig. 1 c). They were observed regularly twice
a day.
All these explants developed according to the normal in situ sequence: feather
buds grew out of the already differentiated feather rudiments, the latter being
supplied by blood capillaries of the host membrane from the second day of
culture; new rows of feather rudiments appeared successively in medio-lateral
sequence.
At fixation, from 4 to 7 longitudinal rows of feathers covered the whole width
of the explant; the row containing the longest feather buds was always located
along the initial mid-dorsal edge of the explant (Fig. 8).
(b) In vitro culture in the synthetic medium 199. One hundred and seventy-six
explants were cultured for 3^ days in medium 199 and observed regularly twice
a day. One hundred and twenty-eight fragments of stage A, B or C (48 on
Millipore filter, 43 on vitelline membrane, 27 on agar gel and 10 on lens paper)
were of type c and comprised part of the initial mid-dorsal feather rudiments
along the medial edge and from 0 to 2 lateral rows of feather rudiments. The 48
remaining explants of stage A, B, C or D were of type b and comprised, at
explantation, from 0 to 2\ lateral rows of feather rudiments.
40
E M B 30
616
G. NOVEL
Feather pattern development
617
All explants developed according to the normal medio-lateral sequence,
whether they already contained feather rudiments at explantation or not: the
new lateral rows differentiated one after the other in medio-lateral sequence and
each initial or new rudiment gave rise to one feather bud.
After 3j days of culture all explants showed 3-7 longitudinal rows of feathers,
FIGURES
5-13
Non-dissociated skin fragments cultured for 3-i days. Explants are oriented with
their anterior edge at the top of the pictures. Arrow points towards head of donor
embryos and consequently also indicates the general orientation of longitudinal rows
of feathers. /, Lateral; m, medial; inn, median edges of explant.
Figs. 5-7. Absence of feather pattern reorganization in explants whose middorsal initial row of rudiments was left intact (type a excision).
Figs. 5-6. Left skin fragments, explanted at stage A (Fig. 5) or B (Fig. 6) on Millipore
filter in medium 199. Feather buds are arranged in five or six rows parallel to the
longitudinal edges of the explant. They differentiated in normal medio-lateral sequence
(7, 2, 3, 4, 5) starting with the mid-dorsal initial row (/). Those along the medial
edge correspond to half the prospective area (Fig. 5) or half the rudiments (Fig. 6)
of the first right lateral row of feathers (2'); hence their small size.
Fig. 7. Left skin fragment, explanted at stage B on medium EC. The mid-dorsal row
of rudiments was the first (7) to give rise to feather buds; lateral rows (2, 3, 4,5,...)
differentiated later in medio-lateral sequence. This explant was trypsinized, but not
dissociated, and served as control for the dermo-epidermal recombinants. The
morphogenetic performance (sum of lengths of feather buds) of rows 1-4 was respectively (mm): 1-58, 1-50, 1-36 and 114.
Figs. 8-10. Absence of feather pattern reorganization in right explants whose
mid-dorsal initial row of rudiments was damaged (Fig. 8: type c excision) or
eliminated (Figs. 9-10: type b excisions) at explantation. Explants were cultured
on the CAM (Fig. 8) or on Millipore filter in medium 199 (Figs. 9-10).
Fig. 8. Skin fragment explanted at stage A. The feather filaments along the median
edge were the first (7) to differentiate; they correspond to the first right longitudinal
lateral row. Other longitudinal rows formed in successive medio-lateral order (2, 3,
4, 5, 6). Note decreasing length of feather buds from median to lateral edge of
explant.
Figs. 9-10. Skin fragments explanted at stage A (Fig. 9) or B (Fig. 10). The feather
buds are arranged in rows parallel to the longitudinal edges of the explant. Those
along the medial edge were the first (7) to differentiate. They correspond to half the
prospective area (Fig. 9) or half the rudiments (Fig. 10) of the first right lateral row
of feathers; hence their small size. Rows 2-4 or 2-5 appeared in normal mediolateral sequence.
Figs. 11-13. Feather pattern reorganization in right explants whose mid-dorsal
initial row of rudiments was damaged (type c excision) at explantation. Explants
were cultured on medium EC. The initial feather rudiments (half the mid-dorsal row
in stage A explant of Fig. 11; half the mid-dorsal row and the first lateral row in
stage B explant of Fig. 12; half the mid-dorsal row, the first and the second lateral
rows in stage C explant of Fig. 13) disappeared during the first 24 h in vitro. Newly
formed feather buds differentiated in rows parallel to the longitudinal edges of the
explant: the rows appeared in succession as indicated (7, 2, 3, 4, . . .), starting with
the newly formed primary row (7), the position of which was close to the median
edge in stage A explant (Fig. 11), approximately in the middle of the width in stage
B explant (Fig. 12), and closer to the lateral edge in stage C explant (Fig. 13).
40-2
618
G. NOVEL
arranged in a hexagonal pattern. The row with the longest buds was always
located along the initial medial edge (Figs. 9,10).
The nature of the substratum had no influence on the sequence of formation
of the feather rudiments nor on their final distribution. It only affected the
morphogenetic performance: explants cultured on Millipore filter had the best
morphogenetic performance (0-95 mm on Millipore filter, 0-60 mm on vitelline
membrane, 0-80 mm on agar gel or lens paper) and the highest number of feather
buds.
(c) In vitro culture on natural media (EC and JE). One hundred and twentynine type c fragments of skin of stage A, B, C or D were explanted; 81 on medium
EC and 48 on medium JE.
In explants of stage A, B or C containing less than three lateral rows of feather
rudiments at explantation, the feather pattern underwent a complete reorganization. In contrast, explants of stage D (6 cases, 3 lateral rows at the beginning)
behaved in the same way as all the explants where the initial mid-dorsal row was
left intact: initial rudiments gave rise to feather buds without their arrangement
being reorganized. The behaviour of the explants and the results were similar on
either medium.
The reorganization of the feather pattern of explants of stage A, B or C
consisted in a complete regression and disappearance of the initial rudiments
followed by the differentiation of new rudiments after 24 h of culture. In the
majority of the cases, the newly formed feathers were arranged according to a
hexagonal pattern and aligned parallel to the newly formed primary row. The
latter displayed the best morphogenetic performance at the end of the culture
period (Figs. 11-13). In some of the explants, a few feather rudiments were
placed without apparent order at variance with the surrounding pattern.
Despite these exceptional locations, the hexagonal pattern could, in all cases, be
easily recognized and oriented.
From 2 to 9 longitudinal rows of feather buds formed on all explants after 3%
days of culture. The number of feathers was usually highest in the explants of
stage C, which was probably due to the slightly larger surface of explants of that
stage; it was, however, always lower than the number that an equivalent area of
dorsolumbar skin would have yielded in situ.
The position of the newly formed primary row varied with the stage of the
skin at explantation. The more advanced the stage at explantation, the farther
away from the medial edge of the explant the primary row formed. The subsequent rows differentiated on one or on each side of the newly formed primary
row and parallel to it (Figs. 11-13). For each stage the mean values of the distance
between medial edge and newly formed primary row were significantly different
from one another (Fig. 3). Results obtained with either culture medium were
similar.
It is interesting to note that, in the case of the older (stage C) explants where
the newly formed primary row differentiated close to the lateral edge of the
Feather pattern development
619
explant, the spread of the feather pattern progressed in reverse direction with
respect to the original medio-lateral extension of the spinal pteryla: the lateral
rows were added in a direction moving from the lateral edge towards the middorsal edge of the explant.
Feather pattern reorganization was analysed in more details in 152 type c
fragments of skin of stage A, B, or C: 69 were cultured on medium EC and 83
on medium JE.
Nineteen of those on medium EC and 13 of those on medium IE were
observed regularly every 6 h until 54 h after explantation; the other explants
were fixed after increasing culture periods for histological study and provided
complementary data for the analysis of feather pattern reorganization.
The chronological analysis of the reorganization is given in Fig. 4. The
regression of the initial feather rudiments was total in all cases and affected both
constituents of the skin: the dermal condensations as well as the epidermal
placodes disappeared. Whent he regression was completed, the cell density in
the dermis had become homogeneous and the height of epidermal cells was
uniform again. Thereafter newly formed placodes and condensations rediflferentiated, apparently following the same mechanism as in normal development.
The newly formed feathers were arranged in longitudinal rows and formed,
in most cases, a recognizable, although sometimes distorted, hexagonal pattern.
It can be concluded from this and the previous experimental series that, on
the medium EC as well as on the medium JE, the initial feather rudiments
undergo a transitory regression if the mid-dorsal initial feather rudiments are
cut in half and damaged at explantation.
A number of explants were fixed during the cultivation period for histology
and particularly for the study of the variation of cell density in the plumar and
interpapillar dermis during reorganization of the feather pattern. The histological observations however did not allow to draw any conclusion: on one hand,
the number of cells and the state of differentiation varied to a great extent from
explant to explant, and, on the other hand, under the present culture conditions,
the explants shrunk more or less severely during the first hours of culture. In
consequence, a high cell density did not necessarily result from an increase in
cell number, but might be due to the reduction of intercellular spaces.
II. Role of dermis and epidermis in the establishment of the feather pattern
The previous experimental series showed that an explant of skin, when
cultured on a natural medium, undergoes a complete reorganization of its
feather pattern if the initial mid-dorsal feather rudiments are damaged by an
incision.
The dissociation of the skin into dermis and epidermis likewise causes a
partial destruction of feather rudiments present at the time of explantation. Does
620
G. NOVEL
Feather pattern development
621
a piece of skin constituted by reassociated dermis and epidermis also undergo a
complete reorganization of its initial feather pattern when explanted in vitro or
in vivo'}
1. In vitro culture on the natural medium EC (excision type a)
(a) Control dermo-epidermal reassociation without rotation of the epidermis.
In order to answer that question, pieces of skin of stage A, B, C or D were
excised according to type a. They comprised, at the time of explantation, the
mid-dorsal row of feather rudiments and from 0 to 3 lateral rows of rudiments.
They were separated into their constituents by trypsin digestion, and reasso-
FIGURES
14-24
Feather pattern reorganization in dermo-epidermal type a recombinants cultured
for 3^ days. Epidermis was reassociated to its own dermis without (0°) or with rotation of its antero-posterior axis by 180° or 90° with respect to the cephalo-caudal
polarity of the dermis. Explants are oriented with the anterior edge of dermis at the
top of the pictures. The arrow points towards anterior edge of dermis and consequently also indicates the general orientation of longitudinal feather rows. Da,
anterior; Dm, medial edges of dermis; Ep, posterior edge of epidermis. Except for
Fig. 24, newly formed feather buds are arranged in rows parallel to the cephalocaudal axis of the dermis,, Successive rows are numbered (/, 2,3,.. .) in the order
in which they differentiated.
Figs. 14-17. Left skin fragments cultured on medium EC. The newly formed primary
row (/) differentiated close to the medial edge of dermis in stage A explant (Fig. 14),
approximately in the middle of the width of dermis in stage B explant (Fig. 15),
beyond the middle in stage C explant (Fig. 16), and close to the lateral edge of
dermis in stage D explant (Fig. 17).
Figs. 18-19. Left explants cultured on medium EC. Newly formed primary row (/)
developed close to the medial edge of dermis in stage A explant (Fig. 18), approximately in the middle of the width of dermis in stage C explant (Fig. 19). Note that
buds have their distal tips oriented towards the posterior edge of epidermis.
Fig. 20. Left stage B explant cultured on the CAM. Feather filaments along the
longitudinal edges of the explant grew out first (/) and simultaneously;filamentsin
centre of explant formed later (2). Apices of feather filaments are oriented towards
posterior edge of epidermis.
Figs. 21-22. Right skin fragments cultured on medium EC. Newly formed primary
row (7) developed close to the medial edge of dermis in stage A explant (Fig. 21),
close to the lateral edge of dermis in stage C explant (Fig. 22). Distal tips of buds are
oriented towards posterior edge of epidermis, at 90° to the left (Fig. 21) or to the right
(Fig. 22) with respect to cephalo-caudal axis of dermis. Giant buds (marked by stars)
probably result from the fusion of two adjacent feather rudiments.
Fig. 23. Left stage C explant in medium 199. Newly formed primary row (/)
differentiated within lateral half of explant. Note that buds are individually slanting
towards posterior edge of epidermis, at 90° to the left with respect to cephalo-caudal
axis of dermis.
Fig. 24. Left stage C explant on the CAM. Note the disorderly distribution of
feather filaments. Majority of larger feather filaments (marked by stars) have their
tips oriented towards posterior edge of epidermis. Six otherfilamentsare disoriented,
probably because explants on the CAM tend to round up.
622
G. NOVEL
ciated in correct cephalo-caudal orientation. However, even in orthopolar
recombinations of this kind, it is impossible to cover the dermal condensations
by the corresponding epidermal placodes, because of the shrinkage of the
isolated epidermis. Consequently the integrity of almost all if not all feather
rudiments is destroyed by dissociation.
Seventy-nine explants, of which 21 were observed regularly every 6 h, were
cultured on the medium EC. They behaved in the same way as the explants
whose mid-dorsal row of feather rudiments had been damaged by a median
incision (Fig. 4). The feather pattern underwent a complete reorganization even
in the case of the explants of stage D (comprising 3 lateral rows of feather
rudiments at the beginning of the culture).
In 31 of these explants, the length of the feather buds was measured after 3-£days of culture and the morphogenetic performance of each newly formed row
calculated. After the regression of the initial rudiments, a newly formed primary
row developed whose feathers were longer than those of the adjacent rows. It was
parallel to the longitudinal edges of the explant. Its distance from the medial
edge increased as a function of the stage at explantation (Figs. 14-17); the mean
values of these distances for each of the stages A, B and C were significantly
different from one another (Fig. 3).
Dissociation of dermis and epidermis, and their reassociation thus provide a
means to investigate the respective roles of dermis and epidermis in the reorganization of the feather pattern and in the establishment of its cephalocaudal axis and polarity. Consequently, two other types of reassociation were
performed with type a explants (Fig. 2), in which the epidermis was rotated by
180° or 90° with respect to the cephalo-caudal axis of the dermis. Explants were
cultured in vitro either on the medium EC, or in the medium 199, or as chorioallantoic grafts.
(b) Dermo-epidermal reassociation with 180° rotation of the epidermis. The
epidermis was reassociated with its own dermis after rotation of its anteroposterior axis by 180° with respect to the cephalo-caudal polarity of the dermis.
Forty-three recombinations of this type of stage A, B, C or D were cultured on
the medium EC for 3^ days.
The initial feather rudiments of all explants disappeared progressively and
completely during the first 24 h of culture. After 30 h, newly formed rudiments
appeared. At the end of the culture period they were arranged in 2-7 longitudinal
rows parallel to the lateral edges of the explant (Figs. 18, 19). The apex of the
feather buds was oriented towards the anterior edge of the dermis in conformity
with the cephalo caudal-polarity of the epidermis (Sengel, 1958).
As before, the position of the newly formed primary row of feather rudiments
varied as a function of the stage at explantation. Its distance from the dermal
medial edge was the larger as the stage at which the explants were obtained was
more advanced (Fig. 3). In three recombinants of stage D, the primary row
was closest to the lateral dermal edge of the explant; in six other recombinants
Feather pattern development
623
of stage D, one or two additional lateral rows had developed beyond the
primary row.
(c) Dermo-epidermal reassociation with 90° rotation of the epidermis. The
epidermis was reassociated with its own dermis after a 90° rotation of its
cephalo-caudal axis to the right or to the left with respect to the antero-posterior
polarity of the dermis. Sixty recombinants of this type of stage A, B, C or D were
cultured on medium EC for 3} days.
During the first 30 h of culture the explants underwent a complete reorganization of their feather pattern as described before. From 2 to 7 rows of newly
formed feathers differentiated parallel to the longitudinal edges of the dermis.
The new feathers were individually oriented at approximately 90° with respect
to the cephalo-caudal axis of the dermis; their distal tip pointed towards the
posterior edge of the epidermis in conformity with the antero-posterior polarity
of the latter (Sengel, 1958). Their arrangement in a hexagonal pattern, however,
was in accordance with the cephalo-caudal axis of the dermis (Figs. 21, 22).
The mean position of the newly formed primary row with respect to the
lateral edges of the dermis was similar in this series of experiment as in the
previous ones where the cephalo-caudal axis of dermis and epidermis coincided
or were inversed by 180° (Fig. 3). As in the experiments of 180° rotation, in four
explants of stage D the primary row was closest to the lateral dermal edge;
in 8 other recombinants of stage D, one or two additional lateral rows
differentiated between the primary row and the lateral dermal edge.
2. In vitro culture in synthetic medium 199 (excision type a)
It was shown in the previous experiments, that explants cultured in the synthetic medium 199 did not undergo any reorganization of their feather pattern
when the mid-dorsal initial feather rudiments were damaged by incision or
eliminated at the time of excision. The question then arises whether dermoepidermal recombinants do undergo reorganization of their feather pattern
when cultured in the synthetic medium 199.
Thirty-six recombinants of stage A, B or C in which the epidermis was rotated
by 180° (17 explants) or by 90° (19 explants) were cultured for 3-\ days in medium
199 and observed regularly twice a day.
Results were in every respect similar to those obtained with explants cultured
on medium EC. Dermal condensations and epidermal placodes regressed during
the first 24 h of culture. Subsequently, newly formed rudiments differentiated.
After 3-^ days the newly formed feathers were arranged in longitudinal rows
parallel to the antero-posterior axis of the dermis irrespective of the rotation of
the epidermis (Fig. 23). As before a primary row differentiated whose position
with respect to the lateral edges of the explant's dermis varied as a function of
the stage at explantation. Newly formed lateral rows were added successively on
one or both sides of the newly formed primary row: (1) the latter was located
close to the initial medial dermal edge for explants of stage A; (2) approxi-
624
G. NOVEL
mately in the middle of the width of the explants of stage B; or (3) close to the
lateral edge of the dermis for explants of stage C. As before, the individual
orientation of the feather buds was always in conformity with the cephalo-caudal
polarity of the epidermis, their distal tip pointing towards the anterior (180°
rotations), left (90° rotations to the right), or right (90° rotations to the left)
edge of the explant's dermal component.
3. In vivo culture on the chorioallantoic membrane (excision type a)
The epidermis was reassociated to its own dermis after rotation by 180° or
90° of its antero-posterior axis with respect to the cephalo-caudal polarity of
the dermis. Seventeen 180° recombinants and nine 90° recombinants of stage
A, B, C or D were cultured for 3^ days as chorioallantoic grafts and observed
twice a day.
Epidermal placodes as well as dermal condensations that were already
present at the time of explantation persisted throughout the first 2 days of
culture. The first feather buds arose from these initial structures along the initial
mid-dorsal edges of the epidermal and dermal constituents. New rudiments
differentiated from the initially homogeneous tissues. After 3^ days, the longest
feather buds were located at the site of the initial dermal condensations and
epidermal placodes (Figs. 20, 24).
Whereas in all preceding experimental series, the developing rudiments were,
in the majority of cases, regularly arranged in longitudinal alternate rows
according to the cephalo-caudal polarity of the dermis, the feather buds of
dermo-epidermal recombinants cultured on the chorioallantoic membrane were
distributed on the surface of the explant in a disorderly manner (Figs. 20, 24).
In these recombinants, where the initial dermal condensations and epidermal
placodes persisted, some of the initial dermal condensations induced the formation of an epidermal placode in the overlying epidermis and reciprocally some
of the initial epidermal placodes elicited in the underlying dermis the formation
of a dermal condensation. In addition, it was found that quite a few feather buds
were double structures with bifurcated tips, which probably arose from the
collaboration of partially overlapping dermal condensation and epidermal
placode. It was also possible to observe, in some cases, the overlapping of two
feather patterns: one directed by the dermis, the other one by the epidermis. The
rows of feathers, so far as they could be identified as such, were predominantly
parallel to one another in the case of the 180° recombinants (Fig. 20); they ran
in orthogonal directions to one another in the 90° recombinants (Fig. 24).
Feather pattern
625
development
Table 1. Effects of pre-culture treatment, culture medium and age of skin
explants on feather pattern reorganization
Pre-culture treatment
Medium
Mid-dorsal initial row
preserved
Mid-dorsal initial row
damaged
199, EC or
Stage at
explantation
A, B, C or D
No reorganization
199 or CAM
EC or JE
A, B, C or D
A, B or C
199 or EC
A, B, C or D
No reorganization
Reorganization
No reorganization
Reorganization
CAM
A, B, C or D
No reorganization
JE
D
Dermo-epidermal
dissociation and
reassociation
Effect on
feather pattern
DISCUSSION AND CONCLUSIONS
Embryonic skin explants behave differently according to the culture method
used (Table 1). When blocks of skin are cultured as chorioallantoic grafts, they
continue their differentiation without prior reorganization irrespective of
whether the skin is intact or a dermo-epidermal recombinant. The feather
rudiments that are already present at the time of explantation give rise immediately to feather buds and new longitudinal rows are added progressively to the
first ones. Thus the culture conditions on the chorioallantoic membrane are
particularly suitable for the differentiation and growth of the feather buds and
feather filaments. The behaviour of the explants is quite different in in vitro
cultures, where the medium has a strong influence on the development of the
feather pattern.
Explants of young stages A, B and C, obtained on either side of the middorsal line and whose initial mid-dorsal rudiments are damaged at excision,
develop differently according as they are cultured in the synthetic medium 199
or on natural media EC and JE. In medium 199 these explants form their
feather rudiments in a way similar to the normal development: the initial feather
rudiments give rise progressively to feather buds and the lateral rows appear
successively one after the other in medio-lateral direction. On the media EC and
JE, the feather rudiments that are present at explantation do not continue their
development, but progressively dedifferentiate and disappear. The dedifferentiation affects the dermis as well as the epidermis: in the former, dermal condensations disaggregate and cell density becomes homogeneous; in the latter, placodes
vanish and the height of epidermal cells becomes uniform again. The cutaneous
tissues undergo a complete reorganization, after which newly formed feather
rudiments develop according to a new plan of organization. Explants of advanced stage D, however, continue their development independently of the
culture medium, and do not undergo any reorganization of their feather pattern.
The subtratum has no major role in the construction of the hexagonal feather
626
G. NOVEL
pattern. In medium 199 the explants behave roughly in the same way irrespective
of the substratum used (Millipore filter, agar gel, vitelline membrane or lens
paper): the lateral feather rudiments differentiate successively in longitudinal
rows parallel to the cephalo-caudal axis of the explant in medio-lateral direction.
The substratum merely influences the quality of the differentiation: the feather
buds are longer and more numerous when the explants are placed on Millipore
filter than on any other substratum.
The maintenance of the initial mid-dorsal row results in the orderly and
immediate differentiation of the feather rudiments of the lateral rows. When it is
damaged either by a longitudinal incision in the explants cultured on media EC
and JE, or by trypsin dissociation followed by reassociation for the explants
cultured on medium EC or 199, the already formed feather rudiments dedifferentiate, the explants become optically homogeneous again. This regression
is then followed by the redifferentiation of newly formed feather rudiments.
The site and alignment of the newly formed rudiments are always in conformity with the cephalo-caudal axis of the dermis. The new feathers are
arranged in longitudinal alternate rows in a hexagonal pattern on either side of
the rudiments of the first newly formed (primary) row. The latter appears to
exert an organizing influence on the lateral yet unpatterned dermis. Indeed the
feather rudiments of lateral rows differentiate in an alternate arrangement, each
of them facing an interval between two successive feathers of the preceding row.
The position of the newly formed primary row with respect to the lateral edges
of the explant's dermis varies as a function of stage at explantation. Its distance
from the medial dermal edge is the larger as the explant is obtained at a more
advanced stage.
One of the main features of cultured skin, then, appears to be its ability, under
certain in vitro culture conditions, to undergo a complete regression of its initial
feather rudiments followed by the redifferentiation of a new feather pattern.
This newly-formed pattern organizes itself according to a primary morphogenetic zone whose position varies as a function of the stage at explantation.
Thus, the feather pattern is still capable of regulation provided the integrity of
its first rudiments is destroyed prior to explantation. Consequently, in the spinal
pteryla, the hexagonal feather pattern is not a predetermined one and the
position of each feather rudiment is not preestablished at stages A-D, i.e. at 6 | 1\ days of incubation. These results are in line with those of Linsenmayer (1972),
who showed that the femoral feather pattern likewise was determined late in
development, probably just prior to the formation of the dermal feather
condensations.
In the process of determination of dorsal feathers, two phases must be
distinguished. The first phase is an early one and corresponds to the featherforming determination of the somitic mesodermal cells: indeed, at a stage as
early as 2-2^- days of incubation, the explanted somitic mesoderm is able,
together with its own overlying ectoderm, to form feathers (Straus & Rawles,
Feather pattern development
627
1953). At the same early stage, mesodermal somitic cells are regionally determined to give rise to the different cephalo-caudal levels of the spinal feather
tract (Mauger, 19726). Later in development, just prior to the differentiation of
the rudiments, the second phase leads to the determination of the hexagonal
pattern. The differentiation of the latter begins with the formation of the feather
rudiments of the first row, which is mid-dorsal in the lumbar region of the
spinal pteryla. As soon as these are present, the rudiments of the second, third,
etc., rows progressively arrange themselves parallel to the preceding ones. In
the absence of the first row, the initial hexagonal pattern may temporarily
dedifferentiate and be replaced by a new organization on each side of a new
primary row. It seems clear that the reorganization of the explant occurs step by
step starting with the first redifferentiated rudiments. Each row of feather rudiments, to begin with the primary row, appears to play a double role in the
maintenance and further construction of the hexagonal pattern. The singularity
of the first appearing row resides in the fact that its differentiation apparently is
autonomous, whereas the formation of all following rows is determined by the
position of the feather rudiments in the preceding row. It is important to note
that the position of the newly formed primary row is not random. Indeed the
newly formed rudiments are arranged in rows parallel to the cephalo-caudal
axis of the dermis. It was shown that its distance from the explant's dermal
medial edge increases as a function of the stage at explantation. This may be
explained in the following way: after the regression of the initial structures, the
first cells to begin their redifferentiation are probably those that were on the
verge of forming a condensation at the time of excision; they were then probably
in a phase of morphogenetic activity, which is undetectable at histological
examination and would have led to the differentiation of the overlying epidermal
placode.
Sengel & Rusaouen (1968) showed that the epidermal placodes differentiate
morphologically before any dermal condensations can be detected underneath;
they observed also that the bipolar cells within the dermis are oriented according
to three main directions, namely transverse, oblique, and longitudinal (respectively at right angle, 45° and parallel to the cephalo-caudal axis of the
embryo). Two questions arise: is there a causal relationship between cell
orientation and the fibrous intradermal network described by Stuart & Moscona
(1967)?; what causes cells to concentrate at certain focal points within the
dermis and is there a relationship between the location of these centres and the
nodes of the lattice? For Stuart & Moscona (1967), and Stuart, Garber &
Moscona (1972), the organization of the birefringent lattice in the dermis
slightly precedes the development of the dermal condensations and is required
for their formation; but they could 'not establish, unequivocally, that fiber
alignment precedes dermal cell alignment in lateral areas of the tract'. Thus the
possibility remains that fibre orientation is concomitant with dermal cell polarization. Furthermore it appears that the placodal differentiation of the epidermis
628
G. NOVEL
plays an important role in the geometrical arrangement of the intradermal
fibrous lattice (Goetinck & Sekellick, 1972). However, Ede, Hinchliffe & Mees
(1971) reported that this fibrous collagen lattice forms during and not before the
appearance of the dermal condensations, suggesting that its patterning is
determined by the position and orientation of the dermal cells, rather than vice
versa. Since, in our conditions, the hexagonal pattern is reorganized, the
fibrous network - if already present - either must be labile so that the destruction
of its initial (mid-dorsal) nodes leads to its complete reconstruction, or may
serve during reorganization as a template for the newly formed feather pattern.
Present data do not allow one to choose between these two possibilities, since the
location of newly formed rudiments cannot be ascertained with sufficient
accuracy. The bipolar shape of the cells suggests that the new dermal condensations form by a secondary centripetal cell migration, possibly along the fibres of
the old or of a new lattice. The histological observations of the present investigation are in line with those of Ede et al. (1971), and also of Stuart, Garber &
Moscona (1972), who showed that the dermal condensations do not primarily
result from mitotic foci, as indicated by Wessells & Evans (1968), but rather
from centripetal cell migrations.
The early differentiation of the epidermal placodes and their possible role in
the establishment of the underlying fibrous network may also explain, in part,
the behaviour of the skin cultured in vivo on the chorioallantoic membrane.
Under these particularly favourable culture conditions, the already differentiated
epidermal placodes are visibly maintained and probably able to act on the
reassociated dermis by inducing it to form feather condensations underneath
each epidermal placode.
By the use of in vitro culture techniques, it is possible to establish a relationship between the number of feathers formed and the surface of the skin explant.
It was shown that skin explants shrink during the first two days of culture,
which corresponds to the phase of feather pattern reorganization (Sengel, 1958).
The surface area of the skin, available for the formation of feather rudiments, is
thus reduced and the number of differentiating feather buds is lower than the
number of feathers that would have formed from an equivalent area of noncultured skin. It was also observed that, on medium EC, the number of feather
buds was approximately constant for a given explant size (unpublished results).
It appears, then, that a minimal surface of skin is necessary for the differentiation of one feather rudiment. The larger the available surface, the greater the
number of feathers. In mice, the primary hair follicles are widely separated from
one another at the time of differentiation. According to Claxton (1967), each of
them is thought to be surrounded by an inhibitory zone precluding the formation
of another hair follicle within a certain distance. The situation is quite different
in the chick, where the space between adjacent early feather condensations is all
but non-existent (see fig. 16 in Sengel, 1971). The formation of this close
patterning may result from the conjunction of two mechanisms, namely centri-
Feather pattern development
629
petal cell migration and peripheral cell proliferation, the latter compensating
for the peripheral cell loss due to the former. Thus, within the dermal condensations, the cells would increase in number, until the normal genetically determined
density was reached. Next condensations would develop in tangential contact
with two preceding ones. Histological observations indeed reveal that mitoses
are restricted to the periphery of dermal condensations. The interplumar nondense dermis of older embryos may then result from the slowing down of
marginal proliferation and from overall growth and stretching of the initial
close pattern. It can thus be argued that the diameter of condensations and their
formation at maximal density within a given area of embryonic skin might be a
genetic requirement of pre-dermal cells. This requirement, then, would be the
direct cause of the hexagonal pattern, an arrangement known to allow the
packing of a maximal number of elements within a minimal area.
Since the hexagonal pattern is not predetermined, all dermal cells can, during
reorganization, participate in the construction of a condensation. The question
then arises why a certain cell will end up in the centre of a dermal condensation
and another one will remain at the border between two adjacent condensations.
If feather condensations are formed by centripetal migration of dermal cells, the
following hypothesis may be considered: under some unknown epidermal
influence, a fibrous lattice would build up within the dermis, in a spatial
arrangement in conformity with intrinsic properties of the dermal tissue; then,
starting at an intersection of the fibres, cells would mutually communicate over
a distance equal to the region-specific radius of the future condensation; the
transmitted signal would trigger off their centripetal migration, during which
they would use the network's fibres as a guide. They would thus progressively
concentrate and make up the dermal feather condensations. An alternate but
similar hypothesis may be conceived without the necessity of a pre-established
fibrous network: the unknown epidermal influence could merely confer to the
dermis the ability to build up centres of attraction around which neighbouring
cells would congregate. Determination of the precise location of the centres
would still be entirely dependent on the extension of the feather forming dense
dermis and the intrinsic properties of its cells.
In the present experiments, the new feathers arranged themselves in successive
longitudinal rows on each side of the first newly formed rudiments of the organizing primary row. In the 180° and particularly in the 90° rotation experiments,
it was shown that the newly formed primary row develops at stage-dependent
positions within the width of the explant's dermis. Thus the formation of the
primary row materializes the site of morphogenetic activity within the dermis at the
time of reorganization; this morphogenetic activity then spreads laterally as well
as medially from the primary row towards the edges of the explant's dermis. The
epidermis responds isotropically to the dermal induction and forms placodes in
non-predetermined foci. Consequently the antero-posterior arrangement of the
hexagonal pattern is always in conformity with the cephalo-caudal polarity of
630
G. NOVEL
the dermis, whereas the individual orientation of the buds is controlled solely
by the antero-posterior polarity of the epidermis. It is apparent, then, that the
backward slant (and therefore the bilateral symmetry) of the individual feathers
is independent from the antero-posterior polarity of the hexagonal pattern in
which they participate.
A model of feather pattern formation recently proposed by Ede (1972) is in
close agreement with the present experimental data. According to this model, at
first only a narrow longitudinal band of skin is able to form dermal condensations; later on, this band widens, thus providing the possibility for new rudiments to differentiate. If one looks at a transverse section of the spinal pteryla,
when the first row of rudiments is in the process of being formed, Ede's narrow
band may actually be seen as the only region of dorsal skin where dense dermis
has already formed. This region is just broad enough to accommodate one
feather condensation. Later on, dense dermis expands laterally, and new unpatterned dense dermis becomes available for the formation of additional
lateral rudiments.
In order to generate a regular' diamond' lattice, the model requires, according
to Ede, the diffusion from the forming rudiments of some substance which
inhibits production of a condensation within a defined distance of the already
existing ones. I wish to propose an alternate mechanism by which this inhibition
might be exerted: if one remembers that feather rudiments, when they first
differentiate, are in tangential contact to each other, the constraint to the formation of new rudiments could simply be of steric nature (hence the hexagonal
pattern). Such a mechanism based on spatial restriction could more easily be
applied to feather pattern formation than diffusion, since interplumar distances
(distance from centre of condensation to centre of next condensation) and
rudiment diameters considerably vary from tract to tract and also from one
region to another within the same pteryla. If inhibition is caused by a diffusion
substance, one would have to assume that this substance would diffuse or be
metabolized at different rates from place to place within the skin. In the case of
steric inhibition, however, the only necessary variation would be the regional
value of the rudiment diameter, which could be defined genetically.
However that may be, the present experimental data lead to the following
description of the formation of the spinal pteryla in the chick embryo: each of
the prospective longitudinal rows of feather rudiments is successively the site of
a morphogenetic activity, the preplumar dermis of the presumptive mid-dorsal
row being the first to acquire these properties. The morphogenetic activity leads
to the formation of an epidermal placode, which is followed by the topical
densification of the underlying dermal cells. The position of each feather condensation within a longitudinal row (except those of the mid-dorsal row) is
determined by the rudiments of the preceding row; the rudiments of the newly
formed row in turn control the position of the following ones by a mechanism
of spatial (either chemical or steric) inhibition. As the lateral dermis progres-
Feather pattern development
631
sively becomes established and morphogenetically active, its feather rudiments
form, and the already established median rows lose their activity. It is thought
that a wave of morphogenetic power moves within the dermis from the middorsal line toward the lateral edges of the spinal feather field.
RESUME
1. La formation du patron plumaire a ete etudiee dans des explants de peau prelevee dans
la poition lombaire de la pteryle spinale d'embryons de Poulet ages de 6,5-7,5 jours d'incubation. Les explants ont ete cultives in vivo sur la membrane chorioallantoidienne (MCA) ou
in vitro, soit sur milieux naturels semi-solides [contenant de l'extrait total d'embryon de
Poulet (.IE) ou de l'extrait de cerveau de Poulet (EC)], soit en milieu liquide synthetique (199).
2. Le developpement du patron plumaire depend de la methode de culture ainsi que de la
maniere dont les explants sont excises et traites avant leur mise en culture. Lorsque le rangee
mediodorsale initiale d'ebauches plumaires est preservee lors de Pexplantation, le patron
plumaire est stable quel que soit le milieu et les ebauches plumaires initiales se transforment
progressivement en bourgeons plumaires. Lorsqu'elle est lesee lors de Pexplantation, les fragments de stade jeune (A, B ou C) cultives sur les milieux JE ou EC subissent une complete
reorganisation de leur patron plumaire initial; dans les autres explants (plus ages de stade D,
ou ceux de tous les stades cultives en milieu 199 ou sur MCA), le patron plumaire initial
reste stable. Lorsque les ebauches initiales sont detruites par la dissociation dermo-epidermique suivie de reassociation, on observe une reorganisation du patron plumaire dans tous les
explants cultives in vitro (sur milieu EC ou en milieu 199) quel que soit le stade d'explantation
(Tableau 1, p. 625).
3. La reorganisation du patron plumaire est caracterisee par les processus suivants: les
ebauches plumaires initiales s'estompent progressivement pendant les 24 a 30 premieres
heures de culture; les condensations dermiques et les placodes epidermiques disparaissent.
Apres quoi, une nouvelle rangee 'primaire' se differencie parallelement aux bords longitudinaux de 1'explant; la distance qui la separe du bord median dermique initial est d'autant
plus grande, que l'explant est preleve a un stade plus tardif. Les plumes neoformees sont
disposees en rangees longitudinales paralleles a 1'axe cephalo-caudal du derme, qui est seul
responsable de la polarite du patron plumaire reorganise.
4. La capacite du patron plumaire a se reorganiser montre que ce dernier est encore labile
au moment de Texplantation et que la position de chaque ebauche n'est pas preetablie dans le
derme. Le patron est determine progressivement de rangees presomptives en rangees presomptives a partir de la rangee 'primaire'. Dans le developpement normal in situ on peut
supposer que le role de rangee 'primaire' est assume par la rangee mediodorsale dans la
region lombaire de la pteryle spinale. Peu avant la formation des condensations plumaires,
les cellules dermiques semblent etre le siege transitoire d'un pic d'activite morphogene se
deplacant depuis la ligne mediodorsale vers les bords lateraux de la pteryle spinale.
Ce memoire constitue, pour la part des resultats acquis en culture sur milieux naturels, la
these de doctorat de specialite (3e cycle) que l'auteur a soutenu le 25 juin 1971 devant l'Universite Scientifique et Medicale de Grenoble; les autres resultats feront partie de sa these de
doctorat d'Etat.
The author wishes to express her gratitude to Professor P. Sengel for helpful suggestions
during the course of this investigation and for assistance in translation of the manuscript into
English.
41
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632
G. NOVEL
REFERENCES
CLAXTON, J. H. (1967). The initiation and development of the hair follicle population in
Tabby mice. Genet. Res. 10, 161-171.
EDE, D. A. (1972). Cell behaviour and embryonic development. ////. /. Neuroscience 3, .165—
174.
EDE, D. A., HINCHLIFFE, J. R. & MEES, H. C. (1971). Feather morphogenesis and feather
pattern in normal and talpid3 mutant chick embryos. /. Embryol. exp. Morph. 28, 65-83.
GOETINCK, P. F. & SEKELLICK, M. J. (1972). Observations on collagen synthesis, lattice
formation, and morphology of scaleless and normal embryonic skin. Devi Biol. 28, 636648.
HOLMES, A. (1935). The pattern and symmetry of adult plumage units in relation to the order
and locus of origin of the embryonic feather papillae. Am. J. Anat. 56, 513-537.
LINSENMAYER, T. F. (1972). Control of integumentary patterns in the chick. Devi Biol. 27,
244-271.
MAUGER, A. (1970). Developpement du plumage dorsal de l'embryon de Poulet etudie a
l'aide d'irradiations aux rayons X. Devi Biol. 22, 412-432.
MAUGER, A. (1972a). Role du mesoderme somitique dans le developpement du plumage
dorsal chez Pembryon de Poulet. I. Origine, capacites de regulation et determination du
mesoderme plumigene. J. Embryol. exp. Morph. 28, 313-341.
MAUGER, A. (19726). Role du mesoderme somitique dans le developpement du plumage
dorsal chez l'embryon de Poulet. II. Regionalisation du mesoderme plumigene. J. Embryol.
exp. Morph. 28, 343-366.
MAUGER, A. (1972 c). Role du tube neural dans le developpement du plumage dorsal de
l'embryon de Poulet. Wilhelm Roux Arch. EntwMech. Org. 170, 244-266.
MAUGER, A. & SENGEL, P. (1970). La pteryle spinale de l'embryon de Poulet: territoire
presomptif, arrangement et developpement embryonnaire. Devi Biol. 22, 609-633.
RAWLES, M. E. (1963). Tissue interactions in scale and feather development as studied in
dermal-epidermal recombinations. /. Embryol. exp. Morph. 11, 765-789.
SENGEL, P. (1958). Recherches experimentales sur la differenciation des germes plumaires et
du pigment de la peau de l'embryon de Poulet en culture in vitro. Annls Sci. nat. Zool. 20,
431-514.
SENGEL, P. (196.1). Action morphogene de divers extraits de cerveau sur la peau de l'embryon
de Poulet cultivee in vitro. Archs Anat. Histol. Embryol. norm. exp. 44, suppl., pp. 215239.
SENGEL, P. (1971). The organogenesis and arrangement of cutaneous appendage in birds.
Adv. Morphog. 9, 181-230.
SENGEL, P., DHOUAILLY, D. & KIENY, M. (1969). Aptitude des constituants cutanes de
Papterie medioventrale du Poulet a former des plumes. Devi Biol. 19, 436-446.
SENGEL, P. & KIENY, M. (1963). Sur le role des organes axiaux dans la differenciation de la
pteryle spinale de Pembryon de Poulet. C. r. hebd. Seanc. Acad. Sci., Paris 256, 774-777.
SENGEL, P. & KIENY, M. (1967). Production d'une pteryle supplemental chez Pembryon de
Poulet. I. Etude morphologique. Archs Anat. microsc. Morph. exp. 56, 11-30.
SENGEL, P. & MAUGER, A. (1967). La metamerie de la pteryle spinale, etudiee chez Pembryon
de Poulet a l'aide d'irradiations localisees aux rayons X. C. r. hebd. Seanc. Acad. Sci.,
Paris D 265,919-922.
SENGEL, P. & NOVEL, G. (1970). Sur les mecanismes de la morphogenese du patron plumaire
dans la pteryle spinale de l'embryon de Poulet. C. r. hebd. Seanc. Acad. Sci., Paris, D 271,
2015-2018.
SENGEL, P. & RUSAOUEN, M. (1968). Aspects histologiques de la differenciation precoce des
ebauches plumaires chez le Poulet. C. r. hebd. Seanc. Acad. Sci. Paris, D 266, 795-797.
STRAUS, W. L. & RAWLES, M. E. (1953). An experimental study of the origin of the trunk
musculature and ribs in the chick. Am. J. Anat. 92, 471-509.
STUART, E. S. & MOSCONA, A. A. (1967). Embryonic morphogenesis: role of fibrous lattice in
the development of feathers and feather patterns. Science, N.Y. 157, 947-948.
Feather pattern development
633
E. S., GARBER, B. & MOSCONA, A. A. (1972). An analysis of feather germ formation
in the embryo and in vitro in normal development and in skin treated with hydrocortisone.
J.exp.Zool. 179,97-118.
WESSELLS, N. K. & EVANS, J. (1968). The ultrastructure of oriented cells and extracellular
material between developing feathers. Devi Biol. 18, 42-61.
WOLFF, E. & HAFFEN, K. (1952). Sur une methode de culture d'organes embryonnaires in
vitro. Texas Rep. Biol. Med. 10, 463-472.
ZUST, B. (1971). Le developpement du plumage, d'apres Panalyse des malformations cutanees
produites par l'administration d'hydrocortisone a l'embryon de Poulet. Annls Embryol.
Morph.4, 155-174.
STUART,
(Received 16 March, revised 6 June 1973)
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