J. Embryol exp. Morph. Vol. 25, 1, pp. 65-83, 1971
Printed in Great Britain
65
Feather morphogenesis and feather pattern in
normal and talpid3 mutant chick embryos
By D. A. EDE\ J. R. HINCHLIFFE 2 AND H. C. MEES3
From the Agricultural Research Council Poultry Research Centre, Edinburgh,
the Department of Zoology, University College of Wales, Aberystwyth, and the
Department of Zoology, University of Edinburgh.
SUMMARY
A comparative study of feather morphogenesis and the development of feather pattern in
normal and talpid3 embryos has been carried out.
The development of talpid* CAM grafts shows that the effect of the gene is autonomous in
the skin.
The most striking effect of the gene upon feather morphogenesis is the failure of normal
feather germ condensations to appear within the dermis. This is reflected in the abnormal
distribution of alkaline phosphatase through the dermis.
Dermal cells within and between condensations are not orientated in the mutant as they are
in normal embryos, probably owing to the same defect in cell behaviour which causes
condensation failure in talpid3 precartilage mesenchyme.
The role of dermal cell orientation and movement in generating the overall feather pattern
is examined in both normal and talpid3 embryos.
INTRODUCTION
The three talpid mutants of the fowl are characterized by widespread pleiotropic abnormalities of which the most remarkable is extreme polydactylism.
All three talpid genes are autosomal recessive lethals and death of homozygotes
usually occurs at 8-10 days (d) in talpid1, 10-14 d in talpid2 and at about 6 d in
talpid3. The complex pattern of developmental abnormalities has been described
in talpid1 by Cole (1942) and Inman (1946) and in talpid2 by Abbott, Taylor &
Abplanalp (1960) and Goetinck & Abbott (1964). The development of talpid3
embryos has been described in detail by Ede & Kelly (1964a, b) and by Hinchliffe
& Ede (1967, 1968), who found that the major abnormalities originated in the
failure of precartilaginous mesenchyme to form distinct condensations on the
normal pattern. Abnormalities in cell behaviour which probably account for this
failure were found by Ede & Agerbak (1968) in studies on the reaggregation of
normal and mutant cells in culture.
1
Author's address: Poultry Research Centre, King's Buildings, West Mains Road,
Edinburgh, 9, U.K.
2
Author's address: Department of Zoology, University College of Wales, Penglais,
Aberystwyth, U.K.
3
Author's address: Department of Zoology, University of Edinburgh, King's Buildings,
West Mains Road, Edinburgh, 9, U.K.
5
EMB 25
66
D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
Feather morphogenesis is arrested in all the talpid mutants. In talpid1 there
is no sign of elongation of the feather papillae even at 17 d, and in talpid2
Abbott et al. (1960) report that 'feathering is retarded' at 10 d. In talpid* the
appearance of papillae is retarded, they remain flattened and unelongated and
their overall pattern is disrupted (Ede & Kelly, 19646). Since mesenchymal cell
condensation plays a prominent part in the initial stages of feather formation the
present investigation was undertaken in order to see whether the cellular
abnormality that causes defects of condensation in precartilage mesenchyme
might also account for the defective feather formation. Comparison of the
mutant with normal development also throws light on problems of feather
morphogenesis and feather pattern formation raised by Stuart & Moscona (1967),
Wessells & Evans (1968) and Sengel & Rusaouen (1968).
MATERIALS AND METHODS
Living normal and talpid* embryos were fixed at 6-11 d in Bouin's or Carnoy's
fluid, photographed and then used for whole-mount preparations of skin or
serially sectioned, staining routinely with iron haematoxylin, haematoxylin and
eosin and alcian blue/chlorantine fast red, and in some cases Masson's Ponceauacid fuchsin-light green or iron haematoxylin with van Gieson to show collagen
more clearly.
Since the majority of talpid3 embryos die at 6 d, before feather development
has begun, material obtained from whole embryos was supplemented by material
maintained to the required age as grafts on the chorioallantoic membrane (CAM)
by the method of Hamburger (1960). Skin was removed from embryos at
Table 1. Number of embryos and CAM grafts examined
Embryos
CAM grafts,
dorsal skin
CAM]grafts,
flank skin
A
(days)
Normal
talpid3
Normal
6
7
8
9
10
11
12
13
3
4
4
2
1
2
—
—
3
2
3
1
1
1
—
—
10
12
30
26
—
—
—
—
^t
3
talpid
1
7
36
21
—
—
—
—
Normal
talpid3
2
3
3
4
5
—
—
2
3
5
4
3
5
2
4-}-5 d (1) from the back region, with underlying muscle and vertebral column
still attached, and (2) from the flank area between the anterior and posterior
limb, then grafted on to the chorioallantoic membrane of 8-10 d embryos.
Graft material was sectioned and also used for the preparation of dermis
whole mounts. The latter were made by placing the skin in Ca-Mg-free Tyrode
Feather morphogenesis in talpid3 mutants
67
solution for 10 min, then in trypsin-pancreatin solution (2-25 % Difco trypsin,
0-75 % pancreatin in CMF), stripping the ectoderm off carefully using tungsten
needles and finally flattening the dermis on to a Millipore disc (type THWP
01400) for staining and mounting.
Alkaline phosphatase activity was studied in skin from flank grafts, which
was divided into two halves, one of which was fixed in 80 % alcohol at 0-4 °C
for 12 h and then sectioned and incubated for 4 h using the Gomori (1952)
technique. The other half of each flank graft was fixed in Carnoy, and sections
were stained with iron haematoxylin or by the Trevan modification of the PAS
technique for acid and neutral mucopolysaccharides (described in Hinchliffe &
Ede, 1967) or for RNA by the toluidine blue/ribonuclease method (Barka &
Anderson, 1963). In skin from the back the appearance of cytoplasmic basophilia
with iron haematoxylin was used as an indication of RNA distribution.
The number of embryos and grafts examined at various ages is given in
Table 1.
RESULTS
External appearance of feather papillae
Normal. On the back the first feather papillae appear at 7 d, in a single file
directly over the line of the vertebrae in the posterior (saddle) region and in two
files over the myotome muscles, one on each side of this line, more anteriorly,
producing a tuning-fork pattern. Additional files of papillae are formed in
sequence laterally, each new papilla arising equidistant from its nearest neighbours so that a series of equilateral triangles is marked out, forming eventually
an arrangement of points on a rhomboidal lattice (Fig. 1 A). From 9 to l i d
the papillae, starting with those nearest the mid-line and in sequence laterally,
elongate in a caudal direction (Fig. IB) and barb-ridges are formed within
them. On the flank the development of the papillae is similar but the first files,
which appear ventrally, do not appear until 8 d. Feather papillae are formed in
the same way in grafts but development is retarded by up to half a day and they
do not elongate well.
talpid2. The appearance of feather papillae is delayed and their progressive
production in files from the mid-line is much less marked. There are none visible
in embryos at 7 d or in most grafts at 8 d; in 8 d embryos and 9 d grafts there
are broad flattened papillae over the whole area, arranged much less regularly
than normal, and rather close together, at the points of a very roughly rhomboidal lattice (Fig. 1C). The boundaries of the papillae are less well defined
than in normal embryos. At 10 d the appearance of the skin is not much altered
since there is little elongation and no caudal orientation of the papillae (Fig. 1D).
Development of talpid3 papillae on the flank is similar except that in this region
they are more widely separated than in normal embryos and grafts.
5-2
68
D. A. EDE, J. R. H I N C H L I F F E AND H. C. MEES
Fig. 1. Normal (A, B) and talpid* (C, D) embryos at 8 d and 10 d.
Feather morphogenesis in talpid3 mutants
Fig. 2. Transverse sections of embryos in mid-back region. (A) Normal 6 d, (B)
talpid3 6 d (stained haematoxylin and eosin). (C-E) Normal 7 d, with increasing
magnification of right-hand condensation region. (F-H) talpid31 d, with increasing
magnification of right-hand condensation region. (Stained iron haematoxylin.)
69
70
D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
Histology of early feather development
A. 6-11 days in back skin in embryos and grafts
6 days
Normal (Fig. 2 A) and talpids (Fig. 2B) skin are almost indistinguishable at this
stage. The epidermis consists of a single layer of cells which are not orientated
in any particular way. Beneath it the dermal mesenchyme cells are loosely
packed, not orientated, and there is no collagen present. The neural tube is sunk
more deeply in the mesoderm in talpid3 than in normal embryos.
7-days
Normal (Fig. 2C, D, E). Mesenchyme cells immediately beneath the epidermis
show some orientation transversely across the back. Over the myotomes in the
anterior and mid-back regions and over the vertebrae posteriorly a dense dermal
layer, five to six cells thick, is formed between the epidermis and the loose
mesenchyme. The epidermis consists now of two cell layers, and above the
dense dermis, at each point where a feather papilla is to develop, an epidermal
placode is formed, distinguished by the vertical elongation of the cells comprising its basal layer. Almost simultaneously, the mesenchyme beneath each
placode becomes condensed, i.e. the cells become rounded and closely packed,
with spherical nuclei, and the condensation extends deeper into the mesenchyme
than the surrounding deep dermis. The cells at the periphery of the condensation
are spindle-shaped, with elongate nuclei, orientated tangentially to its surface,
chiefly in concentric rings around the condensation but tending also to follow its
contour upwards towards its summit. No collagen is detectable at this stage.
Fibrils of the type described by Kallman, Evans & Wessells (1967) as anchor
filament bundles extend from the basement membrane into the condensations
and the dense dermis between them, and at the placodes they appear to pull the
basal layer epidermal cells down into the condensation at the points of attachment. The cells of the basal layer show marked basophilia indicating RNA
concentration in the cytoplasm adjacent to the basement membrane, especially
over the condensations.
talpid* (Fig. 2F, G, H). The dermis and epidermis are thrown into a series of
shallow hummocks and valleys across the back. The dense dermis is not well
defined, forming an undulating layer only two to three cells thick. Within it
feather germ condensation is very poor; the cells of each condensation are
loosely packed and those at the periphery are unorientated and not clearly
distinguishable from those of the surrounding dense dermis. The condensations
do not extend much deeper than the latter into the mesenchyme. The epidermal
placodes are formed in the normal way and appear at the hummocks, but the
epidermis is separated from the underlying dermis, especially at the condensations, to which the basal layer remains connected by anchor filament bundles
which bridge the gap. Basophilia in the cells of the basal layer is less marked
than normal and not more evident over the condensations than between them.
Feather morphogenesis in talpid3 mutants
Fig. 3. Transverse sections of 9 d embryos in mid-back region. (A) Normal, left side
omitting the two oldest papillae nearest mid-line. (B) talpid*, left side, including
mid-line at right. (C, E, F) Details of A showing papillae at various stages of
maturation and an intercondensation region in normal. (D, G, H) Details of B
showing papillae and an intercondensation region in talpid*. (Stained haematoxylin
and eosin.)
71
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D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
8 days
Normal. The condensations are now very distinct, and collagen fibres are
present at their base, orientated in the plane of the skin surface. Less collagen
is present at the base of the dense dermis between condensations. The epidermal
placodes are distinct and the interface between each placode and its condensation is now flattened. The anchor filament bundles appear to become connected
to the collagen fibres at the base of the dense dermis, and they are less prominent,
especially in the condensation regions.
talpid3. The dermis remains of uniform thickness, with the condensations
indistinctly marked off from the surrounding dense dermis and not extending
below it. Collagen fibres are not so prominently developed as in normal dermis,
especially between condensations. The epidermal placodes are still separated
from their underlying condensations and the orientation and location of anchor
filament bundles is abnormal; in condensation regions they are sparse and
orientated obliquely to the surface, while between condensations they are
abnormally abundant.
9-11 days
Normal (Fig. 3 A, C, E, F). The feather papillae nearest the mid-line have
begun their outgrowth at 9 d and the dense dermis between condensations
becomes thinner. Basophilia in the basal layer of epidermal cells is now intense,
and there is some palisading of these cells. Few or no anchor filament bundles
are found in the condensation regions, but some still persist between them.
Collagen fibres are increasingly prominent in the lower regions of the dense
dermis both within and between condensations. There is a well-defined system
of blood capillaries in the mesenchyme beneath the deep dermis, with capillaries
extending into the base of each condensation, which has now become the dermal
core of the feather papilla. By 11 d all feather papillae are elongated and barbridge formation is prominent in the epidermal layer.
talpid*(Fig. 3B, D, G, H). The feather papillae at 9 d are broad and shallow;
they do not elongate and those nearest the mid-line are not appreciably more
developed than the most laterally situated. The dense dermis remains uniform
in thickness; the condensations extend no deeper into the mesenchyme and the
dense dermis between them, which is now markedly oedematous, does not
become thinner. The ectodermal placodes are in many cases displaced slightly
away from their condensations, coming to lie in the 'valleys' while the condensations remain at the hummocks. Basophilia is faint in the basal epidermal
layer and is not more pronounced over the condensations. Collagen is present
throughout the entire lower dermis; the fibres are not orientated parallel to the
skin surface, and they are less dense beneath the condensation than in normal
skin. The histological appearance at 11 d is very similar, with no elongation of
the papillae. The dermis at this stage is highly oedematous.
Feather morphogenesis in talpid3 mutants
73
B. 9-13 days in flank skin, grafts only
Events in the flank parallel those in the back region, but are retarded by
about 36 h. The difference between normal (Fig. 4A, B) and talpid3 (Fig. 4C, D)
in the appearance of basophilia in the lower epidermal cell layer showed particularly well in this material. In addition to the histological observations
described above, alkaline phosphatase activity and the appearance of acid
mucopolysaccharides have been investigated in this region.
Fig. 4. Vertical sections of CAM grafts of flank skin (stained iron haematoxylin).
(A) Normal 9 d showing beginning of basophilia in epidermis over the dermal
condensation. (B) Normal 1.0 d, showing intense basophilia (B) in basal cytoplasm of
epidermal cells of the papilla cap. (C) talpid3 9 d, showing absence of basophilia in
epidermis over the dermal condensation. (D) talpid310 d, showing slight basophilia
(B) in basal cytoplasm of epidermal cells of the papilla cap.
Alkaline phosphatase—normal (Fig. 5 A-C). There is no alkaline phosphatase
staining reaction at 7 d, and only an extremely faint positive reaction in the
dermis at 8 d. At 9 d dermal condensations are visible and there is a slight
reaction within them but not elsewhere in the dermis. At 10 d the papillae
project above the general surface of the skin and alkaline phosphatase staining is
intense in the cells of the condensations adjacent to the epidermis but absent
from the dermis condensations. At this stage the basophilia indicating RNA
accumulation in the basal layer of epidermal cells over the condensations is
74
D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
intense. By 11 d the feather papillae are elongated, with barb-ridges formed
from the epidermis, and alkaline phosphatase activity is most intense as a
'collar' in the dermal core beneath the epidermis at the base of each papilla.
Alkaline phosphatase—talpid3. (Fig. 5D-F). No condensations are visible at
9 d and there is only very slight alkaline phosphatase activity distributed homogeneously through the dermis. At 10 d it has increased, but is still distributed
generally throughout the dense dermis and is much less than in normal skin at
this stage. At 11 d the small and widely separated condensations have distinct
D
Fig. 5. Vertical sections of CAM grafts of flank skin stained for alkaline phosphatase.
x 25. (A) Normal 9 d, showing two positive dermal condensations. (B) Normal 10 d,
showing intense positive staining in condensations. (C) Normal l i d , showing
positive staining in dermis in the papilla 'collar' and adjacent to barb-ridges.
(D) talpid3 9 d, showing slight positive staining evenly throughout the dense dermis.
(E) talpid3 10 d, showing that staining is still evenly distributed. (F) talpid3 11 d,
showing staining now limited to the condensations.
activity, which is very slight in the rest of the dermis. No elongation has occurred
at this stage, but at 13 d talpid3 grafts showed development of feather papillae
to the barb-ridge stage, with alkaline phosphatase activity in the dermal
component. Such papillae remained small and did not project above the skin
surface. As in normals, in the skin between papillae the epidermis becomes
stratified and keratinous and the dermis loses all alkaline phosphatase activity.
Acidmucopolysaccharides. Acid mps first appears in the dermal core of normal
papillae at 9 d but the intensity of staining is much increased at 10 and 11 d; in
talpid* dermal condensations acid mps staining appears first at 11 d and is also
present at 12 and 13 d; i.e. in both normal and mutant, acid mps appears in the
dermal condensations almost as soon as they are formed. More diffuse acid mps
staining occurs throughout the dermis.
Feather morphogenesis in talpid3 mutants
75
Dermal cell orientation and collagen distribution
The following observations are derived from whole mounts of skin, either
from embryos or grafts, from which the epidermis has been removed after
trypsinization, and from material sectioned tangentially to the surface. They refer
to the mid-back region, in which feather papillae appear first in two files over
the myotomes, but events are similar in other regions.
Normal. The originally unorientated mesenchymal cells beneath the epidermis
become fibroblastic and lined up transversely across the back before condensations appear, and at 7 d there is an increase of cell density to give the
Fig. 6. (A) Dermis from posterior back region of normal 7 d embryo with a single file
of condensations formed in the mid-line, showing three condensations (stained
haematoxylin). (B) Detail of condensation. (C) Dermis from left side of the posterior
back region of a normal 8 d embryo in which nine files of condensations have
appeared, the mid-line file to the right and the youngest file forming in the dense
dermis to the left. (D) Detail of quartet of condensations showing orientation of cells
between condensations and dermal thinning (stained alcian blue/chlorantine fast red).
(E) Whole skin (dermis + ectoderm) from mid-back region of talpid3 10 d embryo,
showing irregular arrangement of condensations and (the structures appearing
between condensations) blood capillaries (stained haematoxylin).
76
D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
-*
Jin
Feather morphogenesis in talpid3 mutants
11
dense dermis. No collagen fibres are detectable by histological methods at this
stage.
Condensations arise as clusters of rounded closely-packed cells in the dense
dermis, condensations within a file (except some at each end which are added
later) appearing almost simultaneously (Fig. 6A). At the same time the dense
dermis extends further laterally on each side, to the position of the next file of
condensations.
During the emergence of the second pair of files, the peripheral cells of the
first condensations and the dermal cells immediately surrounding them become
spindle-shaped and concentrically arranged, with their elongate nuclei tangential
to the condensation (Fig. 6B); beyond this the cells of dense dermis become
orientated radially to the condensations. This pattern is always found in the
two most recently formed lateral files.
No distinct lattice arrangement of cells connecting neighbouring condensations or preferential distribution of collagen appears before 4-6 files of condensations are established. From this time, at 8 d, a rhomboidal lattice pattern
emerges (Fig. 6C) as the radially orientated cells around the condensations tend
to become concentrated along those radii forming the sides of each lattice unit
which connect one condensation to the next (Figs. 6D; 7A, C). Preferential
collagen staining now occurs within the condensations and along the orientated
cells of the lattice so formed. There is also some tendency for the radiating cells
to concentrate along the vertical and transverse diagonals of each rhomboidal
lattice unit, but in general the cells within the rhomboid are randomly orientated. Cell density, as judged by optical density in the stained preparations,
appears to become much reduced within the rhomboids (Fig. 6C, D), and this
reduction is correlated with the thinning of the dermis between condensations
which is visible in transverse sections (Fig. 3F). This lattice pattern formed by
orientated cells with accompanying collagen extends to the peripheral files of
papillae but from about 10 d, when the papillae are becoming more widely
separated as the embryo increases in size, this cell orientation is lost; the
condensations are then quite distinct from the surrounding dermis, with the
tightly-packed concentrically arranged peripheral cells of the one sharply
marked off from the loose mesenchymal cells of the other (Fig. 7E).
talpid*. Cell orientation and collagen distribution differ markedly from normal.
Fig. 7. Sections through dermis from mid-back region in the plane of skin surface
(stained haematoxylin and eosin). (A) Normal 8 d embryo, snowing condensation
with surrounding dermal fibroblast cells radiating from it and on the left orientated
towards a neighbouring condensation. (B) talpid* 8 d embryo, showing random
orientation of fibrocyte-like cells in the surrounding dermis. (C) Normal 8 d embryo,
showing fibroblasts orientated between condensations at extreme left and right.
(D) talpid* 8 d embryo, showing random orientation of fibrocyte-like cells between
condensations at left and towards bottom right. (E) Normal 9 d CAM graft, showing
condensation distinct from surrounding dermis. (F) talpid3 9 d CAM graft, showing
condensation less clearly demarcated.
78
D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
In 8 d embryos and in grafts where condensations are visible cell clusters are
formed with very roughly concentrically orientated cells at their peripheries,
but there is hardly any radial orientation of cells farther out (Fig. 7B). The
cells between condensations generally have spherical nuclei, show no orientation, and there is little reduction of cell density. Collagen staining is nowhere so
clear as in normals, but is greatest at the condensations and diffuse throughout
the rest of the dermis. No regular lattice pattern of orientated cells and collagen
is formed (Fig. 6E). At 9 d the condensations are much less distinctly marked
off from the surrounding dermis than in normals (Fig. 7F). The peripheral cells
of the condensations are not clearly concentrically orientated and they merge
with the surrounding dermal cells. These appear to adhere closely to each other,
their cytoplasm juxtaposed over much of the cell boundary rather than touching
only at points as in the stellate mesenchymal cells of the normal dermis.
DISCUSSION
Cell-autonomy of defective feather morphogenesis in talpid3
Feather development in both normal and talpid3 material is essentially the
same in CAM grafts as in the embryo. Mutant skin shows the same abnormalities in grafts made at 4 d donor age (i.e. 3 d before feather morphogenesis
begins) as in situ and this strongly suggests that the genetic defect is autonomous,
acting directly on the feather-forming cells, rather than the result of circulating
humoral factors. Hinchliffe & Ede (1968) reported similar findings for cartilage
development in talpid3 and the primary effect of the gene is likely to be on a
common mechanism underlying the mesenchymal condensation process which is
involved in both chondrogenesis and feather morphogenesis.
The site of gene action
The first abnormality to appear in talpid3 skin is the failure of the dermal
cells beneath the epidermis in the back to establish a normal dense dermis at 7 d,
and subsequently the retarded and abnormal appearance of feather germ condensations within it. The basic disturbance in this and subsequent abnormalities
in the development of the condensations and of the dermis between them appears
to lie in themesenchymal cells remaining fibrocytic in form rather than becoming
fibroblastic, and in the random orientation of these cells. Subcutaneous oedema,
which may contribute to the disorientation of the dermal mesenchyme cells at a
later stage, does not begin to appear until 9 d. The formation of placodes in the
ectoderm over the dense dermis at the site of feather papilla formation is normal
in talpid3, and supports the hypothesis that the primary defect is in the mesenchyme.
Interaction between ectoderm and mesoderm
Sengel (1958) and Rawles (1963) have demonstrated a sequence of inductive
interactions between each ectodermal placode and its dermal condensation in
Feather morphogenesis in talpid3 mutants
79
feather development which in some ways, as Wessells (1965) has pointed out,
resembles that found in the apical ectodermal ridge-limb mesoderm system in
the limb-bud. It is still not clear whether the placode or the condensation initiate
papilla formation, though Wessells (1965) and Sengel & Rusaouen (1968) have
shown that it is probably the placode, since this appears first; in either case, it is
clear that the normal development of each is dependent on the presence of the
other. Therefore, though the primary effect of the talpid3 gene may be on the
dermis, the ectoderm will be secondarily affected, and this will in turn react on
the dermal development again. The present results show that although early
placode formation is normal, abnormalities soon appear in it, e.g. in basophilia
and in palisading. A gap appears between ectoderm and mesoderm which may
be due to some defective properties of the basement membrane, and—possibly
related to the last-mentioned—the distribution and orientation of the anchor
filament bundles is abnormal. To what extent these defective relationships
between ectodermal and mesodermal components affect the development of the
talpid3 feather papillae will require reciprocal recombination experiments to
determine. One respect in which the development of the placode ectoderm is
clearly affected is in the virtual absence of basophilia, indicating reduction of
RNA synthesis, in the cytoplasm adjacent to each underlying condensation,
which is correlated with the absence of any intense alkaline phosphatase activity
localized in the condensations. Alkaline phosphatase is known to be active in
cells participating in tissue interactions (Rapola, Vainio & Saxen, 1963; Finn &
Hinchliffe, 1964) and its importance in feather formation has been emphasized
by Hamilton (1965). Specific inhibitors of alkaline phosphatase block epidermal
RNA synthesis and prevent normal feather morphogenesis. Gibley & Hamilton
(1963) found that abortive feather development occurred when they disrupted
the normal pattern of alkaline phosphatase activity and produced a more
homogeneous distribution in the dermis. It appears that one of the consequences
of the abnormal dermal condensation in talpid3 is defective alkaline phosphatase
production, which in itself interferes with normal feather development.
Condensation, dermal cell orientation and the dynamics of
feather pattern formation
Dermal condensations are produced where cells within a certain area become
closely packed and sharply demarcated from the more loosely packed mesenchyme cells surrounding them. Ede & Kelly (19646) showed that the abnormalities in the talpid3 skeleton arose by defective condensation of precartilage
mesenchyme cells. The present studies show that defective mesenchymal cell
condensation is also at the root of developmental failure of the feather papillae
in the same mutant. In both cases the condensations are less well defined and
their boundaries less distinct than in the normal. Further, Hinchliffe & Ede
(1967) showed that the mutant chondroblasts arising from the cells within the
precartilage condensations are not orientated as in the normal, and the present
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D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
study shows that the orientation of the dermal cells within and also between
papilla condensations is likewise disrupted.
Ede & Agerbak (1968) suggested that precartilage condensations arise by
aggregative cell movements in vivo, that the foci of such condensations are
centres of production of some morphogenetic substance leading to cell aggregation in their neighbourhoods, and that talpid3 cells, because of abnormal
adhesiveness and reduced motility, are less effective in forming clearly defined condensations: the basic defect in both cartilage and feather formation is
the failure of mesenchyme cells to move about as freely as usual and hence
failure to form normal ordered arrangements with each other. Time-lapse
cine observations by D. A. Ede & O. P. Flint (unpublished) confirm that in
monolayer culture talpid3 cells are considerably less motile than normal cells.
In the formation of feather papillae Stuart & Moscona (1967) reported that
a fibrous collagen lattice in the skin dermis preceded the appearance of the
dermal condensations and suggested that cell movements and cell aggregations
of the dermal cells are aligned along this lattice in the production of the condensations, thus accounting for the overall pattern of the papillae. But Wessells
& Evans (1968) have shown in an electron microscope study that though
fibroblasts between dermal condensations of early feather germs are indeed
elongated and orientated so as to point from one condensation towards the
next, the collagen fibril bundles in these regions are randomly orientated. Our
present studies show that in normal embryos collagen is laid down in the lower
layers of the dense dermis in the condensations and in the orientated cell
tracts connecting them, forming a rhomboidal lattice, but that this occurs
during and not before the emergence of the corresponding cellular pattern. In
the mutant, collagen staining is greatest in the condensation areas, while
between condensations the dermal cells are randomly orientated and collagen
staining is diffuse. It seems most likely that the position and orientation of the
mesenchyme cells determines that of the collagen rather than vice versa, and
that the presence of the collagen lattice in normal skin and its absence in the
mutant is due to the concentration of cells in orientated lines between condensations in the former and the random arrangement of these cells in the latter.
Using autoradiographic techniques, Wessells (1965) has shown in a very
careful study that feather papillae condensations arise at least partly as a result
of a local increase in mitotic activity beneath the ectodermal placodes and he
believes that this differential mitosis, together with an increase in cell size, is
sufficient to account for the appearance of condensations. His cell counts did
not indicate any decrease in cell density between condensations and he is
inclined to discount any role of cell movement in their formation. Nevertheless,
our material clearly shows the reduction which was noticed by Holmes (1935)
in optical density in these regions of the dermis in the interstices of the lattice
formed by the tracts of elongated cells between condensations, which appears
as the files of condensations are established. Moreover, the cells within the
Feather morphogenesis in talpid3 mutants
81
condensations, around them and in the tracts between them, become orientated
in particular ways, which implies at least a minimal amount of cell movement in
the course of this rearrangement. Unpublished experiments by one of us
(H. C. Mees) have shown that if skin at early condensation stage is organcultured on voile the condensations will quickly disappear as the cells of the
dermis rearrange themselves and become orientated along and between the
voile fibres. Clearly, the pattern of dermal cells is poised at this stage in a
condition of delicately maintained stability and the cells have great capacity for
Fig. 8. Diagrams illustrating the arrangement of the most prominent dermal cells in
and between condensations in (A) normal and (B) talpid* skin. The condensation
regions are indicated in black.
movement. Therefore, while Wessells has clearly established that differential
mitosis plays a predominant role in condensation formation we believe that
cell movement, even if it is perhaps limited to rather small shiftings of cells on
each other, produces the distinct form of the condensations, the concentration
of cells in the lattice tracts and the thinning of cells in the interstices of the
lattice. In talpid* these cells remain unorientated—that is, do not rearrange
themselves on each other, either within or between condensations, to the
extent that normal cells do. It seems likely that it is this defect in cell movement
rather than a difference in mitotic rate that is primarily responsible for the
defective condensation in the mutant.
6
EMB 25
82
D. A. EDE, J. R. HINCHLIFFE AND H. C. MEES
If dermal cell movement is involved in this way, how might cell orientation
and establishment of the corresponding collagen lattice take place? Suppose
that the cells at the foci of the condensations are relatively immobile, and
attractive in some chemotactic way. Suppose that the rest of the cell population
in the dermis is relatively mobile—they will tend to move between and around
the condensation foci. This will lead to some orientation, which may be reinforced by the tendency which has been studied in monolayer culture by Elsdale
(1968) for fibroblasts to align themselves against each other. An analogy would
be the situation at Speakers' Corner in Hyde Park, where each speaker sets up
his pitch at an optimum distance from the others, and people will crowd around
it; those at the periphery will wander concentrically around the edge, and some
people will be moving from one pitch to another. This sort of restless movement
will establish the lines of orientated cells around the condensations and between
condensations (Fig. 8 A). The lines between condensations will be established
at the shortest routes, but much less where routes cross, so that the diagonals of
the lattice units (transverse and antero-posterior to the whole embryo) are not
marked out, though it should be noted that Sengel & Rusaouen (1968) have
found that at certain levels in the dermis transversely orientated cells are more
prominent. Collagen deposition will be densest, and possibly the fibrils aligned
along the cells, where the dermal cells are most densely packed—that is, along
the lines of the lattice and at the condensations—thus producing the collagen
lattice observed by Stuart & Moscona (1967). In talpid*, on the other hand,
skin dermal cell movement is restricted, and orientation and arrangement of the
cells on each other is interfered with so that they are randomly distributed
between condensations (Fig. 8B); this is reflected in the uniform distribution of
collagen, except where it is more densely deposited in the region of the condensations.
RESUME
La morphogenese plumaire et la repartition des plumes chez des
embryons de poulet normaux et talpid3
On a effectue une etude comparative de la morphogenese plumaire et du developpement de
la repartition des plumes chez des embryons normaux et talpid3.
Le developpement de greffons chorio-allantoidiens montre que Feffet du gene est autonome
dans la peau.
L'effet le plus frappant du gene sur la morphogenese plumaire est l'absence d'apparition,
dans le derme, des condensations normales des germes plumaires. Ceci se reflete dans la
repartition anormale de la phosphatase alcaline dans le derme.
Les cellules dermiques dans et entre les condensations ne sont pas orientees chez le mutant
comme elles le sont chez des embryons normaux, probablement a cause du meme defaut
dans le comportement cellulaire, que celui qui provoque l'absence de condensation dans le
mesenchyme precartilagineux talpid3.
Le role de l'orientation et du deplacement des cellules dermiques dans la disposition
generate du plumage est examine a la fois pour les embryons normaux et les embryons
talpid3.
Feather morphogenesis in talpid3 mutants
83
We owe much to discussions with Dr R. A. Kille in the course of this work. We are grateful to Mrs Irene Wilson for technical assistance.
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(Manuscript received 1 May 1970)
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