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DEVELOPMENTAL DYNAMICS 207:157-170 (1996)
Sonic Hedgehog in Feather Morphogenesis: Induction of
Mesenchymal Condensation and Association With Cell Death
SHEREE A. TING-BERRETH AND CHENG-MING CHUONG
Department of Pathology, University of Southern California, Los Angeles, California 90033
Sonic hedgehog is involved in
ABSTRACT
vertebrate tissue interactions during development. During early feather development, Sonic
hedgehog appears very early in epithelial placodes. During late feather development, Sonic
hedgehog expression precedes the development of
the marginal plates and is specifically localized in
the marginal plate epithelium, which will later undergo cell death. By using retroviral vectors, exogenous Sonic hedgehog overexpression in devel.
oping feathers induced enlarged feather buds that
have either lost their anterior-posterior polarity
or exhibited reverse orientation. The enlarged
dermal condensations may be mediated through
broader TGF-P2 expression and reduced protein
kinase C (PKC) expression. Reciprocal mesenchymal interaction is required for the induction and
maintenance of Sonic hedgehog in the epithelial
placodes. In scaleless mutant, Sonic hedgehog is
absent in the apteric region and aberrantly expressed in the mesenchyme of the abnormal
feather ridge. These findings suggest that Sonic
hedgehog mediates key interactions between the
epithelium and mesenchyme during feather morphogenesis. o 1996 Wiley-Liss, Inc.
Key words: Morphogenesis, Hair, Feather, Skin
appendage, Scaleless, Epithelialmesenchymal interaction, Retroviral
gene delivery, Pattern formation, Cell
death.
INTRODUCTION
Pattern formation is a critical process during early
vertebrate development. Coordinated regulation of specific molecular events and cellular signaling is required for the arrangement of functional tissues and
organs. Many of these processes involve secondary inductions that require specific and complex tissue interactions during their development. For example, limb,
tooth, kidney, and skin appendage inductions require
epithelial-mesenchymal interactions (reviewed in Sawyer and Fallon, 19831, and the induction of the floor
plate in the central nervous system requires interactions with the notochord (van Straaten et al., 1988;
Placzek et al., 1990). The molecular basis underlying
the inductive events during these tissue interactions
remains one of the most challenging questions in developmental biology.
0 1996 WILEY-LISS, INC.
Recent studies have shown that Sonic hedgehog
(Shh),a member of the hedgehog (hh)multigene family
that encodes putative signaling molecules, is expressed
in tissues with inductive and polarizing activities in
various vertebrate embryos (Riddle et al., 1993; Krauss
et al., 1993; Echelard et al., 1993). In the developing
limb, Shh is expressed in the zone of polarizing activity
(ZPA). The ZPA is a region of posterior limb bud mesenchyme that is essential for anterior-posterior limb
patterning. Ectopic expression of Shh in the anterior
limb bud induced digit duplication with mirror image
symmetry (Riddle et al., 1993; Chang et al., 1994). Expression of Shh in ventral neural structures such as the
notochord and floor plate suggests a role in the dorsalventral patterning of the neural tube (Echelard et al.,
1993). Indeed, the ectopic expression of Shh can induce
ectopic floor plate formation (Roelink et al., 1994,
1995). Shh has also been implicated in the dorsal-ventral patterning of somites (Fan and Tessier-Lavigne,
1994; Johnson et al., 1994) and in several other sites of
organogenesis (Bitgood and McMahon, 1995). These
studies suggest Shh may play an inductive and fundamental patterning role in various vertebrate tissue interactions.
We are interested in examining the molecular basis
underlying secondary induction using chicken feather
morphogenesis as the model (Chuong, 1993). Skin appendages develop from the inductive interactions between the epithelium and the underlying mesenchyme.
Upon receiving inductive signals from the underlying
mesenchyme, the epithelium segregates into two domains, the placode and the interplacode domains (Sengel, 1976). Mesenchymal cells then condense beneath
the placodes to form dermal condensations. In mature
feathers, the epithelium segregates further into alternating domains destined to become the barbs (barb
plate epithelium) and the space between barbs (marginal plate epithelium). How are these morphogenetic
processes regulated?
With recent studies demonstrating the versatile role
of Shh in different tissue interactions, it is reasonable
that Shh should be involved in feather formation. We
therefore started to explore the roles of Shh in feather
morphogenesis. Recently, Nohno et al. (1995)described
Received May 6, 1996; accepted June 7, 1996.
Address reprint requestdcorrespondenceto Cheng-Ming Chuong,
Department of Pathology, HMR 204,2011 Zonal Avenue, University
of Southern California, Los Angeles, CA 90033,
158
TING-BERRETH AND CHUONG
the presence of Shh in developing feathers. However,
they described only a few stages of feather development. In this report, we examined the expression of
Shh in all the major stages of early feather and filament development. Our detailed studies of the temporal and spatial distribution of Shh provided insight
into the functions of Shh in skin appendage development and the formation of the stripe pattern of Shh in
the feather filament. Furthermore, the functional role
of Shh was explored using retrovirus-rnediated ectopic
expression in ovo. The data suggest that Shh induces
the formation of extra-large feather buds with aberrant
or lost anterior-posterior polarity. This is preceded by
the accumulation of many mesenchymal cells at the
site of Shh overexpression. The regulation of Shh gene
expression was also explored using skin explant cultures with epithelial-mesenchymal recombination. The
results indicate that organized mesenchyme is required to maintain and induce Shh expression in the
epithelium. Shh expression was also examined in
avian scaleless mutants and showed abnormal expression that is consistent with the results from the retroviral experiments. These results show that Shh plays
key roles in different stages of skin appendage morphogenesis.
MATERIALS AND METHODS
Chick Embryos
Chick embryos used were from SPAFAS (Norwich,
CT). These embryos are pathogen free and susceptible
to infection by the A subgroup of RCAS virus. The
embryos were staged according to Hamburger and
Hamilton (1951). Scaleless chicken embryos were from
Department of Poultry Genetics, University of Connecticut a t Storrs.
In Situ Hybridization
Nonradioactive in situ hybridization was performed
according to Sasaki and Hogan (1993). Plasmid PHH-2
containing the full-length coding sequence of chick Shh
(kindly provided by Drs. Randy Johnson and Cliff
Tabin) was linearized with HindIII and transcribed
with T3 RNA polymerase to synthesize the antisense
riboprobe. After hybridization, the tissues were reacted
overnight with preabsorbed antidigoxigenin Fab-conjugated alkaline phosphatase (Boehringer Mannheim).
Color reactions were performed by incubating with
NBT/BCIP substrates (Promega) in alkaline buffer. In
situ hybridization on frozen sections was performed
similarly. The bleaching step was omitted, and hybridization was carried out a t 48°C.
Virus Production and Retroviral
GeneTransduction
RCASBP(A) plasmid containing the full-length coding region of chick Shh in the sense orientation is a
generous gift from Drs. Robert Riddle and Cliff Tabin.
The RCASBP(A)-Shh plasmid was transfected into
stage 34 SPAFAS (line 15b) chicken embryo fibroblasts
using lipofectamine (Gibco/BRL).The transfected cells
were cultured in F-10 media containing 10% fetal bovine serum, 5% chicken serum, l x vitamin for MEM
(Irvine Scientific), 20 pm folic acid, 0.5% dimethylsulfoxide (DMSO), and 1mM L-glutamine. Media containing the viruses were harvested when the cells were
approximately 95% confluent and were stored at
-80°C when not used immediately. Dorsal body ectoderm of stage 20 embryos was injected, in ovo, with
microcapillary needles filled with 3-6 ~1 of virus-containing media. Embryos were harvested at stages
35-37 and analyzed for transgene expression and
changes in skin morphology.
Epithelial-MesenchymalRecombination
Stage 34 dorsal skins were dissected in Hank's balanced salt solution (HBSS) and incubated in 2x calcium- and magnesium-free saline on ice for 5 min. The
epithelium was then dissociated using watchmaker's
tweezers and recombined with the epithelium. For examples shown in this report, the epithelium was rotated 90" to the mesenchyme. Recombined skins were
transferred to culture inserts in six-well culture dishes
(Falcon) containing DMEM with 10% fetal calf serum
and incubated at 37°C in an atmosphere of 5% CO, and
95% air.
Immunostaining
Immunoalkaline phosphatase staining was performed on paraffin sections as described by Chuong et
al. (1990). Rabbit polyclonal antibodies to Pan-PKC
were from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies to TGF-P2 were from Santa Cruz Biotechnology. Antibodies to NCAM and tenascin were
described by Jiang and Chuong (1992).
RESULTS
Early Feather Morphogenesis: Shh Is
Specifically Expressed in Epithelial Placodes
The earliest morphological event detected during
feather development is the formation of epithelial placodes from a flat and homogeneous ectoderm. With the
formation of epithelial placodes, the mesenchymal cells
underneath condense to form visible dermal condensations that will become the feather germs. It is from the
interactions of the epithelium and mesenchyme in
these feather germs that young feather buds develop
(Fig. 7A; Lucas and Stettenheim, 1972). In the dorsal
skin, there is a bilateral growth gradient where feathers at the midline develop first and feathers lateral to
the midline develop subsequently.
Shh is first detected as a tiny dot in the center of
feathers at the early placode stage (Fig. l A , arrowhead).
When the feather placode expands, Shh positive region
also expands. In the short feather bud (the stage at
which the long axis of the feather bud is shorter than the
base; see Widelitz et al., 1996), Shh expression is stronger and is restricted to the tip (Fig. lA, arrow). As the
feather buds develop, Shh expression pattern undergoes
Shh IN FEATHER MORPHOGENESIS
159
Fig. 1. Distribution of Sonic hedgehog (Shh) transcripts during feather
development. A-C and G H are wholemount in situ hybridization of embryonic chicken dorsal skin. D-F are nonradioactive in situ hybridization
on sagittal frozen sections of dorsal skin. A: Top view of stage 35 dorsal
skin. From left to right: early placode stage, placode stage, and short
feather bud stage. The Shh transcript is first detected in a very small
cluster of cells in the center of the early placode (arrowhead). This cluster
enlarges in the short feather bud (arrow). B: Overview of stage 37 dorsal
skin shows feather buds at various stages of development. Arrow indicates the midline of the skin. Inset: Outlined feather buds from the short
bud stage to feather filament stage. C: Side view shows that Shh is
expressed in the tips of the short feather bud epithelium and the mesenchyme is negative. D: Sagittal section of stage 33 dorsal skin. Shh is first
detected in a few cells in the epithelial placode. Dashed lines outline the
epithelium of the feather bud. E: Sagittal section of stage 36 dorsal skin
shows that Shh is localized to the distal and posterior epithelium of the
long feather bud. The expression of Shh has increased both in Intensity
and in the number of positive cells. F: Cross section from the midshaft
region of a feather filament shows that Shh is highly expressed in the
marginal plate (mp) but not in the barb plate (bp). The basal lamina (bl)
is located centripetal to the marginal plate epithelium (Chuong and Edelman, 1985a,b). p, Feather pulp. G: Higher magnification of a feather
filament. Shh expression is in transition from the bud tip to the feather
filament pattern. The arrow points to the transitional zone. Note that one
feather filament stripe is made of two rows of epithelial cells. H: Shh
expression in mature feather filaments from stage 40 chicken skin. Note
the long stripes of Shh expression in the feather filament and the absence
of Shh in the feather collar region (cl, equivalent to hair matrix). Scale
bars = 100 prn in A-E, 50 pm in F-H.
dramatic changes a t different stages of development
(Fig. 1B).The side view showed that Shh is expressed
only in the distal placode epithelium (Fig, 1 0 .
We also performed in situ hybridization on 10 pm
sections of developing feathers. This allowed us to view
Shh expression at a higher resolution and to rule out
artifacts due to inadequate probe penetration. The pat-
tern is consistent with the wholemount in situ hybridization data. Shh is first detected at the center of the
epithelial placode (Fig. 1D).The Shh signal increased
as the feather developed. In the long feather bud (the
stage a t which the long axis of the feather bud is longer
than the base; see Widelitz et al., 19961,Shh expression
is shifted to the distal end and posterior side of the
160
TING-BERRETH AND CHUONG
feather bud (Fig. 1E). Shh was not detected in feather
mesenchyme a t any of the stages studied,
Late Feather Morphogenesis: Shh Is Expressed
in the Marginal Plates
Following the long feather bud stage, the developing
feathers invaginate into the skin to form feather follicles. At this stage, feather growth is at the collar region
of the follicle base and the morphogenesis of feather
filaments begins. The feather epithelium starts to form
alternating barb ridges. For each barb ridge, the epithelium produces a barb plate in the center and marginal plates bilaterally. The barb plate epithelium will
later keratinize to become feather barbs, and the marginal plate epithelium will undergo programmed cell
death to become the spaces between the barbs (summarized in Fig. 7A; Lucas and Stettenheim, 1972). Wholemount in situ hybridization of feather filaments showed
that the pattern of Shh expression changed dramatically during the transition from the long bud to a filamentous feather. Prior to filament formation, Shh is
localized in the distal tip epithelium. With the development of feather filaments, Shh is expressed in alternating “stripes” as epithelial compartmentalization begins (Fig. 1E). The “striped pattern suggests that Shh
is either in the barb plate or in the marginal plate. A
cross section through the feather filaments revealed
that Shh is in the marginal plate (Fig. lF,G). This
pattern of expression is maintained through stage 40
feathers, in which the filaments are longer and more
mature. However, Shh has by now disappeared in the
distal feather filament. Shh is negative in the collar
epithelium (in which the new feather epithelial cells are
added and compartmentalization has not started; Fig.
1H).
We attempted to analyze the relationship between
marginal plate formation and Shh expression. Because
the feather is more mature toward the distal end,
semiserial cross sections of feather filaments were cut
from the base to the tip so that the different stages of
barb ridge formation could be viewed. Barb ridges are
initiated from the anterior feather by a series of epithelial invaginations a t the site of future marginal
plates. Minute amounts of Shh are first detected in the
smooth epithelium just prior to invagination (Fig. 2A,
arrowhead). This is followed by the gradual increase of
Shh in the invaginating cells that initiate the formation of a marginal plate (Fig. 2A, arrow). At this stage,
the posterior side of the epithelium remains cylindrical
and completely negative of Shh (Fig. 2A, asterisk). The
epithelial invaginations propagate bilaterally around
the circumference of the feather and Shh is turned on
(Fig. 2B,C). As the bilateral waves of marginal plate/
barb ridge formation spread towards the posterior
feather (Fig. 2D, solid curved arrows), a ring of Shh
expression is seen in each marginal plate (Fig. 2E). The
earliest Shh-expressing cells are in the valley of each
marginal plate invagination, specifically in the basal
layer of the stratified epithelium. As the invagination
of the marginal plates deepens, Shh expression propagates bilaterally in the marginal plates toward the tip
of the ridge (Fig. 2F, curved open arrows). Eventually
the entire marginal plate epithelium becomes strongly
positive for Shh expression (Fig. 2F). As the adjacent
barb plate epithelium begins to keratinize, Shh-positive marginal plate epithelial cells will undergo cell
death and disintegrate. This process leaves the space
between the keratinized cells that creates the branched
barbs (Lucas and Stettenheim, 1972). Our data suggest
that Shh is associated with the epithelial compartmentalization process in feather filament morphogenesis.
Ectopic Expression of Shh-Induced
Mesenchymal Condensation
To test the functional role of Shh in feather development, we adopted the retroviral gene delivery technique developed in chicken embryos (Morgan et al.,
1992; Riddle et al., 1993; Fekete and Cepko, 1993). We
first injected RCAS alkaline-phosphatase (Alk-P) virus-containing media into the dorsal ectoderm of stage
20 (embryonic day 3) chicken embryos. Embryos were
sacrificed after 5 days and assayed for Alk-P activity
(Fig. 3A). The patch of blue-purple staining represents
the extent of transgene expression. Higher magnification revealed no abnormal feather development. This
served as a control to show that the RCAS virus with
an innocuous gene does not cause abnormal feather
development.
Ectopic overexpression of Shh was induced by injecting 3-6 ~1 of RCASBP(A1-Shh viral media into the
dorsal ectoderm of stage 20 embryos. The embryos were
incubated for an additional 7 days, harvested, and the
skins examined. The development of enlarged feather
buds was observed in the affected patch in 11 of 36
embryos that survived the injection. Many of the enlarged feather buds appeared to be symmetrical and
Fig. 2 Expression sequence of Shh during the formation of marginal
plates in the feather filament. In situ hybridization of serial cross sections
from the base (A) towards the tip (F) of one feather filament. A: Shh
expression is first detected (arrowhead) just prior to the invaginationof the
epithelial sheet to form barb ridges and increases slightly as the epithelial
invagination is more evident (arrow). The posterior side of the feather that
has yet to undergo barb ridge formation is marked by the asterisk. B: Shh
expression is initially seen when the filament epithelium is still smooth
(arrowhead), and it is slightly stronger in the shallow indentations (arrows).
C: The indentationsdeepen, and the developing marginal plates are more
evident (arrows). D: Shh is enhanced in the developing marginal plates
(straight arrows) between the forming barb ridges (bp). At this time, the
posteriorside is still without barb ridges and is negative of Shh. In a similar
fashion, a wave of barb ridge formation will spread bilaterally (curved
arrows) toward the posterior side of the feather. E: Marginal plates (mp)
have formed along the entire circumference of the feather, and Shh is
expressed in all areas destined to become marginal plates. The marginal
plates at the anterior side are more mature and are stronger in Shh
expression. F: Within a more mature marginal plate, Shh expression
spreads from the cells in the “valley” of the marginal plates towards the
top of barb ridge (arrows). Later, these Shh- positive cells become the
space between feather barbs. a-tp, Anterior-posterior axis of feather
filament. Scale bars = 30 pm.
Shh IN FEATHER MORPHOGENESIS
Fig. 2.
161
162
TING-BERRETH AND CHUONG
stunted in growth (Fig. 31, and some exhibited reversed
anterior-posterior axis (Fig. 4).
We verified the ectopic expression of Shh in the affected skin by wholemount and section in situ hybridization. Increased expression of Shh in and around the
enlarged buds confirms the ectopic overexpression of
Shh (Figs. 3B, 4A). It should be noted that, even
though Shh is normally expressed only in the feather
epithelium, our injection procedure ectopically expressed Shh in both the epithelium and the mesenchyme. The area occupied by this enlarged skin appendage in Figure 3B represents the space that would
normally accommodate approximately eight feather
buds. The mesenchymal cells in that area may all be
incorporated into the enlarged bud. The size of the enlarged buds varied in different experiments, reflecting
the degree of transgene expression. Some of the skins
harvested (approximately 10%)showed no visible phenotypic changes even though they had some degree of
Shh overexpression as judged from small patches of
intense signals when hybridized with Shh antisense
probe (not shown). Controls included embryos that
were injected with RCASBP(A) virus only or with
RCANBP(A)-Shhvirus (vector unable to produce transcripts due to the absence of a splice acceptor). These
did not exhibit abnormal feather phenotype (not
shown).
Histological analysis of enlarged feather buds was
performed. Sagittal sections stained with hematoxylin
and eosin showed that there is a large accumulation of
mesenchymal cells in the enlarged feather buds. This
accumulation extended deeply into the dermis and the
underlying muscle layer (compare Fig. 3C, a normal
feather bud, and Fig. 3D).In situ hybridization with
Shh antisense probe showed that there is overexpression of Shh in the accumulated mesenchymal cells and
in the deep dermis (Fig. 4A).
Downstream to Shh: Loss of Polarized NCAM
and Tenascin Localizations, Increased
Distribution of TGF-P2, and Suppressed
PKC Expression
To search for potential downstream molecular
changes caused by Shh overexpression, sections were
stained with various antibodies. Previously, we reported that tenascin and NCAM are expressed in developing feather buds (Jiang and Chuong, 1992). Tenascin is initially expressed in the anterior epithelium
of early feather germs. In the short bud stage, both
tenascin and NCAM are localized in the anterior
feather mesenchyme. In the symmetrically enlarged
bud, the asymmetric localization of these molecules is
lost. Both tenascin and NCAM became symmetrically
distributed compared to a normal bud (Fig. 3E,F, open
arrow). The expression patterns of NCAM and tenascin
remained intact in the reverse-oriented feather buds,
but they were polarized to the new feather axis (not
shown).
Recently, we showed that TGF-p2 message is
strongly expressed in the placode epithelium, and
TGF-P2 protein can exogenous substitute for the epithelium by inducing dermal condensations from a mesenchyme stripped of its epithelium (Ting-Berreth and
Chuong, submitted for publication). In the Shh- induced enlarged bud, there is a wider distribution of
TGF-P2 (Fig. 4B, arrowheads) than in normal feather
bud (Fig. 4C). However, the intensity of expression (per
cell) between the normal and abnormal bud did not
increase. We have also shown that enhanced PKC activity favored the expansion of the interbud domain
(Noveen et al., 1995b). When TGF-P2 was delivered
locally by an Mi-Gel Blue bead, PKC expression was
suppressed in the induced mesenchymal condensation
(Ting-Berreth and Chuong, submitted for publication).
These results are consistent with our observation that
PKC expression is suppressed in the region of Shh
overexpression (compare Fig. 4D and El.
Shh Is Induced and Maintained by the
Feather Mesenchyme
We explored the possible regulatory mechanisms of
Shh expression in the skin. Feather formation requires
continuous and reciprocal interactions between the epithelium and the underlying mesenchyme. When the
epithelium and mesenchyme are separated, ongoing
cellular and molecular organization is lost. However, if
the epithelium and mesenchyme are recombined after
separation, feather growth will resume (reviewed in
Sengel, 1978). The position of these reformed feather
germs depends on the mesenchyme (Sengel, 19761,
whereas the orientation is determined by the epithelium (Novel, 1973). The regeneration capacity of the
skin provides an excellent opportunity to determine
whether the epithelial expression of Shh is autonomous or mesenchyme dependent.
The epithelium and mesenchyme of stage 34 dorsal
skins were dissociated and immediately recombined
with a 90" rotation of the epithelium with respect to the
mesenchyme. As a result, these two components of the
skin were out of phase from their original positions and
any existing interactions that may have been initiated
prior to dissociation were reorganized. In addition, a
new feather A-P orientation was established, allowing
us to examine whether Shh induction is associated
with feather orientation. Figure 5A shows that stage
34 skin expresses Shh in the posterior bud. Immediately after the recombination, Shh in the epithelium
was still visible. However, the epithelium was out of
phase and did not overlap with the condensed mesenchyme (Fig. 5B). Within 3 hr, Shh transcripts in the
epithelium had completely disappeared (Fig. 5C), suggesting that close contact with organized mesenchyme
is essential for the maintenance of Shh expression. Six
hours after the recombination, new Shh expression was
induced in the center of newly formed epithelial placodes (Fig. 5D). Subsequent expression patterns of
specimens harvested a t 8, 10, 12, and 18 hr recapitu-
Shh IN FEATHER MORPHOGENESIS
163
Fig. 3 Ectopic Shh expression, mediated by retroviral vectors, induces enlarged buds, accumulation of mesenchymal cells, and abnormal
expression of adhesion molecules. Stage 20 SPAFAS chicken embryos
injected with 3-6 pl viral media at the dorsal body wall. A: Chicken
embryo injected with RCAS(A)-alkalinephosphatase viral media (RCASAlkP) was harvested at stage 34. The transgene expression is shown by
a patch of skin expressing ectopic alkaline phosphatase (purple color,
indicated by the arrow). The head is toward the right margin. 8: Wholemount in situ hybridization of a stage 37 embryo skin that was injected
with RCASBP(A)-Shh. An enlarged bud is the result of Shh overexpression. Shh overexpression was verified by wholemount in situ hybridiration. The bud is abnormally large, round, and stunted. It is surrounded by
normal buds that have already entered the feather filament stage. C: H
and E staining of a normal feather bud (normal; H and E). D: H and E
staining of the Shh-induced bud (RV; H and E). Note the accumulation of
large numbers of mesenchymal cells in the Shh-induced bud. The accumulation extended deeply into the dermis (small open arrow). E: Sagittal
section through an enlarged and symmetrical Shh-induced bud showed
that NCAM (RV-NCAM), normally in the anterior of feather buds (solid
arrows) has lost its polarized distribution. Adjacent normal feather is indicated by large open arr0w.F: Sagittal section of the enlarged and symmetrical Shh-induced bud showed that tenascin, also normally expressed
in the anterior portion of normal buds, is induced in the mesenchyme of
both anterior and posterior portions of the abnormal bud (RV-Tn). In the
abnormal bud, tenascin is also lost in the epithelium. Anterior-posterior
axis is designated a-p. Scale bars = 100 prn.
lated the expression sequence in vivo (not shown).
Thirty hours after recombination, the reformed feather
buds were elongated and asymmetrical. A new anterior-posterior axis was established by the epithelium
and Shh was located in the posterior-distal ends of the
new bud orientation (Fig. 5E). The orientation of Shh
and newly formed feather buds was still determined by
the epithelium. These results showed that epithelial
expression of. Shh is mesenchyme dependent. The entire piece of the epithelium is competent to respond to
the mesenchymal signals to form placodes and express
Shh.
Shh Is Abnormally Expressed in
Scaleless Mutants
Scaleless is an avian autosomal recessive mutant
that fails to develop scales on its feet and is mostly bald
except for a few patches of feathers along the back,
wing, gluteal, and head areas (Abbott and Asmundson,
1957). The genetic defects of scaleless have been localized in the epithelium using epithelial-mesenchymal
recombination experiments (Sengel and Abbott, 1963;
McAleese and Sawyer, 1981). At stage 34, Shh is
mostly absent from the dorsal skin of the scaleless embryo (Fig. 6A). Some embryos at this stage developed
164
TING-BERRETH AND CHUONG
Fig. 4 Ectopic Shh expression induces enlarged buds with abnormal
orientation, wider TGF-gi2 distribution, and suppressed PKC expression.
Sagittal sections through an embryo with enlarged feather growth due to
Shh overexpression. A, overview. B,D, region of abnormal buds. C,E,
region of normal buds. A: Shh induces a feather bud with reversed anterior-posterior polarity. The original anteroposterior direction is indicated
by the straight open arrow. In situ hybridization with Sbh antisense
probes was performed to verify the degree of overexpression. In the
induced bud (open curved arrow), Shh is expressed in both the epithelium and mesenchyme, and extends into the muscle layer. In the normal
buds (solid curved arrow), Shh is present only in the distal feather bud
epithelium. B.C: In the enlarged bud, TGF-p2 protein expression is similar to the normal buds, but th6 area of expression is wider, The border of
high TGF-p2 expression is flanked by large arrowheads in the abnormal
buds and small arrowheads in the normal feather buds. In normal feather
buds, TGF-P2 is limited to the distal-posterior feather epithelium.
D,E: PKC is suppressed in the mesenchymal region of the enlarged bud
(large star) as well as in the normal buds (small star). PKC is normally
expressed in the interbud region of chick skin. Note that the region of
PKC suppression correlates well with the region of Sbb overexpression
(cf. A and C). Scale bars = 100 km.
ridges of fused, feather-like structures along the lateral skin. It is possible that the scaleless gene is upstream of
part of the dorsal skin. Interestingly, the mesenchyme Shh and plays a role in regulating Shh.
of these fused structures strongly expressed Shh (Fig.
DISCUSSION
6A, arrow), but the epithelium did not. At stage 38,
bald regions of the scaleless skin remain negative of
The elaborate morphogenetic processes in vertebrate
Shh expression. A few normal feathers that had devel- epithelial-mesenchymal interactions create various oroped in the gluteal regions appeared normal and ex- gans with different functions in different species. Howpressed Shh in the normal striped pattern (Fig. 6B). ever, the fundamental molecular mechanism underlyThese results show that Shh is affected in the scaleless ing these processes is probably the same. To understand
Shh IN FEATHER MORPHOGENESIS
165
Fig. 5 Feather mesenchyme induces Shh expression in the epithelium. Epithelial-mesenchymal recombinants with 90" rotation were prepared from stage 34 skin. Orientations of epithelium (E)and mesenchyme (M) are indicated by arrows in the lower left corner. The
recombinants were cultured for the time indicated in the upper right corner, then fixed and processed for wholemount in situ hybridization with
Shh. A: Stage 34 skin without recombination at time 0 showed that the
hexagonal expression pattern of Shh is in the posterior-distal end of
feather germs (arrow). The margin of some feather buds is outlined.
6: Immediately after E-M recombination, the epithelium and the mesenchyme are out of phase. The old Shh transcripts are still present in the
epithelium. The mesenchyme not covered by the recombined epithelium
(asterisk) does not express Shh. C: Three hours after the recombination,
Shh expression has completely disappeared. D: Six hours after the recombination, Shh is reinduced in the epithelium at the center of each
newly organized feather germ. Polarity is not clear at this stage. Note that
Shh expression is more advanced in the midline region (arrow) of the
epithelium. E: Thirty hours after recombination, normal Shh expression
pattern is reestablished in the posterior-distalend of regenerated feather
buds (arrow, pointing to posterior end of bud). The position of the buds is
determined by the mesenchyme, but the orientation of the buds is determined by the epithelium. Scale bars = 250 pm.
166
TING-BEFtFtETHAND CHUONG
Fig. 6 Shh in the skin of the scaleless mutant. Wholemount in situ
hybridization of scaleless embryonic skins with antisense Shh probe.
A: Stage 34, lateral and rniddorsal skin region. The middorsal region is
towards the bottom of the figure. Shh is negative in the portion of the skin
that does not form skin appendages (star). Large, fused skin appendages
are formed in the lateral dorsal regions of some mutants (arrow). These
ridges contain abnormally fused dermal condensations that express Shh
in their mesenchyme. 8: Stage 37. gluteal skin region. The bare region
remains Shh negative. A few morphologically normal feathers have
formed. These exhibit the normal Shh alternating stripe pattern in the
feather filament. This suggests that, in scaleless, Shh can be expressed
normally in areas with normal feather buds, but it is deregulated in most
regions. Scale bar = 150 pm.
this mechanism, we have been studying various morphogenetically related molecules using the feather
model (Chuong, 1993; Widelitz et al., 1996). For this
report, we studied the roles of Shh during feather development. The process of feather morphogenesis and
various Shh expression patterns are summarized in
Figure 7A. Contrary to the mesenchymal expression
pattern reported for the limb (Riddle et al., 19931, Shh
is expressed exclusively in the epithelial structures of
feather buds in all stages that we studied. This may be
reasonable given the fact that the limb consists mainly
of mesenchymal cells, whereas the feather consists
mainly of epithelial cells. If Shh plays a major role in
skin morphogenesis, it should be present in the epithelium, where major morphogenetic events occur. This is
clearly evident during late feather development, when
Shh is found in the marginal plates of feather filament
epithelium with alternating stripes. Upon closer exam-
ination, we found that Shh expression precedes the initiation of marginal plates, suggesting that Shh may be
involved in specifying epithelial compartments that are
destined to die.
Shh Is an Early Event in the Molecular Cascade
of Skin Appendage Induction
We observed that, in the process of early feather development, Shh message is first detected during epithelial placode formation (summarized in Figs. 1, 7A).
In reorganized epithelial-mesenchymal recombinations, old Shh transcripts in the epithelium disappeared while new Shh was induced by the feather mesenchyme. These findings suggest that 1)mesenchymal
factor(s) is required to maintain the expression of Shh,
2 ) mesenchymal factors in the organized feather mesenchyme can initiate the expression of Shh, and 3 ) the
entire epithelium a t this stage, whether it is placodal
Shh IN FEATHER MORPHOGENESIS
A
B
-
Ectoderm
Mcsenchvnie
1 Induction 1
1
1
Dermal
Condensation
Expression of
Sonic Hedgehog
in placode epithelia
I___I__+
Placode
1
(E)
1
Axis and
4
Shon Fealher Bud
1
Growth
167
1
TGF Beta (E) and or
other grow:h factors
1
AA&.
+I
1
1
Enhanced NCAM
Pr
TGF Beta receptors (M)
Long Feather Bud
IInvagination I
I
feather hlament
dermal papilla
expression (M)
and/or other adhesion
molecules
collar
Feather Follicle
1
-Formation of dermal condensations (M)
.
_
I
I
basement membrane
r-zLJ
Feather Filament
Differentiation and
1
I
,---
rachis
Matun: Feather
or interplacodal, is competent to respond to the mesenchymal factor(s) to express Shh and form new placodes.
These data are consistent with earlier findings that the
pattern is determined by the mesenchyme (reviewed in
Sengel, 1978).
Our expression data of Shh during early feather development showed that Shh appears after the formation of epithelial placodes. This implies that Shh is
probably not the initiator of the molecular cascade of
skin appendage induction but that it is one of the early
events in the cascade. The early requirement of Shh
during skin induction that came from the avian autosoma1 recessive mutant scaleless (Abbott and Asmund-
Fig 7. Summary of Sbh expression pattern and a working model.
A: Shh expression is schematically shown for different stages of feather
development. Following induction, the formation of epithelial placodes
creates the first recognizable morphological structure. Cells in the placode domain will proliferate, migrate, invaginate, and differentiate in ways
totally different from those used by cells remaining in the interappendage
domain. See text and Chuong (1993) for further explanation of the process of feather morphogenesis. The filament depicted is a cross section
at the level shown for the feather follicle stage. The location of Shh
transcripts is shown in black. Note that the marginal plate is an "altered"
basal layer epithelium. Shh is expressed periodically in these basal epithelial cells. B: Hypothetical model of the epithelial-mesenchymal interaction during the formation of skin appendages that includes Shh and
molecules recently studied in our laboratory (for NCAM and tenascin, see
Jiang and Chuong, 1992; for TGF-pP, see Ting-Berreth and Chuong,
submitted for publication). The epithelial or mesenchymal location of the
molecular and cellular events is indicated by (E) or (M), respectively.
Each arrow may include more than one step. See text for discussion.
son, 1957) supports this hypothesis. The origin of the
scaleless defect is in the epithelium; the mesenchyme is
initially normal. However, if the scaleless mesenchyme
does not receive the proper signals from the epithelium, it will become abnormal as well (Sengel and Abbott, 1963; McAleese and Sawyer, 1981). Because Shh
is present in the placode epithelium and is involved in
feather induction, we expect that Shh will be absent
in the bare region. This is indeed the case (Fig. 6). In
these mutants, there are fused skin appendage ridges
in the dorsal regions that morphologically resemble the
enlarged bud induced by ectopic Shh expression. We
found abnormal Shh expression in the mesenchymal
168
TINGBERRETH AND CHUONG
region of these ridges. Shh may act to induce mesenchymal cells to form the abnormal ridges. These data
suggest that the scaleless gene probably acts upstream
of Shh, and the defect may cause deregulation of Shh
distribution and subsequent skin appendage defects.
Shh May Induce Mesenchymal Condensations
Through Wider Distribution Expression of
TGF-62 and Suppression of PKC
A major event during the placode and short bud
stages of feather development is the condensation of
mesenchymal cells toward the placode regions. 3H-thymidine and bromodeoxyuridine (BrdU) labeling experiments showed that cell proliferation has ceased during
these stages (Wessels, 1965; Noveen et al., 1995a).
Therefore, dermal condensations are mostly due to cell
migration. When Shh was ectopically expressed in both
the epithelium and the mesenchyme by retrovirus-mediated gene transduction, there was an enormous accumulation of mesenchymal cells toward this region
that extended deeply into the muscle layer. This observation suggests that Shh transcript in the placode epithelium may be used to make Shh peptides that act on
the surrounding epithelium and mesenchyme to induce
the formation of dermal condensations (Fig. 3B). Morphologically, the Shh-induced feather buds appeared to
be the result of the fusion of several feather fields.
Therefore, Shh may have a strong ability to induce
mesenchymal condensation. Recent studies have
shown that Shh protein is proteolytically cleaved to
produce amino- and carboxyl-terminal proteins that
may play distinct biological roles (Lee et al., 1994; Porter et al., 1995; Johnson and Tabin, 1995). The aminoterminal end of Shh protein appears to be responsible
for floor plate, motor neuron, and sclerotome induction
(Roelink et al., 1995; Fan et al., 1995). In future studies, we will try to detect Shh peptides to see whether
they are secreted into the mesenchyme from the placodes.
What are the possible molecular events downstream
of Shh? Recently we found that the activation of PKC
expands the interbud domains (Noveen et al., 199513).
We then searched for the extracellular signals that can
induce dermal condensations and act on PKC. TGF-PZ
protein-coated beads can substitute for epithelial placodes to induce dermal condensations. Around the
bead, there is an inhibition of PKC expression (TingBerreth and Chuong, submitted for publication), suggesting that TGF-P2 may be involved in the induction
of dermal condensations, at least partially, through the
inhibition of PKC. In regions of Shh overexpression, we
observed a wide distribution of cells that exhibit
TGF-P2 and inhibit PKC expression (Fig. 4I3,D).
Therefore, Shh may act early in the signaling cascade
of mesenchymal condensations, upstream of TGF-P2
(Fig. 7B). With the current experimental procedure,
both the epithelium and the mesenchyme are made to
produce ectopic Shh. However, the ectopic expression
data do demonstrate that Shh has a functional role in
feather morphogenesis. In the future, higher resolution
studies will be performed by directing overexpression
only to the mesenchyme or epithelium.
Shk and the Anterior-PosteriorPolarity of
Feather Buds
As the developing feathers grew from a symmetric
short bud into an asymmetric long bud, Shh expression
shifted from a symmetric, centrally localized pattern to
a polarized, distal-posterior pattern. Therefore, Shh
may also be involved in determining the anterior-postenor axis of feather buds. Among the Shh-induced
abnormal buds, some are round and have lost asymmetry, whereas others exhibit reversed anterior-posterior
orientation. In the round buds, the anterior localization
of NCAM and tenascin in feather mesenchyme became
symmetrical. The anterior expression of tenascin in the
anterior bud epithelium (Jiang and Chuong, 1992; Fig.
3)was also lost in the symmetric buds. In the reverseoriented buds, NCAM and tenascin are distributed in a
polarized manner, in accordance with the new anteriorposterior orientation.
In the feather, there is a wider variation in the phenotypes observed than in the reported limb bud studies
(Riddle et al., 1993). This is partially due to the fact
that the feather field is much smaller than the limb
bud. As a result, the region of ectopic expression influenced several feather fields rather than a single
feather field. Future refinements in retroviral gene delivery may allow more specific and detailed studies of
Shh function. The results presented here show that
ectopic expression of Shh can cause abnormal anteroposterior orientation during feather morphogenesis.
Shh Expression and
Epithelial Compartmentalization
A major cellular event in feather filament morphogenesis is the invagination of the cylindrical filament
epithelium. This in essence is the compartmentalization of the feather filament epithelium into the barb
plate and marginal plate. Among the numerous molecules we examined during feather morphogenesis
(Widelitz et al., 1996), NCAM is the only molecule that
is specifically expressed in the marginal plate (Chuong
and Edelman, 1985b1, in a pattern similar to that of
Shh. However, the onset of NCAM expression in the
marginal plate lagged behind that of Shh by three marginal plates. Therefore, the order of appearance is Shh
+ marginal plate initiation + NCAM.
What are the roles of Shh and NCAM in the marginal plate? Previous studies have shown that NCAM
in the marginal plate epithelium will eventually “zip
in” and form a collective cell group (Chuong and Edelman, 1985b). We hypothesized that the adhesive property of NCAM is used to define a group of cells to undergo a different developmental fate. The new fate can
be cartilage, kidney epithelium, keratinization, or, as in
this case, programmed cell death (Chuong, 1990). Shh
may act upstream to regulate adhesion molecules to
169
Shh IN FEATHER MORPHOGENESIS
define epithelial compartments. In this regard, it will
be most exciting to study whether Shh and related molecules can be used to regulate the spacing and number
of feather barbs and, therefore, the shape of the mature
feathers.
Nohno et al. (19951, using an independently derived
Shh antisense probe, describe their observation of Shh
expression in chicken limb feathers. The results from
the representative stages of feather development that
they studied are basically consistent with our data. In
describing their data, the authors referred to the “zone
of Shh expression” in the wing feathers. This, in fact, is
the valley region of marginal plates, and its significance is discussed above.
A WORKING MODEL FOR
EPITHELIAL-MESENCHYMAL
INTERACTION DURING SKIN
APPENDAGE MORPHOGENESIS
In this paper, we present a hypothetical model of how
Shh may be involved in dermal condensations based on
the data reported here and on our previous work (Fig.
7B). There are other molecules and signaling pathways
involved, which remain to be elucidated. Our results
indicate that initially unidentified molecule(s) from
specific locations of the feather mesenchyme induce the
epithelium to form epithelial placodes and that Shh
message is subsequently turned on in these placodes.
Secreted Shh proteins in the placode can induce dermal
condensations through TGF-P2 (Ting-Berreth and
Chuong, submitted for publication). The condensed
mesenchyme is enriched in NCAM, tenascin, and other
molecules and is distinctly different from the intercondensation mesenchyme. The condensed mesenchyme
can signal back to the epithelium to maintain and induce Shh expression.
In conclusion, during early feather development,
Shh is a major component of feather placode epithelium and a major mediator of dermal condensations.
Here, we have analyzed part of this molecular mechanism. In late feather development, Shh is a major component of the marginal plate epithelium and is associated with cell death. The distinct pattern exhibited by
the feather provides a powerful model for dissecting the
multifaceted roles of Shh in tissue interactions.
ACKNOWLEDGMENTS
We are grateful to Drs. Cliff Tabin, Robert Riddle,
and Randy Johnson for providing us with RCASBP(A)Shh, RCANBP-Shh, and PHH-2 plasmids, and Dr.
Bruce Morgan for RCASBP-Alk-P virus, We thank
Dr. Ting-Xin Jiang for contributing Figure 3A and Dr.
Randall B. Widelitz, Dr. Kevin Moses, and Ms. Florence Miyagawa for critical reading of the manuscript.
This work was supported by NIH grant AR 42177, NSF
grant IBN 9317397, a USC Zumberg Faculty Research
Award, and a grant from the Council for Tobacco Research.
REFERENCES
Abbott, U.K., and Asmundson, V.S.(1957) Scaleless, an inherited
ectodermal defect in the domestic fowl. J . Hered. 48:63-70.
Bitgood, M.J., and McMahon, A.P. (1995) Hedgehog and BMP genes
are coexpressed at many diverse sites of cell-cell interaction in the
mouse embryo. Dev. Biol. 172:126-138.
Chang, D.T., Lopez, A., von Kessler, D.P., Chiang, C., Simandl, B.K.,
Zhao, R., Seldin, M.F., Fallon, J.F., and Beachy, A. (1994) Products,
genetic linkage and limb patterning activity of a murine hedgehog
gene. Development 120:3339-3363.
Chuong, C.-M., Oliver,G.,Ting, S.A., Jegalian,B.G., Chen,H.-M., and
De Robertis, E.M. (1990) Gradients ofhomeoproteins in developing
feather buds. Development 110:1021-1030.
Chuong, C.-M. (1993) The making of a feather: Homeoproteins and
retinoids and adhesion molecules. Bioessays 15:513-521.
Chuong, C.-M., and Edelman, G.M. (1985a) Expression of cell-adhesion molecules in embryonic induction. I. Morphogenesisof nestling
feathers. J. Cell Biol. 101:1009-1026.
Chuong, C.-M., and Edelman, G.M.(1985b) Expression of cell adhesion molecules in embryonic induction. 11. Morphogenesis of adult
feathers. J. Cell Biol. 101:1027-1043.
Echelard, Y., Epstein, D.J., StJacques, B., Shen, L., Mohler, J., McMahon, J.A., and McMahon, A.P. (1993) Sonic hedgehog, a member
of a family of putative signaling molecules, is implicated in the
regulation of CNS polarity. Cell 751417-1430.
Fan, C.-M., and Tessier-Lavigne,M. (1994) Patterning of mammalian
somites by surface ectoderm and notochord: Evidence for sclerotome
induction by a hedgehog homolog. Cell 79:1175-1186.
Fan, C.-M., Porter, J.A., Chiang, C., Chang, D.T., Beachy, P.A., and
Tessier-Lavigne, M. (1995) Long-range sclerotome induction by
Sonic hedgehog: Direct role of the amino-terminal cleavage produd
and modulation by the cyclic AMP signaling pathway. Cell 81:457465.
Fekete, D., and Cepko, C. (1993) Replication-competent retroviral
vectors encoding alkaline phosphatase reveal spatial restriction of
viral gene expressiodtransduction in the chick embryo. Mol. Cell
Biol. 13:2604-2613.
Hamburger, V., and Hamilton, H. (1951)A series of normal stages in
the development of the chick embryo. J. Morphol. 88:49-92.
Jiang, T.-X., and Chuong, C.-M. (1992) Mechanism of feather morphogenesis: I. Analyses with antibodies to adhesion molecules tenascin, N-CAM and integrin. Dev. Biol. 15082-98.
Johnson, R.L., and Tabin, C. (1995) The long and short of hedgehog
signaling. Cell 81:313-316.
Johnson, R.L., Laufer, E., Riddle, R.D., and Tabin, C. (1994) Ectopic
expression of Sonic hedgehog alters dorsal-ventral patterning of
somites. Cell 79:1165-1173.
Krauss, S., Concordet, J.P., and Ingham, P.W. (1993) A functionally
conserved homolog of the Drosophila segment polarity gene hh is
expressed in tissues with polarizing activity in zebrafish embryos.
Cell 75:1431-1444.
Lee, J.J., Ekker, S.C., von Kessler, D.P., Porter, J.A., Sun, B.I.,and
Beachy, P.A. (1994)Autoproteolysisin hedgehog protein biogenesis.
Science 266:1528-1537.
Lucas, A.M., and Stettenheim, P.R. (1972) Avian anatomy. Integument. In: “Agriculture Handbook 362.” Washington, DC: Agricultural Research Services, U.S.Department of Agriculture.
McAleese, S.R., and Sawyer, R.H. (1981) Correcting the phenotype of
the epidermis from chick embryos homozygous for the gene scaleless
(sc/sc).Science 214:1033-1034.
Morgan, B.A., Izpisua-Belmonte, J.-C., Duboule, D., and Tabin, C.J.
(1992) Targeted misexpression of Hox-4.6 in the avian limb bud
causes apparent homeotic transformation. Nature 358236-239.
Nohno, T., Kawakami, Y., Ohuchi, H., Fujiwara, A,, Yoshioka, H.,
and Noji, S. (1995)Involvement of the Sonic hedgehog gene in chick
feather development. Biochem. Biophys. Res. Commun. 206:33-39.
Noveen, A., Jiang, T.-X., Ting-Berreth, S.A., and Chuong, C.-M.
(1995a) Homeobox genes Msx-1 and Msx-2 are associated with induction and growth of skin appendages. J. Invest. Dermatol. 104:
711-719.
Noveen, A., Jiang, T.-X., and Chuong, C.-M. (1995b)Protein kinase A
170
TING-BERFETH AND CHUONG
and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis. Dev.
Biol. 1713377-693.
Novel, G. (1973)Feather pattern stability and reorganization in cultured skin. J. Embryol. Exp. Morphol. 30605-633.
Placzek, M., Tessier-Lavigne, M., Yamamda, T., Jessell, T.M., and
Dodd, J. (1990)Mesodermalcontrol of the neural cell identity: Floor
plate induction by the notochord. Science 250985-988.
Porter, J.A., Ekker, S.C., Young, K.E., von Kessler, D.P., Lee, J.J.,
Moses, K., and Beachy, P.A. (1995)The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature 374:363-366.
Riddle, R.D., Johnson, R.L., Laufer, E., and Tabin, C. (1993)Sonic
hedgehog mediates the polarizing activity of the ZPA. Cell 7514011416.
Roelink, H.,Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S.,
Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T.M.,
and Dodd, J. (1994)Floor plate and motor neuron induction by
vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 76761-775.
Roelink, H., Porter, J.A., Chiang, C., Tanabe, Y., Chang, D.T.,
Beachy, P.A., and Jessell, T.M. (1995)Floor plate and motor neuron
induction by different concentrations of the amino-terminal cleavage product of Sonic hedgehog autoproteolysis. Cell 81:445-455.
Sasaki, H., and Hogan, B.L. (1993)Differential expression of multiple
fork head related genes during gastrulation and axial pattern formation in the mouse embryo, Development 118:47-59.
Sawyer, R.H., and Fallon, J.F. (eds.; 1983)“Epithelial-Mesenchymal
Interactions in Development.” New York: Praeger Publishing.
Sengel, P. (1976)“Morphogenesisof Skin.” Abercrombie, M., Newth,
D.R., and Torrey, J.G. (eds). Cambridge: Cambridge University
Press.
Sengel, P. (1978)Feather pattern development. Ciba Found. Symp.
2961-70.
Sengel, P., and Abbott, U.K. (1963)In vitro studies with the scaleless
mutant: Interaction during feather and scale differentiation. J.
Hered. 54:254-262.
van Straaten, H.M.W., Hekking, J.M.W., Wiertz-Hoessells, E.L.,
Thors, F., and Drukker, J. (1988)Effect of the notochord on the
differentiation of a floor plate area in the neural tube of the chick
embryo. Anat. Embryol. 177:317-324.
Wessels, N.K. (1965) Morphology and proliferation during early
feather development. Dev. Biol. 12131-153.
Widelitz, R.B., Jiang, T.-X., Noveen, A., Ting-Berreth, S.A., Yin, E.,
and Chuong, C.-M. (1996)Molecular histology in skin appendage
morphogeneeis. Microsc. Res. Tech. (in press).
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