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DEVELOPMENTAL DYNAMICS 210:274–287 (1997)
CHOXC-8 and CHOXD-13 Expression in Embryonic Chick
Skin and Cutaneous Appendage Specification
BENOÎT KANZLER, FABRICE PRIN, JACQUES THELU, AND DANIELLE DHOUAILLY*
Biologie de la Différenciation Epithéliale-UMR CNRS 5538, Etude de la Différenciation et de l’Adhérence Cellulaires,
Institut Albert Bonniot, Université Joseph Fourier, Grenoble, France
ABSTRACT
We studied the expression of
two distantly clustered Hox genes which could,
respectively, be involved in specification of dorsal feather- and foot scale-forming skin in the
chick embryo: cHoxc-8, a median paralog, and
cHoxd-13, located at the 58 extremity of the HoxD
cluster. The cHoxc-8 transcripts are present at
embryonic day 3.5 (E3.5)in the somitic cells, which
give rise to the dorsal dermis by E5, and at
E6.5–8.5 in the dorsal dermal and epidermal cells
during the first stages of feather morphogenesis.
The cHoxd-13 transcripts are present at E4.5–9.5
in the autopodial mesenchyme and at E10.5–12.5
in the plantar dermis during the initiation of
reticulate scale morphogenesis. Both the cHoxc-8
and cHoxd-13 transcripts are no longer detectable after the anlagen stage of cutaneous appendage morphogenesis. Furthermore, heterotopic
dermal–epidermal recombinations of dorsal, plantar, and apteric tissues revealed that the epidermal ability or inability to form feathers is already
established by the time of skin formation. Retinoic acid (RA) treatment at E11 induces after 12
hr an inhibition of cHoxd-13 expression in the
plantar dermis, followed by the formation of
feather filaments on the reticulate scales. When
E7.5 dorsal explants are treated with RA for 6
days, they form scale-like structures where the
Hox transcripts are no more detectable. Protein
analysis revealed that the plantar filaments, made
up of feather b-keratins, corresponded to a homeotic transformation, whereas the scale-like structures, composed also of feather b-keratins, were
teratoid. These results strengthen the hypothesis
that different homeobox genes play a significant
role in specifying the regional identity of the different epidermal territories. Dev. Dyn. 1997;210:
274–287. r 1997 Wiley-Liss, Inc.
Key words: chick; epidermis; feather; homeobox
genes; keratin; retinoic acid; scale
mal keratinocytes synthetize specific keratin polypeptides according to their regional origin, thus allowing
the molecular identification of the protein content
inherent to a given morphology. The a-keratins range
from 40 to 73 kD in molecular weight and are expressed
in all epithelial cells from fish to human (Moll et al.,
1982). The expression of b-keratin polypeptides, which
range from 10 to 25 kD, is restricted to reptiles and
birds and characterizes such terminally differentiated
tissues as scales, claws, beak, and feathers (Gregg et
al., 1984). Feathers, which characterize avian skin,
present some differences in their morphology and distribution pattern according to the body regions. Furthermore, in the chick, the type of appendage changes from
zeugopodial feathers to autopodial scales in the hindlimb. The scales themselves display two main subtypes:
the scuta, which are large overlapping scales from the
anterior tarsometatarsal region, and the reticula, which
are small and roundish scales covering the plantar
surface of the foot. Their structural protein content
characterizes these three types of appendages: Feathers express lighter b-keratin polypeptides than scutate
scales, while the reticulate epidermis is composed
mostly of a-keratins (Kemp and Rogers, 1972; Dhouailly et al., 1978; Haake et al., 1984; Sawyer, 1983).
These cutaneous appendages develop as a result of
successive interactions between the two tissues comprising the skin, an epithelium, the epidermis, and an
underlying mesenchyme, the dermis; their regional
diversity depends both on the inductive properties of
the dermis and on the competence of the epidermis
(Sengel et al., 1969; Sengel, 1976; Dhouailly and Sengel, 1983, Cadi et al.; 1983; Dhouailly, 1977, 1984;
Sawyer, 1983; Viallet and Dhouailly, 1994).
The skin regionalization specification could involve
transcription factors belonging to the homeoprotein
family. Vertebrate homeobox genes, which are homologous to the first identified genes of the Drosophila
antennapedia and bithorax complexes are organized
into four clusters, HoxA, B, C, and D, located on
different chromosomes (for reviews, see MacGinnis and
INTRODUCTION
The vertebrate skin provides a powerful model system to study cellular interactions which govern organ
morphogenesis because of its distinct developmental
pathways and the heterogeneity of its appendages.
Furthermore, during integument development, epiderr 1997 WILEY-LISS, INC.
Grant sponsor: ARC; Grant number: 6233; Grant sponsor: Fondation de la Recherche Médacale.
*Correspondence to: D. Dhouailly, Institut Albert Bonniot, LEDAC
UMR CNRS 5538-Biologie de la Différenciation Epithéliale, Domaine
de la Merci, 38706 La Tronche Cedex, France. E-mail: danielle.
dhouailly@ujf.grenoble.fr
Received 18 February 1997; Accepted 1 August 1997
cHoxc-8, cHoxd-13 AND EPIDERMAL SPECIFICATION
Krumlauf, 1992; Krumlauf, 1994). Within each cluster,
genes most similar in sequence to a particular Drosophila gene, or paralogous genes, occupy the same
relative positions within their respective cluster. In
addition, the rostro-caudal boundary of one given Hox
expression domain correlates with the position of the
gene within its cluster, a property termed colinearity
(Duboule and Dollé, 1989; Duboule and Morata, 1994).
The same Hox sets have been shown to regulate the
developmental processes within antero-posterior and
proximo-distal specification in patterning of the vertebrae and limb segments (among others: Kessel and
Gruss, 1991; Duboule, 1992). Previous results have
shown that the cHoxc-6 and cHoxd-4 homeoproteins are
differentially expressed during dorsal chick skin formation (Chuong et al., 1990, 1993). Likewise, in mouse,
two different homeobox gene families, namely Otx and
Hox, identify the facial and body skin territories,
respectively, (Kanzler et al., 1994).
In order to investigate the putative involvement of
Hox genes in providing positional information for skin
cells, and consequently in specification of chick featherversus scale-forming skin, we chose to study the expression of a median paralog, of order 8, which may be
expressed at its highest level in the middorsal skin and
that of the most distal one, of order 13, which similarly
could be expressed in foot skin. A chick Hoxd-13 probe
was provided to us by D. Duboule (Izpisùa-Belmonte et
al., 1991). In order to obtain a chick Hox gene belonging
to the 8th paralog, we screened a E8.5 dorsal skin cDNA
library (Michaille et al., 1994) using a mouse Hoxc-8
probe obtained from H. Le Mouellic (Le Mouellic et al.,
1992) and consequently isolated and characterized the
chick Hoxc-8 cDNA. The developmental expression
pattern of these two genes, analyzed by in situ hybridization, suggests that the cHoxc-8 and cHoxd-13 proteins play a role in the specification of dorsal and
plantar skin morphogenesis. To further define the
involvement of both genes, we carried out two complementary types of experiments. First, dermal–epidermal recombinants were performed between chick dorsal and plantar skin, at stages during which Hoxc-8 and
Hoxd-13 are expressed in skin and which correspond to
the initiation of appendage morphogenesis. To further
test the morphogenetic abilities of the plantar dermis,
some of the recombinants involved apteric midventral
epidermis. Second, as it is well known that retinoic acid
(RA) is both able to modulate homeobox gene expression (Simeone et al., 1991; Boncinelli et al., 1991) and to
promote the ectopic formation of feathers on chick feet
(Dhouailly et al., 1980) and scale-like structures in
dorsal skin (Chuong et al., 1992), we repeated these two
types of RA treatment in order to analyze the possible
correlative changes in cHoxc-8 and cHoxd-13 expression.
RESULTS
Overview of Chick Tegument Morphogenesis
In the chick, while most of the body is covered with
feathers, the feet bear scales, and some areas, referred
275
to as apteria, remain bare. All the epidermis, with the
exception of the median facial epidermis (Couly and Le
Douarin, 1988), originates from the ectoderm, whereas
the origin of the dermis varies according to the different
regions. The back dermis is formed by E5 by the
migration of somitic dermatomal cells (Mauger, 1972),
whereas the limb dermis is formed at E8.5 by mesodermal cells which originate from the somatopleure. The
first feather primordia (Fig. 1; stage f1 as referred by
Michaille et al., 1994) appear at E7 along the midline of
the lumbar region and consist of a circular epidermal
thickening, or placode, which then becomes associated
with an underlying dermal condensation (Sengel, 1976;
Dhouailly, 1984). The epidermal and dermal cells then
proliferate to form the feather bud (stage f3) by E8.5,
which slants backward. The feather bud then elongates
and invaginates at its base to form the feather filament
(stage f7) by E14.5. Feather keratinization (a-keratins
in the outer epidermal sheath and b-keratins in the
feather itself) is first detectable at E12.5 at the apex of
the filament and then proceeds downward (Haake et
al., 1984). For a better comparison with the corresponding stages of feather morphogenesis, the three main
stages of scuta and reticula formation were called
stages 1, 3, and 7, respectively (Fig. 1). The first
indication of scutate scale formation in the anterior
tarsometatarsal region is the appearance of oval placodes (stage s1) during E9. The scuta rudiments are
composed of an epidermal thickening covering a lightly
marked dermal condensation. By E12, the placodes give
rise to the scale buds (stage s3), then to the scutate
scales (stage s7), which express a- and b-keratins by
E16. Scutate scale b-keratins (Fig. 8, lane 5) involve
three major polypeptides (21 to 18 kD), numbered
b1–b3. The polypeptides 1 to 3 are also present but
barely detectable in feather, whereas feather specific
b-keratins (Fig. 8, lane 6) include a minor (b4) and four
major (b5–8) 16- to 10-kD polypeptides. The first step of
reticula formation (stage r1) occurs in the center of the
plantar region by E11.5 and is characterized by an
epidermal dome without any associated dermal condensation. Reticulate scale differentiation reaches stage r7
by E16. These structures express a-keratins (Fig. 8,
lane 1) with the exception of the embryonic peridermal
and subperidermal shedding layers which express a
25-kD b-keratin (Sawyer and Borg, 1979; Knapp et al.,
1993).
cHoxc-8 cDNA Cloning
Three distinct positive clones were obtained by screening the chick skin cDNA library under high-stringency
hybridization conditions with the mouse Hoxc-8 cDNA
probe, and their sequences were determined. The longest cDNA clone (Fig. 2A) is 2,800 bp in length and
encompasses the complete open reading frame of
cHoxc-8, as revealed by sequence comparison with the
known Hox genes belonging to the 8th paralog. The
partial sequence of this cDNA (Fig. 2B) has been
submitted to the EMBL/GenBank Data Libraries and
276
KANZLER ET AL.
Fig. 1. Main stages of chick feather (A), anterior tarsometatarsal scale
(scuta) (B), and plantar scale (reticula) morphogenesis (C) (see text). br,
barb ridge; d, dermis; dc, dermal condensation; dp, dermal pulp; e,
epidermis; ec epidermal collar; ep, epidermal placode; fb, feather bud; ff,
feather filament; ies, inner epithelial sheath; oes, outer epithelial sheath;
p, papilla; r, reticula; rb, reticulate scale bud; s, scuta; sb, scutate scale
bud.
received the accession number X94179. The predicted
corresponding protein contains 242 amino acids and
exhibits a Mr of 28,200 kD. Sequence comparison with
the human (Boncinelli et al., 1989) and mouse (Le
Mouellic et al., 1988) Hoxc-8 amino acid sequences
reveal a complete interspecies conservation of the homeodomain; the chick sequence (Fig. 2B) is 100%
identical with its human and mouse homologues. Two
other regions of the predicted cHoxc-8 protein are
strongly conserved when compared with the consensus
sequence, namely a hexapeptide (MYPWMK) located
five amino acids upstream of the homeodomain and a
peptide located at the N-terminal end. The YPWM
motif has been shown to be essential for establishing
cooperative interactions between a subset of Hox proteins and Pbx proteins (Chang et al., 1995). The Nterminal region contains a high number of hydrophilic
residues, particularly serine and threonine. As reported
for the mouse amino-acid sequence, the carboxyterminal region comprises an acidic region that is
particularly rich in glutamate residues (15 out of 21
amino acids). Such a region has been previously identified in the Hoxa-7 gene, where 15 glutamate codons
preceed a stop codon (Colberg-Poley et al., 1985).
Expression of the cHoxc-8 and cHoxd-13 Genes
During Chick Skin Morphogenesis
Using in situ hybridization, we studied the spatiotemporal distribution of cHoxc-8 and cHoxd-13 transcripts
in 3.5-, 4.5-, and 6.5-day whole embryos, as well as in
8.5- to 10.5-day whole hindlimbs. The tissue distribution of the cHoc-8 and cHoxd-13 transcripts were
studied on serial sagittal sections of 6.5- to 14.5-day
embryos and of 9.5- to 16-day hindlimb, that is during
dorsal and plantar skin morphogenesis. Antisense RNA
probes, labeled either with digoxygenin-UTP or 35SCTP, were synthesized from the 38 cDNA flanking
region of the cHoxc-8 homeobox, as well as from the
similar region of the cHoxd-13 cDNA.
Whole-mount in situ hybridizations performed at
E3.5 and at E4.5 (Fig. 3A) show that cHoxc-8 transcripts are present in the neural tube, in the mesodermaly derived cells of somitic origin from the midthoracic level to the caudal extremity, as well as in the
antero-proximal region of the wing bud, and more
lightly of that of the hindlimb bud. This gene displays
an anterior expression limit in the somites 23 and 24, a
level which is posterior to that observed in the neural
tube. These two somites later give rise to the fifth
thoracic vertebra and to the overlying thoracic dermis.
By 6.5 days of incubation (Figs. 3B, 4A), the chick skin
(stage f0) has formed in the dorsal region and the
cHoxc-8 transcripts are detectable at the same level in
the vertebrae and the overlying mesenchymal, dermal,
and epidermal cells. This expression reaches anteriorly
to the fifth thoracic vertebra, thus appearing colinear
with the limit observed earlier in the somites. The
cHoxc-8 signal is significantly more intense in the
dorsal skin region corresponding to its anterior expression boundary and decreases toward the caudal region.
This dorsal antero-posterior pattern is conserved at
E7.5 (stage f1), where transcripts are present in the
dorsal feather primordia (Fig. 4B,C), both in the dermal
condensation and the epidermal placodes. By E8.5, the
epidermal and dermal signal remain uniformly distributed in the feather bud (stage f3) (Fig. 4E). This signal
is also present in the mid-thoracic and at a less degree
in the abdominal ventral skin, comprising the midventral apterium. At stage f7 (E14.5), the cHoxc-8 transcripts are no longer present in the interfollicular skin
or in the feather follicle (Fig. 3G). No specific signal was
detected with the corresponding sense probes on consecutive sections (Fig. 4D,F,H). No expression of cHoxc-8
was found with the antisense probe in the head and
anterior thoracic skin regions at any of the studied
stages.
At E3.5, the cHoxd-13 transcripts were present in
somites 39–40 at the distal end of the trunk (data not
shown). They were never detected in either the dermis
or the epidermis during trunk skin morphogenesis, a
result which is confirmed by their presence in the
lombar spinal cord and caudal vertebrae in the same
sections. At E4.5 and E6.5 (Fig. 3C,D) the cHoxd-13
transcripts were detected in both the wing and hindlimb autopodes and then at E9.5 (Fig. 5A) in the distal
region of the feet, including the digits. On sagittal
sections, it appears that these transcripts display an
antero-posterior to proximo-distal gradient, being par-
Fig. 2. A: Organization of the cHoxc-8 cDNA clone. The isolated
cDNA contains the complete cHoxc-8 coding region (hatched box; nt
439–1168). The black box demarcates the position of the homeodomain
(HD; nt 883–1063). Nucleotide positions of several restriction sites are
indicated. E, EcoRI; P, PstI; K, KpnI. B: Partial nucleotide sequence of the
chick cHoxc-8 cDNA and predicted amino acid sequence of the coding
region. The arrow points to the 38 end of the cDNA clone. Amino acid and
nucleotide positions are indicated on the left and right sides, respectively.
The sequence of the putative cHoxc-8 protein is indicated below the open
reading frame. Amino acid changes between the chick and mouse protein
sequences are underlined. Three consensus regions, i.e., the N-terminus,
the conserved hexapeptide, as well as the homeodomain (HD) are boxed.
The position of the intron as deduced from the mouse Hoxc-8 cDNA
sequence is indicated by a black arrowhead. The splicing site (ACG/CT)
fits well with the consensus sequence (Cech, 1983). These sequence
data are available from EMBL/GenBank/DDBJ sequence database under
the accession number X94179.
278
KANZLER ET AL.
Fig. 3. Comparison of the distribution of cHoxc-8 and cHoxd-13
transcripts in 4.5- and 6.5-day chick embryos. Whole-mount in situ
hybridization. In profile (A,C) and dorsal views (B,D) with rostral at the top.
At E4.5, cHoxc-8 transcripts (A) are present in the somites from the
caudal extremity to the mid-thoracic level (arrowhead) as well as in the
anterior proximal part of the forelimb. Labeling is more intense in the
somites preceding the anterior expression limit. At E6.5, cHoxc-8 transcripts (B) form in the skin a thoracic shield and a circumference at the
insertion of the wing. The cHoxd-13 transcripts are present in the distal
autopodial part of both limbs, shown here at E4.5 (C) and E6.5, as well as
in the genitalia (not shown). Bars: 1 mm.
ticularly abundant in the mesenchymal cells surrounding the bones and in the chondrocytes located at the
extremities of the bones. From E10.5, cHoxd-13 expression is present in the mesenchymal cells which give rise
to the plantar dermis. This signal reaches its highest
expression level by E11.5 (Fig. 5B), the transcripts
being significantly more abundant in the central area of
the plantar region, precisely at the place where the first
reticulae appear (stage r1). At E12.5 (Fig. 5C), the
cHoxd-13 signal observed in the plantar dermis decreases markedly and cannot be detected afterward
(Fig. 5D). No significant signal was detected in the
epidermal cell layer at any of the stages examined.
Because of a high death rate in 7-day RA-treated
embryos, and in order to study the effect of RA treatment on cHoxc-8 expression dorsal skin morphogenesis, we turned to in vitro organotypic culture and
repeated the experiments previously performed by
Chuong and coworkers (1992). Dorsal skin explants
from 7.5-day chick embryo were cultured in vitro for
either 2 or 6 days with or without added RA and then
grafted onto the chick chorioallantoic membrane (CAM)
for 6 additional days. Control explants differentiated
feathers normally. Stable morphogenetic alterations
were obtained only with the longer period of RA treatment (6 days); thus the results reported below will
concern only this experimental series. In 12% of cases,
the explants exhibited no apparent modification of
feather morphogenesis (Fig. 7A,B). In 20% of cases,
abnormal feather filaments developed, characterized
by a dilated spherical extremity (Fig. 7C,D), and in 68%
of cases structures morphologically resembling scutate
scales were obtained (Fig. 7E,F). The latter were predominant in the posterior region of the skin explants. In
situ hybridization performed on longitudinal sections
from explants after 6 days of in vitro culture with both
antisense and sense cHoxc-8 probes revealed that the
cHoxc-8 transcripts were no longer specifically detectable (data not shown).
In order to discriminate between a single shape
convergence and a real transformation from feather to
scale or the reverse, the keratin content of both the
RA-induced feather filaments and scale-like structures
was subsequently analyzed and compared with that of
hatchling feather, scutate, and reticulate scales (Fig. 8).
The control keratin composition of hatchling scale (Fig.
8, lane 5) and feather (Fig. 8, lane 6) revealed characteristic, previously reported b-keratin profiles (Dhouailly
et al., 1978; Sawyer, 1983), while reticulate scale epidermis contained mostly a-keratins about 65 to 67 kD, and
a 25-kD (peridermal) b-keratin (Fig. 8, lane 1). The
Effect of Retinoic Acid (RA) Treatment
on cHoxc-8 and cHoxd-13 Expression and
the Resulting Skin Phenotypes
and Keratin Expression
Chick embryos treated in ovo with RA at E11 showed
by E17 enlarged and short feather filaments which look
like scales in the cephalic, cervical, alar, and femoral
tracts, and abnormal feather location on the reticulate
scales from the center of the plantar region of the feet
(Fig. 6A), as previously observed (Dhouailly et al.,
1980). In situ hybridization was consequently performed with the cHoxd-13 probe on longitudinal frozen
sections of feet of treated embryos at 11.5 and 12 days of
incubation, thus 12 and 24 hr after the RA injection.
This analysis revealed a lack of cHoxd-13 transcripts in
the plantar dermal cells at both stages (Fig. 6B),
whereas the controls showed a normal reticula morphogenesis (Fig. 6C) and cHoxd-13 plantar dermal expression (Fig. 6D). Conversely, chick embryos treated with
RA at E10 never formed feathered reticulae (ectopic
feathers only formed on the anterior face of the tarsometatarsus). In this case, the appearance of cHoxd-13
transcripts was just delayed by about 12 hr, and thus
the transcripts were present by the time of reticulate
morphogenesis.
cHoxc-8, cHoxd-13 AND EPIDERMAL SPECIFICATION
279
Fig. 4. Distribution of cHoxc-8 transcripts during dorsal chick skin
morphogenesis. In situ hybridization of sagittal sections with the antisense probe (A–C,E,G) and the sense probe (D,F,H). At E6.5 (stage fo) (A),
transcripts are present in the vertebrae (vt) as well as in the overlying skin,
both in the epidermis (e) and the dermis (d), in the midthoracic region. The
arrowhead marks the anterior level. At E7.5 (stage f1) (B), the newly
formed dorsal feather primordia (fp) express the cHoxc-8 gene. The
transcripts are located in the epidermis (e), the placode (p), and the
dermal condensation (dc). They display the same anterior expression
boundary (arrowhead) (C), both in the skin (s) and the vertebrae, while the
section hybridized with the sense probe (D) does not show any specific
labelling. At E8.5 (stage f3), transcripts are present (E) in the feather bud
(fb) epidermis, in the dermal condensation, and in the interappendage
epidermis and superficial dermis. Compare with the sense control (F). At
E14.5 (stage f7), the cHoxc-8 transcripts are no longer detectable in the
skin (G). The labeling which is confined to the epidermal cells (arrowhead)
surrounding the dermal papilla at the base of the feather filament (ff) is an
artifact. Compare with the sense control (H). Darkfield illumination. Bars 5
210 µm (A,B), 3 mm (C,D), 150 µm (E–H).
analysis of the protein content of scutate scale-like
structures (Fig. 8, lane 4), obtained by treating E7
dorsal skin explants with RA, as well as of the ectopic
feather filaments (Fig. 8, lane 3) formed on plantar skin
of treated E11 embryos, showed a similar protein
content: specific feather b4- to b8-keratins together
with nonspecific b1- and b2-keratins as well as akeratins, making an embryonic feather-type keratin
profile. It should be noted that, contrary to the feather
keratin profile, the scutate scale keratin profile does not
comprise any bands in the lower part of the gel. The
presence of a minor b4-keratin and that of a-keratins,
280
KANZLER ET AL.
the latter originating either from the interfollicular
epidermis or the epidermal sheath of feather filaments,
is usual when analyzing embryonic feathers or feathered skin explants.
Analysis of Epidermal and Dermal Abilities
Using Heterotopic Skin Recombinants (Table 1)
When E7 dorsal epidermis was associated with either
an E7 dorsal dermis (Fig. 9A) or an E10–E11 plantar
dermis (Fig. 9B), it developed numerous long feather
filaments, whereas it remained bare when it was
recombined with a midventral apterium dermis (Fig.
9C; Sengel et al., 1969). Three types of differentiation
were obtained with the E10–E11 plantar epidermis,
depending on its associated dermis: reticulate scales
with a plantar dermis (Fig. 9D), arrested buds distributed according to the feather hexagonal motif, plus a
few hypomorphic feather on the edges of the explant,
with a dorsal dermis (Fig. 9E), glabrous skin when
recombined with a midventral apterium dermis (Fig.
9F). The reverse association of E10–11 plantar dermis
and E10 midventral apterium epidermis resulted in the
formation of numerous reticula-like dome-shaped buds,
diplaying a tight distribution pattern with 2–3 days
after grafting. However, after 8 days of culture, a few
cases showed abnormal but short feather filaments,
recognizable by their barb ridges (Fig. 9G). In the other
cases (Fig. 9H), the epidermal top of the reticula-like
structures contained b-keratins within 8 days as shown
by immunofluorescent analysis (Fig. 9I,J). These were
feather-type b-keratins, added to the 25-kD b peridermal and the a-keratins of the reticulate scales, as
determined by electrophoretic analysis (Fig. 8, lane 2).
In situ hybridization with both the cHoxc-8 and
cHoxd-13 probes was performed 1, 12, 24, 36, and 48 hr
after the recombination. No transcripts were detected
for any of the dermal–epidermal recombinants.
DISCUSSION
We report the isolation and characterization of the
chick homeobox gene cHoxc-8. The predicted sequence
of the cHoxc-8 coding region analyzed is strikingly
similar in sequence to the corresponding region of the
mouse Hoxc-8 paralog (Le Mouellic et al., 1988). Particularly, the homeodomain appears identical with its mouse
and human (Boncinelli et al., 1989) cognates. This
evolutionary conservation probably extends to the developmental role of this gene during mouse and chick
embryonic development, as its restricted expression
pattern during early embryogenesis and the first stages
of skin morphogenesis appear to be very similar in
these two species. Indeed, the pattern of expression of
the cHoxc-8 gene in the neural tube, somites, and
Fig. 5. Distribution of cHoxd-13 transcripts during plantar chick skin
morphogenesis. A: In situ hybridization of a longitudinal section of a E9.5
foot (stage r0). The transcripts are present in the perichondrial mesenchymal cells in the distal region of the foot and the digits, as well as in the
plantar mesenchyme. B: Longitudinal foot section at the plantar level at
E11.5 (stage r1): the cHoxd-13 transcripts are present in the perichondrial
mesenchyme (pm) and in the plantar dermis (d). C: At E12, on a similar
section, the dermal cHoxd-13 signal appears significantly decreased and
is no longer detectable at E.16 (D). tmt, tarsometatarse; e, epidermis; r,
reticula. Darkfield illumination, Bars 5 25 mm (A); 125 µm (B–D).
cHoxc-8, cHoxd-13 AND EPIDERMAL SPECIFICATION
Fig. 6. Effects of retinoic acid (RA) treatment on plantar skin phenotype and cHoxd-13 expression. A: Plantar view of a foot from a 17-day
embryo, treated with RA at E11, showing ectopic feather filaments (f) that
formed on the reticulate scales. Compare with the untreated control (C).
B: In situ hybridization of a longitudinal section of a 11.5-day foot
RA-treated at E11, showing the lack of cHoxd-13 expression in the plantar
281
dermis (d). Compare with the untreated control (D), in which the cHoxd-13
transcripts are present in the plantar dermis and in the perichondrial
mesenchyme (pm), whereas the cHoxd-13 signal does not exceed the
background level in the anterior skin region which forms scutate scales
(ss). B,D: Darkfield illumination. Bars 5 1.3 mm (A,C); 280 µm (B,D).
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KANZLER ET AL.
Fig. 7. Effects of retinoic acid (RA) treatment on dorsal skin phenotype. Whole explants (A,C,E) and their corresponding sections (B,D,F).
Dorsal RA-treated skin explant (dissected at E7.5) after 6 days of in vitro
culture, followed by 6 days on the chick chorioallantoic membrane,
displays either almost normal feather filaments (A,B), abnormal enlarged
feather filaments in which the barb (b) and barbule cells (bl) are still
recognizable (C,D), or even scutate scale-like structures (sl) in the caudal
region and arrested very short feathers (af) in the anterior region of the
explant (E,F). e, epidermis; d, dermis. D,E,F, Hematoxylin/Biebrich
Scarlet; Bars 5 1.3 mm; (A–C) 120 µm (D–F).
proximodistal region of the limb buds of the 3-day chick
embryo is similar to the pattern previously reported for
the mouse at a comparable stage of development (E
10.5) (Le Mouellic et al., 1988, 1992). Likewise, the
pattern of expression of cHoxc8 in the skin at E7.5
corresponds with the overall expression of this gene
along the cephalo-caudal and dorso-ventral axes previously reported in embryonic murine skin at a similar
stage (Kanzler et al., 1994). The cHoxc-8 transcripts are
present in both the epidermis and the dermis of the
feather primordia and feather buds and disappear
afterward. These results suggest that cHoxc-8 expression plays a role in the thoracic region during the
initiation and first stages of feather embryonic morphogenesis. In contrast, no cHoxd-13 expression was found
in the trunk skin over the period of morphogenesis in
the different feather pterylae, including the caudal
tract. The cHoxd-13 expression characterizes the ventral skin of both wing and leg autopode, and particularly the plantar dermis during skin morphogenesis.
This is in concordance with the restricted pattern of
this gene in the distal part of the limb, already shown
by D. Duboule and coworkers (Dollé et al., 1991, 1993).
In the mouse, the Hoxd-13 gene is expressed in a
slightly more distal skin region, namely in the anterior
and plantar dermis from the digits when skin differentiation takes place (unpublished results from Dr. J.P.
Viallet in our group). It should be noted that in both
species, the cHoxd-13 transcripts were only found in
the dermis. Given their distinct expression pattern in
skin, the cHoxc-8 and cHoxd-13 could be part of different homeoproteins sets, providing positional information specifying the dorsal thorax and autopodial ventral
skin territories, respectively. Nevertheless, the difference between the ventral skin of the wing, which is
feathered, and the plantar skin implies that the formation of reticulate scales must involve a further set of
instructions which prevents the plantar epidermis from
developing into feathers and which remains to be
identified. Indeed, the results of heterotopic recombi-
cHoxc-8, cHoxd-13 AND EPIDERMAL SPECIFICATION
Fig. 8. Molecular identification of the protein content of chick cutaneous appendages formed in normal and experimental skin. (MW) Molecular weight; (lane 1) hatchling foot pad epidermis (reticulate scales); (lane
2) reticulate-like structures formed after 8 days of culture by recombinants
composed of E11 plantar dermis and E10 apteric midventral epidermis
(see Fig. 9,H,I,J); (lane 3) 18-day ectopic feathers formed on foot pad
after RA-treatment at E11 (see Fig. 6A); (lane 4) scutate-like structures
formed by E7 dorsal skin cultured for 6 days with added RA, and grafted
for 6 more days on chick CAM (see Fig. 7,E,F); (lane 5) hatchling anterior
tarsometatarsal epidermis (scutate scales); (lane 6) hatchling dorsal
feathers. The a-keratins are designated as a-K, the b-keratins 1–8 as b-K;
the histidine-rich proteins by an asterisk. 12% acrylamide gel stained with
Coomassie blue.
nants show that the plantar epidermis acquires a
restricted ability to only differentiate reticulate scales
on E11, which coincides with the expression of cHoxd-13
in the underlying dermis, while the dorsal epidermis,
and to a lesser degree the midventral epidermis, which
both express cHoxc-8, are endowed with featherforming ability, as shown by their ability to interpret
inducing clues originating either from a dorsal or a
plantar dermis to go over the feather differentiation
program. The morphogenesis of such heterotopic dermal–
epidermal recombinants is consistent with the hypothesis of an early regional specification of the epidermal
abilities (Dhouailly and Sengel, 1983; Viallet and Dhouailly, 1994). The fact that we were unable to detect
Hox gene expression in the dermal–epidermal recombinants, following the recombination, supports this hypothesis. The transcripts present in the dissected skin
are likely to be destroyed by the enzymatic treatment of
the skin, which is required to split it into its two
components. They may just be some leftovers, without
the need to be replaced. At the time of skin recombination, 7 days for the dorsal, 10 days for the midventral,
and 11 days for the plantar skin, the Hox genes have
already played their role in specifying epidermal abili-
283
ties. This may even occur earlier, at the time of skin
formation, or even when the global pattern of hox gene
expression within the body is established by E3.
Phenotypic changes following RA treatment add indirect proof, which nevertheless supports our hypothesis.
Retinoic acid treatment at E10 or E11 resulted in an
inhibition of cHoxd-13 expression in the plantar dermis. Embryos treated on day 11 developed feather
filaments on the reticulate scales. In contrast, in embryos treated on day 10, the cHoxd-13 expression
reappeared by E11, followed by normal reticulate scale
morphogenesis. The ectopic plantar feather filaments
displayed a feather b-keratin electrophoretic profile,
suggesting that this is a real homeotic transformation.
A possible explanation is that retinoic acid acts by
inhibiting the posterior paralogs and respecifying the
plantar cells to a more proximal positional identity,
thus allowing the formation of feathers. It cannot be
excluded that RA may also act by enhancing the
expression of more anterior paralogs. The RA-treated
dorsal skin explants formed abnormal structures which
can be interpreted as abnormal short and enlarged
feather filaments, since the expressed set of b-keratins
was of feather type. Similar malformed feathers were
already obtained in the cephalic, alar, and femoral
pterylae by in ovo RA treatment at E11 (Dhouailly et
al., 1980). By the time of treatment the feathered
specification of the dorsal skin might have already
occurred. Furthermore, it should be noted that the RA
effect on cHoxd-13 expression in embryonic skin is
consistent with the known effects of RA on Hox genes
expression in teratocarcinoma cells in vitro (Boncinelli
et al., 1991; Simeone et al., 1991), during vertebrae
differentiation (Kessel and Gruss, 1991), and during
limb morphogenesis (Hayamizu and Bryant, 1994): The
expression of Hox genes belonging to the median paralogs 4 to 8 is generally not modified by RA, whereas the
58 clustered genes are downregulated.
Further experiments of mis-expression of both Hoxc8
and Hoxd13 are required to refine our hypothesis. In
particular, the question still remains to know whether
the thoracic skin is specified by the expression of
cHoxc8 as well as other median paralog Hox genes, or
by the absence of expression of posterior paralogs as
cHoxd13. Likewise, is the autopodial plantar skin
specified by the expression of cHoxd13 or the absence in
a sufficient amount of homeoproteins of more anterior
paralogs? Finally, bird scale morphogenesis must require, in addition to the proximodistal clues provided by
the Hox genes, still-unknown information involved in
the specification of the hindlimb versus the wing.
EXPERIMENTAL PROCEDURES
cDNA Cloning
Approximately 1.5 3 106 recombinant phages of a
LZapII (Stratagene) cDNA library prepared from E8.5
chick dorsal skin RNA (Michaille et al., 1994) were
screened under high-stringency conditions (50% formamide; 53 standard saline citrate [SSC]; 1% standard
284
KANZLER ET AL.
TABLE 1. Skin Morphogenesis in Chick Heterotopic Epidermal–Dermal Recombinants
Cultured 6–8 Days on Chick Chorioallantoic Membrane
Dermis
Dorsal E7
Dorsal E7
(number of cases)
Feather filaments (5)
Plantar E11
Feather filaments (10)
Midventral apterium
E10
Glabrous skin (2) (and
Sengel et al., 1969)
saline sulfate [SDS]; 50 mM Tris-HCl pH 7.5; 0.1
mg · ml21 denatured salmon sperm DNA at 44°C). The
probe, a gift from Dr. H. Le Mouellic, was a 840-bp
SalI-EcoRI probe comprising the main part of the
mouse Hoxc-8 homeodomain sequence and that extended to the 38 end (Le Mouellic et al., 1988). We
followed the automatic excision protocol with helper
phage and recircularization to generate the subclones
containing the different chick cDNA inserts in
pBluescripttII SK phagemid vector, as stated in the
STRATAGENE Kit instructions.
DNA Sequencing
The nucleotide sequences were determined with the
dideoxy chain-termination method, using (35S)-dATP
and the Pharmacia T7-sequencingy Kit, according to
the manufacturer’s instructions. The sequence of the
cDNAs were read on both DNA strands.
Embryos
Warren breed fertile chick eggs were obtained from
the ‘‘Centre d’aviculture de Cerveloup’’ (Moirans,
France). Some Bresse breed eggs were obtained from
the ‘‘Centre de Sélection de la Race Bressane’’
(Béchanne, France) as we have previously shown (unpublished data) that this scaled-feet breed is more
prone than the Warren breed to develop feathers on
their feet through RA treatment (70% instead of 10% of
treated embryos), which facilitates the corresponding
in situ hybridization analysis. The eggs were incubated
at 38°C. For in situ hybridization, embryonic and
postnatal samples were embedded in Tissue Teckt
medium and frozen.
In Situ Hybridization
A 600-bp XhoI-PstI fragment of the cHoxd-13 cDNA
(a gift from Dr. D. Duboule) containing the homeodomain region and the following 38 sequences and a 1,500
bp KpnI-EcoRI fragment of the isolated chick Hoxc-8
cDNA clone were used as templates for synthesis of
either (35S)-CTP or digoxygenin-11-UTP–labeled riboprobes for in situ hybridization on frozen sections or
whole-mount embryos, respectively. In vitro transcription reactions were performed using either T7 or T3
Epidermis
Plantar E10–11
(number of cases)
Arrested buds (18)
Arrested buds plus
hypomorphic
feathers (18)
Reticulate scales (5)
Glabrous skin (4)
Midventral apterium
E10 (number of cases)
Feather filaments
(Sengel et al., 1969)
Abnormal reticulae (12)
Abnormal feathers and
reticulae (11)
Glabrous skin (2)
RNA polymerase as directed by the manufacturer (Boehringer Mannheim Biochemicals).
Whole-mount in situ hybridization protocol was based
on the Conlon and Rossant (1992) procedure with minor
modifications. Following fixation, bleaching, and proteinase K treatment, either 3.5-, 4.5-, or 6.5-day embryos as well as 9.5-day feet were hybridized overnight
at 70°C with 1 µg · ml21 of probe. After several washes
under stringent conditions and RNase A and T1 treatment, embryos and feet were incubated with antidigoxigenin Fab conjugated to alkaline phosphatase
(Boehringer Mannheim). Staining was allowed to proceed at room temperature by the addition of alkaline
phosphatase substrates NBT/BCIP. In situ hybridization on frozen sections was carried out essentially as
follows. Serial cryostat sections of 6–10 µm thickness
were treated successively with acetone, 4% formaldehyde at 4°C, 0.1 M triethanolamine/0.25% acetic anhydride, 50% formamide/13 SSC at 60°C, and two ethanol
washes. The hybridization buffer included 50% formamide, 10 mM DTT, 500 µg/ml tRNA, 13 saltsDenhardts, 10% dextran sulfate, and heat-denatured,
(35S)-labeled antisense riboprobe (specific activity of
5 3 108 c.p.m.21 · mg21; final concentration: 2 · 104
c.p.m./µl) or the corresponding opposite control probe.
After overnight hybridization at 54°C, slides were
washed under stringent conditions and treated with
RNase A and T1. After ethanol dehydration, slides were
dipped in Kodak NTB-2 nuclear track emulsion and
exposed for about 3 weeks before developing. Sections
were stained with propidium iodide, mounted in Surgipatht, and then analyzed with an AX70 Olympus
microscope using both darkfield and fluorescence illuminations.
In Vivo and In Vitro Retinoic Acid Treatment
Retinoic acid (125 µg of all-trans retinoic acid, a gift
from Hoffmann-La Roche, Basel, Switzerland), previously dissolved in absolute ethanol (50 µl) was injected
into the amniotic cavity of 10- or 11-day chick embryo
(Bresse breed). Preliminary experiments (Dhouailly et
al., 1980) showed that 125 µg was the most suitable
dose to obtain a high percentage of both surviving
Fig. 9. Homotopic and heterotopic skin recombinants after 8 days of
culture on the chick chorioallantoic membrane. A–C: Recombinants
involving E7 dorsal thoracic epidermis, associated with a dermis from (A)
E7 dorsal thorax, (B) E11 plantar foot pad, (C) E10 midventral apterium:
formation of feather filaments when the dermis originates from an
appendage-forming region. D–F: Recombinants involving E11 plantar
epidermis, associated with a dermis from (D) E11 plantar foot pad, (E) E7
dorsal thorax, (F) E10 midventral apterium: formation of reticulate scales
with a plantar dermis, of reticulate abnormal structures distributed
according to the feather hexagonal motif, of a few hypomorphic feathers
with a dorsal dermis, and of nude skin when the dermis originates from an
apteric region. G–J: Recombinants involving an E10 apteric midventral
epidermis associated with an E11 plantar dermis. Formation of reticulate
structures (G,H) and in a few cases (G) of a few hypomorphic feather
filaments recognizable by their barb ridges (arrow). Even in most of the
cases which only differentiate reticulate structures as in (H), the immunofluorescent staining with specific polyclonal antibodies to b- (I) and a- (J)
keratin polypeptides shows that the epidermis located at the top of the
reticulate-like structures (arrows) elaborates both a- and b-type keratins.
Bars 5 0.5 mm (A–H); 0.2 mm (I,J).
286
KANZLER ET AL.
embryos and morphogenetic alterations. Feet from
control and treated embryos were collected 12 and 24 hr
after injection for in situ hybridization, as well as 6
days later to study the resulting skin phenotype and to
determine the keratin composition of the appendages
by SDS-polyacrylamide gel electrophoresis (PAGE).
Dorsal skin tissues from 7.5-day chick embryo (Warren breed) were microdissected from the level of the
wing to the caudal extremity. Cultures were then set up
by placing the explants onto a grid in a Falcon dish with
DMEM (Dulbecco’s modified Eagle medium) supplemented with 20% fetal calf serum and either all-trans
retinoic acid previously dissolved in ethanol (final
concentration: 5 µg/ml) or an equivalent volume of
ethanol alone. The medium was changed every 2 days.
After 2 or 6 days of in vitro culture, the explants were
grafted onto the chick chorioallantoic membrane (CAM)
for 6 more days. Some of the control and RA-treated
grafts were conserved at 220°C for further protein
content analysis, and some of them were embedded in
Tissue Teckt medium for cryostat sectioning and in situ
hybridization analysis.
Heterotopic Dermal–Epidermal Recombinations
Midthoracic dorsal, ventral, and plantar skin fragments were dissected from 7-, 10-, and 11-day Warren
chick embryos, respectively, in Ca21- and Mg21-free
Earle’s saline. Dermis and epidermis were separated in
Tyrode’s solution after incubation in 2% trypsin and 1%
pancreatin in Earle’s saline for 10 min at 4°C; protease
digestion was stopped in 50% fetal calf serum. Tissues
were reassociated in heterotopic recombinations (referred in Table 1) on a semisolid nutritive agar and
placed for 30 min at 37°C to obtain sufficient mutual
adhesion between dermis and epidermis. Recombinants were transferred to the CAM of 10-day chick
embryos, cultured for 6 to 8 days, photographed, and
then some of them were processed for electrophoretic
keratin analysis or immunofluorescent staining with a
polyclonal rabbit antibody which recognizes all bkeratins (Dhouailly and Sawyer, 1984).
Keratin Analysis
Control skin fragments were dissected from the
tarsometatarsus, central foot pad, and feather plucked
from the back of a 21-day hatchling chicken. Skin
recombinants and RA-treated dorsal explants were
retrieved from the CAM 7 or 8 days after grafting.
Ectopic feather filaments formed after RA-treatment on
the reticula were plucked at E19. For gel electrophoresis, keratin polypeptides from control and experimental
specimens were isolated and S-carboxymethylated according to the procedure of Dhouailly et al. (1978). Then
one-dimensional SDS-PAGE (12% acrylamide) was performed according to Laemmli (1970).
ACKNOWLEDGMENTS
We are grateful to Dr. D. Duboule for the gift of the
chick Hoxd-13 probe and to Dr. H. Le Mouellic for the
mouse Hoxc-8 probe, as well as for their advice, and to
Dr. R.H. Sawyer, in the laboratory of whom D. Dhouailly became interested in chick reticulate morphogenesis. We thank Dr. J.P. Viallet for his advice in performing the whole mounts, Mr. Ray Dunn and Mrs. G.
Chevalier for their help in keratin analysis, and Mrs. B.
Peyrusse for the illustrations. We are indebted to Dr.
Bernigaud for providing the Bresse breed eggs and to
Dr. Jennifer Seed for critical reading of the manuscript.
This work was supported by ARC grant 6233 and a
‘‘Fondation de la Recherche Médicale’’ grant to D.
Dhouailly.
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