www.elsevier.com/locate/ydbio

Developmental Biology

cDermo-1 misexpression induces dense dermis, feathers, and scales

Christoph Hornik1, Kewal Krishan1, Faisal Yusuf, Martin Scaal, Beate Brand-Saberi*

Institute of Anatomy and Cell Biology II, Albert-Ludwigs-Universitat, D-79104 Freiburg, Germany

Received for publication 26 June 2003, revised 26 August 2004, accepted 26 August 2004

Abstract

Reciprocal epithelio-mesenchymal interactions between the prospective epidermis and the underlying dermis are the major driving forces

in the development of skin appendages. Feather development is initiated by a still unknown signal from the dermis in feather-forming skin.

The morphological response of the ectoderm to this signal is the formation of an epidermal placode, which signals back to the mesenchyme to

induce dermal condensations. Together, epidermal and dermal components constitute the outgrowing feather bud. The bHLH transcription

factor cDermo-1 is expressed in developing dermis and is the earliest known marker of prospective feather tracts. To test its function during

feather development, we forced cDermo-1 expression in embryonic chicken dermis using a retroviral expression vector. In featherless

(apteric) regions, cDermo-1 misexpression induced dense, thickened dermis normally observed in feathered skin (pterylae), and leads to the

development of regularly spaced and normally shaped ectopic feather buds. In pterylae, cDermo-1 misexpression enhanced feather growth.

In hindlimb skin, according to the local skin identity, misexpression of cDermo-1 induced ectopic scale formation. Thus, we show that forced

cDermo-1 expression in developing dermis is sufficient to launch the developmental program leading to skin appendage formation. We

propose a role of cDermo-1 at the initial stages of feather induction upstream of FGF10.

D 2004 Elsevier Inc. All rights reserved.

Keywords: cDermo-1; bHLH transcription factor; Feather development; FGF10; Skin development; Twist2

Introduction

Morphogenesis of skin appendages is driven by a series

of reciprocal epithelio-mesenchymal interactions between

the prospective epidermis and the dermal mesenchyme. In

avian embryos, the development of cutaneous appendages is

restricted to certain areas of the skin. Feather buds are found

in feather-forming areas of the integument, the pterylae,

which are separated from each other by primary featherless

regions, known as apteria or semi-apteria. Accordingly, in

the integument that covers the feet and the distal legs, scale-

bearing regions and scaleless regions can be distinguished.

Transplantation experiments have shown that the first

signal initiating feather development is given by the dermis

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2004.08.050

* Corresponding author. Institute of Anatomy and Cell Biology II,

Albert-Ludwigs-Universit7t, Albertstrasse 17, D-79104 Freiburg, Germany.

Fax: +49 761 203 5091.

E-mail address: beate.brand-saberi@anat.uni-freiburg.de

(B. Brand-Saberi).1 These authors have contributed equally to this work.

(reviewed in Dhouailly, 1977; Sengel, 1976). However, the

molecular identity of this putative primary inducer is

unknown. The epidermal placodes, local epidermal thicken-

ings, are formed in response to the primary inductive signal

from the dermis and represent the first morphological

manifestation of feather bud formation. In a next step, the

feather placodes signal back to the underlying mesenchyme

to induce dermal condensations. Ongoing molecular cross-

talk between epidermal and dermal components orchestrates

the coordinated spatial arrangement and regular outgrowth

of the feather anlagen to form feather filaments and, finally,

functional feathers.

A number of molecules that participate in feather

development have been identified (reviewed in Chuong

and Widelitz, 1998; Oro and Scott, 1998; Pispa and

Thesleff, 2003). The earliest known marker of the newly

formed epidermal placodes is b-catenin. Forced expression

of stabilized h-catenin is sufficient to induce ectopic feather

buds in normally apteric regions (Noramly et al., 1999;

Widelitz et al., 2000). In the further course of development,

277 (2005) 42–50

C. Hornik et al. / Developmental Biology 277 (2005) 42–50 43

application of various factors like Shh (Morgan et al., 1998;

Ting-Berreth and Chuong, 1996), the BMP antagonist

Follistatin (Patel et al., 1999), and FGFs (Jung et al.,

1998; Song et al., 1996; Tao et al., 2002; Widelitz et al.,

1996) has been shown to promote feather bud formation.

Conversely, application of BMP2 has been reported to

inhibit feather formation at placode stages, suggesting a

possible function of BMPs in the spacing of nascent buds

(Jung et al., 1998; Noramly and Morgan, 1998). So far,

however, no inducer of feather formation prior to the

formation of feather placodes (i.e., during the activity of the

hypothetical primary dermal inducer) has been found.

The Twist-like bHLH transcription factor cDermo-1 is

expressed in subectodermal mesenchyme from the earliest

stages of dermal differentiation, and is subsequently

associated with the dermal mesenchyme underlying the

prospective feather buds (Scaal et al., 2001). As it

demarcates the presumptive feather anlagen prior to the

formation of epidermal placodes and b-catenin expression,

cDermo-1 is the earliest known marker of feather develop-

ment, isochronic with primary dermal induction. Earlier

experiments on the regulation of cDermo-1 expression have

shown that early application of BMP2 and 4 to the

subectodermal mesenchyme upregulates cDermo-1 expres-

sion. Strikingly, in contrast to the inhibitory function of

BMPs at later stages of feather development, early

upregulation of cDermo-1 by BMPs was followed by local

upregulation of epidermal b-catenin and the formation of

ectopic feather buds (Scaal et al., 2002).

Here, we investigated whether cDermo-1 has an active

role in early feather development. We overexpressed

cDermo-1 in the chick embryo using a retroviral expression

vector (Logan and Tabin, 1998). We found that misexpres-

sion of cDermo-1 induces ectopic feather buds in apteric

regions and augments feather growth in the pterylae.

Furthermore, according to the local properties of the

integument (Dhouailly, 1977), cDermo-1 misexpression in

the integument overlying the feet and distal legs induced

ectopic scale formation. We could show that cDermo-1

function in skin development is restricted to mesenchymal

cells that underlie the ectoderm. Our results indicate that

cDermo-1 is a positive regulator of the earliest signaling

events leading to the primary induction of the development

of cutaneous appendages in the avian embryo.

Materials and methods

Embryos

Fertilized eggs of Gallus gallus (White Leghorn) were

obtained from a local breeder. For RCAS injection, virus-free

SPF eggs were obtained from Charles River Laboratories.

All eggs were incubated at 388C and 80% relative humidity

for the required time period. Stages of embryos were

determined according to Hamburger and Hamilton (1951).

Retroviral infection

RCAS cDermo-1 and RCAN cDermo-1 were con-

structed to contain the entire 483 bp of the avian cDermo-

1 coding region (Scaal et al., 2001). RCAN cDermo-1 is a

retrovirus of identical length that does not produce cDermo-

1 protein due to a lack of the appropriate splice acceptor and

can therefore be used as control. Chick embryo fibroblasts

and concentrated viral stocks were prepared according to

Fekete and Cepko (1993). Concentrated viral titers were in

the range of 109 to 4� 109 cfu/ml. Viral supernatant was

injected into the paraxial mesoderm of 2-day chick embryos

that were subsequently reincubated for 1–8 days.

Microsurgery

Eggs at HH stages 12–18 were windowed and the

vitelline membrane and the amnion were slit open in the

operation field. For injections of virus-containing super-

natant, a small tunnel was pierced into the embryo at the

desired injection position using a tungsten needle. With the

help of a pulled out Pasteur pipette, the supernatant was then

injected into the tunnel. To monitor success of injection, the

viral supernatant was stained with methylene-green.

The operated eggs were sealed with medical tape and

reincubated for the desired period of time from 6 h to 8

days. Embryos were then inspected, sacrificed, and fixed in

4% paraformaldehyde in PBS overnight at 48C, dehydratedin graded methanol series, and stored at �208C.

In situ hybridization

In situ hybridization was performed as previously

described (Nieto et al.,1996) using a cDermo-1 probe of

1.029 kb (Scaal et al., 2001) and a b-catenin probe of 0.800

kb obtained from C. Chuong. A fgf10 probe of 0.684 kb was

obtained from Hideyo Ohuchi.

Antiviral antibody staining

Effectivity of viral infection in ovo was monitored by

antibody staining using an AMV-3C2 antibody obtained

from the Developmental Studies Hybridoma Bank, identi-

fying cells infected with all strains of the avian sarcoma/

leukemia virus complex, including RCAS and RCAN.

Results

cDermo-1 overexpression leads to ectopic and enhanced

feather growth

Previous work from our laboratory showed that BMPs

induce ectopic cDermo-1 expression, which is followed by

the formation of ectopic feather buds in primary apteric

regions and leads to enhanced feather development in the

C. Hornik et al. / Developmental Biology 277 (2005) 42–5044

pterylae (Scaal et al., 2002). However, it remained unclear

whether cDermo-1 is a mere marker of feather-forming

dermis or is functional in feather development.

To test this, we overexpressed cDermo-1 in the chick

embryo using an RCAS retroviral expression vector. Forty-

eight hours after injection of the virus into the segmental

plate, in situ hybridization revealed cDermo-1 overexpres-

sion in the paraxial mesoderm and in the subectodermal

mesenchyme (Figs. 1a and b; n = 20). Transverse sections

showed that cDermo-1 overexpression was correlated with

increased thickness and high cell density of the dermal

mesenchyme (Figs. 1c and e) as compared to the dorso-

lateral, nontransfected region (Fig. 1d).

Five days after infection, that is, 3 days after the onset

of ectopic cDermo-1 expression, the ectoderm in the

Fig. 1. cDermo-1 overexpression leads to dense and thickened dermis in developin

leads to misexpression of cDermo-1, as monitored by in situ hybridization. Stron

subectodermal mesenchyme on the manipulated side (arrows). (b) Sixty-micromete

1 expression is detectable in the subectodermal mesenchyme overlying the derm

Paraffin sections with HE staining through the trunk of a stage HH 29 chick embry

causes a thickened dermal layer in the embryonic back skin prior to feather differen

dorsolateral skin. The bracket indicates the thickness of the dermal layer. (e) Epid

Note the excessive thickness (bracket) and density of the dermis, while the thickn

regions of cDermo-1 overexpression showed high levels of

b-catenin transcripts, which is a marker of nascent feather

tracts and the onset of overt feather differentiation

(Widelitz et al., 2000) (Fig. 2a; n = 10). Moreover, these

regions displayed high levels of dermal fgf10 expression,

which has been described as an early dermal factor

involved in feather formation (Tao et al., 2002) (Fig. 2d;

n = 4).

Six days after infection, feather buds were observed in

ectopic feather tracts located in primary apteric regions (Fig.

3a; n = 15). Size, shape, and pattern of the ectopic feather

buds were identical with those in the neighboring pterylae,

and markers of feather induction like b-catenin (Fig. 2b; n =

10) and fgf10 (Tao et al., 2002; not shown) were duly

expressed. Within the regular pterylae, misexpression of

g skin. (a) Injection of cDermo-1-containing RCAS retrovirus at HH12–18

g cDermo-1 expression is observed within the paraxial mesoderm and the

r-thick vibratome section through the manipulated area shown in a. cDermo-

omyotome (arrow) and the medial portion of the dermomyotome. (c–e)

o transfected with cDermo-1 RCAS. (c) Dorsal misexpression of cDermo-1

tiation (arrow). (d) Epidermis (arrowhead) and dermis (D) of nontransfected

ermis (arrowhead) and dermis (D) of cDermo-1 overexpressing dorsal skin.

ess of the epidermis remains largely normal (d and e are of the same scale).

Fig. 2. (a) Three days after infection with a cDermo-1 retrovirus, high levels

of b-catenin mRNA were observed in a region corresponding to the

scapular feather tract (arrow). (b) After 5 days of reincubation, ectopic

feather buds display a normal b-catenin expression pattern (arrow). (c)

Injection of a control RCAN virus at similar positions into the embryo

induces no changes in the development of feather buds seen here with b-catenin expression pattern (arrow). (d) Days (7.5) after transfection with

cDermo-1-RCAS, upregulation of dermal fgf10 is observed in primary

apteric regions (arrow).

Fig. 3. cDermo-1 overexpression leads to ectopic feathers and scales. (a)

cDermo-1 misexpression in the dorsal apteria leads to the ectopic

formation of regularly sized, normally shaped and patterned feather buds

as seen here with c-Dermo-1 in situ hybridization (arrow and arrow-

head). (b and c) When misexpressed in pteric regions of the integument,

cDermo-1 enhances feather growth, leading to feather filaments of

excessive length (arrow in b, arrowhead in c). (c and e) After injection

of cDermo-1-containing RCAS virus into the developing hind limbs of

a stage HH19 chick embryo, ectopic scales overlying the intertarsal joint

and distal leg region (at stage HH37) are formed (arrows); ectopic

feather buds of excessive length are formed in more proximal leg

regions (arrowhead in c). (d and f) Control embryos injected with

inactive cDermo-1-containing RCAN virus. Scales are restricted to

normal sites (c–f, in situ hybridization with b-catenin probe).

C. Hornik et al. / Developmental Biology 277 (2005) 42–50 45

cDermo-1 resulted in longer feather primordia without

perturbing pattern and shape (Figs. 3b and c; n = 5).

Injections of a control retrovirus (RCAN) had no effect

on the expression of cDermo-1 or b-catenin and did not lead

to changes in feather development (Figs. 2c and 3d,f; n = 9).

Thus, we could show that cDermo-1 overexpression in

the dermal mesenchyme is sufficient to induce normal

feather development. We demonstrated that dermal cDermo-

1 acts upstream of epidermal h-catenin and dermal FGF-10,

hence being the earliest known positive regulator of feather

development.

cDermo-1 overexpression promotes scale development in

distal leg and foot skin

In the integument of the distal legs and feet, cDermo-1 is

expressed in scale-forming dermis prior to and during the

development of scales (Scaal et al., 2001).

To test the effect of forced cDermo-1 expression on the

formation of scales, we ectopically expressed cDermo-1 in

C. Hornik et al. / Developmental Biology 277 (2005) 42–5046

the developing leg using the same retroviral expression

vector as reported above. Eight days after infection of

intertarsal joint and distal leg areas, we observed ectopic

scales that expressed high levels of b-catenin (Figs. 3c and

e; n = 8). In contrast, cDermo-1 misexpression in more

proximal areas in the leg induced the formation of feather

buds of excessive length (Fig. 3c).

Infection with control RCAN virus did not elicit the

formation of ectopic skin appendages (Figs. 3d and f; n = 8).

Thus, dependent on the regional quality of the skin

(Dhouailly, 1977; Sengel, 1976), cDermo-1 misexpression

induces the formation of various qualities of cutaneous

appendages. This suggests a role of cDermo-1 during

primary dermal induction, which determines position but

not quality of skin appendages.

cDermo-1 overexpression does not interfere with the

process of somite patterning

In the experiments described above, cDermo-1-express-

ing retrovirus was injected into the segmental plate prior to

somite formation. To exclude that the effects on feather

formation observed are due to alterations in the develop-

ment of somites, which give rise to dorsal dermis, we

analyzed the development of the somites after cDermo-1

misexpression.

We infected the paraxial mesoderm of embryos with

cDermo-1-RCAS and checked for the expression pattern of

dermomyotomal marker genes Pax-3 (Goulding et al., 1994;

Williams and Ordahl, 1994) and Wnt-11 (Marcelle et al.,

1997), the sclerotomal marker gene Pax-1 (Brand-Saberi et

al., 1993; Ebensperger et al., 1995), and the myotomal

markers MyoD, Myf-5, and Myogenin (reviewed in Mol-

kentin and Olson, 1996). Muscle differentiation was

assessed by anti-desmin staining.

Neither of these marker genes displayed any alteration in

expression compared to uninfected embryos (Figs. 4a–g),

nor did we observe changes in somite morphology. We can

therefore exclude that the feather-promoting phenotype after

cDermo-1 overexpression is a secondary effect due to

alterations in somite development. In particular, these

experiments rule out a role of cDermo-1 during the

recruitment of dermal precursor cells from the dermomyo-

tome, which is thought to be mediated by Wnt11 (Olivera-

Martinez et al., 2002), and exclude a functional overlap of

cDermo-1 with the closely related inhibitor of myogenesis

Twist (Hebrok et al., 1997; Li et al., 1995). We conclude

that the function of cDermo-1 during feather formation is

restricted to the subectodermal mesenchyme.

Discussion

Feather development is a well-studied example illustrat-

ing the importance of epithelio-mesenchymal interactions in

development. Pioneering studies in the laboratory of

Philippe Sengel and others have dissected the reciprocal

inductive interactions within the embryonic integument that

converge in skin appendage formation (reviewed in

Dhouailly, 1977; Sengel, 1976, 1990) In recent years, many

of the signals involved in the complex crosstalk between

dermal mesenchyme and epidermal epithelium in feather-

forming skin have been identified (reviewed by Pispa and

Thesleff, 2003). However, the molecular nature of the initial

steps in skin appendage formation are unknown. Classical

experiments established that the presence of dermis con-

stituted of densely associated cells is necessary to enable the

epidermis to participate in appendage development (Sengel,

1958). Accordingly, dermal cell density in feather tracts is

higher than in apteric skin (Wessells, 1965). More recently,

Jiang et al. (1999) have suggested from in vitro data that a

certain threshold level in dermal cell density will cause local

cell aggregates, which seem to be a prerequisite for the

emission of an unknown dermal signal to the epidermis

leading to epidermal placode differentiation. They propose a

reaction–diffusion model based on the work of Turing

(1952) assuming that the primary dermal signal be

uniformly distributed in the prospective feather tracts, and

subsequently localized to the feather anlagen by competitive

promotors and repressors of placode formation. The identity

of this unknown bfirst dermal messageQ (Hardy, 1992;

Sengel, 1976) or primary dermal inducer will be of major

interest to understand skin appendage development.

Very little is known about early dermal signaling. Several

lines of evidence point to Wnt molecules as potential

candidates. Wnt11 is expressed in dorsal dermal precursor

cells (Olivera-Martinez et al., 2002, our own data), and

inhibition of Wnt signaling in mice impairs hair develop-

ment (Andl et al., 2002; van Genderen et al., 1994) while

overexpression of the Wnt-mediated transcription factor h-catenin promotes feather growth. So far, however, the nature

of the primary inducer remains speculative, and no upstream

regulators have been identified.

In earlier work, we have described the bHLH tran-

scription factor cDermo-1 as an early marker of differ-

entiating dermis that demarcates the dense dermis of the

future feather tracts and shows but weak expression in

prospective apteria. Prior to the onset of feather bud

formation, expression in the feather tract regions is uniform.

Subsequently, during early stages of feather development,

its expression concentrates on the dermal compartment of

the feather anlagen (Scaal et al., 2001, 2002). This

expression pattern is highly reminiscent of the distribution

of the postulated primary inducer (Sengel, 1976; see above).

Considering the dynamics of its expression, we have

speculated that cDermo-1 plays an active role in feather-

forming dermis, possibly via cell–cell interactions during

dermal densification (Scaal et al., 2002).

Here, we overexpressed cDermo-1 in the dermal

mesenchyme of chick embryos. Our results confirmed our

hypothesis that cDermo-1 is involved in feather and scale

development. We found that in developmental stages prior

Fig. 4. cDermo-1 misexpression does not affect differentiation of the somite. Embryos were infected with cDermo-1-containing retrovirus and analyzed by in

situ hybridization. (a) Dermomyotomal development remains unaffected, as assessed by Pax-3 expression. (b) The dorsomedial lip of the dermomyotome, the

origin of dermal precursor cells, expresses Wnt11 and is not altered in size after cDermo-1 misexpression. (c) Sclerotomal development remains unaffected, as

assessed by Pax-1 expression. (d–f) Myotomal development is not altered by cDermo1 misexpression, as assessed by MyoD (d), Myf5 (e), and Myogenin (f)

expression. (g) Immunohistochemical analysis using anti-desmin antibodies and in situ hybridization for cDermo-1. Ectopic expression of cDermo1 in the

somite does not affect the distribution of desmin. Arrowheads show in situ hybridization for the overexpression of cDermo-1 and arrows show desmin staining

of the myotome (black bars show the injection area).

C. Hornik et al. / Developmental Biology 277 (2005) 42–50 47

to feather bud differentiation, forced cDermo-1 expression

in the subectodermal mesenchyme of chick embryos leads

to thickened and dense subectodermal mesenchyme, which

differentiated into dense dermis typical for feather tracts.

This was observed not only in feather tract regions, which

display endogenous cDermo-1 expression, but also in

apteria, where endogenous cDermo-1 expression is low.

Thus, cDermo-1 expression is sufficient to induce the

dermal properties of prospective feather tracts.

With the onset of feather development, we observed

different effects of forced cDermo-1 expression in pterylae

and apteria. Within regular feather tracts, the pterylae,

feather growth was augmented so that feathers on the

manipulated side of the embryo were longer than on the

control side. This argues for a dose-dependent feather-

promoting activity of cDermo-1, leading to excess or

premature onset of feather growth at locations where

overlap of endogenous and experimental cDermo-1 expres-

C. Hornik et al. / Developmental Biology 277 (2005) 42–5048

sion occurs. In primary apteric regions, overexpression of

cDermo-1 induced development of ectopic feathers in a

regular hexagonal array. Thus, cDermo-1 expression is

sufficient to induce ectopic feather tracts. In most cases, we

observed an expansion of pterylic skin into apteric regions,

as virus-mediated overexpression was rather widespread.

There, the hexagonal pattern of feather buds was continuous

between endogenous and ectopic pterylae. These findings

establish that cDermo-1 enables feather formation at early

stages, prior to the patterning processes within pterylae. We

propose a role of cDermo-1 in the establishment of dermal

competence to induce feather development. This is sup-

ported by the timing of endogenous subectodermal cDermo-

1 expression that precedes epidermal placode formation. We

postulate that cDermo-1 is a positive regulator of the

primary dermal inducer. The question if cDermo-1 acts

directly on the dermal inducer, or enables its activity

indirectly, perhaps through a role in dermal cell assembly,

remains speculative.

Further evidence in favor of this hypothesis comes from

our findings that cDermo-1 acts upstream of the early

dermal marker fgf10 and the early epidermal marker h-catenin. Both FGF10 and h-catenin have been shown to be

sufficient to induce feather formation when overexpressed;

however, feather patterning and morphology in these

ectopic feathers are abnormal (Noramly et al., 1999; Tao

et al., 2002). In contrast, after cDermo-1 overexpression,

patterning and morphology of ectopic feathers are normal.

fgf10 and b-catenin are upregulated following cDermo-1

overexpression and participate normally in feather develop-

ment within ectopic pterylae. This argues for a role of

cDermo-1 to convey dermal feather-forming competence,

without interfering with later steps of feather patterning and

morphogenesis.

We could show that cDermo-1 overexpression not only

induces ectopic feather tracts, but also ectopic scale

formation in the skin of distal legs and feet of chicken

embryos. The local identity of skin appendages depends on

local properties of the dermis and the epidermis, depending

on the stage of skin maturation (reviewed in Dhouailly,

1977; Sengel, 1976). cDermo-1 is expressed in the dermis

of both feather and scale tracts (Scaal et al., 2001). We

conclude that cDermo-1 is a positive regulator of skin

appendage morphogenesis irrespective of the local quality

of appendages, which acts prior to the specific differ-

entiation programs of feathers and scales.

Our results demonstrate that cDermo-1 overexpression

is sufficient to launch the developmental program leading

to skin appendage formation. From our data, we do not

know if cDermo-1 is also a necessary factor. Data in

mouse, however, are strongly supportive of a vital role of

Dermo-1 in skin and hair development. The cDermo-1

murine homologue Dermo-1, also known as Twist2, was

recently analyzed by loss of function experiments. Mice

homozygous for a Twist2 null mutation showed elevated

expression of proinflammatory cytokines, resulting in

perinatal death from cachexia. Importantly, these animals

displayed multiple skin defects, which included absence of

subcutaneous fat, decreased dermal thickness, and few hair

(Sosic et al., 2003). These defects are in line with the

overexpression phenotype described here in chick. Never-

theless, it remains to be addressed whether the observed

changes in murine skin result directly from a loss of

Dermo-1 in the skin or are secondary effects caused by

severe cachexia. Loss-of-function experiments in the chick

will be needed to clarify if similar phenotypes are observed

in the avian integument.

In our experimental setup, we infected cells of the

segmental plate of 2-day chick embryos with retroviral

supernatant. The time point of infection needed to be chosen

early to ensure sufficient retroviral infection of tissue. The

segmental plate gives rise to somites, which in turn form

multiple mesodermal derivatives including the axial skel-

eton, ribs, skeletal muscle, endothelium, and dorsal dermis

(reviewed in Brand-Saberi and Christ, 2000). Dorsal dermis

derives from the dorsomedial dermomyotome. As the dorsal

dermis, like other somitic derivatives, is determined by

complex signaling networks from surrounding tissues

(reviewed in Marcelle et al., 2002), notably Wnt1 from

the neural tube and possibly Wnt11 within the dorsomedial

lip of the dermomyotome (Olivera-Martinez et al., 2001,

2002), it needed to be tested if the observed effects of

cDermo-1 on feather tract formation were secondary effects

due to cDermo-1 overexpression in somites. We could rule

out this possibility as we neither observe any changes in

morphology nor abnormal expression of various marker

genes within different somitic compartments. Most impor-

tantly, we did not find an altered expression of Wnt11,

which is a marker of emigrating the dermal precursor cells

(Olivera-Martinez et al., 2002; own results). It is thus very

unlikely that dermal thickening and enlarged feather tracts

observed after cDermo-1 overexpression are consequences

of excess emigration of dermal precursor cells from the

dermomyotome. We therefore conclude that the feather-

promoting activity of cDermo-1 overexpression is restricted

to the dermal mesenchyme in situ. Interestingly, we neither

observed any effect of cDermo-1 overexpression on

myogenic markers. This is in contrast to findings in vitro

where cDermo-1, like its close homologue Twist (Hebrok et

al., 1997), has been reported to inhibit MyoD-dependent

muscle differentiation (Fuchtbauer, 2002). It remains to be

addressed if this is due to differences between in vivo and in

vitro conditions.

In summary, we report here that overexpression of

cDermo-1 in dermal mesenchyme is sufficient to induce

feather-forming dermis and to launch feather development

even in primary featherless regions of avian skin. In the

scale-bearing skin regions, cDermo-1 induces scale devel-

opment, accordingly. Our findings indicate that cDermo-1 is

an early positive regulator of dermal competence to

participate in skin appendage formation and the earliest

known marker of prospective feather tracts. We hypothesize

C. Hornik et al. / Developmental Biology 277 (2005) 42–50 49

that cDermo-1 is directly or indirectly promoting the activity

of the primary dermal inducer of feather development.

Acknowledgments

The authors thank Meike Ast, Ellen Gimbel, Susanna

Konradi, and Ulrike Pein for excellent technical assistance.

The generous gift of murine Dermo-1 by Dr. Eric Olson is

gratefully acknowledged. We furthermore thank Eric Olson

and Drazen Sosic for valuable discussion of our findings

and communication of details on Twist2-knockout mice. We

thank Dr. C. Chuong for the b-catenin probe and Dr. H.

Ohuchi for the avian fgf10 plasmid. The AMV 3C2

antibody was obtained from the Developmental Studies

Hybridoma Bank. We wish to thank Annette Neubqser forthe communication of unpublished data. The project was

generously supported by the Centre of Excellence

bSignaling Mechanisms in Embryogenesis and Organo-

genesisQ (SFB 592) Project B4.

References

Andl, T., Reddy, S.T., Gaddapara, T., Millar, S.E., 2002. WNT signals are

required for the initiation of hair follicle development. Dev. Cell 2,

643–653.

Brand-Saberi, B., Christ, B., 2000. Evolution and development of distinct

cell lineages derived from somites. Curr. Top. Dev. Biol. 48, 1–42.

Brand-Saberi, B., Ebensperger, C., Wilting, J., Balling, R., Christ, B., 1993.

The ventralizing effect of the notochord on somite differentiation in

chick embryos. Anat. Embryol. (Berl.) 188, 239–245.

Chuong, C.M., Widelitz, R.B., 1998. Feather morphogenesis: a model of

the formation of epithelial appendages. In: Chuong, C.M. (Ed.),

Molecular Basis of Epithelial Appendage Morphogenesis. RG Landes

Co., Austin TX, pp. 55–74.

Dhouailly, D., 1977. Dermo-epidermal interactions during morphogenesis

of cutaneous appendage in amniotes. Front. Matrix Biol. 4, 86–121.

Ebensperger, C., Wilting, J., Brand-Saberi, B., Mizutani, Y., Christ, B.,

Balling, R., Koseki, H., 1995. Pax-1, a regulator of sclerotome

development is induced by notochord and floor plate signals in avian

embryos. Anat. Embryol. (Berl.) 191, 297–310.

Fekete, D.M., Cepko, C.L., 1993. Retroviral infection coupled with tissue

transplantation limits gene transfer in the chicken embryo. Proc. Natl.

Acad. Sci. U. S. A. 15, 2350–2354.

Fuchtbauer, E.M., 2002. Inhibition of skeletal muscle development:

less differentiation gives more muscle. Results Probl. Cell Differ. 38,

143–161.

Goulding, M., Lumsden, A., Paquette, A.J., 1994. Regulation of Pax-3

expression in the dermomyotome and its role in muscle development.

Development 120, 957–971.

Hamburger, V., Hamilton, H., 1951. A series of normal stages in the

development of the chick embryo. J. Morph. 88, 49–92.

Hardy, M.H., 1992. The secret life of the hair follicle. Trends Genet. 8,

55–61.

Hebrok, M., Fuchtbauer, A., Fuchtbauer, E.M., 1997. Repression of

muscle-specific gene activation by the murine twist protein. Exp. Cell

Res. 232, 295–303.

Jiang, T.X., Jung, H.S., Widelitz, R.B., Chuong, C.M., 1999. Self-

organization of periodic patterns by dissociated feather mesenchymal

cells and the regulation of size, number and spacing of primordia.

Development 126, 4997–5009.

Jung, H.S., Francis-West, P.H., Widelitz, R.B., Jiang, T.X., Ting-Berreth, S.,

Tickle, C., Wolpert, L., Chuong, C.M., 1998. Local inhibitory action of

BMPs and their relationships with activators in feather formation:

implications for periodic patterning. Dev. Biol. 196, 11–23.

Li, L., Cserjesi, P., Olson, E.N., 1995. Dermo-1: a novel twist-related

bHLH protein expressed in the developing dermis. Dev. Biol. 172,

280–292.

Logan, M., Tabin, C., 1998. Targeted gene misexpression in chick limb

buds using avian replication-competent retroviruses. Methods 14,

407–420.

Marcelle, C., Stark, M.R., Bronner-Fraser, M., 1997. Coordinate actions of

BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite.

Development 124, 3955–3963.

Marcelle, C., Lesbros, C., Linker, C., 2002. Somite patterning: a few more

pieces of the puzzle. Results Probl. Cell Differ. 38, 81–108.

Molkentin, J.D., Olson, E.N., 1996. Defining the regulatory networks for

muscle development. Curr. Opin. Genet. Dev. 6, 445–453.

Morgan, B.A., Orkin, R.W., Noramly, S., Perez, A., 1998. Stage-specific

effects of sonic hedgehog expression in the epidermis. Dev. Biol. 201,

1–12.

Nieto, M.A., Patel, K., Wilkinson, D.G., 1996. In situ hybridization

analysis of chick embryos in whole mount and tissue sections. Methods

Cell Biol. 51, 219–235.

Noramly, S., Morgan, B.A., 1998. BMPs mediate lateral inhibition at

successive stages in feather tract development. Development 125,

3775–3787.

Noramly, S., Freeman, A., Morgan, B.A., 1999. Beta-catenin signaling can

initiate feather bud development. Development 126, 3509–3521.

Olivera-Martinez, I., Thelu, J., Teillet, M.A., Dhouailly, D., 2001. Dorsal

dermis development depends on a signal from the dorsal neural tube,

which can be substituted by Wnt-1. Mech. Dev. 100, 233–244.

Olivera-Martinez, I., Missier, S., Fraboulet, S., Thelu, J., Dhouailly, D., 2002.

Differential regulation of the chick dorsal thoracic dermal progenitors

from the medial dermomyotome. Development 129, 4763–4772.

Oro, A.E., Scott, M.P., 1998. Splitting hairs: dissecting roles of signaling

systems in epidermal development. Cell 95, 575–578.

Patel, K., Makarenkova, H., Jung, H.S., 1999. The role of long range, local

and direct signalling molecules during chick feather bud development

involving the BMPs, follistatin and the Eph receptor tyrosine kinase

Eph-A4. Mech. Dev. 86, 51–62.

Pispa, J., Thesleff, I., 2003. Mechanisms of ectodermal organogenesis. Dev.

Biol. 15, 195–205.

Scaal, M., Fuchtbauer, E.M., Brand-Saberi, B., 2001. cDermo-1 expression

indicates a role in avian skin development. Anat. Embryol. (Berl.) 203,

1–7.

Scaal, M., Prols, F., Fuchtbauer, E.M., Patel, K., Hornik, C., Kohler, T.,

Christ, B., Brand-Saberi, B., 2002. BMPs induce dermal markers and

ectopic feather tracts. Mech. Dev. 110, 51–60.

Sengel, P., 1958. Recherches experimentales sur la differenciation des

germes plumaires et du pigment de la peau de lTembryon de poulet en

culture in vitro. Ann. Sci. Nat., Zool. 20, 432–514.

Sengel, P., 1976. Morphogenesis of the Skin. Cambridge Univ. Press,

Cambridge.

Sengel, P., 1990. Pattern formation in skin development. Int. J. Dev. Biol.

34, 33–50.

Song, H., Wang, Y., Goetinck, P.F., 1996. Fibroblast growth factor 2 can

replace ectodermal signaling for feather development. Proc. Natl. Acad.

Sci. U. S. A. 93, 10246–10249.

Sosic, D., Richardson, J.A., Yu, K., Ornitz, D.M., Olson, E.N., 2003. Twist

regulates cytokine gene expression through a negative feedback loop

that represses NF-kappaB activity. Cell 112, 169–180.

Tao, H., Yoshimoto, Y., Yoshioka, H., Nohno, T., Noji, S., Ohuchi, H.,

2002. FGF10 is a mesenchymally derived stimulator for epidermal

development in the chick embryonic skin. Mech. Dev. 116, 39–49.

Ting-Berreth, S.A., Chuong, C.M., 1996. Sonic Hedgehog in feather

morphogenesis: induction of mesenchymal condensation and associa-

tion with cell death. Dev. Dyn. 207, 157–170.

C. Hornik et al. / Developmental Biology 277 (2005) 42–5050

Turing, A.M., 1952. The chemical basis of morphogenesis. Philos. Trans.

R. Soc. Lond., Ser. B 237, 37–72.

van Genderen, C., Okamura, R.M., Farinas, I., Quo, R.G., Parslow, T.G.,

Bruhn, L., Grosschedl, R., 1994. Development of several organs that

require inductive epithelial–mesenchymal interactions is impaired in

LEF-1-deficient mice. Genes Dev. 15, 2691–2703.

Wessells, N.K., 1965. Morphology and proliferation during early feather

development. Dev. Biol. 12, 131–153.

Widelitz, R.B., Jiang, T.X., Noveen, A., Chen, C.W., Chuong, C.M., 1996.

FGF induces new feather buds from developing avian skin. J. Invest.

Dermatol. 107, 797–803.

Widelitz, R.B., Jiang, T.X., Lu, J., Chuong, C.M., 2000. Beta-catenin

in epithelial morphogenesis: conversion of part of avian foot

scales into feather buds with a mutated beta-catenin. Dev. Biol. 219,

98–114.

Williams, B.A., Ordahl, C.P., 1994. Pax-3 expression in segmental

mesoderm marks early stages in myogenic cell specification. Develop-

ment 120, 785–796.