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Connexins in feather morphogenesis

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Connexins in feather morphogenesis

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CONNEXINS IN FEATHER MORPHOGENESIS

by

Chun-Chih Tseng

—————————————————————————————

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

May 2011

Copyright 2011 Chun-Chih Tseng

ii

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vi

Chapter 1: Introduction 1

Chapter 2: Objectives and Hypothesis 8

Chapter 3: Materials and Methods 9

Chapter 4: Results

1. Gap junction intercellular communication in fresh chicken skin 17
2. The inhibition of GJIC in early feather development 26
3. Molecular expressions and functions of gap junction isoforms 34

Chapter 5: Discussion

1. LY dye transfer, connexin expression, and compartmental development 51
2. Drug treatment and the formation of new bud-like structures 54
3. Inhibition and promotion of bud growth by connexin 30 56
4. Roles of connexin 43 57
5. Roles of connexin 40 58
6. Future direction 59

Chapter 6: Conclusion 62

References 63

iii

LIST OF TABLES
Table 1: Primers used for RNA probe making 10
Table 2: Oligonucleotides for Cx30 functional perturbations 11
Table 3: Travel distances of LY along the A-P axis on the H&H stage 28 20
chicken skin
Table 4: Travel distances of LY along the A-P axis on the H&H stage 31 23
chicken skin

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LIST OF FIGURES
Figure 1: The life cycle of a feather in the midline of the spinal tract 1 6
Figure 2: Periodic patterning of feather field 6
Figure 3: Dorsal and ventral skin of H&H stage 36 (E10) chick embryo 7
Figure 4: Scrape-loaded lucifer yellow dye transfer assay on H&H stage 28 (E6) 19
embryos
Figure 5: Scrape-loaded lucifer yellow dye transfer assay on H&H stage 31 (E7) 21
embryos
Figure 6: Scrape-loaded lucifer yellow dye transfer assay on H&H stage 34 (E8) 24
embryos
Figure 7: Scrape-loaded Lucifer yellow dye transfer assay on St 35 and St 36 26
chicken skins
Figure 8: 18 alpha-GA, glycyrrhizic acid (18 alpha-GA analog) and their vehicle 28
(DMSO) treated skin explants
Figure 9: Characterization of 18 alpha-GA and DMSO treated skin explants by 30
WM-ISH (shh and beta-catenin) and scrape-loaded LY dye transfer assay
Figure 10: BrdU incorporation in 18 alpha-GA and DMSO treated skin explants 31
Figure 11: Possible new bud-like structures forming sites 33
Figure 12: Expression of Cx30 transcripts shown by whole-mount in situ 1 36
hybridization
Figure 13: Functional perturbations of connexin 30 by siRNAs 38
Figure 14: Functional perturbations of connexin 30 by RCAS virus overexpression 39
Figure 15: Cx30 mRNA expression in adult flight feathers (regeneration day 8 after 40
plucking)
Figure 16: Expression of Cx43 transcripts shown by whole-mount in situ 1 42
hybridization
Figure 17: Immunostaining of Cx43 and proliferating cell nuclear antigen (PCNA) 44
in adult flight feathers (regeneration day 8 after plucking)

v

Figure 18: Expression of Cx40 transcripts shown by whole-mount in situ 46
hybridization
Figure 19: Expression of other connexins transcripts shown by whole-mount in situ 1 48
hybridization
Figure 20: Schematic summary of molecular expressions of other connexins 49
Figure 21: Schematic summary of molecular expressions of connexins in the spinal 50
tract

vi

ABSTRACT
Gap junctions are formed by the direct docking of two hexamers of connexins
(Cxs) which allow the exchange of cellular contents, such as ions, second messengers and
small molecules, between neighboring cells. Some studies have revealed that connexins
could have gap junction intercellular communication (GJIC) independent roles by
interacting with diverse proteins (reviewed in Herve et al., 2004; Herve et al., 2007).
Previous work done by Serras (et al., 1993) suggested that GJIC may be involved in the
compartmental development during embryonic feather morphogenesis, as demonstrated
by dye couplings of microinjected lucifer yellow in different domains of skin explants. A
recent study (Alibardi, 2010) also demonstrated the existence of gap junctions in adult
branching feathers. Here, I investigated the expression patterns of 12 connexin isoforms
according to the sequences published on chicken nucleotide database on the NCBI
website. I found that only 7 connexin isoforms (Cx30, Cx31.1, Cx31.9, Cx32, Cx40,
Cx43, and Cx46) were expressed during early feather morphogenesis. Among them,
Cx30 , Cx40 and Cx43 were dynamically expressed while only Cx43 was expressed in
the mesenchyme in the spinal feather tract. In addition, Cx31.1and Cx32 were expressed
in both the bud and interbud epithelium while Cx31.9 and Cx46 were only expressed in
the bud epithelium. Moreover, I found the patterns of lucifer yellow, a small molecule
which can pass through gap junctions, reflected the compartmental development during
feather morphogenesis, as demonstrated by the scrape-loaded lucifer yellow dye transfer
assay. In functional studies, inhibition of GJIC by the small molecule, 18 α-
Glycyrrhetinic acid (18 alpha-GA), led to accelerated feather growth and outgrowths of

vii

new bud-like structures in skin explants. Feather patterning was not affected by this drug,
as illustrated by the treatment of 18 alpha-GA at the early stage (St28) before any visible
cues of feather development were detectable. Furthermore, I demonstrated that knock
down of Cx30 expression by siRNA caused aberrant feather development, including
absence of feather buds, smaller bud sizes, and abnormal filament formation.
Overexpression of Cx30 resulted in both bigger and smaller feather sizes. Collectively
speaking, these results suggest that connexins are dynamically involved in the regulation
of compartmental development, pattern formation, and bud growth and differentiation.

1

CHAPTER 1: INTRODUCTION
Gap junctions are composed of two hexamers, also called hemichannels, of
connexins (named connexons) and pannexins (named pannexons) in vertebrates. Each
connexin and pannexin contains one intracellular N-terminus, four transmembrane
domains, one intracellular loop, two extracellular loops, and one intracellular C-terminus.
Gated gap junctions allow adjacent cells to directly share their cellular contents such as
secondary messengers, small ions, siRNA , amino acids, and other small molecules with
molecular weighs less than 1 kDa (Schwarzmann et al., 1981). The nomenclature of
members of connexin family are denoted by connexin (abbreviated as Cx for mouse and
chicken, CX for human) and appended with the predicted molecular mass of the
polypeptide by kDa. Pannexins are simply abbreviated as PANX followed by a number
representing the sequence they were found. Connexins (Cxs) and Pannexins (PANXs)
form distinct families of gap junction subunits. There are 3 PANXs and 21 CXs within
the human genome and at least 3 PANXs and 12 Cxs identified or predicted within the
chicken genome. The conserved domains, including the N-terminus, extracellular loops
and transmembrane domains in connexin families, have shown great sequence homology
across vertebrates (Cruciani & Mikalsen, 2007). PANXs share some sequence homology
with the invertebrate innexins, which form invertebrate gap junctions, but show no
homology with connexins (Yen and Saier, 2007). It has been known that PANX 1
hemichannels can modulate the transmission of calcium waves in astrocytes (Scemes et
al., 2007). However, little is known so far if PANXs are able to form functional channels.
On the other hand, Cxs, except for Cx31.1 (Bruzzone et al., 1996), have been shown to

2

form GJIC in diverse systems. In the present study, I am only focusing on the expression
and function of the connexin family during feather morphogenesis.
The feather is one of the major skin appendages of avian species and feathered
dinosaurs (Hu et al., 2009; Xu et al., 2010). It has been confirmed that gap junctions exist
in adult branching feathers (Alibardi, 2010) and GJIC does exist in early feather
development (Serras et al., 1993) in chickens. The latter work also suggests a spatial and
temporal specific coupling of functional gap junctions. A similar finding was obtained in
embryonic mouse epidermis and hair follicles (Choudhry et al., 1997). In addition,
multiple connexins have been identified during keratinocyte differentiation in humans (Di
et al., 2001) and in murine species (Brissette et al., 1994; Butterweck et al., 1994; Goliger
and Paul, 1994; Risek et al., 1992). Many of them have shown tissue specific and
overlapping expression patterns. Therefore, it would be important to know which
connexin isoforms are mediating specific functions of gap junctions in tissue-specific and
spatiotemporal manners.
Adding to the complexity, it was found that heterotypic and heteromeric types of
connexins are able to form functional channels. The evidence has shown a selective
permeability to cytoplasmic molecules by gap junctions formed by different types of
connexins (reviewed in Harris, 2007). Furthermore, mouse “knock in” experiments
showed individual connexins may play distinct and shared roles in specific cellular
processes (Plum et al., 2000). Many recent discoveries also demonstrated that gap
junctions not only mediate intercellular exchange of hydrophilic molecules but also act as
regulatable signaling scaffold called "nexus." Connexins can be regulated by
posttranslational modifications such as phosphorylation and ubiquitination (reviewed in

3

Kjenseth et al., 2010). Studies (reviewed in Giepmans, 2004; Herve et al., 2004; Herve et
al., 2007) have shown that multiple protein kinases can phosphorylate the C-terminus of
human CX43 and either promote GJIC (PKA/ S364, 365, 368, 369, 373) or inhibit GJIC
(Src/Y247, 265, 274, 277, 280, 283; PKC/ S368, 372; PKG/S257, rat; MAPK/ S255, 275,
279,282; cdc2/ S255; CK1/ S325, 328, 330). Diverse interacting proteins can also
modulate connexin behaviors. The direct associations of Src (non-receptor tyrosine
kinase) and ZO-1 (PDZ domain containing protein; interacts with C-terminus of CX43;
bridges actin filaments and tight junctions) with CX43 have been well established.
Immunoprecipitation and GST protein pull-down experiments also suggested a direct
and/or indirect interactions between connexins and PKC (serine kinase), CK1 (serine
kinase), RPTPµ (receptor-like protein tyrosine phosphatase µ, dephosphrylate Y265 of
human CX43), β-Catenin (cadherin-associated protein; key transcription factor in
canonical wnt signaling pathway), Caveolin (the component of caveolae membranes),
α/β-Tubulin (microtubule subunits), occludin (main tight junction component), and
claudin (important tight junction component). Gradients of biological chemicals are able
to affect permeabilities of gap junction channels as well (reviewed in Harris, 2001; Harris,
2007). Taken together, it is not too surprising that connexins have exhibited highly
dynamic trafficking and short half-life (1-6 h).
Thus far, at least ten connexins, CX26, CX30, CX30.3, CX31, CX31.1, CX32,
CX37, CX40, CX43, and CX45, have been identified in human skin (Di et al., 2001).
Mutations in connexins have been implicated in many human skin diseases, and five
connexins, including CX26, CX30, CX30.3, CX31, and CX43, are associated with
inherited skin diseases (Richard, 2005; van Steensel, 2004; Zoidl and Dermietzel, 2010).

4

Dominant mutations in CX26 result in thickening of the skin, hearing loss, and increased
risk of skin cancer. Recessive mutations in CX26 cause nonsyndromic deafness.
Mutations in connexin 30 contribute to Clouston syndrome, which is characterized by
severe hair loss, nail hypotrophy, and palmoplantar hyperkeratosis. Mutations in CX30.3
and CX31 cause hyperkeratosis and inflammatory responses leading to migratory
erythema. Mutations in CX43 result in oculodentodigital dysplasia (ODDD)
characterized by small eyes, sparse hair growth, abnormal dentition, brittle nails, and a
webbing of the skin. Sometimes, curly hair and hypotrichosis can be seen in ODDD
patients.
The feather is a specialized appendage of the skin which has demonstrated
impressive regional specificity and complex developmental processes. The life cycle of a
feather is shown in Figure 1. Briefly speaking, as far as we know, the initiation of feather
morphogenesis starts from an unspecified inductive signal from the dermis. Some studies
suggested that exogenous bmp-2 can induce the dense dermis marker (cDermo-1, basic
helix-loop-helix transcription factor) and promote the formation of an ectopic feather
tract at early development (Scaal et al., 2002). This group also demonstrated that
expression of cDermo-1 in the dermis occurs earlier than epidermal beta-catenin.
Different feather tracts (fields) are shown in Figure 3. In the spinal tract, the earliest
inducted (competent) field comes from the midline, and the pteric field is deployed
bilaterally to allow buds to periodically form (Fig. 2). Evidence has shown that cell
proliferation and migration play important roles in this early phase of feather
morphogenesis (Michon, et al, 2008). Moreover, neural cell-adhesion molecule (NCAM)
is expressed at a basal level in the mesenchyme and is essential for the formation of

5

dermal cell aggregates (Chuong & Edelman, 1985; Jiang & Chuong, 1992; Jiang et al.,
1999). On the other hand, the study on rat liver epithelial cells had shown that E-cadherin
can facilitate Cx43 to form gap junctions (Govindarajan et al. 2010) and connexon-
mediated cell adhesion is able to drive microtissue self-assembly in several human cell
lines (Bao et al., 2010). This led us to further explore the roles of Cx43 or other Cxs and
E-cadherin in the process of cell condensation during feather development. The small
aggregates in the mesenchyme will crosstalk with the overlaying epithelium and the
dermal condensation will be stabilized during this process. In addition, the diffusive
activator and inhibitor (reaction-diffusion) signals between the buds will specify the
periodic patterns of the feather primordia. The entire process of field induction and
periodic patterning will conclude before late Hamburger-Hamilton (H&H) stage 31 (St
31). Then, the individual feather bud will begin asymmetric development (H&H stage 31-
34), though the actual cellular determinants may come out earlier. About H&H stage 35,
the individual feather bud starts to elongate and initiate the development of feather
filament from the polarized distal site. Beyond H&H stage 36, invaginations can be
observed around the base of feathers, and the polarized distal basal epithelium will
longitudinally invaginate into the developing feathers to form the edge of barb ridges,
called marginal plates (Fig. 1A, A'). The cells in the sheath, marginal plates, and axial
plates will eventually undergo apoptosis, thus allowing feathers to open up and form
spectacular branching.

6

Fig. 1 The life cycle of a feather in the midline of the spinal tract. Adapted and revised from Lin
et al., 2006.

Fig. 2 Periodic patterning of feather field. Adapted and revised from Lin et al., 2009.

7

Fig. 3 Dorsal and ventral skin of H&H stage 36 (E10) chick embryo. Pteric (feather forming)
regions include spinal tract, femoral tract, scapular tract, pectoral tract, ventral tract, and caudal
tract. The spinal tract can be subdivided into cervical, thoracic, lumbar, and sacral regions.
Apteric, no feather formation, region (medioventral apterium) and semi-apteric, forming sparse
feathers, region (semi-apterium) can also be seen here. A-P axis is from left to right. Scale bar,
300μm.

8

CHAPTER 2: OBJECTIVES AND HYPOTHESIS
Our lab has been using feathers as a model to understand principles of
morphogenesis, of particular interest and one of the major aspects of which is
intercellular communication. In this model, studies on cell communication have been
focused on secreted growth factors and cell-adhesion molecules. In the present study, I
hypothesize that direct and/or indirect cellular communications mediated by gap
junctions are also involved in patterning feather primordia, coordinating feather bud
growth, and mediating compartmental differentiation of feather follicles. To evaluate
roles of connexins and gap junction intercellular communication (GJIC) in feather
morphogenesis, I studied the expression patterns of known chicken connexin isoforms
during early feather development. For functional studies, I explored the effect of the
small molecular inhibitor, 18 alpha-glycyrrhetinic acid, in skin explants. Furthermore, I
used in ovo siRNA electroporation and RCAS virus overexpression to perturb Cx30
normal functions, in an effort to decipher the roles of connexins in a more detailed
fashion.

9

CHAPTER 3: MATERIALS AND METHODS
EMBRYOS
Specific pathogen-free eggs used in the virus experiments were purchased from
Charles River. Local farm eggs were used for non-viral experiments. The eggs were
cultured at 38.5 °C in a humidified chamber until the desired stages were reached.
PRIMER DESIGN AND PROBE MAKING
The mRNA coding sequence was submitted to the PCR design program, Primer3.
The final primer sequences adopted in this study are shown in Table 1. The promoter
sequence for T7 RNA polymerase (5'- TAATACGACTCACTATAGGG- 3') was added
on the 5' end of the antisense primer. Gene products of around 500bp were cloned from
chicken embryonic cDNAs. The cloned products were sequenced for verification and
transcribed with T7 RNA polymerase to obtain the antisense probes labeled with
digoxigenin.

10

Table 1 Primers used for RNA probe making.

siRNA DESIGN
The mRNA coding sequence was submitted to the siRNA design program,
Ambion siRNA Target Finder. Sequences having 4 or more Gs in a row were avoided.
The candidate sequences of 30-50% GC were analyzed by the Basic Local Alignment
Search Tool (BLAST) to ensure that they only annealed with their intended cognate
sequence. The final sequence was used only if there were no other similarities in the
chicken genome. For a control, the randomized sequence with the same ATCG
composition was analyzed by the BLAST. The final sequence was used only if there were
no similarity to other sequences in the chicken genome. The siRNAs were synthesized

11

and desalted by Thermo Scientific®. The oligonucleotides for Cx30 siRNA and the
randomized control can be seen in the Table 2.
Table 2 Oligonucleotides for Cx30 functional perturbations.

IN OVO siRNA ELECTROPORATION
The siRNAs were dissolved in RNase- free water at the concentration of 100µM
and stored in 10µl stocks at -80℃. Right before use, a small drop of FastGreen was
added to the stock. The siRNA stocks containing FastGreen were directly used for
injections. The siRNAs were injected into the subepidermal layer of embryos at H&H
stage 25 with 1-2µl. The electrodes were put on the flank of embryos. The siRNAs were
transferred to cells using electroporation at 16V (the actual output is 12V), 50ms three
times. The siRNA sequences used in this study are shown in Table 2.
CONSTRUCTION OF RCAS Cx30 PLASMID
The RCAS cloning was performed according to the manual of the manufacturer
(Invitrogen). In brief, the full-length coding sequence of Cx30 was ampilified by PCR
with the primers shown in Table 2. The cloned fragment was sequenced to ensure the
accuracy of the sequence. The accurate full-length sequence was subcloned into the

12

empty RCAS plasmid by B-P and then L-R reactions with the Gateway® Cloning system.
The resulting plasmids were transformed into One Shot® OmniMAX 2-T1R Chemically
Competent cells (Invitrogen, C8540-03) by heat shock. The transformed cells were
grown overnight on Lysogeny Broth (LB)- agar plates containing kanamycin and
penicillin after B-P and L-R reactions, respectively. The cells containing the desired
plasmids were picked up and amplified by Qiagen Highspeed Maxi kit (Qiagen, 12663).
The purified plasmids were stored at -20ºC until use.
CULTURING CHICKEN EMBRYONIC FIBROBLASTS
Specific pathogen- free (SPF) H&H stage 31 chicken embryos were collected and
decapitated. Internal organs and limbs were removed with sterile forceps. The remaining
tissue was macerated with sterile scissors and trypsinized with 0.05% trypsin and EDTA.
The disassociated cells were filtered through a 70µm nylon filter and plated on 100mm
culture dishes supplemented with DMEM/high glucose containing 10% fetal calf serum
(FCS), 2% chicken serum, and 10µg/mL Gentamycin.
TRANSFECTION OF CHICKEN EMBRYONIC FIBROBLASTS (CEFs)
When CEFs reached 80-90% confluence, RCAS connexin 30 plasmids and RCAS
plasmids were transfected into cells by treatment with 250mM CaCl
2
and 2X HeBS for 4
hours at 37 ºC. Cells were then briefly treated with 15% sterile glycerol in phosphate-
buffered saline (PBS) and washed with PBS. Then, they were incubated at 37ºC
overnight in DMEM with 10% FCS, 2% CS, and 10µg/mL Gentamycin.

13

VIRUS COLLECTION
The day after transfection, the culture media was replaced with 10ml DMEM with
1% FCS and 0.2% CS. On the three subsequent days, the media was collected and filtered
through sterile Acrodisc® syringe filters (0.22μm pore, Nylon, PALL Life Sciences). The
collected media was temporarily stored at –80ºC until the third day of collection. The
collected media for individual virus were pooled together and centrifuged at 4ºC at
20,000 rpm for three hours. Nine-tenths of the supernatant was pipetted out and the
remaining one-tenth of the culture media containing viral particles was gently shaken at
4ºC overnight. The media was then aliquoted in 100µl and stored at -80ºC until use.
VIRUS INJECTION
The aliquoted virus was thawed briefly at 37ºC. Volumes of about 5-10μl were
injected into the amniotic cavity around the SPF embryonic day 3 (E3) chicken embryos.
GJIC INHIBITOR TREATMENT
The small molecule inhibitors 18 alpha-glycyrrhetinic acid (Sigma, G8503) and
its analog, glycyrrhizic acid (Sigma, 50531), were dissolved in DMSO at a concentration
of 50mM. The concentrated stock was diluted in DMEM with 10% FBS and 2% CS to
make 100μM working solutions right before applying to skin explants. The skin explant
culture medium was replaced every other day with the same working solutions.

14

SCRAPE-LOADED LUCIFER YELLOW DYE TRANSFER ASSAY
Lucifer Yellow CH dipotassium salt (Sigma, L0144) and Rhodamin Dextran
(Invitrogen, D1824) were dissolved in distilled water to make a 1% stock. The working
solution was made by mixing 100µl lucifer yellow, 100µl Rhodamine Dextran stock
solution and 300 µl PBS. 20-40ul of working solution was applied to each skin before
scraping and 8 minutes was allowed for dye transfer. Then, the skins were washed with
PBS briefly and fixed with 4% paraformaldehyde for 20minutes at room temperature.
The samples were imaged with either Zeiss 510 confocal microscope or an epifluorescent
microscope.
SAMPLE PROCESSING
Samples for whole-mount in situ hybridization (WM-ISH) were collected in
DEPC-treated PBS with 0.1% Tween 20 (DEPC-PBT) and fixed with 4%
paraformaldehyde (PFA) in DEPC-H
2
O at 4ºC overnight. Then, samples were dehydrated
through a methanol gradient and stored at –20ºC. Samples collected for sectioning were
fixed with 4% PFA and dehydrated through an ethanol gradient. Then, samples were
cleared in xylene twice and embedded in paraffin. 14μm sections of WM-ISH samples,
20μm of Cx40 probe hybridized samples, and 7μm sections of Immunohistochemistry
(IHC) and in situ hybridization on sections were collected.

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IN SITU HYBRIDIZATION (ISH)
Whole-mount and deparaffinized (xylene treated) sections were rehydrated
through methanol and ethanol gradients, respectively. Then, samples were bleached with
6% H2O2 in DEPC-PBT and treated with 20μg/mL Proteinase K in DEPC-PBT. Samples
were post-fixed in freshly prepared 0.25% glutaraldehyde /4% PFA solution and pre-
blocked in the hybridization buffer. Digoxigenin-labeled probes were applied at 65ºC
overnight. Samples were then washed thoroughly with 2X and 0.2X Sodium Chloride-
Sodium Phosphate-EDTA Buffer (SSC buffer) for 4 hours. Samples were pre-blocked
with 20% heat-inactivated goat serum in DEPC-PBT for two hours and treated with pre-
absorbed anti- digoxigenin alkaline phosphatase conjugated antibody at 4ºC overnight.
The next day, samples were washed in DEPC-PBT containing 1% levamisole for 3 hours
followed by NTMT (100mM NaCl, 100mM Tris-HCl, 50mM MgCl, and 0.1% Tween 20)
containing 1% levamisole washes for 2 hours. Colors were developed with NBT/BCIP ,
and reactions were stopped by PBS.
IMMUNOHISTOCHEMISTRY (IHC)
Sections were briefly heated at 65 ℃ for 5 minutes and deparaffinized in xylene
twice. Sections were subsequently rehydrated through an ethanol gradient and 6% H
2
O
2

in methanol was used to block the endogenous peroxidase. Antigen retrieval was done by
treatment of 10mM citric acid buffer (pH 6.0) for 30min at 95℃. Sections were allowed
to cool down for 30minutes at room temperature. Then, the sections were pre-blocked
with Zeller's solution for two hours at room temperature. Primary antibodies against the

16

desired antigens were diluted in Zeller's solution and applied to the sections at 4ºC
overnight. Then, biotin-linked secondary antibodies were applied at 4ºC overnight. The
next day, streptavidin-horseradish peroxidase were labeled for two hours at room
temperature. Color was developed with AEC (Vector, SK-4200) for 5 to 15 minutes.
WHOLE-MOUNT BrdU STAINING
BrdU stock solutions were made by dissolving BrdU powder in Hank's Buffered
Salt Solution (HBSS) at the concentration of 1.5 mg/ml and were stored at -20℃. The
skin explants were labeled with 150µg/ml BrdU for 4 hours and fixed with 100%
methanol for two hours. Then, 10% H
2
O
2
in 1:4 DMSO: Methanol was used to block the
endogenous peroxidase for 2 hours. Samples were washed with PBT for 15 minutes, 4
times. Then, samples were treated with 20μg/mL Proteinase K in PBT for 7 minutes at
RT. Samples were briefly washed and fixed again with 4% paraformaldehyde/ 0.1%
glutaraldehyde in PBS for 20 minutes at RT. Samples were washed and treated with 2N
HCl in PBT for 1 hour. Then, samples were washed and treated with 0.1 M sodium
borate buffer, pH=8.5 for 10 minutes, 4 times. Samples were briefly washed and then
labeled with the mouse anti-BrdU antibody (Millipore, MAB3424) at 1:1000 dilution,
biotin-linked anti-mouse antibody, and streptavidin-horseradish peroxidase for two hours,
respectively. Color was developed with DAB (Vector, SK-4100) for a few minutes.

17

CHAPTER 4: RESULTS
GAP JUNCTION INTERCELLULAR COMMUNICATION IN FRESH
CHICKEN SKIN
The previous work done by Serras (et al., 1993) had shown that during early
embryonic feather development, GJIC was very dynamically coordinated and might be
involved in compartmental development. To gain more knowledge about how GJIC is
working in the feather system, I examined lucifer yellow (LY; -2 charge, 452 Da) dye
transfer relative to rhodamine dextran (10 kDa) control in fresh embryonic skin from
H&H stage 28 to 36. At H&H stage 28, I found GJIC is intensively expressed between
mesenchymal cells, and the lucifer yellow seemed to transfer through the gap junctions
faster along the midline compares to the travel distance on either the right or left side (Fig.
4, 4F). The raw data of travel distances of LY are shown in Table 3. In addition, the
distances of LY dye transfer in the flank regions were similar. However, this data was
collected from only one skin explant, and more samples will have to be analyzed to
determine the significance. At H&H stage 31 (Fig. 5), the superimposed image (Fig. 5D)
showed decreased LY intensity at certain distances if I made a scrape across a bud. This
result was further confirmed by analyzing the LY intensity cross the yellow line (Fig. 5G,
H). The two lower points around the middle of the wave indicated two separate buds
impeded the LY dye transfer. However, the signals in both of the lower points were still
higher than the background. The distance of dye transfer was gradually reduced
bilaterally at this stage (Fig. 5E, F). The raw data of travel distances of LY are shown in

18

Table 4. At H&H stage 34 (Fig. 6), the dye transfer was less effective across a bud and
very effectively cross the interbud (Fig. 6B, D). In fact, there was nearly none, if there
was still some, LY dye transfer in the bud, as I could see the overlapping patterns of
rhodamine dextran and Lucifer yellow (arrow, Fig. 6D). Image J analysis also showed
that the LY signal in the bud is close to the background level (Fig. 6E, F). The LY
intensity analysis cross two buds showed similar results as well (Fig. 6G, H).
Interestingly, there were two LY intensity decreased zones present between the buds
(arrowheads, Fig. 6G; site 1 and 4, Fig. 6G; site 1’ and 4’, Fig. 6H), which might suggest
bigger compartmental differences in the tissue level. At H&H stage 35 (Fig. 7), the dye
transfer was very strong in bud mesenchyme and very limited in the bud epithelium. The
overlapping signals of rhodamine and LY in the interbud (arrowheads, Fig. 7A)
suggested that there was no LY dye transfer in the interbud at this stage. This result
contradicts earlier studies (Serras et al., 1993), which may due to the reduced detectable
signals in each section plane of confocal microscopy. At H&H stage 36 (Fig. 7B), I could
see significant dye coupling in the barb plate and, interestingly, I can also see some cells
(arrows) in the marginal plate holding higher LY signals.

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Fig. 4 Scrape-loaded lucifer yellow dye transfer assay on H&H stage 28 (E6) embryos. The dye
transfer seems faster in the midline and is similar in the flank regions. Dyes were allowed to
transfer for eight minutes. A-P axis is from left to right. (A) Bright view. Dotted line indicates the
midline of spinal tract. (B-E) Epifluorescent images taken after scrape-loading of dyes. (B)
Lucifer yellow dye transfer. (C) Rhodamine dextrans. The white circle shows the edge of signals.
(D) Superimposition of the images B and C. (E) The extension of lucifer yellow was measured in
every 50µm from the midline, as shown in Table 1. The position of midline is marked by the red
dot on the ruler. The minimum unit of the ruler is 10 µm. (F) The distance of lucifer yellow dye
transfer along the A-P axis was quantified by comparing to the edge of rhodamine dextran. This
figure was drawn according to the data in table 3. Scale bar, 300µm for A.

20

Table 3 Travel distances of LY along the A-P axis on the H&H stage 28 chicken skin. Threshold
of gray value is eight for calculations of significant LY signals in the image analysis software,
ImageJ.

Distances from the
midline (µm)
Travel distances of LY
on the Posterior Side
(µm)
Travel distances of LY
on the Anterior Side
(µm)
-300 213 290
-250 231 288
-200 233 271
-150 236 264
-100 249 289
-50 256 264
0 289 330
50 281 319
100 259 288
150 242 291
200 243 273
250 235 288
300 262 286

21

Fig. 5 Scrape-loaded lucifer yellow dye transfer assay on H&H stage 31 (E7) embryos. Dye
transfer is largely impeded by buds and gradually reduced bilaterally. Dyes were allowed to
transfer for eight minutes. A-P axis is from left to right. (A) Bright view. (B-E and G)
Epifluorescent images taken after scrape-loading of dyes. (B) Lucifer yellow dye transfer. (C)
Rhodamine dextrans. The white circle shows the edge of signals. (D) Superimposition of the
images B, the white line in image C, and feather primordia, as indicated by the dots, in image A.
(E) The fluorescent image of the left side of skin was cropped to calculate the distance of lucifer
yellow dye transfer. The extension of lucifer yellow was measured in every 50µm starting from
the reference point (the red dot) shown on the ruler. The minimum unit of the ruler is 10 µm. (F)
The distance of lucifer yellow dye transfer along the A-P axis was quantified by comparing to the
edge of rhodamine dextran. This figure was drawn according to the data in table 4. (G) The
intensity of lucifer yellow cross the yellow line was calculated in image analysis software, Image
J, and shown in image H. The red dot is the starting point. The length of the yellow line
represents the length of the whole X-axis in Fig. 5H. Scale bars, 300µm.

22

Figure 5, continued

23

Table 4 Travel distances of LY along the A-P axis on the H&H stage 31 chicken skin. Threshold
of gray value is eight for calculations of significant LY signals in the image analysis software,
ImageJ.

24

Fig. 6 Scrape-loaded lucifer yellow dye transfer assay on H&H stage 34 (E8) embryos. Dye
transfer is nearly inhibited in the buds and is extensive in the interbuds. Dyes were allowed to
transfer for eight minutes. A-P axis is from left to right. Asterisks show the same feather bud. (A)
Bright view. (B-E and G) Epifluorescent images taken after scrape-loading of dyes. (B) Lucifer
yellow dye transfer. (C) Rhodamine dextrans. The white circle shows the edge of signals. (D)
Superimposition of the images B, the white line in image C, and feather primordia, as indicated
by the dots, in image A. The arrow indicates one of the feather buds which has an artificial wound
in the bud domain. (E) The intensity of lucifer yellow cross the yellow line was measured starting
from the reference point, the blue dot, and the fluorescent intensity was translated into image F by
image analysis software, Image J. The length of the yellow line represents the length of the whole
X-axis in Fig. 6F. (G) The intensity of lucifer yellow cross the yellow line was measured starting
from the reference point, the blue dots in image G and H, and the fluorescent intensity was
translated into image H by image analysis software, Image J. The red dots show the comparable
sites in image G (1- 6) and image H (1'- 6'). Dots number 1 (1') and number 4 (4') demonstrated
zones, as indicated by the arrowheads, with less stronger signals. The distances (pixels) between
dots were shown in green numbers. The length of the yellow line represents the length of the
whole X-axis in Fig. 6H. Scale bar, 300µm.

25

Figure 6, continued

26

Fig. 7 Scrape-loaded Lucifer yellow dye transfer assay on St 35 and St 36 chicken skins. Dye
transfer is much more effective in the bud mesenchyme than the epithelium, and it is largely
inhibited in the interbuds on St 35 skin explants. Dye transfer is extensive in the bud epithelium
on St 36 skin explants. Dyes were allowed to transfer for eight minutes. These images are
visualized by Zeiss LSM 510 confocal microscope. A-P axis is from left to right. (A) H&H stage
35 (E9) skin. The extension of lucifer yellow (Green) in the interbud is indicated by arrowheads.
Rhodamine dextran (Red) shows the wound made by the scrape. DAPI is blue. The green and red
signals are largely overlapped. (B) H&H stage 36 (E10) skin. Lucifer yellow signal is green,
rhodamine dextran signal is red, and the overlapping signal is yellow. Dotted signals in the distal
feather bud are presented close to marginal plates (basal layers). The arrows show the comparable
sites in the section plane and the Z-stack image cross the green line. Scale bars, 100µm.

THE INHIBITION OF GJIC IN EARLY FEATHER DEVELOPMENT
To understand the importance of GJIC in early feather development, I used 18
alpha-glycyrrhetinic acid (18 alpha-GA), a gap junction blocker, to inhibit GJIC in the
chicken skin explants. Treated skins were compared to 18 alpha-GA analog, glycyrrhizic
acid, and their vehicle, DMSO, treated samples (Fig. 8). The glycyrrhizic acid does not
block gap junctions but reproduces its non-specific effects, GJIC could be extensively
seen in glycyrrhizic acid treated skin explants (data not shown). The skin staged H&H 34
was found to have a higher potential to produce phenotypes. The H&H 31 skins treated

27

with serial concentrations of 18 alpha-GA alone showed slightly inhibited growth
potential (data not shown). The 100µM 18 alpha-GA treated H&H 34 skins exhibited
tapered feathers and extra cell masses started to form around the base of feathers upon
day 3 of drug treatment. The glycyrrhizic acid controls did not show any of these
phenotypes and their appearances were similar to the DMSO controls. I also tested the 18
alpha-GA at the concentration of 25 µM and 75 µM, neither of which inhibited GJIC
significantly in this system nor produced the phenotypes mentioned above (data not
shown). The extra cell masses formed in the 18 alpha-GA treated samples were able to
grow bigger, however, only a very limited number of them developed into advanced
feather-like structures (long buds) in this system. According to my observation, only
some cell condensations developed at site 1 (Fig. 11) are capable of stepping forward in
development. For cell condensations developed at the other sites, most of them will
disappear if I cultured for eight days.

28

Fig. 8 18 alpha-GA, glycyrrhizic acid (18 alpha-GA analog) and their vehicle (DMSO) treated
skin explants. 18 alpha-GA treated skin explants show accelerated feather growth and the
emergence of new bud-like structures but not in the analog control and DMSO control. Dorsal
skins were collected from H&H stage 34 (E8) embryos and cultured for additional 5 days. The
same culture medium is replaced on every other day. Pictures were taken under bright view in
thoraciclumbar regions. A-P axis is from left to right. Scale bars, 300µm.

29

The effectiveness of 18 alpha-GA was tested by scraped-loaded LY dye transfer
(Fig. 9) and the results showed that GJIC were significantly inhibited in 18 alpha-GA
treated skin explants through the cultural days but not in DMSO controls. The 18 alpha-
GA treated original feathers displayed similar molecular characterization with DMSO
treated samples (Fig. 9). By comparing the sonic hedge hog (shh) expression patterns
between the 18 alpha-GA-treated samples and DMSO treated samples at day 2, I found
that feathers in the treated skin developed faster than the controls. Shh expression pattern
of treated skins was located in the proximal epithelium, which was similar to the shh
expression pattern at day 3. While, the shh expression pattern in the controls at day 2 was
located in the distal epithelium. Beta-catenin characterization samples gave us a similar
conclusion. The newly formed bud-like structures demonstrated very similar or the same
shh and beta-catenin expression patterns as normal feather development at earlier stages.
Interestingly, after three days of culture, 18 alpha-GA treated samples demonstrated an
intensive beta-catenin positive region surrounding the base of feathers with the newly
formed structures located in the middle of the anterior side of this region. Beta-catenin
was absent at the very posterior end of the bases (data not shown). The "ring" would
gradually disappear.

30

Fig. 9 Characterization of 18 alpha-GA and DMSO treated skin explants by WM-ISH (shh and
beta-catenin) and scrape-loaded LY dye transfer assay. The original buds show similar shh and
beta-catenin expression patterns as DMSO control but the developmental time between each
stages is shortened. GJIC is effectively inhibited with the treatment of 18 alpha-GA, as shown by
the LY dye transfer assay. The confocal images in LY dye transfer assay are visualized by Zeiss
LSM 510 confocal microscope. The representative section planes from Z-stacks are shown here.
LY signal is green and Rhodamine dextran is red. A-P axis is from left to right. Scale bars,
300µm for WM-ISH samples and 100µm for confocal images.

Then, I wondered where the cells that form new condensations come from. I
treated H&H stage 28 skin explants, which showed not any visible cues indicating that
cell condensations had formed at this stage, with 18 alpha-GA and labeled them with
BrdU for 4 hours at day 4 in culture (Fig. 10). I discovered that those cell masses are
highly active in proliferation. Interestingly, I noticed that the BrdU labeling could be

31

patterned like a ring (arrows) or stay on the whole bud (arrowhead). I also tried to use DiI
labeling to see if those condensations could come from the "old" buds or go back to them.
However, I have not had conclusive results yet. This experiment on early embryonic
skins also showed that inhibition of GJIC, as demonstrated by LY dye transfer, did not
stop the formation of "original" cell condensations nor the deployment of the feather field.

Fig. 10 BrdU incorporation in 18 alpha-GA and DMSO treated skin explants. The new bud-like
structures show increased proliferation activities. The skins were collected from H&H stage 28
(E6) embryos and cultured for additional 4 days. Culture medium was not changed. BrdU
solution was applied into the medium and the top of skin explants at the concentration of
150µg/ml for 4 hours before collections. The arrows and arrowhead indicate the places of new
bud-like structures. A-P axis is from left to right. Scale bars, 300µm.

New bud-like structures can form at multiple sites, as summarized in Fig. 11. The
numbers in the schematic picture indicate the sequence that new bud-like structures can
come out in a timely manner. According to my experience, the relative frequency that I
can see new bud-like structures come out at each sites is site 2> site 3=site 3’> site 1>
site 4= site 4’. In addition, if I see a bud at site 1, it always comes earlier than other sites,

32

about 24 hours in advance (day 2 in culture). Buds at site 4 and 4’ always come after
others, and do not show up independently. Notely, newly formed buds at site 3 and 3’ can
come out at the same time or come out independently, and buds at site 4 and 4’ always
come out together. In a rare case, a seventh new bud-like structure can appear (arrowhead,
Fig. 11B).

33

Fig. 11 Possible new bud-like structures forming sites. The new bud-like structures can come out
around the base of original feather buds or the interbuds. Dorsal skins were collected from H&H
stage 34 (E8) embryos and cultured for additional 5 days. A-P axis is from left to right. The
schematic picture summarizes the possible sites for the cell condensations to form. The numbers
represent the timely sequence that they may come out. The new bud-like structures usually
emergence symmetrically at sites number 3 and 3', and number 4 and 4'. (A) This image shows
that new bud-like structures can form at site number 2 or sites number 3 and 3' or sites number 2,
3, and 3'. (B) New bud-like structures are able to form at least five (as indicated by arrows and
corresponding numbers) new bud-like structures and an additional one (as indicated by the
arrowhead) around the base of feather buds. The asterisk shows the same feather bud. The image
on the right corner represents the same skin after WM-ISH with the shh probe. (C) The arrows
and the number indicate the new bud-like structures can also form at site number 1. The skin,
stained with beta-catenin by WM-ISH, on the right corner is different from image C but the new
bud-like structures come from a similar site. Scale bar, 300µm.

34

MOLECULAR EXPRESSIONS AND FUNCTIONS OF GAP JUNCTION
ISOFORMS
Thus far, we know that GJIC may play some roles in chicken skin morphogenesis.
To understand the mechanisms, I further explored the roles that the gap junction may
play for each connexin isoforms. At least 12 connexin isoforms have been identified or
predicted so far. To determine the expression pattern of each connexin isoform, it would
be important to avoid cross reactivity in our experiments. Therefore, I designed primer
sets for each connexin and made sure that the similarities between each of the probe
sequences for whole mount in situ hybridization was less than 55% (Table 1).
CONNEXIN 30
Connexin 30 was expressed dynamically through the stages I investigated. It was
strongly expressed and formed a midline (arrowhead, Fig. 12A, J) at H&H stage 28 and
then this midline breaks into separate buds. At H&H stage 31, it can be seen that the
expression in younger bud primordia (arrowhead, Fig. 12B, K) was bigger in size and
gradually shrank into small dots (arrow, Fig. 12B, L). Shortly after H&H stage 31, the
dots seemed to separate into two parts (Fig. 12E, F), and then Cx30 expression became
bigger again with stronger expression in the more anterior side of the bud epithelium. I
could also observe a "transverse line" in the expression regions (Fig. 12G, H). Notably, a
stronger expression region extended from the region above the "line" on the right side of
the anterior bud towards the left side (Fig. 12H, I). Surprisingly, Cx30 expression was

35

nearly if not completely absent at H&H stage 35 (Fig. 12D, N). The schematic pictures
summarizes the expression patterns of Cx30 on sections after WM-ISH (Fig. 12O-S).

36

Fig. 12 Expression of Cx30 transcripts shown by whole-mount in situ hybridization. Cx30 is
expressed dynamically between St 28 and St 35. A-P axis is from left to right. (A-I) Embryos
were stained with Cx30 by WM-ISH and the colors were developed by NBT/BCIP. (A) H&H
stage 28 (E6) embryos. The arrowhead indicates the sectioning site shown on Fig. 12J. (B) H&H
stage 31 (E7) embryos. The arrowhead and arrow indicate the sectioning sites shown on Fig. 12K
and 12L, respectively. (C) H&H stage 34 (E8) embryos. (D) H&H stage 35 (E9) embryos. (E-I)
Images cropped from left cervical tract of E8 embryos, which represent the development of
feather buds in the midline between H&H stage 31 and 34. (J-S) Longitudinal sections (14µm)
after WM-ISH and schematic diagrams show the dynamic expressions of Cx30 from H&H stage
28 to stage 35. Scale bars, 300µm for A-I, 50µm for J-N.

37

Perturbation of Cx30 mRNA stability at H&H stage 26 (E5) caused irregular
expression of Cx30 at H&H stage 31 (Fig. 13B) compared to the siRNA random control
(Fig. 13A). I could observe a region lacking the expression of Cx30 (dotted circle, Fig.
13B) and a line pattern (arrowhead, Fig. 13B). By H&H stage 35 (Fig. 13C), the shh
staining on siRNA perturbed embryo showed an absence of shh staining (dotted circle), a
smaller bud with aberrant shh expression (arrowhead), and abnormal developing feather
filaments (arrows). The random control of Cx30 siRNA failed to show the phenotypes
mentioned above (n=23, data not shown). The overexpression of Cx30 by RCAS virus
(Fig. 14) resulted in bigger (arrow, Fig. 14B) as well as smaller (arrowheads, Fig. 14B;
arrows, Fig. 14C) feather buds compared to the comparable sites of RCAS virus control
(Fig. 14A). Interestingly, an area absent of feather buds was noticed after RCAS
overexpression of Cx30 (dotted circle, Fig. 14C). In addition, Cx30 overexpression could
transform part of scales into feathers (arrowheads, Fig. 14D; arrows, Fig. 14E).

38

Fig. 13 Functional perturbations of connexin 30 by siRNAs. Knock-down of Cx30 causes
abnormal feather development or no bud phenotypes. Cx30 siRNA and random control were
injected in ovo into subepidermal layers of thoraciclumbar regions of H&H stage 26 (E5)
embryos and electroporated into epidermal and subepidermal cells. The colors were developed by
NBT/BCIP. (A) Cx30 siRNA random control (100µM) electroporated embryo, collected at H&H
stage 31 (E7). The embryo was stained with Cx30 by WM-ISH. (B) Cx30 siRNA (100µM)
electroporated embryo, collected at H&H stage 31 (E7). The embryo was stained with Cx30 by
WM-ISH. The arrowhead indicates a fusion or an incomplete segregation of feather primordia.
The dotted line circles an area that is absent with feather primordia. (C) Cx30 siRNA (100µM)
electroporated embryo, collected at H&H stage 35 (E9). The embryo was stained with shh by
WM-ISH. The arrowhead indicates a feather bud in smaller size and aberrant shh expression
pattern. The arrows point out abnormal shh expression in differentiating feather filaments. The
dotted line circles an area that is absent with feather primordia. Scale bars, 300µm.

39

Fig. 14 Functional perturbations of connexin 30 by RCAS virus overexpression. Cx30
overexpression causes both bigger and smaller bud sizes and transforms part of scales into
feathers. Condensed viruses were in ovo injected into amniotic cavities in the H&H stage 18 (E3)
embryos. (A-D) Embryos were stained with Cx30 by WM-ISH and the colors were developed by
NBT/BCIP. (A-B) Embryos were collected at H&H stage 35+ (E9+) after the injections of
viruses containing RCAS plasmid control (A) or RCAS plasmid with the insertion of Cx30
coding sequence (B). The feather bud is enlarged (arrow) or decreased (arrowheads) in size after
Cx30 overexpression (B) comparing to comparable sites in the control (A). (C) Cx30
overexpressed embryos show shorter feather buds (arrows) and the absence of feather buds (the
area in dotted circle) at H&H stage 38+ (E12+). (D-E) Cx30 overexpression can transform a
portion of scale into feathers, as shown in H&H stage 35+ (E9+) embryos (arrowheads; D) and
H&H stage 38+ (E12+) embryos (arrows; E). Scale bars, 300µm.

Adult feather filaments have well-defined compartments. In regenerating day 8
adult flight feather (Fig. 15), Cx30 was expressed in the axial plates in younger stages
(Fig. 15A, Aa), and then the expression disappeared in axial plates (Fig. 15B, Ba). Cx30
was expressed in the basal and suprabasal layers of feather walls at both younger and
older stages.

40

Fig. 15 Cx30 mRNA expression in adult flight feathers (regeneration day 8 after plucking). The
expression of Cx30 in basal and suprabasal layers of feather walls indicates Cx30 may be
involved in the cell proliferation. The expression of Cx30 in the axial plates, which is negative
with cell proliferation at this stage, suggests Cx30 could be involved in the other cellular
processes. The colors were developed by NBT/BCIP. (A-B) The transverse sections (7µm) were
obtained from the same feather follicle. Cx30 is expressed in the axial plates, and basal and
suprabasal layers of feather walls in less mature (less distal) section planes (A and Aa). Cx30 is
only expressed in basal and suprabasal layers of feather walls in more mature (more distal)
section planes (B and Ba). Scale bars, 50µm.

CONNEXIN 43
Cx43 was expressed dynamically through the stages I investigated. At H&H
stages 28, it was expressed in the pteric regions (Fig. 16A, F, G, Fa-e) and negative in
apetric regions (arrows, Fig. 16Fc, Fe). At this stage, it is expressed in both skin
mesenchyme and epithelium of the spinal tract (Fig. 16F, Fa-b, H) and ventral tract (Fig.
16Fe). It was only expressed in the epithelium of the pectoral tract at this stage (Fig.
16Fd). At H&H stage 31, Cx43 was expressed in both bud and interbud epithelium, and
was intensified in the bud domain (Fig. 16B, I). Cx43 was undetectable in the

41

mesenchyme at this stage. Swiftly after the beginning of H&H stage 31, the Cx43
expression was largely reduced if not completely absent in the bud domain and appeared
in both the epithelium and mesenchyme of the interbud domain (arrowhead, Fig. 16C, J).
At H&H stage 34, Cx43 was solely expressed in the bud domain, and the expression
extended from the distal epithelium towards the proximal mesenchyme (Fig. 16D, K, L).
At the later long bud stage, Cx43 was merely expressed in the outer layers of the bud
epithelium (Fig. 16M). In the caudal tract, the expression of Cx43 was similar with the
spinal tract, however, at H&H stage 34, a ring can be observed surrounding the bases of
tail buds, and then gradually disappears bilaterally (Fig. 16E). The schematic pictures
summarizes the expression patterns of Cx43 on sections after WM-ISH (Fig. 16N-S).

42

Fig. 16 Expression of Cx43 transcripts shown by whole-mount in situ hybridization. Cx43 is
expressed dynamically from St 28 to St 35, and it is the only Cx isoform that can be expressed in
the mesenchyme. A-P axis is from left to right. (A-E and G) Embryos were stained with Cx43 by
WM-ISH and the colors were developed by NBT/BCIP. (A and G) The same H&H stage 28 (E6)
embryo with dorsal (A) and ventral (G) view. The arrowhead in Fig. A indicates the sectioning
site shown on image 16H. (B) H&H stage 31 (E7) embryos. The arrowhead indicates the
sectioning site shown on image 16I. (C) H&H stage 34 (E8) embryos. The arrowhead and arrow
indicate the sectioning sites shown on image 16J and 16K, respectively. (D) H&H stage 35 (E9)
embryos. (E) Left caudal tract of H&H stage 34 (E8) embryos. (F) Transverse section (14µm)
around the thoracic region of the embryo shown in image A and G. Cropped images can be seen
in images Fa-Fe. Fa-b, spinal tract. Fd, pectoral tract. Fe, ventral tract. The arrows in Fig. Fc, Fe
point out the apteric regions. (H-S) Longitudinal sections (14µm) of dorsal feather tracts for H&H
stage 28 to 35 embryos after WM-ISH. The schematic diagrams show the dynamic expressions of
Cx43 in those sections. Scale bars, 300µm for A-E and G, 50µm for F, Fa-Fe and H-M.

43

In the regenerating day 8 adult flight feathers, the immunostaining of Cx43 and
PCNA showed nicely juxtaposed patterns at both the earlier stage (Fig. 17A, Aa for Cx43;
Fig. 17B, Ba for PCNA) and later stage (Fig. 17C, Ca for Cx43; Fig. 17D, Da for PCNA).
This result suggested a possible roles of Cx43 might be in growth inhibition and/or
cellular maintenance.

44

Fig. 17 Immunostaining of Cx43 and proliferating cell nuclear antigen (PCNA) in adult flight
feathers (regeneration day 8 after plucking). The protein expressions of Cx43 and PCNA show
juxtaposed patterns in the differentiating feather filaments. The transverse sections (7µm) were
obtained from the same feather follicle. The majority of Cx43 is expressed on the cell membrane
in the axial plates, the inner sheath, and outer layers of feather walls in less mature (less distal)
section planes (A and Aa). In more mature (more distal) section planes (C and Ca), the expression
of Cx43 can also be found in the barbule plates and marginal plates. The expression of PCNA
seems opposite to Cx43, in the less mature status, PCNA can be expressed in the ramus, marginal
plates, and the basal layers of the feather walls (B and Ba). In the more mature status, the staining
of PCNA in the marginal plates are absent (D and Da). Scale bars, 50µm.

45

CONNEXIN 40
The expression of connexin 40 showed an unexpected dotted pattern. The dots
were initially evenly distributed at H&H stage 31 (Fig. 18A, E) and then became negative
in the domains of feather primordia (asterisk in Fig. 18D; Fig. 18F). From H&H stage 34
to H&H stage 35, the expression of Cx40 started from the proximal posterior end and
gradually covered the entire epithelium of feather buds (Fig. 18B, C, D, G, H). Each dot
could be a single cell or adjacent two cells under the inspection of 100X oil lens in bright
field on a microscope (data not shown). The schematic pictures summarized the
expression patterns of Cx40 on sections after WM-ISH (Fig. 18I-L).

46

Fig. 18 Expression of Cx40 transcripts shown by whole-mount in situ hybridization. Cx40 is
expressed dynamically from St 31 to St 35 in dotted patterns. A-P axis is from left to right. (A-D)
Dorsal views of thoraciclumbar regions of embryos. Embryos were stained with Cx40 by WM-
ISH and the colors were developed by NBT/BCIP. (A) H&H stage 31 (E7) embryos. (B) H&H
stage 34 (E8) embryos. (C) H&H stage 35 (E9) embryos. (D) The image was cropped from left
femoral tract of a E9 embryo, which represented the development of feather buds in the midline
between H&H stage 31 (asterisk) and 34. (E-L) Longitudinal sections (20µm) after WM-ISH and
schematic diagrams show the dynamic expressions of Cx40 from H&H stage 31 to stage 35.
Scale bars, 300µm for A-D, 50µm for E-H.

47

OTHER CONNEXINS
As can be seen in Fig. 19, only Cx31.1, Cx31.9, Cx32, Cx46 were expressed in
the embryonic chicken skin. The others might be expressed at very low levels, if not
completely absent. Those expressed were all in the epithelium throughout the stages
investigated. The expression of Cx31.9 was found in the outer epithelium of buds at
H&H stage 31 and afterwards. Both the Cx31.1 and Cx32 were weakly expressed, if not
completely absent, at H&H stage 34 and strongly expressed in both bud and interbud
domains at H&H stage 35. Cx46 was weakly expressed in the epithelium of buds at H&H
stage 34 and more strongly expressed in the epithelium of buds at H&H stage 35. The
schematic picture summarizes the expression patterns of other connexins on sections after
WM-ISH (Fig. 20). The schematic summary for the expressions of all connexins viewed
from the top can be seen in Fig. 21. These differential expressions of other connexins in
different layers of bud and interbud epithelium suggest that other connexins might play a
role in epidermal differentiation and compartmental development.

48

Fig. 19 Expression of other connexins transcripts shown by whole-mount in situ hybridization.
Among these Cx isoforms, only Cx31.1, Cx31.9, Cx32, and Cx46 are expressed in early feather
development. A-P axis is from left to right. Dorsal view for WM-ISH embryos. Colors were
developed by NBT/BCIP. Longitudinal sections (14µm) represented feather primordia or feather
buds in the midline, and they were obtained after WM-ISH of Cxs. Scale bars, 300µm for WM-
ISH, 50µm for sections.

49

Fig. 20 Schematic summary of molecular expressions of other connexins. The differential
expressions of Cxs suggest they may play a role in the epidermal differentiation. A-P axis is from
left to right. Cx31.1, Cx32, and Cx46 were detectable in the epithelium of short (St 34) and long
(St 35) feather bud stages, however, Cx31.1 and Cx32 were expressed in bud and interbud at the
same time while Cx46 were only expressed in the bud region. Cx31.9 can be detected in outer
epitheliums of the earlier stage (St 31) as well as later stages (St 34 and St 35). None of the
transcrips were detectable by WM-ISH and subsequent sectioning in the pre-placode stage (St 28).
Cx36, Cx37, Cx40.1, Cx50, and Cx52.6 were undetectable in all stages (St 28- St 35) in this
investigation.

50

Fig. 21 Schematic summary of molecular expressions of connexins in the spinal tract. The
differential expressions of Cxs suggest they may be involved in the pattern formation,
compartmental development, and feather development. St 31+ stands for the H&H stages
between St 31 and St 34. Viewed from the top. Cx30 was expressed very early in feather tracts
(St 28) and lately (St 31) became restricted to the bud primordia. The expression gradually
disappeared in the marginal regions of buds (St 31+) and eventually show up at the anterior side
of buds (St 34) and are almost absent, if there was still any, at long bud stage (St 35). Cx43 was
expressed in the entire future feather forming field (St 28) and gradually increased in the bud
domain (St 31). The expression inside bud domains were extremely weak if not completely
absent at early short bud stage (St 31+). At H&H stage 34, the expression outside buds
disappeared and became intensified at more posterior side, then (St 35) the signal would occupy
the whole bud domains. The rest of Cxs did not show early (St 28) expressions, among them,
Cx40 scattered around the entire epithelium at H&H stage 31 (E7). This expression would be
absent transiently in bud domains and gradually come out again from the posterior side of bud
domains. At nearly the same time, the expression in interbuds were markedly lost. At H&H stage
35, Cx40 was expressed in whole bud domains and only sporadic expressions can be seen in
interbuds. Cx31.1 and Cx32 were expressed in similar patterns in bud as well as interbud domains
starting from H&H stage 34 and beyond. Cx31.9 and Cx46 were expressed in bud domains
starting from H&H stage 31+ and 34, respectively. Cx36, Cx37, Cx40.1, Cx50, and Cx52.6 were
undetectable in all stages (St 28- St 35) in this investigation.

51

CHAPTER 5: DISCUSSION
LUCIFER YELLOW DYE TRANSFER, CONNEXIN EXPRESSION, AND
COMPARTMENTAL DEVELOPMENT
In this study, I have investigated the broad lucifer yellow dye transfer in freshly
prepared H&H stage 28-36 (E6-E10) chicken skin by the scrape-loaded method. This
method has been widely adopted in cell cultures, and I showed that this method is also
effective in the chicken skin. At H&H stage 28, before any visible cues of bud formation,
it seems that lucifer yellow can be transmitted faster in the midline (Fig. 4F). At this stage,
Cx30 is strongly expressed in the epithelium of the midline (Fig. 12A, J), and Cx43 is
expressed in both the epithelium and mesenchyme (Fig. 16Fa, H) of the spinal tract. The
other connexins are undetectable at this stage. This observation raised an intriguing
question that if the Cx30 and Cx43 can form heteromeric or heterotypic gap junctions in
the midline ? Moreover, if different combinations of Cx30 and Cx43 can specify different
potentials of cells in the midline at this stage? Or, more simply, can say what Cx30 is
doing in the midline at this stage? Cx43 is expressed more evenly in the spinal tract at
this stage, and I can see the distance of dye transfer is similar on the right side and the left
side (Fig. 4F). Therefore, I may conclude that Cx43 alone is important for the cellular
communication in the future feather-forming field. On the other hand, the development of
the midline involves cell proliferation and migration (Michon, et al, 2008). I speculate
that Cx30 is important for cell proliferation before cell condensations can periodically
form.

52

At H&H stage 31 (Fig. 5), GJIC is gradually reduced bilaterally at this stage (Fig.
5E, F). Although the buds largely impede the dye transfer, I can still see some lucifer
yellow signals transferred through the bud domains (Fig. 5D, G, H) and transferred
effectively in the interbud domains. At this stage, the expression of Cx30 is gradually
centered on the older buds (Fig. 12B, K, L) and Cx43 is expressed in the entire
epithelium and intensified in the buds (Fig. 16 B, I). In addition, Cx31.9 may start to be
expressed in the older bud domains. These results partially explain the relationship
between the expression patterns of connexins/ GJIC and compartmental specifications.
At H&H stage 34 (Fig. 6), lucifer yellow dye transfer in the bud domains is close
to the background level (Fig. 6E, F). Interestingly, this coincides with the disappearance
of the expression of Cx43 in the bud domains at this stage (Fig. 16J). Although some
other connexins may be expressed in the buds at this stage (Cx30, Cx31.1, Cx31.9, Cx32,
Cx40 and Cx46), they cannot promote the lucifer yellow dye transfer effectively. Cx31.1,
Cx32, and Cx43 are expressed in the interbud at this stage, so they may form
homo/hetero-meric, homo/hetero-typic gap junctions in the interbuds and facilitate the
specification of compartmental development.
At H&H stage 35 (Fig. 7A), LY dye transfer is only effective in the bud
mesenchyme and Cx43 is the only isoform that can be expressed in the mesenchyme
during feather development. Meanwhile, although Cx31.1, Cx31.9, Cx32, Cx40, and
Cx46 can be expressed in the bud epithelium at this stage, the LY dye transfer is very
limited there. Therefore, I expect that the Cx43 gap junction is able to mediate long-range
communication cross cells and other composition of gap junctions may mediate short-

53

range cellular communications, if they are able to do so. The only two connexins that are
expressed in the interbud at this stage are Cx31.1 and Cx32. However, there is no LY dye
transfer in the interbud at this stage.
By H&H stage 36 (Fig. 7B), LY dye transfer is more restricted to the epithelium,
and the dotted pattern observed in Fig. 7B may represent proliferating cells because this
pattern is similar with BrdU pulse labeling results (data not shown).
Collectively speaking, scrape-loaded LY dye transfer can effectively reflect the
compartmental differences during feather morphogenesis and may also be specific for
certain cell types or cellular behaviors. Although the differences of connexin expression
patterns can be seen along with the differences of LY dye transfer between different
compartments, more detailed analyses should be done in the future to see what kinds of
isoforms are actually responsible for those differential development and cellular
behaviors.
Furthermore, evidences have shown that different compositions of gap junctions
have different permeability to small molecules (Nitsche & Nicholson, 2004). In the
present study, I use negatively charged Lucifer yellow which weighs 452Da. For the
future, I can use positively charged small molecules (ex. biotin) or other small molecules
with different sizes (ex. Alexa 350, 488, and 594) to test the relationship between GJIC
and compartmental development.

54

DRUG TREATMENT AND THE FORMATION OF NEW BUD-LIKE
STRUCTURES
In normal skin explant cultures, I can occasionally see the formation of ectopic
buds. However, they normally form in the flanking regions around the base of feathers or
in the interbud region between adjacent buds. The anterior side of the base rarely or never
forms new bud-like structures in normal culture conditions. In the present study, I have
shown that, under the inhibition of GJIC, the new bud-like structures can arises at certain
sites (Fig. 11) suggesting there are common rules for them to follow. There are at least
three possibilities to allow the formation of new bud-like structures. First, the cells in the
newly formed condensations may come from interbud regions or adjacent old feathers,
which may be accounted for by the loss of lateral inhibition (lateral inhibion in feather
morphogenesis was described by Noramly & Morgan, 1998). Second, the cells in
"original" buds may migrate out to form the condensations. The third possibility is that a
group of cells in the anterior side around the base were reset to begin proliferation and
then form the new condensations. I have tested the possibilities of these speculations. For
the first speculation, the cells in new bud-like structures at the site 1 in Fig. 11 may come
from the interbud due to the loss of lateral inhibition. Because the buds at site 1 come
very quickly, the underlying mechanism for them seems different from the buds that
come from the other sites. However, I do not have any proliferation or lineage-tracing
evidences yet to support this speculation. In addition, DiI-labeled cells in the
mesenchyme of adjacent buds never go into the newly formed cell condensations located
at the adjacent original feather buds. For the second possibility, I do find that cells in the

55

original bud mesenchyme are able to migrate out to the location of newly formed buds
around the base. Also, when I replace the culture medium with normal culture medium,
the cells in the newly formed buds are able to migrate back after 48 hours of the
replacement of culture medium. However, those are very rare situations. I labeled over 20
buds for each of the experiments and only observed one bud in each experiment in which
cells were able to migrate out and return (data not shown). For the rest of the labeled cells
dispersed into interbuds and did not show a clear direction. This is probably due to the
fact that the skins were too old. I labeled the cells when the skins were already cultured
for at least four days.
For the third possibility, 18 alpha-GA has been known to promote the interaction
of Src protein kinase with connexin 43 (Chung et al., 2007) and cause the inhibition of
GJIC. It is not fully understood if this kind of interaction will activate Erk2 and PI3
kinase, which are two important downstream kinases of Src kinase. In the present study, I
have found ectopic beta-catenin expression around the anterior part of the base of
feathers after 3 days in culture. Also, the cell proliferation is largely increased around the
base. Beta-catenin is one of the major effector proteins in the canonical wnt pathway and
nucleated beta-catenin/Tcf-4 complex plays a major switch role controlling cell
proliferation in healthy and malignant intestinal epithelial progenitor cells (van de
Weteringet al., 2002). Therefore, I can speculate that beta-catenin is closely related to the
increased cell proliferation around the base in 18 alpha-GA treated skins. On the other
hand, before or during the formation of new bud-like structures, I can see a line and a cell
condensation overlap. Then, about one day later in culture, I can see a "ring" by BrdU

56

incorporation and staining. This is very similar to normal early feather development. At
H&H stage 26, if we the embryos with beta-catenin by in situ hybridization, we can see a
clear midline. This is the earliest stage that we can tell that the feathers are starting
morphogenesis. One day later in the normal development, we can also see a BrdU "ring"
around the feather primordia. So, the big question is how similar are the "drug induced"
and the normal feather buds?
We have to remember, the inhibition of GJIC is not equal to the inhibition of all
of the functions of gap junctions. The connexins may still exert GJIC independent
functions. Also, the mono-ions such as sodium ions and potassium ions may still
penetrate "closed" gap junctions.
INHIBITION AND PROMOTION OF BUD GROWTH BY CONNEXIN 30
In this study, overexpression of connexin 30 caused the inhibition as well as the
promotion of feather formation. On the other hand, down-regulated Cx30 by siRNA only
caused the inhibited phenotypes. Although we should also consider the stage specific
effects that the siRNA can only be effective transiently at early stages and RCAS may
work after H&H stage 31. The positive in situ hybridization staining of Cx30 in the basal
and suprabasal layers in the feather wall also indicate that Cx30 may be involved in
proliferation primarily in the epidermis. Ozawa et al. in 2009 pointed out that the C-
terminus of Cx30 can promote cell proliferation independent of GJIC. In another case,
Cx30 is able to slow down proliferation as well (Princen et al., 2001). Moreover, CX26
and CX30 expression were upregulated in the two most common types of skin cancer,

57

basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) (Haass et al., 2006).
This data suggest that Cx30 may play roles in both positively and negatively mediating
cell proliferation. However, I can also see positive Cx30 in situ hybridization staining
during early development of the axial plate in adult feathers, which does not overlap
PCNA staining at this stage. This result indicats that Cx30 may play other roles during
feather morphogenesis and this remains a big question for us to answer.
ROLES OF CONNEXIN 43
Connexin 43 is the most widely expressed connexin isoform in vertebrates. In the
present work, I found that Cx43 is the only connexin isoform that can be expressed in the
mesenchyme of the dorsal feather tract between H&H stage 28 and 35 and also the only
one that is expressed on the apical ectodermal ridge (AER) of the limb bud, which
confirms the previous study of Cx43 on the AER (Makarenkova & Patel, 1999). When I
look at the patterns of GJIC, I also find that GJIC is largely overlapped with Cx43
expression pattern. For example, the GJIC in the interbud is significantly reduced after
late H&H stage 34. That is right around the time when the expression of Cx43 is absent
in the interbud. I may conclude that Cx43 is the most widely used connexins for GJIC in
early feather development.
It has been known that the C-terminus of Cx43 can regulate cell cycle by
promoting the degradation of S phase kinase-associated protein 2 (skp2), which regulates
ubiquitination of the cell cycle inhibitor p27 (Zhang et al., 2003). Overexpression of
Cx43 in HeLa cells can also increase the expression of the cell cycle inhibitor p21

58

(Johnstone et al., 2010). In our model, I observed complementary protein expression
patterns of Cx43 and proliferating cell nuclear antigen (PCNA) at two different time
points during cellular differentiation in adult feathers. This suggests that Cx43 may also
play a growth inhibition role in feather morphogenesis. Some studies have pointed out
that Cx43 is critical in polarized cell movement and the directional migration of cardiac
neural crest cells (Xu et al., 2006) and primordial germ cells (Francis & Lo, 2006). For
the polarized cellular behaviors, there are some interesting questions that I can ask: Can
the spatial distribution of gap junctions control the direction of the cellular differentiation
in a single cell? Can these cells be grouped to function well in developmental processes
such as planar cell polarity? For the first question, Shelly et al. in 2010 probably has
already given us some cues on this. They found localized reciprocal regulation of cAMP
and cGMP can promote and suppress axon and dendrite formation, respectively, in
undifferentiated neurites. Interestingly, cAMP dependent protein kinase (PKA) and
cGMP dependent protein kinase (PKG) have opposite functions in the regulation of Cx43
as well (Imanaga et al., 2004).
ROLES OF CONNEXIN 40
I speculate that the dynamic and dotted patterns of Cx40 could be a de novo
expression associated with certain migratory events accounted for by a specific cell type.
The Cx41.8 has been implicated in the pigmentation in zebrafish (Watanabe et al, 2006)
and is an ortholog of human and chicken connexin 40 (Cruciani & Mikalsen, 2007).
Although there is not yet any evidence that shows the relationship between human
connexin 40 and human pigmentation disorders, it is still interesting to think about the

59

relationship between chicken connexin 40 and color patterning. The work done by
Cruciani and Mikalsen (2007) also revealed a closer phylogenetic relationship of
conserved domains between chicken Cx40 and zebrafish Cx40 than human CX40 and
zebrafish Cx40.
FUTURE DIRECTION
One of the major goals of this study was to see if the patterns of GJIC can be
explained by the expression patterns of connexins and the tissue compartments. Thus far,
I have seen that the expression of Cx43 is markedly overlapped with GJIC. For other
connexins or different compositions of connexins, I do not have conclusive results yet.
To answer this question, I need fine tools such as single cell injections to investigate the
GJIC in specific layers of tissue. Then, I should be able to study each connexin in a more
detailed fashion. I can begin with connexins that I am particularly interested in, such as
Cx30, Cx43, Cx40, and Cx31.1 (maybe related to apoptosis, data not shown). For GJIC
dependent functions, I can use fluorescence resonance energy transfer (FRET) to
decipher the interaction of small molecules and specific connexins. For GJIC independent
roles, I can overexpress C-terminus truncated or point mutated connexins to see the
consequences of aberrant molecular interactions. I can also pull down the connexin
interacting proteins with columns and then differentiate their roles with each connexins as
well as in animal development. I would be very interested to know if connexins can
bridge the gap between the expression of epigenetic enzymes and the environment
through the interacting proteins.

60

18 alpha-GA-treated skins show accelerated feather development and the
emergence of new bud-like structures. For the next steps, I need to develop good cell
tracing techniques and see where the cells in those structures are exactly coming from,
though, they can also be cells that proliferate locally. If they from a localized growth
domain, it would be interesting to know if that can be a new feather tract? Thus far, the
comparative molecular characterizations (shh, beta-catenin and bmp2, data not shown for
bmp2) of new feather-like structures and normal feathers are the same. For the
accelerated phonotype, I can investigate the expression of cell cycle genes and connexins.
One interesting question is if the inhibition of GJIC is harmless to feather development.
In 18 alpha-GA-treated samples, I can see original feather buds still develop nice
branching (Fig. 9 shh staining, D4; beta-catenin staining, D4 and D5). If they can grow
well without GJIC, then I may ask what would be required for feather development other
than GJIC? In addition, knock-down of Cx30 did not show any of the phenotypes like 18
alpha-GA-treated skin explants. This may be due to the redundancy of Cxs which can
compensate the GJIC functions of Cx30. It is also possible that Cx30 is not involved in
the cellular functions related to those phenotypes. However, knock-down of Cx30 did
show sever phenotypes during feather development. I can speculate that the GJIC-
independent functions of Cx30 is critical for normal feather morphogenesis. Since the
cells in 18 alpha-GA treated skins still have the N/C-terminus and intracellular loop of
connexins, I can assume that the GJIC-independent functions are already enough to
support feather development, at least until the early branching morphogenesis. It is also
possible that other ion channels can facilitate the feather development and buffer the
effects caused by the inhibition of GJIC. I will speculate that this is a good chance to

61

further decipher the differences between GJIC-dependent and -independent functions and
explore the potential interactions between Cxs and ion channels.

62

CHAPTER 6: CONCLUSION
From this study, I have seen that lucifer yellow dye transfer can reflect the gross
compartmental development, and the differential expression patterns of different
connexin isoforms during early feather morphogenesis may account for the ability
allowing the dye to be transferred. The inhibition of GJIC accelerates but does not alter
the developmental program of original feather buds and induces the formation of new
bud-like structures in specific sites. Meanwhile, the feather patterning of original buds is
normal. However, the feather patterning is changed in Cx30 knock-down experiments
suggesting the roles that Cx30 or other Cxs may play in early feather pattern formation is
GJIC-independent. In addition, individual connexins have shown their potentials in
regulating cellular functions, especially Cx30 in cell proliferation, and Cx43 in the
inhibition of growth as well as the promotion of cellular differentiation. New insights of
the roles of Cx40 in pigmentation and Cx31.1 in apoptosis during feather morphogenesis
are gained from this study as well. Collectively speaking, this study has opened a new
field of view regarding the crosstalk between GJIC-dependent and - independent
functions in the coordination of pattern formation and feather development. Further
studies still need to be done in order to understand and answer those interesting and basic
questions unveiled in this study.

63

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Abstract (if available)

Abstract Gap junctions are formed by the direct docking of two hexamers of connexins (Cxs) which allow the exchange of cellular contents, such as ions, second messengers and small molecules, between neighboring cells. Some studies have revealed that connexins could have gap junction intercellular communication (GJIC) independent roles by interacting with diverse proteins (reviewed in Herve et al., 2004

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Asset Metadata

Creator Tseng, Chun-Chih (author)

Core Title Connexins in feather morphogenesis

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2011-05

Publication Date 03/03/2013

Defense Date 01/28/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag connexin,feather,gap junction,GJIC,morphogenesis,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Chuong, Cheng-Ming (committee member), Maxson, Robert E. (committee member)

Creator Email be_in_one@hotmail.com,tsengchu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3676

Unique identifier UC1145740

Identifier etd-Tseng-4324 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-416056 (legacy record id),usctheses-m3676 (legacy record id)

Legacy Identifier etd-Tseng-4324.pdf

Dmrecord 416056

Document Type Thesis

Rights Tseng, Chun-Chih

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu