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Expression and functional studies of the Notch signaling pathway in feather development

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Expression and functional studies of the Notch signaling pathway in feather development

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EXPRESSION AND FUNCTIONAL STUDIES
OF THE NOTCH SIGNALING PATHWAY
IN FEATHER DEVELOPMENT
by
Chia-Wei Janet Chen
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment o f the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Pathobiology)
May 2000
Copyright 2000 Chia-Wei Janet Chen
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UM I Number: 3054856
Copyright 2000 by
Chen, Chia-Wei Janet
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
...........................................................
under the direction of hstc Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Dean of Graduate Studies
D a t g March _ 31,_ 2000
DISSERTATION COMMITTEE
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ACKNOWLEDGMENTS
I would like to thank:
* My advisory committee chairman, Dr. Cheng-Ming Chuong, whose immense
enthusiasm and optimism inspired me to pursue this work.
* My advisory committee members, Dr. Henry Sucov, Dr. Michael Stallcup and Dr.
Malcolm Snead, for their kind support and constructive suggestions. Dr. Malcolm Snead
had provided me financial support for two years through his National Institute o f Dental
and Craniofacial Research training grant DE-07211.
* Dr. Randall Widelitz for always being available to provide advices and assistance. He
has been my constant source o f solutions.
* Dr. Ting-Xin Jiang for teaching me delicate embryological procedures and collaborating
with me on many projects. I truly admire his expertise and greatly value his technical and
emotional support throughout the years.
* All the colleagues whom I have worked with, in particular Dr. Sheree Ting-Berreth, Dr.
Yun-Shain Lee, Dr. Eric Yin, Ms. Jianfen Lu and Mr. Ying-Hsien Kao for sharing their
knowledge and experiences.
* My brothers for accompanying me to the laboratory in those late nights when I was
doing time-course experiments and while preparing this dissertation. Their patience and
support are deeply appreciated.
* My parents for providing me the environment and opportunity to attain higher
education. I am thankful for their love and encouragements which have sustained me
through difficult times.
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iii
TABLE OF CONTENTS
ACKNOWLEDGMENTS..................................................................................................... ii
LIST OF FIGURES............................................................................................................... iv
ABSTRACT........................................................................................................................... vi
CHAPTER 1: Introduction......................................................................................................1
CHAPTER 2: Asymmetric expression o f Notch/Delta/Serrate is associated with
the anterior-posterior axis o f feather buds................................................... 20
CHAPTER 3: Dynamic expression o f Lunatic fringe during feather
morphogenesis: a switch from medial-lateral to anterior-posterior
asymmetry...................................................................................................... 47
CHAPTER 4: Functional studies o f the Notch signaling pathway during feather
morphogenesis................................................................................................61
CHAPTER 5: Isolation o f Chicken Suppressor o f Hairless I and 2.................................. 86
CHAPTER 6: Conclusion.....................................................................................................101
REFERENCES.....................................................................................................................106
TABLE l.l: Comparison o f feather and hair........................................................................3
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LIST OF FIGURES
Figure
1.1. Diagram o f the life cycle o f a feather.............................................................................5
1.2. Examples o f molecular localization as in vivo expression pattern or
following experimentation............................................................................................ 12
2.1. Expression pattern o f C-Notch-l, C-Delta-l and C-Serrate-1 in developing
feather buds as shown by whole-mount in situ hybridization.................................. 26
2.2. Expression pattern o f C-Notch-l, C-Delta-l and C-Serrate-1 in developing
feather buds as shown by paraffin section in situ hybridization................................ 28
2.3. BrdU labeling................................................................................................................ 32
2.4. Expression o f C-Notch-l, C-Delta-l and C-Serrate-1 in experimentally
rotated feather buds......................................................................................................35
2.5. Expression o f C-Notch-l, C-Delta-l and C-Serrate-1 in experimentally
manipulated branched feather buds..............................................................................37
2.6. Expression o f C-Notch-l, C-Delta-l and C-Serrate-1 in experimentally
manipulated symmetric feather buds........................................................................... 40
2.7. A working model.......................................................................................................... 44
3.1. The first two phases o f L-fng expression in dorsal feather morphogenesis...............51
3.2. The first two phases o f L-fng expression in femoral feather morphogenesis........... 54
3.3. R-fiig expression in feather morphogenesis.................................................................57
3.4. The third phase o f L-fhg expression in feather morphogenesis................................. 59
4.1. Constitutively active forms o f Notch.......................................................................... 69
4.2. Viral transcripts were found on embryos infected with RCAS(A)CDN...................71
4.3. Reconstituted skin explants overexpressing C-Delta-l and activated Notch 1......... 73
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4.4. Viral transcripts were found on reconstituted skin explants infected with
RCAS(A)AP, RCAS (A)C-Delta-l, RCAS(A)CDN and RCAS(A)nCDN..............76
4.5. Expression o f PCNA in reconstituted skin explants overexpressing
C-Delta-l and activated Notch 1..................................................................................79
4.6. Expression ofNCAM and Tenascin C in reconstituted skin explants
overexpressing C-Delta-l and activated Notchl........................................................81
5.1. Primary nucleotide and amino acid sequence o f chicken Suppressor o f
Hairless 1..................................................................................................................... 91
5.2. Primary nucleotide and amino acid sequence o f chicken Suppressor o f
Hairless 2..................................................................................................................... 94
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ABSTRACT
vi
To study the role o f the Notch signaling pathway in feather morphogenesis, the
expression patterns o f C-Notch-l, C-Delta-l, C-Serrate-1 and Lunatic fringe were
examined. It was found that from symmetric short buds to asymmetric long buds, C-
Delta-l and C-Serrate-I are expressed in the posterior bud mesenchyme in a nested
fashion, while C-Notch-l is expressed as a stripe perpendicular to the anterior-posterior
axis and positioned posterior to the midpoint. Lunatic fringe is also dynamically
expressed, exhibiting three phases throughout development. First, Lunatic fringe is
expressed in the epithelium as a ring bordering the feather primordium when it is initially
induced. Shortly after, it shows a polarized pattern, first toward the lateral side o f the
feather primordium and then makes a 90°C switch toward the posterior side o f the short
bud. It then becomes weakly expressed in the long bud stage. Finally, it is expressed in the
marginal plate epithelia o f feather filaments. To examine the relationship between
molecules o f the Notch pathway and anterior-posterior axis determination, feather bud
orientation was perturbed experimentally and the expressions o f C-Notch-l, C-Delta-Iand
C-Serrate-1 were examined. The results showed that polarized expression correlated with
polarized growth; moreover, this molecular asymmetry was detected prior to
morphological asymmetry. To study the function o f the Notch signaling pathway, C-
Delta-l and two active forms o f rat Notchl were misexpressed in vivo and in the
reconstituted skin explants. No irregular phenotypes were observed when rat Notchl was
misexpressed using the described reagents and procedures. C-Delta-l misexpression
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inhibited feather bud formation in the reconstituted feather buds, similar to the in vivo
results reported by other groups. An expansion o f proliferating cells is observed in the C-
De/m-/-misexpressing feather buds, although their posterior outgrowth is normal. These
data suggest that C-Delta-1 plays an essential role in the inductive phase o f feather bud
formation, but is not sufficient in establishing anterior-posterior polarity. The effort
toward studying the downstream effects o f Notch signaling led to the isolation o f two
chicken Suppressor o f Hairless genes by RT-PCR. The partial coding sequences show
that C-Su(H)-l and C-Su(H)-2 respectively share 95% and 60% amino acid identity to the
Xenopus homologs, X-Su(H)-l and X-Su(H)-2.
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1
CHAPTER 1: INTRODUCTION
SUMMARY
In this introductory chapter, the feather model in which this work is based on is
discussed. The development o f a feather through its various stages is summarized and
compared to that o f the hair. The cellular and molecular approaches used to study feather
morphogenesis are also introduced; many o f these approaches were employed in the
course o f this study. Finally, the strengths and weaknesses o f this model are presented.
1.1 The Feather Model
To analyze the morphogenic events during skin appendage formation, it is
important to have an animal model that offers distinct patterns at various stages o f
development and is accessible to analysis using state o f the art technology. The avian
integument is such a model. Combining experimental embryological approaches, organ
cultures and gene transduction technology, we are now able to begin to address the
molecular basis o f pattern formation, primordium initiation, anterior-posterior axis
formation, proximo-distal axis formation, phenotypic determination and others. Parallel
mechanisms are usually found in feathers and hairs, and the avian integument model has
matured to be a major source o f new findings in the study o f skin appendage
morphogenesis.
In order to manage disease conditions involving skin and skin appendages, we
need to understand the molecular and cellular events underlying skin appendage
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2
morphogenesis (Chuong, 1998; Oro and Scott, 1998). The avian integument has been
used as a major model in developmental biology (Sengel, 1976). Although the avian
integument includes feathers and scales, the extent o f my research focused more on the
feather. I will first describe the development o f feather, and compare it with mammalian
hair. Although there are differences, both share many common features as skin appendages
(Table 1.1).
Both feathers and hairs are complex structures that originate from a flat piece o f
skin. Their formation starts with the interaction between the epidermis and the
mesenchyme. The epithelium has to be competent but the signals to initiate an individual
primordium from a specific location comes from the mesenchyme. The result is the
formation o f an epithelial placode and a dermal condensation (Fig. 1.1). The dermal
condensation is at first radially symmetric. Its anterior-posterior (A-P) asymmetry is
endowed by the epithelium and with proliferation, the skin appendage elongates along the
proximo-distal (P-D) axis. The early stages o f feather development, up to the long bud
stage, is growth above the skin surface. Later on, the epithelium invaginates into the
dermis to form the follicle. The hair, however, initiates its primordium beneath the skin
surface. At the follicle stage, both feather and hair are composed o f layers o f epithelial
cells ensheathing a cluster o f dermal cells called the dermal papilla. They continue to
lengthen when new post-mitotic cells are added at the proximal end. Depending on the
number o f available cells, the rate these cells enter cell cycles, and the length o f the anagen
period, their length can be regulated. Both feather and hair can regenerate, called molting
hi feathers, under the influence o f seasons and hormones (Lucas and Stettenheim, 1972).
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Table 1.1. Comparison of feather and hair
Feather and hair share many common features as skin appendages; however, there
are also many differences.
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Events Feather Hair
Induction based on
epithelia-mesenchymal
interactions
Yes. The thickened
epithelium is called feather
placode
Yes. The thickened
epithelium is called hair
germs
Appendage primordia Feather buds protruding out
o f skin surface
Hair pegs invaginate into
dermis
A-P asymmetry in
primordia
Yes Yes
Follicle Yes. Proliferating epithelium
is called feather collar.
Yes. Proliferating epithelium
is called hair matrix.
Within the filament Pulp filled with mesenchyme
tissues transiently
No pulp. Cortex and
medulla made o f epithelial
cells.
Branch formation o f the
filament
Yes. go through differential
cell death to form barbs and
barbules.
No such process
Cycling o f appendages Yes. Called molting. Yes
Color o f appendages Complex pigment patterns,
also have iridescences
Solid color based on density
and size o f melanin granules
Regulation by sex
hormones
Yes Yes, but much less obvious
Regeneration induced by
dermal papilla
Yes Yes
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FIG. 1.1. Diagram of the life cycle of a feather.
The various stages o f feather development. Revised from Fig. 1. in Chuong, 1993.
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Feathers serve many purposes for the avian species; soft down feathers keep them warm,
colorful feathers allow them to communicate, and strong wing feathers enable them to
soar in the sky. These different types o f feather are constructed by different types o f
keratins (Rogers, 1985). There are also many different types o f hair found in nature, but
they are less diverse structurally. Feathers are also distinct for their branching structure,
which involves up to three levels; the rachis, the major branch; the barbs, the secondary
branches; and the barbules, the tertiary branches. The branches are formed when the
epithelial sheet, with basal layer being the innermost layer, invaginates periodically to
segregate regions that will either keratinize or apoptose. The death o f cells in the marginal
plate epithelia creates spaces between barb plate epithelia. A similar fractal like process
takes place to form the barbules. In hairs, there is no branch formation. Most
conspicuously, feathers have elaborate color patterns. Besides the biochrome pigments
that give them the spectrum o f chemical colors, they also have structural colors that render
iridescences (Gill, 1995). Thus the feather is also a superb model to study how mother
nature dresses up in colorful magnificence.
Feather formation involves many fundamental cellular processes that are widely
studied by basic scientists. We, the feather biologists, wish to understand how the
molecular and cellular events are utilized to construct fascinating structures. But besides
catering to the wonders o f the curious minds, our research presents relevance to the
practical world. Recent studies employing molecular biology techniques have shown that
the findings from the feather parallel those from human disease research. One example o f
such a link is that while retrovirus mediated overexpression o f SHH causes extra large
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8
feather buds (Ting-Berreth and Chuong, 1996b; Morgan et al., 1998), the activation of
SHH pathway is involved in Gorlin syndrome and human basal cell carcinoma (Bale et al'.,
1998; Johnson et al., 1996; Oro et al., 1997; Xie et al., 1998).
1.2 Cellular and Molecular Approaches
1.2.1 Experimental Embryology
The earliest efforts to learn how feathers or other skin appendages are formed
involved manipulating the embryonic tissues with classical experimental procedures,
namely transplantation and recombination (Sengel, 1976; Dhouailly, 1973). There are
many kinds o f skin appendages on different species o f organisms (eg. mouse hair vs. chick
feather) and on different regions o f the same organism (eg. chick dorsal feather vs. chick
midventral apterium vs. chick foot scales). The early transplantation experiments sought to
discover what determines the regional specificity o f the various types o f skin appendages.
Blocks o f tissue were dissected from early embryos and transplanted elsewhere in the body
to find that the determination factors o f the diverse characteristics originate from the
paraxial mesoderm very early in embryogenesis (reviewed in Sengel, 1990; Dhouailly et
al.„ 1998).
To discover the roles o f epidermis and dermis, skins from different sources were
separated into epidermis and dermis and then cross cultured or recombined. Heterospecific
recombinations o f lizard dorsal epidermis and chick dorsal dermis resulted in growth o f
scale primordia arranged hexagonally (the feather pattern) (Dhouailly, 197S). This result
demonstrates three points. One, the size and distribution pattern o f the primordia are
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determined by the dermis. Two, the dermal signal “to make an appendage” is recognizable
among different classes o f vertebrates. Three, the type o f skin appendages on
heterospecifically recombined skins is dictated by the epidermis. In contrast to the third
point, heterotopic recombinations between chick midventral apteric epidermis and chick
dorsal dermis resulted in growth o f feathers. In the latter case, the epidermis has the innate
ability to make feather, and upon obtaining the dermal signal, it makes feathers. In the
former case, the lizard epidermis only knows how to make lizard skin appendages, so
upon receiving the same signal, scales are formed.
So what role does the epidermis play then? When recombination experiments are
done between scaleless epidermis and normal dorsal dermis, no feathers are formed, but
the vice versa recombinations o f normal dorsal epidermis and scaleless dermis produce
feathers. Scaleless chick mutants have no scales and very few feathers. These results not
only direct the scaleless gene activity to the epidermis (Brotman, 1977), they illustrate the
importance o f epidermis competence and suggest the presence o f epidermal signals. When
these similar experiments are performed in mature skin, the results are more complex,
depending on the competence and determined state o f germinative epidermal cells (Jahoda
and Reynolds, 1993). From them, we hypothesize that epithelial precursor cells go
through stages o f differentiation, and they become less flexible and more determined as
they progress (Chuong, 1998). To understand skin appendage morphogenesis
comprehensively, we must keep in mind that it is a chronological event, thus studying at
different temporal points and integrating those results are important. And while these
classical experiments have provided us with much information, more molecular studies are
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needed. These experiments have shown that there is crosstalk between the two tissues.
However, they did not tell us what the molecules are. In recent years, many development
related signaling molecules have been identified and it becomes possible to study the roles
o f these molecules in skin appendage morphogenesis (Chuong et al., 1996). In the
following, we summerize some o f these approaches.
1.2.2 Searching for the Molecular Basis
To elucidate which molecular signals are involved, one can either assay for the
possible role o f a known molecule or search for new candidates. Because other more
genetically accessible systems have already discovered many interesting genes and the fact
that many genes or genetic pathways have repeatedly been shown to be conserved across
species (Noveen et al., 1998), we often select genes o f interest from these other systems.
Many o f the genes we studied are homologs to Drosophila genes that play important
developmental roles. Other sources o f interesting molecules come from mouse mutants
with skin abnormalities and human genetic diseases involving the skin.
Besides the “borrowed genetics” strategy, it is possible to use non-biased
differential expression screening to search for candidate genes. Under this category, it is
possible to use a subtraction library or differential display-like techniques to fish out
molecules whose expression is changed before and after a morphogenetic process. With
human genome project approaching the end and new molecular technology being
developed, we can expect many more molecules waiting to be studied.
Once we have candidate genes, we need to have chicken clones, either through our
own efforts or as generous gifts from other laboratories. The chick ho mo logs are often
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1 1
cloned through RT-PCR (eg. the cloning o f chicken Suppressor o f Hairless described in
Chapter 5) and cDNA library screening. With a chick probe, we first analyze their
expression at various stages o f feather development. We often employ in situ hybridization
to analyze expression at the RNA level. This can be done on the intact embryo or tissue
(whole mount) or on histological sections (Fig. 1.2A, Nieto et al., 1996, Jiang et al.,
1998). The advantage o f whole mount versus section in situ is that one can obtain a three-
dimensional overview o f gene expression at different stages because there is a feather
growth gradient on a single tissue or embryo. The in situ results tell us not only whether a
gene is expressed, but also when in development and where in the tissue it is expressed.
With those information, we can then start making hypotheses and perform functional
studies. For molecules that are better characterized and against which we have antibodies,
immunohistochemical staining can be done to detect expression at the protein level. Often
a comparison o f the expression o f two genes is helpful in providing clues to the
relationship o f their roles. This can be achieved through double staining, such as double in
situ (Nieto et al., 1996, Jiang et al., 1998).
1.2.3 Perturbation of Molecular Function
After we discover that a certain gene is expressed in an interesting manner, we
wish to know its function. The chick skin is useful for in vitro functional studies because
there are a variety o f culture techniques that allow us to address different issues in skin
appendage morphogenesis. A typical chicken skin tissue culture uses chicken dorsal skin
from stage 30 embryos. At the beginning, it is a homogeneous piece o f skin. After four
days, many feather buds will have formed and are regularly arranged in a hexagonal
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FIG. 1.2. Examples of molecular localization as in vivo expression pattern or
following experimentation.
(A) Non-radioactive whole mount in situ hybridization o f Msx-1 to show the overall
feather pattern. Alkaline phosphatase are used to develop the color (Chen et a l. 1997).
Lateral view o f embryonic day 8 chicken embryo.
(B) Demonstration o f exogenous gene transduction in embryonic chicken skin using
retrovirus. Chicken embryos were injected with RCAS-alkaline phosphatase virus
(Morgan and Fekete, 1996) at day 4 and incubated for 7 more days before fixation. The
dark patches represent areas with virus infection and expressing alkaline phosphatase
gene.
(C) “Chicken Blaschko lines”. Chicken embryos were injected with replication defective
spleen necrosis virus carrying beta galactosidase between embryonic day 1 - 2 (Chuong et
al., 1999). The parallel lines represent progenies o f the same lineages. Human Blaschko
lines have been implicated in congenital and acquired skin disorders (reviewed in Happle,
1985).
Size bar = 3 mm
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pattern. This culturing system can be modified so as to reset the developmental process to
earlier stages. One modification is called recombination, in which the epithelium and the
mesenchyme are separated and recombined with rotation. Doing so, we learned that the
location o f feather buds is determined by the mesenchyme, while the orientation is
determined by the epithelium. Preexisting epithelial placodes are unstable and will
disappear following separation. The mesenchyme will then induce new feather buds from
the epithelium (Chuong et al., 1996). Another method is called reconstitution. In this
method, after the separation o f mesenchyme from epithelium, mesenchymal cells are
disassociated into single cells and then plated at high density. Then a piece o f epithelium is
placed above. In this case, the mesenchyme is set back to the stage when the cells are
more equivalent and forced to reorganize, but it retains the ability to form evenly-spaced
feather buds (Jiang et al., 1999).
These three ways o f culture allow us to study feather bud morphogenesis at
different developmental stages. They also enable us to easily add exogenous agents and
study their effects on the developmental process. There are also different ways to
administer exogenous factors. To add a factor globally, one simply dilutes the factor into
the culturing media. For example, we have added antibodies against adhesion molecules
and extracellular matrix molecules to culturing media to perturb cell surface interaction
and study the effect o f adhesion in various stages o f feather development (Jiang and
Chuong, 1992; Chuong et al., 1994). To test the effect more locally, one can deliver the
reagent via a coated bead so a gradient o f the tested molecule is formed from the source.
This is ideal because in many cases cells sense the difference in relative concentration more
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15
effectively than the presence o f the molecule in the media. Depending on the nature of the
molecule, different types of beads are employed. For example, growth factors such as FGF
and TGFP are coated on AfBgel blue beads (Widelitz et al., 1996; Ting-Berreth and
Chuong, 1996a), whereas pharmacological reagent such as cAMP is coated on AGI-X8
anion exchange beads (Noveen et al., 1995a).
To perturb molecular function at the genetic level, it is possible to use virus
mediated gene therapy-like technology to cause misexpression. One can infect cultures in
vitro or embryos in vivo. For in vitro experiments, one can culture the skin explants in the
presence o f virus. For in vivo infection, one can inject virus into the amniotic cavity or
into the early limb buds which will later develop into wings or legs covered with feathers
or scales (Fig. 1.2B). The infected tissues or embryos are incubated for a length o f time
before assaying for changes in the size, number and spacing of feather primordia,
orientation of feather buds and phenotypic transformations such as the conversion of
feathers into scales. The timing o f the injection should be noted for consideration when
one analyzes the result because the same virus administered at different developmental
stages can produce different phenotypes (Morgan et al., 1998). When sonic hedgehog is
overexpressed prior to stage 22 (Hamburger and Hamilton, 1951), there are disorganized
ectodermal growths and an overall inhibition o f feather buds; at stages 22 and 23, large
feather buds formed; and after stage 23, there is no apparent defect. These differences
suggest the dynamic nature of feather morphogenesis and remind one o f the importance o f
timing and competence in developmental studies.
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Genes do not work in isolation. In most cellular processes, many genes and
molecular pathways are involved. Thus to determine the role o f a pathway, evidences from
both overactivation and inhibition are helpful. To activate a pathway, overexpression of a
gene or its constitutively active form is used. For inhibition, one can overexpress a
dominant negative mutant form or an antagonist. Sometimes antisense is also used. Thus
far, most o f the viral transduction experiments used an avian Rous Sarcoma virus derived
vector called RCAS (Replication-Competent Avian Sarcoma Virus) (Morgan and Fekete,
1996). Another virus o f choice is the adenovirus (Leber et al., 1996). We have more
experience using RCAS; however, preliminary experiments have shown that adenovirus
can also infect chicken skin cells without perturbing feather development (Wang et al.,
unpublished data).
The downstream effects of activating a molecular pathway are cellular processes.
We have observations and evidences that processes such as cell proliferation, adhesion,
migration, differentiation and apoptosis occur in feather morphogenesis. To study cell
proliferation, we have used BrdU labeling (Chen et al., 1997). For cell migration, we can
use Dil labeling or defective virus carrying reporter genes to track cells (Chuong et al.,
1996). Cells at different stages and locations can be injected and traced for their
distribution at different stages of development. An example is using spleen necrosis virus
carrying LacZ (Fig. 1.2C and Mikawa et al., 1991). It is fascinating to observe that the
labeled avian epidermal cell distribution resembles those o f human Blaschko lines (Chuong
et al., 1999). Many skin diseases have been shown to present along this linear distribution
(reviewed in Happle, 1985). To study differentiation, we can examine expression of
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feather-specific keratin markers (Rogers, 1985). And for examining apoptosis, we can do
TUNEL assays and various apoptosis molecular markers.
Several examples studying signaling molecules and adhesion molecules in early
stages o f feather morphogenesis are summarized in Widelitz and Chuong, 2000a.
1.3 Strengths and Weaknesses of the Avian Integument Model
As has been stated above, the avian skin has many strengths as a research model,
particularly the accessibility o f experimental embryological manipulations. Another
advantage is the presence of a wide spectrum o f apparently different sizes and shapes of
feathers in one single bird. While human beings also have these body regional differences
and mouse hairs have various types (Sundberg, 1994), they are not easy research models
to study region-specific appendage size/shape or sexual dimorphism.
However, this model also has its weaknesses. One of them being that information
on genetics and integument of chickens are not as complete as those of mice (Sundberg et
al., 1998) and humans. While information on the human genome project and mouse are
rapidly accumulating, chicken genome project has started but is lagging behind (Burt et
al., 1995). On the other hand, there are mutations affecting chicken integument
appendages such as scaleless and wingless. Another potential which have not been
exploited is the large collections o f chicken or bird variants and breeds raised by poultry
fanciers (Smyth 1990; Somes, 1990). They show a wide spectrum of appendage shapes
and pigment patterns ranging from the bizarre to the beautiful, but many of them are
controlled by single genes! They also show how molecular processes can be pushed to the
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extreme to create morphology that can only be imagined in Nature. This is a treasure box
yet to be opened.
The ability to produce transgenic and knock-out mice is still the most powerful
aspect o f the mouse model. Although attempts were made (Ono et al., 1994), the
technology to produce transgenic birds is not yet mature. For now, it is possible to take a
“pseudo-genetic approach” using viral vectors such as those used in gene therapy. The
introduction of active or dominant negative genes to early chicken epithelial precursor
cells has contributed to new understanding o f skin development as stated in the text.
However, the application and interpretation are still limited by the incomplete transduction
and we hope future work from the research community will make transgenic chicken
available.
Another practical issue is that the background information on avian skin cell
biology requires more work. For example, the identification o f the equivalent of bulge
stem cells (Cotsarelis et al., 1990) in feather follicles, or the expression mapping o f keratin
markers (Rogers, 1985) need to be further pursued. This should be possible once more
investigators join to use the avian integument model. More information on the avian
integument model can be found at website http://www-hsc.usc.edu~cmchuong. More
information on the chicken genome can be found at the US Poultry Gene Mapping website
http://poultry.mph.msu.edu, and the Roslin Institute ChickMap website
http://www.ri.bbsrc.ac.uk/chickmap.
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1.4 Conclusion
Skin appendage formation is a complex morphogenic phenomenon. While many
biomedical researches are carried out in the hope of enhancing human health and curing
diseases, many unexpected clues and novel findings have come from basic developmental
biology studies. Among the many examples, avian embryology is one o f the most powerful
models. It is also advantageous to compare and contrast the role o f signaling molecules in
different model systems so we can appreciate the conservation and variation of
developmental pathways. The study of avian integument, in conjunction with transgenic
mouse studies (Sundberg et al., 1998) and human diseases such as ectodermal dysplasia
(Slavkin et al., 1998), wfll bring our knowledge in skin appendage morphogenesis to a
new level in the next decade.
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CHAPTER 2: ASYMMETRIC EXPRESSION OF NOTCH / DELTA / SERRATE
IS ASSOCIATED WITH THE ANTERIOR-POSTERIOR AXIS OF FEATHER
BUDS
SUMMARY
We studied the roles of Notch, Delta and Serrate in vertebrate epithelial appendage
morphogenesis using feather as a model and found the following. 1) C-Notch-I, C-Delta-1
and C-Serrate-1 are not expressed at the early placode stage and are therefore not
involved in the determination of bud versus interbud compartments. 2) From symmetric
short buds to asymmetric long buds, C-Delta-l and C-Serrate-1 are expressed in the
posterior bud mesenchyme in a nested fashion, while C-Notch-1 is expressed as a stripe
perpendicular to the anterior-posterior (A-P) axis and positioned posterior to the
midpoint. 3) Epithelial - mesenchymal recombination with rotation led to the
disappearance o f these gene transcripts followed by their re-appearance with new
positions appearing to predict their new morphological orientation. 4) Conditions leading
to branched buds (e.g., recombination of later buds) show polarized staining patterns
before branching occurs. S) Conditions leading to symmetrical round buds (e.g., treated
with the protein kinase A agonist forskolin) suppress expression of all three genes. These
results lead us to hypothesize that Notch, Delta and Serrate are involved in establishing
the A-P asymmetry o f feather buds.
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2.1 Introduction
Epithelial appendage morphogenesis involves a series of fate determination
processes that progressively transform a homogeneous epithelial sheet into complex
structures. We have been using feather development as a model to study this process
(Chuong, 1993). At the placode stage, the fate of the epithelium is separated into placodal
and interplacodal regions. At the short bud stage (when the bud height is shorter than the
base), the buds protrude out of the surface but remain symmetrical. At the long bud stage
(when the bud height is longer than the base), the buds elongate and become asymmetrical
by slanting posteriorly. Therefore during these developmental stages, the presumptive skin
is first divided into periodically arranged bud and interbud domains, then the bud domain
is subdivided into anterior and posterior domains. What are the molecules involved in
these processes?
Notch and its ligands Delta and Serrate are transmembrane proteins shown to be
involved in regulating cell fate commitment in several developmental models (reviewed in
Artavanis-Tsakonas et al., 1995; Lewis, 1996). In a field o f equivalent precursor cells,
activated Notch maintains cells in an undifferentiated state, while cells with inactive Notch
are allowed to differentiate. In Drosophila, the Notch pathway is involved in neurogenesis
through lateral inhibition (Artavanis-Tsakonas et al., 1995) and in wing patterning by the
control o f position-specific cell proliferation (Speicher et al., 1994). Notch and related
genes are expressed in developing (Weinmaster et al., 1991, 1992; Shawber et al., 1996a)
and mature (Kopan and Wemtraub, 1993) rodent hair follicles. These results suggest that
Notch may play roles in vertebrate epithelial fete determination- How Notch and related
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molecules may be involved in the early stages o f skin appendage development has not been
explored. In this report, we studied their roles in early feather development and found
evidence suggesting that the Notch signaling pathway may be involved in anterior-
posterior (A-P) axis determination in chicken feather buds.
2.2 Materials and Methods
2.2.1 Embryos
White leghorn fertilized chicken eggs were purchased from SPAFAS (Preston,
CT). Embryos were incubated at 37.5°C and were staged according to Hamburger and
Hamilton, 1951.
2.2.2 Plasmids and probes
C-Notch-l, C-Delta-1, and CSerrate-1 plasmids were obtained from Drs. Julian
Lewis, Domingos Henrique and Anna Myat and were described in Henrique et al., 1995
and Myat et al., 1996. Antisense C-Notch-I probes were transcribed with T7 RNA
polymerase from the HindiII- linearized plasmid. Antisense C-Delta-1 probes were
transcribed with T3 RNA polymerase from the EcoRI-Iinearized plasmid. Antisense C-
Serrate-1 probes were transcribed with T7 RNA polymerase from the Hindlll-linearized
plasmid.
2.2J Whole-mount in situ hybridization
This procedure was carried out according to procedures described in Nieto et al.,
1996. Briefly, embryos or skins were dissected in Rnase-free phosphate-buffered saline
(PBS) and fixed in 4% paraformaldehyde at 4°C for 2 hr. to overnight. Tissues were
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dehydrated, rehydrated, bleached, digested with proteinase K and then fixed again in 0.2%
glutaraldehyde / 4% paraformaldehyde. Samples were then hybridized at 65-70°C
overnight in hybridization buffer containing 0.2pg/ml digoxigenin-Iabeled riboprobes.
Samples were washed the following day in 2X SSC/0.1%CHAPS solution 3-5 times,
0.2XSSC/0.1%CHAPS solution 3 times and PBT twice. They were then blocked in PBT
containing 20% goat serum before incubation with alkaline phosphatase-conjugated anti-
digoxigenin Fab fragment antibody (Roche) at 4°C overnight. Samples were washed in at
least 5 changes o f PBT containing ImM levamisole for 1 hr. each. Samples were
equilibrated in NTMT solution (lOOmM NaCL, lOOmM Tris-HCL, pH 9.5, 50mM MgCl:,
0.1% Tween-20) before the addition of the alkaline phosphatase substrates NBT (4.5pl
per ml) and BCIP (3.5pl per ml).
2.2.4 Paranin section in situ hybridization
This procedure was carried out according to procedures described in Nieto et al.,
1996. Briefly, embryos were fixed in 4% paraformaldehyde, dehydrated through an
ethanol series (70%, 80%, 95%, 100%), embedded in paraffin wax, sectioned at 7-10pm
thickness and then mounted on TESPA-coated slides. These paraffin sections were then
dewaxed in xylene, rehydrated through an ethanol series (100%, 95%, 90%, 80%, 70%,
50%, 30%) and postfixed in 4% paraformaldehyde for 30 min. The specimens were then
digested with proteinase K (lOpg/ml) for 5 min., refixed with 4% paraformaldehyde and
washed in Tris/glycine buffer. The tissue sections were then hybridized overnight at 60°C
in section in situ hybridization buffer (40% formamide, 5X SSC, IX Denhard’s solution,
lOOpg/ml salmon testis DNA, lOOpg/ml tRNA) containing Ing/pl of digoxigenin-Iabeled
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riboprobes. After post-hybridization washes and digestion with RNAse A (lOpg/ml), the
slides were blocked with blocking solution (1% Roche blocking reagent in lOOmM maleic
acid and 150mM NaCl, pH 7.5) and incubated with alkaline phosphatase conjugated sheep
anti-digoxigenin Fab fragment antibody (Roche). The bound antibody was detected with
the BM purple substrate (Roche).
2.2.5 Skin explant and recombined skin explant cultures
The methods for skin explant and recombined explant culture are described in
Jiang et al., 1998. Briefly, dorsal skins from stages 28-34 chicken embryos were dissected
in Hank’s buffered saline solution (HBSS). When recombination was done, the skin was
incubated in 2X calcium-magnesium-free saline (274mM NaCl, 8mM KC1, 0.8mM
NaH,P04 , 0.4mM KH2 P 04 , 24mM NaHC03 , 20mM glucose, pH 7.3) containing 0.25%
EDTA (weight/volume) on ice packs for 10 min. The loosened epithelium was then
separated from the mesenchyme using watchmaker’s forceps. The free epithelium was
placed back on top o f the mesenchyme after a 90°C rotation with respect to its original
position. The skin explants and recombinants were then transferred to culture inserts in 6-
well culture dishes and cultured in Dulbecco's modified Eagle’s medium (DMEM)
containing 10% fetal calf serum. Forskolin-treated skin explants were cultured in DMEM
containing 10% fetal calf serum and 20pM forskolin.
2.2.6 BrdU labeling and detection in whole mount tissue
Chicken embryonic dorsal skins were dissected and pulsed with 20pM BrdU in
DMEM at 37°C for 30 min. After labeling, the tissues were fixed in absolute m e t h a n o l at
4°C overnight, bleached with 10% hydrogen peroxide in 1:4 DMSO/Methanol for 2 hr.
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and rehydrated. Then the tissues were incubated in 2N HC1 at RT for 1 hr. to denature
DNA. After neutralization with 0.1 M borate buffer and washing, the tissue was incubated
in PBS/0.1%BSA containing anti-bromodeoxyuridine antibody (Roche) at 4°C overnight.
After washing with PBS, the tissues were incubated with biotinylated horse anti-mouse
antibody at RT for 3 hr. After washing, ABC kit (Vector) was applied and allowed to
incubate for 2 hr. The color development was revealed after addition of diaminobenzidine
(DAB). To observe histologically, the tissue were embedded in wax and sectioned. To
embed, the tissues were dehydrated through an EtOH series, cleared with xylene and then
infiltrated with melted wax. After embedding, lOpM sections were cut, dewaxed and
mounted on slides for observation under the microscope.
2.3 R esults
2.3.1 C-Notch-l, C-Delta-1, and C-Serrate-l are absent in the placode stage but are
expressed in restricted patterns in posterior compartments when feather buds appear
We used whole mount and paraffin section in situ hybridization to examine the
expression of C-Notch-l, C-Delta-1, and C-Serrate-1 from stages 29 to 36 chicken
embryos (Figs. 2.1- 2.2). Since feathers start from the midline and propagate laterally,
there is a spectrum o f developmental stages starting from the lateral edge. C-Notch-l is
absent in the flat epithelium and placodes and does not appear until the short bud stage
(Figs. 2.1 A-C, 2.2A-B). From the top view, C-Notch-l is expressed most remarkably as a
center stripe (about 70pm in width, or 5-7 cells wide). This stripe is perpendicular to the
future A-P axis o f the bud, which is still round and symmetrical at this stage (Figs. 2.1A
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FIG. 2.1. Expression pattern of C-Notch-l, C-Delta-1 and C-Serrate-1 in developing
feather buds as shown by whole-mount in situ hybridization.
In situ hybridization o f chicken dorsal skin explants. C-Notch-l (A - C), C-Delta-1
(A’ - C') and C-Serrate-1 (A” - C”). Feather buds from placode stage (PL), short bud
stage (SB), and long bud stage (LB) are shown. Anterior is to the left o f the panel.
A-A”, Stage 33. B-B”, Stage 36. White arrows in A-B” line up with the middle
row o f each skin explant. Note there is a maturation gradient from the midline to the
lateral rows. C-C’\ Enlarged short buds, with dotted lines placed outside the epithelial
border o f the bud. Note feather primordia o f different stages show different expression
patterns. Most dramatic is the mid C-Notch-l stripe, and the posterior mesenchymal
nested pattern o f C-Delta-1 and C-Serrate-1 at the short bud stage.
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Notch Delta Serrate
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FIG. 2.2. Expression pattern of C-Notch-l, C-Delta-1 and C-Serrate-l in developing
feather buds as shown by paraffin section in situ hybridization.
A-A”, Placode stage. C-Notch-l, C-Delta-1 and C-Serrate-1 are absent. B-B”,
Short bud stage. C-Notch-l is mainly in the mesenchyme and weakly in the epithelium. C-
Delta-l and C-Serrate-1 are only in the mesenchyme. In the C-Notch-l panel, the straight
arrow indicates the midline stripe, and the arrowheads point to the posterior crescent. The
dotted lines mark the epithelial boundary over a feather bud. Anterior is to the left of the
panel.
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Notch Delta Serrate
A A r t _____ pad PL A’ A”
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and C). In addition, there is faint expression throughout the posterior half and in a
stronger crescent along the posterior margin. This expression is transient. As feather buds
elongate, C-Notch-l expression becomes weak and is diffusely distributed (Fig. 2. IB).
C-Delta-1 is also absent in the placodes and starts to appear at the short bud stage
(Figs. 2.1A’-C \ 2.2A’-B’). It starts in the mesenchyme near the posterior edge and
extends in the anterior direction, which is manifested as a transition from a crescent to a
half-moon staining pattern (Fig. 2.1 A’). As the buds start to elongate, C-Delta-1
disappears completely (Fig. 2.IB’). C-Delta-1 is always negative in the epithelium.
C-Serrate-1 is also absent until the short bud stage, then appears in the posterior
mesenchyme of short buds in a fashion similar to that of C-Delta-1 (Figs. 2.1 A”-C”,
2.2A”-B”). From the top, C-Delta-1 and C-Serrate-1 appear to have overlapping
expression domains at the short bud stage (Figs. 2.1C’-C”). These particular sections
show that C-Serrate-1 occupies a smaller domain than C-Delta-1 (Figs. 2.2 B’-B”).
However, the nested relationship shifts during development and comparison of precise
compartment borders will depend on future double in situ hybridization. At the long bud
stage, C-Serrate-1 becomes localized to the proximal posterior region of the bud (Fig.
2. IB”).
The absence of these three genes at the early placode stage precludes the
possibility that C-Notch-l, C-Delta-1 and C-Serrate-1 are involved in the initiation o f
feather primordia. However, it is possible that other forms of Notch, Delta and Serrate
(Weinmaster et al., 1992; Myat et al., 1996; Shawber et al., 1996a) may be involved at
the early stages.
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Section in situ hybridization showed that C-Delta-1 and CSerrate-l are restricted
to the mesenchyme (Figs. 2.2B’-B”). C-Notch-l is mainly in the mesenchyme with weaker
staining in the epithelium (Fig. 2.2B). The molecular subdivision o f feather buds into
several anterior - posteriorly arranged compartments by Notch, Delta and Serrate strongly
suggests that they are involved in establishing A-P asymmetry. These compartments may
then show characteristic cellular behavior such as differential cell proliferation that can
contribute to the asymmetric shaping o f the buds. For example, if cells in the posterior bud
proliferate faster than cells in the anterior bud, there will be more expansion in the
posterior domain and the bud will slant. In accord with this hypothesis, we observed
preferential distribution o f S phase cells in the posterior feather bud epithelia and
mesenchyme using BrdU labeling (Fig. 2.3; also see Desbiens et at., 1992).
Several experimental conditions can perturb feather A-P axial orientation. If
molecules o f the Notch pathway are involved in determining the A-P axis, their expression
should be polarized before altered morphological asymmetry.
2.3.2 C-Notch-l, C-Delta-1 and C-Serrate-1 expression is regulated by epithelial and
mesenchymal interactions and the new polarized expression pattern precedes
morphological asymmetry following recombination
Rotated buds Epithelium and mesenchyme from skin explants can be separated
and recombined. Old feather buds will disappear and new buds will form. To test the
origin o f the orientation activity, we can rotate the relative positions of the epithelia and
mesenchyme, recombine them, and then culture the recombined explants. When this was
done on skins from stages 29 to 33, the locations o f the new feather buds are in accord
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FIG. 2.3. BrdU labeling.
Populations of S phase cells are preferentially localized in the posterior epithelium
and mesenchyme of the short feather buds.
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with the original dermal condensations, but their A-P orientation is in accord with the
original epithelium (Novel, 1973; Chuong et al., 1996). We examined the expression of C-
Notch-1, C-Delta-1 and CSerrate-l following a 90° rotation (Figs. 2.4A-B”). Three
hours following the recombination, C-Notch-l and C-Delta-1 have disappeared, and C-
Serrate-I transcripts are reduced and will disappear (Figs. 2.4A-A”). Naked mesenchyme
(to the right o f the dashed lines in Figs. 2.4A-B”) also lose their C-Notch-l, C-Delta-1 and
C-Serrate-1 transcripts but, surprisingly, at a slower rate. These results suggest that the
maintenance o f C-Notch-l, C-Delta-1 and C-Serrate-1 requires the interactions between
epithelia and mesenchyme, and that epithelium not directly above the Notch stripe may
have a negative effect on the mesenchymal expression of C-Notch-l.
Thirty hours after recombination, when the buds are still round and symmetrical.
C-Notch-l, C-Delta-1, and C-Serrate-1 are expressed asymmetrically (Figs. 2.4B-B”).
For C-Notch-l, the central stripe is now re-orientated to be perpendicular to the new
cephalic - caudal orientation o f the epithelium. Similarly, C-Delta-1 and C-Serrate-1 re
appeared in a polarized fashion. In 72 hrs, the regenerated buds elongate and become
morphologically asymmetric (Fig. 2.4B’ inset). The A-P axis is set in the direction as
predicted by the positions of C-Notch-l, C-Delta-1, and C-Serrate-1.
Branched buds When recombination / rotation was carried out on explants from
stage 34 embryos, many branched feather buds formed (Fig. 2.5A). We then examined the
expression o f these genes under this experimental condition. At 10 hours after
recombination, we observed bipolar and even tripolar expression o f C-Delta-1 in the
round buds (Fig. 2.5A’), as a prelude for the subsequent branched feather bud formation.
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FIG. 2.4. Expression of C-Notch-l, C-Delta-1 and C-Serrate-1 in experimentally
rotated feather buds.
Epithelial (E) - mesenchymal (M) recombination with 90° rotation were performed
on skin from stage 33 embryos. Whole-mount in situ hybridization using C-Notch-l (A,
B), C-Delta-1 (A’, B’) and C-Serrate-l (A”, B”) probes on explants fixed at 3 hr (A-A”)
and 30 hr (B-B”) after recombination. Thin arrows point to the posterior end of the
epithelium or mesenchyme. The dashed lines mark the border o f the recombined
epithelium. Regions to the right o f the line are naked mesenchyme. Broad arrows point to
the posterior o f regenerated feather buds when they elongate in 3 days (inset in B’). Note
the disappearance and reappearance of C-Notch-l, C-Delta-1 and C-Serrate-1.
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FIG. 2.5. Expression of C-Notch-l, C-Delta-1 and C-Serrate-1 in experimentally
manipulated branched feather buds.
E-M recombination with rotation was carried out on skin from stage 34 embryos.
A, Phase contrast photograph o f the explant after S days o f culture show many branched
feather buds (*). Whole-mount in situ hybridization using C-Delta-1 probe (A’, 10 hr after
recombination) show bipolar (*) and tripolar (V) expression. A”, E-M recombination with
90° rotation was carried out on skin from stage 35 embryos. Mesenchyme flanking the
midline (white arrow) are more mature and maintain original orientations. Mesenchyme
toward the lateral edge (upper panel) are still flexible and follow the orientation of the
epithelia.
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C-Delta-1
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When recombination / rotation was carried out on skins from stage 35 embryos, the
mesenchymal orientation o f the more mature buds (those flanking the midline of the
explant) becomes resistant to change; however, the lateral mesenchyme is still receptive to
the orientation signals from the epithelia (Fig. 2.5A”). At stage 35, long feather buds have
formed in regions flanking the midline o f the explant, while the rest of the skin is mainly
composed of short feather buds. This suggests that dermal condensations up to the short
bud stage are flexible and can respond to epithelial signals to set up new orientations. As
they mature into long feather buds, the flexibility o f the mesenchyme is gradually lost and
the orientation signals shift from the epithelium to the mesenchyme. In the middle o f this
transition, when the epithelium and the mesenchyme have about equal orientation signals,
branched buds form.
2.3.3 Protein kinase A (PKA) agonists lead to round symmetric buds that lack the
expression o f C-Notch-l, C-Delta-1 and C-Serrate-1
Symmetric buds PKA agonists such as cAMP and forskolin can enhance dermal
condensations and inhibit feather bud elongation. The phenotype is discrete short round
buds (Noveen et al., 1995a). If the Notch pathway is involved in setting up the A-P axis,
we predict that there should be a suppressed or diffuse expression of C-Notch-l, C-Delta-
1 and C-Serrate-1 in the round feather buds in this type o f skin explants. Indeed we
observed the normal stripe and posterior expression o f C-Notch-l, C-Delta-1, C-Serrate-1
in control explants, but a lack o f their expression in forsko Iin-treated explants 18 hr after
culture (Figs. 2.6A’-B” and not shown). Five days later, while control buds have
elongated (Fig. 2.6A), the forskolin-treated buds are still round and short (Fig. 2.6B).
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FIG. 2.6. Expression of C-Notch-l, C-Delta-l and C-Serrate-l in experimentally
manipulated symmetric feather buds.
Skin explants were not treated (A- A”) or treated (B-B”) with the PKA agonist,
forskolin (20pM). The explants were hybridized with C-Notch-l (A’, B’) and C-Delta-l
(A”, B”) 18 hrs after culture. Note the disappearance o f C-Notch-l and C-Delta-l. The
treated buds remain round and short 5 days after culture (B) while the feather buds on the
control explant elongate normally (A).
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(< u |D 0 3 n g o i|u o i
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Thus, the polarized expression o f C-Notch-l, C-Delta-l and C-Serrate-1 at the short bud
stage appears to predict the elongation and the axial orientation o f this elongation at the
long bud stage.
The phosphorylated cyclic AMP responsive enhancer binding protein (pCREB) can
be detected by antibody staining and used as an indicator of PKA activity. pCREB is
preferentially expressed in the anterior short bud (Fig. 1C in Noveen et al., 1995a) at
about the time C-Notch-l, C-Delta-l and C-Serrate-l are preferentially expressed in the
posterior mesenchyme. Thus it is possible that activation of PKA may down-regulate
Notch, Delta and Serrate expression in the anterior bud. Speculatively, we may view the
cAMP-treated buds as completely “anteriorized” by eliminating the Notch pathway and
the posterior compartment.
2.4 Discussion
This report sets the base to pursue many interesting issues regarding the
establishment of A-P asymmetry in feather buds. It is apparent now that this event does
not occur until the transition from short bud to long bud stage. A review of previous data
showed that NCAM and Hox C6 are expressed all over the bud mesenchyme at the short
bud stage, and only become localized to the anterior mesenchyme in long buds when bud
morphology is asymmetrical (Chuong and Edelman, 1985; Chuong et al., 1990). Similarly,
Shh is symmetrically expressed in the central placode at the short bud stage, and only
becomes localized to the posterior placode at the long bud stage (Ting-Berreth and
Chuong, 1996). Notch, Delta and Serrate are unique because they are already polarized
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when buds are still round. Two other molecules are asymmetrically expressed at a
comparable early stage. One is pCREB in the anterior mesenchyme (Noveen et al.,
1995a). The other is Wnt 7a in the posterior placode epithelium (Chuong et al., 1996).
Whether these molecules are involved in regulating the expression o f Notch, Delta and
Serrate in vivo and which molecule(s) are the initiators of A-P axis determination remain
to be determined.
Supposing that Notch, Delta and Serrate are involved in establishing the A-P axis,
what may be the mechanism? In the imaginal wing disc of Drosophila, Serrate has been
shown to mediate position-specific cell proliferation and contributes to the control of
dorsal / ventral patterning (Speicher et al., 1994; Diaz-Benjumea and Cohen, 1995). In the
short feather buds, BrdU labeling is originally homogeneously distributed, but later shifts
to the posterior region (Fig. 2.3; Desbiens et al., 1992; Noveen et al., 1995b). One
compelling hypothesis is that differential growth o f the anterior - posteriorly arranged
compartments can lead to an asymmetric morphology (Figs. 2.3 and 2.7). A higher
proliferation potential may be maintained in cells with an activated Notch pathway. To test
this hypothesis, in the future we will test whether feather symmetry is altered by mis-
expressing molecules in the Notch pathway with retroviral gene transduction. We will also
analyze cell lineage and trafficking in the forming buds with lineage tracer labeling.
It is surprising that C-Notch-l, C-Delta-l, and C-Serrate-l are not involved in the
early specification between primordia and inter-primordial regions. However, there are
multiple members o f Notch and related signaling molecules (Weinmaster et al., 1992;
Myat el al., 1996; Shawber et al., 1996a) that may act at different stages during feather
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FIG. 2.7. A working model.
Events are listed in time sequence. Question marks indicate that a causal
relationship is not established. The nature o f epithelial A-P positional information remains
to be determined and may involve PKA, Wnt 7a, gap junction communication (Serras et
al., 1993) or other molecular activities.
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Dermal condensation
without polarity
Epithelial ____ ^
signal
Morphologically symmetric
bud with asymmetric
expression of Notch,
Delta and Serrate
Transient preferential
proliferation in posterior
bud mesenchyme
Morphologically asymmetric
bud slants caudally
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46
morphogenesis. Further work is required to determine how the expression domains of
these molecules are lined up (juxtaposed, overlapped, or apart?) and interpreted by local
cells. The sharing o f Notch signaling molecules in Drosophila and avian epithelial
appendage morphogenesis further demonstrates the fundamental nature o f this signaling
mechanism. Elucidation o f these processes will advance our understanding in the intricate
patterning of feathers.
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CHAPTER 3: DYNAMIC EXPRESSION OF LUNATIC FRINGE DURING
FEATHER MORPHOGENESIS: A SWITCH FROM MEDIAL-LATERAL TO
ANTERIOR-POSTERIOR ASYMMETRY
SUMMARY
Expression of Lunatic fringe mRNA was studied during feather morphogenesis
and showed three stages o f dynamic expression pattern. (1) Lunatic fringe was first
expressed in the epithelium as a ring bordering the feather primordium when it was initially
induced. (2) Shortly after, it showed a polarized pattern, first toward the lateral side of the
feather primordium and then made a 90°C switch toward the posterior side of the short
bud. It then becomes weakly expressed in the long bud stage. (3) Finally, it is expressed in
the marginal plate epithelia of feather filaments. In contrast, Radical fringe is weakly
expressed in the feather bud and is also present in the marginal plate epithelia of feather
filaments.
3.1 Introduction
The fringe gene was first isolated in Drosophila (Irvine and Wieschaus, 1994) and
has since been studied in many systems, including Drosophila wing (Irvine and Wieschaus,
1994) and eye (Cho and Choi, 1998), vertebrate neurogenesis (Sakamoto et al., 1997),
vertebrate somitogenesis (Evard et al., 1998; Forsberg et al., 1998; McGrew et al., 1998;
Zhang and Gridley, 1998), vertebrate limb bud development (Rodriguez-Esteban et al.,
1997; Laufer et al., 1997) and mouse skin (Thelu et al., 1998). In many of these models,
Fringe is found to be involved in boundary determination via the Notch s i g n a l i n g pathway
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by modulating the interaction of Notch receptor and its ligands (Panin et al., 1997). Notch
and its related molecules have been found to be expressed in various stages o f feather
morphogenesis (Chen et al., 1997; Crowe et al., 1998; Viallet et al., 1998). It is thus of
interest to examine whether the two chick fringe homologs, Lunatic and Radical fringe,
are also expressed in the feather to set up boundaries. Although L-fng was used as a
marker to show that it is present in the posterior domains o f both normal and noggin
transduced feather buds (Noramly and Morgan, 1998), a complete study focused on L-frig
has not been presented. This paper presents a complete report on the dynamic expression
of L-fng in different stages o f feather development, particularly the surprisingly novel
finding that its expression goes through a transition from medial-lateral asymmetry to
anterior-posterior asymmetry.
3.2 Materials and Methods
3.2.1 Embryos
White leghorn fertilized chicken eggs were purchased from SPAFAS (Preston,
CT). Embryos were incubated at 37.5°C and were staged according to Hamburger and
Hamilton, 1951.
3.2.2 Plasmids and probes
The chicken Lunatic fringe, Radical fringe and C-Serrate-2 plasmids were
obtained from Dr. Cliff Tabin and were described in Laufer et al., 1997. Antisense Lunatic
fringe and Radical fringe probes were transcribed with T3 RNA polymerase from the
Clal-linearized plasmids. Antisense C-Serrate-2 probes were transcribed with T3 RNA
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polymerase from the EcoRI-linearized plasmid. The C-Serrate-1 plasmid and probe were
discussed in section 2.2.2.
3.2.3 Whole-mount in situ hybridization
see section 2.2.3.
3.2.4 Paraffin section in situ hybridization
see section 2.2.4.
3.2.5 Tissue sectioning
Embedding and sectioning tissue samples after whole-mount in situ hybridization
were done as described in Nieto et al. 1996. Briefly, the samples were fixed in 4%
paraformaldehyde overnight at 4°C. The samples were then washed with PBS, MeOH,
isopropanol and tetrahydronaphthalene before they were embedded in paraffin wax. The
embedded samples were then cut on a microtome at 7- 10pm thickness and mounted on
clean slides. The slides were then dewaxed with xylene and mounted under coverslips
using mounting media (Richard Allan).
3.3 Results
Whole-mount in situ hybridization on chick embryos shows that Lunatic fringe (L-
fn g ) is expressed in dorsal feather primordia dynamically. The expression pattern goes
through three phases: 1) a circular pattern in feather primordia, 2) a polarized pattern in
feather buds, first laterally then posteriorly and 3) a striped pattern in marginal plates of
feather filaments.
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The first two phases are illustrated in Figs. 3.1A-E, where embryos of
progressively more mature stages are shown. The feather primordia rows are numbered in
their order of appearance. Feather placodes appear sequentially from the midline (row 0)
of the dorsal skin so that the most lateral rows are the most nascent. In a H&H stage 30
embryo (Hamburger and Hamilton, 1951) which has only one row of feather placodes, L-
Jhg is expressed as rings encircling the placodes (Fig. 3.1 A). Similar ring expression
pattern in feather primordia was observed in Wnt-7a (Widelitz et al., 1999). In another
stage 30 embryo where rows 2/2’ are starting to appear, rows 2/2' show feint circular L-
Jhg expression, while rows 1/1’ have L-fng in the lateral domain (lateral o f the spinal tract,
or the side facing the apteric region) and row 0 has posterior expression (Fig. 3.1B). In a
stage 31 embryo with 7 rows o f feather primordia forming, a similar pattern is observed.
In the boxed region in Fig. 3.1C, L-fng has just begun to be expressed as feint circles
bordering the feather placodes in rows 3/3'. But feather buds in rows 1, 1', 2 and 2' have
polarized L-fng expression, with rows 2/2' having localized expression toward the
right/left lateral side respectively and rows 1/1' having localized expression toward the
posterior side. The middle row feather buds have polarized L-fng expression toward the
posterior domain. For comparison, an embryo o f equivalent stage and probed with C-
Serrate-1 is shown (Fig. 3. IF). C-Serrate-1 is expressed in the posterior domain of all
seven rows of feather buds. Enlarged views o f the boxed regions in Figs. 3.1C and 3. IF
and are shown respectively in Figs. 3.1C’ and 3.IF’. The arrows indicate the polarity of
gene expression. L-fng has the lateral-posterior switch but C Serrate-l doesn’t. This
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FIG. 3.1. The first two phases of L-fng expression in dorsal feather morphogenesis.
Whole mount in situ hybridization to show L-fng expression in the dorsal tract of
early embryos. Arrowhead in A points to the circular L-fng expression. Arrows in B, C \
D and F’ point to the localization of polarized L-jhg expression. Cross lines in C and E
show the section planes for the insets respectively. C’ and F’ are enlargements of the
boxed areas in C and F. The numbers designate the sequential rows. Phase 1: Circular
expression along the border o f a feather primordium. Phase 2: A novel expression mode
where the polarized expression undergoes a 90°C lateral-posterior switch. This was not
observed in other genes such as in C-Serrate-1. Se = C-Serrate-1, a = anterior, p =
posterior, size bar = 1mm.
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intriguing switch of medial-lateral to anterior-posterior polarization is seen in many
different embryos (n=15).
As can be observed from Figs. 3.1A-C and further supported by Figs. 3.1D-E,
feather buds that initially have lateral L-fng expression later express L-fng in the posterior
domain. In Figs. 3.ID and 3.IE, embryos of stages 32 and 34 have many rows o f feather
buds. While the most lateral rows show feint circular expression or lateral expression
(rows 4/4* in Fig. 3.1D or rows 8/8' in Fig. 3.1E), the center rows have L-fng in the
posterior domain. The feather buds in rows 2/2' and 3/3' that expressed L-fng laterally at
stage 31 later express L-fng posteriorly when the embryo reaches stage 32. As feather
placodes continue to grow into short and then long bud stages, L-fng expression
decreases. The feather buds in the center rows of a stage 34 embryo have weaker L-fng
expression (Fig. 3.IE).
In situ hybridization on longitudinal sections of short feather buds shows that only
the posterior epithelium o f the feather placode expresses L-fng (inset in Fig. 3.1C). As
feather buds elongate, L-fng expression slowly decreases but remains localized distal-
posteriorly (inset in Fig. 3.IE).
L-fng is also expressed in the feather buds in the femoral tract (Figs. 3.2A-C). It is
also first expressed in a ring pattern (Fig. 3.2A). As feather placodes advance into the
short bud stage, L-fhg expression intensifies in the posterior domain o f the feather buds in
the posterior lateral region (where feathers started to form and therefore are more mature)
o f the femoral tract. However, in newly forming feather primordia, the polarization is
toward the midline or the apteric region (Fig. 3.2B). By stage 34, all feather buds have the
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FIG. 3.2. The first two phases of L-fng expression in femoral feather morphogenesis.
Whole mount in situ hybridization to show L-fng expression in the femoral tract o f
early embryos. Arrowhead in A points to the circular L-fng expression. Arrows in B’
points to the localization o f polarized L-fng expression. B’ is an enlargement of the boxed
area in B. Phase 1: Circular expression along the border of a feather primordium. Phase
2: A novel expression mode where the polarized expression undergoes a 90°C lateral-
posterior switch. Size bar = I mm.
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expression pattern as posteriorly localized (Fig. 3.2C). Thus in both spinal and femoral
tracts, the initial localization o f L-fng expression appears to be toward the side where the
apteric region resides.
In comparison to L-fng, R-fng expression is weak and much less dynamic. It is
expressed within the placodes in both the dorsal tract and femoral tract feather buds (Figs.
3.3A-B), and later disappears. An enlarged view of a stage 33 skin probed with R-fng
shows that it is present in the center of the placodes (Fig. 3.3C).
As feather buds advance to the filament stage, the epithelium invaginates and later
differentiates into barb plates and marginal plates. Whole-mount in situ hybridization of L-
fitg, R-fng, C-Notch-l and C-Serrate-2 on H&H stage 35-36 skins shows that all four
genes are expressed in feather filaments as striking stripes (Fig. 3.4A-D). Sectioning the
feather filaments or performing in situ hybridization on sections shows that L-fng and R-
fn g appear periodically in the marginal plate epithelial region similar to C-Serrate-2 (Figs.
3.4A\ B’ and D’ and Crowe et al., 1998). This is in contrast to C-Notch-l, which is
expressed in the barb plate (Fig. 3.4C’ and Crowe et al., 1998). The stage for the R-fng
specimen is younger, and a low level o f R-fng expression is seen beyond the marginal plate
epithelia.
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FIG. 3.3. R-fng expression in feather morphogenesis.
Whole mount in situ hybridization to show R-fng (A-C) expression in the dorsal
(A, C) and femoral tracts (B) of early embryos. Dotted circles in C demarcate the feather
placode and the R-fng expression boundary. R-fng is only weakly expressed. Size bar =
500pm (A-B) or 100pm (C).
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FIG. 3.4. The third phase o f L-fng expression in feather morphogenesis.
Whole mount and section views o f R-fng, L-fng, C-Notch-l and C-Serrate-2
expression in feather filaments. Cross lines in A-D show the section planes for A’-D \ A’,
B’ and D’ are sections o f skins after whole-mount in situ hybridization. C’ is from section
in situ hybridization. Dotted markings delineate the basal layer o f the epithelium giving rise
to barb plates and marginal plates (A’-D’). Phase 3: R-fng, L-fng, and C-Serrate-2 are
mainly in the marginal plates while C-Notch-l is in the barb plate. Bp=barb plate,
mp=marginal plate, size bar = 100pm (A-D) or 50pm (A’-D’)
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CHAPTER 4: FUNCTIONAL STUDIES OF THE NOTCH SIGNALING
PATHWAY DURING FEATHER MORPHOGENESIS
SUMMARY
To examine the role o f the Notch signaling pathway in feather morphogenesis, two
active forms of rat Notchl and C-Delta-l were misexpressed in vivo and in the
reconstituted skin explants. No irregular phenotypes were observed when rat Notchl was
misexpressed using the described reagents and procedures. C-Delta-l misexpression
inhibited feather bud formation in the reconstituted feather buds, similar to the in vivo
results reported by other groups. An expansion o f proliferating cells is observed in the C-
Delta-/ -misexpressing feather buds, although their posterior outgrowth is normal. These
data suggest that C-Delta-l plays an essential role in the inductive phase o f feather bud
formation, but is not sufficient in establishing anterior-posterior polarity.
4.1 Introduction
The results in chapters 2 and 3 have shown that C-Notch-l, C-D elta-l, C-Serrate-
1 and Lunatic fringe are expressed dynamically in the early stages o f feather development.
They suggest that the Notch pathway plays an essential role in feather morphogenesis. To
study the function of a gene or pathway in development, it is often necessary to perturb its
normal activity level and assess the effect o f this perturbation. In studying the Notch
signaling pathway, work in other systems have discovered different methods o f its
overactivation. One method is by misexpression o f the ligand Delta (Henrique et al., 1997,
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Crowe et al., 1998, Viallet et al., 1998). Another method is by expression of a
constitutively-active form o f Notch (Rebay et al., 1993).
Misexpression o f the C-Delta-l ligand in chick embryo skins was first
accomplished by two independent groups and they showed that it leads to an inhibition of
feather placodes (Crowe et al., 1998, Viallet et al., 1998). Both groups reported that C-
D elta-l begins to be expressed in the forming feather buds at a stage earlier than what we
observed. However, the expression patterns shown are very weak and differ between the
two groups. Crowe et al. 1998 reported that C-Delta-l is expressed in the mesenchyme
underlying the forming epithelial placodes, while Viallet et al. reported that C-Delta-l first
appears as a '‘ wreath” and then becomes restricted to the posterior feather primordium.
Perhaps as a result of the lower concentration o f proteinase K . used in our whole-mount in
situ hybridization procedure which offered less sensitivity, we only detected C-Delta-l
expression as it appeared in a posterior crescent in the short feather buds (Chapter 2).
Despite the feet that C-D elta-l appears to be expressed at a low level in early feather
primordia, the misexpression results suggest that it plays a role in the inductive phase of
feather development. When C-Delta-l was misexpressed in a large region o f the skin, a
loss o f feather buds was observed. In this region, C-Notch-l and C-Notch-2 expressions
were upregulated (Crowe et al., 1998). However, when C-Delta-l was misexpressed in a
small region o f the skin, cells overexpressing C-Delta-l formed bigger feathers while their
adjacent cells failed to form feathers. Thus it was proposed that C-Delta-l plays a dual
role; it is involved in feather promotion while also participates in lateral inhibition. Crowe
et al. claimed that C-Deha-1 promotes feather growth through its interaction with C-
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Notch-1, whose RNA transcripts were found in the epidermal placodes. A high level o f C-
Delta-1 can overactivate C-Notch-l and accelerate feather growth. But C-Delta-l also has
an inhibitory role through its interaction with C-Notch-2, whose RNA transcripts were
found in the interbud dermis. In normal development, C-Delta-1 in the bud dermal cells
delimits the feather bud region by signaling to neighboring cells that express C-Notch-2.
When C-Delta-l is misexpressed via retroviral transduction to a small group o f cells, this
lateral inhibition affects only the neighboring cells. However, when a large group o f cells
overexpresses C-D elta-l, C-Delta-l induces C-Notch-2 and their interaction then leads to
an inhibition of feather bud formation over the entire region o f misexpression.
This model for the dual role o f C-Delta-l in feather bud development is based on
the hypothesis that C-Notch-l and C-Notch-2 each carry out one of the two distinct roles.
Yet it remains to be tested whether the phenotypes observed is a direct consequence of
Delta activation of Notch. To address this question, we attempted to express an activated
form of Notch both in ovo and in reconstituted skin explants. Another puzzling question is
that if C-Delta-l have these two seemingly antagonistic roles, how does it mediate and
balance these two actions? In Chapter 2, we also hypothesized another role for C-Delta-l,
that in establishing the anterior-posterior axis o f feather buds. To facilitate easier analysis,
we overexpressed C-Delta-l in our reconstituted skin explants model. We then analyzed
the effect o f misexpression on cell proliferation and cell adhesion by examining the
expression o f PCNA and cell adhesion molecules.
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4.2 Materials and Methods
4.2.1 Embryos
White leghorn fertilized chicken eggs were purchased from SPAFAS (Preston,
CT). Embryos were incubated at 37.5°C and were staged according to Hamburger and
Hamilton, 1951.
4.2.2 Plasmids and probes
RCASBP(A) and RCAS(A)AP plasmids were obtained from Dr. Donna Fekete.
RCAS(A) C-Delta-l and pBS-RCAS/w/ plasmids were obtained from Dr. Lee Niswander
and were described in Crowe et al., 1998. Antisense RCAS polymerase probes were
transcribed using T7 polymerase from the Xbal-linearized plasmid.
4.2.3 Construction of the RCAS-constitutively active Notch retrovirus
The pBOS-CDNl and pBOS-nCDNl plasmids were obtained from Dr. Gerry
Weinmaster. The pBOS-CDNl construct contains nucleotides 5716-8221 o f rat Notchl
and encodes for a constitutively active form of Notch that does not transactivate CBF-1.
The pBOS-nCDNl plasmid contains nucleotides 5727-8221 of rat Notchl and encodes
for the entire cytoplasmic domain o f Notchl that is able to transactivate CBF-1.
The CDN1 and nCDNl inserts were isolated from the pBOS plasmids by
restriction digest with Xbal and subcloned into the Clal2 shuttle vector. The resulting
plasmids were then digested with Clal and the CDN1 and nCDNl fragments were cloned
into RCAS(A)BP vectors. Clones with the inserted gene in the sense direction from the
viral promoter were selected by PCR and confirmed by sequencing.
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4.2.4 RCAS virus infection
The protocols for RCAS (Replication-Competent Avian Sarcoma Virus) virus
production, concentration, titration and injection are described in Morgan and Fekete,
1996. The chicken embryo fibroblasts (CEF) were used as retrovirus producing cells. The
retroviral vector DNA was transfected into 80% confluent CEFs in 10-cm plates using
calcium phosphate. The cells were cultured in DMEM containing 10% fetal bovine serum
and 2% chicken serum and passaged two times. After the second passage, cells were
grown to confluency and the culturing media containing retrovirus were collected 3 times
every 18-24 hrs. If a more concentrated retroviral stock is desired, the retroviral media
was ultracentrifuged to achieve up to a 100 fold concentration. After concentration, the
viral titer was usually around 107 infectious units (IU)/mI. The retroviral media were kept
in the -70°C freezer for long term storage and thawed in a 37°C water bath immediately
before usage. The retrovirus were either injected in ovo into the chicken embryo (in the
amnionic cavity or into the early limb buds) or used to infected dissociated mesenchymal
cells in reconstituted skin explants. For in ovo injection, fast green dye (1/40 volume of
1% stock) was added to aid visualization. The embryos were injected at embryonic day
3.5 (E3.5), around H&H stages 19-21. Eggs were resealed with tape and the embryos
were allowed to develop for 5-6 days at 37.5°C before they were sacrificed for
observation o f phenotypes.
The method for reconstituted skin explant culture is described in Jiang et al.. 1999.
For reconstitution, the epithelium and mesenchyme were first separated as in
recombination, but then the mesenchyme was incubated in 0.1% collagenase/trypsm
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66
solution for 5 min. at 37°C to dissociate it into single cells. The epithelium and the
dissociated mesenchymal cells were then incubated in retroviral media for 2 hr at 4°C.
After pelleting, the mesenchymal cells were allowed to reaggregate for I hr at 37°C in the
culture inserts. The epithelium was then placed on top o f the aggregated mesenchymal
cells resulting in a reconstituted skin explant that can be cultured in DMEM containing
10% FCS.
4.2.5 Immunohistochemistiy
Immunohistochemistry o f embryos sections was performed as described (Jiang and
Chuong, 1992). The paraffin sections were prepared as described in section 2.2.4. The
paraffin sections were dewaxed in xylene and rehydrated through an ethanol series and
distilled water. After circling around each tissue section with a PAP pen, the sections were
incubated with Zeller’s solution (lOmM Tris, lOOmM MgCl,, 5% fetal calf serum, 1%
BSA and 0.5% Tween-20, pH 7.4) for 30 min. Then the specimens were incubated with
primary antibody (diluted in Zeller’s solution) overnight in a humidified chamber at room
temperature. The sections were washed with TBST, incubated with secondary antibody
for 2 hr, washed again with TBST and incubated with tertiary antibody for 2hr. The
sections were washed with PBT and then the bound antibody was detected with the DAB
substrate. Secondary antibodies used were biotinylated goat anti rabbit or biotinylated
horse anti mouse antibodies (Vector). Streptavidin-HRP (Zymed) was added as the
tertiary antibody. Monoclonal antibody clone PC 10 to proliferating cell nuclear antigen
(PCNA) was used (DAKO. Denmark). Rabbit polyclonal antibodies to Tenascin C and
NCAM were from Jiang and Chuong, 1992.
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4.2.6 Quantitative measurements
Mocha image analysis program was used to measure the bud size, explant size and
number of feather buds (Fig. 4.3E-G). The bud size was determined by the area of the
feather bud. The bud density was calculated as the number o f buds per unit area. The
interbud space was calculated as the quotient o f the difference o f explant area and total
bud area and the bud number. The density o f proliferating cells was calculated as the
number o f PCNA-positive cells per unit area (Fig. 4.5E). A line was drawn at the mid line
of the feather bud to divide it into the anterior and posterior domains. The areas of these
regions were also measured by Mocha.
4.3 Results
4.3.1 Misexpression of activated Notch in vivo and in vitro resulted in
phenotypically normal feather buds
Structural and functional analyses have found that truncated Notch proteins are
dominantly activated and ligand-independent (Rebay et al., 1993). Work m vertebrate
systems has used this approach to study the effect o f gain-of-function o f Notch (Coffinan
et al., 1993; Nye et al., 1994). Since full-length chicken Notch genes have not yet been
reported, we decided to use available constructs from other species. Partial DNA
sequence indicates that C-Notch-1 shares 86% amino acid identity with the corresponding
part o f rodent Notchl (Myat et al., 1996). This relatively high degree o f conservation
predicts that rat Notchl protein would function in chicken cells. We obtained two
activated forms o f rat Notchl from Dr. G. Wemmaster. Both o f these are intracellular
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68
forms o f Notch but differ in their ability to transactivate CBF-1 (Shawber et al., 1996b).
Their work using these constructs found that Notch signaling can go through a
CBFl/Su(H) independent pathway (see section 5.1 for more discussion). A schematic
diagram o f the two forms o f Notchl is shown in Fig. 4.1. Both forms were subcloned into
retroviral vector RCAS for retrovirus production and gene transduction in ovo and in
reconstituted skin explants. The RCAS(A)AP (alkaline phosphatase) virus was used to
control for the effect o f virus infection.
We misexpressed both forms o f Notchl in chicken embryos by injecting retrovirus
into the amnionic cavity o f stages 21-22 embryos. At embryonic days 9 or 10 when
feathers were developed, the embryos were sacrificed for observation. In more than 20
embryos injected, I was not able to detect any consistent abnormalities. To verify
retroviral delivery o f foreign genes, whole-mount in situ hybridization was used to detect
viral transcripts. As one example shown in Fig. 4.2, viral polymerase transcripts were
expressed all over the chicken embryo in large and small patches (Fig. 4.2A). The
detection was specific as the expression was heterogeneous (Fig. 4.2B). There was
preferential infection to the feather bud regions compared to the interbud skin. The reason
for this phenomenon is unclear, but it was also observed by other groups who performed
similar procedures (Noramly et al., 1999).
We also misexpressed both forms o f activated Notch in reconstituted skin explants.
However, the feather buds on the reconstituted skin cultures resembled those on the
control (Figs. 4.3 A, C and D). Infection efficiency was determined by section in situ
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FIG. 4.1. Constitutively active forms of Notch.
69
Full-length Notchl protein is schematically drawn at the top. Notchl contains 36
tandem epidermal growth factor (EGF)-like repeats and 3 cysteine-rich repeats (LNR) in
the extracellular region. The 0 indicates a potential proteolytic cleavage site. The
cytoplasmic domain contains 6 ankyrin (ANK) repeats. ST sequences are required for
CBF1 binding to Notchl. All Notch proteins have a PEST sequence rich in pro line,
glutamic acid, serine and threonine at the terminal end. The nCDN construct contains the
entire cytoplasmic region and thus can transactivate CBF1. The CDN construct contains
all of the cytoplasmic region except the ST sequences and cannot transactivate CBF1. This
figure is modified from Fig.l A in Shawber et al., 1996b.
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CDN1
71
FIG. 4.2. Viral transcripts were found on embryos infected with RCAS(A)CDN.
The embryo was infected with RCAS(A)CDN in ovo at E3.5 and fixed at E 10 for
whole-mount in situ hybridization to detect RCAS polymerase transcripts. The patchy
purple staining shows areas where viral transcripts were present. The transgene was found
in most feather regions although no abnormal phenotype was observed. B is an
enlargement of the dorsal region of the embryo shown in A.
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72
:
m
w r w m m
V m * * *
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FIG. 4 J . Reconstituted skin explants overexpressing C-Delta-1 and activated
Notchl.
The reconstituted skin explants were infected with the RCAS(A)AP (A),
RCAS(A)C-Delta-1 (B), RCAS(A)CDN (C) and RCAS(A)nCDN (D) retrovirus and were
cultured for 3 days. RCAS(A)AP virus served as a control. Fewer feather buds formed on
the RCAS(A)C-Oe/fa-/-infected explant compared to the control (B, A). The
RCAS(A)CDN and RCAS(A)nCDN explants resemble the control (C, D, A). The
reconsitution procedure was performed by Dr. Ting-Xin Jiang.
Quantitative analysis o f bud size (E), bud density (F) and interbud space (G) was
done using data from three sets o f RCAS(A)AP and RCAS(A)C-Delta-1 explants. The
average size o f the Delta-transduced feather buds appears to be slightly smaller than the
control buds (E). However, the bud density is decreased more than two-fold. As a result,
the interbud space is increased more than three-fold in the Delta-transduced explants (E).
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B u d S ize (xlO^m 3 )
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A RCAS(A)AP B RCAS(A)C-Delta-I
C RCAS(A)CDN
RCAS(A)nCDN
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hybridization using the RCAS polymerase gene as a probe. Exogenous gene expression
was detected in high levels both in the epidermal and dermal layers (Fig. 4.4D, E).
4.3.2 Misexpression of C-De/ta-l in reconstituted skin explants resulted in a
reduction of the number of feather buds
Misexpression of C-Delta-l in chicken embryos resulted in ectopic inhibition of
feather formation (Crowe et al., 1998, Viallet et al., 1998). To facilitate further studies,
we transduced C-Delta-l in reconstituted skin explants using the RCAS retrovirus. Similar
to the in vivo results, misexpression of C-Delta-l resulted in a loss of feather buds,
especially on the outer region of the skin explant (Fig. 4.3 A-D). This result was seen in
triplicates in three independent experiments. Minor heterogeneity in the size of the forming
feather buds was observed, but this was also seen on the control explant. Results from
quantitative analysis show that the average size of the Delta-transduced feather buds
appears slightly smaller than the control buds (Fig. 4.3 E). However, the Delta-transduced
explants have a more dramatic decrease in bud density and increase in interbud space (Fig.
4.3F-G). The feather buds also all pointed to the same orientation, regardless of their size
and location. As sections o f the reconstituted skin explants show, the infected feather buds
appeared morphologically similar to those uninfected ones on the control explant (Fig.
4.4 A, B). To examine the localization o f the infection, section in situ hybridization was
used to detect exogenous gene expression. When C -D elta-l was used as a probe, an
abundant level o f exogenous transcripts was present throughout the entire RCAS-C-
Z)e/ta-/-transduced skin explant while no endogenous transcripts was detected in the
control (Fig. 4.4A, B). When RCAS polymerase was used as a probe, similar result was
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FIG. 4.4. Viral transcripts were found on reconstituted skin explants infected with
RCAS(A)BP, RCAS(A)C-Delta-l, RCAS(A)CDN and RCAS(A)nCDN.
Section in situ hybridization was done to determine the expression of exogenous
genes, C-D elta-l (A, B) or RCAS polymerase (C-E) in 3-day old reconstituted skin
explants. No endogenous C-Delta-l was expressed in the control RCAS-infected feather
buds (A). The exogenous C-Delta-l transcripts were found at high levels throughout the
explant (B). The viral polymerase transcripts were also found at high levels in
RCAS(A)C-£>e//a- / (C), RCAS(A)CDN (D) and RCAS(A)nCDN-infected (E) skin
explants.
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A RCAS
C-Delta-l
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B RCAS(A)C-Delta-1 C-Delta-l
C RCAS(A)C-Delta-1
pol
D RCAS(A)CDN
pol
E RCAS(A)nCDN
pol
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obtained except that a slightly higher level of expression was seen compared to the
neighboring section probed with C-Delta-l (Fig. 4.4C, B).
4.3.3. A loss of asymmetric PCNA expression is seen in the C-Delta-l overexpressing
skin explants but the localization of NCAM and Tenascin C is retained.
To examine the effect o f misexpressing C-D elta-l and activated N otchl on
proliferation, the expression of PCNA was detected using immunostaining (Fig. 4.5). In
control short feather buds, a higher percentage o f proliferating cells was observed in the
posterior vs. the anterior region (Fig. 4.5 A). This was also seen in the RCAS(A)CDN and
RCAS(A)nCDN skin explants (Fig. 4.5C, D). In contrast, this asymmetric expression was
lost in the C-Delta-1-overexpressing feather buds (Fig. 4.5B). More proliferating cells
were found throughout the entire feather bud. Quantitative analysis shows that the density
o f proliferating cells is higher in the posterior than the anterior domain o f control buds (E).
The difference in the density o f proliferating cells in the anterior vs. posterior domain in
the Delta-transduced buds is decreased. The expression patterns of NCAM and Tenascin
C were also examined and were found to be similar in the control, C -D elta-l, CDN and
nCDN skin explants (Fig. 4.6).
4.4 Discussion
We had expected to see a feather promoting effect when activated N otchl was
misexpressed. We also had expected to observe abnormally orientated feather buds if the
Notch pathway is involved in establishing anterior-posterior (A-P) asymmetry. However,
neither result was observed in vivo or in the reconstitution model. These results perhaps
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FIG. 4.5. Expression of PCNA in reconstituted skin explants overexpressing C-Delta-
1 and activated Notchl.
Immuno histochemistry using antibodies against PCNA was done to examine the
proliferation status o f the 3-day old reconstituted skin explants infected with the
RCAS(A)AP (A), RCAS(A)C-Delta-1 (B), RCAS(A)CDN (C) and RCAS(A)nCDN (D)
retrovirus. More proliferating cells were found in the posterior region of the feather buds
in the AP (A), CDN (C) and nCDN (D) samples. An equal distribution o f proliferation
cells was observed throughout the C-Delta-l-infected feather bud (B). Quantitative
analysis shows that the density of proliferating cells is higher in the posterior than the
anterior domain o f control buds (E). The difference in the density of proliferating cells in
the anterior vs. posterior domain in the Delta-transduced buds is decreased. Each mark on
the graph represents one data point. Ant=Anterior, Post=Posterior.
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80
A RCAS(A)AP B RCAS(A)C-Delta-1
C RCAS(A)CDN D RCAS(A)nCDN
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FIG. 4.6. Expression o f NCAM and Tenascin C in reconstituted skin explants
overexpressing C-Delta-l and activated Notchl.
I mmuno histoc hemisty was used to examine the localization o f NCAM and
Tenascin C in the 3-day old reconstituted skin explants infected with the RCAS(A)AP,
RCAS(A)C-Delta-1, RCAS(A)CDN and RCAS(A)nCDN retrovirus. NCAM was
expressed at the base o f the feather buds in the mesenchyme. Tenascin C was expressed in
the anterior and posterior mesenchyme flanking the feather bud. No apparent differences
o f NCAM or Tenascin C expression were observed in these specimens.
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2 ;
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underline the complex interaction between C-Delta-l and different Notch iso forms and
reflect the possibility that the feather promoting effect elicited by C-Delta-l is not directly
due to Notchl activation. Activation o f the Notchl pathway alone is not sufficient to
determine A-P polarity. It is possible that Notch2 or other molecules and pathways are
required or that the role o f Notch is to maintain polarity after it is established. To address
whether Notch signaling is necessary for the A-P outgrowth, one would need to abrogate
Notch signaling, for example, by expressing a dominant negative form o f Notch. However,
one technical difficulty at the present time is that the RCAS has a 2.4 kb upper limit on the
size o f the foreign gene that can be inserted into its genome and that the size of a
dominant negative form o f Notch exceeds this limit. An appropriate replication defective
retrovirus, adenovirus or in vivo transfection may be used when these procedures are
optimized.
It is also possible that there exist technical problems that remain to be identified
and solved. Potential problems can reside in the possibility that rat Notchl may not be
folly functional in chicken. It is also possible that rat Notchl is not expressed at the same
level as the polymerase transcripts because the inserted gene can get deleted during the
replication rounds. This possibility is noteworthy since a lower amount o f exogenous C-
Delta-1 transcripts is detected compared to the RCAS polymerase transcripts (Figs. 4.4B,
C). However in this case, exogeneous C-Delta-l is still expressed at a fairly high level.
The level o f exogenous protein may also vary from that of the viral gene products because
exogenous proteins are translated from alternatively spliced transcripts. One can either
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84
perform in situ hybridization to detect rat Notchl transcripts or immunostaining to detect
the presence of exogenous Notch proteins.
When C-Delta-l was misexpressed in the reconstituted skin explants, a loss of
feather buds was observed like in the in vivo experiments. This result is consistent with the
finding that C-Delta-l plays an important role in the inductive phase o f feather bud
formation. The exact mechanism o f the inhibition remains to be elucidated. The
preferential inhibition on the outer region of the skin explant is probably due the fact that
there is more infection in the outer area (whole-mount in situ hybridization result, figure
not shown). However, the forming bud region and the interbud region appear to have an
equal level o f exogenous C- Delta-1 distribution (Fig. 4.4B, C). Perhaps C-Notch-1 and/or
C-Notch-2 are induced differentially in these regions, and this remains to be examined.
Notch expression is induced as a result of a positive feedback loop in a receiving cell
(Artavanis-T sakonas et al., 1999). If C-Delta-l signals via binding to Notch, cell-cell
interaction between two abutting cells is required. Then, how does C-Delta-l demarcate
the boundary between a bud region and an inhibitory region? It is possible that a small
difference in the level o f C-Delta-l is important. It is also possible that effect seen is not a
direct consequence o f Notch activation, and this would explain why the same effect was
not observed when activated Notch was misexpressed.
The PCNA immunostaining results showed that the C-Delta-l overexpressing
feather buds had an elevated level o f proliferating cells m the anterior compartment. This
is reminiscent of the result seen in the Wnt-7a-transduced feather buds. However, in this
case the feather buds did not show a loss o f orientation morphologically. Examination of
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85
expression of adhesion molecules NCAM and Tenascin C also showed that their patterns
of expression were not altered. A number of anterior (BMP2, M sxl) and posterior (Wnt
7a, C-Serrate-1, Lunatic fringe) domain markers can be examined to further confirm this
finding. An effect of in vivo C-Delta-l misexpression on feather polarity was also not
observed (Crowe et al., 1998). These results suggest that perhaps C-Delta-l has a
transient effect on the polarized outgrowth of feather bud through a direct or indirect
effect on proliferation. However, to achieve a morphological change, other signals are
required. Good candidate signals include Wnt7a, Lunatic fringe or Serrate.
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CHAPTER 5: ISOLATION OF CHICKEN SUPPRESSOR OF HAIRLESS 1 AND 2
SUMMARY
Two chicken homologs of the Suppressor o f Hairless gene were isolated by RT-
PCR. The partial coding sequences show that C-Su(H)-l and C-Su(H)-2 respectively
share 95% and 60% amino acid identity to the Xenopus homologs, X-Su(H)-l and X-
Su(H)-2. Attempts to detect expressions o f both genes in different stages of embryos and
tissues were made using whole-mount in situ hybridization and RT-PCR.
5.1 Introduction
Signaling activity at the cell membrane is often relayed intracellularly to cause a
change in cellular information such as activation o f nuclear transcription. Notch signaling
is found to transmit its intercellular signals from the cell surface to the nucleus through a
network of activities that may involve a DNA-binding protein called Suppressor o f
Hairless (Fortini and Artavanis-Tsakonas, 1994; Artavanis-Tsakonas et al., 1999).
Suppressor of Hairless [Su(H)] was found to participate in Notch signaling from results of
Drosophila genetic screens. Studies in Drosophila ceil lines suggested that Su(H) is usually
sequestered in the cytoplasm by binding to the intracellular cdclO/ankyrin repeats o f
Notch but translocates into the nucleus when Notch is activated by ligand binding. In the
cell nucleus, Su(H) then acts as a transcriptional activator for downstream genes, such as
Enhancer o f Split [E(spl)]. But as more studies were done, they revealed the complexity of
Notch signaling. Reports about a proteolysis model emerged, which states that upon
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ligand activation, Notch is proteolytically cleaved to release an intracellular fragment that
can enter the nucleus and associate with Su(H) to act as a co-activator of transcription
(reviewed in Chan and Jan, 1998). Later on, it was reported that Notch signaling may also
go through a Su(H)-independent pathway (Matsuno et al'., 1997).
In mammals, the Su(H) homolog was first isolated as the recombination signal
binding protein Jk (RBP-Jk) (Matsunami et al., 1989) and then found to be identical to
CBF1 (C-promoter binding factor 1) ((Henkel et al., 1994). Mammalian homologs of
E(spl) have also been found and are called Hairy enhancer of split (HES-1). The complex
mechanism o f Notch signaling appears to be conserved from Drosophila to mammals.
Studies have found that activated mouse Notch can associate with CBF1 to activate HES-
1 (Jarriault et al., 1995). Membrane-tethered activated forms o f mouse Notch were later
found to also undergo proteolytic processing and nuclear translocation (Kopan et al.,
1996). Later study using activated forms of Notch that differ in their ability to interact
with CBF1 concluded that Notch can also signal through a CBF-1 independent pathway
(Shawber et al., 1996b).
Our study o f the role Notch signaling in feather morphogenesis has led us to
wonder its mechanism of activation. Does Notch signaling in the feather require the
activity o f Su(H)? What are the downstream targets? To address these questions, it is
useful to have chicken homologs o f the related genes. I selected to clone the chicken
homolog o f the Suppressor o f Hairless gene because several homologs had been identified
and their sequence similarities suggest that it is a gene that is conserved across species.
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5.2 Materiab and Methods
5.2.1 Embryos
White leghorn fertilized chicken eggs were purchased from SPAFAS (Preston.
CT). Embryos were incubated at 37.5°C and were staged according to Hamburger and
Hamilton, 1951.
5.2.2 RT-PCR
To clone the chicken homolog o f the Suppressor o f Hairless gene, RT-PCR was
used. To design degenerate PCR primers, a desired region of amplification must be first
determined. From Genbank, five Su(H) genes from four different species were found.
They include:
1. Drosophila Su(H), complete cds. 2928bp
2. Ascidian RBP-Jk, complete cds. 3022bp
3. Xenopus Su(H )-l, complete cds. 2535bp
4. Xenopus Su(H)-2, complete cds. 2319bp
5. Rodent RBP-Jk, partial cDNA. 1581 bp
Using sequence analysis software IntelliGenetics, a highly conserved region o f 1271 bp
was found, this region corresponds to nucleotides 735-2005 (1), 305-1575 (2), 404-1674
(3), 146-1416 (4) and 164-1434 (5) of these five genes respectively. Two flanking 23bp
stretch o f sequences were selected to serve as PCR primers that will amplify a product of
an expected length of approximately l.2kb.
Total RNA was extracted from 2-day-old chicken embryos using TRIzol Reagent
(Life Technologies) and reverse transcribed into cDNA using oligo-dT priming and AMV
reverse transcriptase. The sequence o f the 23bp sense primer was 5-CAYGCHAARG-
TKGCHCARAARTC-3', and the 23bp antisense primer was 5'-CCDGGYTCWGGB-
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GTRWABGTGAA-3', where Y stands for C or T, H for A or C or T, R for A or G, K for
G or T, D for A or G or T, W for A or T and B for G or C or T. These primers were
synthesized by DNAgency. PCR was performed using these primers for 35 cycles of
amplification at 94°C for 1 min; 50°C for 1 min; 72°C for 1 min 30 sec. Platinum Taq
polymerase from Gibco Life Technologies was used. Amplified PCR fragments were
ligated to the pCR2.1 vector from the Original TA Cloning Kit (Invitrogen). Positive
clones were selected by restriction digestion or PCR. Eight positive clones were
sequenced by the dideoxynucleotide sequencing method using Sequenase v2.0 (US
Biochemical). After analyzing the preliminary sequencing results, it was concluded that
two chicken homologs were cloned. The complete sequences of the cloned fragments
were then obtained using the Automated Sequencing services provided by the Norris
Microchemical Facility.
5.2.3 Whole-mount in situ hybridization
Antisense C-Su(H)-l and C-Su(H)-2 probes for hybridization were transcribed
with T7 RNA polymerase from the Hindlll linearized plasmids. Please see section 2.2.2
for the whole-mount in situ hybridization procedure.
5.3 Results
53.1 Isolation of Two Chicken Su(H) homologs
Out o f eight positive clones, two homologs were identified and named C-Su(H)-l
and C-Su(H)-2. The C Su(H )-l fragment is 1193bp long and its sequence is shown in Fig.
5.1. The sequence o f the 1210bp fragment o f C-Su(H)~2 is shown in Fig. 5.2. Open
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reading frame analysis was done using the program Gene Runner to reveal potential amino
acid sequences in all reading frames. These sequences were then entered into the program
BLAST available through the National Center for Biotechnology Information (NCBI)
website to search for sequence similarity. The sequences that are homologous to Su(H)/
RBP J/rare selected and are shown above the nucleotide sequences in Figs. 5.1 and 5.2.
The analyses show that the entire 1193bp C-Su(H)-l fragment encodes for 397 amino
acids. There is probably a sequencing artifactual error in the nucleotide sequence o f C-
Su(H)-2 at positions 754-755. Upstream o f this position, one polypeptide in one reading
frame shows similarity to the N-terminal o f Su(H). while downstream from this position
another polypeptide in a different reading frame matches Su(H) at the C-terminus. The
sequence shown in Fig. 5.2 reflects a hybrid o f these two polypeptides and consists o f 402
amino acids.
Suppressor o f Hairless is highly conserved across species. C-Su(H)-l shares 95%
amino acid sequence identity to Xenopus Suppressor o f Hairless proteins 1 and 2 [X-
Su(H)-l and -2] and mouse CBF1/RBP- Jk transcription factor and 94% amino acid
sequence identity to human RBP- Jk protein. At the nucleotide level, C-Su(H)-I is 86%
identical to human RBP-Jrcand 85% identical to Xenopus Suppressor o f Hairless I and
mouse RBP-Jk. C-Su(H)-2 shares 76% amino acid sequence identity to human and mouse
RBP-L transcription factors and 60% amino acid sequence identity to X-Su(H)-l and -2.
At the nucleotide level, C-Su(H)~2 is 63% identical to human RBP-L and 64% identical to
mouse RBP-L transcription factor. C-Su(H)-l and C-Su(H)-2 are 62% identical in
nucleotide sequences but are 58% identical in amino acid sequences. Both proteins
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FIG. 5.1. Primary nucleotide and amino acid sequence of chicken Suppressor o f
Hairless 1 .
The sequence o f the 1193bp fragment ofC -Su(H )-l is shown. This fragment
encodes for 397 amino acids.
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H A K V A Q K S Y G N E K R F F C P P P
1 CATGCTAAGG TTGCACAAAA ATCATATGGA AATGAAAAAA GGTTCTTTTG TCCCCCTCCG
C V Y L M G S G W E C E C E C E C E Q M E R D G
61 TGTGTGTATC TTATGGGCAG TGGATGGAAG AAAAAAAAAG AGCAAATGGA ACGGGATGGT
C T E Q E S Q P C A F I G I G N S D Q E
121 TGCACTGAGC AAGAGTCACA GCCTTGCGCC TTCATTGGAA TAGGAAACAG TGACCAAGAA
M Q Q L N L E G K N Y C T A E C T L Y I S
181 ATGCAGCAAC TGAACTTGGA AGGAAAGAAT TATTGCACTG CCAAAACGTT ATACATATCA
D S D K R EC El F M L SVECM F Y G N S D
241 GATTCAGACA AGAGAAAGCA CTTCATGCTA TCAGTAAAAA TGTTCTATGG CAATAGTGAT
D I G V F L S EC R I ECVI S K P S K K K
301 GACATCGGTG TGTTCCTCAG TAAACGAATC AAAGTCATCT CCAAGCCTTC TAAAAAGAAG
Q S L K N A G L C I A S G T K V A L F N
361 CAGTCACTGA AAAATGCAGG CTTATGTATT GCATCAGGGA CAAAAGTGGC ACTATTTAAT
R L R S Q T V S T R Y L H V E G G N F H
421 AGACTTCGAT CCCAAACAGT TAGCACCAGA TATTTGCACG TAGAAGGCGG TAATTTCCAT
A S S Q QWG A F Y I H L L O D D E S E
481 GCCAGTTCAC AACAGTGGGG AGCATTTTAC ATTCACCTCT TGGATGATGA TGAATCGGAA
G E E F T V R D G Y I H Y G Q T V K L V
541 GGAGAAGAAT TCACAGTGAG AGACGGCTAC ATTCATTATG GGCAGACTGT CAAACTTGTA
C S V T G M A L P R L I I R K V D K Q T
601 TGCTCCGTTA CTGGCATGGC ACTCCCAAGA CTGATAATTC GAAAAGTGGA TAAACAAACA
A L L D A D D P V S Q L H K C A F Y L K
661 GCATTATTGG ATGCAGATGA TCCGGTATCG CAGCTCCATA AATGTGCATT TTACCTTAAA
D T E R M Y L C L S Q E R I I Q F Q A T
721 GACACTGAGA GAATGTATTT GTGCCTTTCC CAGGAGAGAA TAATCCAATT TCAAGCCACT
P C P K E P N K E M I N D G A S W T I I
761 CCATGCCCAA AAGAACCAAA TAAAGAAATG ATTAATGATG GAGCTTCTTG GACAATCATT
S T D K A E Y T F Y E G M G P V H A P V
841 AGCACAGAT A AAGCAGAGTA CACATTTTAT GAGGGGATGG GACCGGTCCA TGCTCCAGTG
T P V P V V E S L Q L N G G G D V A M L
901 ACACCTGTGC CTGTTGTAGA AAGTCTTCAA TTGAATGGCG GCGGGGATGT AGCAATGTTG
E L T G Q N F T P N L R V W F G G V E A
961 GAACTTACAG GACAGAATTT CACTCCAAAT TTACGTGTCT GGTTTGGGGG TGTGGAAGCC
E T M Y R C A E S M L C V V P D I S A F
1021 GAAACCATGT ACAGATGTGC AGAGAGCATG CTATGCGTTG TTCCAGATAT TTCTGCATTT
R E G W R W V R Q P V Q V P V T L V R N
1081 CGAGAGGGTT GGAGGTGGGT CCGTCAACCA GTCCAAGTTC CAGTAACTTT GGTCCGTAAC
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Figure 5.1 Continued
D G I I Y S T S L T F T Y T P E P
1141 GATGGCATAA TTTACTCCAC CAGCCTTACC TTCACATACA CCCCAGAACC CGG
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FIG. 5.2. Primary nucleotide and amino acid sequence of chicken Suppressor of
Hairless 2.
The sequence o f the I210bp fragment o f C-Su(H)-2 is shown. This fragment
encodes for 402 amino acids. The * indicates the location where there is probably an error
in the nucleotide sequence that resulted in a frameshift.
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95
H A K V A Q EC S Y G N E K R F F C P P P
1 CACGCTAAAG TGGCACAGAA ATCGTATGGC AATGAGAAAA GGTTTTTTTG CCCCCCACCT
C V Y L G G P G WK L K Q E Q L T A R D
61 TGTGTTTACC TGGGCGGGCC AGGCTGGAAG TTAAAGCAGG AGCAACTAAC AGCCAGGGAC
V G E A G L R V W G Y M G L D T M G S S
121 GTGGGAGAGG CAGGGCTCCG TGTGTGGGGC TACATGGGGC TGGACACCAT GGGCAGCAGC
L M E T Q EC L S F E E Q P D A E C G F G C
181 CTGATGGAGA CACAGAAGCT CAGCTTTGAG GAGCAGCCAG ATGCAAAGGG GTTTGGCTGT
A R A L Y I S D A D E C R E C H F R L V L K
241 GCCAAGGCTC TGTACATCTC AGATGCGGAT AAGCGCAAGC ATTTCCGCCT GGTCCTGAAG
L F F S N G Q E I G T F H S E C L I E C V I
301 CTCTTCTTCA GCAATGGGCA GGAGATCGGC ACCTTCCACA GCAAGCTGAT CAAGGTCATC
S K P S Q EC EC Q S L K N T D L C I S S G
361 TCCAAGCCCT CGCAGAAGAA GCAGTCACTG AAGAACACGG ATCTGTGCAT CTCCTCGGGC
S E C V S L F N R L R S Q T V S T R Y L S
421 TCCAAGGTGT CTCTCTTCAA CCGCCTGCGC TCACAGACCG TCAGCACCCG CTACCTGTCC
V E G G A F I A S A R Q W A A F T L E i L
481 GTGGAGGGGG GAGCCTTCAT CGCCAGTGCC AGGCAATGGG CAGCCTTCAC CCTCCACCTG
A D E R C T Q S E F P L R E G Y I R Y G
541 GCTGATGAGC GCTGCACCCA AAGCGAGTTC CCCCTGCGGG AAGGGTACAT CCGCTATGGC
S V V Q L i e T A T G I T L P P L I I R
601 TCTGTGGTTC AGCTCATCTG CACGGCCACC GGCATCACCC TGCCACCCCT GATCATTCGG
E C V S E C Q Y A M L Y V D E P I S Q L E l E C
661 AAGGTGAGCA AGCAGTACGC TATGCTGTAC GTGGACGAGC CCATCTCCCA GCTCCACAAG
C A F Q F Q G S D H M * L C L S T E E C V I
721 TGTGCCTTCC AGTTCCAGGG CAGCGACCAC ATGACTTGTG TCTGTCCACA GAGAAAGTCA
Q F Q A S P C P E C E A N R E L L N D G S
781 TCCAGTTCCA GGCATCACCC TGTCCAAAGG AAGCCAACCG GGAGCTGCTG AACGATGGCT
C W T I I G T E T V E Y S F S E S L A C
841 CCTGCTGGAC CATCATTGGC ACCGAGACGG TGGAGTACAG CTTCAGTGAG AGCCTGGCCT
A R E P V S P V P L I T A L Q L S G G G
901 GTGCCCGTGA GCCCGTCAGC CCTGTGCCAC TCATCACAGC CCTGCAGCTC AGTGGTGGAG
D V A M L E V Q G E H F H A H L E C V WF
961 GGGATGTGGC CATGCTGGAG GTGCAGGGAG AGCATTTCCA TGCTCACCTC AAGGTCTGGT
G D V E A E T M Y R SPEC S L M C V V P
1021 TCGGTGACGT GGAAGCAGAG ACGATGTACA GGAGCCCCAA ATCCCTGATG TGTGTCGTCC
D V S A F S S D W R W L R Y P I T V P L
1081 CCGACGTCTC TGCCTTCAGC AGCGACTGGA GGTGGCTGCG GTACCCCATC ACTGTCCCAC
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96
Figure S.2 Continued
L L V R D D G L I Y S S S F T F T Y T P
1141 TCCTGCTGGT CAGGGATGAT GGCCTCATCT ACTCCAGCTC CTTCACCTTC ACATACACCC
E P
1201 CAGAACCCGG
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encoded by the cloned fragments o f C-Su(H)-l and C-Su(H)-2 coincide to amino acids
53-449 o f Xenopus X-Su(H )-l, which has a complete coding sequence o f 501 amino acids
(Wettstein et al., 1997). Thus both gene fragments are partial coding sequences that lack
the most 5’ and 3’ ends o f the full-length gene. Studies using X-Su(H)-l found that four
residues required for DNA binding are located between amino acids 193-203 (Wettstein et
al., 1997). These residues are completely conserved in C-Su(H)-l and C-Su(H)-2.
5.3.2 Expression of C-Su(H)-l and C-Su(H)-2
To determine the expression patterns o f C Su(H )-l and C-Su(H)-2, whole mount
in situ hybridization was performed on stage 15 embryos and stages 31 and 33 chicken
dorsal skins. C-Notch-l is reported to be present throughout the neural tube in stage 15
chick embryos and is suggested to be involved in regulating neuronal commitment
throughout vertebrate neurogenesis (Myat et al., 1996). As has been discussed, C-Notch-
1, C-Delta-1 and C-Serrate-1 are expressed in the feather primordia and feather buds in
stages 31 and 33 chicken dorsal skins (Chen et al., 1997; Crowe et al., 1998; Viallet et al.,
1998). C-Su(H)-l and C-Su(H)-2 were thus examined at these stages in the selected
tissues under the assumption that C-Notch-l signals via one or both o f these gene
products. However, using the 1.2kb probe, both C-Su(H)-l and C-Su(H)-2 transcripts
were not detected above background level in three independent experiments.
As an alternative to examine expression, RT-PCR was performed on total RNA
from different stages o f chick embryos. A first-year rotation student Jennifer Chen Zhong
carried out this part o f the experiment under my guidance. She only examined expression
o f C-Su(H)-l. A new set o f PCR primers that are similar to the degenerate primers used
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98
but are specific for C-Su(H)-I was made. The sequence o f the sense primer was 5'-
CATGCTAAGGTTGCACAAA-AATC-3’ and that o f the antisense primer was 5’-
CCGGGTTCTGGGGTGTATGTGAA-3’. Using this set o f primers, PCR products o f
expected size were not obtained when E2 (embryonic day 2), E3, E4 or E8 cDNA was
used as the template. PCR products were obtained however when C-Su(H)-l plasmid
DNA was used as the template. It was odd that there was no PCR product from the E2
cDNA because C-Su(H)-l was cloned using the same approach. Chicken tenascin primers
were used to verify the integrity o f the cDNA template and a PCR product was obtained.
To change parameters, we designed a second set o f primers that flank a 550bp region in
the middle o f C-Su(H)-l. The sequence o f the sense primer was 5'-ATCGGTGTG-
TTCCTCAGTAA-3’ (a 20mer that spans nucleotides 304-323). The sequence o f the
antisense primer was 5’-ACTCTGCTTTATCTGTGCTA-3’ (a 20mer that spans
nucleotides 840-859). Preliminary results using this set o f primers showed that PCR
products o f expected size were obtained when E2 and E3 embryo, E4 head, E4 body and
E8 skin cDNA were used as templates (preliminary data not shown).
5.4 Discussion
Throughout evolution, gene duplications often give rise to two or more vertebrate
isoforms o f a single Drosophila gene. The sequence similarities between the different
isoforms suggest that they came from the same ancestral gene. This is probably how the
two chicken Suppressor o f Hairless genes arose. Xenopus also has two homologs, but
they are almost identical, differing only in the length o f their N-termini (Wettstein et al.,
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99
1997). The two chicken homo logs are only 58% identical. Perhaps each gene product has
specificity in its interaction with Notch, which also has several isoforms. Or they may
interact with different molecules through mechanisms that are not yet identified.
To determine the expression o f chicken Supressor o f Hairless in embryonic
tissues, whole-mount in situ hybridization and RT-PCR were performed. Several attempts
have not led to positive data. The negative results point to the possibility that Su(H) is not
expressed or is expressed at an undetectable level at the stages examined. However, this
conclusion cannot be affirmatively made because o f the lack o f a positive control. Thus we
decided to use a different set o f primers to repeat the RT-PCR experiment. Preliminary
data from this experiment gave a contrary, but positive result. It showed that C-Su(H)-l is
expressed in various stages o f chicken embryos and skins. But this result still remains to be
repeated and verified. To verify, the PCR products can be cloned and sequenced to show
that they are indeed C-Su(H)~l. Another set o f PCR primers can also be tested. Northern
blots can be performed. A different probe can also be synthesized to use for whole-mount
in situ hybridization to see where the expression is localized in the tissues.
The reason that we examined expression o f C-Su(H)-l is that the presence o f
transcripts offers the possibility that Notch signaling in feather morphogenesis goes
through a Su(H)-dependent pathway. It should be mentioned, however, that the activity o f
Su(H) is controlled post-translationally. The presence o f RNA transcripts does not
indicate that the protein is active as a transcription factor. The current model states that
for Su(H) to exert its function as a transcription activator, it requires a co-activator,
namely activated Notch. It appears that Su(H) is in excess in the cells because
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overexpression o f only activated Notch is sufficient to induce the expression of
downstream genes. Overexpression o f only Su(H) is not sufficient to induce the same
effect, in accordance with the idea that Notch is the limiting coactivator (Jarriault et al.,
1995; Wettstein et al., 1997). These results indicate that the presence o f Su(H) RNA or
protein does not predict its activity. A better marker o f a functioning Su(H) is its nuclear
localization. Thus to address whether Su(H) plays a role in Notch signaling in feather
morphogenesis, one need to examine whether Su(H) and Notch colocalize in the nucleus
using immunohistochemistry.
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CHAPTER 6: CONCLUSION
In this study, expression patterns o f C-Notch-l, C -D elta-l, C-Serrate-l and
Lunatic fringe in feather buds were carefully mapped. The data showed these molecules to
be dynamically expressed in various stages o f feather bud development. Moreover, C-
Notch-1 was found to be a good marker for the middle and posterior domain o f the short
feather bud, while C-D elta-l, C-Serrate-l and Lunatic fringe are good markers o f the
posterior domain. Their polarized expression patterns were also found to precede and
correlate with the caudal outgrowth o f the feather buds. To investigate their function, C-
Delta-1 and active forms o f Notchl were misexpressed in the in vitro reconstitution model
or in vivo. Although the misexpression results failed to give the hypothesized phenotypes,
they helped us to refine our hypotheses and provide a direction for future experiments.
The effort toward studying the downstream effects o f Notch signaling led to the isolation
o f two chicken Suppressor o f Hairless genes.
As common as the revelation that one molecule or pathway can be used in different
developmental settings, many studies have found that one developmental process often
requires the actions o f several molecules or pathways. As this study was progressing,
work done by other people in the laboratory and other groups have shown that similar
phenotypic changes can be obtained when different pathways were perturbed. These
results suggest that pathways crosstalk during the same morphogenetic process to help
shape intricate structures. Based on data presented in this study and those discovered by
other people, it is evident that the Notch signaling pathway is involved in the inductive and
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morphogenetic phases o f feather development and it directly o r indirectly interacts with
FGF, BMP and Wnt7a/p-catenin pathways (Widelitz and Chuong, 2000).
From functional experiments, it appears that the C -D elta-l, P-catenin, FGF and
BMP pathways are all involved in the periodic patterning o f the feather buds in a
hexagonally-arranged array. FGF and BMP act respectively as local activators and
inhibitors o f the formation o f cell aggregates, which can later become dermal
condensations (Jung et al., 1998). Local activators such as FGF directly or indirectly
activate the P-catenin pathway in the epidermal placodes and subsequently induce other
placodal genes (Noramly et al., 1999). C-Delta-l can also be induced by FGF and are
expressed in dermal cells underneath feather placodes (Viallet et al., 1998). C-Delta-l
appears to be involved later in the periodic patterning event by consolidating the
demarcation o f dermal condensations through lateral inhibition. A failure o f this periodic
reinforcement, as in the case o f misexpression o f C-Delta-l or scaleless mutants, results in
a loss o f feather buds (Crowe et al., 1998; Viallet et al., 1998). Therefore, C-Delta-l
seems to exert a reinforcing, rather than an establishing, role in periodic patterning. The
mechanism through which it carries out this effect still require further experiments
(Chapter 4).
Similar to its involvement in periodic patterning, the role o f Delta or Notch
pathway in anterior-posterior axis formation may also be one o f maintenance rather than
establishment. Moreover, just as periodic patterning is achieved by the integrated effort o f
several signaling pathways, the asymmetric outgrowth is coordinated by the interaction o f
several pathways. Misexpression o f Wnt7a or a stabilized form o f P-catenin resulted in
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symmetrically posteriorized or abnormally oriented feather buds (Widelhz et al., 1999;
Noramly et al., 1999; Widelitz et al., 2000). In these feather buds, the polarized
expressions o f C-Notch-l, C-D elta-l, C-Serrate-l and Lunatic fringe were disrupted.
These results suggest that the Wnt7a and/or p-catenin pathways are involved in anterior-
posterior asymmetry and they can directly or indirectly affect the expression o f the Notch
related molecules. Recombination experiments show that the orientation o f the feather
buds is determined initially by the epithelium, where Wnt7a and P-catenin are expressed
(Widelitz et al., 1999, Widelitz et al., 2000). Thus, Wnt7a and P-catenin are good
candidates o f the polarity signal. Since C-Delta-l misexpression did not produce effects
on feather polarity, C-Delta-l is unlikely to be the determining factor in A-P axis
formation. However, the polarized expressions o f C-Notch-l, C-Delta-l and C-Serrate-l
are observed prior to morphological asymmetry (Chen et al., 1997). Thus they are likely
candidates to be involved in maintaining the polarity instructed by the epidermal signals,
but they alone are not sufficient to determine polarity. Loss o f function o f Notch activity
experiments can be done in the future to determine whether it is necessary in A-P axis
formation.
Molecules o f the Notch pathway are also expressed in the differentiation phase o f
feather development. C-Serrate-2, Radical fringe and Lunatic fringe are expressed in the
marginal plates while C-Notch-l is expressed in the barb plates (Chen and Chuong, 2000).
The functional roles o f these molecules have not been examined at this stage. But we can
predict that this pathway, if involved, interacts with the BMP pathway and Wnt7a/P-
catenin pathway to shape the feather filaments and feather follicles. Evidences that these
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two pathways play a role during this phase have been presented. Abnormally shaped and
enlarged feathers were observed when noggin was overexpressed (Noramly and Morgan,
1998). The enlarged feathers had abnormal filaments and failed to form follicles (data not
shown). When mutated p-catenin was misexpressed, abnormal growth on the filaments
were seen and the filament diameter and follicle size were increased (Noramly and
Morgan, 1998; Widelitz et al., 2000). It is very possible that these pathways crosstalk to
structure the feather follicles and feather filaments.
Another phenotype that recurred when perturbation experiments were done is
scale to feather transformation. Feather-like appendages were found on the scales when
dominant negative BMPR-IB, C-D elta-l, mutated P-catenin and noggin were
misexpressed in the chicken limbs (Zou and Niswander, 1996; Crowe and Niswander,
1998; Widelitz et al., 2000; data not shown). In the first three scenarios, feather markers
were expressed or feather structures were present on these appendages to identify them as
feathers. These results suggest that a higher level o f BMP and lower levels o f C-Delta-l
and P-catenin activity favor the formation o f scales, while the vice versa would favor the
formation o f feathers, except in the case o f C-Delta-l which is hypothesized to have a dual
role (Chapter 4). Therefore, the activity level o f a signaling pathway can modulate the
formation o f different skin appendage formation and its regulation may be achieved by
interactions between pathways.
Thus for, this study has centered on the role o f the Notch pathway in feather
morphogenesis. As these findings are incorporated with those from other pathways, the
importance o f integration stands out. While more work remains to be done on the role o f
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Notch, another challenge is to study how this pathway crosstalk with other pathways. To
try to decipher the epistatic relationship o f different pathways, one can study the effect on
the expression o f other genes when only one pathway is perturbed. To observe possible
synergistic effects, it may be necessary to perturb two or more pathways simultaneously.
Double misexpression using retrovirus is difficult due to a cellular mechanism that inhibits
superinfection. Perhaps new methods, such as in ovo transfection, can be developed in the
near future that will make this kind o f study feasible.
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Asset Metadata

Creator Chen, Chia-Wei Janet (author)

Core Title Expression and functional studies of the Notch signaling pathway in feather development

School Graduate School

Degree Doctor of Philosophy

Degree Program Pathobiology

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

Tag biology, cell,biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Chuong, Cheng-Ming (committee chair), Snead, Malcolm (committee member), Stallcup, Michael R. (committee member), Sucov, Henry (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-171640

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Legacy Identifier 3054856.pdf

Dmrecord 171640

Document Type Dissertation

Rights Chen, Chia-Wei Janet

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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