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Shifting topology of stem cells and molecular expression during feather regenerative cycling

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Shifting topology of stem cells and molecular expression during feather regenerative cycling

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Content SHIFTING TOPOLOGY OF STEM CELLS AND MOLECULAR EXPRESSION
DURING FEATHER REGENERATIVE CYCLING
by
Syuzanna Avetyan
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(EXPERIMENTAL AND MOLECULAR PATHOLOGY)
December 2008
Copyright 2008 Syuzanna Avetyan
Acknowledgements
I would like to express my deep and sincere gratitude to my advisor Dr. Cheng
Ming Chuong. I could not have imagined having a better advisor and mentor for my
thesis. Always abundantly helpful, understanding, encouraging, Dr. Chuong helped
and guided me throughout this thesis, and gave me many lessons which will be invalu-
able in my future career.
I would like to eagerly thank Dr. Randall Widelitz who shared with me lots of his
insights and helped in many stages of the project development as well as my thesis.
Our endless late discussions were huge stimuli for my mind and encouraged me to
continue and finalize this work.
My sincere thanks are due to Dr. Ping Wu, my direct mentor of hands on laboratory
work. He has taught me most of the lab work that I know today. He has put his valuable
time and given much patience to train me on everything from how to handle a chicken
to how to do molecular biology.
I would like to thank Dr. Lingtao Wu and Dr. Saverio Bellusci for helping me
with the thesis writing and the time they put in discussing with me what possible roles
different moleucles and cell cyclins may have in my system.
This work would not have been possible without the support of all my laboratory
members, past and current ones. Each one of them has taught me something very
valuable during my study and research.
I must thank my family and friends who encouraged me to pursue a Masters pro-
gram and have been supporting me every step of the way.
ii
TableofContents
Acknowledgements ii
List of Tables v
List of Figures vi
Abstract ix
Chapter 1: Introduction 1
Chapter 2: Methods and Materials 20
2.1 Collecting feathers . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Fixation and Embedding of feathers . . . . . . . . . . . . . . . . . . 23
2.3 Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.4 BrdU Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.5 In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.6 Three-dimensional reconstruction . . . . . . . . . . . . . . . . . . . 26
Chapter 3: Cellular events 27
3.1 Morphology of feathers in different stages . . . . . . . . . . . . . . . 27
3.1.1 Early Growth phase . . . . . . . . . . . . . . . . . . . . . . . 28
3.1.2 Late Growth phase (Fig 1.5) . . . . . . . . . . . . . . . . . . 29
3.1.3 Resting phase (Fig 1.5) . . . . . . . . . . . . . . . . . . . . . 29
3.2 Rationale for observing LRC and TA . . . . . . . . . . . . . . . . . . 32
3.3 Long term label retaining cells (Fig 3.3) . . . . . . . . . . . . . . . . 32
3.4 Transiently Aplifying cells (PCNA) . . . . . . . . . . . . . . . . . . 41
Chapter 4: Molecular expression 42
4.1 Molecules tested in feather follicle . . . . . . . . . . . . . . . . . . . 42
4.2 Adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.1 Beta Catenin . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.2 Neural Cell Adhesion Molecule (NCAM) . . . . . . . . . . . 47
4.2.3 Integrin alpha 5 . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2.4 Integrin alpha 6 . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.3 Extracellular molecules . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3.1 Tenascin-C . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3.2 Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.3.3 Collagen type 5 anti rabbit . . . . . . . . . . . . . . . . . . . 63
iii
4.3.4 Collagen 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.4 Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.4.1 Transforming growth factor beta (TGF-2) . . . . . . . . . . 69
4.4.2 Transforming growth factor beta (TGF-3) . . . . . . . . . . 71
4.5 Signaling molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.5.1 Extracellular signal-regulated kinase (ERK) . . . . . . . . . . 73
4.6 Growth of feather . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Chapter 5: Discussion 78
5.1 Growth of Follicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
5.2 Physical Changes the Follicle Undergoes . . . . . . . . . . . . . . . . 79
5.3 Shifting Topology of Stem Cells and Molecular Expression During
Feather Regenerative Cycling . . . . . . . . . . . . . . . . . . . . . . 81
5.3.1 Long term label retaining cells . . . . . . . . . . . . . . . . . 83
5.3.2 Transiently amplifying cells . . . . . . . . . . . . . . . . . . 83
5.4 Molecular Expression in the Feather Follicles . . . . . . . . . . . . . 86
5.5 Collagen 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
5.6.1 Cellular Events . . . . . . . . . . . . . . . . . . . . . . . . . 92
5.6.2 Molecular Events . . . . . . . . . . . . . . . . . . . . . . . . 93
5.6.3 Regional Differences: Neck vs Saddle . . . . . . . . . . . . . 93
References 94
iv
ListofTables
Table 2.1 Growth Rate and Length of Feathers . . . . . . . . . . . . . . . 21
Table 3.1 Measurements of Neck Dermal Papilla . . . . . . . . . . . . . 30
Table 3.2 Measurements of Saddle Dermal Papilla . . . . . . . . . . . . . 30
Table 4.1 Measurements of Feather . . . . . . . . . . . . . . . . . . . . . 76
Table 5.1 Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
v
ListofFigures
Figure 1.1 Different tracts displayed on a head of a chicken . . . . . . . . 4
Figure 1.2 Different tracts displayed on body of a chicken . . . . . . . . . 5
Figure 1.3 Images of Downy Contour and Flight Feathers . . . . . . . . . 6
Figure 1.4 Interscapular and Dorsopelvic tract . . . . . . . . . . . . . . . 7
Figure 1.5 Feathers in different stages . . . . . . . . . . . . . . . . . . . 8
Figure 1.6 Neck and Saddle Regions . . . . . . . . . . . . . . . . . . . . 9
Figure 1.7 Molts and Plumages . . . . . . . . . . . . . . . . . . . . . . . 11
Figure 1.8 Afterfeather . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Figure 1.9 Cartoon drawing of Feather follicle . . . . . . . . . . . . . . . 14
Figure 1.10 Feather follicle . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Figure 1.11 Location of LRC, TA cells . . . . . . . . . . . . . . . . . . . 17
Figure 2.1 Feather Growth Rate . . . . . . . . . . . . . . . . . . . . . . 21
Figure 2.2 Neck Feathers . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Figure 2.3 Saddle Feathers . . . . . . . . . . . . . . . . . . . . . . . . . 23
Figure 3.1 Represents a midsection from a feather follicle in its growth stage 27
Figure 3.2 Size Difference in Dermal Papilla . . . . . . . . . . . . . . . . 31
Figure 3.3 BrdU label of neck & saddle feather . . . . . . . . . . . . . . 32
Figure 3.4 Schematic for BrdU . . . . . . . . . . . . . . . . . . . . . . . 33
Figure 3.5 3D reconstruction of early growth neck feather follicle . . . . . 35
Figure 3.6 3D reconstruction of late growth neck feather follicle . . . . . 36
Figure 3.7 3D reconstruction of resting neck feather follicle . . . . . . . . 37
Figure 3.8 3D reconstruction of early growth saddle feather follicle . . . . 38
Figure 3.9 3D reconstruction of late growth saddle feather follicle . . . . 39
vi
Figure 3.10 3D reconstruction of resting saddle feather follicle . . . . . . . 40
Figure 4.1 Beta Catenin . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Figure 4.2 Schematic for Beta Catenin . . . . . . . . . . . . . . . . . . . 47
Figure 4.3 N-CAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Figure 4.4 Schematic for N-CAM . . . . . . . . . . . . . . . . . . . . . 50
Figure 4.5 Integrin alpha 5 . . . . . . . . . . . . . . . . . . . . . . . . . 52
Figure 4.6 Schematic for Integrin alpha 5 . . . . . . . . . . . . . . . . . 53
Figure 4.7 Integrin alpha 6 . . . . . . . . . . . . . . . . . . . . . . . . . 55
Figure 4.8 Schematic for Integrin alpha 6 . . . . . . . . . . . . . . . . . 56
Figure 4.9 Tenascin-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Figure 4.10 Schematic for Tenascin . . . . . . . . . . . . . . . . . . . . . 60
Figure 4.11 Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Figure 4.12 Schematic for Fibronectin . . . . . . . . . . . . . . . . . . . . 63
Figure 4.13 Collagen 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Figure 4.14 Schematic for Collagen 5 . . . . . . . . . . . . . . . . . . . . 66
Figure 4.15 Collagen 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Figure 4.16 Schematic for Collagen 6 . . . . . . . . . . . . . . . . . . . . 69
Figure 4.17 TGF-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Figure 4.18 Schematic for TGF-2 . . . . . . . . . . . . . . . . . . . . . 71
Figure 4.19 TGF-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Figure 4.20 Schematic for TGF-3 . . . . . . . . . . . . . . . . . . . . . 73
Figure 4.21 Extracellular signal-regulated kinase (ERK) . . . . . . . . . . 75
Figure 4.22 Schematic for ERK . . . . . . . . . . . . . . . . . . . . . . . 76
Figure 5.1 Cell Counts for Neck . . . . . . . . . . . . . . . . . . . . . . 82
Figure 5.2 Cell Counts for Saddle . . . . . . . . . . . . . . . . . . . . . 82
vii
Figure 5.3 PCNA on TA . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Figure 5.4 Schematic for PCNA on TA . . . . . . . . . . . . . . . . . . . 86
viii
Abstract
Feather is known for its robust regenerative ability. Stem cells in the feather have
recently been mapped. However different parts of the chicken body have different size
feathers. How stem cells are managed differently in these different feathers has not
been elucidated. Here we analyze the growth pattern of feathers from neck and saddle
feather. We analyze the topological arrangement of stem cell, TA cell, differentiated
cell during different regenerative feather cycling. We found the stem cell is in a ring
configuration during growth phase but shifted down to smaller ring or to assume a U-
shape flanking the dermal papilla. The number of stem cells remains rather constant
during stem cell cycling but also changes around three folds. On the contrast the num-
ber of TA cells change up to twenty thirty fold differences. There also appear to have
higher stem cell number and a larger dermal papilla in the saddle compared to the neck
feather. We found N-CAM and Tenascin-C expressed in the niche next to the stem
cells. We also found TGF-2 enriched in the stem cells in the resting phase of the
dermal papilla. This work suggests there is a dramatic shifting of stem cell topology
during feather cycling, probably to accommodate the different homeotic relationship
between stem, TA and differentiated cells. The dynamic changes of molecules also
refract the dynamic changes of the microenvironment in the stem cell niche. Further
investigation would be required to understand their molecular mechanism and regula-
tion.
ix
Chapter1
Introduction
Epithelial appendages arise from the epithelial sheet and serve many functions for
a given animal. Two categories of epithelial appendages exist, ones that evaginate
and ones that invaginate. Evaginated organs are those that protrude out of the epithe-
lium, such as feathers, hair, nails, scales claws, beaks, horns, teeth, or intestinal villi.
Whereas invaginated ones are the organs that grow into the mesenchyme such as the
mammary gland, lung, sweat glands, sebaceous glands and etc (Chuong, 1998). All
of the epithelial organs are topological isoforms of the two dimensional epithelia, and
they all form through a similar developmental process, using similar molecular tools.
The choice of tools used, and the time of use are what makes each organ so unique. As
an analogy to this concept, one can think of a construction worker making a building.
The common tools used for construction are hammers, pliers, nails, cement, wood,
water etc. To begin one would lay down the foundation of the home, put up the frame
and then add details. The same tools can be used to build homes, apartment buildings,
schools, and even skyscrapers, but the manner in which the tools are applied and the
timing of their use differs for each of these cases.
In science, to make an epithelial appendage, in most cases the epithelia and mes-
enchyme must interact (Chuong, 1998). In order for these organs to form, they must
go through the different stages of development, which are: induction, morphogenesis
and differentiation (Chuong, 1998). Some of the different molecular tools involved in
1
the formation of all the organs listed above are growth factors, cell cycle genes, extra-
cellular matrix components, inhibitory components, and many more that either have
been identified or still remain unidentified.
Stem cells are pluripotent, retaining the ability to differentiate into many different
cell types. Theoretically they can divide indefinitely and give rise to either more stem
cells, or a more specialized cell. Adult stem cells can generate new stem cells to
replace cells that have been lost due to any number of reasons, for example, injury,
disease or normal wear and tare (NIH, 2001). As stem cells in the feather proliferate
and differentiate, their progeny is displaced upward to create a feather. Transiently
amplifying cells are the cells that populate the growth zone, and they are pushed up
by the new cells that arise from the proximal follicle. Differentiated cells are those TA
cells that get differentiated. They form the distal filament, the barb ridges and the more
keratinized layers of the feather. These three cell types work together to continuously
form the feather. They are used, for the initial building, repair and regeneration of the
organ. The stem cells in the collar bulge give rise to the TA cells which are abundant
in the collar region. The TA cells move up, distally, and differentiate becoming the
differentiated cells which form the barb ridges, ramogenic zone and so on.
My goal is to study the homeostasis of stem cells, transiently amplifying cells, and
differentiated cells in an epithelial appendage. The cells listed above are the key factors
involved for making many different organs, therefore understanding where these cells
are located at different time points of organogenesis, how these cells interact with
each other, how they transition from one stage of development to another, and what
molecules are expressed in different stages of development of an epithelial appendage
can lead to a better understanding of organogenesis. The knowledge that we gain from
this study can be applied to a wide range of fields. It will give insight to how a feather
2
grows out to be of certain shape, what happens in the hair follicle of humans that
causes alopecia, and what can be done to regenerate hairs. It can be applied to what
we can do to help regenerate an amputated leg, or how a lizard regenerates its tail. As
one can imagine, the applications of this study can be applied to any organ system in
any species. Hence the results from this study will have wide ranging ramifications to
stem cell research, organogenesis, wound healing, etc.
In studying organogenesis, I use the chicken as my model and its feathers as
the experimental system because the feather is a dynamic organ. It can regenerate
repeatedly by cycling through its three different stages. Feathers are integumentary
appendages used to identify the avian system. They serve many functions for the
chicken, providing flight (Parkes (1966), Maderson (1972), Feduccia (1993), Feduc-
cia (1996)) waterproofing (Dyck, 1985), cushioning, protection for the deeper tissues
by regulating temperature (Lucas and Stettenheim, 1972), and heat shielding (Regal,
1975). The name integument derives from the Latin integumentum, which means ’a
covering’ (Kardong, 2001). Chicken feathers are a great organ to study for a num-
ber of reasons. They are easily obtained, they continually regenerate, and the speed
of regeneration is fast. Chickens are inexpensive to purchase, maintain and do not
require constant care. The feather follicles from the chicken are big compared to fol-
licles from mouse hair, and this makes all the technical aspects of experiments much
easier to conduct. Avian feathers are complex and their evolutionary origin remains
poorly understood (Brush (1993), Brush (1996)). Prum (1999) describes 5 stages of
the development of the feather. He explains that the feather follicle originated with a
cylindrical epidermal invagination. That it was a hollow cylinder and it evolved to be a
complex structure with a rachis barbs and barbules. This complexity is another factor
that makes the chicken a useful animal to study experimentally.
3
Chicken feathers show diverse phenotypes in different tracts of a single bird
whereas mouse hair is similar over the surface of mice. Feathers can be radially sym-
metric, bilaterally symmetric or asymmetric (Sengel, 1976) (Fig 1.3).
The different forms provide different functions. According to Lucas and Stet-
tenheim (1972), the chicken has over thirteen different tracts just in the head alone
(Fig 1.1).
Figure 1.1: Different tracts displayed on a head of a chicken (Lucas and Stettenheim,
1972)
Some of which are the Frontal tract that lies in the forehead area. The coronal
tract is related to the comb and the head. The occipital tract covers the dorsal surface
of the head, and the auricular tract covers the external ear opening. Then there is
pterylosis which excludes the head. Some of these are the spinal tracts, dorsal cervical
4
tract, interscapular tract, dorsopelvic tract, abdominal tract and so on. The body of a
chicken has over 14 tracts (Fig 1.2).
Figure 1.2: Different tracts displayed on body of a chicken (Lucas and Stettenheim,
1972)
Feathers differ in different tracts. They differ in length, basic morphology and
types. The percentage of plumulaceous to pennaceous portions differ as well (Fig 1.3).
For example, in the tracts of the head, the feathers are short, and moderately erect
(Lucas and Stettenheim, 1972). In the tracts under the wings, the feathers are downy,
mainly plumulaceous. In the tracts of the trunk they are mainly contour feathers long,
and asymmetrical. Feathers differ within a tract as well, some are short, some long,
5
some are contour and some are flight feathers. In the interscapular tract for exam-
ple, which is the region before the tail, one can find short contour feathers and long
flight feathers (Lucas and Stettenheim, 1972). The number of feathers distributed over
the surface of a chicken range anywhere from 7.5 thousand to nine thousand feathers
(Lucas and Stettenheim, 1972). The feathers for this study come from the dorsopelvic
tract and interscapular tract (Fig 1.4).
(a) Neck (Yu et al., 2004)
(b) Tail (Yue et al., 2005)
Figure 1.3: Images of Downy Contour and Flight Feathers
6
Figure 1.4: Interscapular and Dorsopelvic tract (Lucas and Stettenheim, 1972)
The dorsal tract itself harbors many types of feathers to be discussed later.
Given the diversity of feather types, we chose to use the chicken as a source of
feathers to conduct our study. In the past several studies on feathers have given us
abundant information on what types of feathers exist on the chicken, what types of cells
7
are found in the feather follicle, where they are localized, and what their functions are.
These will be discussed later in detail. Important mechanisms have been elucidated as
well which also will be covered in great detail. Many aspects of the feather have been
thoroughly examined, however to date no one has looked at the homeostasis of the
cells involved in the regeneration of the feather. My aim is to study the homeostasis of
long term label retention cells, transient amplifying cells, and differentiating cells in
three distinct stages of the feather cycle. These stages are initiation, growth and resting
(Fig 1.5).
Figure 1.5: Feathers in different stages. Row 1: Images of different size feathers with
a size bar. Row 2: H&E section of feather follicles corresponding to each stage
To see what growth factors, signaling molecules and extacelullar matrix related
genes are involved in the making of the feather. To identify what they are, their
expression pattern, and if and how their expression pattern changes in the three dif-
ferent stages. The feathers I study come from the neck and saddle region of the male
leghorn (Fig 1.6).
8
Figure 1.6: Male white Leghorn displaying Neck and Saddle region where feathers
were collected from
These regions were chosen for this study because feathers from the neck are shorter
than feathers from the saddle region, and we believed that there would be a difference
in the molecular expression explaining the difference in the length of the feather, and
its growth.
The shape of a feather is established during its growth and remains that way except
for wear and tear (Lucas and Stettenheim, 1972). As noted by (Lucas and Stettenheim,
1972), all feathers of fully grown birds are replaced by molting, usually at regular
intervals. Molting involves the shedding of an old part (ecdysis) and the growth of a
new one (endysis). During molting, the older feather is replaced by the pushing out of
9
the new feather (Lucas and Stettenheim, 1972). Molting in chickens appears to be reg-
ulated by a natural annual photoperiod, but is commonly suppressed or altered in those
housed under conditions with an artificial photoperiod (Lucas and Stettenheim, 1972).
The generation of feathers that is brought in after each molt is known as plumage, and
a chicken may wear feathers of more than one generation at the same time, therefore on
certain tracts the feathers of a given plumage may remain the same, whereas on other
tracts, they are replaced by those of a new plumage (Lucas and Stettenheim, 1972).
As Lucas and Stettenheim (1972) discuss the first generation of feathers in the
chicken is produced during embryonic life. The chicken hatches with these feathers
already protruding above the skin. These feathers are termed natal (or nestling) down.
Natal down is found on the bird when they hatch, or a few days after hatching. Natal
down is similar around the entire body of a chicken, but a second generation of juvenile
feathers begins to form in the follicle late in embryonic life. As the juvenile feather
grows it pushes the natal down out of the follicle. The new feather that is to emerge
is enclosed inside its sheath while it forms. As it pushes out above the skin, it has a
long conical shape with a blunt tip (Lucas and Stettenheim, 1972). According to Lucas
and Stettenheim (1972), at this stage, these feathers are known as pin feathers. They
represent an early immature stage of development. With time, the sheath of this feather
dries and flakes off. What remains is the feather that is growing out. The portion of the
feather that is in the sheath can be seen as containing distinct regions. The distal region
is opaque white because of air that is inside and around the forming barbs and rachis.
The more proximal region is the medium pink zone that houses the rich vasculature of
the pulp. With the progression of growth a pulp cap forms, some parts of the feather
keratinize, the feather continues to grow and emerge from its sheath, the pulp recedes
and gets resorbed. At this point the feather is mature, fully grown, because its vanes
10
are completely free of the sheath and the pulp disappears. It takes nearly 10 days for
the pulp to be completely resorbed into the calamus. This time period varies with the
length of the calamus (Fig 1.7).
Figure 1.7: Molts and Plumages (Lucas and Stettenheim, 1972)
(Lucas and Stettenheim, 1972) state that there are 4 generations of feathers within
the first year, and that the third and fourth are the same as the second generation.
The chicken has different types and sizes of feathers. Some of the major types are
down, contour, and flight (Fig 1.3). Contour feathers populate the major portion of the
chickens body. The contour feather incorporates almost all parts found on other types
of feathers (Lucas and Stettenheim, 1972). The major parts of a body contour feather
are the shaft, the plates or vanes on either side of the shaft, and an afterfeather on the
undersurface (Fig 1.8).
11
Figure 1.8: Afterfeather (Lucas and Stettenheim, 1972)
The afterfeather is an integral part of the feather. It is a small feather under the
large main feather. It is common to most plumaceous and pennaceous feather follicles
(Lucas and Stettenheim, 1972).
The shaft of a feather contains two regions, the calamus and the rachis (Fig 1.8).
The calamus is the short unbranched tube structure at the base of the feather, the por-
tion that immediately emerges from the skin. Lucas and Stettenheim (1972) describe it
to be circular in cross section and slightly tapered toward the ends. At its base is a hole
also known as the inferior umbilicus through which the pulp enters the feather while
it is growing. It is composed of stratified Squamous epithelium. It is transparent as
it does not have pigment. During the early phase of feather growth, the calamus gets
12
filled with vascularized pulp, and later is resorbed and pulp caps are formed. Towards
the top of the calamus is the superior umbilicus. The superior umbilicus marks the
division between the calamus and the rachis. The rachis is the long solid portion of the
shaft above the skin. The the barbs branch off from each side of the shaft. Collectively
they are referred to as a vane. The rachis has a dorsal convex surface, and a ventral
concave surface.
The proximal end of the feather which is the portion closer to the skin is fluffy.
It is referred to as the plumulaceous barbules. Plumulaceous barbules are seen in the
downy part of the contour feathers (Lucas and Stettenheim, 1972). The distal end of
contour feathers are coarse and flat and the barbules at the distal end are referred to
as pennaceous barbules. Pennaceous barbules make up the closely knit portions of
the vane (Lucas and Stettenheim, 1972). The ratio of plumulaceous to pennaceous
regions on each feather is a way to distinguish feather types. For example, a contour
feather has both pennaceous and plumulaceous portions. Remiges and retrices have
only pennaceous vanes. Natal down has only plumulaceous vanes.
A vane can have varying quantities of barbs. According to (Lucas and Stettenheim,
1972) a White Leghorn chicken feather vane that is 88mm long can have 266 barbs.
The spacing of barbs differs among feathers and along the rachis of a single feather. In
body contour feathers the barbs are close together at the base, and spread out toward
the tip.
The remiges and rectrices are the larger feathers on the wings and the tail of a
chicken. Their size range is from a few mm to 445 mm in length. They are large, stiff
and asymmetric. Their vanes are mostly pennaceous they do not have an afterfeather.
Their function is to help in flight. The calamus of a pennaceous feather is longer
thanthe calamus found in contour feathers. Their outer vane is narrower than the inner
13
vane. Down feathers are radially symmetric, they provide body warmth. Contour
feathers have a weak bilateral symmetry, flight feathers can be bilaterally symmetric
and asymmetric (Chuong, 1998). Contour feathers are parted into distal pennaceous
and proximal plumulaceous regions. The pennaceous region maintains optimal body
temperature to the chicken and the pennaceous portion aids in flight. So far I have
discussed in some detail the physical appearance of the portion of the feather that
protrudes out of the skin, and its variations. I will now cover how the follicle forms
and what the components of the follicle are.
A great portion of how the follicle forms comes from Jiang et al. (1999) and Prum
(1999). To form a feather follicle, the epidermis and dermis must interact. During this
time, the mesenchymal cells located in the dermis form periodically arranged bud and
interbud domains (Jiang et al., 1999). The dermis sends a signal to the epidermis to
form a feather primordium (Jiang et al. (2004), Fig 1.9).
Figure 1.9: Cartoon drawing of Feather follicle (Yu et al., 2004)
14
Once dermal condensations are in place, epithelial condensations follow (Sengel,
1976). These epidermal condensations are where the feather primordia will form
(Jiang et al., 2004). Once the feather primordia are established, they start to differ-
entiate, express different signals, growth factors and cell adhesion molecules in the
different regions of the bud, interbud and junction between these two domains (Chuong
(1990), Jiang and Chuong (1992)).
Between E9-E16, the feather buds are actively proliferating; the cells inside the
buds migrate and differentiate for the feather to form. The follicle invaginates into
the epidermis and encloses itself into its own capsule. The follicle wall epidermis
is composed of three layers (Fig 1.10): the germinative layer, the intermediate layer
and the corneous layer. The feather filament has three layers as well. They are the
outermost layer called the feather sheath, the intermediate layer, and the innermost
layer which forms the rachis and the barbs.
15
Figure 1.10: Feather follicle (Yu et al., 2004)
The feather filament is filled with the pulp. The pulp is the mesenchyme, it is pro-
duced by the dermal papilla. Some of the components of the pulp identified are fibrob-
lasts, fibronectin and laminin (Chuong and Edelman, 1985). An hourglass shaped
structure sits at the base of the follicle. This structure is called the dermal papilla.
According to Lillie and Wang it has two main functions. One is to produce the vascu-
lar pulp which gives nutrition to the growing feather, and the other is the development
of the feather. The dermal pulp at the center of follicle supplies nutrients for the growth
of the feather and is also the source of feather pigments (Prum, 1999). Surrounding
the dermal papilla is the collar, which forms the epidermis.
16
The cells in the collar which are keratinocytes proliferate, and differentiate to form
intracellular feather keratin filaments. The collar can be broken up into segments. The
bottom segment is the proliferation zone, and the segment above this zone is the ramo-
genic zone (Jiang et al., 2004). Later work by Yue et al. (2005) show that the collar
houses slow-cycling long-term label retaining stems cells (LRCs) transient amplifying
cells, and differentiating keratinocytes (Fig 1.11).
Figure 1.11: Location of LRC, TA cells (Yue et al., 2005)
In the growing follicle the LRCs are positioned in a location called the collar bulge,
in the moulting follicle these LRCs move into the papillar ectoderm niche adjacent to
the dermal papilla (Fig 1.5). The transient amplifying cells (TA) are located above the
LRC zone. These cells give rise to cells that that become the feather filaments, father
sheaths, follicle sheaths, and interfollicular epidermis (Fig 1.9). The keratinocytes are
housed above the TA cells (Yue et al., 2005). As new cells are formed from the base of
17
the follicle the older keratinized cells are pushed up, compressed on top of each other
until they slough off.
The three main stages that the follicle goes through during its cycling is growth,
resting and molting. During growth, the follicle must regenerate what was lost in the
previous stage of the regeneration cycle. A thick collar starts to grow. The bulge
must reform, and move back into its normal location. The LRCs are diffuse in the
beginning, but with time migrate to the bulge, and stay there until the next phase. The
resting phase follows the growth. During resting, the calamus becomes keratinized
and the collar reduces in size (Yue et al., 2005). The calamus continues to shrink
and gets pushed out. During resting since the collar goes through drastic changes
and is continuously reduced, the LRCs move down toward the base of the collar and
eventually come in contact with the dermal papilla (Yue et al., 2005). Once resting
phase is completed, the moulting phase begins. During moulting the feather dislodges
but the LRCs are not lost with it. They rest by the dermal papilla, until growth is
initiated (Yue et al., 2005).
Different investigators have examined which molecules are responsible for differ-
ent regions of the feather. For example, bone morphogenic protein BMP4 is primarily
expressed in the dermal papilla and pulp (Yu et al., 2004). BMP2 is identified to be
in the marginal plate and later expressed in the barbule plate. Shh is expressed in
the marginal plate (Yu et al., 2004). Noggin is found in the pulp at the ramogenic
zone. Other molecules identified are NCAM, LCAM (Chuong and Edelman, 1985),
Shh (Ting-Berreth and Chuong, 1996b) the Wnt pathway (Yu et al., 2004) and notch
pathway (Chen et al., 1997). This work represents only a minute fraction of some of
the work done in regards to molecular biology.
18
In looking at the homeostasis of LRC, TA cells and Differentiating cells, I will
attempt to look for molecular differences in the dorsal neck region of a chicken and
compare with the feathers of the dorsal saddle region. Using immunostaining we hope
to identify different molecular markers. The dermal papilla markers to compare and
contrast are NCAM. The marker used to look at proliferating regions is PCNA. In addi-
tion to some of these we will look at varying isoforms of collagen and integrin. We will
examine L-CAM, N-CAM, ERK, TGF-2 and TGF-3, Fibronectin and Tenascin-C.
We hope to explain their expression pattern in the distinct regions and stages of feath-
ers. The findings from immunohistochemistry will give insight on the homeostasis of
these cells, and on a bigger scale, it will contribute to the knowledge of what it takes to
form epithelial organs whatever the organ may be. In addition to the molecular aspect
of this study, using BrdU labeling, we will trace the LRC, TA cells and differentiating
cells throughout their lifetime of feather generation in different stages of follicles that
come from the dorsal neck and saddle. It is shown by Yue et al. (2005) that the LRC
populate a different niche in the growing vs the moulting stage, but how they do that
remains unknown. Through a three dimensional reconstruction program called recon-
struct we will trace these different cells, and see how their distribution changes from
the early growth stage to the late growth and resting stages of the feather. The results
are spectacular; these cell populations undergo drastic changes in their location within
the feather follicle. The 3D reconstruction not only helps one visualize the changes of
the cell populations but maps out their new location from the bulge to the base of the
collar, adjacent to the dermal papilla.
19
Chapter2
MethodsandMaterials
2.1 Collectingfeathers
To look for dermal papilla size and shape differences of the feather from the dorsal
neck region and the dorsal saddle region of a chicken we had to first obtain chicken
feathers of different stages. We used a male white leghorn chicken for this experiment.
To get different growth stages of the feathers, we plucked the chicken feathers out twice
a week, every Tuesday and every Friday for a period of three months. We allowed the
chicken to replace the plucked feathers, to regenerate feathers at many different stages
in their growth cycles. To analyze the feathers, we needed to know the growth rate,
so we conducted a parallel study to measure the time it takes a feather to grow out to
a given length. Measurements of the feather shaft growing above the surface of the
skin were made once a week. This was done by restraining the chicken and measuring
the length of the feather with a ruler. The ruler was placed on the skin at the base of
the feather and the length of the feather was recorded. The following graph shows the
measurements of feather length for each of these regions, and the rate of their growth
(Fig 2.1).
20
DAYS Neck Length
(cm)
Std. Dev. for
Neck
Saddle Length
(cm)
Std. Dev. for
Saddle
14 0.4 0.03 0.24 0.006
21 1.6 0.32 1 0
31 3.5 0.23 2.1 0.5
36 4 0 3 0.17
40 4.5 0.5 3 0
45 6 0 4.5 0.28
52 7 0 6 0
57 7.5 0.8 6 0.5
65 9 0 8.5 0.3
90 10 0.3 10 0.3
110 10 0.06 12 0.3
120 10 0.3 14 0.06
Table 2.1: Growth Rate and Length of Feathers
Figure 2.1: Feather Growth Rate
21
As this table 2.1 and figure 2.1 depict, the saddle feathers grow at a slower rate but
they grow taller than the neck feathers.
Once we got sufficient amount of feathers in different stages of regeneration, we
treated the bird with BrdU to label the slow cycling cells. The chicken was given BrdU
water to drink for one week, and simultaneously given injections of 1% BrdU for one
week. The BrdU drinking water was prepared by diluting 10ml of 1% BrdU in 500ml
of tap water. The BrdU for injection was prepared by dissolving 1gram of BrdU in
100ml of HBSS.
To look at slow cycling cells we did a one week chase and then anesthetized the
bird and collected the feathers needed (Fig 2.2, 2.3). For transiently amplifying cells,
we did a short term labeling for two hours only and then again anesthetized the bird and
collected the feathers. For anesthesia, we injected the bird with one part xylazine and
two part ketamin. The bird is given one ml of anesthesia per one kg of body weight.
Figure 2.2: Collected Neck feathers that were in different stages in their growth cycles
(early growth-moulting)
22
Figure 2.3: Collected Saddle feathers that were in different stages in their growth
cycles (early growth-moulting)
2.2 FixationandEmbeddingoffeathers
Fix the feathers in 4% PFA overnight at four degrees. Wash with PBS twice for five
minutes. Rinse once in 70% ethanol, and leave in 70% ethanol overnight. Rinse in
80% ethanol for two hours, then in 95% ethanol for two hours and finally in 100%
ethanol for two hours. Clear the feathers in xylene for 45 minutes twice, then incu-
bate in paraffin at 65 degrees for forty five minutes twice. Embed and cut sections 7
micrometers thick.
2.3 Immunostaining
Incubate slides at 60 degrees for 20 minutes. Allow to air dry for 5 minutes. Place
slides in xylene for 10 minutes. Replace xylene with 100% ethanol for 6 minutes, then
dehydrate in 100%, 90%, 80%, 70% and deionzed water for 2 minutes each. Place
23
slides in 3% hydrogen peroxide and methanol for 20 minutes. Wash the slides with
PBS to remove excess reagent. Make borders for samples with pap pen. Place slides
in humidified chamber, add zeller solution to slides for blocking purposes, allow to
block for 20 minutes. Drain slides and add primary antibody and leave overnight in
four degrees. Each antibody has a specific dilution for optimum results. The following
day, wash the slides with PBS for 5 minutes 3 times. Block with zeller solution for 10
minutes, add secondary antibody for two hours. Wash the slides again with PBS three
times 5 minutes each time. Add Strepavididn HRP for one hour, was with PBS 3 times
5 minutes each time, then wash with deionized water for 2 minutes. Develop color
with AEC. Wash with deionized water, counterstain for 10 seconds, wash again with
deionized water, this brings out the red stain. Then wash with PBS for 15 seconds,
wash again with deionized water and mount the slides. It is important to note that with
some antibodies it is critical to do antigen retrieval to get results. With feather sec-
tions sometimes this process takes the sample off the slide, and to better the situation
sometimes it is helpful to bake the slides in the incubator overnight.
2.4 BrdUImmunostaining
Deparaffinize sections in xylene tree times five minutes each. Place slides in graded
alcohol washes (100%, 90%, and 80%) to water at two minute intervals. Circle sec-
tions with PAP pen and allow to dry. Wash in PBS for 5 minutes. Treat with PK in
PBS (1:1000) for 15 minutes. Wash with PBS for 3 times 5 minutes each time. Incu-
bate in pre-warmed (37 degree) 1N HCL for up to one hour. Wash with PBS three
times five minutes each. Incubate sections in 4% hydrogen peroxide for 10 minutes.
Wash in PBS twice five minutes each time. Block using Zeller solution or 20% goat
serum for 20 minutes at room temperature. Incubate with primary antibody overnight
24
at four degrees using (1:200) dilution. Wash in PBS three times five minutes each time.
Block again with either 20% goat serum or zeller for 10 minutes at room temperature.
Incubate slides with secondary anti mouse biotinylated antibody (1:200) for one hour
at room temperature. Wash in PBS for three times, five minutes each time. Incubate
slides with Strepavididn HRP for thirty minutes at room temperature. Wash in PBS for
three times five minutes each. Develop color using AEC for 2-5 minutes. Wash with
deionized water twice two minutes each time to stop the reaction. Counterstain with
hematoxylin. Wash in deionized water and mount using glycerol mounting media.
2.5 InSituHybridization
In situ Hybridization is a three day process. It is critical to use everything fresh, and
sterilized to avoid contamination. On the first day incubate slides at sixty five degrees
for 10 minutes. Dewax slides in Xylene twice for 5 minutes each time. Wash in
100% ethanol twice for five minutes each time. Dehydrate with the ethanol series
down to water five minutes each time. Rinse in PBT for five minutes twice. Treat
with Proteinase K in PBT (1:1000) or 50 microliter of PK in 50 milliliter of PBT. For
10 minutes. Rinse with PBT for five minutes. Refix with fresh 0.2% gluteraldehyde
and 4% PFA in PBS for 20 minutes. Wash with PBT twice for five minutes twice.
Apply 500 microliter of prehybe solution per slide, and leave at sixty five degrees for
one to two hours. Incubate with probe at 65 degrees overnight. Use 12.5 microliter
of probe per one ml of prehybe solution. On the second day float off the coverslip
in 2X SSC with 0.1% chaps and prewarm the same solution. Wash with Prewarmed
2X SSC containing chaps at 65 degrees for three times 20 minutes each time. Agitate
occasionally. Wash with prewarmed 0.2X SSc containing Chaps at 65 degrees for three
times twenty minutes each time. Agitate. Rinse with PBT at room temperature twice
25
ten minutes each time. Preblock with 20% goat serum in PBT for one to two hours at
room temperature. Use PAP pen to draw circled around smple. Preabsorb anti DIG-
AP antibody (1:1000) at room temperature for one to two hours. Add anti-DIG-AP
antibody solution to slides at four degrees overnight. The third day wash with PBT
containing levamisole (12mg/50mL PBT) at room temperature for thirty minutes or
more for a total of five times. Wash with NTMT containing 1 mM levamisole twice
for fifteen minutes each. PrepareNBT (4.5 microliter) and BCIP (3.5 microliter) per
mL NTMT. Add NBT/BCIP/NTMT solution to slides in wet box and develop color for
1-24 hours. If you need to leave the development overnight, put slides at four degrees.
Stop the reaction when needed in PBS, dehydrate, treat with xylene and mount.
2.6 Three-dimensionalreconstruction
We used different number of sections for each follicle in the three different stages
of the neck and saddle follicles (28 microns between each) for reconstruction. We
collected every third section 7 microns thick from the follicle from the beginning to the
end. Sections were digitized using medscan microscope. These images were imported
into reconstruct and aligned. LRC and the follicle was traced using reconstruct, and
rendered as a three-dimensional viow of LRC using Reconstruct (Fiala, 2005).
26
Chapter3
Cellularevents
3.1 Morphologyoffeathersindifferentstages
The morphology of the feather is examined by cutting seven micron sections and by
staining them with Hematoxylin and Eosin (Fig 3.1).
Figure 3.1: Represents a midsection from a feather follicle in its growth stage (Lucas
and Stettenheim, 1972)
27
The morphology of the proximal feather follicle taken from the neck region in
its growth stage contains the anterior and posterior collar, the dermal papilla lies at the
base of the follicle collar, the pulp is located above the dermal papilla within the collar.
A line drawn vertically down the center of the dermal papilla from its most apical
point to its most basal point divides the dermal papilla into its anterior and posterior
portions. A horizontal line drawn from the base of the left collar to the base of the right
collar divides the dermal papilla into its apical portion (above the horizontal line) and
the basal dermal papilla, (below the horizontal line). The dermal papilla can be divided
into four portions: anterior apical, anterior basal dermal papilla, and the posterior
apical, posterior basal dermal papilla.
3.1.1 EarlyGrowthphase
Looking at a middle section taken from the center of an early growth phase neck feather
that is a half a month old, the dermal papilla stains dark pink. The apical dermal papilla
is round, resembling a dome shaped sphere. The basal dermal papilla is round as well,
but more pointy toward the base. It resembles the letter u. The collar stains dark purple.
The anterior and posterior collar surround the dermal papilla. Neither the anterior nor
the posterior collar regions show a prominent bulge (the site of the epithelial stem cell
niche (Yue et al., 2005)). The posterior collar bulge is more elevated than the anterior
collar bulge. Measuring the diameter of the thickest portion of the collar from the tip
of the bulge down to the other end of the collar, at 10x magnification the diameter
measures 1mm thick. The ramogenic zones extend out from the most upper portion of
the collar to the top of the feather follicle. The dermal pulp stains light purpl and fills
in the region between the two collars above the dermal papilla.
28
3.1.2 LateGrowthphase(Fig1.5)
The dermal papilla of a late growth phase follicle representing a follicle that is two
months old resembles the one in the early growth phase. It stains dark pink. Its apical
portion is dome shaped, round, and the basal portion is round and pointy. The collar
stains dark purple, and surrounds the dermal papilla. Measuring the diameter of the
thickest portion of the collar from the tip of the bulge down to the other end of the col-
lar, at 10x magnification the diameter measures 1.5mm thick. The bulge is prominent
in both the anterior and posterior collar. The ramogenic zone extends from the bulge
upward until the tip of the feather. The pulp is present between the collar and stains
lighter purple.
3.1.3 Restingphase(Fig1.5)
The dermal papilla in this phase representing a feather follicle that is three to four
months old stains dark purple. It no longer assumes a spherical dome shape. It is
more square in shape. The apical portion is leveled and does not protrude out into the
portion that contains the pulp. The collar stains pink. It is much slimmer, tapering
down toward the base of the dermal papilla, resembling a curved arrow. Measuring
the diameter of the thickest portion of the collar from the tip of the bulge down to the
other end of the collar, at 10x magnification the diameter measures 0.3mm thick. The
cells in the collar are keratinized. Both the collar bulge and pulp are missing.
The table 3.1 provides measurements of the dermal papilla from the neck region:
29
Feather
Stage
Height of
dermal
papilla
(mm)
Length of
dermal
papilla
(mm)
Height of apical
dermal papilla
(mm)
Height of basal
dermal papilla
(mm)
Early
Growth
3.5 2.3 1.7 1.7
Late Growth 4 3 2 2
Resting 3 2 1.7 1.2
Table 3.1: Measurements of Neck Dermal Papilla
I measured three different feathers for each stage. The standard deviation for the
height of the dermal papilla is 0.4. The standard deviation for the length of the dermal
papilla is 0.5. The standard deviation for the height of the apical dermal papilla is 0.2.
The standard deviation for the height of the basal dermal papilla is 0.2.
The table 3.2 provides measurements of the dermal papilla from the saddle region:
Feather
Stage
Height of
dermal
papilla
(mm)
Length of
dermal
papilla
(mm)
Height of apical
dermal papilla
(mm)
Height of basal
dermal papilla
(mm)
Early
Growth
3.6 3 2.2 1.4
Late Growth 4.8 3.5 2.5 2.3
Resting 2.3 2.7 1.3 1
Table 3.2: Measurements of Saddle Dermal Papilla
I measured three different feathers for each stage. The standard deviation for the
height of the dermal papilla is 0.8. The standard deviation for the length of the dermal
papilla is 0.4. The standard deviation for the height of the apical dermal papilla is 0.2.
The standard deviation for the height of the basal dermal papilla is 0.6.
In general the difference observed in the dermal papilla for the following stages
as depicted by graph (Fig 3.2) are the following: For the growth phase there does not
30
appear to be a big difference in the height and length of the dermal papilla. The other
measurement is the height of the vertical line dividing the dermal papilla into its ante-
rior and posterior portions, For the early growth stage, the dermal papilla dimensions
differ. The saddle feather has a longer and wider dermal papilla the apical dermal
papilla is taller, and the basal dermal papilla is shorter. For the resting stage between
the neck and saddle feather, the saddle feather has a taller and wider dermal papilla.
In the saddle feather the apical dermal papilla is longer and basal portion is shorter.
The saddle feathers have a slightly larger dermal papilla, this may be due to the saddle
feathers being longer than the neck feathers.
Figure 3.2: Size Difference in Dermal Papilla
31
3.2 RationaleforobservingLRCandTA
We hypothesize that there is a homeostasis of stem cells, transient amplifying cells,
and differentiating cells between feathers that grow from different regions in different
stages. We look at LRC with BrdU, and TA with PCNA to study whether the location
of these cells change with each stage and whether the numbers of these cell types
vary as well. Through Immunohistochemistry we show that LRC cells are located
in the collar bulge in the growth stages of the follicle, and move down to populate the
papillar ectoderm adjacent to the dermal papilla during the resting stage. We also show
that the TA cells are abundant and located in the collar during the growth stage, and
decrease in numbers and location during the resting stage.
3.3 Longtermlabelretainingcells(Fig3.3)
Figure 3.3: BrdU label of neck & saddle feather : 4x magnification
32
Figure 3.4: Schematic for BrdU
In the feathers of the neck region, BrdU labeled cells are present in the bulge of the
initiation and growth stage feather follicles. However since the resting phase follicle
is missing the bulge these cells are expressed close to the dermal papilla. The BrdU
labeled cells move down to the base of the posterior and anterior collar. Early growth
stage of the neck feather shows the BrdU labeled cells spread out uniformly in the
bulge. They appear to be in closer proximity to the dermal papilla. In addition some
of these cells line the anterior and posterior collar, and the surface layer of the apical
dermal papilla. They appear to be sprinkled in the base of the collar and the surface
layer of the apical dermal papilla. This pattern changes in the late growth phase. In
the late growth phase of the feather follicle the BrdU labeled cells are still in the bulge,
however they are not uniformly distributed in the bulge, they instead appear to have
moved higher up in the bulge. They appear to concentrate in the higher end of the bulge
closer to the ramogenic zone, away from the dermal papilla. This pattern changes
more drastically in the resting follicle. In the resting follicle the BrdU labeled cells are
concentrated at the lowest portion of the collar. They outline the lower anterior and
33
posterior collar. They are also present in the apical dermal papilla in one layer, just
outlining the surface of the apical dermal papilla.
Sectioning and looking at all the sections in order and tracing the BrdU cells from
one section to the next, I notice a particular pattern that resembles a semi circle. The
BrdU cells are at first at the same level in the follicle, then a shift in this balance
occurs where the ones in the bulge closer to the anterior position are higher and more
spread out in the bulge and the ones in the bulge closer to the posterior side are lower.
At some point these cells appear to be out of the bulge, and in the surface layer of
the apical dermal papilla and then they appear again in the bulge and form a semi
circle conformation. The three dimensional reconstruction shows where the LRCs
are located in the early growth, late growth and resting stages of the neck and saddle
feathers (Figures 3.5, 3.6, 3.7, 3.8, 3.9, 3.10)
34
Figure 3.5: 3D reconstruction of early growth neck feather follicle
35
Figure 3.6: 3D reconstruction of late growth neck feather follicle
36
Figure 3.7: 3D reconstruction of resting neck feather follicle
37
Figure 3.8: 3D reconstruction of early growth saddle feather follicle
38
Figure 3.9: 3D reconstruction of late growth saddle feather follicle
39
Figure 3.10: 3D reconstruction of resting saddle feather follicle
40
3.4 TransientlyAplifyingcells(PCNA)
Neckearlygrowthstage
Proliferating Cell Nuclear Antigen is used to detect transiently amplifying cells. In the
early growth stage is expressed in the collar closer to the pulp rather than closer to the
outer follicle. It appears to be expressed in the first four layers of the collar. It is in the
barb ridges on the posterior feather but not as much in the barb ridge in the anterior
side. The PCNA is expressed all along the collar, but not in the dermal papilla, or the
pulp. In the barb ridge, it is expressed in the layer closest to the pulp, and in the outer
root sheath just in one layer.
Necklategrowthstage
In the late growth phase PCNA is expressed in the collar closest to the pulp but is not
expressed in the collar closes to the outer root sheath. It is in the bulge and surrounds
the neck of the collar next to the dermal papilla, however comparing the PCNA con-
centration in the early growth stage vs the late growth stage there is a decrease in the
percentage of cells that stain with PCNA during the growth stage.
Neckrestingstage
In the resting stage of the feather PCNA expression decreases drastically compared to
the growth stages. There remain a few cells that express PCNA near the base of the
collar and some in the surface layer of the apical dermal papilla. It looks like sprinkles
on the lower follicle rather than a clump of cells or a uniformly spread out group of
cells.
41
Chapter4
Molecularexpression
4.1 Moleculestestedinfeatherfollicle
I have tried to scan a number of molecules in my model, to see whether
they are expressed in my system, and where there expression is. The
following list shows all the names of the molecules that gave positive
expression, and all the ones that did not give expression in my system.
Antibodiessuccessfullyusedtotrackmolecularexpressionduringthefeathercycle
Proliferation
BrdU (long and short)
PCNA
ECM
Collagen 5
Collagen 6
Fibronectin (in pulp of growth stage only)
Adhesion molecules
NCAM
Tenascin C
Integrin alpha 5
Integrin alpha 6
42
Signaling molecule
Beta-catenin
ERK1
Growth factor molecule
TGF-2
TGF-3
Triedbutdidn’tworkinchicken
LCAM (works for John) CDK2, 4, 7
Notch 1 Col I, II, IV , IX
Integrin beta 1 Cyclin D1, E, H
PDGF IGF
IGFR Mat1
pERK1 FGF
CD31 CD34
CD133 RAR alpha
RB pRB
Sca1 Laminin
Pax 7 FLK1
N-cadherin
For the molecules that did not appear to be expressed in my system, it is not clear
why they were not expressed. One possibility is that the immunohistochemistry tech-
nique was not at its optimum for each molecule. Another possibility for some of them
is that the antibodies were old and maybe they lost their strength. Another possibility
may be that they were not avian specific. Further investigation is needed to rule out
the non existence of these molecules in the feather system.
43
4.2 Adhesionmolecules
4.2.1 BetaCatenin
NeckInitiationstage
Absent from this stage.
NeckGrowthstage
It is present in the dermal papilla, the collar, the outer root sheath, and in a single layer
of cells that border the pulp.
Neckrestingstage
It is present in the outer root sheath. It is expressed in one layer lining the apical
dermal papilla. It is expressed in the zone between the neck of the collar and the
dermal papilla. It is also expressed in the muscle tissue outside the follicle.
SaddleInitiationstage
Absent from this stage.
SaddleGrowthstage
It is expressed in the dermal papilla. The basal dermal papilla expresses more Beta
Catenin then the apical dermal papilla. This molecule is expressed in the papillar
ectoderm, in the outer and inner root sheath. It is also expressed in the barbule plates,
and in the outer muscle tissues surrounding the follicle. It is absent from the zone
between the neck of the collar and the dermal papilla.
44
Saddlerestingstage
It is expressed in the apical dermal papilla, but is absent from the basal dermal papilla.
It is expressed at the neck of the collar, in the region between the base of the collar and
the dermal papilla. It is expressed in the outer root sheath, and is present in a single
row of cells inside the epithelium that surrounds the tip of the follicle. It is also seen
in the muscle tissues that surround the follicle.
45
(a) Neck
(b) Tail
Figure 4.1: Beta Catenin
46
Figure 4.2: Schematic for Beta Catenin
4.2.2 NeuralCellAdhesionMolecule(NCAM)
Neckinitiationstage
Expressed only in the dermal papilla; both in the apical and basal portion.
NeckGrowthstage
It is expressed in the dermal papilla. It is expressed periodically spaced in the basal
layer in the valleys between the barb ridges. It is also present in the zone between the
dermal papilla and the neck of the collar.
Neckrestingstage
It is expressed in one layer covering the apical layer of the apical dermal papilla. It is
absent from the dermal papilla, and the collar.
47
Saddleinitiationstage
Expressed only in the dermal papilla; both in the apical and basal portion.
Saddlegrowthstage
It is expressed in the zone between the neck of the collar and the dermal papilla. It is
expressed periodically spaced in the basal layer in the valleys between the barb ridges.
Saddlerestingstage
It is expressed in the neck of the collar, in the zone between the collar and the dermal
papilla. It is also expressed in one layer covering the apical dermal papilla.
48
(a) Neck
(b) Tail
Figure 4.3: N-CAM
49
Figure 4.4: Schematic for N-CAM
4.2.3 Integrinalpha5
Neckinitiationstage
Integrin alpha 5 is faintly expressed on the edge of the collar. It appears to be in the
extracellular matrix, not inside the cells.
Neckgrowthstage
It is expressed in the upper portion of the follicle. It is diffusely in the outer most root
sheath of the follicle, and is seen in little patches at the barb ridges.
Neckrestingstage
It is not expressed in the resting follicle.
50
Saddleinitiationstage
Integrin alpha 5 is faintly expressed on the edge of the collar. It appears to be in the
extracellular matrix, not inside the cells.
Saddlegrowthstage
In the section represented, Integrin alpha 5 is not expressed.
Saddlerestingstage
It is not expressed in the resting follicle.
51
(a) Neck
(b) Tail
Figure 4.5: Integrin alpha 5
52
Figure 4.6: Schematic for Integrin alpha 5
4.2.4 Integrinalpha6
Neckinitiationstage
Integrin alpha 6 is expressed in one or two layers surrounding the pulp. This molecule
outlines the inner and outer collar. It is not expressed in the dermal papilla, or in the
epithelium.
Neckgrowthstage
At the base of the feather, the portion that contains the dermal papilla , the bulge and
the ramogenic zone, Integrin alpha 6 is expressed at the edge of the collar facing the
epithelium. In this zone it is absent at the edge of the collar that is adjacent to the pulp
or the dermal papilla. As you move up the follicle, past the ramogenic zone, starting
from the mid pulp, Integrin alpha 6 begins to be expressed in one or two layers adjacent
53
to the pulp, in immediate contact to the pulp, and on the outer root sheath. Expression
is also seen in the barb ridges at the ramogenic zone.
Neckrestingstage
Not expressed at this stage.
SaddleInitiationstage
At this stage, this molecule lines the inner and outer root sheath. It is seen surrounding
the collar, however it is absent from the dermal papilla.
Saddlegrowthstage
It is expressed in the upper follicle at the outer root sheath. It is also expressed in the
layer adjacent to the top portion of the pulp, and not the lower portion of the pulp. It
is also not expressed in the dermal papilla.
Saddlerestingstage
At this stage, this molecule appears to be absent from the follicle.
54
(a) Neck
(b) Tail
Figure 4.7: Integrin alpha 6
55
Figure 4.8: Schematic for Integrin alpha 6
4.3 Extracellularmolecules
4.3.1 Tenascin-C
Neckinitiationstage
It is expressed in the base of the basal dermal papilla, in the outer root sheath, and in
the inner root sheath. It is present in the epithelium that surrounds the follicle. It is
absent from the collar. It is absent from the majority of the pulp, however it is present
at the top of the pulp, the portion that pushes against the outer root sheath. It is also
expressed in the portion in between the dermal papilla and the lower collar.
56
NeckGrowthstage
It is present in the layer outside of the collar bulge, covering the collar bulge, and its
expression extends upward until it reaches the ramogenic zone. It is absent from the
collar, the pulp, and the dermal papilla. It appears to be expressed capping the neck of
the lower collar.
NeckGrowthstage
It is absent from the follicle, however it is present in the epithelium that surrounds the
follicle.
Saddleinitiationstage
It is expressed in the base of the basal dermal papilla, in the outer root sheath, and in
the inner root sheath. It is present in the epithelium that surrounds the follicle. It is
absent from the collar. It is absent from the majority of the pulp, however it is present
at the top of the pulp, the portion that pushes against the outer root sheath. It is also
expressed in the portion in between the dermal papilla and the lower collar.
SaddleGrowthstage
It is expressed lightly at the basal dermal papilla. It is expressed in the region between
the dermal papilla and the lower collar. It also appears to be present in the lower portion
of the outer root sheath extending from the base of the feather to the ramogenic zone.
It is absent from the collar and the pulp.
57
SaddleRestingstage
It is present in the epithelium surrounding the basal dermal papilla. It appears to be
concentrated at the neck of the collar, in the region between the dermal papilla and
the lower neck of the collar. It is absent from the apical and most of the basal dermal
papilla.
58
(a) Neck
(b) Tail
Figure 4.9: Tenascin-C
59
Figure 4.10: Schematic for Tenascin
4.3.2 Fibronectin
NeckInitiationStage
Fibronectin is not expressed in the initiation stage of the follicle.
NeckGrowthStage
At this stage Fibronectin is present in a few layers outlining the collar bulge. It is
present in the pulp starting at the level of the ramogenic zone. It is also expressed
on the outer root sheath starting at the level of the ramogenic zone and up. It is not
expressed in the outer root sheath beneath the ramogenic zone.
Neckrestingstage
Fibronectin is not expressed in the resting stage of the follicle.
60
SaddleInitiationStage
Not expressed at this stage.
SaddleGrowthStage
This molecule is expressed in the lower pulp; its expression in the pulp appears to begin
surrounding the outer portion of the apical dermal papilla. It is expressed diffusely in
the upper pulp, and it is specifically in the layer adjacent to the pulp. It appears as
though it is lining some vessels.
SaddleRestingStage
It is absent from this stage.
61
(a) Neck
(b) Tail
Figure 4.11: Fibronectin
62
Figure 4.12: Schematic for Fibronectin
4.3.3 Collagentype5antirabbit
Neckinitiationstage
Collagen type 5 is expressed in the epithelium surrounding the follicle. It is not
expressed in the dermal papilla, pulp or the collar.
Neckgrowthstage
Collagen type 5 is expressed in the epithelium surrounding the follicle. It is diffusely
expressed in the pulp starting at the level of the collar and higher up. It is also seen
in the dermal papilla midline dividing the dermal papilla into its anterior and posterior
portions.
63
Neckrestingstage
Collagen type 5 expression reduces. It is still seen in the epithelium however in much
lower levels. It is seen in the apical dermal papilla surface layer but not spanning the
apical dermal papilla from left to right. It’s concentrated in the left surface layer of the
apical dermal papilla.
Saddleinitiationstage
It is expressed similar to the neck growth stage. It is abundant in the pulp above the
collar. It appears to be present in the inner root sheath and in the epidermis surrounding
the follicle.
Saddlerestingstage
It is expressed at the surface layer of the apical dermal papilla and in the epidermis
surrounding the lower follicle.
64
(a) Neck
(b) Tail
Figure 4.13: Collagen 5
65
Figure 4.14: Schematic for Collagen 5
4.3.4 Collagen6
Neckearlygrowthstage
It is expressed in the tissue surrounding the follicle. It appears to be present in the
layer lining the collar facing not the pulp, but the epithelium.
NeckGrowthstage
It is present in the dermal papilla, and in the region between the neck of the collar and
the dermal papilla. It is present in a single layer lining the collar that faces the pulp, and
this extends upward all the way to the tip of the follicle. It appears to encapsulate the
pulp. It is present in the pulp above the ramogenic zone. It is present in the epithelium
that surrounds the follicle.
66
Neckrestingstage
It is expressedn in the epithelium surrounding the follicle.
SaddleInitiationstage
It is expressed in the tissue surrounding the follicle. It appears to be present in the
layer lining the collar facing not the pulp, but the epithelium.
SaddleGrowthstage
It is expressed in the dermal papilla, more condensed in the basal dermal papilla than
the apical dermal papilla. It is expressed in the region between the neck of the collar
and the dermal papilla. It is present in the outer root sheath that extends from the
dermal papilla to the ramogenic zone. It is expressed in the epithelium that surrounds
the top portion of the follicle and the bottom portion of the follicle, but not the mid
portion of the follicle.
Saddlerestingstage
It is expressed in the region between the neck of the collar and the dermal papilla. It is
expressed in the outer root sheath, and in the epithelium that surrounds the top portion
of the follicle and the bottom portion of the follicle, but not the mid portion of the
follicle.
67
(a) Neck
(b) Tail
Figure 4.15: Collagen 6
68
Figure 4.16: Schematic for Collagen 6
4.4 Growthfactors
4.4.1 Transforminggrowthfactorbeta(TGF-2)
Neckinitiationstage
It is expressed in the dermal papilla, in the collar, in the outer root sheath and the inner
root sheath surrounding the pulp. It is absent in the zone in between the dermal papilla
and the collar. Its expression appears to be extracellular.
NeckGrowthstage
Expression is in the dermal papilla, it is expressed heavier in the basal dermal papilla
and lighter in the apical dermal papilla. It is absent from the zone in between the
dermal papilla and the lower collar. Starting at the region of the barb ridge formation,
69
it is expressed in the layer adjacent to the pulp, just encapsulating the pulp, and in the
outer root sheath.
NeckRestingstage
It is absent from the follicle, but is present in a single layer in the epithelium surround-
ing the follicle.
(a) Neck
Figure 4.17: TGF-2
70
Figure 4.18: Schematic for TGF-2
4.4.2 Transforminggrowthfactorbeta(TGF-3)
Neckinitiationstage
It is expressed in the dermal papilla, the collar, the inner and outer root sheath.
NeckGrowthstage
It is expressed in the dermal papilla and the collar, but it is absent from the zone in
between the dermal papilla and the collar. It appears to be present in the pulp and
surrounding the blood vessels. It is also present in the layer encapsulating the pulp.
71
NeckRestingstage
To be examined
(a) Neck
Figure 4.19: TGF-3
72
Figure 4.20: Schematic for TGF-3
4.5 Signalingmolecules
4.5.1 Extracellularsignal-regulatedkinase(ERK)
NeckInitiationstage
It is expressed in the collar extending upward and surrounding the pulp. It is expressed
in the outer and inner root sheath. It is present in the pulp and the dermal papilla,
however it is absent in the layers between the collar and the dermal papilla, this zone
is also referred to as the buffer zone. Expression of ERK appears to be extracellular.
NeckGrowthstage
It is expressed in the collar, in the pulp surrounding the vasculature, in the layer adja-
cent to the pulp, and in the outer root sheath.
73
NeckRestingstage
It is absent from the follicle, but it is present in the epithelium in a row of cells sur-
rounding the follicle.
SaddleInitiationstage
It is expressed in the collar extending upward and surrounding the pulp. It is expressed
in the outer and inner root sheath. It is present in the pulp and the dermal papilla,
however it is absent in the layers between the collar and the dermal papilla, this zone
is also referred to as the buffer zone. Expression of ERK appears to be extracellular.
SaddleGrowthstage
It is expressed in the collar, the pulp and the outer root sheath. It is not present in the
dermal papilla.
SaddleRestingstage
It is expressed in the outer root sheath.
74
(a) Neck
(b) Tail
Figure 4.21: Extracellular signal-regulated kinase (ERK)
75
Stage of
feather
Feather
height (cm)
Area of
Proximal
follicle (Micron
squared)
V olume of
Proximal
follicle (micron
cubed)
Duration of
regeneration
(days)
Early growth 0.8 27 186 21
Late Growth 7.7 30 210 52
Resting 7.7 12 81 52
Table 4.1: Measurements of Feather
Figure 4.22: Schematic for ERK
4.6 Growthoffeather
We initially hypothesized that there was a linear relationship between the feather length
and duration of growth. However the data I collected does not support this hypothesis.
The table 4.1 shows the data:
Our data shows that it takes a feather about three weeks to grow to the early growth
stage (stage that the feather has just budded out of the skin), this stage according to
76
Sttetenheim is defined as the portion that pushes out above the skin, it has a long
conical shape with a blunt tip. According to Lucas and Stettenheim (1972), at this
stage these feathers are known as pin feathers. They represent early immature stage of
development. With time, the sheath of this feather dries and flakes off, what remains
is the feather that is growing out. It takes a feather about eight weeks to be in a
growth stage (the stage where the feather has little plumulaceous and more pennaceous
portions, and still has room to grow to the point where it no longer grows) and the
same amount of time for a feather to be in a moulting stage (Sttetenheim defines this
stage as the mature stage, and describes it as the stage where some parts of the feather
keratinize, the pulp recedes and gets resorbed). My data suggests that from initiation
stage of a feather to growth there is a big increase in the time it takes a feather to get
from one stage to another. This could be due to several factors. One being that there is
damage to the proximal follicle due to the plucking, and that damage creates a variance
in growth rate. Depending on the extent of damage, the feather can regenerate faster
or slower.
A Second factor could be that the chicken used for the parallel study was a different
chicken. It could have been a stronger chicken, a younger chicken, could have had
better genes to regenerate feathers, therefore giving different results, results that show
a different rate of growth. It could have been a chicken with less potential, so that
would change my results as well.
In the future to get a more sound understanding of the rate of growth and it’s
relationship with the volume of cells in the proximal follicle that gives rise to the whole
feather, a study could be conducted with a better protocol to take the measurements of
a feather and to trace that feather from time zero until it’s time of dissection.
77
Chapter5
Discussion
5.1 GrowthofFollicle
The feather follicle from the early growth stage which measured to have a volume
of 186 microns cubed gave rise to a small feather that had just broken through the
skin. That small feather measured 0.8cm in length and measured to have a volume of
0.001mL cubed. Its density was 0.1 g/mL. The feather follicle from the growth stage
with a volume of 210 microns cubed gave rise to a feather that measured to be 7.7cm
long with a volume of 0.024mL cubed and a density of 0.12g/mL. The feather follicle
from the neck resting stage with a volume of 81 microns cubed cave rise to a feather
that was 7.7cm long, had a volume of 0.2ml cubed and a density of 0.025g/mL. The
volume for each feather may be used to represents the volume of cells that are present
in that feather, in which case going from an initiation stage to a growth stage there is
a big increase of volume of cells (.001ml cubed to 0.2ml cubed), however from the
growth stage to the resting stage the volume of cells stays the same (0.2ml cubed).
This data may indicate that during the growth stage there is a significant increase in
the amount of cells in the follicle, the cells are actively dividing, and proliferating,
and differentiating, however in the resting phase these cells are no longer actively
proliferating and multiplying, but they are just being compacted one layer on top of
another. We had hypothesized that in the resting follicle the volume of cells would
decrease due to the fact that the cells are keratinized, and no longer hold the same
78
dimensions as they did in the growth stage, meaning they are no longer plump and
round, instead they are flattened out, however our data shows that the volume remains
the same as the volume in the growth stage. This may be because these cells are
compressed, there may be more number of cells but since they are flattened out and
compacted, they still measure to have a similar volume. The feathers we collected
from the neck region all measure to be about the same height once they are at their
full growth stage and will no longer grow to be any taller in length and the ones in the
resting stage are all about the same height as well. But when dissecting the feather out
I have no way of telling how late in its growth stage this feather is in, therefore the
volume of cells in the follicles would change with a growing follicle or a follicle that
is not in its complete resting phase.
5.2 PhysicalChangestheFollicleUndergoes
One of our goals was to discuss the changes that the follicle undergoes throughout
the three distinct stages being the initiation stage, the growth stage and the resting
stage. Our aim was to study the homeostasis of stem cells, transiently amplifying cells
and differentiated cells in the feather, meaning to understand where these cells are
located during the three different stages, how they interact with each other and how
they transition from one stage of development to another.
The changes that the follicle undergoes during the three stages are as follows: Dur-
ing the initiation phase the follicle has a large dermal papilla that takes up the lower
half of the follicle, the pulp is just beginning to form, and fills up the upper half of the
follicle, collar begins at the dermal papilla and moves upward surrounding the dermal
the pulp. At this stage the collar bulge, the ramogenic zone and the barb ridges are
79
not present. The stem cells are believed to be in the papilla ectoderm in direct con-
tact with the dermal papilla. Data regarding the location of LRC and TA cells for this
stage are absent from my study because the earliest stage of the feathers we collected
were 2 weeks old, and this is already considered early growth, in order to see what
happens in the initiation stage one needs to collect feathers three days after plucking.
We have obtained samples that represent the initiation stage on which we have con-
ducted molecular studies on, but not BrdU labeling nor PCNA. Furthermore for all
the molecules we conducted Immunohistochemistry with, we used the same initiation
stage follicle is used to represent the neck and saddle region. This is because initially
the experiment was set up to collect feathers that had emerged from the skin, and those
feathers tend to be 2 weeks old or older, however the true initiation stage follicles
emerge 3 days after plucking.
As the feather proceeds from the initiation stage to the growth stage the follicle
undergoes drastic physical changes. In the growth stage the dermal papilla changes
from its prior shape to an hour glass shape, the collar becomes plump, a bulge appears
in the collar, the pulp becomes abundant, a ramogenic zone forms above the bulge area,
and barb ridges begin to form above the ramogenic zone. The resting stage shows a
significant change in morphology as well. From the growth stage to the resting stage,
the dermal papilla loses its hour glass shape, it appears to look somewhat square in
shape, the collar changes as well, the bulges disappear the cells in the collar become
keratinized, the collar itself gets narrower and tapers down as it progresses from the
top toward the bottom, toward the dermal papilla. The ramogenic zone is not present
at this stage, and the pulp completely disappears because it gets reabsorbed.
The regional differences between the neck and saddle feathers ar the following:
Saddle feathers are longer than neck feathers. The saddle feather follicles contain
80
more long term label retention cells. Similarly the dermal papilla of saddle feathers
are bigger than that of the neck. How these are regulated remain to be investigated.
5.3 Shifting Topology of Stem Cells and Molecular
ExpressionDuringFeatherRegenerativeCycling
The shifting topology of the LRC, the TA cells and the differentiated cells are as fol-
lows: During the early growth phase it is believed that the LRC are in the base of the
follicle directly in contact with the dermal papilla. The dermal papilla gives rise to the
pulp, and the epithelium gives rise to the collar. The location of the LRC changes as
the follicle progresses from early growth stage to late growth stage. The LRC move
up to the bulge and give rise to the TA cells. The TA cells slowly differentiate to
become the differentiated cells forming the keratinized portions of the feather such as
the barb ridges, and the barbs. As time progresses and the feather changes into its rest-
ing stage, the stem cells move down to populate the papillar ectoderm, the TA cells are
lost, because they have become differentiated and lost, and the differentiated cells have
formed the keratinized portions of the feather. In conclusion the topology of stem, TA
and differentiated cells change drastically druing regenerative feather cycling. LRCs
form a ring in the collar bulge position during the grwoth stage, but shift down to pap-
illar ectoderm nested by dermal papilla during the resting stage. The number of LRCs
vary a few folds, but that of TA cells fluctuate tens of folds, both being higher in the
growth phase (Fig 5.1 and 5.2).
81
Figure 5.1: Cell counts in different stages of the follicle for the neck
Figure 5.2: Cell counts in different stages of the follicle for the saddle
82
5.3.1 Longtermlabelretainingcells
Long term BrdU labeled cells are those that retain the label for a long time. These slow
cycling cells represent the feather stem cells as shown by Yue et al. (2005). These stem
cells are located in the collar bulge. Yue et al. (2005) describe that the stem cells in the
resting phase shift toward the lower collar and come in direct contact with the dermal
papilla. In the growth phase, these stem cells take the position of the papillar ectoderm.
During the transition from the resting phase to the growth phase, the stem cells are at
first restricted in the resting phase, but later repopulate the collar bulge in the growth
phase. From the data we collected, and the data presented by Yue et al. (2005). We
observed that in the early growth phase where the follicle is less than a week old, the
slow cycling cells are in the collar bulge, in the papillar ectoderm and some are even
in the dermal papilla. There are a few possibilities as to what these slow cycling cell
population represent.
The first possibility is that these cells represent two different populations. One
may be epithelial and a separate and distinct population may be mesenchymal. The
second possibility is that the cells may represent a single population and there is either
a mesenchymal to epithelial transition, or epithelial to mesenchymal transition or both.
Long term label retention cells form a ring in the collar bulge position during the
growth stage, but shift down to papillar ectoderm nested by dermal papilla during the
resting stage. The number of LRCs vary a few fold between the greowth and resting
stage but still remain higher during grwoth stage (Table 5.1, Fig 5.1 and 5.2).
5.3.2 Transientlyamplifyingcells
Proliferating Cell Nuclear Antigen is an antigen expressed in the nuclei of cells during
the DNA synthesis phase of the cell cycle. For this project it is used to locate the
83
REGION & STAGE # OF LRC # OF TA
NECK EARLY GROWTH 800 13000
NECK LATE GROWTH 700 10000
NECK RESTING 300 1000
SADDLE EARLY GROWTH 1020 15000
SADDLE LATE GOWTH 730 10000
SADDLE RESTING 150 500
Table 5.1: Cell Counts for Neck and Saddle Feathers
transient amplifying cells in the feather follicle. In the early growth stage of the neck
feather, PCNA is expressed in the collar bulge, in the elongated cells. As figure 5.3(a)
and 5.3(b) show, PCNA is expressed surrounding the collar and in the barb ridges
closest to the bulge. They are also observed in the outer root sheath layer. Their
expression in the collar suggests that these cells are in their synthesis stage of the
cell cycle and that once the cell cycle is completed, these cells will be pushed up
toward and become differentiated cells and eventually shed off. During the growth
stage of the neck feather PCNA is expressed in the collar as well, but compared to the
early growth stage of the neck feather PCNA is expressed less surrounding the base
of the collar, and it seems as though the PNCA expression overall has decreased in its
intensity. This suggests that the growth was more robust in the early growth stage and
is starting to slow down in the late growth stage. Finally in the resting stage of the
neck feather, PCNA expression is minimal, it is a few cells surrounding the base of the
collar suggesting that active proliferation is not occurring, that the majority of cells in
a resting follicle have completed the cell cycle and are in dormant state.
84
(a) Neck
(b) Tail
Figure 5.3: PCNA on TA
85
Figure 5.4: Schematic for PCNA on TA
5.4 MolecularExpressionintheFeatherFollicles
Different molecules were expressed in different regions of the different stages of the
follicle, however there was not a significant difference observed between the neck and
saddle feathers within the same stages. Initially we were looking for a possible change
in the molecular expression between the feathers from the neck and saddle region that
would help understand why the saddle feathers grow longer in a similar period of time
than the neck feathers but a major difference was not observed. The difference between
the longest neck feather and the longest saddle feather is approximately 4cm. This
difference in feather size did not give a difference in molecular expression between
these two regions. In future research it may be more informational to compare the
longest neck feather with the longest wing feather because there tends to be at least a
10cm difference between the feathers that come from these regions. There were some
notable changes observed in molecular expression between the initiation growth and
86
resting stages of feathers, but these expressions did not differ between the neck and
saddle feathers. Some molecules were absent from the initiation phase, appeared in
the growth stage and then decreased in the resting phase, and some were present in the
initiation phase but were absent toward the resting phase.
Beta Catenin for example was absent from the initiation stage follicle, but began
to be expressed during the growth stage in the dermal papilla and in the collar and in
the layer in between the pulp and the collar. As the follicle is in its resting stage, Beta
Catenin expression appears to populate the outer root sheath, it forms a covering on
top of the dermal papilla, and it populates the same area that the stem cells do during
this stage. Beta Catenin has its effects on lots of pathways, and depending on what is
activated, in different cellular contexts, it could be promoting growth or suppressing
growth perhaps keeping stem cells in a self renewal state. Beta Catenin has been shown
to act on BMP. Early on it was found that you need to have bmp briefly present and
then disappear. They show that if BMP stays around past a certain time you get an
apteric region. So maybe its involved in triggering initiation but then it forces cells to
prematurely differentiate. Maybe in resting phase it is helping stem cells stay as stem
cells.
Tenascin-C for example is present in the epithelium during the initiation phase sur-
rounding the follicle, it is also present in the basal dermal papilla, in the papilla ecto-
derm and in the top portion of the pulp pushing against the collar, however this expres-
sion decreases in the growth stage. As the follicle is in its growth stage, Tenascin-C
expression remains in the papillar ectoderm and it moves into the region of the stem
cells, it appears to be covering the collar bulge where the stem cells are located during
this stage. And then as the follicle enters the resting stage, Tenascin-C is expressed
outside of the papilla ectoderm. This is the region adjacent to where the stem cells
87
are located during the resting stage. This expression of Tenascin-C appears to be in
parallel locations to where the stem cells appear to be in the follicle.
Tenascin-C in the embryo is made by migrating cells like the neural crest; it is
also abundant in developing tendons, bone and cartilage. Tenascin-C is an extracel-
lular matrix protein that plays an important role in cell proliferation, migration and
tumor invasion in various types of cancers (Ohno et al., 2008). In other studies as well
Tenascin-C has been shown to promote cell migration and mesenchymal condensa-
tion during development (Mackie et al. (1988), Epperlein et al. (1988)). In a different
study by Kloepper et al. (2008) Tenascin-C was found to be upregulated in the bulge
of the human hair follicles where the stem cells are located. This group concluded that
tenascin-C may constitute a component of the bulge stem cell niche of human hair fol-
licles. Tenascin has been shown to be enriched in develping feathers and is thought to
be involved in their formation (Jiang and Chuong, 1992). Noveen et al. (1995) showed
that during skin morphogenesis, tenascin is restricted to the anterior placode epithe-
lium by the feather plocode satge and spreads to the mesenchyme by early feather bud
formation. In addition inhibiting tenascin causes feather buds to remain round and
stop growth. It is believed that tenascin may belong to the same molecular pathway
as cAMP. The role of Tenascin-C as guiding in migration and proliferation shown in
other studies and in combination with its expression in the feather follicle suggests that
Tenascin-C in the feather may be playing a role in protecting the stem cells, or keeping
the stem cells in their reserved state.
Transforming growth factor-beta 2 (TGF-2) is a secreted protein known as a
cytokine that performs many cellular functions and functions during embryonic devel-
opment (Lodish et al., 1995). The TGF- family of growth factors controls growth in
a variety of different organisms (Moses et al., 1994). TGB-B may have a stimulatory
88
or an inhibitory effect on cell proliferation. They are also shown to regulate cell migra-
tion. This molecule may even have an effect on downstream adhesion and extracellu-
lar matrix molecules (Nakashima et al., 1994). TFGB-2 in earlier stage is expressed
in the bud and interbud epithelium of the placode stage (Jakowlew et al., 1994), by
stage 35 it is restricted to the feather epithelium and mesenchyme, in addition TGF-
2 was shown to induce feather buds (Ting-Berreth and Chuong, 1996a). Widelitz
and Chuong (1998) concludes that TGF-2 induced the expression of tenascin-C and
NCAM, both of which are involved in the formation of dermal condensation.
Our data shows that in the initiation stage TGF-2 and TGF-3 are expressed
in the dermal papilla and in the collar, as the feather changes into its growth stage,
these molecules remain in the dermal papilla, but decreases in expression in the collar,
instead it becomes concentrated in the boundary between the pulp and the barb ridges.
And in the resting stage its expression decreases even more to be only expressed in the
papillar ectoderm and covering the surface layer of the apical dermal papilla where the
stem cells are seen to localize. This gradual decrease from the initiation stage to the
resting stage suggests that maybe TGF-2 in its initiation stage and growth stage is
causing the cells to proliferate, its allowing the mesenchyme to grow and become the
dermal papilla, so that the dermal papilla can support the feather to grow larger in size,
and the decrease in expression of TGF-2 as the feather enters it later stages in growth
may mean that it is being suppressed in its function, that there must be a molecular
switch that occurs for the feather to stop its growth and start to retract. As the fol-
licle is retracting, and getting ready to fall of, the pulp sheds and gets resorbed, the
dermal papilla changes in size, the stem cells retract and so does TGF-2 expression.
Tenascin-C and N-CAM are seen to co-localize with TGF-2 and TGF-3 in the ini-
tiation stage but in the growth stage TC and N-CAM change their location to populate
89
the papillar ectoderm and to flank the collar bulge where the stem cells are located. As
sown by earlier literature, the transforming growth factors in the early initiation stage
may recruit TC and N-CAM to the dermal papilla, but later these molecules seem to
take a different role other than proliferation and growth, they begin to guard the stem
cells, and they begin to move up and down the collar with the stem cells perhaps keep-
ing the stem cells in their self renewal state.
Fibronectin is an extracellular matrix protein that can bind to different Integrin
receptors through an Arg-Gly-Asp peptide sequence (Widelitz and Chuong, 1998).
Specifically Fibronectin binds to Integrin alpha 5 Beta 1 (Lodish et al., 1995). My data
does not show a strong parallel expression of Fibronectin with Integrin alpha 5 nor
with Integrin alpha 6. Fibronectin and Integrin are only co-localized during the growth
stage in the inner layer between the pulp and the barb ridges. They are all absent from
the resting stage of the feather. This pattern of expression in these stages may mean
that in the feather Fibronectin is not playing a role of proliferation or migration. It
is expressed in the upper pulp next to the areas that are differentiated. It is possibly
playing a role in differentiation and apoptosis.
Integrins are cell surface receptors that mediate adhesion to the extracellular matrix
and cell-cell interactions. They are heterodimers of alpha and beta subunits. In gen-
eral the integrins containing B1 subunits bind components of the extracellular subunit
(Reece and Campbell, 2001). In the feather integrin B1 is expressed in the epithelial
cells and localizes to the epithelial mesenchymal interface of the developing placode
(Widelitz and Chuong, 1998). Inhibiting integrin B1 from stage 33 embryos results
in the separation of epithelium from the mesenchyme in younger feather buds and
inhibits their further development Widelitz and Chuong (1998). In the three different
stages that I have the integrins in the initiation stage are bordering the epithelium, as
90
the feather transitions to the growth stage they are found in the epithelium and border-
ing the mesenchyme and the epithelium. They are expressed at the border of the collar
and the epithelium, and at the junction of the pulp and the barb ridges. In the resting
stage they present in minute amounts. They are present in the border of the dermal
papilla and the keratinized collar. This expression along with prior history of what
roles integrins play suggests that the integrins in the feather are holding the epithelium
and mesenchyme together and this makes sense that they are reduced during the resting
stage because most of the mesenchyme has shed away. The molecules involved with
the LRC niche change dynamically during feather cycling. Beta Catenin and TGF-2
ae enriched in stem cells in the resting phase. Tenascin-C and NCAM appear to be in
the niche adjacent to the stem cells, and remain so during the shifting of stem cells.
How these are regulated or what their function is during the feather cycle remais to be
investigated.
5.5 Collagen5
Collagen is a fibrous protein that provides structure secreted by connective tissue cells
as and other cell types (Lodish et al., 1995). Collagen is the most abundant protein in
mammals, it is a major component of the skin and bone (Lodish et al., 1995). Collagen
type 5 is one of the 20 collagens that is specifically found in skin and bone (Lodish
et al., 1995). As figure 4.13(a) and 4.13(b) show collagen 5 in the initiation stage of the
neck and tail feather is expressed in the outer root sheath only. It is not present in the
pulp as it is in the growth stage of the feather follicle. Collagen 5 in the growth stage
of the neck and tail feather is abundantly expressed in the pulp, and in the outer root
sheath. It is also expressed in the center of the dermal papilla but not as much as it is in
91
the pulp. Since the general function of collagen is to provide structure and its expres-
sion changes from the initiation stage to the growth stage from not being expressed in
the pulp to being expressed in the pulp suggests that collagen in the growth stage of
the feather follicle plays a role in providing strength and structure to the follicle. In the
initiation stage it is not present maybe because the feathers calamus in the initiation
stage is small, it has just begun to form, it does not have much structure to support but
as it grows, now the pennaceous and plumulaceous portions begin to form, barbs begin
to form and at this stage the calamus must support a lot more feather mass, so colla-
gen starts to be expressed and is abundant in the pulp. The feather follicle in its resting
stage looses its pulp, and expression of collagen is only present faintly in the outer root
sheath. The feather is keratinized at this stage and collagen has a minimal function, at
this stage it is still expressed in the outer toot sheath again probably playing its role to
provide structure to the follicle.
5.6 Conclusion
5.6.1 CellularEvents
The topology of stem, TA and differentiated cells change drastically during regenera-
tive feather cycling. LRCs form a ring in the collar bulge position during the growth
stage, but shift down to papillar ectoderm nested by dermal papilla during the resting
stage. The number of LRCs vary a few folds, but that of TA cells fluctuate tens of
folds, both being higher in the growth phase.
92
5.6.2 MolecularEvents
They change dynamically during feather cycling. Beta catenin and TGF beta2 are
enriched in stem cells in the resting phase. Tenascin C and NCAM appear to be in the
niche adjacent to the stem cells, and remain so during the shifting of stem cells.
5.6.3 RegionalDifferences: NeckvsSaddle
Saddle feathers are longer than neck feathers. The saddle follicles contain more LRCs.
Similarly the dermal papilla of Saddle feathers are bigger than that of the neck. How
this are regulated remain to be investigated.
93
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96

Abstract (if available)

Abstract Feather is known for its robust regenerative ability. Stem cells in the feather have recently been mapped. However different parts of the chicken body have different size feathers. How stem cells are managed differently in these different feathers has not been elucidated. Here we analyze the growth pattern of feathers from neck and saddle feather. We analyze the topological arrangement of stem cell, TA cell, differentiated cell during different regenerative feather cycling. We found the stem cell is in a ring configuration during growth phase but shifted down to smaller ring or to assume a Ushape flanking the dermal papilla. The number of stem cells remains rather constant during stem cell cycling but also changes around three folds. On the contrast the number of TA cells change up to twenty thirty fold differences. There also appear to have higher stem cell number and a larger dermal papilla in the saddle compared to the neck feather. We found N-CAM and Tenascin-C expressed in the niche next to the stem cells. We also found TGF-beta2 enriched in the stem cells in the resting phase of the dermal papilla. This work suggests there is a dramatic shifting of stem cell topology during feather cycling, probably to accommodate the different homeotic relationship between stem, TA and differentiated cells. The dynamic changes of molecules also refract the dynamic changes of the microenvironment in the stem cell niche. Further investigation would be required to understand their molecular mechanism and regulation.

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Asset Metadata

Creator Avetyan, Syuzanna (author)

Core Title Shifting topology of stem cells and molecular expression during feather regenerative cycling

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 12/10/2008

Defense Date 10/20/2008

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

Tag differentiated cells,OAI-PMH Harvest,stem cells,transient amplifying cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chuong, Cheng-Ming (committee chair), Bellusci, Saverio (committee member), Hofman M., Florence (committee member), Widelitz, Randall B. (committee member), Wu, Lingtao (committee member), Wu, Ping (committee member)

Creator Email savetyan@gmail.com,savetyan@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1902

Unique identifier UC147780

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Document Type Thesis

Rights Avetyan, Syuzanna

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

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Repository Location Los Angeles, California

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