Dynamic expression of trk receptors during sensory neuron differentiation
by Jason Thomas Rifkin
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biological Sciences
Montana State University
© Copyright by Jason Thomas Rifkin (1996)
Abstract:
Studies over the past several years have provided evidence for the functions of neurotrophins during
development of the nervous system prior to their well characterized role in target-mediated cell death.
To define the potential functions of neurotrophins during this early period of development we looked at
the spatial and temporal expression patterns of neurotrophin receptors throughout the formation of
motorneurons, within the ventral horn of the spinal cord, and sensory neurons of the dorsal root
ganglion (DRG) in an avian model. To this end we have used a highly specific panel of antibodies to
the avian trkA, trkB, and trkC receptors and p75 and compared their expression to that of known
neuronal cellular markers. We have found that p75 is the first of the neurotrophin receptors to be
expressed (at stage 14), followed by TrkC (stage 15/16), then trkA (stage 18), and finally trkB (stage
22). At stage 18 trkA is expressed only on a subpopulation of trkC+ cells, and trkA+ and trkC+ cells
show a reduction of the HNK-1 carbohydrate moiety. At stage 24, trkC is expressed by the vast
majority of cells within the developing DRG, while by E6 (and beyond) trkA becomes the most
prominently expressed trk receptor in the DRG. At ES, trkB and trkC are expressed primarily by cells
in the VL region of the DRG while trkA predominates in the DM region of the DRG. Early in
development, trk receptors are expressed in the distal regions of peripheral axon afferents. Both trkC
(at stage 24) and p75 (at stage 18, 24) receptors are expressed by mitotically active cells. Finally, at E6,
BEN is now coexpressed with trkA and is no longer coexpressed with trkC; while, at E13, NF ( which
was expressed throughout the DRG) is now localized to the VL region of the DRG. In conclusion, our
data point to two additional potential functions for neurotrophins during the development of the dorsal
root ganglion: regulating proliferation of sensory neuroblasts and influencing patterns of axon
outgrowth during target innervation. DYNAMIC EXPRESSION OF TRK RECEPTORS DURING
SENSORY NEURON DIFFERENTIATION
by
Jason Thomas Rifkin
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Biological Sciences
MONTANA STATE UNIVERSITY-BOZEMAN
Bozeman, Montana
December 1996
g 441
ii
APPROVAL
of a thesis submitted byJason Thomas Rifkin
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Frances Lefcort
(Chairperson)
Date
Approved for the Department of Biology
Ernest Vyse
(Department Head)
Approved for the Cplleg
R . L . Brown
(Graduate Dean)
Date
f Graduate Studies
OS'
Date
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the
requirements for a master's degree at Montana State
University-Bozeman, I agree that the Library Shall make it
available to borrowers under rules of the Library.
If I have indicated my intention to copyright this
thesis by including a copyright notice page, copying is
allowable only for scholarly purposes, consistent with "fair
use" as prescribed in the U.S. Copyright Law.
Requests for
permission for extended quotation from or reproduction of
this thesis in whole or in parts may be granted only by the
copyright holder.
/
iv
"For the real amazement, if you wish to be amazed, is this
process.
You start out as a single cell derived from the
coupling of a sperm and an egg; this divides in two, then
four,
then eight, and so on, and at a certain stage there
emerges a single cell which has as all its progeny the human
brain.
The mere existence of such a cell should be one of
the great astonishments of the earth.
People ought to be
walking around all day, all through their waking hours
calling to each other in endless wonderment,
talking of
nothing except that cell."
Lewis Thomas
V
ACKNOWLEDGEMENTS
I would like to thank the members of my committee for
all of their support and advice and encouragement throughout
my graduate program.
I would especially like to thank my
Major Advisor, Frances Lefcort, for her support and
friendship, and for enhancing my ability to think for myself.
vi
TABLE OF CONTENTS
Page
INTRODUCTION..............................................I
EXPERIMENTAL OBJECTIVES AND RATIONALE.................. 10
METHODS....................................!............ 13
Embryos.............................................. 13
Fixation/Tissue Sectioning..... •................... 13
Fluorescense Immunocytochemistry................... 14
Antibodies...........................................15
BrdU Injections..................................... 16
BrdU Immunocytochemistry............................ 17
RESULTS.......................................... '.......19
Trk Expression of Developing Spinal Motor Neurons..19
Sensory Neurogenesis............................ ; ..20
Stage 1 4 ................................ ,...........20
Stage 15/16......................................... 22
Stage 1 8 .............................................25
Stage 2 3 .............................................28
Trk Expression of Mitotically Active Cells.........28
Trk Receptor Expression Within the Distal
Growing Axon Tract and Growth Cone................. 33
Stage 2 7 .............................................33
E8/E13 .
35
DISCUSSION............................................... 39
LITERATURE CITED..................
49
vii
LIST OF TABLES
Page
Table
1.
Stage 18 cell populations coexpressing trkA and
trkC with known antigenic determinants.......... 26
2.
Neurotrophin receptor expression on proliferative
cells during neural crest migration and DRG
formation. . ...................................... 32
?
viii
LIST OF FIGURES
Page
Figure
1.
Critical periods in the development of the
sensory neurons of the D R G ................... '.....9
2.
Trk expression patterns in the ventral
horn of the E6 embryo..................... ;...... 21
3.
Early expression of p75 within the stream of
migrating neural crest cells, stage 1 4 .......... 21
4.
TrkC coexpression with several known antigenic
determinants within the migrating neural crest
at stage 15/16.................................... 23
•5.
Expression patterns of trkA and trkC in the
neural crest of the stage 18 embryo..............27
6.
Expression of trkA, B, and C in association with
the neuronal marker BEN, at stage 2 4 ............ 29
7.
The presence of neurotrophin receptors on
mitotically active cells......................... 31
8.
Expression of trk receptors in the distal growing
axons of the developing sensory neurons......... 34
9.
Expression of trkA and trkC with BEN at E 6 ....... 3 6
.
10. Expression of trk receptors in ES D R G ............ 37
11. E13 D R G ............................... L........... 3 8
12. Expression of trk receptors in relation to
critical events during DRG formation....
48
ix
ABSTRACT
Studies over the past several years have provided
evidence for the functions of neurotrophins during
development of the nervous system prior to their well
characterized role in target-mediated cell death.
To define
the potential functions of neurotrophins during this early
period of development we looked at-the spatial and temporal
expression patterns of neurotrophin receptors throughout the
formation of motorneurons., within the ventral horn of the
spinal cord, and sensory neurons of the dorsal root ganglion
(DRG) in an avian model-. To this end we have used a highly
specific panel of antibodies to the avian trkA, trkB, and
trkC receptors and p75 and compared their expression to that
of known neuronal cellular markers. We have found that p75
is the first of the neurotrophin receptors to be expressed
(at stage 14), followed by TrkC (stage 15/16), then trkA
(stage 18), and finally trkB (stage 22). At stage 18 trkA is
expressed only on a subpopulation of trkC+ cells, and trkA+
and trkC+ cells- show a reduction of the HNK-I carbohydrate
moiety. At stage 24, trkC is expressed by the vast -majority
of cells within the developing DRG, while by E6 (and beyond)
trkA becomes the most prominently expressed trk receptor in
the D R G . At ES, trkB and trkC are expressed primarily by
cells in the VL region of the DRG while trkA predominates in
the DM region of the D R G . Early in development, trk
receptors are expressed in the distal regions of peripheral
axon afferents . Both trkC (at stage 24) and p75 (at stage
18, 24) receptors are expressed by mitotically active cells.
Finally, at E6, BEN is now coexpressed with trkA and is no
longer coexpressed with trkC; while, at E13, NF ( which was
expressed throughout the DRG) is now localized to the VL
region of the D R G . In conclusion, our data point to two
additional potential functions for neurotrophins during the
development of the dorsal root ganglion: regulating
proliferation of sensory neuroblasts and influencing patterns
of axon outgrowth during target innervation.
I
INTRODUCTION
Development of the nervous system is a complex,
intricate process involving cell proliferation, migration,
differentiation, survival, and synapse formation (Chao,
1992).
Subsequent to gastrulation, neurulation ensues and
involves the changing of the shape of the ectodermal cells
within the dorsal portion of the developing embryo, which
will form the neural plate (Jacobson, 1981; Smith et'al.,
1991).
The cells of the neural plate begin to invaginate,
while the adjacent neural folds begin to rise and finally
fuse converting the neural plate into the neural tube
(Erickson et a l ., 1983; Nichols, 1981).
Many theories have
been proposed as to the mechanisms underlying the initiation
of the shape changes that result in the neural groove, and
subsequent neural tube.
These include:
pushing and pulling
forces (His, 1874); adhesion interactions (EdeIman, 1984);
and structural changes in the size and shape of the neural
epithelial cells (Trinkhaus, 1969).
The neural tube gives
rise to the central nervous system (CNS) including the spinal
cord and all of the spinal cord's various cell types.
This
occurs via the differentiation of neuroepithelial cells
within the wall of the neural tube giving rise to the various
populations of neurons and supportive cells in the spinal
cord (Gilbert, 1994).
2
Neurons generally develop in a ventral to dorsal
gradient within the spinal cord, with the motorneurons
developing first and projecting their axons into the
periphery (Lumsden et a l ., 1991; Placzek et a l ., 1991).
In
the chick, the first motorneurons appear .(leave the mitotic
cycle) at stage (St.) 15, with all of the population being
born by St. 23 (HoIlyday et al., 1977).
By approximately St.
29 one half of the motorneuron population has died due to
programmed cell death (Hamburger, 1975; Oppenheim et al.,
1978; Lanser et al., 1984). Considerable research has
implicated the involvement of the notochord and floor plate
in the development of the motorneurons through cell-cell
contacts and extrinsic factors (Van. Straten et al., 1985;
Smith et al., 1989) .
Placzek et al., 1991, found that
following removal of the notochord early in development, the
floor plate did not form and motorneurons failed to properly
develop.
Placzek et al., 1991 also discovered that adding an
additional notochord would lead to the formation of an
another- floor plate within the spinal cord.
The extrinsic
factor mediating these ventral patterning effects is most
likely sonic hedgehog (Ericson et al., 1995).
The peripheral nervous system develops from migrating
neural crest cells which give rise to the DRG, sympathetic
and parasympathetic ganglia, and supportive cells
(Lallier et
al., 1988; Vogel et al., 1992; Marusich et al., 1991; Weston,
1991; Serbedzija et al., 1990).
The neural crest cells form
3
between the ectoderm and the dorsal region.of the recently
closed neural tube, and their migration occurs in a rostral
to caudal gradient (Holmdahl, 1928).
There are two streams
of neural crest cell migration:
a dorsolateral route
(I)
which will give rise to the melanocytes and has been studied
in relation to pigmentation patterns (Rawles, 1948); and (2)
a ventrolateral route which travels lateral to the neural
tube and both medial to and through the developing somites
(Weston, 1963; Bronner-Fraser, 1986).
This second migratory
pathway travels only along the anterior portion of the
adjacent somite (Bronner-Fraiser, 1986).
This stream, of
cells will give rise to the autonomic ganglia, glial cells,
adrenomedulIary cells, and of most interest in this study, to
the cells of the D R G .
The mechanisms involved in this
migration are not completely understood, but it is believed
that the extracellular matrix through which the neural crest
cells migrate is of utmost importance (Pratt et a l ., 1975;
Toole, 1976; Derby, 1978; Rovasio et al., 1983) .
Many different intrinsic and extrinsic factors play a
role in this migration and subsequent formation of the
peripheral nervous system. These include various cell
‘
adhesion molecules and cell surface receptors.
There are
also a multitude of exogenous ligands acting via receptor
proteins to activate intracellular signaling cascades, which
will lead to changes in the expression patterns of various
gene products.
These include numerous trophic factors which
4
act on populations of cells in the process of development and
promote survival, proliferation, or differentiation of these
cells.
In the development of the nervous system these
trophic factors are collectively referred.to as neurotropic
factors.
Throughout the development of the central arid
peripheral nervous systems these neurotrophins and their
associated receptors play an important role, and a role that
appears to expand as more research in this area is
undertaken.
The first of the neurotrophins to be discovered was
Nerve Growth Factor (NGF), discovered by Rita Levi-Montalcini
and Victor Hamburger in association with Stanley Cohen in the
early 1950's (Levi-Montaleini et a l ., 1956; Levi-Montalcini
et al., 1960).
It was found that mouse sarcoma tissue
transplanted into a chick embryo promoted-outgrowth of
sympathetic and sensory neurons into the sarcoma tissue.
This growth was also induced by an extract of snake venom.
44kD protein responsible for this "growth promoting effect"
was termed nerve growth factor, and so began the fascination
with neurotrophins in the development of the nervous system.
Brain derived neurotrophic factor (BDNF) was subsequently
discovered by Barde and his group in 1982 by purifying the
protein through homogenization of thousands of swine brains.
It was found that there existed a considerable sequence'
homology (approximately 50%) between NGF and BDNF (Hohn et
al., 1990; Jones et al., 1990; Maisopierre et al., 1990;
A
5
Rosenthal et a l ., 1990), and this fact was used to identify a
third neurotrophin.
Hohn et al., 1990; Jones et a l . , 1990;
Maisopierre et al., 1990; and Rosenthal et al., 1990 all
utilized degenerate oligonucleotides (from homologous regions
of the two previously discovered neurotrophins) in PCR
reactions using rat or human genomic DNA to discover a novel
neurotrophin called Neurotrophin-3 (NT-3).
The three
previously mentioned neurotrophins will be the major focus,
in this thesis, because of their prominent effect on cell
populations within the developing nervous system. .
The neurotrophins are exogenous ligands and must
therefore act at a receptor on or within the cell to exert
their effects.
Previous data had identified a low-affinity
receptor for N GF, p75, which does not contain a tyrosine
kinase domain (Johnson et al., 1986: Radeke et al., 1987).It
was discovered that NGF bound with a high affinity to an
unknown receptor, which was thought to be p75.
Since p75
binds NGF with a low affinity it was therefore realized that '
there must also be another receptor binding NGF (Weskamp et
al., 1991).
It was theorized that the tyrosine protein
kinase superfamily could contain a likely candidate for the
receptor (Barbaeid et al., 1991), given that a large number
of the genes within this superfamily act as growth factor
receptors (Yarden et al., 1988; Ullrich et al., 1990) .
Since
the trks fall into this tyrosine protein kinase superfamily
they were considered a candidate for this role.
The trk
6
receptors are a family of proto-oncogenes
(Barbaeid et a l .,
1991), and' were originally detected in 3T3 cells (MartinZanca et al., 1986).
They derive their name from a
rearrangement of nonmuscle tropomyosin fused to the
cytoplasmic and transmembrane domain of a kinase receptor
(therefore trk - tropomyosin receptor kinase).
Trk receptor
proteins display a C-2 type extracellular domain similar to
that of an immunoglobulin (WiIlimas et al., 1987), which
makes them members of the immunoglobulin superfamily.
Intracellularly they contain a tyrosine kinase domain, and'
upon binding ligand (neurotrophin)
(Klein et al., 1991) the
trk receptor dimerizes forming a homodimer complex (Eide et
al., 1996) which leads to the autophosphorylation of the
tyrosine kinase domain.
This event initiates an
ihtraqellular signaling cascade that directly or indirectly
acts on transcriptional events within the cell (Jing et al.,
1992; Fantl et al., 1993).
Klein et al., 1991 determined that NGF bound to the
human proto-oncogene trk (to be later deemed "trkA", a 140 kD
glycoprotein) by cross linking NGF to trk, and through
immunoprecipitation experiments.
This binding of trk
receptor to NGF ligand induced rapid phosphorylation of the
trk intracellular kinase domain (Klein et al., 1991; Kaplan
et al., 1991).
Trk B was subsequently isolated by molecular
characterization of trk-relate'd transcripts.
It was found to
be a glycoprotein of 145 kD with a 57% homology to the
7
extracellular domain of trk A and 88% homology with the
intracellular kinase domain (Klein et a l ., 1989).
It was
later determined that BDNF, and to a lesser extent NT-3,
bound with high affinity to trk B (Squinto et al., 1991).
Lamballe, et. al.,
(1991) identified a third member of this
family, trk C, as a 145 kD glycoprotein
and showed that trk
C specifically bound NT-3 and not NGF or BDNF.
These
receptor tyrosine kinases are believed to have the ability to
promote growth and proliferation, or to halt growth and
promote differentiation depending on the receptor, its
ligand, and the type of cell in which it is expressed
(Schlessinger et al., 1992).
For a long period of time it was believed that the sole
role of neurotrophin/trk receptor interactions was one that
promoted survival during a period of target-mediated cell
death (Purves, 1988).
During the formation of the nervous
system many potential neurons die in a competitive struggle
to gain access to a limited supply of survival promoting
neurotrophins which are produced by the target tissue
(Hamburger et al., 1981; Oppenheim, 1991).
Those cells which
receive these nourishing ligands will survive and set up the
neural network, while those that do not will die through the
mechanism of apoptosis (Hamburger et al., 1982).
Apoptosis
is a neat and orderly process which results in the
condensation of the cells chromatin followed immediately by
phagocytosis by neighboring cells (McConkey et al., 1992).
8
Various genes (depending on the cell type involved) are
activated or repressed and this leads to cell death.
•
It is
termed programmed cell death because these genes are
systematically turned on or off by endogenous o r .exogenous
cues resulting in survival or death.
Recent evidence points to the involvement of
neurotrophins and their receptors in the development of the
nervous system prior to target mediated cell death (Davies,
1994).
Migrating neural crest cells express trk C mRNA and
will proliferate in response to NT-3 in vitro (Kalcheim et
a l ., 1992).
Moreover, NT-3 has been shown to accelerate 1
differentiation of newly born spinal sensory neurons
(Wright
et al., 1992), as well as induce the proliferation of
precursor populations
al., 1993).
(DiCicco-Bloom et al., 1993; Pinco et
Most strikingly, the number of DRG neurons is
dramatically reduced in NT-3 knockout mice (Farinas et al.,
1996).
Farinas et al., 1996 (in press) have shown that an
increase in apoptotic figures and a depletion of the
neuroblast pool leads to this reduction of 60-70% of DRG
neurons compared to controls.
The DRG develops from the ventral-lateral migratory
pathway of the neural crest cells
(Raible et al., 1995),
differentiating into sensory neurons, as well as supportive
cells (Figure I).
During development, the DRG will grow in
size and cell number and the sensory neuron cell bodies will
send afferent extensions into the spinal cord and to their
9
peripheral targets, where they will synapse onto their
appropriate targets.
Finally, target-mediated cell death
occurs, pruning the number of sensory neurons in the DRG to
the level that will persist in the adult.
<______>
peak of neural crest migration
<
>
peak of neurogenesis
<
>
target-mediated cell death
<
0
I
2
3
4
5
6
7
(embryonic day)
8
9
10
>
11
Figure I Critical periods in the development of the sensory neurons of the DRG (Carr,
1978; Lallier, 1988).
10
EXPERIMENTAL
OBJECTIVES
AND
RATIONALE
The goal of this study was to understand the role trk
receptors play in development of the nervous system.
To
accomplish this task we have looked at single cell resolution
of trk receptor proteins utilizing immunofluorescense to
determine their temporal and spatial expression patterns.
Previous studies have utilized in situ hybridization to
determine the expression patterns of trk receptors, and a
more definitive study determining protein expression needed
to be performed.
The highly specific antibodies utilized in
this study provide a far greater amount of cellular
resolution than in situ hybridization.
We have focused our
attention on the DRG and the interaction between
neurotrophins and their respective trk receptors because
neurotrophins are known to promote survival of neurons within
the developing DRG (Davies et a l ., 1985; Lindsay et a l .,
1985; Davies et al., 1986; Farinas et al., 1996; Lefcort et
al., 1996; Oakley et al., 1995).
that correspond to:
(I)
We examined several ages
migration of the neural crest cells
to the region of DRG formation (Marusich et al., 1991),
(Vogel et al., 1992)
(st. 14-19),
(2)
the peak of
neurogenesis within the developing DRG (st. 22-25)
et al., 1988), and (3)
(Lallier
E6-E13, the period of target mediated
cell death (Purves, 1986)(Figure I ) .
To examine this
question we used a highly specific panel of antibodies to the
11
trk receptors (Lefcort et a l ., 1996), as well as antibodies'
to well known antigenic determinants including:
BEN,
recognizing a cell-adhesion molecule present on sensory and
motorneuron populations
(Pouquie et al., 1990); Tuji,
recognizing a neuronal specific B -tubulin subunit (Memberg et
al., 1994); NE, recognizing the 160 kD neurofilament subunit
(Bennett et al., 1984); HNK-I, recognizing a carbohydrate
moiety associated with several celI-adhesion molecules
(Nordlander et al., 1993; Schachner et al., 1995); Hu,
recognizing a vertebrate RNA binding protein (Marusich et
al., 1993); and p75, recognizing the low affinity growth
factor receptor (Weskamp et al., 1991).
Use of these known
antigenic determinants allowed observations of the expression
of trk receptors in relation to events occurring throughout
development of the peripheral nervous system.
For instance,
HNK-I is present on the majority (but not all) of the
migrating neural crest cells.
NP, Tuji, and Hu will stain
neuronal cells which may still be mitotically active; while
BEN is believed to stain mature neurons' within the DRG and
ventral horn of the spinal cord.
To identify mitotically
active cells we labeled sections with 4',6-diamidino-2phenyIindoIe (DAPI) or injected BromodeoxyUridine (BrdU) and
stained with an anti-BrdU antibody (Yoshida et al., 1987;
Waid et al., 1995) .
In addition, it was of interest to determine the
expression patterns of trk receptors within the developing
12
motorneuron populations.
This is critical because it could
help us to understand why the motorneurons develop where they
do and how their axonal extensions find their targets.
This
was accomplished by viewing sections at various axial levels
early on in development.
The axial level is of importance
due to an anterior to posterior gradient of development over
time (HoIlyday et a l ., 1977).
13
METHODS
Embryos
Fertilized White Leghorn chicken embryos were obtained
from Truslow Farms
(Chestertown , Maryland) and incubated at
37° C in a rocking incubator (Kuhl, Flemington, N J ) .
Eggs
were then windowed and embryos staged at various timepoints
according to Hamburger and Hamilton, 1951.
Staging could
generally be determined by the development of the limb buds.,
but counting the somites was necessary for the youngest of
the embryos.
This is accomplished by injecting India Ink
underneath the embryo to produce a background contrast
suitable for determining the number of somites present.
Fixation/Tissue
Sectioning
Whole embryos were fixed in 4% paraformaldehyde/
Phosphate-Buffered Saline (PBS) from 8 hours to overnight
(depending on the age).
This fixing procedure crosslinks the
proteins within the tissue and therefore keeps the embryo in
a "preserved" state for future examination.
Embryos were
then rinsed in PBS and cryoprotected in 30% sucrose/PBS
overnight.
This helps protect the tissue from temperature
extremes that occur in the freezing process and future
14
cryosectioning.
They were then placed in 1:1 OCTzPBS' for
several hours, followed by straight OCT (Miles, Elkhart, IN).
The embryos were then oriented in a plastic block containing
OCT and flash frozen in an ethanol/dry ice bath.
Tissue used
for BrdU immunocytochemistry was fixed in Carnoy1s solution
(60% ethanol, 30% chloroform, and 10% glacial acetic acid).
The Tissues were then cryosectioned at 10 um on a Frigocut
2800 (Reichert-Jung) and sections were placed on gelatin
subbed slides.
Slides were subbed in chromium/gelatin
solution (300ml de-ionized (dl) H 2O, 2.Sg Knox gelatin, and
0.Sg chromium potassium sulfate), to ensure minimal loss of
sectioned material.
For motorneuron studies the axial level
of the embryos was determined by the presence of limbs and
the size of the ventral horn.
Generally, in the DRG studies,
brachial sections were obtained for the sake of consistency.
Fluorescense
Immunocytochemistry
Tissue was initially rehydrated in PBS for 20 minutes
and then placed in blocking buffer (10% normal goat serum, 1%
glycine, 3% BSA, 0.4% Triton X-100 in Tris-Buffered Solution
(TBS)) for one hour.
This buffer contains a detergent to
expose antigenic sites within the cell membrane, as well as
large globular proteins to block any non-specific binding of
antibodies.
Slides were initially treated with an Avidin/
Biotin blocking kit (Vector Laboratories, Burlingame, CA)
15
prior to incubation with primary antibodies overnight at 4 C
(approximately lug/ml), until it was found that the
Avidin/Biotin blocking step was unnecessary.
Following
incubation in primary antibodies, slides were rinsed in block
4 times at 10 minutes/rinse and then incubated in either
biotinylated or fluorescent conjugated secondary antibodies
for I hour.
Sections exposed to biotinylated secondary
antibodies were then rinsed 4 times at 10 minutes/rinse in
block and incubated in fluorescent conjugated avidin for I
hour.
This was followed by 4 more 10 minute rinses in block
and finally slides were cover slipped utilizing a Prolong
Anti-Fade Kit (Molecular•Probes) to minimize loss of
fluorophore signal over time.
Antibodies
Rabbit polyclonal antibodies to the trkA, B, and C
receptors were produced as described in Lefcort, et a l .,
1996.
These were used at approximately lug/ml.
To amplify
the signals of these antibodies biotinylated goat-anti rabbit
H&L IgG (Vector Laboratories, Burlingame, CA) was used as
second antibody at 1:450.
For the polyclonal fluorescence
signal, Fluorescein Avidin DCS/Cell Sorting Grade or
Streptavidih-CyS
used at I :1000.
(Vector Laboratories, Burlingame, CA) was
16
For double labelling experiments appropriate mouse
monoclonal antibodies,were used in association with- the trk
receptor antibodies.
Supernatant containing BEN, an antibody
which specifically recognizes an adhesion molecule expressed
by post-mitotic neurons, was used at 1:50 (Developmental
Hybridoma Bank, U. of Iowa).
HNK-I.supernatant, which
recognizes a carbohydrate moiety associated with many cell
adhesion molecules, was used at 1:10
Bank, U. of Iowa).
(Developmental Hybridoma
NFM supernatant, which recognizes the
phosphorylated and non-phosphorylated form of the 160 kD
neurofilament protein, was used at 1:100 (Virginia Lee,
University of Pennsylvania).
Tuji, recognizing a neuronal'
specific 6-tubulin isoform, ascites fluid was used at 1:5000
(A. Frankfurter, U. of Virginia).
A monoclonal antibody
supernatant against Hu, a vertebrate neuron specific family
of RNA binding proteins, was used at .1:100 (J. Weston,
University of Oregon).
Also, an anti-BrdU antibody
(Novocastra Laboratories) was used at 1:500.
Fluorescent
conjugated secondary antibodies were used to visualize these
antigens.
Finally, the nuclear stain, DAPI, was applied to
visualize individual nuclei.
BrdU
Injections
Embryos were injected, in ovo, with IOul of a 6.6mg/ml
solution of Bromodeoxyuridine (BrdU) in sterile Tyrodes
17
buffer (composed of CaCl, MgCl2, KCl, NaCl, NaPC>4 , and Dglucose), on the chorioallantoic membrane.
This was
generally accomplished by producing a small tear in the
allantoic membrane, followed by application of the BrdU with
a micropipette.
BrdU is a thymidine analogue incorporated
into the DNA of cells during DNA replication (S phase) and
can be visualized using monoclonal antibodies to it
(Gratzner, 1982) .
BrdU
Iimunocytochemistry
BrdU staining was accomplished using the above mentioned
immunocytochemical procedures, but the tissue was initially
pretreated with a denaturing agent to expose the recentlyincorporated BrdU.
This was accomplished by exposing the
tissue to either 2N HCl for 30 minutes (Yoshida et a l .,
1987), or to 0.07M NaOH for 2 minutes
(Waid et al., 1995).
Both treatments are followed by numerous rinses in PBS.
We
found that the HCl pretreatment worked well with the BrdU
stain, but somehow destroyed the antigenic epitope of the trk
receptors and therefore inhibited the binding of our trk
antibodies.
On the other hand, the NaOH did not disrupt the
binding sites of our antibodies, but destroyed the internal
portion of the tissue being stained.
This destruction almost
always resulted in the loss of the tissue in the area of DRG
18
formation and was therefore unsatisfactory.. Postfixing the
tissue in 1% paraformaldehyde for 2 minutes prior to the
denaturing treatments (personal communication with David
Waid, U. of Minnesota), only partially overcame this problem.
19
Results
Trk Expression on Developing Spinal Motor Neurons
It was of interest to examine, the expression patterns of
the trk receptors within the ventral horn of the spinal cord,
where motorneurons will develop and reside, throughout
development of the avian embryo.
Thus both the stage of
development and the axial level of the embryo were
experimental variables.
We looked at time points in early
development prior to the onset of target-mediated c e l l xdeath
while members of Ronald Oppenheim's laboratory (Bowman-Gray
Medical School, Wake Forest University) examined the later
stages.
We chose three stages:
E2.75,. E4.5, and E 6 .
Motorneuron populations were identified using a
monoclonal antibody (BEN) to a cellular adhesion glycoprotein
present on motorneurons (Pourguie et a l ., 1990) .
By double
labelling with different fluorescent secondary antibodies to
the trk and BEN primary antibodies we could determine if the
trk receptors were indeed present on the motorneuron
population.
At E 2.75 (Stage 18) there was no trk expression within
the ventral horn of the spinal cord at any axial level.
By ■
E4.5 (Stage 25) faint trkB staining was observed within all
axial levels of the ventral horn, but this could not be
20
clearly distinguished from background.
At E6 the. pattern of
trk expression became more interesting with trkB present only
at the lumbar level, and only over the medial column of the
ventral horn (Figure 2c).
There was a distinct lack.of
staining over the lateral column of the ventral horn.
TrkC
appeared to be present on the motorneuron population
throughout all axial levels at E6 (Figure 2a), yet showed the
same selective pattern of expression at the lumbar level
where it is distinctively not expressed in the lateral column
of the ventral spinal cord.
(Figure 2b).
The lack of either
trkB or trkC expression in the lateral column of the ventral
horn was more apparent in horseradish-peroxidase staining
experiments performed by Frances Lefcort.
Sensory
Neurogenesis
Stage 14
The earliest timepoint examined in these experiments was
Stage 14, corresponding to day 2.25 of development (E2.25).
Here we noticed no trk receptor expression, but did observe
the widespread expression of the p75 receptor (Figure 3a).
Of our panel of monoclonal antibodies, only HNK-I showed
expression in the region of the migrating neural crest
(Figure 3b).
p75 expression was present on the majority of
the tissue, including the HNK-I positive cells and therefore
within the migrating neural crest.
21
Figure 2 TrkC expression patterns in the ventral horn of
the E6 embryo.
(a) TrkC (green) and BEN (red) coexpress in
the cervical ventral horn producing a yellow color during
double exposure photography (arrow). (b) Absence of trkC
and trkB (c) staining observed over the lateral motor column
of the lumbar ventral horn (arrow). (v = ventral, m =
medial, 1= lateral column of ventral horn).
Figure 3 Early expression of p75 within the stream of
migrating neural crest cells, stage 14.
(a) Migrating
streams of neural crest identified with HNK-I . (b) P75
positive cells seen within the neural crest, as well as the
surrounding tissue.
(v = ventral, s = spinal cord, so =
somite).
22
Stage 15/16
At approximately E 2 .5/Stage 15-16 days- we noticed the
first evidence of trk'expression within the migrating neural
crest.
TrkC is expressed on only a very small population of
cells, and is coexpressed with Eu (Figure 4a,b) BEN (Figure
4c,d) and NF (Figure 4e,f).
N F , the antibody we used, labels
the I6OkD neurofilament protein which is believed to be
present in differentiated as well as proliferating neuronal
progenitor cells (Bennett et a l ., 1984; Bennett et a l .,
1985).
BEN is a transiently expressed cell adhesion
molecule present on the cell surface of several different
groups of peripherally projecting neurons (Pourquie et al.,
1990), and is believed to be present on post-mitotic neuronal
cells.
Eu is a family of vertebrate RNA binding proteins
that are found in mitotically active cells within the
developing DRG (Marusich et al., ,1993).
Therefore the trkC
receptor coexpresses with antigens recognizing both
mitotically active neuronal progenitor cells and post-mitotic
neurons.
We also noticed that there was no Tuji staining in the
periphery and trkC positive cells were Tuji negative (Fig.
4g,h).
There was no trk A or trk B expression present at
this age.
23
Figure 4 TrkC coexpression with several known
determinants within the migrating neural crest
(a) TrkC (arrow) seen coexpressing with (b) Hu
(arrow).
(c,d) TrkC (arrow) seen to coexpress
(arrow), and (f) NF (arrow). (d = dorsal, s =
antigenic
at stage 15/16.
positive cell
with both (d) BEN
spinal cord).
24
Figure 4
(continued)
(g) trkC expressing cell with process
extending ventrally (arrow).
(h) Note that tuji is not
coexpressed with trkC (arrow), and does not appear until stage
18. (d = dorsal,s = spinal cord).
25
Stage 18
This age (day 2.75) in the avian embryo, represents a
peak period of neural crest cell migration (Lallier et a l .,
1988) which can be easily visualized with the HNK-I antibody
(Figure 5e,f)
1993) .
(Schachner et al., 1995; Nordlander et al.,
The trkC receptor is now coexpressed with the 6-
tubulin isoform labeled by Tuji (Figure 5a,b) and is also
believed to be present on proliferating as well as
differentiated cells (Memberg et al., 1994).
TrkA is also coexpressed with NF (Fig. 5c,d), Hu, Tuji ,
and BEN, but only on a subpopulation of these cells.
Since
trkA and C are expressed in conjunction with neural specific
markers it seems' evident that these trks are expressed only
on neurons or neuronal precursors within the migrating neural
crest during this period of development.
Of special interest
is the subpopulation of trkA expressing cells, our data show
must also coexpress the trkC receptor (Table I ) .
Hu+ , NF+ , and Tuji+ cells express trkC.
also BEN/Hu/NF/Tuji+ .
All BEN+ ,
All trkA+ cells were
Therefore this suggests the trkA
positive cells must exist as a subpopulation of trkC positive
cells.
26
Table I
Stage 18 cell populations coexpressing trk A and trk C
with
mAb
Total
known
#
antigenic
Trk C+
%
determinants.
Total
#
Trk A+
Sfi
9
22.5
24
72.7
NF
39
38
97.4
40
Tuj i
51
51
100
33
BEN
36
36
100
68
38
55.9
Hu
46
44
35
7
20
95.7 '
'
Our data indicates that trkA and trkC positive migrating
neural crest cells either express HNK-I at much reduced
levels or do not express it at all.
This antibody labels the
majority (but not all) migrating neural crest cells (BronnerFraser, 1986)
(Fig. Se,f).
In these double exposure
photomicrographs of HNK-I with either trkA or trkC there is
clearly a region of low expression of the HNK-I antigen that
coincides with the expression of either of these trk
receptors.
At later stages in development this mutually
exclusive pattern of immunoreactivity becomes more difficult
to determine as more cells are present in the developing DR G ,
and the two signals exist at different focal planes
shown).
(data not
There exists the possibility that the trk antibodies
interfere with HNK-I binding and this could result in the •
apparent reduction of HNK-I expression levels.
This
27
Figure 5 Expression patterns of trkA and trkC in the neural
crest of the stage 18 embryo.
(a) trkC (arrow) is now
coexpressed with the (b) tuji marker (arrow) on a small
population of cells. (c) trkA (arrow) is now present within the
neural crest and is seen coexpressed here with NF (d;arrow). (e)
trkC (arrow) and (f) trkA (arrow) positive cells are now clearly
seen to have a reduction in the amount of HNK-I (red) expression,
(d = dorsal, s = spinal cord).
28
possiblity will be examined in future experiments.
Also at
this time the p75 receptor is present on all the migrating
neural crest as well as the majority of the cells in the
surrounding sclerotome (Figure 7a,c).
Stage 23
By 4.5 days of development the peak of neurogenesis in
the developing DRG is achieved (Lallier et a l ., 1988).
TrkB
positive neurons are now present, having first appeared at
approximately stage 22.
At this age the vast majority, if
not all, of the neurons of the DRG are NF + , Hu+, Tuji+, and
BEN+
(Figure 6a).
TrkC appears to be coexpressed with these
other antigens and therefore is also present on the vast
majority of the DRG neurons
(Figure 6b).
TrkA (Figure 6c)
and trkB (Figure 6d) are also present in the DRG at this age,
but exist only on a small'subpopulation of neurons.
The
ubiquitous expression of trkC at this time is further
evidence of multiple trk receptor expression in the trkA and
trkB subpopulations of cells within the developing D R G .
Trk Expression on Mitotically Active
Cells
Another important question in this study was to
determine whether mitotically active cells express trk
receptors during DRG formation.
If so, this would suggest a
29
Figure 6 Expression of trkA, B, and C in association with the
neuronal marker BEN, at stage 24.
(a) BEN expression of the DRG
region.
(b) trkC is on the obvious majority, if not all, of the
DRG neurons, while (c) trkA and (d) trkB exist only on
subpopulations. (e) Coexpression of trkC and BEN showing trkC on
the vast majority of DRG neurons.
(d = dorsal, s = spinal cord,
drg = dorsal root ganglia).
30
possible role for the trk receptors in the generation of
sensory neurons during development.
Utilizing the nuclear
stain DAPI, which stains chromatin, the nucleus could be
visualized and mitotically active cells could be identified.
This approach yields only a "snapshot" of mitotically active
cells and therefore many sections were observed to examine
this question.
Only stage'18 and 24 embryos were analyzed
for the presence of mitotically active cells.
At stage 18
the only neurotrophin receptor expressed on mitotically
active cells was p75
(Figure 7a,b ) .
In fact, all of the
cells in the various stages of mitosis expressed the p75
receptor, while only some expressed HNK-I (Fig. 7c).
In
addition, and of more interest to us, was the presence of
mitotically active cells at stage 24 expressing trkC (Fig.
7d,e).
However, only a subpopulation of the mitotically
active cells were trkC+ (19.7%, Table 2).
This could be due
to the. "snapshot" nature of the DAPI stain (as it only
represents the instant of tissue fixation); or, more
impressively, may represent identification of an actual
subpopulation of neural progenitors.
Consistent with these
data, we have found that only 20% of DRG cells that had
incorporated BrdU, during a short 4 hour pulse, were also
trkC positive.
Alternatively, trkC may be expressed by all
neural progenitors, but be down-regulated during mitosis.
No
trkA or trkB positive cells were found to be in any stage of
mitosis.
31
Figure 7 The presence of neurotrophin receptors on mitotically
active cells.
(a) Stage 18, p75 (arrow) present on cell in
anaphase (b;arrow) of cell cycle, while only (c) one daughter
cell expresses HNK-I (arrow). (d) Stage 24, trkC expression
(arrow) on cell in anaphase (e;arrow) of mitosis. (d = dorsal,s
= spinal cord, drg = dorsal root ganlia).
32
Table 2
Neurotrophin
cells
receptor
expression
during neural
and DRG
Mitotically
Active
on
crest
migration
formation
Trk A+
O
Active
Trk B+
O
O
O
Active
Trk C+
(st.
122
10 (BrdU incorporated)
2
Active
97
Mitotically
Active
19
p75+
(st.
96
p7 5 +
(st. 18)
19 (100%)
(prophase-4)
(metaphase-4)
(anaphase-10)
%
24)
24
(prophase-I4)
(metaphase-5)
(anaphase-5)
Mitotically
%
(st.24)
85
Mitotically
%
(st. 24)
118
Mitotically
proliferative
19.7
20
%
24)
99.0
HNK-I+
(St.
18)
10 (52.6%)
(prophase-2)
(metaphase-3)
(anaphase-5)
33
Trk Receptor Expression Within the
Growing Axon Tract
Distal
and Growth Cone
To determine whether neurotrophin receptors may play a
role in axon guidance we looked for their presence on the
distal tips of the growing sensory fiber tracts.
This is ,
where the neuronal growth cones are found that serve a
critical role in helping axon extensions find their target.
These extend ventralIy and lateral to the developing DRG and
are a fasiculated bundle of axons projecting to their
peripheral targets.
We examined the expression of trkA, B
and C with BEN (Figure 8a-f).
BEN was utilized because they
readily stain these axon extensions and are easily
visualized.
We found trk receptor expression on distalIy
projecting axons.
This evidence suggests a possible role for
trk receptors in axonal outgrowth and guidance, as expression
occurs approximately three days prior to target-mediated cell
death and well before the axons have innervated their
targets.
Stage 27
At 6 days of development the process of target-mediated
cell death has commenced.
By this age the trk positive cells
are beginning to segregate into patterns that will persist
34
Figure 8 Expression of trk receptors in the distal growing axons
of the developing sensory neurons. (a) trkA, (b) trkB, and (c)
trkC shown respectively with (d-f) BEN. (d = dorsal, T = distal
growing tip of axons).
35
into the mature animal.
As opposed to the earlier ages, trkA
(Figure 9a) is now present on a majority of the cells within
the DRG (especially in the dorsal medial pole), while trkB
and C are present on only a small minority of cells located
primarily in the ventral lateral pole.
Also of interest is
the change of expression of the BEN cellular adhesion
molecule which appears to now be limited to the dorsal medial
pole of the DRG, coexpressing with the trk A+ cells
9b).
(Figure.
BEN appears to be absent from the ventral lateral pole
and therefore no longer coexpressed with trk C, as was seen
at stage 24.
E8/E13
By ES the characteristic location of trk receptors in
the DRG is now apparent.
TrkA is located on the majority of
neurons and localized to the dorsal medial region (Figure
10a).
TrkB (Figure 10b) and trkC (Figure 10c) are now
expressed primarily by the large diameter neurons located in
the ventral lateral portion of the DR G .
This pattern persists at E13, and now the NF antibody
labels a subset, of cells primarily within the ventral lateral
portion of the DRG (Figure 11a).
This corresponds to
coexpression of this NF subunit with trkB and trkC, but to a
lesser extent with the trk A receptors.
p75 remains present
on the vast majority of cells within the DRG (Figure 11b).
36
Figure 9 Expression of trkA and trkC with BEN at E6.
(a) trkA
is now present on the majority of the cells within the dorsal
medial pole of the DRG, while (b) trkC (green) is becoming
segregated to a small population of cells within the ventral
lateral region.
Note that BEN (b;red) is now expressed in the
dorsal medial region of the DRG, where trkA is now most widely
expressed; and no longer coexpresses with trkC.
(d = dorsal, m
medial).
37
Figure 10 Expression of trk receptors in ES D R G . Here the
characteristic pattern of trk expression is evident that will
continue into adult life.
(a) trkA within the DM pole, (b) trk B
and (c) trkC segregated to the large diameter neurons in the VL
region of the D R G . (d = dorsal, m = medial).
38
Figure 11 E13 D R G . (a) NF is now localized to the the VL pole,
while (b) p75 remains ubiquitous throughout the D R G . (D =dorsal).
39
DISCUSSION
Our goal in this study was to identify the full spectrum
of developmental events in which neurotrophin-trk receptor
interactions could function during the differentiation of
spinal sensory neurons and motorneurons.
To accomplish this
task we utilized immunocytochemistry to compare trk receptor
expression with that of known markers for cell types involved
in DRG genesis.
• One important aspect of this research lies in the
association of different trk receptors with different sensory
modalities.
TrkA is expressed by the small diameter
noiciceptive (pain and temperature) cells and knockout mice
lacking this receptor and/or its ligand NGF display a loss of
noiciceptive neurons (Smeyne et a l ., 1994,- Silos-Santiago et
a l ., 1995) .
Moreover, NT-3 and trkC are required for the
survival of the large diameter proprioceptive neurons located
in the VL sector of the DR G . ' NT-3 and trkC knockouts show a
distinct loss of these proprioceptive neurons
(Ernfors et
al., 1994; Farinas et al., 1994; Tojo et al., 1995).
Although less well defined, trkB seems to be expressed by
sensory neurons projecting to visceral targets
al., 1994).
(McMahon et
If we understood the developmental factors
involved in organizing these cells into different modalities,
40
we could hope to apply this knowledge to various conditions
that affect the loss of these associated senses.
The temporal and spatial pattern of trk expression was
examined in the developing motorneurons within the ventral
horn of the spinal cord.
We looked at three ages,, all prior
to target-mediated cell death, and found the presence of trkB
and trkC only on the medial column of the motorneuron
population within the ventral horn.
We observed a distinct
lack of trk receptors in the lateral motor column.
This
implies that the motorneurons innervating the axial
musculature, but not those innervating the more distal limb
■
musculature rely on trk receptors for survival, or possibly
guidance, during development.
Three significant ages were utilized to investigate trk
expression during DRG formation.
At stages 14-19 neural
crest cells migrate to the area of future DRG formation.
i
Stage 24-25 corresponds to the peak period of •neurogenesis
within the DRG, and at E6 target mediated cell death has
commenced.
We have determined that p75 is the first of the
neurotrophin receptors to be expressed at stage 14 (2.25
days), nearly three days prior to the onset of targetmediated cell death.
It is present on the majority of the
tissue in the region of the migrating neural crest, as well
as the crest itself, and will continue to be expressed by all
DRG sensory neurons throughout development.
p75 has recently
41
been implicated as an apoptotic signal, in part due its
partial homology to Fas and tumor necrosis factor B (Frade et
a l ., 1996) .
This function does not seem likely due to the
ubiquitous pattern of p75 expression, and would cast doubt on
this present theory of p75 as an apoptotic factor.
We found that the trkC receptor was first expressed at
stage .15/16 (day 2.5) and appeared solely on a small
subpopulation of the migrating neural crest cells'.
At this
time trkC is coexpressed in full with the 160 kD NF subunit,
Hu, and BEN, but not yet with neuron specific B-tubulin
(recognized by the Tuji antibody).
Thus these trkC positive
cells may not be fully determined as neurons at this point,
and may exist as some sort of intermediate pluripotent or
bipotent neural crest cell.
Alternatively, they may comprise
a precocious pool of differentiated neurons.
Also, since NF
has been shown to be expressed in mitotically active
sympathetic neurons and Hu is seen on mitotically active
sensory neurons, we can postulate that these trkC expressing
cells are still proliferative.
TrkC is still present at stage .18 (day 2.75), and is now
coexpressed with neuron specific B -tubulin, as well as the
former markers.
Trk A is now present on a subpopulatiqn of
the N F , Hu, BEN, and Tuji positive cells; and therefore
exists on a subpopulation of trkC positive cells as well.
This is approximately 2 days prior to the earliest presence
of trkA seen by Schropel, et. al., 1995, who were looking at
42
trkA IriRNA expression.■ Interestingly, Zhang et a l ., 1994,
found the presence of trkC iriRNA through in situ hybridization
at.this early age, but did not see trkA mRNA until E 6 .
It
has been proposed that developing neuronal cells may express
more than one member of the trk receptor family, or that they
may switch from one type of receptor to another at some point
in the formation of the nervous system (Davies, 1994b), and
here we have provided rather strong evidence to support this
claim of coexpression.
This trkA+/trkC+ subpopulation of
cells may represent a distinct subset of neural crest cells.'
They may possibly proliferate at a different rate, or halt
proliferation altogether and enter into differentiation.
We
believe that there exists a temporal progression of the
expression of NP, Hu, BEN, and Tuji; and the coexpression of
trkA with these antigenic determinants may- help to explain
this progression.
TrkA is expressed on
approximately 20% of
NP+ and Hu+ cells, and on approximately 55% of BEN+ cells.
TrkA is expressed on approximately 73% of Tuji positive
cells. . Possibly NP and Hu are expressed prior to BEN and
then is followed by the expression of Tuji, which we know to
come on later than the previous three.
Our results provide
evidence for the expression of both trkA and trkC within
early (St. 18) embryos.
These trk positive cells showed -a reduction, or possibly
mutual exclusivity, in the expression of the HNK-I marker.
This reduction could possibly represent a mechanism whereby
43
early cells expressing trk receptors bind 'neurotrophins and
then turn off the expression of HNK-1, resulting .in a loss of
pluripotency in a subpopulation of the neural crest.
This
may allow for migration or process extension, or some other
differentiative effect.
Stage 24-25 represents a time point during the peak of
neurogenesis within the developing DRG (Lallier et a l .,
1988).
This is when the majority of the sensory neurons are
created and the vast majority of these cells express the trkC
receptor, which is consistent with evidence provided by
Lefcort, et al., 1996.
TrkA and trkB are present on only a
subpopulation of the developing sensory neurons at this time,
providing further evidence for the coexpression of two or
more trk receptors on a single cell.
When Minichiello et
al., 1995, looked at double homozygous negative mutants for
the trkA and trkB receptors they found that these mice showed
the same deficiencies as trkA negative mice, within the D R G .
This lends support to the thought that these trkB positive
cells expressed trkA at an earlier time point, and adds
support to trk receptor coexpression or switching during
development of the D R G .
Neurotrophin receptor switching is
supported further by the switching of receptors from p75 to
trkB on motorneurons within the ventral horn.
McKay et. al.,
1996, found by in situ hybridization that trkB mKNA was not
present in these cells until after the period of programmed
cell death, which is consistent with the change in
■
44
organization of trk receptor expression in the DRG that we
have observed.
Roskams et a l ., 1 9 9 6
, found
that trkA mRNA
expression was followed by that of trkB and then trkC in
mature neurons of the regenerating olfactory neuroepithelium,
which is the opposite of the pattern we have observed within
the differentiating DRG, and could possibly account for
regional developmental differences.
Of most interest were our findings of mitotically
active, neurotrophin receptor expressing cells, within the
region of the'developing D RG.
At stage 18 we saw the
expression of p75 on mitotically active cells in the region
where the DRG will arise.
One of these cells also contained
the neural crest marker HNK-I, which supports our finding
that these cells comprise part of the migrating neural crest
and a ire not part of the surrounding mesenchyme.
The daughter
cell lacking HNK-I expression could have been removed from
its pluritotency, moving into a more differentiated state.
We have also seen the presence of trkC, but not trkA or trkB,
on mitotically active cells.
This evidence is consistent
with a direct role for neurotrophins in proliferation of
early sensory neuroblasts.
This is extremely interesting,
because it goes beyond the model of target-mediated cell
death to provide direct evidence that cells expressing
neurotrophin receptors are proliferating, and suggests that
an additional proliferative function for neurotrophins exists
during DRG formation.
45
We have also observed the expression of trk receptors
within the distal growing sensory axons, with possible
expression in the growth cones.
This suggests that
neurotrophins could promote growth of and/or guide axon
projections as they navigate towards their targets. ' The
binding of target-derived neurotrophins could provide a
directional cue to these extensions and help them find their
respective target..
This possibility is also reinforced by
the finding that the sensory neuron modalities within the DRG
coincide with expression of particular trk receptors.
By E6 the DRG has begun to take on a more characteristic
pattern of trk expression that will persist into the adult
animal.
TrkC and B are now expressed only on a small
population of large diameter cells in the ventral lateral
(VL) pole of the DRG, while
Trk A is on the majority of
neurons and confined to the dorsal medial (DM) pole.
This
organizational pattern of separate populations of neurons
expressing different trk receptors is consistent with data
provided by Kashiba et a l ., 1995, utilizing in situ
hybridization in adult rat D R G . .This pattern may be
established as the sensory neurons begin to innervate their
target and bind the target derived neurotrophins.
The
retrograde transport of neurotrophins that follows, once'
bound to trk receptors, as seen by Von Bartheld et al. 1996,
may produce this change in the organization of trk receptors
within the D R G .
46
Interestingly, double labeling with BEN and trkC at E6
revealed a reduction in the coexpression of these two
proteins, indicating that the relationship between these two
proteins changes throughout the development of the DRG.,
One
and a half days prior to this time point trkC and BEN were
completely coexpressed, while now they appear to be almost
mutually exclusive.
This switching of antigenic determinants
throughout development is most likely due to an intracellular
signaling mechanism initiated by the binding of neurotrophins
to particular populations of cells.
At approximately 8 days in development the DRG is now
well organized and the populations of sensory neurons
expressing the different trk receptors is well defined.
It
is now clear that a subpopulation of the small diameter DM
neurons express trkC, as well as trkA.
This was also seen by
Wright et a l ., 1995, who found that a small population of DRG
neurons coexpress trkA and trkC mRNA, which is consistent
with our results.
Airaksinen et al., 1996,, showed that NT-3,-
in vivo/ is critical for the survival of Merkel cell
innervating sensory fibers that provide fine, tactile
discrimination.
These may be the trkC+/trkA+ small diameter
perikarya residing in the DM pole of the D R G .
These trkC
expressing cells might follow the path of the trkA
noiciceptive cells to their targets in the skin but innervate
separate targets once they have arrived.
47
These patterns of trk expression hold into the E13
embryo.
At this age p75 continues to be present throughout
the DRG, while the 160kD NE subunit (on the majority of DRG
neurons at stage 24) is now primarily on a small
subpopulation of cells in the VL region.
It has ceased its
coexpression with trkA and now coexpresses with the trkB+ and
trkC+ cells within the.VL region of the DR G .
Trk receptor
expression throughout the formation of the DRG is summarized
in figure 12.
In conclusion, the goal of this study was to provide
evidence for the early role of trk receptors and their
respective neurotrophin ligands in the development of the
sensory neuron populations within the DRG, prior to the onset
of target-mediated programmed cell death.
It has been well
established that neurotrophins play an extensive role in
target-mediated cell death, but from the present data it is
clear that the extent of action of neurotrophins and trk
receptors goes far beyond.
Hopefully, this enhanced
knowledge of trk receptor expression will lead to a better
understanding of the series of developmental events involved
in sensory neuron differentiation, as well as to the sequence
of events involved in the development of other areas of the
nervous system.
48
►
trkA
trkB
trkC
neural crest migration
DRG formation
peak of neurogenesis
NP, Hu,
BEN
..
Tuji
target-mediated programmed cell death
I I
st. 14
st. 16
st. 18
st.24
E6
ES
13
E m bryonic D ay
Figure 12 - Expression of trk receptors in relation to critical events
during DRG formation (Carr, 1978; Lallier, 1988).
49
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