1. J Anat. Soc. India 51(2) 239-243 (2002)
Neural Crest Cell Migration-Cell Tracing Techniques-A Review
Jain, S.K.; Anand, C; Sood, K.S.
Department of Anatomy, Dr. R.P. Govt. Medical College, Kangra at Tanda, H.P. INDIA
Abstract. Neural crest a distinctive feature of vertebrate embryo had attracted much attention of development biologists. A number
of creative approaches have been devised and have yielded various types of information regarding their origin, migration and fate. The aim
of present review article is to discuss, compare and to critically analyse the various techniques used for neural crest cell migration. The
article ends with the perspective section which deals with the challenges of future in the study of neural crest cell migration.
Key words : neural crest, explant, chimera, retrovirus, transgene, LRD, MEBL-1, TRP, C-kit.
Introduction :
The main areas of concern of development
biologists today are inextricably linked with those
topics which were of compelling interest to the
embrylogists of the past.
The neural crest is a transient structure found
only in vertebrate embryos. The significance,
migration and the fate of neural cest cells has been
and is currently of great interest. The importance of
the neural crest can not be underestimated, as it
appears to be a unique feature of vertebrates
distinguishing them from their chordate ancestors.
(Gans & Northcutt; 1983).
The fact that the neural crest yields
mesenchymal cells was first proposed at the turn of
century by Katschenko (1888), Goronowitsch (1892)
and later by Platt (1893) who showed that it
contributes to the cartilage of pharyngeal arches and
to the dentine of the teeth in lower vertebrates.
Since these early times the neural crest has
attracted much attention from embrylogists who,
during the earlier part of this century investigated
the fate of this structure mainly in amphibian
embryo. Hostradius (1950) described that neural
crest cells have such a range of diverse derivatives
that it was very difficult to follow them
developmentally. In this review article various
techniques for studying the neural crest cell
migration have been discussed and compared. The
highlight is being kept on the interpretative
problems, which are being critically discussed and
analyzed.
Techniques Used For Tracing Neural Crest Cell
Migration :
1. Classic Ablation Experiments :
In this experiment a thin slice of neural fold is
resected from an experimental animal at the
appropriate stage of development and then to see
what structures do not develop. On one hand this
technique is simple to perform but at the same time
it presents the problems of interpretation. For
example :
(a) Some embryos have some capacity of
functionally replace the ablated crest e.g. Rat
neural crest has got the property of self
renewal (Stemple & Anderson., 1992).
(b) It is difficult to define precisely the boundaries
of presumptive neural crest cell subpopulation,
so the results of diferent experiments are often
hard to compare.
(c) It can not be known that structure which does
not develop is due to ablation of the progenitor
crest cell or due to accidental ablation of other
cells essential for their differentiation e.g.
ablation of cells secreting trophic substances.
2. Explantation experiments : Refined and
sterile media are available for culturing many
different cell lines that are available for research.
Cell culture procedures require considerable
attention, the constituents of the nutritive media, as
well as optimal temperature and sterile conditions.
Culture conditions can be provided which allow
neural crest cells to grow and express their
development potentialities even when seeded as
single cells (Sieber-Blum & Cohen, 1980, Baroffio,
Dupin and Le Dourain; 1988). The abilities of
individual neural crest cells to proliferate and
differentiate are highly variable. Differentiating
potentialities of neural crest cells vary from
unipotent (neuronal or glial) to totipotent (cephalic
neural crest cells). It has been argued that neural
crest may be totipotent (whether their migration
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Neural Crest Cell Migration
pattern follows in vitro or in vivo) or alternatively it
may be interepreted that an explant that gives rise
to several derivatives actually consists of a
heterogenous population of cells each of which is
unipotent.
Explantation studies suggest plasticity of
neural crest cell development and assign control of
the patterning mainly to the embryonic territories
that the crest cells colonize.
3. Cell Marking Techniques : Early studies of
embryonic cell lineage were based on natural
markers found in certain cells, such as pigment
granules, distinctive nuclear morphologies, or yolk
inclusions, which could be used to trace the
migration of and differentiation of cells. But this
approach didn't prove useful as crest cells have got
no distinctive natural markers.
In 1963 Weston and 1967 Chibon devised a
more precise technique by labelling the DNA of
migrating neural crest cells with tritiated thymidine.
Migration of cells labelled with tritiated thymidine
can be followed by autoradiography
(Autoradiography is used for visualizing a newly
synthesized cell product after cells are exposed to
radioactive precursor molecules for varying lengths
of time either under in vitro or in vivo condition).
However because the radioactive label is
diluted each time a cell divides & synthesizes new
DNA, the technique can trace a cell lineage through
only a limited number of generations. So this
technique is not stable and furthermore reuptake of
label released by dead cells compromised
specificity. The problem of label dilution was solved
by development of quailchick chimera system.
4. Quail-Chick Chimera Technique : The
technique is based on the observation by Le Douarin
(1969) on the structure of the interphase nucleus in
the Japanese quail. The cells of this species are
characterised by the condensation of constitutive
heterochromatin into a single mass, generally
centronuclear, associated with the nucleolus. Such a
characteristic is rare in animal kingdom. In most
species the constitutive hetrochromatin is evenly
distributed in the neucleoplasm in small
chromocenters e.g. in chick. Quail and chick cells
can thus easily be distinguished by DNA staining or
by electron microscopy, where the large DNA rich
nucleolus of the quail is easily recognisable. These
inter-species differences have been used to study
the migration of embryonic cells and to analyse the
contribution of cells of different embryonic origins to
complex tissues or organs during ontogeny. Since
the quail and the chick are closely related in
taxonomy, the substitution of definite territories
between embryos of these two species in ovo
results in viable chimeras which develop normally
and can hatch. Thus when defiinite fragments of
either the neural fold or the neural tube and
associated neural crest of the quail are grafted
isotopically or isochronically into the chick (or vice
versa) the migration and fate of their constituted
cells can be followed. This approach has been
systematically applied to whole neuraxis to establish
the fate map.
By grafting cranial paraxial mesenchyme and
the 6 rostral somites Couly, Coltey and Le Douarin
(1992), were able to delineate precisely the
respective contributions of the somites, paraxial
mesenchyme and the neural crest to skull.
The question whether crest cells are,
unipotent, pleuripotent or totipotnet has been partly
answered by the experiments in which quail cell
from one part of the neural crest were transplanted
to various locations along the chick neural tube.
The interpretive problems of thymidine and
quail-chick marking experiments can be avoided by
studying single cells marked with fluorescent dyes.
5. Cell Lineage Study Using Fluorescent Dye :
It is now possible to follow the development of a
single neural crest cell by injecting it with a highly
fluorescent, nontoxic marker that remains visible in
the injected cell and its descendents. In some
cases, a single neural crest cell injected with
lysinated rhodamine dextron (LRD), before or during
migration gives rise to daughter cells that develop
into a variety of neural crest derivatives. Clonal
analysis of LRD-injected trunk neural crest cell
precursors of the mouse shows that their
developmental potential depends, in part, on the
time that they emigrate from the neural tube. Single
cells injected during early neural crest migrations
may give rise to neural tube cells as well as neural
crest derivatives that migrate along both dorsal

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Jain, S.K. et al
pathways and ventral pathways. Derivatives of the
dorsal pathway include melanocytes and cells of the
dorsal root ganglia, while cells of the ventral
pathway form sympathetic ganglia and
adrenomedullary cells. In contrast migration of late-
emigrating neural crest cells is restricted to dorsal
pathway (Larsen; 1997).
6. Cell Lineage Studies Using Retroviruses :
In amphibians and avians direct cell injection of
fluorescent lineage tracers is possible. This is so far
impossible in mammals owing to inaccessibility of
the embryos in utero. Instead, lineage analysis have
relied on the injection of progenitor cells in the
ventricular zone on rodent embryos using replication
defective retorviruses (Sanes, Rubinstein and
Nicolas, 1986). The retroviral DNA incorporates into
the genome of the infected cell providing an
indelible marker that is transmitted to all progeny
durinig cell divison, but not to surrounding cells.
Retroviral techniques have yielded some
information about lineage in the various regions of
the nervous system. For example an electron
microscopic study performed by Parnavelas,
Barfield, Franke and Luskin; 1992 of the synaptic
morphology and ultrastructure of retovirally marked
cells has reported seprate clones of pyramidal and
nonpyramidal neurons as well as of astrocytes and
oligodendrocytes.
Very recently techniques using replication
defective virus have been used for developing
heart. In this study a replication defective avian
spleen necrosis virus which lacks the viral structural
genes gag, pol and env, (which have been replaced
with the bacterial B-galactosidase gene lac Z), has
been used to mark NC cells. Its results were
compared with those of chicken-quail chimera used
as a gold standard. Retrovirus infected cells
expressed the Lac Z gene and could be
distinguished both in whole mount preparation and
in the histological sections. Because of the
possibility of whole mount preparations retroviral
approach using B-gal for staining is far superior to
the chick-quail chimera to obtain overall picture of
labelled cell progenitor in the target organs
(Polemann, Mikawa, Gittenbergen and Groot; 1998).
7. Transgenic Techniques : Recombinant DNA
techniques can now be used to study differentiation
in specific tissue lineages. In an experiment a
bacterial transgene was introduced into the mouse
genome in such a way that it caused certain cell of
neural crest origin - specifically neurons of dorsal
root ganglia to stain themselves blue. The lac Z
gene was inserted into the transgene in a position
that put it under the control of the same regulatory
DNA region that controls the expression of the gene
for a neurofilament protein called peripherin. Copies
of this engineered sequence were then injected into
the male pronucleus of pronuclear stage mouse
embryos, where they become incorporated into the
embryonic genome. Peripherin is normally
expressed in a number of tissues of neural crest
origin. The lac Z gene in the transgenic mice should
be activated in all cells that express the endogenous
peripherin gene, with the result that all peripherin
producing tissue would stain blue. (Larsen, 1997).
8. Marker Technique : Recently a number of
marker have become available that label migratory
neural crest cells. These include antibodies or
probes to the receptor tyrosine kinase - Ret
(Pachnis, Manko and Constantini; 1993, Tsuzuki., et
al 1995., Dubec et al 1996., Wantanabe et al 1997)
the low affinity neurotrophin receptor, P 75 NTR
(Baetge & Gershon; 1990; Chalazonitis, et al 1994;
Lo and Anderson, 1995), the endothelin-B receptor
(Gariepy et al 1998) the 5-Ht2 B receptor (Florica-
Howells et al 1998) the transgene DBH n lac z
(Kapur, et al 1992) the catecholamine synthetic
enzyme, tyrosine hydroxylase TH (Cochard, et al
1978; Teitelman et al 1978; Jonakait et al 1979) and
the transcription factors MASH1 (Lo, et al 1991; Lo
and Anderson, 1995; Lo, et al 1997), Phox
2a(Tiveron, et al 1996), phox 2b (Pattyn et al 1997),
and sox 10 (Herbaerth, et al 1998). All the above-
mentioned antibodies or probes are concerned with
enteric nervous system.
The markers or probes for melanocytes are
MEBL-1 (Kitamura et al 1992), TRP-1 (Reddy,
Faraco & Erickson; 1998), TRP-2 (Steel, et al 1992)
Mitf (Opdecam et al 1997) and C-Kit (Kitamura et
al., 1992; Wehrle-Haller and Weston., 1996.)
For Neural crest derived neurons the markers
are Hu (Marusich and Weston., 1992; Marusich, et
al 1994), SC-1/BEN (Pourguie, et al 1990), Neuron
specific Tubilin (Lee, et al 1990).

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Neural Crest Cell Migration
For Neural crest derived Glial cells-7B3
(Henion, et al 2000) and for Trunkal neural crest
cells – NC1/HNK1 (Tucker et al 1984) are the
markers.
Perspective
After having compiled the above mentioned
techniques for neural crest cell migration, the topics
which still remain unsolved are :
(a) Whether the fate of a crest cell is
predetermined before the crest cell leaves the
neural fold or is determined by clues along the
root of migration and at the site of
differentiation.
(b) Identification of signals that guide migrating
neural crest cells.
(c) Mechanisms by which the cells migrate.
(d) Nature of cell-cell interaction that regulate their
differentiation.
In the last decade, a remarkable progress has
been made in evolution of markers, to trace the
various neural crest cell derivatives. It is expected
from the above observation that plethora of
information will be generated in the very near future.
Acknowledgement :
Authers wish to acknowledge Principal, Dr.
Rajendra Prasad Govt. Medical College, Kangra at
Tanda H.P. India for providing all types of help
needed during this project.
Authors wish to acknowledge Mr. Amir Chand
for computerized typing of this manuscript.
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