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Journal of the Anatomical Society of India

Neural Crest Cell Migration-Cell Tracing Techniques-A Review

Author(s): Jain, S.K.; Anand, C; Sood, K.S.

Vol. 51, No. 2 (2002-07 - 2002-12)

Department of Anatomy, Dr. R.P. Govt. Medical College, Kangra at Tanda, H.P. INDIA


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.


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:

  1. 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).
  2. 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.
  3. 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 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 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).

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.


After having compiled the above mentioned techniques for neural crest cell migration, the topics which still remain unsolved are:

  1. 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.
  2. Identification of signals that guide migrating neural crest cells.
  3. Mechanisms by which the cells migrate.
  4. 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.


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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