In mammals the intermediate neurofilament Nestin is a well established marker for neuronal stem cells and proliferating precursor cell populations, as well as for precursor cell populations in some other tissues (reviewed in ). Previously, nestin expression has only been characterized in mammalian systems [2, 10, 11], and chick . However, while a gene prediction for a zebrafish nestin has been placed by ENSEMBL on linkage group 16 at 27.11 Mb, until now it has not been demonstrated that this intermediate filament represents a true nestin ortholog in teleosts. Here we provide two lines of evidence that this zebrafish intermediate filament is the true nestin ortholog: (1) Zebrafish Nestin protein sequence clusters with higher vertebrate Nestin proteins, and is clearly separated from other vertebrate intermediate neurofilaments, based on phylogenetic tree analysis. (2) nestin is expressed at embryonic, larval, and juvenile stages in cell populations which in the CNS largely represent stem and precursor cells. Subsets of nestin positive cells co-express the proliferating cell nuclear antigen PCNA – zebrafish nestin expression thus correlates with the expression pattern described in mouse .
We found that outside of the nervous system nestin is expressed in mesodermal muscle precursor cells and in craniofacial mesenchyme. This correlates with nestin expression in mammals at comparable developmental stages, which has been reported for head mesenchyme and muscle precursors . In contrast to the reported nestin expression in mouse, we could not detect nestin expression neither in developing somites nor in the myotome of more mature somites at any stage. Our analysis was focused on nestin expression in the nervous system, and therefore we did not test whether there may be correlates to nestin expression reported in mammalian epidermis, heart, pancreas, kidney, and lung (reviewed in ).
During late somitogenesis stages zebrafish nestin is widely expressed throughout the developing nervous system. A similar widespread expression has been reported for comparable stages of mouse development where nestin expression and protein distribution were investigated by in situ hybridization and immunohistochemistry [1, 3]. Further, the comparison of zebrafish nestin expression with GFP expression in nestin promoter-GFP transgenic mice, which has been described in great detail [4, 27, 28], strengthens the notion of evolutionary conserved roles of Nestin in stem and precursor cell development. With progressive development of the nervous system in zebrafish, like in mouse, nestin expression becomes gradually restricted to regions of the CNS which have previously been identified as zones of proliferating stem and precursor cells: the ventricle walls in the CNS, the ciliary marginal zone in the retina and some scattered cells in the medulla oblongata [16–18].
In the peripheral nervous system nestin is strongly expressed in cells associated with the octaval ganglion, the lateral line ganglia, the facial and trigeminal ganglia, and the ganglia of the nervous vagus. It has been reported for mice that nestin in the PNS is expressed in astrocytes associated with neurons of the cranial ganglia , therefore it is likely that the nestin expressing cells associated with zebrafish ganglia are also glia.
The zebrafish brain grows throughout post-embryonic stages as well as in mature adult fish. To analyze nestin expression in the maturing post-embryonic and post-larval brain, we studied the brain of four weeks old zebrafish. In the maturing nervous system nestin expression was detected in all areas, which were recently described as stem cell niches and zones of proliferating precursor cells . In the forebrain, these are: the ventricular zones of the tel- and diencephalon, the ventral habenular nucleus (Hav), the periventricular pretectal nucleus (PP), the thalamic nuclei (TN) including the dorsal thalamic nucleus (DT), and the ventromedial thalamic nucleus (VM). In the mid- and hindbrain, these are: the mesencephalic areas of the torus longitudinalis and optic tectum adjacent to the ventricle, the MHB, proliferative zones in the cerebellum, and ventricular zones of the hindbrain and the spinal cord. In summary, nestin expression in the maturing, post-embryonic CNS correlates with the previously described proliferation zones [16–18]. Thus, the expression of the intermediated neurofilament nestin in zebrafish, like in mammals, may serve as a marker for stem cells and proliferating precursors in the developing embryonic nervous system as well as in the adult brain.