Analysis of cerebro‐spinal fluid protein composition in early developmental stages in chick embryos

Abstract

Foetal cerebro-spinal fluid (CSF) has a very high protein concentration when compared to adult CSF, and in many species five major protein fractions have been described. However, the protein concentration and composition in CSF during early developmental stages remains largely unknown. Our results show that in the earliest stages (18 to 30 H.H.) of chick development there is a progressive increase in CSF protein concentration until foetal values are attained. In addition, by performing electrophoretic separation and high-sensitivity silver staining, we were able to identify a total of 21 different protein fractions in the chick embryo CSF. In accordance with the developmental pattern of their concentration, these can be classified as follows: A: high-concentration fractions which corresponded with the ones described in foetal CSF by other authors; B: low-concentration fractions which remained stable throughout the period studied; C: low-concentration fractions which show changes during this period. The evolution and molecular weight of the latter group suggest the possibility of an important biological role. Our data demonstrate that all the CSF protein fractions are present in embryonic serum; this could mean that the specific transport mechanisms in neuroepithelial cells described in the foetal period evolve in very early stages of development. In conclusion, this paper offers an accurate study of the protein composition of chick embryonic CSF, which will help the understanding of the influences on neuroepithelial stem cells during development and, as a result, the appropriate conditions for the in vitro study of embryonic/foetal nervous tissue cells.

Embryonic cerebrospinal fluid regulates neuroepithelial survival, proliferation, and neurogenesis in chick embryos

“…Moreover, an anti-FGF2 antibody introduced by microinjection into cephalic ventricular cavities in vivo impairs cell replication and neurogenesis in the subventricular region (Tao et al, 1997), which is the fetal and adult successor of embryonic neuroepithelial cells (Bruni, 1998;Tramontin et al, 2003). These findings support the hypothesis that the CSF is an alternative fluid way to control the cellular behavior of the cells that are in close contact with brain cavities (Tao et al, 1997;Gato et al, 1998Gato et al, , 2004Vaccarino et al, 1999a, 199b). Therefore, our data show that the cell proliferation and neurogenesis in neuroepithelial explants cultured with serum-free medium clearly decreased compared with controls, and that the apoptosis rate greatly increased.…”

“…After dissecting the embryos out of extraembryonic membranes, the E-CSF was aspirated as previously described (Gato et al, 2004). To minimize protein degradation, E-CSF samples were kept at 4°C, aliquoted, lyophilized, and frozen at Ϫ40°C until used.…”

“…Although these organotypic cultures were prepared with mesencephalic neuroectoderm from embryos cultured in vivo until HH20 and left to develop for 24 hr (corresponding chronologically to stage HH23), the E-CSF was obtained from embryos at stage HH25 to obtain a reasonable amount of this fluid (approximately 5 l/embryo). This temporal mismatch may not significantly affect the in vitro behavior of the mesencephalic neuroepithelial explants, as the protein composition of the E-CSF is not qualitatively modified between HH20 and HH25 (Gato et al, 2004).…”

“…On the other hand, E-CSF has a complex protein composition, and the exposure of the apical surface of neuroepithelial cells to this fluid could affect their behavior (Dziegielewska et al 1980a(Dziegielewska et al , 1980b(Dziegielewska et al , 1981(Dziegielewska et al , 1991(Dziegielewska et al , 2000Checiu et al, 1984;Gato et al, 2004). Experimental data demonstrate that the loss or modification of the composition of the E-CSF strongly decreases the neuronal cell population (Desmond and Jacobson, 1977;Desmond, 1985).…”

“…In this way, several studies have demonstrated that, during development, the most prevalent components of the E-CSF are proteins. Moreover, the fact that the protein fraction of the CSF is more complex in embryos than in adults, and that it is detected at higher concentrations, has led some authors to suggest that the E-CSF is involved in the regulation of neuroepithelial cell behavior (Dziegielewska et al, 1980b(Dziegielewska et al, , 2000Ojeda and Piedra, 2000;Miyan et al, 2003;Gato et al, 2004). In addition, it has been shown that E-CSF plays a key role in the expansion of the embryonic brain primordium at the earliest stages of development (Desmond and Jacobson, 1977;Desmond, 1985;Gato et al, 1993;Alonso et al, 1998Alonso et al, , 1999Desmond and Levitan, 2002).…”