Auat Embryol (1996) 193:131-144 9 Springer-Verlag 1996 Amale Moujahid 9 Julio Navascu6s Jos6 L. Marin-Teva 9 Miguel A. Cuadros Macrophages during avian optic nerve development: relationship to cell death and differentiation into microglia Accepted: 10 July 1995 Abstract Cell death is frequent during the development of the nervous system. In the developing optic nerve of chicks and quails, neuroepithelial cell death was first ob- servable on the third day of incubation, slightly after the first cell ganglion axons appeared in the stalk. Special- ized phagocytes were observed within the stalk in chro- nological and topographical coincidence with cell death. These cells were identified as macrophages because of their morphological features, intense acid phosphatase activity and, in quail embryos, labeling with QH1, a monoclonal antibody recognizing quail hemangioblastic cells. Macrophages in areas of cell death were round and actively phagocytosed cell debris. We used electron mi- croscopy and histochemical and immunocytochemical labeling to study macrophagic cells of the optic nerve in avian embryos of 3-6.5 days of incubation. As develop- ment proceeded, phagocytosing, round macrophages be- came ameboid macrophages that migrated from areas of cell death toward regions occupied by optic axonal fasci- cles. Macrophages in these locations were thin and elon- gated, with a few processes. To elucidate the final fate of macrophagic cells in the optic nerve, sections taken from older embryonic and hatched quails were stained with the QH1 antibody. On the 8th day of incubation some slightly ramified QHI+ cells were present among axonal fascicles. In subsequent stages these cells increased in number and acquired more complex ramifications. In adult optic nerves, QHI+ cells had a small body and sent out slender processes, sometimes with secondary and ter- tiary branches, which were frequently orientated parallel to the course of the optic axons. These cells were consid- ered to be microglial cells. The appearance of macro- phages within the developing optic nerve at the same time as neuroepithelial cell death suggests that cell death influences the recruitment of macrophages into the nerve. When macrophages reach the areas invaded by A. Moujahid - J. Navascu6s( ~ ) 9 J. L. Marfn-Teva M. A. Cuadros Departamento de BiologfaCelular, Facnltadde Ciencias, Universidad de Granada, E-18071 Granada, Spain Fax: +34-58-243078 optic axonal fascicles, they undergo structural and proba- bly also physiological changes that appear to signal dif- ferentiation into microglia. Key words Macrophage 9 Microglia 9 Cell death 9 Chick optic nerve 9 Quail optic nerve Introduction The optic nerve develops from the optic stalk, which is composed of neuroepithelial cells stretching from the pi- al surface to the ventricular lumen. The early develop- ment of the optic nerve in chick embryos has been previ- ously described (Rager 1983; Navascu6s et al. 1985, 1989). From developmental stage 20 (HH20) of Ham- burger and Hamilton (1951) onward, ganglion cell axons invade the optic stalk, and the neuroepithelial organiza- tion progressively disappears, first in ventral regions and then dorsally. At HH29-30 the ventricle is no longer ob- servable, and the optic nerve is almost completely occu- pied by optic fiber fascicles, delimited by glial cells. On- ly a thin cellular rim persists beneath the pia on the dor- sal surface of the nerve, where no axons are identifiable. Cell death takes place in many regions of the central nervous system (CNS) during development (Oppenheim 1991; Raft et al. 1993), including the early stages of vi- sual system development in chick embryos (Ulshafer and Clavert 1979; Rager 1980; Cuadros and Rfos 1988; Na- vascu6s et al. 1988; Martfn-Partido and Navascu6s 1990). Specialized phagocytes, which have been charac- terized as macrophages, are frequent during the first days of incubation in birds (Cuadros et al. 1992a) and are also present within the developing CNS (Cuadros et ai. 1993). The presence of such cells in the CNS during ear- ly stages of development has been linked to cell death (Hume et al. 1983; Perry and Gordon 1988; Ashwell 1991). This hypothesis can be easily tested in the optic nerve, because it is well delimited and relatively small and because cell death occurs during its early develop- ment (Navascu6s et al. 1985). We have studied the