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Abstract
CD44 is a transmembrane protein that acts as a receptor for an adhesion molecule, hyaluronic acid. The type of cells expressing CD44 and roles of CD44 are still controversial and need to be elucidated. The aim of the present study was to examine the type of cells expressing CD44 and the changes in their distribution in the retina and the cerebellum of the developing and adult chicken. Embryonic day 14 (E14) and post-hatch day 90 (P90) chickens were used in this study. CD44-immunoreactive (ir) cells were observed both in the retina and the cerebellum of the two developmental stages examined. In the retina of E14, CD44-ir cells were mainly located in the nerve fiber layer. In adults, most of the CD44-ir cells were in the nerve fiber layer and some were dispersed in other layers of the retina. In the cerebellum of E14, CD44-ir cells were distributed throughout the cerebellar cortex, including the external and internal granular layers. CD44-ir cells were more frequently found in the cerebellum of P90 adult chickens than in that of E14 embryos. At higher magnification, CD44-ir cells showed ramified cytoplasmic processes irradiating from their cell bodies. In the retina and in the cerebellum of all ages examined, double staining showed that most of the CD44-ir cells also expressed RCA-1, a marker of microglia. In contrast to that, at the same locations, GFAP and CD44 were not co-expressed in cells. When the adult retina was stimulated by LPS, CD44 immunoreactivity increased, and CD44-ir cells were also RCA-1-positive. The present results indicated that CD44 was expressed in microglia of the retina and the cerebellum of the developing and adult chicken even in normal conditions, and microglial CD44 expression was increased upon LPS stimulation.
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Fig. 1.
CD44 expression in the retina (A-C, G-I) and cerebellum (D-F, J-L) of E14 chick embryo and adult chicken. Cresyl violet staining shows layers of the retina (A, G) and cerebellum (D, J). Images in small boxes of B, E, H, and K are magnified to C, F, I, and L, respective-ly. At E14 day, CD44-ir cells in the retina are mainly located in the nerve fiber layer (B). At P90 CD44-ir cells are also observed mainly in the nerve fiber layer (H). In the cerebellum, CD44-ir cells are distributed throughout the cerebellar cortex of E14 embryo (E) and P90 adult (K). CD44-ir cells show ramified cytoplasmic processes from their cell bodies in higher magnified images (C, F, I, L). nf, nerve fiber layer; gc, ganglion cell layer; ip, inner plexiform layer; in, inner nuclear layer; eg, external granular layer; ig, internal granular layer; m, molecular layer; g, granule cell layer. Bars represent 100 μm (A, B, D, E, G, H, J, K) and 30 μm (C, F, I, L).
Fig. 2.
Double immunofluorescent staining for CD44 and RCA-1 in the retina (A, C) and cerebellum (B, D) of E14 chick embryo and adult chicken. All merged images (A3, B3, C3, D3) represent that CD44-ir cells are colocalized with RCA-1-immunoreactivity, a marker of mi-croglia. nf, nerve fiber layer; gc, ganglion cell layer; ip, inner plexiform layer. Bar indicates 50 μm.
Fig. 3.
Double immunofluorescent staining for CD44 and GFAP in the retina (A, C) and cerebellum (B, D) of E14 chick embryo and adult chicken. GFAP-immunoreactiviry is observed only in adult cerebellum (D2), not in E14 (A2) and P90 retina (C2), and E14 embryonic cer-ebellum (B2). All merged images (A3, B3, C3, D3) represent that CD44-ir cells are not labeled with GFAP, a marker of astrocytes. (A) E14 embryonic retina, (B) E14 embryonic cerebellum, (C) P90 adult retina, (D) P90 adult cerebellum. nf, nerve fiber layer; gc, ganglion cell lay-er; ip, inner plexiform layer. Bar indicates 50 μm for all figures.
Fig. 4.
CD44 expression in P90 adult chicken retina after LPS stimulation and double immunofluorescent staining for RCA-1 and GFAP, respectively. At 24 hours after LPS injection, CD44 is overexpressed and its immunoreactivity is observed mainly in the nerve fiber lay-er of the retina (A1, B1). CD44-immunoreactivity is colocalized with RCA-1 (A3) but not with GFAP (B3). Despite of LPS stimulation, GFAP-immunoreactivity is not observed (B2). These results represent that CD44 is expressed both in resting and activated microglia of adult retina. Bar indicates 50 μm for all figures.
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