Morphological characterization of pecteneal hyalocytes in the developing quail retina

Abstract

The periphery of the vitreous body contains a population of cells termed hyalocytes. Despite the existence for more than one century of publications devoted to the pecten oculi, a convoluted coil of blood vessels that seems to be the primary source of nutrients for the avian avascular retina, little information can be found concerning the pecteneal hyalocytes. These cells are situated on the inner limiting membrane in close relationship with the convolute blood vessels. To characterize the origin and macrophagic activity of pecteneal hyalocytes, we have analysed two different stages of quail eye development using histochemistry and immunohistochemistry. Pecteneal hyalocytes express the QH1 epitope and cKit, confirming that these cells belong to the haematopoietic system. They also express vimentin, an intermediate filament protein present in cells of mesenchymal origin and very important for differentiation of fully active macrophages. However, similarly as described in porcine hyalocytes, pecteneal hyalocytes express the glial fibrillary acidic protein, a recognized neuroglial marker. Pecteneal hyalocytes did not express other neuroglial markers, such as glutamine synthetase or S100. Acidic phosphatase was activated and Lep100 was found in secondary lysosomes, confirming phagocytic activity of pecteneal hyalocytes during ocular development. Pecteneal hyalocytes strongly react with RCA-I, WFA, WGA, PNA, SNA, LEA and SBA lectins, whereas other avian macrophages from thymus and the bursa of Fabricius did not bind PNA, SNA and LEA lectins. Interestingly, WGA lectin reacts with all kinds of avian macrophages, including pecteneal hyalocytes, probably reflecting the specific binding of WGA to components of the phagocytic and endocytic pathways. In conclusion, pecteneal hyalocytes are a special subtype of blood-borne macrophages that express markers not specifically associated with the haematopoietic system.

Fig. 1

Pecten morphology and hyalocyte localization at 37 (a) and 43 (b) HH stages. The pecten was projected into the enormous vitreous cavity (Vi) from the retina (Re) at the point of exit of the optic nerve (ON). The pecten was more pleated at 43 HH (b) than at 37 HH (a). The bridge of the pecten (Br) was seen as a swelling of the apical edge at HH stage 43 (b). Hyalocytes (Hy) were seen isolated or forming clusters both in the internal and external faces of the pleats. Insets show the areas magnified. Haematoxylin and eosin stain. Scale bars: (a) 822 µm (left panel), 127 µm (middle panel) and 16.6 µm (right panel); (b) 1135 µm (left panel), 174 µm (middle panel) and 14.5 µm (right panel).

Fig. 2

Hyalocytes express markers belonging to the haematopoietic and mesenchymal cell lineages. (a) QH1 and cKit were co-expressed in hyalocytes (Hy), endothelial cells (EC) and angioblasts (Ang). QH1 signal was observed in the cytoplasm and cytoplasmic membrane of hyalocytes and, as expected, cKit signal was mainly found in the cytoplasmic membrane. Pigmented glial cells (PG) only expressed cKit. (b,c) Vimentin was expressed in hyalocytes (Hy) and in undifferentiated pigmented glia (PG). Nuclei were counterstained with TO-PRO-3. Scale bars: (a) 13 µm (upper panel) and 6.5 µm (bottom panels); (b) 17.1 µm; (c) 7.6 µm.

Fig. 3

Hyalocytes express a characteristic marker of the neuroglial cell lineage. (a) No GS signal was observed in hyalocytes (Hy). Endothelial cells (EC) showed a low expression of GS, whereas in contrast pigmented glial cells (PG) had a stronger expression of GS. (b) GFAP was expressed by hyalocytes at both HH stages 37 and 43. Pigmented glial cells (PG) also express GFAP. (c) GFAP and vimentin do not co-localize in the cytoplasm of hyalocytes. Arrowhead clearly indicates two granules of vimentin (green) and GFAP (red) that are very close but do not co-localize. (d) All pecteneal cells, including hyalocytes, do not express S100 either at 37 HH or 43 HH. Insets show the magnified areas. In negative controls (Control–) the primary antibody was omitted. S100 expression in the auditory epithelium was used as a positive control (Control+). Nuclei were counterstained with TO-PRO-3. Scale bars: (a) 14 µm (left panel) and 7.5 µm (right panels). (b) 32 µm (37 HH, left and right panels) and 4 µm (37 HH, middle panel); 19.9 µm (43 HH, left and right panels) and 6 µm (middle panel). (c) 9 µm. (d) 27 µm (37 HH and 43 HH) and 19 µm (Control+).

Fig. 4

Hyalocyte macrophagic activity at 37 HH. (a) Positive AcPase lysosomes (arrowheads) were observed in the cytoplasm of hyalocytes (Hy). (b) Lep100 was only expressed in hyalocytes, confirming the presence of activated secondary lysosomes (arrowheads). (c) In some cases, hyalocytes presented granules of pigment (arrowheads) in their cytoplasm, suggesting that hyalocytes had phagocytosed cellular debris of pigmented glial cells (PG). Insets show the areas magnified. Nuclei were counterstained with TO-PRO-3 in laser scanning confocal micrographs. Histological section in (c) was counterstained with Nuclear fast green. Scale bars: (a) 17.5 µm (left panel) and 3.1 µm (right panel); (b) 16 µm (left panel) and 3 µm (right panel); (c) 14 µm (left panel) and 5.4 µm (right panel).

Fig. 5

Hyalocyte lectin binding pattern. (a) Ricinus communis agglutinin-I (RCA-I); (b) Wisteria floribunda agglutinin (WFA); (c) Wheat germ agglutinin (WGA); (d) Peanut agglutinin (PNA); (e) Sambucus nigra agglutinin (SNA); (f) Lycopersicon esculentum agglutinin (LEA); (g) Soya bean agglutinin (SBA); (h) Griffonia simplicifolia-I agglutinin (GSA-I). EC, endothelial cell; PG, pigmented glial cells. Nuclei were counterstained with TO-PRO-3. Scale bars: 4 µm.