Characterization of Progenitor Cells during Canine Retinal Development

Abstract

We identify the presence of progenitor cells during retinal development in the dog, as this species represents a natural model for studying several breed-specific degenerative retinal disorders. Antibodies to detected progenitor cells (Pax6, C-kit, and nestin) and ganglion cells (BDNF, Brn3a, and Thy1) were used in combination with H3 for the purpose of identifying proliferating cells. Pax6, nestin, C-kit, and H3 were localized mainly in the neuroblastic layer of the retina during the embryonic stage. During the fetal stage, proteins were expressed in the inner neuroblastic layer (INL) as well as in the outer neuroblastic layer; BDNF, Thy1, and Brn3a were also expressed in the INL. During the neonatal stage only C-kit was not expressed. Proliferating cells were present in both undifferentiated and differentiated retina. These results suggest that, during canine retinogenesis, progenitor cells are distributed along the retina and some of these cells remain as progenitor cells of the ganglion cells during the first postnatal days.

Figure 1

Histological analysis of the retina in embryos ((a)–(d)), fetuses ((e)–(h)) and neonatal ((i)–(l)) dogs. (a) Transversal section of the eye showing the retina (R) and lens (L). (b) In longitudinal section, retina consists of a neuroblastic layer (NBL) formed by an inner marginal (arrow head) and outer nuclear zone (arrows). (c) The retinal pigment epithelium (RPE) is observed to consist of a single layer with oval shaped melanosomes on the apical surface. (d) Electron micrograph showing cells with mitotic activity (*) localized in the NBL. (e) Eye from the fetal stage where retina (R) and lens (L) are evident. (f) Cell migration has formed the inner neuroblastic layer (INBL) and outer neuroblastic layer (ONBL). (g) Some rudimentary photoreceptors were evident at the fetal stage (arrow head). (h) The INBL and ONBL are separated by the transitional space of Chievitz where high resolution exposes a space between the INBL and ONBL without any cells (*). (i) Eye from the postnatal stage where part of the lens (L) and the retina (R) are visible. (j) During the postnatal stage the ganglion cell layer (GCL) was apparent. (k) The RPE was observed as a row of cubic cells. (l) Electron micrograph showing the GCL where the axons (arrow head) are forming the nerve fiber.

Figure 2

Immunofluorescent staining of C-kit (a and d), Pax6 ((b) and (e)) and Nestin ((c) and (f)) in the retina from dog embryos. The expression was observed in the NBL, examined in combination with Nomarski optics. Inserts present a panoramic view of the pattern of expression for the C-kit, Pax6 and Nestin. (g) Confocal image showing the proliferation of cells stained with H3. (h) The same figure presented in (g) but here the C-kit positive cells are evident. (i) Merged image showing cells expressing both C-kit (blue) and H3 (red) proteins in combination with Nomarski optics.

Figure 3

C-kit (a), Pax6 (b) and Nestin (c) expression in the retina from fetal stage. The expression pattern was evident in the INBL. At this stage the differentiation of ganglion cells was evident as were the markers BDNF (d), Thy1 (e) and Brn3a (f). (g) Confocal image showing the Thy1 positive cells. (h) The same image was presented in (g) but made evident with H3. (i) Merged image, but the proliferation of Thy1 cells is evident, localized in INBL (arrows). (j) Merged image illustrates the colocalization of Pax6 positive cells with H3 positive cells (arrows). (k) Merged image illustrating proliferation of BDNF positive cells in the INBL made evident by H3 (yellow stained). (l) Colocalization of BDNF and Brn3a proteins distributed along the INBL (arrows).

Figure 4

Immunofluorescent staining of Nestin (a) and Pax6 (b) during neonatal stage was observed in the INBL. (c) H3 only revealed in the GCL and did not co-localize with stem cell markers. BDNF (d), Thy1 (e) and Brn3a (f) staining showed the typical morphology of ganglion cells localized in the GCL (red cells). A number of cells marked with BDNF ((g)–(i)) and Thy1 ((j)–(l) were evidently proliferating as they were positive to H3 protein (arrows).