Anat Cell Biol.  2012 Mar;45(1):38-46. 10.5115/acb.2012.45.1.38.

Nestin expressing progenitor cells during establishment of the neural retina and its vasculature

• Lee JH
• Park HS
• Shin JM
• Chun MH
• Oh SJ

• KMID: 1447451
• DOI: http://doi.org/10.5115/acb.2012.45.1.38

Abstract

In order to test if nestin is a useful marker for various types of progenitor cells, we explored nestin expression in the retina during development. Nestin expression was co-evaluated with bromodeoxyuridine (BrdU) labeling and Griffonia simplicifolia isolectin B4 (GSIB4) histochemistry. Nestin immunoreactivity appears in cell soma of dividing neural progenitor cells and their leading processes in retinas from embryonic day (E) 13 to E20, in accordance with a BrdU-labeled pattern. At postnatal day (P) 5, it is restricted to the end feet of Muller cells. BrdU-labeled nuclei were mainly in the inner part of the inner nuclear layer in postnatal neonates. The retinal vessels demarcated with GSIB4-positive endothelial cells were first distributed in the nerve fiber layer from P3. Afterward the vascular branches sprouted and penetrated deeply into the retina. The endothelial cells positive for GSIB4 and the pericytes in the microvessels were additionally immunoreactive for nestin. Interestingly, the presumed migrating microglial cells showing only GSIB4 reactivity preceded the microvessels throughout the neuroblast layer during vascular sprouting and extension. These findings may suggest that nestin expression represents the proliferation and movement potential of the neural progenitor cells as well as the progenitor cells of the endothelial cell and the pericyte during retinal development. Thus, Muller glial cells might be potential neural progenitor cells of the retina, and the retinal microvasculature established by both the endothelial and the pericyte progenitor cells via vasculogenesis along microglia migrating routes sustains its angiogenic potential.

Keyword

Neurogenesis; Vasculogenesis; Retina; Nestin; Proliferation; Migration

MeSH Terms

Bromodeoxyuridine
Carisoprodol
Endothelial Cells
Foot
Griffonia
Humans
Infant, Newborn
Intermediate Filament Proteins
Lectins
Microglia
Microvessels
Nerve Fibers
Nerve Tissue Proteins
Neurogenesis
Neuroglia
Pericytes
Plant Lectins
Retina
Retinal Vessels
Retinaldehyde
Stem Cells
Bromodeoxyuridine
Carisoprodol
Intermediate Filament Proteins
Lectins
Nerve Tissue Proteins
Plant Lectins
Retinaldehyde

Figure

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  Fig. 1 Confocal microscopic views of the developing retinas processed for nestin immunofluorescent staining. E13 (A), E18 (B), E20 (C), P3 (D), P7 (E), and P14 (F). Nestin immunoreactivity appeared first in the proximal radial fibers of the neuroepithelium (NE) at E13 (A). Subsequently it expanded distally to P3 (D). The nestin immunoreactivity was gradually restricted to proximal fibers (E) and finally to the end feet of Müller glial cells (F). E, embryonic day; HB, hyaloid body; INL, inner nuclear layer; IPL, inner plexiform layer; NBL, neuroblast layer; ONL, outer nuclear layer; P, postnatal day. Scale bar=50 µm.

• 

  Fig. 2 Light micrographs of BrdU labeling in the developing retinas. Proliferating cell nuclei labeled with BrdU were distributed throughout the whole depth of the neuroepithelium (NE) at E13 (A). BrdU labeling was localized in the inner part of the neuroblast layer (NBL) at E15 (B). There was almost no BrdU labeling at E20 (C) through the main body of the retina, except in the ciliary margin (caret). BrdU labeling (arrows) was detected in the inner nuclear layer (INL) postnatally (D-F) and reached a peak at P14 (E). E, embryonic day; GCL, ganglion cell layer; IPL, inner plexiform layer; LV, lens vesicle; ONL, outer nuclear layer; P, postnatal day; PE, pigment epithelium. Scale bar=50 µm.

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  Fig. 3 Confocal microscopic views of the developing retinas processed for nestin immunofluorescence (red) and lectin (GSIB4, green) histochemistry. Lectin- and nestin-positive endothelial cell-lined retinal vessels developed in the nerve fiber layer from P3 (A), and lectin-positive presumed microglial cells (arrows) were distributed sporadically along the radial fibers with red fluorescence throughout the neuroblast layer. Microglial cells (arrows) with green fluorescence were restricted to the inner half of the inner nuclear layer at P5 (B) and P7 (C). At P14 (D), the microvessels lined with lectin- and nestin-positive endothelial cells were distributed in the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, and the outer plexiform layer. P, postnatal day; GSIB4, Griffonia simplicifolia isolectin B4. Scale bar=50 µm.

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  Fig. 4 Electron microphotographs taken from parts of the developing retina processed for nestin immunohistochemistry. Nestin immunoreactivity appeared in the dividing cells (solid arrows) in the ventricular zone of E18 (A) and in the radial fibers (arrows) throughout the neuroblast layer and inner plexiform layer (IPL) (P3) (B) of the developing retina. Nestin immunoreactivity was strongly demarcated the end feet (EF) of the Müller glial cells at P7 (C). Additional nestin imminoreactivity was detected in the endothelial cells (arrowhead) of the retinal vessel in the nerve fiber layer at P7 (C). After the establishment of the capillary networks, the endothelial cells in the retinal vessels showed no nestin immunoreactivity (P14) (D). While in the developing capillary networks of the outer plexiform layer (OPL) at P14 (E) and P21 (F), nestin was also labeled in the pericytes (open arrow) underneath the endothelial cells (arrowhead). E, embryonic day; INL, inner nuclear layer; P, postnatal day. Scale bars=2 µm (A-D, F), 1 µm (E).

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