Abstract
Adult rat hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitor cells (NPCs) that can differentiate into neurons, oligodendrocytes, and astrocytes. AHPCs contact a variety of molecular cues within their surrounding microenvironment via integrins. We hypothesize that integrin receptors are important for NPCs. In this study, we have examined the distribution of integrins in neuronal-like, oligodendrocyte-like, and astrocyte-like AHPCs when grown on substrates that support integrin-mediated adhesion (laminin, fibronectin), and those that do not (poly-l-ornithine, PLO) using immunocytochemistry as well as characterized the phenotypic differentiation of AHPCs plated on laminin and fibronectin. Focal adhesions were prominent in AHPCs plated on purified substrates, but were also found in AHPCs plated on PLO. The focal adhesions observed in AHPCs plated on PLO substrates may be formed by self-adhesion to the endogenously produced laminin or fibronectin. We have demonstrated that integrins contribute to the initial morphological differentiation of AHPCs, as inhibition of fibronectin binding with the competitive inhibitor echistatin significantly decreased the number of processes and microspikes present in treated cells, and also decreased overall cell area. Finally, we have characterized the genetic profile of a subset of integrins and integrin-related genes in the AHPCs using reverse transcriptase polymerase chain reaction. These results demonstrate an important role of integrins, in vitro, for the initial morphological differentiation of AHPCs.
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Acknowledgments
The authors would like to acknowledge W. Law, J. Callahan, and D. Au for technical assistance. The authors would also like to thank Dr. R. Doyle and the Roy J Carver Laboratory for Ultrahigh Resolution Biological Microscopy for use of facilities and equipment essential for genetic analysis. The authors thank Drs. F.H. Gage (for the gift of the AHPCs), K. Yamada (for the gift of the anti-Beta 1 antibody), E. Rouslahti (for the gift of the anti-Alpha 2 antibody), T. Joos (for the gift of the anti-Alpha 5 antibody), and E. Engvall (for the gift of the anti-laminin antibody). Finally, the authors thank Dr. C.J. Jeon, J. Oh, J-Y. Yeo, and H-H. Kim for their critical reading of this manuscript. This work was supported by a grant from NIGMS R01-GM072005-01.
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Supplemental Fig. 1
β1 integrin subunit expression by AHPCs plated on poly-l-ornithine 7 days after plating. β1-IR in Neuro-L AHPC was present throughout the cell body and processes (a). Merged images (b, c) reveal little evidence of focal adhesion formation, and expression of β1 in discrete clusters (c, arrows). Oligo-L cells also expressed high levels of β1 in the cell body and processes (d). Merged images revealed few focal adhesions were present (e). While there were some focal adhesions present (f, arrows), a majority of β1 was not present in focal adhesion complexes (f, arrowheads). Astro-L AHPCs had prominent β1 expression throughout the cell (g). Merged images (h, i) demonstrate that focal adhesions were present at the periphery of the cell (i, arrows), but not in the interior (i, arrowheads). Scale bar: 20 µm in a for images a, b, d, e, g, and h. Scale bar: 10 µm in c for images c, f, and i. Talin—red; F-actin cytoskeleton—green; β1—blue
Supplemental Fig. 2
α5 integrin subunit expression by AHPCs plated on poly-l-ornithine for 7 days. α5-IR was present in Neuro-L and Oligo-L AHPCs in the cell bodies and processes (a, d). Merged images (b, e) demonstrate some evidence of focal adhesions in these cells. Digital magnification of Neuro-L cells showed some focal adhesion near the cell body (c, arrowhead), but not in the processes (c, arrow). Oligo-L cells also showed focal adhesions near the cell body (f, arrow), but not in the processes (f, arrowhead). Astro-L cells displayed α5-IR near the nucleus with decreasing staining intensity toward the periphery (g, h), but only had modest focal adhesions in the interior of the cell (i, arrow). Scale bar: 20 µm in a for images a, b, d, e, g, and h. Scale bar: 10 µm in c for images c, f, and i. Talin—red; F-actin cytoskeleton—green; α5—blue
Supplemental Fig. 3
α5 integrin subunit expression by AHPCs plated on laminin for 7 days. α5-IR was observed in the cell bodies and processes of Neuro-L and Oligo-L AHPCs (a, b, d, e). Digital magnification revealed focal adhesions in the cell body (c, f, arrowhead), but not in the processes (c, f, arrows). Robust expression of α5 was observed in Astro-L AHPCs (g, h). Very few focal adhesions (i, arrow) were present in Astro-L cells. Scale bar: 20 µm in a for images a, b, d, e, g, and h. Scale bar: 10 µm in c for images c, f, and i. Talin—red; F-actin cytoskeleton—green; α5—blue
Supplemental Fig. 4
α2 integrin subunit expression by AHPCs plated on poly-l-ornithine for 7 days. α2 was highly expressed in AHPCs (a, d, g). Clusters of focal adhesions were present in Neuro-L cells (b, c, arrows). No significant focal adhesions were present in Oligo-L or Astro-L cells (e, f, h, i, arrows). Scale bar: 20 µm in a for images a, b, d, e, g, and h. Scale bar: 10 µm in c for images c, f, and i. Talin—red; F-actin cytoskeleton—green; α2—blue
Supplemental Fig. 5
α2 integrin subunit expression by AHPCs plated on fibronectin. α2 was highly expressed in AHPCs (a, d, g) growing on a fibronectin substrate. Clusters of focal adhesions were present in Neuro-L cells (b, c, arrows). Focal adhesions were also present in Oligo-L or Astro-L cells (e, f, h, i, arrows). Scale bar: 20 µm in a for images a, b, d, e, g, and h. Scale bar: 10 µm in c for images c, f, and i. Talin—red; F-actin cytoskeleton—green; α2—blue
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Harper, M.M., Ye, EA., Blong, C.C. et al. Integrins Contribute to Initial Morphological Development and Process Outgrowth in Rat Adult Hippocampal Progenitor Cells. J Mol Neurosci 40, 269–283 (2010). https://doi.org/10.1007/s12031-009-9211-x
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DOI: https://doi.org/10.1007/s12031-009-9211-x