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Daniel Elefant, Ignacio Hernandez-Morato, Jaliya Kumaratilake, Sansar C. Sharma, Stuart A. Newman; Alkaline Phosphatase Enriched Exosomes as Guidance Cues in the Developing Avian Optic Chiasm. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3616a.
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© ARVO (1962-2015); The Authors (2016-present)
Many molecules at the site of the developing optic chiasm have been associated with guiding the contralateral and ipsilateral navigation of developing axons of retinal ganglion cells (RGCs). Yet, the factors that attract RGC axons to the midline are not known. The midline attractants present in the developing spinal cord are absent from the developing diencephalon. We have localized a high concentration of enzymatically active, levamisole resistant (LR), alkaline phosphatase (AP) closely associated with the developing chiasm that we believe is involved in creating this uncharacterized midline attraction. Transmission EM indicates that the AP resides on the surfaces of exosome-like particles. Exosomes are densely packed signaling particles released by cells.
Chick embryos were incubated at 38.5°C. 15μ sections from 3d-10d embryos were utilized. Standard IHC protocol was used for labeling. Selected sections were labeled with either BCIP/NBT-levamisole or 1° Ab of either 1:100 rabbit anti-CD81 or 1:100 rabbit anti-Intestinal AP (IAP), and 1:200 anti-rabbit AF-488 as fluorescence-labeled 2° Ab. Photos were taken either on fluorescence or confocal microscopes.
From day 3 of optic chiasm development onward, positive detection of BCIP/NBT-levamisole at the chiasm (Fig 1) indicates the presence of LR AP (a characteristic previously only attributed to IAP). The IHC detection in 5d-7d embryos of IAP at the chiasm following a similar pattern to BCIP/NBT labeling identifies the LR AP as IAP. Transmission EM shows that this AP covers the outer surface of exosome-like particles. Labeling with anti-CD81, an exosome specific antibody (Fig 2), in 5d-7d embryos shows an almost identical pattern of labeling to the IAP antibodies. The matching pattern of putative exosomes and IAP at the developing chiasm suggests that the two are associated.
We have identified and localized a specific AP that appears to be deposited on the outer surface of exosomes in the midline of the developing diencephalon. Work in progress in our laboratory suggests that the AP on these exosomes serves as a guidance molecule for growing RGCs. Further experiments are necessary to establish the exact molecular mechanisms of this guidance pathway. Finally, the possibility that this exosmal AP serves as a guidance molecule has great potential impact on future axon regeneration studies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Fig 1- BCIP/NBT-lev
Fig 2- CD81
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