Cellular and developmental distribution of human homologues of the Drosophilia rdgB protein in the rat retina.
OBJECTIVE
The Nirs (Nir1, Nir2, and Nir3), human homologues of Drosophila retinal degeneration B (rdgB), have been considered candidate genes for human inherited retinal degeneration diseases. To gain a better understanding of their functions in the retina and their putative roles in retinal degeneration diseases, this study was undertaken to determine their distribution profile in developing and mature rat retinas.


METHODS
Specific antibodies against each of the Nir proteins were raised in rabbits and used in indirect immunofluorescence analysis to determine the distribution profile of the three proteins. Eyes from Wistar rats at various developmental stages (embryonic day [E]18 to postnatal day [P]16) were sectioned vertically and immunostained with anti-Nir antibodies. Coimmunostaining for Nirs and several specific cellular and subcellular markers was used to determine precisely the cellular and subcellular distribution of the Nirs. Sections were observed under a confocal laser microscope, and image analysis was performed with the standard operating software provided with the microscope.


RESULTS
Confocal microscopic analysis of Nir1 immunoreactivity revealed that it was predominantly expressed in premature Müller cells at birth and that it was upregulated during Müller cell maturation. In contrast, Nir2 and Nir3 were homogeneously distributed in undifferentiated neuroblasts and ganglion cells at birth and later became distinctly distributed in newly differentiated neuronal cells. From P4, Nir2 and Nir3 were highly expressed in neuronal cells and their processes, coinciding with the formation of synaptic layers and ongoing synaptogenesis. From P12, Nir2 was uniformly expressed in all classes of retinal neuronal cells, including ganglion cells, horizontal cells, amacrine cells, bipolar cells, and photoreceptor cells. In the adult rat retina, Nir2 was preferentially localized to the somata of all classes of retinal neurons, whereas Nir3 was highly expressed in the synaptic terminals. This specific localization of Nir3 was confirmed by double immunostaining with the presynaptic protein synaptosomal-associated protein (SNAP)-25. In photoreceptor cells, both Nir2 and Nir3 were found to be highly expressed in the inner segments but were not detectably expressed in the outer segments.


CONCLUSIONS
These findings suggest that the three Nir proteins are highly expressed in the developing retina, each exhibiting a distinct distribution profile. The different distribution patterns of these closely related proteins during development and at maturity may reflect their different cellular functions in vivo and their different roles in retinal cell survival or degeneration.
Tian
Donghua
D
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
Lev
Sima
S
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005136:Eye Proteins; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D015854:Up-Regulation
D002135:Calcium-Binding Proteins; D029721:Drosophila Proteins; D005136:Eye Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C117888:PITPNM2 protein, human; C117885:PITPNM3 protein, human; C068848:rdgB protein, Drosophila