Gene discovery in genetically labeled single dopaminergic neurons of the retina.
In the retina, dopamine plays a central role in neural adaptation to light. Progress in the study of dopaminergic amacrine (DA) cells has been limited because they are very few (450 in each mouse retina, 0.005% of retinal neurons). Here, we applied transgenic technology, single-cell global mRNA amplification, and cDNA microarray screening to identify transcripts present in DA cells. To profile gene expression in single neurons, we developed a method (SMART7) that combines a PCR-based initial step (switching mechanism at the 5' end of the RNA transcript or SMART) with T7 RNA polymerase amplification. Single-cell targets were synthesized from genetically labeled DA cells to screen the RIKEN 19k mouse cDNA microarrays. Seven hundred ninety-five transcripts were identified in DA cells at a high level of confidence, and expression of the most interesting genes was confirmed by immunocytochemistry. Twenty-one previously undescribed proteins were found in DA cells, including a chloride channel, receptors and other membrane glycoproteins, kinases, transcription factors, and secreted neuroactive molecules. Thirty-eight percent of transcripts were ESTs or coding for hypothetical proteins, suggesting that a large portion of the DA cell proteome is still uncharacterized. Because cryptochrome-1 mRNA was found in DA cells, immunocytochemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.
Gustincich
Stefano
S
Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.
Contini
Massimo
M
Gariboldi
Manuela
M
Puopolo
Michelino
M
Kadota
Koji
K
Bono
Hidemasa
H
LeMieux
Julianna
J
Walsh
Pamela
P
Carninci
Piero
P
Hayashizaki
Yoshihide
Y
Okazaki
Yasushi
Y
Raviola
Elio
E
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004298:Dopamine / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012160:Retina / Q000166:cytology / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D004298:Dopamine