Figure 2: Triple-label in situ hybridization shows that two identified neurons, I1 and B3, express DCC, Neogenin and UNC5 mRNA. (A) Schematic drawing of the large larval lamprey brainstem showing major anatomical features and the locations of identified neurons and neuron groups. I, isthmic; M, mesencephalic; B, bulbar; Mth, Mauthner cell; mth, auxiliary Mauthner cell; hab.-ped. tr., habenulopeduncular tract; inf., infundibulum; isth.retic., isthmic (anterior rhombencephalic) reticulospinal nucleus; s.m.i., sulcus medianus inferior; Vm, trigeminal motor nucleus; IX, glossopharyngeal motor nucleus; X, vagal motor nucleus. The view is from the dorsal (ventricular) surface. (B) DCC mRNA detection by DCC-digoxigenin probe followed by Alkaline Phosphatase (AP)-Fast Red staining. (C) Second step - Neogenin mRNA detection by in situ hybridization with Neogenin-biotin probe and developed with the AP-INT/BCIP system. (D) Third step – UNC5 mRNA detection by in situ hybridization with UNC5-fluorescein probe and developed with AP-NBT/BCIP system. (E–J) Magnifications of the images in the boxes in B, C and D. The large identified reticulospinal neurons I1 and B3 co-express mRNAs for all three receptors: DCC, Neogenin and UNC5.