Fig. 7. Parapineal mediates habenular laterality. After ablation of the parapineal (pp) or contralateral (control) region at 28-32 h, larvae were allowed to develop and assayed at 4 d for expression of diencephalic markers by in situ hybridization as indicated or (A,E,I) by labeling with an acetylated tubulin antibody (white) and an otx5 RNA probe. Under fluorescence microscopy, the pineal and parapineal (outlined in white) appear black because of quenching by the NBT/BCIP precipitate. Wild-type larvae showed (A) dense neuropil labeling in the left habenula (white arrowhead), an otx5-labeled parapineal (white asterisk in A, black arrowhead in B), (B,C) bilateral expression of cpd2 and f-spondin2, and (D) the characteristic asymmetric lov expression pattern in the dorsal habenular nuclei. (E-H) Control-ablated larvae (see Fig. 6G,H) were indistinguishable from wild type. (I-M) Parapineal-ablated larvae. Destruction of the parapineal was confirmed by the later absence of the otx5 parapineal domain (black arrowhead in J), and resulted in (I) the absence of dense neuropil in the left habenula (n=12/12). (J,K) Bilaterally expressed habenular markers were unaffected (cpd2, n=18/18; f-spondin2, n=8/8), but (L-M) the laterality of lov expression was lost. Instead, weak expression resembling the right side was found in both habenulae (n=24/24). (N) By contrast, ablation of the pineal anlage, with the parapineal intact, did not affect habenular asymmetry (n=4/4). Scale bar: 50 µm.