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doi: 10.1242/10.1242/dev.00270

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The parapineal mediates left-right asymmetry in the zebrafish diencephalon

¹ Carnegie Institution of Washington, Department of Embryology, 115 W. University Parkway, Baltimore, MD, 21210, USA
² IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP10142, CU de Strasbourg, 67404 Illkirch cedex, France

View larger version (101K): [in a new window]   Fig. 1. Laterality of the pineal complex. The zebrafish pineal complex consists of the pineal organ and the parapineal (black arrowhead), which both express otx5 (blue). (A,B) The parapineal lies to the left of the pineal anlage at 2 d, at the same dorsal level as the pineal, but (C,D) becomes more caudally and ventrally located during larval development. (E) In a frontal view of a 15 d larval brain, the parapineal lies in a more ventral position relative to the pineal. (F) In the adult brain, the parapineal is found at the base of the pineal stalk (arrow), next to the left habenula (Lh). (A,C,F) Dorsal views; (B,D) cross-sections counterstained with basic fuchsin (pink). Rh, right habenula. Scale bars: 50 µm for A-E; 150 µm for F.

View larger version (73K): [in a new window]   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.

View larger version (61K): [in a new window]   Fig. 2. Asymmetric expression of leftover in the habenular nuclei. (A) The left habenula (white arrowhead) expresses lov (blue) at higher levels and in more cells than the right. (B) Asymmetric lov expression persists in the adult zebrafish brain. (C-E) lov expression in the left habenula coincides with a region of dense neuropil (black arrowhead on left side in D, compare with right side) labeled with anti-acetylated tubulin antibody. (F) The lov gene encodes a 288 amino acid protein that contains a conserved oligomerization domain (T1-like domain) at the N-terminus. (G) The Leftover oligomerization domain is 47% similar to the T1 tetramerization domain of the Drosophila Shaker K+ channel (Papazian, 1999) and 41% similar (not shown) to the POZ/BTB domain of human promyelocytic leukemia zinc finger protein (PLZF) (Collins et al., 2001). Scale bars: 50 µm for A,C-E; 150 µm for B.

View larger version (51K): [in a new window]   Fig. 3. LR randomization of habenular laterality. The laterality of lov expression is altered in mutant larvae. (A-D) Approximately half of casanova (casta56), no tail (ntlb160), floating head (flhn1) and rescued one-eyed pinhead (oepm134) homozygotes showed the same lov expression pattern as wild type at 4 d. (E-H) The pattern was reversed in the other half. (I-J) Infrequently, both habenulae showed equivalent levels of expression (and refer to Fig. 4C,F,I). Scale bar: 50 µm. See also Table 1.

View larger version (65K): [in a new window]   Fig. 4. Correspondence between parapineal and habenular laterality. (A) In the majority of wild-type 4 d larvae, the parapineal (black arrowhead, otx5 expression) is on the left, beside the habenula with high lov expression (red). (B) LR reversal of parapineal and habenular laterality is sometimes observed in wild type (n=20/395). (C) Paired parapineal organs and bilateral lov expression in the habenulae is found infrequently (n=1/395). (D) Almost half of rescued MZoep larvae show the wild-type pattern, while the other half (E) show a LR reversal in laterality. (F) Bilateral expression of lov in rescued MZoep mutants also occurs when two parapineals form (partially obscured by intense otx5-expressing pineal). (G) flh mutants have a greatly reduced pineal anlage (Masai et al., 1997). The majority of otx5-expressing cells in flh mutants are presumed to be parapineal because of their lack of flh expression (Gamse et al., 2002). In half of flh mutants, the parapineal and stronger lov expression are found on the left side, and (H) in the other half, laterality is reversed. (I) The parapineal is sometimes found medially, adjacent to both habenulae (5/84). In these flh mutants, lov is expressed strongly in both habenulae. Scale bar: 50 µm. See also Table 2.

View larger version (71K): [in a new window]   Fig. 5. Parapineal formation precedes asymmetric expression of lov. Expression of otx5 (blue) and lov (red) in wild-type sibling embryos at the indicated times post-fertilization. The otx5-expressing parapineal (blue arrowhead) can be distinguished at 28 h. Expression of lov (red arrowhead) is first detected at 40 h and is always found at higher levels on the left side of the brain. Scale bar: 50 µm.

View larger version (61K): [in a new window]   Fig. 6. Selective ablation of the parapineal in flh:GFP transgenic embryos. (A-D) Integrity of the parapineal before and after laser-mediated cell ablation. (A) The pineal anlage and parapineal can be recognized by their specific fluorescent labeling in 30 h Tg(flh:eGFP) embryos, and (C) visualized by Nomarski optics (black arrowhead; parapineal). The parapineal was destroyed by laser pulses, as evidenced by (B) the loss of fluorescence and (D) abnormal morphology immediately after ablation. Cell death was not observed in neighboring regions after the laser treatment. (E-H) As a control, an equal number of cells were destroyed to the right of the pineal organ (white arrowhead in H), but the parapineal was left intact (black arrowhead in G and H). Scale bar: 25 µm.