Laterotopic representation of left-right information onto the dorso-ventral axis of a zebrafish midbrain target nucleus

Abstract

The habenulae are part of an evolutionarily highly conserved limbic-system conduction pathway that connects telencephalic nuclei to the interpeduncular nucleus (IPN) of the midbrain . In zebrafish, unilateral activation of the Nodal signaling pathway in the left brain specifies the laterality of the asymmetry of habenular size . We show "laterotopy" in the habenulo-interpeduncular projection in zebrafish, i.e., the stereotypic, topographic projection of left-sided habenular axons to the dorsal region of the IPN and of right-sided habenular axons to the ventral IPN. This asymmetric projection is accounted for by a prominent left-right (LR) difference in the size ratio of the medial and lateral habenular sub-nuclei, each of which specifically projects either to ventral or dorsal IPN targets. Asymmetric Nodal signaling directs the orientation of laterotopy but is dispensable for the establishment of laterotopy itself. Our results reveal a mechanism by which information distributed between left and right sides of the brain can be transmitted bilaterally without loss of LR coding, which may play a crucial role in functional lateralization of the vertebrate brain .

Figure 1. Laterotopic Projections from the Habenular Nuclei to the IPN

(A) Schematic illustration of a lateral view of an adult zebrafish brain showing the telencephalo-habenulo-interpeduncular pathway that connects the telencephalon with the ventral midbrain. (B–G) Confocal images of sections of brains of adult fish in which left habenular axons are anterogradely labeled with DiO (green) and right habenular axons are labeled with DiI (red). (B and C) Transverse sections at the level of the habenulae (B) and the IPN (C) showing left habenular axons (green) terminating in the dorsal IPN and right habenular axons (red) terminating in the ventral IPN. (D and E) Low- (D) and high (E)-magnification views of a parasagittal section showing axonal projections from the right habenula (red) through the FR to the IPN. Within the IPN, dorsal and intermediate regions (green) are predominantly innervated by axons from the left habenula. (F and G) Horizontal sections showing projections from the left habenula (green) terminating in the intermediate IPN (F) and projections from the right habenula (red) terminating in the ventral IPN (G). In both cases, the labeled axons surround the fluorescent Nissl-stained cell bodies (blue) at the center of the nucleus. (H) Dorsal view of the intact ventral midbrain in a 5-day-postfertilization (dpf) larva, showing a 3D reconstruction of projections from the left (green) and right (red) habenulae. The oculomotor nucleus is visualized with a foxD3:GFP transgene (blue). Some FR axons on both left and right sides bypass the IPN and terminate more caudally (arrows). (I and J) Transverse sections through the IPN of 4-dpf larvae showing projections from the left (I) and right (J) habenulae labeled with photoconverted DiI.

Figure 2. Asymmetries between Left and Right Habenular Sub-Nuclei Account for the Laterotopy of Projections to the IPN

(A) Dorsal view of a brain of a 4-dpf Tg(brn3a-hsp70:GFP) larva showing GFP labeling of the entire length of the habenulo-interpeduncular projection (arrows). (B) Frontal view of an adult brain from a Tg(brn3a-hsp70:GFP) fish. (C and D) Transverse sections through the habenulae (C) and the IPN (D) of an adult Tg(brn3a-hsp70:GFP) fish showing GFP expression (green) and SYTOX orange counter staining of cellular nuclei (red). (E–H) Transverse sections through the habenulae (G and H) and IPN (E and F) of adult Tg(brn3a-hsp70:GFP) fish in which DiI was applied to the dIPN (E and G) or vIPN (F and H). DiI application sites and retrogradely labeled cells are indicated by asterisks. (I) Expression pattern of lov mRNA (red) in the habenulae of an adult Tg(brn3a-hsp70:GFP) fish. (J) Summary schematic of habenular organization indicating brn3a-hsp70:GFP expression (green) and lov (red) in restricted sub-nuclei. Two additional, bilaterally symmetric sub-nuclei (asterisks) are present in the dorso-medial and ventro-medial regions of the habenulae. Abbreviation: Chi, optic chiasm.

Figure 3. Reversal of lefty1:GFP Activation Correlates with Reversal of Habenular Laterality and Inversion of Projections in abf Fish

(A–D) abf embryos and larvae having normal (A and C) and reversed (B and D) laterality, shown by anti-light meromyosin antibody (MF20) labeling of the heart (brown in [A] and [B], frontal views) and BODIPY FL-C5 labeling of the gall bladder (green in [C] and [D], ventral views). (A and B) 48 hr postfertilization (hpf); (C and D) 4.5 dpf. (E and F) Dorsal views of live wild-type; Tg(lefty1:GFP) (E) and abf;Tg(lefty1:GFP) (F) 28-hpf embryos showing expression of GFP in the left epithalamus (E) and in the right epithalamus (F). (G and H) Dorsal views of 3D reconstructions of confocal images of habenular projections in the IPN of a wild-type 5-dpf larva (G) and an abf 5-dpf larva with reversed lefty1:GFP expression (H). Axons from the left habenula have been labeled with DiA (green), and those from the right habenula have been labeled with DiI (red). (I and J) Dorsal views of the habenulae of a wild-type (I) and a heart-reversed abf mutant (J) 4-dpf larva showing prominent lov expression in the left habenula of the wild-type and in the right habenula of the heart-reversed abf mutant. (K and L) Confocal images of transverse sections of the IPN of a wild-type adult fish (K) and a heart-reversed abf fish (L) in which DiA (green) was applied to the left and DiI (red) to the right habenula. Abbreviations: H, heart; GB, gall bladder; and WT, wild-type.

Figure 4. Laterotopic Habenular Projections Are Still Established in Embryos Laking Lateralized Nodal Activity in the Epithalamus

(A) A cartoon viewing dorsally onto the epithalamus showing the parapineal organ innervating the left habenula. (B–K) LZoep^−/−;Tg(foxD3:GFP) (B–E), wild-type (F), and ntl morphant (G–K) embryos and larvae in which Nodal-pathway activity (F and G) or parapineal (asterisks) laterality (B, C, H, I) was assessed by GFP-transgene (indicated bottom left) expression and left and right habenular projections were subsequently assessed by DiI and DiA labeling at 4 dpf (D, E, J, and K). LZoep^−/− embryos show an equal number of cases of parapineal migration to the left (B) and to the right (C). Position of the parapineal is concordant with the orientation of laterotopic innervation of the IPN (D and E). The left-sided activation of lefty1:GFP in the wild-type embryo (F) is normally associated with subsequent migration of the parapineal organ to the same side of the brain (indicated by the gray arrow). lefty1:GFP is activated bilaterally in ntl morphants (G), leading to randomization of parapineal position (black arrows to [H] and [I]), which is 100% concordant with the orientation of laterotopic habenular connectivity (J and K). (L) Orientation of laterotopic habenular projections in mutants and morphants that are defective in the expression of Nodal-pathway genes in the epithalamus. Habenular projection patterns were assessed in 4-dpf larvae by carbocyanine-dye axon tracing. The orientation of laterotopy is described as “normal” when left habenular neurons innervate the dorsal IPN and as “reversed” when right-sided axons terminate in the dorsal IPN. (M) Nodal activity determines the orientation of laterotopy in habenulo-interpeduncular projections. The top panels are cartoons of dorsal views of late-somite-stage embryos showing activation of Nodal signaling (green) in the epithalamus. The bottom panels show sub-nuclear organization of the habenulae and laterotopic projections from the habenulae to the IPN in normal and reversed-laterality adult fish. In wild-type embryos, we propose that Nodal activity in the left epithalamus indirectly leads to expansion of the lateral sub-nucleus (red) in the left habenula and the medial sub-nucleus (green) in the right habenula, resulting in projection of the left habenula predominantly to the dorsal IPN and of the right habenula to the ventral IPN. This pattern of sub-nuclear organization is completely reversed (right lower panel) in those abf mutant embryos in which Nodal signaling is activated on the right side. When Nodal signaling is activated bilaterally (ntl) or is absent (abf; LZoep), habenular asymmetry is still established, but laterality of the sub-nuclear organization and consequently of the laterotopic innervation of the IPN becomes randomized. Abbreviations: norm, normal laterality; PO, pineal organ; PP, parapineal; and rev, reversed laterality.