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, 29 (45), 14309-22

Brn3a and Nurr1 Mediate a Gene Regulatory Pathway for Habenula Development

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Brn3a and Nurr1 Mediate a Gene Regulatory Pathway for Habenula Development

Lely A Quina et al. J Neurosci.

Abstract

The habenula is a dorsal diencephalic structure consisting of medial and lateral subnuclei and a principal output tract, the fasciculus retroflexus, which together form a link between the limbic forebrain and ventral midbrain. Here, we have used microarray and bioinformatic approaches in the mouse to show that the habenula is a distinctive molecular territory of the CNS, with a unique profile of neurotransmitter, ion channel, and regulatory factor expression. Neurons of the medial habenula and part of the lateral habenula express the transcription factor Brn3a/Pou4f1, and Brn3a-expressing habenular neurons project exclusively to the interpeduncular nucleus in the ventral midbrain. In Brn3a mutant embryos, the fasciculus retroflexus is directed appropriately, but habenular neurons fail to innervate their targets. Microarray analysis of Brn3a null embryos shows that this factor regulates an extensive program of habenula-enriched genes, but not generic neural properties. The orphan nuclear receptor Nurr1/Nr4a2 is coexpressed with Brn3a in the developing habenula, is downstream of Brn3a, and mediates expression of a subset of Brn3a-regulated transcripts. Together, these findings begin to define a gene regulatory pathway for habenula development in mammals.

Figures

Figure 1.
Figure 1.
Brn3a is expressed in habenular neurons projecting specifically to the IPN. A–D, Expression of a Pou4f1tLacZ transgene in the habenula and its projections revealed by xgal staining in the adult mouse brain. Afferent habenular fibers of the striae medularis are also indicated. E, F, Colocalization of Brn3a protein expression and βgal immunoreactivity in the MH and LH. Brn3a is expressed in nearly all MH and a subset of LH neurons. The bracketed area in E is enlarged in F. G–I, Relationship of habenular projections to serotonergic neurons and fibers revealed by Tph2 immunoreactivity. G, βgal-labeled habenular fibers terminate in the IPN. Tph2 staining at this level is predominantly ascending serotonergic fiber tracts. The plane of the section is similar to D. H, I, Progressively more caudal sections showing that βgal-labeled fibers do not associate with cell bodies of serotonergic neurons of the raphe nuclei. J–L, Relationship of habenular projections to dopaminergic neurons and fibers, marked by TH immunoreactivity. βgal-labeled fibers do not project to the dopaminergic areas of the basal midbrain. CLI, Central linear nucleus raphe; CSm, superior central nucleus raphe, medial part; DR, dorsal raphe; fr, fasciculus retroflexus; Hip, hippocampus; Hyp, hypothalamus; IPL, IPN, lateral subnucleus; opt, optic tract, RRF, retrorubral field; sm, striae medularis; SNr, substantia nigra, pars reticulata; Thal, thalamus. Scale bars: E, G–L, 100 μm; F, 50 μm.
Figure 2.
Figure 2.
Neurotransmitter phenotype of Brn3a-expressing habenular neurons. A–D, Immunofluorescence in adult brain sections for ChAT and βgal expressed from the Pou4f1 locus showed colocalization in the ventral two-thirds of the MH. The bracketed area in A appears enlarged in B–D. E, ISH signal for SP/Tac1 was restricted to the dorsal third of the MH (ABA Tac1_187_2030). F, G, ChAT strongly colocalizes with βgal in the principal part of the IPN, but not in the lateral subnucleus. H, I, SP immunoreactivity strongly colocalizes with βgal in the lateral subnucleus of the IPN. fr, fasciculus retroflexus; IPL, IPN, lateral subnucleus. Scale bars: A–D, 100 μm; F–I, 200 μm.
Figure 3.
Figure 3.
Microarray and bioinformatic analysis of habenula gene expression in the embryonic and adult brain. A, Microarray analysis of the E16.5 habenula, cortex, and thalamus revealed 112 unique transcripts with >10-fold enriched expression in the habenula compared with the mean expression level in the cortex/thalamus. Of these transcripts, 91 had available data in the ABA. Quantitative analysis of the ABA data showed that 42 of these transcripts also had enriched expression in the adult brain, whereas 30 were expressed but not enriched and 19 were not detectable. Microarray enrichment of transcripts in all three categories were verified by quantitative RT-PCR (qPCR; mean and SD of 3 assays are shown). B, Control transcripts were identified in the microarray analysis that had equal expression in the habenula and cortex/thalamus. Of 88 control transcripts, 82 had available data in the ABA. Three of the control transcripts exhibited habenula-enriched expression in the adult brain, whereas 50 did not show enriched expression and 29 were not detected. C, Characteristic expression patterns of habenula-enriched transcripts. Recurring patterns observed included MH plus LH (Pou4f1, Vav2), MH only (Tac2), and the ventral part of the MH only (Slc18a3). Enriched expression in the dorsal half of the MH or in the LH alone was much less frequently observed (Nhlh2, Prokr2). See also data for ChAT and SP/Tac1 in Figure 2. Hab, habenula; cortex, Ctx; Thal, thalamus; Pou4f1, Brn3a; Vav2, Vav2 oncogene; Tac2, Tachykinin 2; Slc18a3, solute carrier family 18, member 3/vescicular acetylcholine transporter; Nhlh2, nescient helix loop helix 2; Prokr2, Prokineticin receptor 2. ISH data are derived from the ABA. Scale bar, 200 μm.
Figure 4.
Figure 4.
The habenula develops from Dbx1-expressing precursors in the presumptive dorsal thalamus. A, Schematic of the brain at E12.5 showing approximate plane of section for subsequent views. B, C, Immunofluorescence for Pax3, Dbx1, and Olig3 reveals nearly discrete domains in the neuroepithelium of the presumptive thalamus. D–F, Immunofluorescence for Brn3a with each of the progenitor domain markers reveals an association of the habenula with Dbx1-expressing precursors. Scale bars: B, C, 100 μm; D–F, 50 μm. thal, Thalamus; hab, habenula.
Figure 5.
Figure 5.
Displacement of habenular neurons and defective target innervation in Brn3a knock-out mice. A, Sagittal view of the brain at E16.5 showing the location of the habenula, FR, and IPN and the plane of sections shown in subsequent views. B, C, Midline view of habenula, FR, and IPN in hemisected, xgal-stained E16.5 embryos. D, E, Structure of the habenula in Pou4f1tLacZ/+ and Pou4f1tLacZ/ E16.5 embryos. Habenular neurons and axons are displaced toward the midline in the knock-out. F–I, Coronal sections showing the course of the FR in Pou4f1tLacZ/+ and Pou4f1tLacZ/ P0 mice, taken near the middle of its course (F, G) and just before termination in the IPN (H, I). J, Cross-sectional area of the FR from the caudal end of the habenula (zero coordinate) to the interpeduncular nucleus in P0 mice. Average values of right and left measurements are shown. The paired t test for the comparison of all sections from Pou4f1tLacZ/+ and Pou4f1tLacZ/ mice yields p = 1.0 × 10−4. K–N, Termination zone of habenular axons in the IPN at P0. The majority of βgal-immunoreactive fibers are missing in the Pou4f1tLacZ/ specimen (K, L), and the remaining expression appears to be associated with a few Brn3a+ neurons that are intrinsic to the IPN (M, N). O–S, Robo3 expression in the habenulopeduncular system. Robo3 is undiminished in the caudal FR (O, P; plane of section is similar to H). Robo3-expressing fibers innervate the IPN in a crossing pattern in a Pou4f1tLacZ/+ embryo (Q; detail R; plane of section is similar to K), but are nearly absent in a Pou4f1tLacZ/ specimen (S). CP, Choroid plexus; fr, fasciculus retroflexus; Hab, habenula; Mes, mesencephalon; R, red nucleus; rs, rubrospinal tract; vtgx, ventral tegmental decussation. Scale bars: D–I, 100 μm; K, L, O–S, 100 μm; M, N, 50 μm.
Figure 6.
Figure 6.
Molecular phenotype of the habenula in Brn3a null mice. Expression of potential Brn3a target genes identified by microarray were examined in Pou4f1+/+ and Pou4f1−/− embryos at E16.5 using ISH. In general, the magnitude of the changes in expression detected by ISH appeared to be greater than the changes observed on the array, and transcripts with more than fourfold decreased expression on the microarray were generally undetectable by ISH in Pou4f1−/− embryos. This may be in part because the use of heterozygous controls in the microarray study somewhat reduced the magnitude of the changes in expression measured by that method. DCC expression was verified but restricted to cells immediately adjacent to the ventricle (arrowheads). Nrp2 expression appeared unchanged, but Nrp2-expressing cells were displaced toward the midline, as observed for other markers (arrows). Expanded gene names appear in Table 2. Hip, Hippocampus; CP, choroid plexus. Scale bar, 100 μm.
Figure 7.
Figure 7.
Coexpression of transcription factors regulating habenula development. Expression of Brn3a, Nurr1, and Etv1 were examined by immunofluorescence at E16.5 and E12.5. A, Diagram depicting the planes of section in subsequent views. B–E, Coexpression of Brn3a and Nurr1 in E16.5 embryos. D, Nearly all neurons that express Brn3a in the MH and LH also express Nurr1, although relative expression levels vary. E, Nurr1 is almost undetectable in the Pou4f1−/− habenula. F–I, Coexpression of Brn3a and Etv1 in E16.5 embryos. In the Pou4f1−/− habenula, a small residual population of Etv1+ neurons is noted near the ventricle. J–L, Brn3a and Nurr1 expression in early habenular neurons at E12.5. J, Expression of the tauLacZ transgene faithfully replicates the expression of Brn3a protein in the first habenular neurons to differentiate in Pou4f1tLacZ/+ embryos. K, L, All or nearly all of these neurons coexpress Nurr1 (K), and Nurr1 expression is not initiated in the absence of Brn3a (L). Scale bars: B–I, J–L, 100 μm.
Figure 8.
Figure 8.
Regulation of habenular gene expression by Nurr1. A–D, Brn3a and LacZ expression in the habenula of E16.5 Nr4a2+/+ and Nr4a2−/− embryos bearing a Pou4f1tLacZ reporter allele. Neurons and axons do not display the medial displacement observed in Pou4f1−/− embryos, and Brn3a expression is not affected by the loss of Nurr1. E, F, Innervation of the IPN in P0 Nr4a2+/+ and Nr4a2−/− mice carrying a Pou4f1tLacZ reporter allele. Nurr1 expression also identifies dopaminergic neurons in the VTA adjacent to the IPN. G, Changes in gene expression in Nr4a2−/− embryos at E16.5. Nurr1 mediates a subset of the gene expression changes observed in the habenula of Brn3a null embryos. Complete gene names appear in Table 2. fr, Fasciculus retroflexus; hip, hippocampus; R, red nucleus; rs, rubrospinal tract; vtgx, ventral tegmental decussation. Scale bars: A–D, 50 μm; E, F, 100 μm; G, 100 μm.
Figure 9.
Figure 9.
Brn3a is not sufficient to induce ectopic expression of Nurr1 in the diencephalon. A, Plasmids encoding Brn3a plus cytoplasmic or nuclear targeted GFP, or a control plasmid expressing GFP alone, were electroporated into habenular precursors by injection into the third ventricle at E13.5. Embryos were harvested at E16.5. B, Expression of GFP from a control plasmid was observed within the habenula, and in more ventral and caudal domains within the developing thalamus. The control plasmid did not alter the pattern of endogenous Brn3a expression. C, Low-power image of the habenula and periventricular thalamus in sagittal section, following electroporation of a Brn3a-GFP expression vector. Immunofluorescence for Brn3a and GFP with 4′,6-diamidino-2-phenylindole staining reveals colocalization of the expressed proteins in nearly all cells for which the nucleus resides within the plane of section. The inset box indicates the area enlarged in D and G. D–I, Expression of Brn3a (D–F) and Nurr1 in adjacent sections (G–I) after electroporation of a Brn3a-GFP plasmid. Nurr1 expression was observed in 4% of GFP+ cells in this region electroporated with Brn3a-GFP (n = 400) and 2% of cells electroporated with GFP alone (n = 400; data not shown), which did not represent a statistically significant difference. 3V, Third ventricle; Pi, pineal; fr, fasciculus retroflexus; Hb, habenula; Th, thalamus; LV, lateral ventricle.

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