Supplementary Figure 1. Transgene guided dissection and microarray analysis of habenula gene expression.

(A) Xgal staining of a hemisected E16.5 Pou4f1tLacZ/+ embryo illustrating the area

dissected for microarray analysis of habenula gene expression.

(B-C) Microarray analysis of the habenula compared to embryonic thalamus and cerebral

cortex. (D-F) Microarray comparison of replicate assays of the habenula, cortex and

thalamus. Parallel lines represent 3-fold change. Only transcripts determined to be

present (P call) in both replicates of at least one brain region are shown.

Ctx, cerebral cortex; fr, fasciculus retroflexus; HB, habenula; Mes, mesencephalon; Thal,

thalamus.

Supplementary Figure 2. ABA expression data for transcripts enriched in the microarray analysis of the E16.5 habenula.

In situ hybridization results are shown in the order of decending enrichment in the habenula, corresponding to Table 1. The plane of section in coronal views corresponds to level 70-72 in the Allen Reference Atlas.

HIP, hippocampus; LH, lateral habenula; MH, medial habenula; PVT, paraventricular nucleus of the thalamus; TRS, triangular nucleus of septum.

Supplementary Figure 3. ABA expression data for habenula-enriched transcripts identified by bioinformatic search.

In situ hybridization results are shown in alphabetical order by gene name. The plane of section in coronal views corresponds to level 70-72 in the Allen Reference Atlas. Legend

appears in Figure S2.

Supplementary Figure 4. Initial differentiation of habenula neurons and early

trajectory of fasciculus retroflexus at E12.5 in Pou4f1tLacZ/+ and Pou4f1tLacZ/-

embryos. Coronal sections were stained for tauLacZ expressed from the Pou4f1 locus and the pan-neuornal marker tubulin beta-3 (Tubb3). The plane of section in the most rostral view corresponds to that illustrated in Figure 7A/7J. At this stage, the FR projects caudally within the thalamus but does not yet reach its target in the interpeduncular

nucleus, and the FR of Pou4f1tLacZ/- and control embryos appeared similar in trajectory

and extent.

Supplementary Figure 5. ChAT and SP immunoreactive axons fail to innervate the

IPN in Brn3a knockout mice.

tLacZ/+ tLacZ/- Pou4f1 and Pou4f1 mice were examined at P0 in coronal section for the expression of βgal and ChAT or SP/Tac1. (A,B) Immunostaining for βgal and ChAT.

ChAT expressing neurons are not present in the MH of Pou4f1tLacZ/- mice (not shown), and few ChAT immunoreactive axons innervate the IP. (C,D) Immunostaining for βgal and SP. Innervation of the IPN with SP immunoreactive fibers is markedly diminished in the Pou4f1tLacZ/- sample. IPN, interpeduncular nucleus; R, red nucleus; rs, rubrospinal tract; vtgx, ventral tegmental decussation. Scale: 100μm.

Supplementary Figure 6. RT-PCR confirmation of transcripts decreased at E16.5 in the habenula of Pou4f1tLacZ/- embryos. RT-PCR assays were performed on RNA samples from Pou4f1tLacZ/- and Pou4f1tLacZ/+ control embryos. The Y-axis scale shows cycle threshold difference (Δct) and fold-change values for the RT PCR assays (mean +/- s.d. of 3 determinations), and values at the top of each bar show the mean fold change detected by microarray. High concordance is noted between the two methods, except for

Sstr4, in which the microarray significantly underestimated the fold change, probably due to background signal on the array, and for Efcbp2, for which RT-PCR did not replicate the ~2-fold change observed by microarray. P20Rik represents 3110047P20Rik. Other gene names appear in Table 2.

Supplementary Figure 7. Mis-expression of Brn3a does not induce Nurr1 in the neocortex, ventral thalamus, hypothalamus or superior colliculus. E13.5 embryos were electroporated in ovo with an expression construct encoding Brn3a plus GFP and examined at E16.5 for GFP + Brn3a, and GFP + Nurr1 expression in adjacent sections.

(A-C) Misexpression of Brn3a in the neocortex. (D-F) Misexpression of Brn3a in the hypothalamus. (G-I) Misexpression of Brn3a in the tectum (midbrain superior colliculus). (J-L) Misexpression of Brn3a in a population of cells which originate adjacent to the third ventricle and migrate to contribute to the ventral thalamus. 3V, third ventricle; ventricle, Hb, habenula; LH, lateral habenula; LV, lateral ventricle; MH, medial habenula; R, red nucleus; SC, superior colliculus, Thal, thalamus.