At E14.5, the rostral thalamic pool of GFP-positive cells initiates a new wave of tangential migration in a rostroventral direction to colonize the developing vLGN ( Figures 3B and 3C; Movie S4). In summary, we define the rostral thalamic domain of Sox14-expressing

cells as a source of tangentially migrating GABAergic neurons that form nuclei of the SVS at the thalamus-epithalamus border (next to the LHa), at the thalamus-pretectum border (PLi), and in the vLGN. The bulk of the Sox14-positive cells does not migrate tangentially and instead forms the IGL ( Figure 3A). We consider the PLi and the region lateral to the LHa a continuous structure that shares with the IGL and part of the vLGN a common origin. In the pretectum, Sirolimus clinical trial Sox14-positive cells TGF-beta inhibitor coalesce in the CPA that continues laterodorsally with the OPN. Minor tangential migration from this region results in spreading of Sox14-positive cells along the NOT and scattered throughout the anterior pretectum ( Figure 2C). Some pretectal Sox14-positive cells also appear to reach the PLi ( Figure 3C; Movie S2). Gene expression analysis and live imaging indicate that

nuclei of the SVS arise from two progenitor domains, the r-Th and the caudal pretectum. We identified Helt as an early lineage-specific transcription factor expressed by both pools. We therefore investigated whether Helt function is required for SVS development. Analysis of the diencephalon at E12.5 in the Helt knockout mouse (MgntZ/tZ) revealed a strong downregulation of Sox14, Tal1, and the GABAergic marker Gad1 in the pretectum, but not in the r-Th ( Figures 4A and 4B and data not shown). By E16.5, Sox14 is nearly absent from the pretectum, with only a few cells visible in the presumptive CPA ( Figure 4C). In contrast to Helt function in the midbrain, where it acts to promote the GABAergic fate by suppressing the alternative glutamatergic lineage

determinants Neurog1 and Neurog2 ( Nakatani et al., 2007), progenitors in the MgntZ/tZ pretectum do not upregulate Neurog2 expression ( Figure 4A). Yet, the alternative lineage marker Lhx9 expands into the posterior pretectum, suggesting PDK4 that MgntZ/tZ pretectal progenitors have switched to an excitatory fate ( Figure 4A). The failure to induce the genetic program underlying SVS development in the pretectum, but not in the r-Th, gave us an opportunity to further investigate the contribution of these two domains to the nucleus PLi that forms at the boundary between thalamus and pretectum. At E16.5, the MgntZ/tZ diencephalon displays a normal accumulation of Sox14-positive cells at the thalamus-pretectum border ( Figure 4C), further confirming that this segment of the SVS develops largely from the r-Th via tangential migration, with only minimal contribution from the pretectum. At E12.