Figure 7: Fate mapping of tectal Sox14⁺ precursors.

Labelling of Sox14⁺ precursors via in utero focal injection of AAV2/1-EF1α-DIO-tdTomato virus into the SC of Sox14^cre/+ embryos at E15.5. The time line of the experiment is summarized in e. (a) At P14, the cre-dependent expression of tdTomato labels the site of injection in the outer-most region of the left SC. Scale bar, 100 μm. (b) Coronal sections at thalamic level reveal an enrichment of tdTomato-expressing cells within the boundaries of the dLGN. Scale bars, 100 μm. Higher-magnification confocal imaging (insets 2 and 3) reveals the typical morphology of local Ins; scale bar, 10 μm. (c) The location of Sox14⁺ neurons in a comparative section from an age-matched Sox14^Gfp/+ brain illustrates the expected labelling pattern if the injected AAV-containing solution had passively spread from the site of injection to the thalamus. Scale bar, 100 μm. (d) Although not visible in b, in some brains tdTomato-labelled cells could be detected in the vLGN. The distribution of tdTomato-labelled Sox14⁺ tectal precursors in the LGN subdomains is highly correlated to that of Sox14⁺Otx2⁺ neurons in the same regions (R²=0.98). (e) Time line of the in utero AAV injection and indicative view of the morphological landmarks used to select the unilateral injection site on the SC. (f) Neurogenesis in the E12.5 Sox14^Gfp/+ dorsal midbrain displaying the sequential activation of Ascl1 (Mash1), Gata2 and Sox14 (also see inset 1), and lack of Nkx2.2, which is instead expressed in a Sox14⁺ ventral midbrain domain. Scale bars, 100 μm, and 10 μm for the inset. (g) Detection of the Gad1 transcript in P7 control and Gata2 conditional knockout brains. The En1^cre/+ driver selectively recombines the floxed (flx) Gata2 allele in the midbrain, but not in more rostral forebrain structures. The marked reduction of Gad1⁺ neurons in the SC is accompanied by a similarly strong reduction of Gad1⁺ cells in the dLGN. In contrast, the density of Gad1⁺ neurons in the intervening pretectum, as well as vLGN/IGL remains grossly normal. Scale bar, 100 μm.