Fig. 6.

Ascl1 controls TRN cell fate and PTA outgrowth and is essential for Isl1 expression in the prethalamus. (A) In situ hybridization for prethalamic markers on control (Ascl1^KI/+) and mutant (Ascl1^KI/KI) coronal sections at E14.5 and E16.5. In Ascl1 mutants, Dlx1 expression was lost in the TRN as well as in the rZI, vLG, and zli (A6, A16). Loss of Ascl1 resulted in severe downregulation of Meis2 expression in the TRN (A7, A17). Arx, Pax6, or Lhx1 expression in the vLG and zli of Ascl1^KI/KI mutants was downregulated but ectopically induced in the TRN (A8-A10, A18-A20). (A21) Quantification of in situ hybridization. The pixel intensity values of TRN or vLG were grouped and averaged from three different sections per embryo (n = 5 embryos; Student’s t-test; **p < 0.01, ***p < 0.001, ****p < 0.0001). (B) Immunohistochemistry for NF-M on coronal sections of control (Ascl1^KI/+) and mutant (Ascl1^KI/KI) embryos at E14.5 and E16.5. In control embryos, NF-M⁺ thalamic axons extended in ordered and parallel arrays that were less fasciculated in the thalamus and prethalamus (B1,B2) than in mutant embryos (B3,B4). Moreover, in Ascl1 mutants, thick NF-M⁺ bundles crossed each other with abnormal overlapping course of axons (arrows in B3,B4). (C) Immunohistochemistry for tdTomato on coronal sections of control (Dlx5/6-Cre; R26-TdTomato; Ascl1^KI/+) and mutant (Dlx5/6-Cre; R26-TdTomato; Ascl1^KI/KI) embryos at E13.5 and E14.5. The control prethalamus formed ordered and parallel tdTomato⁺ projections to the thalamus (C1,C3), while very few PTAs were detected in the mutant embryos (C2,C4). (C5) Examples of the measurements taken for the quantification. Yellow dashed lines in C1-C4 indicate the position of the lines used to quantify the number of axon bundles crossing the thalamus. (n = 3 embryos; two-way ANOVA with Sidak’s multiple comparison test; **p < 0.01, ***p < 0.001, ****p < 0.0001). (D) Implanting the chemical tracer Neurovue into the prethalamus of control (Ascl1^KI/+) and mutant embryos (Ascl1^KI/KI) at E13.5. Compared to those in controls (D1), no Neurovue⁺ PTAs were labeled in the thalamus in the absence of Ascl1 (D2). (E) In situ hybridization for Isl1 and Shh on control (Ascl1^KI/+) and mutant (Ascl1^KI/KI) coronal sections at E12.5. Shh is a specific marker for the zli. Loss of Ascl1 abrogated Isl1 expression in the TRN. (E5) Quantification of in situ hybridization. The pixel intensity values of TRN were grouped and averaged from four different sections per embryo (n = 4 embryos; Student’s t-test; ****p < 0.0001).Pt, prethalamus: Hyp, hypothalamus; Pt, prethalamus; rZI, rostral zona incerta; Th, thalamus; TRN, thalamic reticular nucleus; vLG, ventral lateral geniculate; zli, zona limitans intrathalamica. Scale bars, 200 μm