DEV02717 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression of Pax2 and Lhx1/5 in the developing spinal cord. (A) Lhx1/5 (green) is expressed in early-born Pax2⁺ (red) inhibitory neurons. Glutamatergic dI2 neurons express only Lhx1/5 (arrowhead in A). (B,C) Lhx1/5 is also extensively co-expressed with Pax2 in dIL_A neurons in the dorsal horn at E12.5 and E13.5 (arrowheads in B and C).

• Supplemental Figure 2 -

  Fig. S2. Expression of Lhx1 and Lhx5 in the E11.5 spinal cord. (A-D) In situ analysis of Lhx1 (A) and Lhx5 (B) expression showing nearly identical expression patterns in early-born neurons. Panels C and D show high magnification images of the dI4 neuron domain. Note the expression of Lhx1 and Lhx5 in all cells emerging from this domain. (E-M) Immunohistochemistry using antibodies that recognize Pax2 (green), which marks dorsal inhibitory neurons, and both the Lhx1/Lhx5 proteins (red) was performed on sections from E11.5 wild-type, Lhx1^−/− and Lhx5^−/− spinal cords. Extensive co-expression of Pax2 and the Lhx1/5 proteins was observed in dI4 neurons (E-G). In the Lhx1-mutant cord, Lhx5 is co-expressed with Pax2 in all dorsal inhibitory neurons (H-I). Likewise, in the Lhx5-mutant cord, Lhx1 co-localizes with Pax2 in the same neurons (K-L), indicating that Lhx1 and Lhx5 are co-expressed in dorsal inhibitory neurons.

• Supplemental Figure 3 -

  Fig. S3. Neuronal specification in the Lhx1 and Lhx5 single-mutant spinal cord. Multiple cell-type-specific markers were used to analyze the fate of early-born neurons in the cords of Lhx1^−/− and Lhx5^−/− knockout mice. (A-C) Expression of Brn3a (red) was not altered in dI1-dI3 and dI4 neurons. (D-F) Expression of Isl1 in motor neurons and dI3 neurons was also unchanged. Pax2 expression (green), which marks inhibitory neurons, was normal in the Lhx1- (E) and Lhx5- (F) mutant cord. (G-I) Lbx1⁺ Class B neurons were generated in normal numbers in the Lhx1 (H) and Lhx5 (I) single-mutant cord. dI4 and dI6 neurons that express a combination of Lbx1 and Lhx1/5 showed a normal pattern of genesis in each of the single mutants, as did dI5 neurons that express Lmx1b.

• Supplemental Figure 4 -

  Fig. S4. Pax2, GAD1 and VGluT2 expression in the E14.5 Lhx1/5 DKO cord. (A,B) Pax2 (red) and GAD1 (green) expression is downregulated in the DKO spinal cord. GAD1 expression (pseudo-colored green) is strongly reduced in the lateral dorsal horn in regions where Pax2 expression is lower (arrowheads in B). (C,D) Regions of the dorsal horn where Pax2 (red) is no longer expressed do not show a concomitant increase in VGluT2 (green) expression (arrowhead in D).

• Supplemental Figure 5 -

  Fig. S5. Programmed cell death in wild-type and Lhx1/5 DKO spinal cords. (A,B) Cross sections through an E17.5 wild-type and DKO spinal cord showing TUNEL staining for apoptotic cells. (C) TUNEL⁺ cell counts from E14.5-E13.5 spinal cords. The cell counts represent the average number of TUNEL⁺ cells in six representative sections. Error bars: s.d.