Figure 4. Lmx1b is temporally required for 5-HT projection pathway formation.

(A) Schematic of tamoxifen-inducible approach to target Lmx1b at postnatal day (P)1. (B) RT-qPCR of flow sorted TdTomato⁺ neurons from postnatal targeted mice (n = 3, iControl; n = 4, Lmx1bicKO mice). Unpaired t-test with Welch's correction, *p<0.05. Data are represented as mean ± SEM. (C) Coronal sections of P1 targeted Lmx1bicKO hippocampus compared to iControls analyzed at P31. *, incomplete formation of SCS and cingulum in P1 targeted Lmx1bicKO brain. Scale bars, 200 µm. (D) Coronal sections of P1 targeted Lmx1bicKO cortex compared to iControls analyzed at P31. Scale bars, 200 µm. (E) Coronal sections at level of corpus callosum showing incomplete formation of major 5-HT axon routes, SCS and cingulum, in P1 targeted Lmx1bicKO forebrain compared to iControls (above dotted line). Scale bars, 100 µm. cg, cingulum; SCS, supracallosal stria; cc, corpus callosum; m, midline. (F) Quantification of axons within SCS and cingulum tracts (n = 3, iControl; n = 3, Lmx1bicKO mice). Unpaired t-test with Welch's correction, p=0.0112. Data are represented as mean ± SEM.

Figure 4—figure supplement 1. Efficiency of postnatal tamoxifen inducible targeting of Lmx1b.

(A) TdTomato⁺ cells co- localized with serotonergic marker Tph2 in the DRN of iControl mice. Scale bars, 100 µm. (B) Tph2-CreER efficiency: 94% of TdTomato+ cells expressed Tph2 (Tph2+/RFP+) and 94% of Tph2+ cells expressed TdTomato+ (RFP+/Tph2+) in DRN/MRN/B9 nuclei (n = 4, iControl mice). Data are represented as mean ± SEM. (C) DRN of P1 targeted Lmx1bicKO and iControl mice. Scale bars, 100 µm. (D) Cell counts confirmed comparable numbers of TdTomato+ cells in Lmx1bicKO and iControl mice. (E) Normal cell body volume in postnatal targeted Lmx1bicKO mice (n = 464, control cells; n = 651, P3 Lmx1bicKO cells; p=0.9353). Unpaired t-test with Welch's correction. Data are represented as mean ± SEM.