LMX1A and LMX1B are evolutionary conserved human mDAN transcription factors required for early neurogenesis. (A) Phylogenetic analysis by the Maximum Likelihood method based on the JTT matrix-based model. Initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 0.6637]). The analysis involved 23 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 186 positions in the final dataset. (B) Bootstrap consensus tree inferred from 2,000 replicates. Only branches that appeared in more than 50% bootstrap replicates are shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. (C–E) Incucyte analysis of neurite length using automated tracking software. Bar = 100 µm. (C) iPSC-derived mDANs were imaged over 4 d following transduction with hsyn-GFP/mcherry-U6-shRNA lentiviruses as indicated at (D) day >35 and (E) day <17. mDANs were virally transduced on the day of plating. Shown are means from three wells of a single representative experiment. For clarity, error bars have been omitted. (F) qRT-PCR analysis of mRNA levels following shRNA suppression of LMX1A or LMX1B in young (day 17) cultures. Significant reductions in TH, TUJ1, NURR1, and MSX1 expression following LMX1A shRNA. LMX1B suppression increases LMX1A levels. mRNA levels normalized to shControl. Data show means ± SE of triplicate wells from a single representative experiment. Student’s t test: *P < 0.05 and **P < 0.01.