Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 41 Figure 2. Transcriptomic and proteomic profiling of early developmental stages in human iPSC-derived neurons A. Volcanoplot of differentially expressed transcripts between day 7 and day 1 (false discovery rate (FDR) p < 0.05, Benjamini & Hochberg corrected). B,C. Top 10 most significantly enriched GO terms of downregulated ( B ) and upregulated ( C ) genes at day 7. Representative cellular components (CC, cyan), biological processes (BP, yellow), molecular function (MF, green), FDR p < 0.05, Benjamini & Hochberg corrected. D,E. Six clusters with distinct protein expression profiles, divided in upregulated ( D ) and downregulated ( E ) protein expression, obtained by unsupervised clustering, and the GO enrichment analysis for each cluster. Representative cellular components (CC, cyan), biological processes (BP, yellow), molecular function (MF, green). F. Correlative analysis of relative transcriptomic and proteomic expression levels (day7/day1) (Pearson’s correlation, R = 0.51, p < 0.0001). Highlighted are some typical stem cell (blue), neuron (yellow), and axon (red) markers. G,H . Heatmaps showing the relative expression of RNA ( G ) and protein ( H ) levels of typical stem cell, neuron, and axon markers at different timepoints. 3 neurons were morphologically defined by the appearance of a single elongated neurite, the future axon, which has grown at least twice as long as the other neurites. Interestingly, we found that AIS proteins in stage 3 neurons first appeared as noncontinuous structures consisting of multiple smaller puncta and stretches that cover distal parts of the axon (referred to as stage 3a), prior to their more conventional localization in the AIS at the proximal axon (referred to as stage 3b) (Fig 3A,B). Quantification of the abundance of these neurodevelopmental stages over time revealed a developmental decline of stage 2b neurons that was accompanied with an increase of stage 3a neurons, as well as an increase of stage 3b neurons with a relatively later onset (Fig 3C). We further characterized the distribution of AIS proteins by measuring their lengths and distances to the soma at each stage. Developmental changes in the length of AnkG and Trim46 structures were observed, as the total neurite length covered by Trim46 or AnkG signals was strongly increased by ~40% in stage 3a neurons, and significantly reduced by ~55% in stage 3b neurons (Fig 3D; Fig S3A). Movereover, the axonal Trim46 and AnkG structures were localized more distally in stage 3a neurons, as the distance from the soma to both the start as well as the end of the Trim46 and AnkG appearance was significantly larger compared to stage 3b neurons (Fig 3E; Fig S3B-D). The axonal localization of NaV during development shows a similar dynamic profile as Trim46 and AnkG (Fig S3D). These data imply that axon specification (transition stage 2-3) in human iPSC-derived neurons can be subdivided in 4 steps (stage 2a, 2b, 3a and 3b) based on the subcellular localization of AIS proteins. Here, AIS proteins first