Feline Lindhout

2 48 upregulated, which is in line with changes previously observed in mouse iPSCs and human ESCs (Wu et al. 2010; Chen et al. 2013). To determine if regulatory changes of transcripts are reflected in protein expression, we performed quantitative proteome analysis. We identified 2,218 proteins with more than two-fold expression changes during the first stages of neurodevelopment, indicating that significant remodeling of the proteome takes place during early stages. We identified six clusters of expression profiles and conducted GO enrichment analysis, which revealed a coordinated proteomic rearrangement during neurodevelopment. Proteins related to neuronal differentiation are upregulated and proteins related to cell proliferation are downregulated. Similar changes are observed in dissociated rodent neurons, differentiating neural crest and immortalized human neural progenitor cells, confirming the neuronal identity adopted by our human iPSC-derived cells (Kobayashi et al. 2009; Frese et al. 2017; Song et al. 2019). Similar to changes in protein expression levels, RNA expression also showed a strong increase of factors involved in neuronal differentiation and polarization, such as TUBB3 and TRIM46. Indeed, RNA and protein dynamics were generally correlated, indicating a coordinated cellular reprogramming into neurons. Furthermore, reorganization of the microtubule cytoskeleton is reflected in both transcriptome and proteome profiles during early neurodevelopment. However, differences between transcriptome and proteome dynamics during early neurodevelopment are also observed. This may reflect a temporal shift in regulation of transcription and translation. In addition, possible regulatory mechanisms on the protein level might be at play. Together, these data provide a rich resource for both the transcriptome and proteome dynamics in developing human neurons, which can be used in future studies to advance our understanding of the molecular mechanisms involved in neuronal differentiation and polarity. Distal to proximal relocation of AIS proteins during axon development A critical event in early neurodevelopment is the polarization of a symmetric cell into a neuron, which is initiated by the formation of a single axon (Dotti, Sullivan, and Banker 1988; Craig and Banker 1994). Here, we studied the onset of polarity in human iPSC- derived neurons in detail, and found that axon specification proceeds through a multistage process with multiple intermediate steps. We observed that AIS proteins Trim46, AnkG and NaV first appear as long noncontinuous structures in distal regions of the axons at the onset of stage 3 (stage 3a), and later relocate and cluster at proximal regions to form the stable AIS structures (stage 3b). In conjunction, we also found a distal-to-proximal reorganization of the microtubule cytoskeleton network in axons. This is marked by a developmental shift towards the characteristic uniform, plus-end out orientation in growing axons. Although the precise relation between AIS formation and microtubule remodeling in early axon development is unclear, various studies have shown the cooperative interaction between AIS components and microtubule structures during axon development (Leterrier et al. 2011; Freal et al. 2016). Recently Fréal et al., described a feedback-based mechanism that drives AIS assembly, in which membrane, scaffolding, and microtubule(-associated)