3 4 5 Running title: Human LUHMES neurons are ciliated 6 7 8

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing.LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain.In LUHMES, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

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Peptidyl arginine deiminases (PADIs) are enzymes that change the charge of proteins through citrullination. We recently found Padi2 was expressed exclusively in fetal Sertoli cells. In this study, we analyzed the transcriptional regulation of Padi2 and the role of PADI2 in testicular development. We showed SOX9 positively regulated Padi2 transcription and FOXL2 antagonized it in TM3 cells, a model of Sertoli cells. The responsive region to SOX9 and FOXL2 was identified within the Padi2 sequence by reporter assay. In fetal testes from Sox9 knockout (AMH-Cre:Sox9flox/flox) mice, Padi2 expression was greatly reduced, indicating SOX9 regulates Padi2 in vivo. In vitro analysis using siRNA suggested PADI2 modified transcriptional regulation by SOX9. However, Padi2−/− XY mice were fertile and showed no apparent reproductive anomalies. Although, PADI2 is known as an epigenetic transcriptional regulator through H3 citrullination, no significant difference in H3 citrullination between wildtype and Padi2−/− XY gonads was observed. These results suggest Padi2 is a novel gene involved in testis development that is specifically expressed in Sertoli cells through the regulation by SOX9 and FOXL2 and PADI2 supports regulation of target genes by SOX9. Analysis of the Padi2−/− XY phenotype suggested a redundant factor compensated for PADI2 function in testicular development.

Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and a mutant with altered cilia (RFX2 -/-), we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.