bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451124; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Direct neuronal reprogramming by temporal identity factors Camille Boudreau-Pinsonneault1,2, Awais Javed1,3, Michel Fries1,3, Pierre Mattar1,#, and Michel Cayouette1,2,3,4,5,* 1 Cellular Neurobiology Research Unit, Montreal Clinical Research Institute (IRCM), Montreal, Canada, H2W1R7 2 Integrated Program in Neuroscience, McGill University, Montreal, Canada 3 Molecular Biology Program, Université de Montréal, Montreal, Canada 4 Department of Anatomy and Cell Biology, McGill University, Montreal, Canada 5 Department of Medicine, Université de Montréal, Montreal, Canada # current address: Ottawa Health Research Institute (OHRI), Ottawa, ON, K1H 8L6 and Department of Cell and Molecular Medicine, University of Ottawa, Ottawa, ON, K1H 8M5 * corresponding author: [email protected] ! 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451124; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. SUMMARY Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors, but whether they could also reprogram the identity of fully differentiated cells is unknown. To address this question, we designed a conditional gene expression system combined with genetic lineage tracing that allows rapid screening of potential reprogramming factors in the mouse retina. Using this assay, we report that co- expression of the early temporal identity transcription factor Ikzf1, together with Ikzf4, another Ikaros family member, is sufficient to directly convert adult Müller glial cells into neuron-like cells in vivo, without inducing a proliferative progenitor state. scRNA-seq analysis shows that the reprogrammed cells share some transcriptional signatures with both cone photoreceptors and bipolar cells. Furthermore, we show that co-expression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons by remodeling chromatin and promoting a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in differentiated cells, opening new opportunities for cell therapy development. ! 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451124; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION Cell reprogramming has revolutionized the way we conceptualize somatic cell states. Previously assumed to be irreversible, we now know that differentiated cell type identity can be altered. Building on decades of work in Xenopus (Gurdon, 1962; Gurdon and Melton, 2008), Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) demonstrated that the combined expression of four transcription factors mediating pluripotency during development (Oct3/4, Sox2, Klf4, c-Myc) reprograms mouse somatic cells to pluripotent cells, which can then differentiate into any cell type. Lineage-specifying transcription factors were also found to convert somatic cells from one identity to another, but without promoting an intermediate pluripotent state. For instance, the bHLH protein MyoD, a transcription factor regulating muscle cell differentiation, can reprogram mouse embryonic fibroblasts (MEFs) directly into myoblasts (Davis et al., 1987). Similarly, a small-scale screen of neural lineage transcription factors has shown that combined expression of Brn2, Ascl1, and Myt1l directly reprograms MEFs to induced neurons (iNs), wherein Ascl1 is sufficient for the conversion, and Brn2 and Myt1l potentiate neuronal differentiation (Chanda et al., 2014; Mall et al., 2017; Vierbuchen et al., 2010; Wapinski et al., 2013). Somatic cell reprogramming offers new opportunities for tissue repair. Endogenous reprogramming is particularly appealing as it could harness the regenerative potential of resident cells, circumventing the need for difficult transplantation procedures and the low integration rate of grafted cells in host tissues (Barker et al., 2018; Li and Chen, 2016). In the central nervous system (CNS), glial cells have been targeted for endogenous reprogramming because they are present in large numbers, and are generally spared in neurodegenerative diseases. Upon injury, some mammalian glia proliferate, but undergo a wound-healing response called reactive gliosis, which is sometimes maladaptive (Bringmann et al., 2009). In contrast, the glia of non-mammalian vertebrates de- differentiate into bona fide neural progenitors capable of regenerating lost neurons (Alunni and Bally-Cuif, 2016). An extensively studied system in lower vertebrates for regenerative response in the CNS is the teleost fish retina, in which Müller glia (MG) acquire stem cell properties and regenerate retinal neurons after injury (Goldman, 2014; Lenkowski and 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451124; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Raymond, 2014). In contrast, the mammalian retina lacks any spontaneous regenerative response. Nonetheless, recent work in the mouse retina has identified a number of factors that can induce reprogramming of mature MG into neuron-like cells (Hoang et al., 2020; Jorstad et al., 2017; Yao et al., 2018; Zhou et al., 2020). Despite the potential of cell reprogramming for regenerative therapies in the CNS, the number of factors with the ability to reprogram fully differentiated somatic cells into therapeutically relevant neurons remains scarce, which is slowing translation efforts. As neuronal reprogramming factors identified to date are generally involved in progenitor cell fate decisions, developmental regulators represent good candidates to identify factors with reprogramming abilities. More specifically, the functional properties of temporal identity factors, which control neural progenitor competence, make them particularly attractive. In most regions of the developing CNS, such as the neocortex, hindbrain, spinal cord, and retina, neural progenitors are temporally patterned, changing their competence to generate different cell types at specific stages (Holguera and Desplan, 2018; Oberst et al., 2019; Rossi et al., 2017). Such temporal patterning is an evolutionary conserved strategy to regulate CNS progenitor output and has been extensively studied in Drosophila neuroblasts. In multiple lineages of the fly ventral nerve cord, for example, a cascade of so called ‘temporal identity factors’ consisting of hunchback (hb), kruppel (kr), pdm, castor (cas), and grainyhead (grh), specify cell fate output and regulate temporal progression of the neuroblasts by cross-regulatory mechanisms (Brody and Odenwald, 2000; Grosskortenhaus et al., 2005; Grosskortenhaus et al., 2006; Isshiki et al., 2001; Kambadur et al., 1998; Pearson and Doe, 2003). Mis-expression of a given temporal factor in neuroblasts outside their normal window of expression is sufficient to confer competence to generate cell types associated with the temporal window of the mis-expressed factor (Cleary and Doe, 2006; Novotny et al., 2002; Pearson and Doe, 2003). In vertebrates, temporal patterning is more intricate, with complex intrinsic and extrinsic cues contributing to temporal progression of neural progenitors (Oberst et al., 2019). Interestingly, vertebrate homologues of Drosophila temporal identity factors have been reported to participate in temporal patterning of retinal and cortical progenitors (Alsio et al., 2013; Elliott et al., 2008; Javed et al., 2020; Mattar et al., 2015). For instance, Ikzf1 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.451124; this version posted July 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. (a.k.a. Ikaros), the homolog of hb, confers early progenitor identity, and is sufficient to induce ectopic production of early-born cell types when mis-expressed in late-stage retinal or cortical progenitor cells (Alsio et al., 2013; Elliott et al., 2008); a process that can be conceptualized as ‘temporal reprogramming’. Although vertebrate temporal identity factors can reprogram the developmental competence of neural progenitors, it remains unknown whether they can also reprogram fully differentiated cells, and whether temporal reprogramming shares common mechanisms with other forms of reprogramming. Here we addressed these questions using the mouse retina and cultured fibroblasts as tractable systems to study cell reprogramming. Our results identify novel properties for temporal identity factors and bridge