Direct neuronal reprogramming by temporal identity factors

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 Muller 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.

A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and identify them in a synapse-scale TEM reconstruction of the Drosophila larval CNS. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

A Casz1–NuRD complex regulates temporal identity transitions in neural progenitors

Neural progenitor cells undergo identity transitions during development to ensure the generation different types of neurons and glia in the correct sequence and proportions. A number of temporal identity factors that control these transitions in progenitor competence have been identified, but the molecular mechanisms underlying their function remain unclear. Here, we asked how Casz1, the mammalian orthologue of Drosophila castor, regulates competence during retinal development. We show that Casz1 is required to control the transition between neurogenesis and gliogenesis. Using BioID proteomics, we reveal that Casz1 interacts with the nucleosome remodeling and deacetylase (NuRD) complex in retinal cells. Finally, we show that both the NuRD and the polycomb repressor complexes are required for Casz1 to promote the rod fate and suppress gliogenesis. As additional temporal identity factors have been found to interact with the NuRD complex in other contexts, we propose that these factors might act through this common biochemical process to regulate neurogenesis.

Pou2f1 and Pou2f2 cooperate to control the timing of cone photoreceptor production in the developing mouse retina

ABSTRACTMultipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated ‘The people behind the papers’ interview.

A novel temporal identity window generates alternating cardinal motor neuron subtypes in a single progenitor lineage

Background: Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. These mechanisms generate cardinal classes of motor neurons, sharing a transcription factor identity and common muscle group targets). In Drosophila , two cardinal classes are Even-skipped (Eve)+ motor neurons projecting to dorsal longitudinal muscles and Nkx6+ motor neurons projecting to ventral oblique muscles. The Drosophila neuroblast 7-1 (NB7-1) lineage generates distinct Eve+ motor neurons via the temporal transcription factor (TTF) cascade Hunchback (Hb)-Krüppel (Kr)-Pdm-Castor (Cas). Methods: Here we use sparse labelling and molecular markers to identify a novel VO motor neuron subtype in the NB7-1 lineage, and birth-date this neuron to a Kr+ Pdm+ temporal identity window. We selectively drive overexpression of Kr and Pdm in the NB7-1 lineage, and assay the production and axonal targeting of ectopic VO neurons. We then use gain- and loss-of-function strategies to show that the identity and targeting specificity of the VO neuron is dependent on the transcription factor Nkx6. Results: Here we show that a newly discovered Kr/Pdm temporal identity window gives rise to an Nkx6+ Eve- motor neuron projecting to ventral oblique muscles, resulting in alternation of cardinal motor neuron subtypes from a single progenitor (Eve>Nkx6>Eve). We show that co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage – the first evidence that this TTF combination specifies neuronal identity. Moreover, we show that the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for ventral oblique muscle targeting, thereby linking temporal patterning to motor neuron synaptic target selection. Conclusions: We show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm>Nkx6 pathway is necessary and sufficient to specify VO motor neuron identity and morphology.

Foxn4 is a temporal identity factor conferring mid/late-early retinal competence and involved in retinal synaptogenesis

During development, neural progenitors change their competence states over time to sequentially generate different types of neurons and glia. Several cascades of temporal transcription factors (tTFs) have been discovered inDrosophilato control the temporal identity of neuroblasts, but the temporal regulation mechanism is poorly understood in vertebrates. Mammalian retinal progenitor cells (RPCs) give rise to several types of neuronal and glial cells following a sequential yet overlapping temporal order. Here, by temporal cluster analysis, RNA-sequencing analysis, and loss-of-function and gain-of-function studies, we show that the Fox domain TF Foxn4 functions as a tTF during retinogenesis to confer RPCs with the competence to generate the mid/late-early cell types: amacrine, horizontal, cone, and rod cells, while suppressing the competence of generating the immediate-early cell type: retinal ganglion cells (RGCs). In early embryonic retinas,Foxn4inactivation causes down-regulation of photoreceptor marker genes and decreased photoreceptor generation but increased RGC production, whereas its overexpression has the opposite effect. Just as inDrosophila, Foxn4 appears to positively regulate its downstream tTF Casz1 while negatively regulating its upstream tTF Ikzf1. Moreover, retina-specific ablation ofFoxn4reveals that it may be indirectly involved in the synaptogenesis, establishment of laminar structure, visual signal transmission, and long-term maintenance of the retina. Together, our data provide evidence that Foxn4 acts as a tTF to bias RPCs toward the mid/late-early cell fates and identify a missing member of the tTF cascade that controls RPC temporal identities to ensure the generation of proper neuronal diversity in the retina.

A novel temporal identity window generates alternating cardinal motor neuron subtypes in a single progenitor lineage

Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. These mechanisms generate cardinal classes of motor neurons (sharing a transcription factor identity and common muscle group targets). In Drosophila, two cardinal classes are Even-skipped (Eve)+ motor neurons projecting to dorsal muscles and Nkx6+ motor neurons projecting to ventral muscles. The Drosophila neuroblast 7-1 (NB7-1) lineage generates distinct Eve+ motor neurons via the temporal transcription factor (TTF) cascade Hunchback (Hb)-Krüppel (Kr)-Pdm-Castor (Cas). Here we show that a newly discovered Kr/Pdm temporal identity window gives rise to an Nkx6+ Eve-motor neuron projecting to ventral oblique muscles, resulting in alternation of cardinal motor neuron subtypes from a single progenitor (Eve>Nkx6>Eve). We show that co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage and that Kr/Pdm act via Nkx6, which itself is necessary and sufficient for VO motor neuron identity. Lastly, Nkx6 is required for ventral oblique muscle targeting, thereby linking temporal patterning to motor neuron morphology and synaptic target selection. In conclusion, we show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm>Nkx6 pathway is necessary and sufficient to specify VO motor neuron identity and morphology.

A Casz1 - NuRD complex regulates temporal identity transitions in neural progenitors

Neural progenitor cells alter their output over developmental time to generate different types of neurons and glia in the correct sequences and proportions. A number of ‘temporal identity factors’ that control transitions in progenitor competence have been identified, but the molecular mechanisms underlying their function remain unclear. Here, we asked how the transcription factor Casz1, the mammalian orthologue of Drosophila castor, regulates competence during retinal neurogenesis. We show that Casz1 is required to control the transition between neurogenesis and gliogenesis. Using BioID proteomics, we reveal that Casz1 interacts with the nucleosome remodeling and deacetylase (NuRD) complex in retinal cells. Finally, we show that both the NuRD and the polycomb repressor complexes are required for Casz1 to promote the rod fate and suppress gliogenesis. As other temporal identity factors have been found to interact with the NuRD complex in other contexts, we propose that these factors might act through a common biochemical process to regulate neurogenesis.