Progenitor competence: genes switching places.

Drosophila neural progenitor cells are competent to give rise to certain neuronal cell types only during a limited period of time. Kohwi et al. link the termination of early competence to changes in subnuclear organization of chromatin.

Ikaros family proteins redundantly regulate temporal patterning in the developing mouse retina.

Temporal identity factors regulate competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc-finger transcription factor Ikzf1 regulates production of early-born cell types, except cone photoreceptors. In this study we show that, during early stages of retinal development, another Ikaros family protein, Ikzf4, functions redundantly with Ikzf1 to regulate cone photoreceptor production. Using CUT&RUN and functional assays, we show that Ikzf4 binds and represses genes involved in late-born rod photoreceptor specification, hence favoring cone production. At late stages, when Ikzf1 is no longer expressed in progenitors, we show that Ikzf4 re-localizes to target genes involved in gliogenesis and is required for Müller glia production. We report that Ikzf4 regulates Notch signaling genes and is sufficient to activate the Hes1 promoter through two Ikzf GGAA-binding motifs, suggesting a mechanism by which Ikzf4 may influence gliogenesis. These results uncover a combinatorial role for Ikaros family members during nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.

Direct neuronal reprogramming by temporal identity factors.

Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors and shift cell fate output, but whether they can also reprogram the identity of terminally differentiated cells is unknown. To address this question, we designed a conditional gene expression system that allows rapid screening of potential reprogramming factors in mouse retinal glial cells combined with genetic lineage tracing. Using this assay, we found that coexpression of the early temporal identity transcription factors Ikzf1 and Ikzf4 is sufficient to directly convert Müller glial (MG) cells into cells that translocate to the outer nuclear layer (ONL), where photoreceptor cells normally reside. We name these "induced ONL (iONL)" cells. Using genetic lineage tracing, histological, immunohistochemical, and single-cell transcriptome and multiome analyses, we show that expression of Ikzf1/4 in MG in vivo, without retinal injury, mostly generates iONL cells that share molecular characteristics with bipolar cells, although a fraction of them stain for Rxrg, a cone photoreceptor marker. Furthermore, we show that coexpression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons in culture by rapidly remodeling chromatin and activating a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in terminally differentiated cells.

Time to see: How temporal identity factors specify the developing mammalian retina.

Understanding how retinal progenitor cells (RPCs) give rise to the variety of neural cell types of the retina has been a question of major interest over the last few decades. While environmental cues and transcription factor networks have been shown to control specific cell fate decisions, how RPCs alter fate output over time to control proper histogenesis remains poorly understood. In recent years, the identification of "temporal identity factors (TIFs)", which control RPC competence states to ensure that the right cell types are produced at the right time, has contributed to increasing our understanding of temporal patterning in the retina. Here, we review the different TIFs identified to date in the mammalian retina and discuss the underlying mechanisms by which they are thought to operate. We conclude by speculating on how identification of temporal patterning mechanisms might support the development of new therapeutic approaches against visual impairments.

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

Multipotent 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.

Ben Barres (1954-2017).

Ben Barres changed our view of glial cell function and impacted the lives of many people who interacted with him. Remembering an outstanding scientist and mentor.

Casz1 controls higher-order nuclear organization in rod photoreceptors.

Genome organization plays a fundamental role in the gene-expression programs of numerous cell types, but determinants of higher-order genome organization are poorly understood. In the developing mouse retina, rod photoreceptors represent a good model to study this question. They undergo a process called "chromatin inversion" during differentiation, in which, as opposed to classic nuclear organization, heterochromatin becomes localized to the center of the nucleus and euchromatin is restricted to the periphery. While previous studies showed that the lamin B receptor participates in this process, the molecular mechanisms regulating lamina function during differentiation remain elusive. Here, using conditional genetics, we show that the zinc finger transcription factor Casz1 is required to establish and maintain the inverted chromatin organization of rod photoreceptors and to safeguard their gene-expression profile and long-term survival. At the mechanistic level, we show that Casz1 interacts with the polycomb repressor complex in a splice variant-specific manner and that both are required to suppress the expression of the nuclear envelope intermediate filament lamin A/C in rods. Lamin A is in turn sufficient to regulate heterochromatin organization and nuclear position. Furthermore, we show that Casz1 is sufficient to expand and centralize the heterochromatin of fibroblasts, suggesting a general role for Casz1 in nuclear organization. Together, these data support a model in which Casz1 cooperates with polycomb to control rod genome organization, in part by silencing lamin A/C.

A link between planar polarity and staircase-like bundle architecture in hair cells.

Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors that crowns hair cells. We previously showed that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the Insc-LGN-Gαi protein complex. We now report that LGN and Gαi also occupy the very tip of stereocilia that directly abut the bare zone. We demonstrate that LGN and Gαi are both essential for promoting the elongation and differential identity of stereocilia across rows. Interestingly, we also reveal that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. We propose that LGN and Gαi participate in a long-inferred signal that originates outside the bundle to model its staircase-like architecture, a property that is essential for direction sensitivity to mechanical deflection and hearing.

The LGN protein promotes planar proliferative divisions in the neocortex but apicobasal asymmetric terminal divisions in the retina.

Cell division orientation is crucial to control segregation of polarized fate determinants in the daughter cells to produce symmetric or asymmetric fate outcomes. Most studies in vertebrates have focused on the role of mitotic spindle orientation in proliferative asymmetric divisions and it remains unclear whether altering spindle orientation is required for the production of asymmetric fates in differentiative terminal divisions. Here, we show that the GoLoco motif protein LGN, which interacts with Gαi to control apicobasal division orientation in Drosophila neuroblasts, is excluded from the apical domain of retinal progenitors undergoing planar divisions, but not in those undergoing apicobasal divisions. Inactivation of LGN reduces the number of apicobasal divisions in mouse retinal progenitors, whereas it conversely increases these divisions in cortical progenitors. Although LGN inactivation increases the number of progenitors outside the ventricular zone in the developing neocortex, it has no effect on the position or number of progenitors in the retina. Retinal progenitor cell lineage analysis in LGN mutant mice, however, shows an increase in symmetric terminal divisions producing two photoreceptors, at the expense of asymmetric terminal divisions producing a photoreceptor and a bipolar or amacrine cell. Similarly, inactivating Gαi decreases asymmetric terminal divisions, suggesting that LGN function with Gαi to control division orientation in retinal progenitors. Together, these results show a context-dependent function for LGN and indicate that apicobasal divisions are not involved in proliferative asymmetric divisions in the mouse retina, but are instead essential to generate binary fates at terminal divisions.

Cell lineage tracing in the retina: Could material transfer distort conclusions?

Recent studies reported the transfer of fluorescent labels between grafted and host cells after transplantation of photoreceptor precursor cells in the mouse retina. While clearly impacting the interpretation of transplantation studies in the retina, the potential impact of material transfer in other experimental paradigms using cell-specific labels remains uncertain. Here, we briefly review the evidence supporting material transfer in transplantation studies and discuss whether it might influence retinal cell lineage tracing experiments in developmental and regeneration studies. We also propose ways to control for the possible confounding occurrence of label exchange in such experiments. Developmental Dynamics 247:10-17, 2018. © 2017 Wiley Periodicals, Inc.

Ikaros promotes early-born neuronal fates in the cerebral cortex.

During cerebral cortex development, a series of projection neuron types is generated in a fixed temporal order. In Drosophila neuroblasts, the transcription factor hunchback encodes first-born identity within neural lineages. One of its mammalian homologs, Ikaros, was recently reported to play an equivalent role in retinal progenitor cells, raising the question as to whether Ikaros/Hunchback proteins could be general factors regulating the development of early-born fates throughout the nervous system. Ikaros is also expressed in progenitor cells of the mouse cerebral cortex, and this expression is highest during the early stages of neurogenesis and thereafter decreases over time. Transgenic mice with sustained Ikaros expression in cortical progenitor cells and neurons have developmental defects, including displaced progenitor cells within the cortical plate, increased early neural differentiation, and disrupted cortical lamination. Sustained Ikaros expression results in a prolonged period of generation of deep layer neurons into the stages when, normally, only late-born upper layer neurons are generated, as well as a delayed production of late-born neurons. Consequently, more early-born and fewer late-born neurons are present in the cortex of these mice at birth. This phenotype was observed in all parts of the cortex, including those with minimal structural defects, demonstrating that it is not secondary to abnormalities in cortical morphogenesis. These data suggest that Ikaros plays a similar role in regulating early temporal fates in the mammalian cerebral cortex as Ikaros/Hunchback proteins do in the Drosophila nerve cord.

Numb regulates the polarized delivery of cyclic nucleotide-gated ion channels in rod photoreceptor cilia.

The development and maintenance of protein compartmentalization is essential for neuronal function. A striking example is observed in light-sensing photoreceptors, in which the apical sensory cilium is subdivided into an inner and outer segment, each containing specific proteins essential for vision. It remains unclear, however, how such polarized protein localization is regulated. We report here that the endocytic adaptor protein Numb localizes to the inner, but not the outer segment of mouse photoreceptor cilia. Rod photoreceptor-specific inactivation of numb in vivo leads to progressive photoreceptor degeneration, indicating an essential role for Numb in photoreceptor cell biology. Interestingly, we report that loss of Numb in photoreceptors does not affect the localization of outer segment disk membrane proteins, such as rhodopsin, Peripherin-rds, Rom-1, and Abca4, but significantly disrupts the localization of the rod cyclic nucleotide-gated (Cng) channels, which accumulates on the inner segment plasma membrane in addition to its normal localization to the outer segments. Mechanistically, we show that Numb interacts with both subunits of the Cng channel and promotes the trafficking of Cnga1 to the recycling endosome. These results suggest a model in which Numb prevents targeting of Cng channels to the inner segment, by promoting their trafficking through the recycling endosome, where they can be sorted for specific delivery to the outer segment. This study uncovers a novel mechanism regulating polarized protein delivery in light-sensing cilia, raising the possibility that Numb plays a part in the regulation of protein trafficking in other types of cilia.

Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions.

In vivo cell lineage-tracing studies in the vertebrate retina have revealed that the sizes and cellular compositions of retinal clones are highly variable. It has been challenging to ascertain whether this variability reflects distinct but reproducible lineages among many different retinal progenitor cells (RPCs) or is the product of stochastic fate decisions operating within a population of more equivalent RPCs. To begin to distinguish these possibilities, we developed a method for long-term videomicroscopy to follow the lineages of rat perinatal RPCs cultured at clonal density. In such cultures, cell-cell interactions between two different clones are eliminated and the extracellular environment is kept constant, allowing us to study the cell-intrinsic potential of a given RPC. Quantitative analysis of the reconstructed lineages showed that the mode of division of RPCs is strikingly consistent with a simple stochastic pattern of behavior in which the decision to multiply or differentiate is set by fixed probabilities. The variability seen in the composition and order of cell type genesis within clones is well described by assuming that each of the four different retinal cell types generated at this stage is chosen stochastically by differentiating neurons, with relative probabilities of each type set by their abundance in the mature retina. Although a few of the many possible combinations of cell types within clones occur at frequencies that are incompatible with a fully stochastic model, our results support the notion that stochasticity has a major role during retinal development and therefore possibly in other parts of the central nervous system.

Numb positively regulates Hedgehog signaling at the ciliary pocket.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina.

In the developing nervous system, cell diversification depends on the ability of neural progenitor cells to divide asymmetrically to generate daughter cells that acquire different identities. While much work has recently focused on the mechanisms controlling self-renewing asymmetric divisions producing a differentiating daughter and a progenitor, little is known about mechanisms regulating how distinct differentiating cell types are produced at terminal divisions. Here we study the role of the endocytic adaptor protein Numb in the developing mouse retina. Using clonal numb inactivation in retinal progenitor cells (RPCs), we show that Numb is required for normal cell-cycle progression at early stages, but is dispensable for the production of self-renewing asymmetric cell divisions. At late stages, however, Numb is no longer required for cell-cycle progression, but is critical for the production of terminal asymmetric cell divisions. In the absence of Numb, asymmetric terminal divisions that generate a photoreceptor and a non-photoreceptor cell are decreased in favor of symmetric terminal divisions generating two photoreceptors. Using live imaging in retinal explants, we show that a Numb fusion protein is asymmetrically inherited by the daughter cells of some late RPC divisions. Together with our finding that Numb antagonizes Notch signaling in late-stage RPCs, and that blocking Notch signaling in late RPCs almost completely abolishes the generation of terminal asymmetric divisions, these results suggest a model in which asymmetric inheritance of Numb in sister cells of terminal divisions might create unequal Notch activity, which in turn drives the production of terminal asymmetric divisions.

Computational prediction of neural progenitor cell fates.

Understanding how stem and progenitor cells choose between alternative cell fates is a major challenge in developmental biology. Efforts to tackle this problem have been hampered by the scarcity of markers that can be used to predict cell division outcomes. Here we present a computational method, based on algorithmic information theory, to analyze dynamic features of living cells over time. Using this method, we asked whether rat retinal progenitor cells (RPCs) display characteristic phenotypes before undergoing mitosis that could foretell their fate. We predicted whether RPCs will undergo a self-renewing or terminal division with 99% accuracy, or whether they will produce two photoreceptors or another combination of offspring with 87% accuracy. Our implementation can segment, track and generate predictions for 40 cells simultaneously on a standard computer at 5 min per frame. This method could be used to isolate cell populations with specific developmental potential, enabling previously impossible investigations.

In vivo evidence for unbiased Ikaros retinal lineages using an Ikaros-Cre mouse line driving clonal recombination.

BACKGROUND: We showed previously that the transcription factor Ikaros is expressed in early but not late retinal progenitors cells (RPCs), and is necessary and sufficient for the production of early-born neurons. Preliminary evidence using retinal explant cultures qualitatively suggested that Ikaros-positive RPCs might share a common lineage with Ikaros-negative RPCs. RESULTS: To explore further this question in vivo in a quantitative manner, we generated BAC transgenic mouse lines expressing Cre recombinase under the regulatory elements of the Ikaros gene, and crossed them with Cre reporter lines. Different transgenic lines labeled a variable number of RPCs, resulting in either dense or sparse radial arrays of reporter-positive progenies. Analysis of over 800 isolated cell arrays, which are most likely clones, confirmed that Ikaros-expressing RPCs generate both early- and late-born cell types in the same lineage, and that the overall cell composition of the arrays closely resembles that of the population of the mature retina. Interestingly, another sparse line did not label arrays, but appeared to specifically reflect Ikaros postmitotic expression in amacrine and ganglion cells. CONCLUSIONS: These mouse lines confirm the unbiased potential of the Ikaros lineage in vivo and provide novel tools for clonal lineage tracing and single neuron tracking in the retina.

Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections.

The balance of contralateral and ipsilateral retinogeniculate projections is critical for binocular vision, but the transcriptional programs regulating this process remain ill defined. Here we show that the Pou class homeobox protein POU3F1 is expressed in nascent mouse contralateral retinal ganglion cells (cRGCs) but not ipsilateral RGCs (iRGCs). Upon Pou3f1 inactivation, the proportion of cRGCs is reduced in favor of iRGCs, leading to abnormal projection ratios at the optic chiasm. Conversely, misexpression of Pou3f1 in progenitors increases the production of cRGCs. Using CUT&RUN and RNA sequencing in gain- and loss-of-function assays, we demonstrate that POU3F1 regulates expression of several key members of the cRGC gene regulatory network. Finally, we report that POU3F1 is sufficient to induce RGC-like cell production, even in late-stage retinal progenitors of Atoh7 knockout mice. This work uncovers POU3F1 as a regulator of the cRGC transcriptional program, opening possibilities for optic nerve regenerative therapies.

Numb regulates Tau levels and prevents neurodegeneration in tauopathy mouse models.

Accumulation of the microtubule-associated protein Tau is linked to neuronal cell death in tauopathies, but how intraneuronal Tau levels are regulated in health and disease remains unclear. Here, we show that conditional inactivation of the trafficking adaptor protein Numb in retinal ganglion cells (RGCs) increases Tau levels and leads to axonal blebbing, which is followed by neuronal cell loss in aged mice. In the TauP301S mouse model of tauopathy, conditional inactivation of Numb in RGCs and spinal motoneurons accelerates neurodegeneration, and loss of Numb in motoneurons also leads to precocious hindlimb paralysis. Conversely, overexpression of the long isoform of Numb (Numb-72) decreases intracellular Tau levels and reduces axonal blebbing in TauP301S RGCs, leading to improved electrical activity in cultured neurons and improves performance in a visually guided behavior test in vivo. These results uncover Numb as a key regulator of intracellular Tau levels and identify Numb-72 as a potential therapeutic factor for tauopathies.