Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.     

Drosophila miR-124 regulates neuroblast proliferation through its target anachronism

MicroRNAs (miRNAs) have been implicated as regulators of central nervous system (CNS) development and function. miR-124 is an evolutionarily ancient, CNS-specific miRNA. On the basis of the evolutionary conservation of its expression in the CNS, miR-124 is expected to have an ancient conserved function. Intriguingly, investigation of miR-124 function using antisense-mediated miRNA depletion has produced divergent and in some cases contradictory findings in a variety of model systems. Here we investigated miR-124 function using a targeted knockout mutant and present evidence for a role during central brain neurogenesis in Drosophila melanogaster. miR-124 activity in the larval neuroblast lineage is required to support normal levels of neuronal progenitor proliferation. We identify anachronism (ana), which encodes a secreted inhibitor of neuroblast proliferation, as a functionally important target of miR-124 acting in the neuroblast lineage. ana has previously been thought to be glial specific in its expression and to act from the cortex glia to control the exit of neuroblasts from quiescence into the proliferative phase that generates the neurons of the adult CNS during larval development. We provide evidence that ana is expressed in miR-124-expressing neuroblast lineages and that ana activity must be limited by the action of miR-124 during neuronal progenitor proliferation. We discuss the possibility that the apparent divergence of function of miR-124 in different model systems might reflect functional divergence through target site evolution.     

Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.     

MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis

Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA--miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs--in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.     

Involvement of miR-9/MCPIP1 axis in PDGF-BB-mediated neurogenesis in neuronal progenitor cells

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.     

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.     

The on/off of Pax6 controls the tempo of neuronal differentiation in the developing spinal cord

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.     

NEUROG2 drives cell cycle exit of neuronal precursors by specifically repressing a subset of cyclins acting at the G1 and S phases of the cell cycle

Proneural NEUROG2 (neurogenin 2 [Ngn2]) is essential for neuronal commitment, cell cycle withdrawal, and neuronal differentiation. Although NEUROG2's influence on neuronal commitment and differentiation is beginning to be clarified, its role in cell cycle withdrawal remains unknown. We therefore set out to investigate the molecular mechanisms by which NEUROG2 induces cell cycle arrest during spinal neurogenesis. We developed a large-scale chicken embryo strategy, designed to find gene networks modified at the onset of NEUROG2 expression, and thereby we identified those involved in controlling the cell cycle. NEUROG2 activation leads to a rapid decrease of a subset of cell cycle regulators acting at G(1) and S phases, including CCND1, CCNE1/2, and CCNA2 but not CCND2. The use of NEUROG2VP16 and NEUROG2EnR, acting as the constitutive activator and repressor, respectively, indicates that NEUROG2 indirectly represses CCND1 and CCNE2 but opens the possibility that CCNE2 is also repressed by a direct mechanism. We demonstrated by phenotypic analysis that this rapid repression of cyclins prevents S phase entry of neuronal precursors, thus favoring cell cycle exit. We also showed that cell cycle exit can be uncoupled from neuronal differentiation and that during normal development NEUROG2 is in charge of tightly coordinating these two processes.     

Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs

BACKGROUND: The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. RESULTS: We report the generation of a Drosophila mutant that lacks let-7 and miR-125 activities and that leads to a pleiotropic phenotype arising during metamorphosis. We focus on two defects and demonstrate that loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. We identify the abrupt (ab) gene, encoding a nuclear protein, as a bona fide let-7 target and provide evidence that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. CONCLUSIONS: Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. Our results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages.     

Xiro homeoproteins coordinate cell cycle exit and primary neuron formation by upregulating neuronal-fate repressors and downregulating the cell-cycle inhibitor XGadd45-gamma

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates, among them are neural plate formation and neural patterning. In this work, we study in detail Xenopus Iro (Xiro) function in primary neurogenesis. We show that misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, we identify XGadd45-gamma as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. We conclude that, during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons.     

A Positive Feedback Mechanism That Regulates Expression of miR-9 during Neurogenesis

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.     

The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.