Prosaposin maintains adult neural stem cells in a state associated with deep quiescence.

In most vertebrates, adult neural stem cells (NSCs) continuously give rise to neurons in discrete brain regions. A critical process for maintaining NSC pools over long periods of time in the adult brain is NSC quiescence, a reversible and tightly regulated state of cell-cycle arrest. Recently, lysosomes were identified to regulate the NSC quiescence-proliferation balance. However, it remains controversial whether lysosomal activity promotes NSC proliferation or quiescence, and a finer influence of lysosomal activity on NSC quiescence duration or depth remains unexplored. Using RNA sequencing and pharmacological manipulations, we show that lysosomes are necessary for NSC quiescence maintenance. In addition, we reveal that expression of psap, encoding the lysosomal regulator Prosaposin, is enriched in quiescent NSCs (qNSCs) that reside upstream in the NSC lineage and display a deep/long quiescence phase in the adult zebrafish telencephalon. We show that shRNA-mediated psap knockdown increases the proportion of activated NSCs (aNSCs) as well as NSCs that reside in shallower quiescence states (signed by ascl1a and deltaA expression). Collectively, our results identify the lysosomal protein Psap as a (direct or indirect) quiescence regulator and unfold the interplay between lysosomal function and NSC quiescence heterogeneities.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation.

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

Mosaic Heterochrony in Neural Progenitors Sustains Accelerated Brain Growth and Neurogenesis in the Juvenile Killifish N. furzeri.

Although developmental mechanisms driving an increase in brain size during vertebrate evolution are actively studied, we know less about evolutionary strategies allowing accelerated brain growth. In zebrafish and other vertebrates studied to date, apical radial glia (RG) constitute the primary neurogenic progenitor population throughout life [1]; thus, RG activity is a determining factor of growth speed. Here, we ask whether enhanced RG activity is the mechanism selected to drive explosive growth, in adaptation to an ephemeral habitat. In post-hatching larvae of the turquoise killifish, which display drastic developmental acceleration, we show that the dorsal telencephalon (pallium) grows three times faster than in zebrafish. Rather than resulting from enhanced RG activity, we demonstrate that pallial growth is the product of a second type of progenitors (that we term NGPs for non-glial progenitors) that actively sustains neurogenesis and germinal zone self-renewal. Intriguingly, NGPs appear to retain, at larval stages, features of early embryonic progenitors. In parallel, RGs enter premature quiescence and express markers of astroglial function. Altogether, we propose that mosaic heterochrony within the neural progenitor population might permit rapid pallial growth by safeguarding both continued neurogenesis and astroglial function.

Life-Long Neurogenic Activity of Individual Neural Stem Cells and Continuous Growth Establish an Outside-In Architecture in the Teleost Pallium.

Spatiotemporal variations of neurogenesis are thought to account for the evolution of brain shape. In the dorsal telencephalon (pallium) of vertebrates, it remains unresolved which ancestral neurogenesis mode prefigures the highly divergent cytoarchitectures that are seen in extant species. To gain insight into this question, we developed genetic tools to generate here the first 4-dimensional (3D + birthdating time) map of pallium construction in the adult teleost zebrafish. Using a Tet-On-based genetic birthdating strategy, we identify a "sequential stacking" construction mode where neurons derived from the zebrafish pallial germinal zone arrange in outside-in, age-related layers from a central core generated during embryogenesis. We obtained no evidence for overt radial or tangential neuronal migrations. Cre-lox-mediated tracing, which included following Brainbow clones, further demonstrates that this process is sustained by the persistent neurogenic activity of individual pallial neural stem cells (NSCs) from embryo to adult. Together, these data demonstrate that the spatiotemporal control of NSC activity is an important driver of the macroarchitecture of the zebrafish adult pallium. This simple mode of pallium construction shares distinct traits with pallial genesis in mammals and non-mammalian amniotes such as birds or reptiles, suggesting that it may exemplify the basal layout from which vertebrate pallial architectures were elaborated.

Embryonic origin and lineage hierarchies of the neural progenitor subtypes building the zebrafish adult midbrain.

Neurogenesis in the post-embryonic vertebrate brain varies in extent and efficiency between species and brain territories. Distinct neurogenesis modes may account for this diversity, and several neural progenitor subtypes, radial glial cells (RG) and neuroepithelial progenitors (NE), have been identified in the adult zebrafish brain. The neurogenic sequences issued from these progenitors, and their contribution to brain construction, remain incompletely understood. Here we use genetic tracing techniques based on conditional Cre recombination and Tet-On neuronal birthdating to unravel the neurogenic sequence operating from NE progenitors in the zebrafish post-embryonic optic tectum. We reveal that a subpopulation of her5-positive NE cells of the posterior midbrain layer stands at the top of a neurogenic hierarchy involving, in order, the amplification pool of the tectal proliferation zone (TPZ), followed by her4-positive RG cells with transient neurogenic activity. We further demonstrate that the adult her5-positive NE pool is issued in lineage from an identically located NE pool expressing the same gene in the embryonic neural tube. Finally, we show that these features are reminiscent of the neurogenic sequence and embryonic origin of the her9-positive progenitor NE pool involved in the construction of the lateral pallium at post-embryonic stages. Together, our results highlight the shared recruitment of an identical neurogenic strategy by two remote brain territories, where long-lasting NE pools serve both as a growth zone and as the life-long source of young neurogenic RG cells.

A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States.

Throughout life, adult neural stem cells (NSCs) produce new neurons and glia that contribute to crucial brain functions. Quiescence is an essential protective feature of adult NSCs; however, the establishment and maintenance of this state remain poorly understood. We demonstrate that in the adult zebrafish pallium, the brain-enriched miR-9 is expressed exclusively in a subset of quiescent NSCs, highlighting a heterogeneity within these cells, and is necessary to maintain NSC quiescence. Strikingly, miR-9, along with Argonaute proteins (Agos), is localized to the nucleus of quiescent NSCs, and manipulating their nuclear/cytoplasmic ratio impacts quiescence. Mechanistically, miR-9 permits efficient Notch signaling to promote quiescence, and we identify the RISC protein TNRC6 as a mediator of miR-9/Agos nuclear localization in vivo. We propose a conserved non-canonical role for nuclear miR-9/Agos in controlling the balance between NSC quiescence and activation, a key step in maintaining adult germinal pools.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs.

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9-5 was validated by 5' RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3'-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay.

Large-scale live imaging of adult neural stem cells in their endogenous niche.

Live imaging of adult neural stem cells (aNSCs) in vivo is a technical challenge in the vertebrate brain. Here, we achieve long-term imaging of the adult zebrafish telencephalic neurogenic niche and track a population of >1000 aNSCs over weeks, by taking advantage of fish transparency at near-infrared wavelengths and of intrinsic multiphoton landmarks. This methodology enables us to describe the frequency, distribution and modes of aNSCs divisions across the entire germinal zone of the adult pallium, and to highlight regional differences in these parameters.

Mechanisms of endoderm formation in a cartilaginous fish reveal ancestral and homoplastic traits in jawed vertebrates.

In order to gain insight into the impact of yolk increase on endoderm development, we have analyzed the mechanisms of endoderm formation in the catshark S. canicula, a species exhibiting telolecithal eggs and a distinct yolk sac. We show that in this species, endoderm markers are expressed in two distinct tissues, the deep mesenchyme, a mesenchymal population of deep blastomeres lying beneath the epithelial-like superficial layer, already specified at early blastula stages, and the involuting mesendoderm layer, which appears at the blastoderm posterior margin at the onset of gastrulation. Formation of the deep mesenchyme involves cell internalizations from the superficial layer prior to gastrulation, by a movement suggestive of ingressions. These cell movements were observed not only at the posterior margin, where massive internalizations take place prior to the start of involution, but also in the center of the blastoderm, where internalizations of single cells prevail. Like the adjacent involuting mesendoderm, the posterior deep mesenchyme expresses anterior mesendoderm markers under the control of Nodal/activin signaling. Comparisons across vertebrates support the conclusion that endoderm is specified in two distinct temporal phases in the catshark as in all major osteichthyan lineages, in line with an ancient origin of a biphasic mode of endoderm specification in gnathostomes. They also highlight unexpected similarities with amniotes, such as the occurrence of cell ingressions from the superficial layer prior to gastrulation. These similarities may correspond to homoplastic traits fixed separately in amniotes and chondrichthyans and related to the increase in egg yolk mass.

A self-organizing miR-132/Ctbp2 circuit regulates bimodal notch signals and glial progenitor fate choice during spinal cord maturation.

Radial glial progenitors play pivotal roles in the development and patterning of the spinal cord, and their fate is controlled by Notch signaling. How Notch is shaped to regulate their crucial transition from expansion toward differentiation remains, however, unknown. miR-132 in the developing zebrafish dampens Notch signaling via a cascade involving the transcriptional corepressor Ctbp2 and the Notch suppressor Sirt1. At early embryonic stages, high Ctbp2 levels sustain Notch signaling and radial glial expansion and concomitantly induce miR-132 expression via a double-negative feedback loop involving Rest inhibition. The changing balance in miR-132 and Ctbp2 interaction gradually drives the switch in Notch output and radial glial progenitor fate as part of the larger developmental program involved in the transition from embryonic to larval spinal cord.

Spatial regionalization and heterochrony in the formation of adult pallial neural stem cells.

Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing, activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging, coupled with gene expression analyses and Notch invalidations, to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary, the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof, activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments.

miR-9: a versatile regulator of neurogenesis.

Soon after its discovery, microRNA-9 (miR-9) attracted the attention of neurobiologists, since it is one of the most highly expressed microRNAs in the developing and adult vertebrate brain. Functional analyses in different vertebrate species have revealed a prominent role of this microRNA in balancing proliferation in embryonic neural progenitor populations. Key transcriptional regulators such as FoxG1, Hes1 or Tlx, were identified as direct targets of miR-9, placing it at the core of the gene network controlling the progenitor state. Recent data also suggest that this function could extend to adult neural stem cells. Other studies point to a role of miR-9 in differentiated neurons. Moreover miR-9 has been implicated in human brain pathologies, either displaying a protective role, such as in Progeria, or participating in disease progression in brain cancers. Altogether functional studies highlight a prominent feature of this highly conserved microRNA, its functional versatility, both along its evolutionary history and across cellular contexts.

[miR-9: a key factor of the physiopathological regulation of the neural progenitor state]. / Les multiples facettes d'un petit régulateur - Le microARN-9 dans le progéniteur neural normal et pathologique.

microRNA are small non-coding RNA that modulate gene expression post-transcriptionally. Discovered 20 years ago, their individual functions start to be unraveled. Collectively, functional studies point to an important functional plasticity of microRNA, along the course of evolution, and across different cellular contexts. This is the case in particular for one of them, miR-9, a key factor of the regulation of the neural progenitor state in Vertebrates.

Pattern and polarity in the development and evolution of the gnathostome jaw: both conservation and heterotopy in the branchial arches of the shark, Scyliorhinus canicula.

The acquisition of jaws constitutes a landmark event in vertebrate evolution, one that in large part potentiated their success and diversification. Jaw development and patterning involves an intricate spatiotemporal series of reciprocal inductive and responsive interactions between the cephalic epithelia and the cranial neural crest (CNC) and cephalic mesodermal mesenchyme. The coordinated regulation of these interactions is critical for both the ontogenetic registration of the jaws and the evolutionary elaboration of variable jaw morphologies and designs. Current models of jaw development and evolution have been built on molecular and cellular evidence gathered mostly in amniotes such as mice, chicks and humans, and augmented by a much smaller body of work on the zebrafish. These have been partnered by essential work attempting to understand the origins of jaws that has focused on the jawless lamprey. Chondrichthyans (cartilaginous fish) are the most distant group to amniotes within extant gnathostomes, and comprise the crucial clade uniting amniotes and agnathans; yet despite their critical phylogenetic position, evidence of the molecular and cellular underpinnings of jaw development in chondrichthyans is still lacking. Recent advances in genome and molecular developmental biology of the lesser spotted dogfish shark, Scyliorhinus canicula, make it ideal for the molecular study of chondrichthyan jaw development. Here, following the 'Hinge and Caps' model of jaw development, we have investigated evidence of heterotopic (relative changes in position) and heterochronic (relative changes in timing) shifts in gene expression, relative to amniotes, in the jaw primordia of S. canicula embryos. We demonstrate the presence of clear proximo-distal polarity in gene expression patterns in the shark embryo, thus establishing a baseline molecular baüplan for branchial arch-derived jaw development and further validating the utility of the 'Hinge and Caps' model in comparative studies of jaw development and evolution. Moreover, we correlate gene expression patterns with the absence of a lambdoidal junction (formed where the maxillary first arch meets the frontonasal processes) in chondrichthyans, further highlighting the importance of this region for the development and evolution of jaw structure in advanced gnathostomes.

miR-9 controls the timing of neurogenesis through the direct inhibition of antagonistic factors.

The timing of commitment and cell-cycle exit within progenitor populations during neurogenesis is a fundamental decision that impacts both the number and identity of neurons produced during development. We show here that microRNA-9 plays a key role in this process through the direct inhibition of targets with antagonistic functions. Across the ventricular zone of the developing zebrafish hindbrain, miR-9 expression occurs at a range of commitment stages. Abrogating miR-9 function transiently delays cell-cycle exit, leading to the increased generation of late-born neuronal populations. Target protection analyses in vivo identify the progenitor-promoting genes her6 and zic5 and the cell-cycle exit-promoting gene elavl3/HuC as sequential targets of miR-9 as neurogenesis proceeds. We propose that miR-9 activity generates an ambivalent progenitor state poised to respond to both progenitor maintenance and commitment cues, which may be necessary to adjust neuronal production to local extrinsic signals during late embryogenesis.

The Enhancer of split transcription factor Her8a is a novel dimerisation partner for Her3 that controls anterior hindbrain neurogenesis in zebrafish.

BACKGROUND: Neurogenesis control and the prevention of premature differentiation in the vertebrate embryo are crucial processes, allowing the formation of late-born cell types and ensuring the correct shape and cytoarchitecture of the brain. Members of the Hairy/Enhancer of Split (Hairy/E(spl)) family of bHLH-Orange transcription factors, such as zebrafish Her3, 5, 9 and 11, are implicated in the local inhibition of neurogenesis to maintain progenitor pools within the early neural plate. To better understand how these factors exert their inhibitory function, we aimed to isolate some of their functional interactors. RESULTS: We used a yeast two-hybrid screen with Her5 as bait and recovered a novel zebrafish Hairy/E(spl) factor--Her8a. Using phylogenetic and synteny analyses, we demonstrate that her8a evolved from an ancient duplicate of Hes6 that was recently lost in the mammalian lineage. We show that her8a is expressed across the mid- and anterior hindbrain from the start of segmentation. Through knockdown and misexpression experiments, we demonstrate that Her8a is a negative regulator of neurogenesis and plays an essential role in generating progenitor pools within rhombomeres 2 and 4--a role resembling that of Her3. Her8a co-purifies with Her3, suggesting that Her8a-Her3 heterodimers may be relevant in this domain of the neural plate, where both proteins are co-expressed. Finally, we demonstrate that her8a expression is independent of Notch signaling at the early neural plate stage but that SoxB factors play a role in its expression, linking patterning information to neurogenesis control. Overall, the regulation and function of Her8a differ strikingly from those of its closest relative in other vertebrates--the Hes6-like proteins. CONCLUSIONS: Our results characterize the phylogeny, expression and functional interactions involving a new Her factor, Her8a, and highlight the complex interplay of E(spl) proteins that generates the neurogenesis pattern of the zebrafish early neural plate.

Her9 represses neurogenic fate downstream of Tbx1 and retinoic acid signaling in the inner ear.

Proper spatial control of neurogenesis in the inner ear ensures the precise innervation of mechanotransducing cells and the propagation of auditory and equilibrium stimuli to the brain. Members of the Hairy and enhancer of split (Hes) gene family regulate neurogenesis by inhibiting neuronal differentiation and maintaining neural stem cell pools in non-neurogenic zones. Remarkably, their role in the spatial control of neurogenesis in the ear is unknown. In this study, we identify her9, a zebrafish ortholog of Hes1, as a key gene in regulating otic neurogenesis through the definition of the posterolateral non-neurogenic field. First, her9 emerges as a novel otic patterning gene that represses proneural function and regulates the extent of the neurogenic domain. Second, we place Her9 downstream of Tbx1, linking these two families of transcription factors for the first time in the inner ear and suggesting that the reported role of Tbx1 in repressing neurogenesis is in part mediated by the bHLH transcriptional repressor Her9. Third, we have identified retinoic acid (RA) signaling as the upstream patterning signal of otic posterolateral genes such as tbx1 and her9. Finally, we show that at the level of the cranial otic field, opposing RA and Hedgehog signaling position the boundary between the neurogenic and non-neurogenic compartments. These findings permit modeling of the complex genetic cascade that underlies neural patterning of the otic vesicle.

Notch activity levels control the balance between quiescence and recruitment of adult neural stem cells.

The limited generation of neurons during adulthood is controlled by a balance between quiescence and recruitment of neural stem cells (NSCs). We use here the germinal zone of the zebrafish adult telencephalon to examine how the frequency of NSC divisions is regulated. We show, using several in vivo techniques, that progenitors transit back and forth between the quiescent and dividing state, according to varying levels of Notch activity: Notch induction drives progenitors into quiescence, whereas blocking Notch massively reinitiates NSC division and subsequent commitment toward becoming neurons. Notch activation appears predominantly triggered by newly recruited progenitors onto their neighbors, suggesting an involvement of Notch in a self-limiting mechanism, once neurogenesis is started. These results identify for the first time a lateral inhibition-like mechanism in the context of adult neurogenesis and suggest that the equilibrium between quiescence and neurogenesis in the adult brain is controlled by fluctuations of Notch activity, thereby regulating the amount of adult-born neurons.

MicroRNAs in brain development and physiology.

The crucial role of microRNAs (miRNAs) in brain development is demonstrated by the dramatic apoptotic phenotypes of mouse mutants abolishing all miRNAs synthesis. Recent advances in Caenorhabditis elegans, Drosophila, zebrafish and mouse moved beyond this global understanding by targeting selected miRNAs. They indicate that single miRNAs successively modulate all steps of brain maturation, including patterning, neurogenesis, neuronal differentiation and subtype specification, and neuronal activity. In detail, they reveal an amazing functional complexity: specific miRNAs can either be used reiteratively, such as miR-9 in patterning, neurogenesis and differentiation, or concomitantly target different mRNAs in distinct cellular compartments, such as Pumilio or Limk1 that can be inhibited by miR-134 to control neuritogenesis or spine growth, respectively. Their regulation can be composite, either through multiple loci (miR-9) or in blocks of numerous miRNAs (miR-134). A major step remains to decipher their impact on brain function, in particular in the control of behaviour.

Towards a synthetic view of axis specification mechanisms in vertebrates: insights from the dogfish.

The genetic mechanisms, which control axis specification, apparently extensively diverge across vertebrates. In amphibians and teleosts, they are tightly linked to the establishment of an early dorso-ventral polarity. This polarity has no equivalent in amniotes, which unlike the former, retain a considerable plasticity for their site of axis formation until blastula stages and rely on signals secreted by extra-embryonic tissues for the establishment of their early rostro-caudal pattern. In order to better understand the links between these seemingly highly divergent mechanisms, we have used an evo-devo approach, aimed at reconstructing the gnathostome ancestral state and focussed on a chondrichthyan, the dogfish Scyliorhinus canicula. A detailed molecular characterization of the dogfish embryo at blastula and gastrula stages highlights striking similarities with all vertebrate model organisms including amniotes. It suggests the presence in the dogfish of territories homologous to the hypoblast and extra-embryonic ectoderm of the latter, which may therefore reflect the primitive condition of jawed vertebrates. In the ancestral state, these territories are specified at opposite sides of an early axis of bilateral symmetry, homologous to the dorso-ventral axis of amphibians and teleosts, and aligned with the later forming embryonic axis, from head to tail. Amniotes have diverged from this pattern through a posterior expansion of extra-embryonic ectoderm, resulting in an apparently radial symmetry at late blastula stages. These data delineate the broad outlines of the gnathostome ancestral pattern of axis specification and highlight an unexpected unity of mechanisms across jawed vertebrates. They illustrate the complementarity of comparative and genetic approaches for a comprehensive view of developmental mechanisms themselves.