Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

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.

Actomyosin is the main driver of interkinetic nuclear migration in the retina.

Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.

From progenitors to differentiated cells in the vertebrate retina.

Multipotent retinal progenitors undergo a varied number of divisions to produce clones of heterogeneous sizes and cell types. We describe the transition from a proliferating progenitor to a differentiated postmitotic cell and discuss how controls of proliferation operate within individual cells as well as in the whole tissue. We discuss how extracellular and intracellular signaling, transcriptional regulation, cell cycle kinetics, interkinetic nuclear migration, orientation of cell division, and epigenetic modifications all interact to regulate a progenitor's transition from division to differentiation. We also propose some directions for future research.

Single-molecule analysis of endogenous ß-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons.

During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that ß-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that ß-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate ß-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.

Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

Genetic control of cellular morphogenesis in Müller glia.

Cell shape is critical for the proper function of every cell in every tissue in the body. This is especially true for the highly morphologically diverse neural and glia cells of the central nervous system. The molecular processes by which these, or indeed any, cells gain their particular cell-specific morphology remain largely unexplored. To identify the genes involved in the morphogenesis of the principal glial cell type in the vertebrate retina, the Müller glia (MG), we used genomic and CRISPR based strategies in zebrafish (Danio rerio). We identified 41 genes involved in various aspects of MG cell morphogenesis and revealed a striking concordance between the sequential steps of anatomical feature addition and the expression of cohorts of functionally related genes that regulate these steps. We noted that the many of the genes preferentially expressed in zebrafish MG showed conservation in glia across species suggesting evolutionarily conserved glial developmental pathways.

The ciliary marginal zone of the zebrafish retina: clonal and time-lapse analysis of a continuously growing tissue.

Clonal analysis is helping us understand the dynamics of cell replacement in homeostatic adult tissues (Simons and Clevers, 2011). Such an analysis, however, has not yet been achieved for continuously growing adult tissues, but is essential if we wish to understand the architecture of adult organs. The retinas of lower vertebrates grow throughout life from retinal stem cells (RSCs) and retinal progenitor cells (RPCs) at the rim of the retina, called the ciliary marginal zone (CMZ). Here, we show that RSCs reside in a niche at the extreme periphery of the CMZ and divide asymmetrically along a radial (peripheral to central) axis, leaving one daughter in the peripheral RSC niche and the other more central where it becomes an RPC. We also show that RPCs of the CMZ have clonal sizes and compositions that are statistically similar to progenitor cells of the embryonic retina and fit the same stochastic model of proliferation. These results link embryonic and postembryonic cell behaviour, and help to explain the constancy of tissue architecture that has been generated over a lifetime.

Inhibitory neuron migration and IPL formation in the developing zebrafish retina.

The mature vertebrate retina is a highly ordered neuronal network of cell bodies and synaptic neuropils arranged in distinct layers. Little, however, is known about the emergence of this spatial arrangement. Here, we investigate how the three main types of retinal inhibitory neuron (RIN)--horizontal cells (HCs), inner nuclear layer amacrine cells (iACs) and displaced amacrine cells (dACs)--reach their specific laminar positions during development. Using in vivo time-lapse imaging of zebrafish retinas, we show that RINs undergo distinct phases of migration. The first phase, common to all RINs, is bipolar migration directed towards the apicobasal centre of the retina. All RINs then transition to a less directionally persistent multipolar phase of migration. Finally, HCs, iACs and dACs each undergo cell type-specific migration. In contrast to current hypotheses, we find that most dACs send processes into the forming inner plexiform layer (IPL) before migrating through it and inverting their polarity. By imaging and quantifying the dynamics of HCs, iACs and dACs from birth to final position, this study thus provides evidence for distinct and new migration patterns during retinal lamination and insights into the initiation of IPL formation.

Hermes Regulates Axon Sorting in the Optic Tract by Post-Trancriptional Regulation of Neuropilin 1.

The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1. SIGNIFICANCE STATEMENT: An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting.

A nutrient-sensitive restriction point is active during retinal progenitor cell differentiation.

In many growing tissues, slowly dividing stem cells give rise to rapidly proliferating progenitors that eventually exit the cell cycle and differentiate. Growth rates are limited by nutrient availability, but it is unclear which steps of the proliferation-differentiation programme are particularly sensitive to fuel supplies. We examined how nutrient deprivation (ND) affects stem and progenitor cells in the ciliary marginal zone (CMZ) of the amphibian retina, a well-characterised neurogenic niche. We show that ND specifically blocks the proliferation and differentiation of progenitor cells through an mTOR-mediated mechanism. By contrast, the identity and proliferation of retinal stem cells are insensitive to ND and mTOR inhibition. Re-feeding starved retinas in vitro rescues both proliferation and differentiation, and activation of mTOR is sufficient to stimulate differentiation even in ND retinas. These results suggest that an mTOR-mediated restriction point operates in vivo to couple nutrient abundance to the proliferation and differentiation programme in retinal progenitor cells.

Spectrum of Fates: a new approach to the study of the developing zebrafish retina.

The ability to image cells live and in situ as they proliferate and differentiate has proved to be an invaluable asset to biologists investigating developmental processes. Here, we describe a Spectrum of Fates approach that allows the identification of all the major neuronal subtypes in the zebrafish retina simultaneously. Spectrum of Fates is based on the combinatorial expression of differently coloured fluorescent proteins driven by the promoters of transcription factors that are expressed in overlapping subsets of retinal neurons. Here, we show how a Spectrum of Fates approach can be used to assess various aspects of neural development, such as developmental waves of differentiation, neuropil development, lineage tracing and hierarchies of fates in the developing zebrafish retina.

Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.

Zic-associated holoprosencephaly: zebrafish Zic1 controls midline formation and forebrain patterning by regulating Nodal, Hedgehog, and retinoic acid signaling.

Holoprosencephaly (HPE) is the most frequently observed human embryonic forebrain defect. Recent evidence indicates that the two major forms of HPE, classic HPE and midline interhemispheric (MIH) HPE, are elicited by two different mechanisms. The only gene known to be associated with both forms of HPE is Zic2. We used the zebrafish Danio rerio as a model system to study Zic knockdown during midline formation by looking at the close homolog Zic1, which is expressed in an overlapping fashion with Zic2. Zic1 knockdown in zebrafish leads to a strong midline defect including partial cyclopia due to attenuated Nodal and Hedgehog signaling in the anterior ventral diencephalon. Strikingly, we were able to show that Zic1 is also required for maintaining early forebrain expression of the retinoic acid (RA)-degrading enzyme cyp26a1. Zic1 LOF leads to increased RA levels in the forebrain, subsequent ventralization of the optic vesicle and down-regulation of genes involved in dorsal BMP signaling. Repression of BMP signaling in dorsal forebrain has been implicated in causing MIH HPE. This work provides a mechanistical explanation at the molecular level of why Zic factors are associated with both major forms of HPE.

RNA-binding protein Hermes/RBPMS inversely affects synapse density and axon arbor formation in retinal ganglion cells in vivo.

The RNA-binding protein Hermes [RNA-binding protein with multiple splicing (RBPMS)] is expressed exclusively in retinal ganglion cells (RGCs) in the CNS, but its function in these cells is not known. Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons. Hermes loss of function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum, and expression of a dominant acting mutant Hermes protein, defective in RNA-granule localization, causes similar defects in arborization. Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability. Surprisingly, Hermes depletion also leads to enhanced early visual behavior and an increase in the density of presynaptic puncta, suggesting that reduced arborization is accompanied by increased synaptogenesis to maintain synapse number.

Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia.

Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.

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.

Retinoic acid receptor signaling regulates choroid fissure closure through independent mechanisms in the ventral optic cup and periocular mesenchyme.

Retinoic acid receptor (RAR) signaling is required for morphogenesis of the ventral optic cup and closure of the choroid fissure, but the mechanisms by which this pathway regulates ventral eye development remain controversial and poorly understood. Although previous studies have implicated neural crest-derived periocular mesenchyme (POM) as the critical target of RA action in the eye, we show here that RAR signaling regulates choroid fissure closure in zebrafish by acting on both the ventral optic cup and the POM. We describe RAR-dependent regulation of eight genes in the neuroepithelial cells of the ventral retina and optic stalk and of six genes in the POM and show that these ventral retina/optic stalk and POM genes function independently of each other. Consequently, RAR signaling regulates ventral eye development through two independent, nonredundant mechanisms in different ocular tissues. Furthermore, the identification of two cohorts of genes implicated in ventral eye morphogenesis may help to elucidate the genetic basis of ocular coloboma in humans.

Reliability of the 2021 National Youth Risk Behavior Survey Questionnaire.

PURPOSE: The Youth Risk Behavior Survey (YRBS) monitors behaviors, experiences, and conditions affecting the health of high school students nationwide. This study examined the test-retest reliability of the 2021 national YRBS questionnaire. DESIGN: Respondents completed a Time 1 and Time 2 paper-and-pencil questionnaire approximately 2 weeks apart during February to May 2022. Data were linked in such a way as to preserve anonymity. SETTING: Convenience sample of high schools. SUBJECTS: High school students (N = 588). MEASURES: Health risk behaviors and experiences assessed on the 2021 national YRBS questionnaire. ANALYSIS: Time 1 and Time 2 responses were compared for each questionnaire item using the McNemar's test. Then, Cohen's kappa coefficients tested the agreement between Time 1 and Time 2 responses overall, and by sex, grade, and Black, White, and Hispanic race and ethnicity. RESULTS: Among the 74 items analyzed, 96% had at least moderate reliability, and 73% had substantial or almost perfect reliability. The mean Cohen's kappa was .68. McNemar's test findings showed Time 1 and Time 2 data significantly differed (P < .01) for 9 items (12%). CONCLUSION: Reliable health behavior measures are important in the development of youth-focused public health programs and policies. Findings suggest the national YRBS questionnaire is a reliable instrument. Such findings lend support to relying on adolescent self-reported data when monitoring health behaviors using the YRBS.

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.