Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease

Regeneration Rosetta: An Interactive Web Application To Explore Regeneration-Associated Gene Expression and Chromatin Accessibility

Time-course high-throughput assays of gene expression and enhancer usage in zebrafish provide a valuable characterization of the dynamic mechanisms governing gene regulatory programs during CNS axon regeneration. To facilitate the exploration and functional interpretation of a set of fully-processed data on regeneration-associated temporal transcription networks, we have created an interactive web application called Regeneration Rosetta. Using either built-in or user-provided lists of genes in one of dozens of supported organisms, our web application facilitates the (1) visualization of clustered temporal expression trends; (2) identification of proximal and distal regions of accessible chromatin to expedite downstream motif analysis; and (3) description of enriched functional gene ontology categories. By enabling a straightforward interrogation of these rich data without extensive bioinformatic expertise, Regeneration Rosetta is broadly useful for both a deep investigation of time-dependent regulation during regeneration in zebrafish and hypothesis generation in other organisms

La visibilidad como factor en la relación copépodos-peces plantívoros

The question we treat in this contribution is whether or not planktivorous fish can use the motion pattern of planktonic copepods to distinguish these animals from other suspended particles in the water. A targeted overview of the predator-prey relationship is followed by a first report of experiments performed in our laboratory where fish selected between two virtual targets on a TV screen, each target showing a different swimming pattern. The results suggest that fish can perceive temporal visual patterns and select their preferred target after two to ten seconds observing the two moving targets. Implications of these preliminary results are discussed and hypotheses for further research formulated.En este trabajo hemos estudiado la capacidad de los peces planctívoros para detectar las trayectorias de los movimientos de los copépodos y para distinguirlas de trayectorias generadas por otras partículas. Una revisión de las relaciones depredador-presa precede la descripción de experimentos realizados en nuestro laboratorio. En estos experimentos el pez seleccionó entre dos trayectorias virtuales diferentes en una pantalla de televisión. Los resultados indican que el pez puede percibir visualmente tendencias temporales y seleccionar sus presas después de observar los movimientos de éstas durante unos segundos (2-10 s). En este trabajo se discute la implicación de estos estudios preliminares y se plantean hipótesis para futuras investigaciones

Cellular reprogramming for successful CNS axon regeneration is driven by a temporally changing cast of transcription factors

In contrast to mammals, adult fish display a remarkable ability to fully regenerate central nervous system (CNS) axons, enabling functional recovery from CNS injury. Both fish and mammals normally undergo a developmental downregulation of axon growth activity as neurons mature. Fish are able to undergo damage-induced "reprogramming" through re-expression of genes necessary for axon growth and guidance, however, the gene regulatory mechanisms remain unknown. Here we present the first comprehensive analysis of gene regulatory reprogramming in zebrafish retinal ganglion cells at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analyses reveal a regeneration program characterized by sequential activation of stage-specific pathways, regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Strikingly, we also find a discrete set of regulatory regions that change in accessibility, consistent with higher-order changes in chromatin organization that mark (1) the beginning of regenerative axon growth in the optic nerve, and (2) the re-establishment of synaptic connections in the brain. Together, these data provide valuable insight into the regulatory logic driving successful vertebrate CNS axon regeneration, revealing key gene regulatory candidates for therapeutic development

Establishment of a murine culture system for modeling the temporal progression of cranial and trunk neural crest cell differentiation

The neural crest (NC) is a transient population of embryonic progenitors that are implicated in a diverse range of congenital birth defects and pediatric syndromes. The broad spectrum of NC-related disorders can be attributed to the wide variety of differentiated cell types arising from the NC. In vitro models of NC development provide a powerful platform for testing the relative contributions of intrinsic and extrinsic factors mediating NC differentiation under normal and pathogenic conditions. Although differentiation is a dynamic process that unfolds over time, currently, there is no well-defined chronology that characterizes the in vitro progression of NC differentiation towards specific cell fates. In this study, we have optimized culture conditions for expansion of primary murine NC cells that give rise to both ectodermal and mesoectodermal derivatives, even after multiple passages. Significantly, we have delineated highly reproducible timelines that include distinct intermediate stages for lineage-specific NC differentiation in vitro. In addition, isolating both cranial and trunk NC cells from the same embryos enabled us to make direct comparisons between the two cell populations over the course of differentiation. Our results define characteristic changes in cell morphology and behavior that track the temporal progression of NC cells as they differentiate along the neuronal, glial and chondrogenic lineages in vitro. These benchmarks constitute a chronological baseline for assessing how genetic or environmental disruptions may facilitate or impede NC differentiation. Introducing a temporal dimension substantially increases the power of this platform for screening drugs or chemicals for developmental toxicity or therapeutic potential. This article has an associated First Person interview with the first author of the paper

MASH1/Ascl1a Leads to GAP43 Expression and Axon Regeneration in the Adult CNS

<div><p>Unlike CNS neurons in adult mammals, neurons in fish and embryonic mammals can regenerate their axons after injury. These divergent regenerative responses are in part mediated by the growth-associated expression of select transcription factors. The basic helix-loop-helix (bHLH) transcription factor, MASH1/Ascl1a, is transiently expressed during the development of many neuronal subtypes and regulates the expression of genes that mediate cell fate determination and differentiation. In the adult zebrafish (Danio rerio), Ascl1a is also transiently expressed in retinal ganglion cells (RGCs) that regenerate axons after optic nerve crush. Utilizing transgenic zebrafish with a 3.6 kb GAP43 promoter that drives expression of an enhanced green fluorescent protein (EGFP), we observed that knock-down of Ascl1a expression reduces both regenerative gap43 gene expression and axonal growth after injury compared to controls. In mammals, the development of noradrenergic brainstem neurons requires MASH1 expression. In contrast to zebrafish RGCs, however, MASH1 is not expressed in the mammalian brainstem after spinal cord injury (SCI). Therefore, we utilized adeno-associated viral (AAV) vectors to overexpress MASH1 in four month old rat (Rattus norvegicus) brainstem neurons in an attempt to promote axon regeneration after SCI. We discovered that after complete transection of the thoracic spinal cord and implantation of a Schwann cell bridge, animals that express MASH1 exhibit increased noradrenergic axon regeneration and improvement in hindlimb joint movements compared to controls. Together these data demonstrate that MASH1/Ascl1a is a fundamental regulator of axonal growth across vertebrates and can induce modifications to the intrinsic state of neurons to promote functional regeneration in response to CNS injury.</p></div

Rats treated with MASH1 exhibit increased regeneration of noradrenergic axons into the SC bridge.

<p>A, A low magnification image of a SC bridge from a MASH1 treated animal showing the rostral (left) and caudal (right) spinal cord/SC bridge interfaces delineated by GFAP-positive astrocytes (white). Note the long astrocyte processes (small arrowheads, A and B) that extend into the bridge. Hoechst-stained nuclei are blue and the large arrowhead indicates the polymer channel. Small holes (arrows) in the top of the channel were created to inject a fluid mixture of SCs and Matrigel (scale bar = 1 mm). Locations of higher magnification images taken on an adjacent section are indicated by letters (B-G). In treated animals, many beaded DβH-positive axons (red, arrows) regenerated 0.25 mm (B), 0.5 mm (C), 1.0 mm (D), 1.5 mm (E), 2.0 mm (F), and more than 2.5 mm (G), beyond the rostral spinal cord/SC bridge interface (scale bar = 20 μm). H, An illustration of the line-transect method of analysis, depicting DβH-positive axons (red) regenerating into a SC bridge. The polymer channel (thick black lines) and the transverse dorso-ventral lines used for quantification (thin purple lines) are diagrammed. The numbers represent mm from the rostral interface. I, The percentage of DβH-positive axons 10 mm rostral to the bridge that regenerated across the bridge was greater in MASH1 treated animals (n = 10) compared to control animals (n = 11; ** = p<0.01, two-way ANOVA; ++ = p<0.01 Bonferroni posttest).</p

Ascl1a is required for RGC axon regeneration in zebrafish.

<p>A-L, Cross sections through the retina focused on the retinal ganglion cell layer. Four days after optic nerve transection, zebrafish RGCs that received MOs (red, arrowheads) were retrogradely traced with dextran (green) 1 mm beyond the site of injury. Animals that received negative control MOs (A-D) were able to regenerate RGC axons as evident by the co-localization of MOs and tracer (yellow, arrow). In contrast few RGCs regenerated axons in animals that received MOs to knockdown GAP43 (E-H) or Ascl1a (I-L). DAPI stained nuclei are blue (scale bar = 10 μm). M, Quantification of the percentage of RGCs that received the MOs and were able to regenerate axons. Compared to controls (n = 6), those that received either GAP43 (n = 6) or Ascl1a (n = 6) MOs exhibited a reduced percentage of RGC axons to regenerate (*** = p<0.001 one-way ANOVA; +++ = p<0.001 Bonferroni posttest). Compared to animals that received GAP43 MOs, those that received Ascl1a MOs exhibited a greater reduction in the percentage of RGC axons to regenerate (*** = p<0.001, t-test).</p