Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of zebrafish retinal development and photoreceptor regeneration to investigate the roles of midkine-a and midkine-b

ABSTRACT: In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult MÃ¼ller glia. Recently, MÃ¼ller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here, we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are co-expressed in horizontal cells and proliferating MÃ¼ller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration. Experiment Overall Design: As a means to identify genes necessary for photoreceptor regeneration, we evaluated transcriptional in the retina of the zebrafish as photoreceptors are regenerated. Albino zebrafish were dark adapted, then half were exposed to bright constant light and half were returned to normal lighting. After 72hrs of light exposure, retinal RNA was isolated and analysis of gene expression was performed using Affymetriz zebrafish gene arrays.

ORGANISM(S): Danio rerio

SUBMITTER: Peter Hitchcock

PROVIDER: E-GEOD-13999 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: MÃ¼ller glia are the only glial cell type produced by the neuroepithelial progenitor cells which generate the vertebrate retina. MÃ¼ller glia are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, they function as an adult stem cell, mediating retinal regeneration among select vertebrate classes. However, the mechanisms which regulate MÃ¼ller development are poorly understood as considerable overlap exists in gene expression between retinal progenitor cells and differentiated MÃ¼ller glia. We investigate the functional role of the LIM homeodomain transcription factor Lhx2 in the specification and development of MÃ¼ller glia in the mouse. Methods: RNA-Seq was performed in collaboration with the Johns Hopkins School of Medicine Deep Sequencing and Microarray Core Facility. Libraries were prepared using Illumina TruSeq RNA Sample kit (Illumina, San Diego, CA) following manufacturerâs recommended procedure. The PCR amplified library was purified using RNAClean XP magnetic beads (Agencourt, Beverley, MA) and run out on a High Sensitivity DNA Chip (Agilent, Santa Clara, CA) for quality check. We used STAR to align RNA-Seq reads onto Ensembl mouse genome GRCm38, release 72. To generate the stand attribute for alignments containing splice junctions, we used the outSAMstrandField intronMotif program. The spliced alignments without strand definition were removed. Number of reads mapped to exons was counted by htseq-count. Genes expressed at very low levels were omitted from further analysis. Gene expression differences between wildtype and mutant samples, significance (p-value) and false discovery rate (FDR) were computed using the generalized linear models based EdgeR. Results: We observed a substantial reduction in expression of Notch pathway genes including Notch1, the Notch ligands Dll1 and Dll3, as well as gliogenic Notch effector genes such as Hes1, Hes5, Id1 and Sox8 and the MÃ¼ller-gliogenic factor Rax. We likewise observe a substantial reduction in expression of progenitor-specific genes such as Vsx2 and Fgf15. Furthermore, we observed a decrease in the expression of early-onset glial markers such as Crym , Spon1, and Car2. Retinal mRNA profiles of post-natal day 0.5 (P0.5) Lhx2 wild type (N=3) and Lhx2lox/lox; PdgfrÎ±-Cre ÎcKO (N=3) mice were generated using Illumina TruSeq and analyzed with Agilent high sensitivity DNA analsis kit.

Project description:Loss of neurons in the neural retina is a leading cause of vision loss. Retinal regeneration in zebrafish is attributed to Müller glia, which are activated, adopt a stem cell-like state, proliferate, and generate new neurons. Despite the same endogenous Müller glia being present in all vertebrates including humans, Müller glia activation in mammals leads to glial scarring instead of neurogenesis. Therefore, understanding Müller glia cellular states and molecular processes during zebrafish regeneration may allow us to improve mammalian regeneration. Since only a subset of Müller glia activate following neuron loss, we asked whether a specialised subset of Müller glia might be genetically primed for activation. Single-cell RNA sequencing (sc-RNAseq) from integrated zebrafish Müller glia samples reveals heterogeneity of gene expression across the quiescent Müller glia pool with 4 main clusters emerging, two of which are genetically similar. Labelling for key differentially expressed markers identified that these main clusters correlated with glia spatially distributed across dorsal, central and ventral retina. Using a genetically driven, chemically induced nitroreductase approach, three distinct photoreceptor types were ablated: the long (Lws2), short wavelength-sensitive (Sws2) and rod (Xops) photoreceptors, which also differ in their abundance. As expected, histological analysis of the three injuries and sc-RNAseq data for the two cone photoreceptor ablations identified common genetic program involved in the transition of Müller glia quiescence to activation, and differential responses to injury related to either injury extent or subtype of photoreceptor targeted. Interestingly, despite differences in the distributed across the retina, biased activation of Müller glia was observed in dorsal and central regions. Gene ontology analysis revealed that these injury-responsive dorsal and central Müller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Müller glia upregulated genes related to homeostasis as well as certain AP-1 and injury-responsive transcription factors, followed by expression of genes involved in cell cycle, chromatin remodeling, and microtubule organisation. Prior to cell cycle entry, Müller glia show a transient upregulation of genes involved in gliogenesis, and Notch signalling. These findings enhance our understanding of heterogeneous states of quiescent Müller glia, and how these differences relate to activation and regeneration potential following neural ablation. A comparison of the distinct quiescent Müller glia with glia states in mammals will reveal key molecular pathways that could be targeted for improved mammalian neural regeneration.

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Transcription profiling of zebrafish retinal development and photoreceptor regeneration to investigate the roles of midkine-a and midkine-b

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