The indispensable role of autophagy in lens morphogenesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA599401 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10843727_1.fastq.gz | Fastqsanger.gz | |||
| SRR10843727_2.fastq.gz | Fastqsanger.gz | |||
| SRR10843728_1.fastq.gz | Fastqsanger.gz | |||
| SRR10843728_2.fastq.gz | Fastqsanger.gz | |||
| SRR10843729_1.fastq.gz | Fastqsanger.gz |
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Project description:FYVE and coiled-coil domain containing 1 (FYCO1) is expressed ubiquitously and is required for microtubule-dependent, plus-end-directed transport of autophagic vesicles. We previously reported loss of function mutations in FYCO1 responsible for cataractogenesis with no other ocular and/or extra-ocular phenotype. Here, we show that Fyco1-/- lenses recapitulated the cataract phenotype and exhibited diminished autophagy, consistent with a critical role of Fyco1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many differentially expressed, autophagy-related, genes, proteins, and lipids, respectively in Fyco1-/- lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial (HLE) cell line confirmed reduced amounts of membrane-bound LC3B-II and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed persistent organelle mass in Fyco1-/-lenses and FYCO1 KI lens-like organoid structures, which were further confirmed through western blot analysis. In summary, our data show that the absence of FYCO1 causes diminished autophagy, delayed organelle degradation, and cataractogenesis. These data support the model of FYCO1 supplementing basal autophagy in the lens to meet the physiological necessity of high autophagy levels required during lens fiber cell differentiation and lack thereof results in delayed organelle removal.
2023-01-02 | GSE143251 | GEO
Project description:FYVE and coiled-coil domain containing 1 (FYCO1) is expressed ubiquitously and is required for microtubule-dependent, plus-end-directed transport of autophagic vesicles. We previously reported the loss of function mutations in FYCO1 responsible for cataractogenesis with no other ocular and/or extra-ocular phenotype. Here, we show that Fyco1-/- lenses recapitulated the cataract phenotype and exhibited diminished autophagy, consistent with a critical role of Fyco1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many differentially expressed, autophagy-related, genes, proteins, and lipids, respectively in Fyco1-/- lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial (HLE) cell line confirmed reduced amounts of membrane-bound LC3B-II and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed persistent organelle mass in Fyco1-/- lenses and FYCO1 KI lens-like organoid structures, which were further confirmed through western blot analysis. In summary, our data show that the absence of FYCO1 causes diminished autophagy, delayed organelle degradation, and cataractogenesis. These data support the model of FYCO1 supplementing basal autophagy in the lens to meet the physiological necessity of high autophagy levels required during lens fiber cell differentiation and lack thereof results in delayed organelle removal.
2023-05-10 | PXD017001 | Pride
Project description:<p>FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in <em>FYCO1</em> cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show <em>fyco1</em> homozygous knockout (<em>fyco1</em><sup><em>-/-</em></sup>) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in <em>fyco1</em><sup><em>-/-</em></sup> mice lenses. Flow cytometry of <em>FYCO1</em> (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in <em>FYCO1</em> (c.2206C>T) KI lens-like organoid structures and in <em>fyco1</em><sup><em>-/-</em></sup> mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.</p>
2024-03-24 | MTBLS206 | MetaboLights
Project description:This study examined the effects of genetic knockdown of autophagy genes on vertebrate cardiac development We performed microarray studies comparing the hearts of control zebrafish embryos to the hearts of embryos with decreased expression of the autophagy genes atg5, becn1 or atg7. The results provide insight into the role of autophagy in developmental morphogenesis. Hearts were purified from 3 day-old zebrafish embryos injected with control or autophagy gene-specific morpholino oligonucleotides. RNA was prepared from all samples and hybridized to zebrafish-specific Affymetricx arrays.
2014-03-01 | E-GEOD-51541 | biostudies-arrayexpress
Project description:This study examined the effects of genetic knockdown of autophagy genes on vertebrate cardiac development We performed microarray studies comparing the hearts of control zebrafish embryos to the hearts of embryos with decreased expression of the autophagy genes atg5, becn1 or atg7. The results provide insight into the role of autophagy in developmental morphogenesis.
2014-03-01 | GSE51541 | GEO
Project description:Autophagy-lysosomal degradation is an evolutionarily conserved process key to cellular homeostasis, differentiation, and stress survival, which is particularly important to the pathophysiology of the cardiovascular system. What is more, both experimental and clinical observations indicate that autophagy-lysosomal degradation affects correct cardiac morphogenesis, and in particular valve development. However, it is still unclear which cells upregulate autophagy-lysosomal degradation and for which specific cellular processes it is required. Here, we introduce novel zebrafish transgenic models to visualize autophagosomes and lysosomes in vivo and to follow their temporal and cellular localization in the larval heart. This allowed us to determine the kinetics of autophagosome and lysosome vesicle formation and to observe significant accumulation of lysosomal vesicles during the development of the atrioventricular and bulboventricular valves. We then addressed the functional role of lysosomal degradation in cardiovascular development using a spns1 mutant as a zebrafish model of lysosomal impairment. We found that spns1 mutants displayed morphologically and functionally abnormal heart development, including abnormal endocardial organization, impaired cardiac valve formation and high incidence of retrograde blood flow. Single-nuclear transcriptome analysis revealed endocardial-specific differences in the expression of lysosome-related genes and alterations of notch1 signaling in the mutant larval heart. Further, endocardial-specific overexpression of spns1 and notch1 rescued features of valve formation and function as well as overall cardiac morphogenesis. Altogether, our study provides an improved description of the autophagy and lysosomal events that take place during zebrafish heart development and reveals a cell-autonomous role of lysosomal processing during cardiac valve formation upstream of notch1 signaling.
2024-10-25 | GSE246850 | GEO
Project description:We report the application of RNA-sequencing technology for the high-throughput profiling of mammalian lens gene expression at embryonic day 15.5. The lens has a particularly biased transcriptome, with the top 50 genes encoding approximately 90% of the protein content. Using RNA-Seq, we have shown that there are over 7,700 genes being expressed in the lens at embryonic day 15.5. As expected, the crystallins and many structural genes were amoung the most highly expressed; however, numerous genes expressed at lower transcript abundance were also identified. This study provides a framework for the application of RNA-Seq technology towards characterization of the mammalian lens transcriptome during development. Using high-throughput RNA-sequencing, gene expression in the mammalian lens was compared between an inbred C56Bl/6<har> strain and a mix background strain at E15.5. This analysis identifies almost 2,000 genes being differentially expressed between the inbred and mixed background lenses, ranging from 6.5 fold upregulated to 5.2 fold downregulated in the mixed background compared to the inbred strain. This list does not include unknown/predicted genes or pseudogenes which are known to change between strains. Further, it appears that approximately 98% of these genes are altered at levels less than 2.5 fold. This study therefore provides a fold change threshold cutoff (2.5 fold) to use in the analysis of differentially expressed lens genes at E15.5 using RNA-Seq technology as it takes into account genetic variation due to background strain differences. SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases where it functionally links TGFb signaling to the loss of epithelial preferred gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes involved in EMT/cancer. Furthermore, 34% of the upregulated genes in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes downregulated in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts. RNA-Seq of inbred background wild type lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and inbred wild type (C57Bl/6<har>) lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and Sip1 conditional knockout lenses at E15.5
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Project description:Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors has not been previously investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, and cataracts. Notch2 mutants also had a persistent lens stalk phenotype at E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2) and Trp63 (p63) that negatively regulates Wnt signaling. Although removal of Notch2 phenocopied the increased proportion of fiber cells of Rbpj and Jag1 conditional mutant lenses, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. Instead, we found that the Notch2 normally blocks progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. We have compared gene expression of ocular lenses of mice that are lens specific conditional mutants of Notch2 gene to that of littermate controls that had no ablation of Notch2 gene in the lens. Two lenses of each of the three conditional mutants and controls were pooled together and total RNA was harvested from embryonic day 19.5 (E19.5) lenses. Gene expression changes caused by absence of Notch2 gene in the lens were analyzed.
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Project description:Genome-wide approach to identify the cell-autonomous role of Brg1 in lens fiber cell terminal differentiation. To examine roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific alphaA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in E15.5 embryonic wild type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous, and Hsf4 homozygous lenses identified multiple genes co-regulated by Brg1, Hsf4 and Pax6. Among them DNase IIbeta, a key enzyme required for lens fiber cell denucleation, was found downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation and indirectly for retinal development but was not essential for lens lineage formation. Wild type and dnBrg1 transgenic lenses, 4 biological replicates each
2011-01-01 | E-GEOD-22322 | biostudies-arrayexpress
Project description:The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that require a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected over 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, over 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic, autophagy, and mitophagy transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells. Differentiation-state transcriptional analysis of embryonic chicken lenses was performed following microdissection of 100 embryonic day 13 (E13) chicken lenses into four distinct regions that represent a continuum of lens cell differentiation states: lens central epithelium (EC), equatorial epithelium (EQ), cortical fibers (FP), and central fibers (FC). Further analysis of the transcriptional content of biologically replicate samples was performed by Illumina directional mRNA sequencing and resulting reads mapped by TopHat and assembled with Cufflinks.
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