Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens - PubMed (original) (raw)

Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens

Daniel Chauss et al. G3 (Bethesda). 2014.

Abstract

The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires 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 more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 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 (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) 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.

Keywords: RNA sequencing; differentiation; eye; lens; mitochondria; mitochondrial dynamics; mitophagy.

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Figures

Figure 1

Lens microdissection, RNA isolation, RNA-sequencing, and data analysis. Lens microdissections were performed as described by Walker and Menko (1999). Two sets (N = 2) of pooled (n = 100) lens differentiation-state specific fractions were subjected to RNA isolation using Trizol and Illumina directional mRNAseq library preparation performed. High-throughput sequencing using the Illumina GAIIx platform with 35-bp unidirectional reads generated millions of sequencing reads that were processed, aligned by Tophat to Galgal4 and biological replicate based statistical modeling and transcript abundance and identity assembled by cufflinks using the maximum transcript abundance likelihood estimate model described by Trapnell et al. (2010). Statistical testing was performed using cuffdiff pairwise statistical gene expression analysis. CummeRbund was used to statistically assess data and literature searches were performed to verify expression results. Ontologically based pathway analysis was performed using DAVID and GenoMatix software packages. Gene clustering was performed that placed mitochondrial-associated transcripts into mitochondrial regulation, biogenesis, homeostasis or degradation functional clusters.

Figure 2

Identification of differentially expressed transcripts. RNA sequencing of microdissected E13 embryonic chicken lenses revealed the expression of more than 16,000 genes, with 3000 (false discovery rate adjusted P < 0.05, termed q < 0.05) genes displaying differential expression between lens cell differentiation-state specific zones (lens central epithelium [EC], equatorial epithelium [EQ], cortical fibers [FP], and central fibers [FC]). (A) Reads generated by Illumina mRNA-sequencing and mapping of reads to the Galgal4 chicken genome (ENSEMBLE). (B) Boxplot analysis of the sum estimate abundance (FPKM) between samples to demonstrate sample skew revealed little to no skew between samples. (C) Differential expression analysis shows differentially expressed genes between each embryonic lens region in pairwise comparison as demonstrated by volcano plot analysis (red indicates differentially expressed). (D) Sum of nonunique differentially expressed gene-specific transcripts between lens differentiation state−specific zones (

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, and

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). FPKM, fragments per kilobase of exon per million fragments mapped.

Figure 3

Transcript comparison to western blot protein level and literature based comparison of select canonical lens development pathways. Transcript levels of (A) beaded filament structural protein 1, filensin (BFSP1) (Ireland et al. 2000, Perng et al. 2007), beaded filament structural protein 2, phakinin (BFSP2/CP49) (Ireland et al. 2000, Perng et al. 2007), and (C) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were compared with protein levels (B, D) determined by western analysis. (E−J) Well-studied lens protein encoding transcripts were examined to assess agreement of E13 spatial RNAseq expression data with previously reported expression patterns in lens differentiation-state specific regions. These transcripts included (E) select lens crystallins encoding transcripts δ−crystallin (CRYD1/ASL1) (Gunhaga 2011), αA-crystallin (CRYAA) (Hawse et al. 2005), and αB-crystallin (Hawse et al. 2005); (F) actin-capping regulator encoding transcript tropomodulin 1 (TMOD1) (Nowak and Fowler 2012); (G) Lensgin (GLULD1/LGSN) (Wyatt et al. 2008); (H) cell-cycle regulator encoding transcript cyclin-dependent kinase 2 (CDK2) (Gao et al. 1999); (I) lens signaling encoding transcripts coiled-coil domain containing 80 (CCDC80/EQUARIN) (Song et al. 2012), EPH receptor type A2 (EPHA2) (Shi et al. 2012; Cheng et al. 2013), fibroblast growth factor receptor 2 (FGFR2) (Robinson 2006), and frizzled class receptor 3 (FZD3) (Dawes et al. 2013); and (J) lens DNA binding encoding transcripts paired box 6 (PAX6) (Cvekl and Piatigorsky 1996), heat shock transcription factor 4 (HSF4) (Fujimoto et al. 2004; Somasundaram and Bhat 2004), SRY (sex determining region Y)-box 2 (SOX2) (Kondoh et al. 2004), prospero homeobox 1 (PROX1) (Duncan et al. 2002), and GATA binding protein 3 (GATA3) (Maeda et al. 2009).

Figure 4

Detected polyadenylated mitochondrial-encoded transcripts display an approximate decrease in expression during lens epithelial to fiber cell differentiation. (A) Detected mitochondrial transcripts (Mt) display an approximate linear decrease in expression during lens fiber cell differentiation. Transcript expression levels of nuclear encoded inner and outer mitochondrial membrane proteins succinate dehydrogenase complex, subunit A (SDHA) and translocase of the outer mitochondrial membrane 20 homolog (TOMM20) display largely unaltered transcript levels (B) with decreased in protein levels detected by western blotting (C) as lens cell differentiation proceeded. (D) TOMM20 (red) immunofluorescence analysis of differentiating lens fiber cells in sections of the embryonic chicken lens at E10, E13, and E15. Results showed largely decreased TOMM20 levels at E13 compared with E10 that expanded at E15 and preceded the loss of nuclei (blue). Scale bars: 20 μm.

Figure 5

Select mitochondrial regulation, mitophagy, and macroautophagy regulatory protein comparison with transcript levels by western blot and immunofluorescent analysis. Protein levels determined by western analysis were compared with respective transcript levels for mitochondrial fusion transcripts encoding proteins mitofusin-1 and mitofusin-2 (MFN-1, -2) (A, B); mitophagy transcripts encoding proteins Parkin E3 ubiquitin ligase (PARK2/Parkin) (C, D) and BCL2/adenovirus E1B 19kDa interacting protein 3-like (BNIP3L/NIX) (E, F); and macroautophagy regulation transcripts encoding proteins (G, H) beclin 1, autophagy related (BECN1), RB1-inducible coiled-coil 1 (RB1CC1), FYVE and coiled-coil domain containing 1 (FYCO1), mechanistic target of rapamycin (serine/threonine kinase) (mTOR), RAB9A, member RAS oncogene family (RAB9A), GABA(A) receptor-associated protein-like 2 (GABARAPL2). Cryosections from embryonic E10, E13, and E15 chicken lenses were immunostained for MFN2 (I, Red) or GABARAPL2/GATE16 (J, red) (nuclei, blue) and analyzed by confocal microscopy. Scale bars: 20 μm.

Figure 6

Identification of nuclear transcribed mitochondrial protein encoding transcripts. Our analysis revealed the expression of more than 650 nuclear transcribed mitochondrial protein encoding transcripts with 94 transcripts preferred to the lens epithelia and 86 transcripts preferred to the lens fibers. Transcripts encoding proteins involved in mitochondrial based apoptotic induction (BID, APAF1) demonstrated high levels of expression in the lens epithelium. Transcripts encoding proteins involved in mitochondrial immobilization (SNPH), respiratory chain complex inhibition (DNAJC15), mitochondrial fragmentation (DNAJA3) and mitochondrial repair and protection (GLRX, MSRA) demonstrated high levels of expression in differentiating lens fiber cells. Direct comparisons of central epithelium to equatorial epithelium or equatorial epithelium to peripheral fibers for this analysis are presented as supplementary (

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, and

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Figure 7

Fold change analysis of detected nuclear encoded mitochondrial-associated degradation and regulation pathways between EC to EQ and EQ to FP transitions. Transcripts encoding proteins involved in mitophagy, autophagy, lysosomal biogenesis, mitochondrial biogenesis, mitochondrial repair/protection, the ubiquitin-proteasome system or heat shock proteins 70/40/27/22 family were compared for the (A) EC to EQ transition and the (B) EQ to FP transition. The FPKMs generated for all genes from these categories are provided as supplementary (

File S3

). Fold change (Δ) was calculated as discussed in the methods. EC, lens central epithelium; EQ, equatorial epithelium; FP, cortical fibers; FPKM, fragments per kilobase of exon per million fragments mapped.

Figure 8

Novel and noteworthy identified mitochondrial regulators between specific lens subregions. (A) A schematic diagram of mitochondrial distribution between the EC, EQ, FP, and FC regions of the day 13 embryonic chicken lens showing active mitochondrial populations and elimination of mitochondria during lens fiber cell maturation. (B) Mitochondrial regulatory transcripts exhibiting increased (+) or decreased (−) levels of expression between indicated lens subregions. (C) Mitochondrial repair and protection transcripts exhibiting increased (+) or decreased (−) levels of expression between indicated lens subregions. (D) Mitophagy transcripts exhibiting increased (+) or decreased (−) levels of expression between indicated lens subregions. (E) Macroautophagy transcripts exhibiting increased (+) or decreased (−) levels of expression between indicated lens sub-regions. APG, autophagic vesicle; EC, central lens epithelium; EQ, equatorial lens epithelium; FP, cortical lens fibers; FC, central lens fibers; LIR, LC3-interacting region; MT, mitochondria(l); OFZ, organelle-free zone; OMM, outer mitochondrial membrane; Pi’s, phosphorylates; ψMT, mitochondrial transmembrane potential.

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Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens - PubMed (original) (raw)