Amplification and sequencing of mature microRNAs in uncharacterized animal models using stem-loop reverse transcription-polymerase chain reaction

Expression of mature microRNA (miRNA) transcripts can be easily measured in many established animal model systems but is difficult to evaluate using conventional methods in new and uncharacterized animal models. In this study, we were able to expand an existing protocol to evaluate miRNA expression in both vertebrate and invertebrate animals for which mature miRNAs remain unsequenced. This method allows the researcher to sequence reverse transcription-polymerase chain reaction products, validating miRNA-specific amplification and providing the opportunity to add to the current body of knowledge of miRNA annotation

MicroRNA Regulation in Extreme Environments: Differential Expression of MicroRNAs in the Intertidal Snail Littorina littorea During Extended Periods of Freezing and Anoxia

Several recent studies of vertebrate adaptation to environmental stress have suggested roles for microRNAs (miRNAs) in regulating global suppression of protein synthesis and/or restructuring protein expression patterns. The present study is the first to characterize stress-responsive alterations in the expression of miRNAs during natural freezing or anoxia exposures in an invertebrate species, the intertidal gastropod Littorina littorea. These snails are exposed to anoxia and freezing conditions as their environment constantly fluctuates on both a tidal and seasonal basis. The expression of selected miRNAs that are known to influence the cell cycle, cellular signaling pathways, carbohydrate metabolism and apoptosis was evaluated using RT-PCR. Compared to controls, significant changes in expression were observed for miR-1a-1, miR-34a and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-125b, miR-29b and miR-2a in foot muscle after freezing exposure at -6. °C for 24. h (P<0.05). In addition, in response to anoxia stress for 24. h, significant changes in expression were also observed for miR-1a-1, miR-210 and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-29b and miR-2a in foot muscle (P<0.05). Moreover, protein expression of Dicer, an enzyme responsible for mature microRNA processing, was increased in foot muscle during freezing and anoxia and in hepatopancreas during freezing. Alterations in expression of these miRNAs in L. littorea tissues may contribute to organismal survival under freezing and anoxia

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

<div><p>Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and <i>dystonia musculorum</i> in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of <i>dystonia musculorum</i>, <i>Dst</i>^{<i>dt-27J</i>}, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of <i>Dst</i>^{<i>dt-27J</i>} oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between <i>Dst</i>^{<i>dt-27J</i>} and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that <i>Dst</i>^{<i>dt-27J</i>} oligodendrocyte progenitor cells were able to migrate normally. Finally, <i>in vivo</i> analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of <i>Dst</i>^{<i>dt-27J</i>} optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.</p></div

<i>Dst</i>^{<i>dt-27J</i>} OLs exhibit normal molecular differentiation.

<p>A. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing maturation marker expression at DD3. B. Quantification of the proportion of NG2⁺, MAG⁺/MBP^-, and MAG⁺/MBP⁺ OLs at DD3. C. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing maturation marker expression at DD6. D. Quantification of the proportion of NG2⁺, MAG⁺/MBP^-, and MAG⁺/MBP⁺ OLs at DD6. B, D: n = 3; all comparisons non-significant by two-tailed Student’s t-test. Data represent mean ± SEM. Arrowheads: yellow = NG2⁺, orange = MAG⁺/MBP^-, grey = MAG⁺/MBP⁺, white = contaminating cell. Scale bars = 50 μm.</p

Apoptosis is not increased in <i>Dst</i>^{<i>dt-27J</i>} OLs.

<p>A. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing colocalization of nuclear CC3 and Olig2 in apoptotic OLs at DD3. B. Quantification of the proportion of CC3⁺/Olig2⁺ relative to total Olig2⁺ OLs at DD3. C. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} showing colocalization of nuclear CC3 and Olig2 in apoptotic OLs at DD6. D. Quantification of the proportion of CC3⁺/Olig2⁺ relative to total Olig2⁺ OLs at DD6. A, C: Arrowheads represent CC3⁺/Olig2⁺ OLs. B, D: n = 3; all comparisons non-significant by two-tailed Student’s t-test. Data represent mean ± SEM. Scale bars = 50 μm.</p

Migration is normal in <i>Dst</i>^{<i>dt-27J</i>} OPCs.

<p>A. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} of OPC aggregates 4 hours post-seeding. B. Quantification of the proportion of NG2⁺ OPCs migrated at 4 hours within rings set at 50 μm increments from the aggregate. C. Immunofluorescence micrographs of WT and <i>Dst</i>^{<i>dt-27J</i>} of OPC aggregate 24 hours post-seeding. D. Quantification of the proportion of NG2⁺ OPCs migrated at 24 hours within rings set at 100 μm increments from the center of the aggregate. B, D: n = 3; all comparisons non-significant by two-tailed Student’s t-test. Data represent mean ± SEM. Scale bars = 50 μm.</p

Proliferating OPCs and differentiating OLs express neuronal <i>Dst</i> transcripts.

<p>Top. Representative RT-PCR products from <i>Dst</i>-<i>A1</i>, <i>-A2</i> and <i>-A3</i> with <i>actb</i> loading control in primary proliferating OPCs (left lanes) and differentiating OLs (right lanes). Bottom. qRT-PCR analysis of <i>Dst</i>-<i>A1</i>, <i>-A2</i> and <i>-A3</i> expression in primary proliferating OPCs and differentiating OLs. n = 4–6; ΔΔCt, <i>Dst</i> normalized to <i>actb</i>. * p<0.05, n.s. = p≥0.05; two-tailed Student’s t-test. Data represent mean ± SEM.</p

Myelination occurs normally <i>in vivo</i> in <i>Dst</i>^{<i>dt-27J</i>} animals.

<p>A. Light microscope images of transected toluidine blue-stained optic nerves from P15 WT and <i>Dst</i>^{<i>dt-27J</i>} mice. B. Electron micrographs of transected optic nerves from P15 WT and <i>Dst</i>^{<i>dt-27J</i>} mice. C. Quantification of the number of myelinated axons per field of view (FOV) in optic nerves from P15 WT and <i>Dst</i>^{<i>dt-27J</i>} mice. D. Quantification of average g-ratio of all myelinated axons per FOV in optic nerves from P15 WT and <i>Dst</i>^{<i>dt-27J</i>} mice. E. g-ratios plotted by axon caliber in optic nerves from P15 WT and <i>Dst</i>^{<i>dt-27J</i>} mice. F, G. Fluorescence Western blot analysis of CNPase, MOG, MBP and α-tubulin (α-tub) from cerebral cortex (CC—top panels) and spinal cord (SC—bottom panels) from P15 WT and <i>Dst</i>^{<i>dt-27J</i>} mice. Protein sizes—CNPase: 48 kDa; MOG: 27 kDa; MBP isoforms, from top to bottom: 21.5 kDa, 18.5 kDa, 17.0 kDa, 14.0 kDa. α-tub: 52 kDa. H, I. Quantification of total CNPase normalized to α-tub (from blots pictured in F, G). J, K. Quantification of total MOG normalized to α-tub (from blots pictured in F, G). L, M. Quantification of total MBP normalized to α-tub (from blots pictured in F, G). C, D: n = 4; H-M: n = 6; all comparisons non-significant by two-tailed Student’s t-test. C, D: data represent mean ± SD. H-M: data represent mean ± SEM. E: n = 100; data represent individual measurements, comparison non-significant by linear regression analysis. A: scale bar = 50 μm. B: scale bar = 2 μm.</p