Edited by: Yajun Wang, Sichuan University, China
Reviewed by: Qinghua Nie, South China Agricultural University, China; Paweł Tomasz Maćkowiak, Poznan University of Life Sciences, Poland
*Correspondence: Kent M. Reed
This article was submitted to Avian Physiology, a section of the journal Frontiers in Physiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Skeletal muscle hypertrophy is a multifaceted process. During embryonic development, undifferentiated cells in the mesoderm (myoblasts) proliferate, differentiate, and fuse to form multinucleated myotubes that further differentiate into muscle fibers. Subsequent muscle growth is dependent upon satellite cells; stem cells located between the basement membrane and sarcolemma of skeletal muscle fibers (Mauro,
In the early post-hatch period, avian satellite cells are highly active (Halevy et al.,
Genetic selection has resulted in poultry with greater breast muscle weights and shorter growth periods (Havenstein et al.,
As satellite cells are the only posthatch myonuclear source, they may directly modify skeletal muscle growth if functionally altered by temperature. Clark et al. (
Previously, we utilized cultured turkey satellite cells to study the effects of thermal challenge on the transcriptome of proliferating satellite cells (Reed et al.,
Satellite cells used in this study were previously isolated from the
Turkey
Total RNA was isolated from each culture by TRIzol extraction (Ambion, Inc.), DNase-treated (Turbo DNA-freeTM Kit, Ambion, Inc.), and stored at −80°C. Initial RNA concentration and quality was measured (Nanodrop 1000) and samples were submitted for library preparation and sequencing at the University of Minnesota Genomics Center (UMGC). Samples were quantified by RiboGreen Assay and RNA integrity was measured on a 2100 Bioanalyzer (Aligent Technologies). All sample had clear peak separation (18S and 28S) and RNA Integrity Numbers (RIN) ranged between 5.1 and 8.8. Indexed libraries were constructed with the TruSeq RNA Sample Preparation Kit version 2 (Illumina, Inc.) from 1 μg of total RNA/sample. Libraries were multiplexed, pooled and sequenced (101-bp paired-end reads) on the HiSeq 2000 using v3 chemistry (Illumina, Inc.). Replicate samples were sequenced from each treatment group (
Trimmomatic (Bolger et al.,
Total RNA isolated from satellite cell cultures was used to construct 12 individual barcoded libraries. Sequencing of the libraries produced over 394 M combined reads with the number of reads per library ranging from 12.4 to 18.5 M (average 16.4 M). After read trimming and filtering, median read quality was consistently high and ranged from 36.8 to 37.3. Replicate libraries for each treatment produced comparable results with the number of reads per treatment group ranging from 29 to 36.4 M (average 32.9 ± 1.25 M reads). Cumulatively, corrected reads comprised 39.9 Gb of sequence for transcriptome analysis. Mean library insert was estimated from mapping results as 213.4 bp (Table
Summary of RNA-seq data.
A | 17836372 | 37.1 | 36.9 | 16326998 | 89.9 | 82.3 | 216 | 16544 | 15351 | 73.1 | ||
B | 18039271 | 37.1 | 37.0 | 16541921 | 89.3 | 82.5 | 218 | 16622 | ||||
A | 16252079 | 37.2 | 36.9 | 14822122 | 89.1 | 82.8 | 213 | 16365 | 15193 | 72.3 | ||
B | 16698643 | 37.1 | 37.0 | 15361039 | 89.2 | 83.3 | 198 | 16532 | ||||
A | 15602450 | 37.2 | 37.0 | 14274919 | 89.7 | 83.4 | 226 | 16244 | 15399 | 73.3 | ||
B | 15303273 | 37.1 | 36.9 | 13986262 | 89.3 | 82.5 | 227 | 16397 | ||||
A | 16169884 | 37.2 | 37.0 | 14865209 | 88.9 | 82.2 | 215 | 16557 | 15314 | 72.9 | ||
B | 17084981 | 37.2 | 37.0 | 15702722 | 89.5 | 82.8 | 215 | 16355 | ||||
A | 18370285 | 37.2 | 37.0 | 16846899 | 89.9 | 83.5 | 217 | 16630 | 15270 | 72.7 | ||
B | 18085832 | 37.1 | 36.8 | 16419915 | 90.2 | 84.1 | 213 | 16575 | ||||
A | 14634877 | 37.2 | 36.9 | 13373887 | 89.2 | 83.0 | 226 | 16498 | 15364 | 73.1 | ||
B | 15356625 | 37.2 | 36.9 | 14035364 | 89.2 | 82.9 | 226 | 16583 | ||||
16619547.7 | 37.15 | 36.94 | 15213104.7 | 89.44 | 82.94 | 213.4 | 16491.8 | 15315.2 | 72.9 |
Approximately 89% of the quality trimmed fragments mapped uniquely to the annotated turkey gene set, and on average, 82.9% mapped concordantly (Table
Variation among groups was evaluated by principal component analysis (PCA) of normalized read counts (Figure
Principal component analysis (PCA) of normalized RNAseq read counts. Sample to sample distances (within- and between-treatments) are illustrated for each dataset on the first two principle components comprising ~98% of the variation. Samples are plotted according to treatment.
Distribution of expressed genes (unique and shared) among treatment groups is summarized in Table
Summary of gene expression and significant differential expression (DE) in pair-wise comparisons of differentiating cells.
33R vs. 38R | 15,885 | 14,659 | 692/534 | 5,963 | 2,608 | 852 (0.337) | |
33F vs. 38F | 15,901 | 14,683 | 631/587 | 8,678 | 3,027 | 969 (0.372) | |
43R vs. 38R | 15,933 | 14,695 | 704/498 | 7,160 | 3,355 | 845 (0.788) | |
43F vs. 38F | 15,951 | 14,757 | 607/513 | 9,039 | 2,905 | 703 (0.752) | |
33F vs. 33R | 15,728 | 14,937 | 414/377 | 873 | 116 | 23 (0.130) | |
38F vs. 38R | 15,652 | 14,811 | 382/459 | 1 | 1 | 1 (0.000) | |
43F vs. 43R | 15,801 | 14,962 | 437/402 | 1,818 | 318 | 52 (0.500) |
Ordering and classification of the genes expressed at the control incubation temperature (38°C, Table
Twenty most significant canonical pathways expressed in satellite cell cultures at 38°C after 48 h of differentiation.
tRNA charging | 8.920 | 0.846 | 8.950 | 0.846 |
Superpathway of inositol phosphate compounds | 6.870 | 0.540 | 7.260 | 0.545 |
3-phosphoinositide degradation | 6.860 | 0.582 | 6.540 | 0.575 |
Colanic acid building blocks biosynthesis | 6.050 | 1.000 | 6.070 | 1.000 |
3-phosphoinositide biosynthesis | 5.960 | 0.543 | 6.020 | 0.543 |
D-myo-inositol (1,4,5,6)-tetrakisphosphate biosynthesis | 5.720 | 0.574 | 5.770 | 0.574 |
D-myo-inositol (3,4,5,6)-tetrakisphosphate biosynthesis | 5.720 | 0.574 | 5.770 | 0.574 |
D-myo-inositol-5-phosphate metabolism | 4.930 | 0.544 | 4.980 | 0.544 |
Superpathway of D-myo-inositol (1,4,5)-trisphosphate metabolism | 4.860 | 0.800 | 5.750 | 0.840 |
Fatty Acid β-oxidation I | 4.200 | 0.719 | 3.600 | 0.688 |
Valine degradation I | 4.130 | 0.833 | 4.140 | 0.833 |
Pyridoxal 5′-phosphate salvage pathway | 3.660 | 0.594 | 3.680 | 0.594 |
Superpathway of cholesterol biosynthesis | 3.650 | 0.714 | 3.670 | 0.714 |
Isoleucine degradation I | 3.590 | 0.857 | 3.600 | 0.857 |
TCA Cycle II (Eukaryotic) | 3.460 | 0.739 | 3.470 | 0.739 |
D-myo-inositol (1,4,5)-trisphosphate degradation | 3.300 | 0.778 | 4.140 | 0.833 |
D-myo-inositol (1,3,4)-trisphosphate biosynthesis | 3.210 | 0.750 | 3.990 | 0.800 |
Phosphatidylglycerol biosynthesis II (Non-plastidic) | 3.080 | 0.692 | 3.090 | 0.692 |
Salvage pathways of pyrimidine ribonucleotides | 2.850 | 0.527 | 2.880 | 0.527 |
GDP-mannose biosynthesis | 2.590 | 1.000 | 2.600 | 1.000 |
Protein ubiquitination pathway | 25.400 | 0.694 | 25.500 | 0.694 |
EIF2 signaling | 24.100 | 0.732 | 24.200 | 0.732 |
Regulation of eIF4 and p70S6K signaling | 18.400 | 0.720 | 18.500 | 0.720 |
mTOR signaling | 16.000 | 0.658 | 16.600 | 0.663 |
NRF2-mediated oxidative stress response | 15.500 | 0.658 | 15.100 | 0.653 |
Estrogen receptor signaling | 12.600 | 0.688 | 12.700 | 0.688 |
Molecular mechanisms of cancer | 12.600 | 0.553 | 12.700 | 0.553 |
Hereditary breast cancer signaling | 12.400 | 0.669 | 12.500 | 0.669 |
Aldosterone signaling in epithelial cells | 12.300 | 0.645 | 12.400 | 0.645 |
Huntington's disease signaling | 11.500 | 0.589 | 11.600 | 0.589 |
Role of BRCA1 in DNA damage response | 10.700 | 0.744 | 10.800 | 0.744 |
PI3K/AKT signaling | 10.400 | 0.661 | 10.500 | 0.661 |
Death receptor signaling | 9.090 | 0.685 | 9.730 | 0.696 |
Integrin signaling | 9.000 | 0.571 | 9.830 | 0.580 |
AMPK | 8.660 | 0.582 | 8.730 | 0.582 |
Protein kinase A signaling | 8.630 | 0.515 | 8.460 | 0.513 |
Mitotic roles of polo-like kinase | 8.440 | 0.727 | 8.480 | 0.727 |
Glioma signaling | 8.420 | 0.645 | 8.470 | 0.645 |
HIPPO signaling | 8.040 | 0.674 | 8.090 | 0.674 |
Apidogenesis pathway | 8.000 | 0.612 | 8.060 | 0.612 |
RNA-Seq affords the opportunity to gain new insights into temporal gene expression patterns of differentiating satellite cells, particularly with respect to muscle fiber-type-specific proteins. Although, the adult turkey
Gaussian-based ANOVA found 12,395 genes with significant (FDR
On average, more genes were significantly affected by cold (33°C) treatment than by heat (43°C; Table
Distribution of differentially expressed genes during differentiation of cultured turkey
The majority of DE genes identified in the treatment comparisons were unique to treatment groups (temperature/line; Figure
Statistical overrepresentation tests (PANTHER) of genes differentially expressed between the 33 and 38°C found greatest enrichment for the GO
Summary of PANTHER Overrepresentation test of the 939 genes differentially expressed in
Myofibril assembly (GO:0030239) | 38 | 8 | 0.96 | + | 8.31 | 4.46E-02 |
Regulation of calcium ion import (GO:0090279) | 56 | 10 | 1.42 | + | 7.05 | 1.40E-02 |
Muscle contraction (GO:0006936) | 106 | 16 | 2.69 | + | 5.96 | 1.38E-04 |
Striated muscle cell development (GO:0055002) | 83 | 12 | 2.1 | + | 5.71 | 1.22E-02 |
Muscle system process (GO:0003012) | 131 | 18 | 3.32 | + | 5.42 | 7.58E-05 |
Muscle cell development (GO:0055001) | 93 | 12 | 2.36 | + | 5.09 | 3.81E-02 |
Striated muscle cell differentiation (GO:0051146) | 123 | 14 | 3.12 | + | 4.49 | 2.73E-02 |
Regulation of metal ion transport (GO:0010959) | 167 | 16 | 4.23 | + | 3.78 | 4.95E-02 |
Striated muscle tissue development (GO:0014706) | 186 | 17 | 4.71 | + | 3.61 | 4.76E-02 |
Muscle tissue development (GO:0060537) | 199 | 18 | 5.04 | + | 3.57 | 2.97E-02 |
Muscle structure development (GO:0061061) | 278 | 24 | 7.04 | + | 3.41 | 1.82E-03 |
Regulation of ion transport (GO:0043269) | 303 | 24 | 7.68 | + | 3.13 | 8.12E-03 |
Anatomical structure formation in morphogenesis (GO:0048646) | 659 | 41 | 16.7 | + | 2.46 | 1.03E-03 |
System process (GO:0003008) | 789 | 49 | 19.99 | + | 2.45 | 6.09E-05 |
Circulatory system development (GO:0072359) | 529 | 32 | 13.4 | + | 2.39 | 4.33E-02 |
Cardiovascular system development (GO:0072358) | 529 | 32 | 13.4 | + | 2.39 | 4.33E-02 |
Tissue development (GO:0009888) | 961 | 51 | 24.35 | + | 2.09 | 3.76E-03 |
regulation of multicellular organismal development (GO:2000026) | 1,017 | 52 | 25.76 | + | 2.02 | 8.59E-03 |
Regulation of transport (GO:0051049) | 1,056 | 52 | 26.75 | + | 1.94 | 2.50E-02 |
Regulation of multicellular organismal process (GO:0051239) | 1,555 | 76 | 39.39 | + | 1.93 | 1.32E-04 |
Anatomical structure morphogenesis (GO:0009653) | 1,390 | 66 | 35.21 | + | 1.87 | 3.73E-03 |
Regulation of developmental process (GO:0050793) | 1,369 | 64 | 34.68 | + | 1.85 | 9.56E-03 |
Animal organ development (GO:0048513) | 1,697 | 79 | 42.99 | + | 1.84 | 5.10E-04 |
System development (GO:0048731) | 2,360 | 108 | 59.79 | + | 1.81 | 2.23E-06 |
Regulation of localization (GO:0032879) | 1,497 | 68 | 37.93 | + | 1.79 | 1.15E-02 |
Multicellular organismal process (GO:0032501) | 3,429 | 153 | 86.87 | + | 1.76 | 3.04E-10 |
Single-multicellular organism process (GO:0044707) | 3,050 | 136 | 77.27 | + | 1.76 | 2.03E-08 |
Multicellular organism development (GO:0007275) | 2,614 | 116 | 66.22 | + | 1.75 | 2.42E-06 |
Anatomical structure development (GO:0048856) | 2,936 | 129 | 74.38 | + | 1.73 | 2.81E-07 |
Single-organism developmental process (GO:0044767) | 3,091 | 131 | 78.31 | + | 1.67 | 2.11E-06 |
Developmental process (GO:0032502) | 3,122 | 131 | 79.09 | + | 1.66 | 4.10E-06 |
Cell communication (GO:0007154) | 2,933 | 114 | 74.3 | + | 1.53 | 5.40E-03 |
Single organism signaling (GO:0044700) | 2,872 | 111 | 72.76 | + | 1.53 | 1.06E-02 |
Signaling (GO:0023052) | 2,875 | 111 | 72.84 | + | 1.52 | 1.12E-02 |
Signal transduction (GO:0007165) | 2,680 | 103 | 67.9 | + | 1.52 | 3.77E-02 |
Response to stimulus (GO:0050896) | 4,290 | 150 | 108.68 | + | 1.38 | 2.62E-02 |
Single-organism process (GO:0044699) | 7,871 | 264 | 199.4 | + | 1.32 | 3.09E-07 |
Regulation of biological process (GO:0050789) | 6,727 | 224 | 170.42 | + | 1.31 | 2.97E-04 |
Single-organism cellular process (GO:0044763) | 6,748 | 221 | 170.95 | + | 1.29 | 1.97E-03 |
Regulation of cellular process (GO:0050794) | 6,374 | 207 | 161.48 | + | 1.28 | 1.67E-02 |
Biological regulation (GO:0065007) | 7,227 | 231 | 183.09 | + | 1.26 | 6.16E-03 |
Biological_process (GO:0008150) | 11,618 | 333 | 294.33 | + | 1.13 | 1.81E-02 |
Unclassified (UNCLASSIFIED) | 4,171 | 67 | 105.67 | − | 0.63 | 0.00E+00 |
Troponin complex (GO:0005861) | 7 | 4 | 0.18 | + | 22.56 | 3.62E-02 |
Striated muscle thin filament (GO:0005865) | 17 | 6 | 0.43 | + | 13.93 | 6.09E-03 |
Myofilament (GO:0036379) | 18 | 6 | 0.46 | + | 13.16 | 8.40E-03 |
A band (GO:0031672) | 21 | 6 | 0.53 | + | 11.28 | 1.99E-02 |
Sarcomere (GO:0030017) | 110 | 25 | 2.79 | + | 8.97 | 3.33E-13 |
Contractile fiber part (GO:0044449) | 120 | 25 | 3.04 | + | 8.22 | 2.33E-12 |
Myofibril (GO:0030016) | 131 | 26 | 3.32 | + | 7.83 | 1.95E-12 |
Contractile fiber (GO:0043292) | 138 | 26 | 3.5 | + | 7.44 | 6.43E-12 |
I band (GO:0031674) | 77 | 14 | 1.95 | + | 7.18 | 1.87E-05 |
Receptor complex (GO:0043235) | 236 | 20 | 5.98 | + | 3.35 | 3.97E-03 |
Proteinaceous extracellular matrix (GO:0005578) | 238 | 20 | 6.03 | + | 3.32 | 4.49E-03 |
Extracellular matrix (GO:0031012) | 305 | 24 | 7.73 | + | 3.11 | 1.54E-03 |
Plasma membrane part (GO:0044459) | 1,442 | 67 | 36.53 | + | 1.83 | 1.09E-03 |
Plasma membrane (GO:0005886) | 2,561 | 97 | 64.88 | + | 1.5 | 2.40E-02 |
Cell periphery (GO:0071944) | 2,651 | 99 | 67.16 | + | 1.47 | 3.39E-02 |
Intrinsic component of membrane (GO:0031224) | 3,877 | 134 | 98.22 | + | 1.36 | 3.72E-02 |
Membrane part (GO:0044425) | 4,536 | 154 | 114.92 | + | 1.34 | 1.69E-02 |
Cellular component (GO:0005575) | 12,385 | 347 | 313.76 | + | 1.11 | 1.41E-02 |
Intracellular membrane-bounded organelle (GO:0043231) | 6,617 | 119 | 167.64 | − | 0.71 | 3.46E-04 |
Unclassified (UNCLASSIFIED) | 3,404 | 53 | 86.24 | − | 0.61 | 0.00E+00 |
Nucleus (GO:0005634) | 4,190 | 64 | 106.15 | − | 0.6 | 3.67E-04 |
Organelle lumen (GO:0043233) | 2,350 | 28 | 59.54 | − | 0.47 | 9.99E-04 |
Intracellular organelle lumen (GO:0070013) | 2,350 | 28 | 59.54 | − | 0.47 | 9.99E-04 |
Membrane-enclosed lumen (GO:0031974) | 2,350 | 28 | 59.54 | − | 0.47 | 9.99E-04 |
Nuclear lumen (GO:0031981) | 2,129 | 25 | 53.94 | − | 0.46 | 2.68E-03 |
Nuclear part (GO:0044428) | 2,465 | 26 | 62.45 | − | 0.42 | 2.26E-05 |
Ribonucleoprotein complex (GO:1990904) | 518 | 1 | 13.12 | − | <0.2 | 2.39E-02 |
Intracellular ribonucleoprotein complex (GO:0030529) | 518 | 1 | 13.12 | − | <0.2 | 2.39E-02 |
Actin binding (GO:0003779) | 285 | 22 | 7.22 | + | 3.05 | 1.07E-02 |
Cytoskeletal protein binding (GO:0008092) | 600 | 42 | 15.2 | + | 2.76 | 9.00E-06 |
Calcium ion binding (GO:0005509) | 493 | 30 | 12.49 | + | 2.4 | 2.41E-02 |
Transmembrane receptor activity (GO:0099600) | 690 | 38 | 17.48 | + | 2.17 | 1.60E-02 |
Receptor activity (GO:0004872) | 886 | 45 | 22.45 | + | 2 | 1.77E-02 |
Molecular transducer activity (GO:0060089) | 886 | 45 | 22.45 | + | 2 | 1.77E-02 |
Protein binding (GO:0005515) | 4,019 | 148 | 101.82 | + | 1.45 | 4.40E-04 |
Unclassified (UNCLASSIFIED) | 4,679 | 87 | 118.54 | − | 0.73 | 0.00E+00 |
Nucleic acid binding (GO:0003676) | 2,522 | 27 | 63.89 | − | 0.42 | 4.12E-05 |
poly(A) RNA binding (GO:0044822) | 835 | 5 | 21.15 | − | 0.24 | 4.04E-02 |
RNA binding (GO:0003723) | 1,108 | 6 | 28.07 | − | 0.21 | 5.56E-04 |
Summary of PANTHER Overrepresentation test of the 856 genes differentially expressed in
Animal organ development (GO:0048513) | 1,697 | 52 | 26.76 | + | 1.94 | 1.37E-02 |
Single-multicellular organism process (GO:0044707) | 3,050 | 88 | 48.10 | + | 1.83 | 1.38E-05 |
System development (GO:0048731) | 2,360 | 67 | 37.22 | + | 1.80 | 4.76E-03 |
Multicellular organism development (GO:0007275) | 2,614 | 72 | 41.22 | + | 1.75 | 4.90E-03 |
Multicellular organismal process (GO:0032501) | 3,429 | 91 | 54.08 | + | 1.68 | 4.14E-04 |
Negative regulation of cellular process (GO:0048523) | 2,565 | 68 | 40.45 | + | 1.68 | 4.28E-02 |
Negative regulation of biological process (GO:0048519) | 2,756 | 73 | 43.46 | + | 1.68 | 1.73E-02 |
Single-organism developmental process (GO:0044767) | 3,091 | 79 | 48.75 | + | 1.62 | 2.24E-02 |
Developmental process (GO:0032502) | 3,122 | 79 | 49.24 | + | 1.60 | 3.29E-02 |
Biological regulation (GO:0065007) | 7,227 | 151 | 113.97 | + | 1.32 | 1.04E-02 |
Single-organism process (GO:0044699) | 7,871 | 160 | 124.13 | + | 1.29 | 1.94E-02 |
Unclassified (UNCLASSIFIED) | 4,171 | 46 | 65.78 | − | 0.70 | 0.00E+00 |
Unclassified (UNCLASSIFIED) | 3,404 | 30 | 53.68 | − | 0.56 | 0.00E+00 |
Unclassified (UNCLASSIFIED) | 4,679 | 58 | 73.79 | − | 0.79 | 0.00E00 |
Analysis of DE genes in IPA outlined several temperature-induced shifts in the satellite cell transcriptomes. The top 10 canonical pathways for each temperature comparison are given in Table
Significant pathway associations identified in IPA Comparison Analysis of thermal challenge vs. control temperature cells. In each pair-wise comparison,
A total of 2,926 significant DE genes (|Log2FC| > 1.0) was shared among all treatment comparisons, but only 70 had |Log2FC| > 2.0 (Figure
Common significant DE genes [FDR
ACTA1 | Actin, alpha 1, skeletal muscle | −3.205 | 2.2E-154 | −3.347 | 0 | −2.861 | 0 | −3.193 | 0 |
APOA1 | Apolipoprotein A-I | −3.110 | 2.87E-36 | −3.113 | 1.19E-45 | −4.200 | 2.2E-53 | −2.404 | 1.29E-28 |
BCAN | Brevican | −4.061 | 1.12E-67 | −3.839 | 1.93E-40 | −2.376 | 4.27E-27 | −2.095 | 9.01E-45 |
C1QL1 | Complement component 1, q subcomponent-like 1 | −2.133 | 2.99E-40 | −2.465 | 4.61E-89 | −2.942 | 8.8E-80 | −2.162 | 1.15E-32 |
CCDC69 | Coiled-coil domain containing 69 | −5.348 | 8.31E-17 | −4.015 | 9.4E-110 | −3.700 | 7.16E-11 | −2.138 | 3.22E-95 |
CD36 | CD36 molecule (thrombospondin receptor) | 2.300 | 2.05E-46 | 2.479 | 9.79E-46 | −2.606 | 3.59E-17 | −2.131 | 3.52E-27 |
DRC7 | Dynein regulatory complex subunit 7 | −4.210 | 2.88E-16 | −3.981 | 4.15E-24 | −2.259 | 7.2E-09 | −2.073 | 3.98E-12 |
EVC | Ellis van Creveld syndrome | 3.026 | 0.005323 | 2.570 | 0.000106 | 3.119 | 0.000946 | 2.024 | 0.007842 |
FABP3 | Fatty acid binding protein 3, muscle and heart | −5.284 | 7.79E-35 | −4.858 | 7.79E-41 | −3.020 | 1.97E-18 | −3.959 | 2.29E-36 |
GUCA1B | Guanylate cyclase activator 1B (retina) | −3.228 | 2.28E-05 | −6.281 | 2.68E-05 | 2.990 | 1.44E-23 | 3.316 | 1.18E-25 |
LNPEP | Leucyl/cystinyl aminopeptidase | 2.589 | 1.2E-07 | 2.319 | 4.07E-13 | 3.110 | 3.88E-16 | 2.267 | 1.53E-12 |
LOC100539445 | Tensin-3 (TNS3) | 2.269 | 0.00123 | 2.321 | 6.04E-08 | 2.791 | 1.56E-09 | 3.026 | 9.17E-16 |
LOC100545344 | Myosin-7-like | −4.975 | 2.4E-142 | −4.716 | 1.5E-45 | −2.988 | 8.58E-75 | −2.169 | 5.61E-42 |
LOC100547876 | Polypeptide N-acetylgalactosaminyltransferase 12-like | 2.823 | 0.001667 | 2.930 | 9.26E-07 | 2.582 | 0.002754 | 2.215 | 0.001923 |
LOC100548077 | Disheveled-associated activator of morphogenesis 2 (DAAM2) | 2.703 | 0.000521 | 2.031 | 0.009045 | 3.218 | 3.7E-06 | 2.329 | 0.000755 |
LOC100548792 | Collagen alpha-1(XII) chain-like | 2.061 | 1.94E-06 | 2.720 | 2.64E-79 | 3.333 | 1.46E-33 | 2.652 | 4.26E-87 |
LOC100549353 | Activating signal cointegrator 1 complex subunit 3 (ASCC3) | 2.127 | 3.37E-06 | 2.379 | 4.43E-16 | 2.699 | 1.16E-13 | 2.276 | 1.47E-16 |
LOC100550192 | HLA class II histocompatibility antigen, DM beta chain-like (DMB2) | 2.263 | 0.003633 | 2.648 | 6.59E-05 | 3.586 | 4.06E-10 | 3.266 | 2.81E-09 |
LOC104909264 | Multidrug resistance-associated protein 4-like | 2.217 | 5.61E-06 | 3.408 | 1.25E-16 | 2.207 | 1.27E-05 | 2.329 | 4.25E-06 |
LOC104909289 | Limbin-like | 2.589 | 1.35E-06 | 3.524 | 3.22E-09 | 2.058 | 0.000628 | 2.740 | 0.000108 |
LOC104909397 | Dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase (probable) | 2.464 | 0.002752 | 2.186 | 1.95E-06 | 3.644 | 1.56E-10 | 2.282 | 1.87E-07 |
LOC104909635 | Latent-transforming growth factor beta-binding protein 1-like | 2.875 | 3.12E-11 | 3.039 | 2.66E-58 | 4.077 | 2.86E-66 | 3.716 | 3.9E-113 |
LOC104909708 | Uncharacterized LOC104909708 (ncRNA) | −3.356 | 0.012361 | −3.268 | 0.000646 | −2.688 | 0.03465 | −3.090 | 0.000878 |
LOC104909779 | Uncharacterized LOC104909779 (ncRNA) | 2.223 | 2.66E-05 | 2.640 | 7.28E-09 | 2.120 | 6.87E-05 | 2.361 | 2.74E-06 |
LOC104909819 | Uncharacterized LOC104909819 (ncRNA) | −3.276 | 0.004612 | −4.031 | 0.000265 | −2.214 | 0.027631 | −2.483 | 0.005267 |
LOC104909922 | Unconventional myosin-VI-like | 3.620 | 0.002294 | 4.227 | 1.24E-05 | 5.139 | 2.25E-08 | 4.453 | 1.39E-06 |
LOC104909972 | Uncharacterized LOC104909972 (ncRNA) | 2.761 | 0.025565 | 3.318 | 7.03E-05 | 3.046 | 0.001915 | 2.946 | 0.002409 |
LOC104910277 | Uncharacterized LOC104910277 (ncRNA) | 2.047 | 0.000265 | 2.011 | 8.08E-06 | 3.469 | 1.52E-14 | 3.345 | 6.5E-30 |
LOC104910488 | Protein EFR3 homolog A-like | 2.207 | 0.001044 | 2.478 | 2.77E-08 | 3.242 | 1.65E-13 | 2.702 | 1.86E-11 |
LOC104911116 | Tetraspanin-18-like | 2.542 | 1.11E-08 | 2.454 | 2.04E-16 | 3.174 | 1.45E-16 | 2.371 | 2.01E-15 |
LOC104911697 | Lymphocyte antigen 75-like | 2.320 | 3.64E-06 | 2.737 | 2.24E-27 | 3.010 | 3.28E-17 | 2.467 | 1.3E-17 |
LOC104912545 | Serine/threonine-protein kinase ATR-like | 2.556 | 0.008755 | 3.134 | 0.000424 | 2.518 | 0.004279 | 2.665 | 0.019456 |
LOC104913390 | Multidrug resistance-associated protein 1-like | 3.731 | 2.77E-06 | 3.871 | 2.66E-12 | 3.832 | 1.01E-06 | 3.542 | 1.1E-09 |
LOC104913470 | Periplakin-like | 6.430 | 2.91E-05 | 6.732 | 2.38E-07 | 6.104 | 0.000567 | 4.863 | 0.040654 |
LOC104913553 | Uncharacterized LOC104913553 (ncRNA) | −2.071 | 7.56E-10 | −2.542 | 1.04E-24 | −2.187 | 8.27E-16 | −3.085 | 1.65E-42 |
LOC104913838 | Uncharacterized LOC104913838 (ncRNA) | −6.959 | 4.68E-07 | −3.967 | 4.63E-07 | −2.318 | 0.000899 | −2.566 | 6.44E-05 |
LOC104914081 | Regulator of G-protein signaling 9-binding protein-like | −3.633 | 1.72E-96 | −3.782 | 1.47E-36 | −4.970 | 1.7E-110 | −4.224 | 3.51E-39 |
LOC104914095 | Probable phospholipid-transporting ATPase IIA | 3.083 | 0.02281 | 3.349 | 0.006467 | 3.225 | 0.018069 | 3.794 | 0.000706 |
LOC104914454 | Importin-9-like | 2.093 | 2E-07 | 2.484 | 6.1E-18 | 2.003 | 1.92E-09 | 2.319 | 3.02E-15 |
LOC104914932 | Protein LAP2-like | 2.253 | 1.33E-07 | 2.614 | 1.59E-18 | 2.978 | 1.19E-13 | 2.505 | 7.31E-17 |
LOC104914983 | Rho guanine nucleotide exchange factor 28-like | 2.756 | 0.021236 | 2.875 | 0.032997 | 3.290 | 0.000905 | 4.226 | 2.95E-05 |
LOC104915185 | Peptidyl-glycine alpha-amidating monooxygenase-like | 2.029 | 2.1E-13 | 2.194 | 1.66E-84 | 3.124 | 5.61E-93 | 2.504 | 3.61E-96 |
LOC104915209 | Cyclin-G-associated kinase-like | 2.718 | 0.003993 | 2.098 | 0.002392 | 2.970 | 0.000509 | 2.078 | 0.002028 |
LOC104915239 | Solute carrier family 12 member 2-like | 2.423 | 0.004607 | 4.386 | 1.2E-06 | 3.172 | 3.58E-07 | 4.979 | 8.76E-10 |
LOC104915240 | Solute carrier family 12 member 2-like | 2.331 | 0.008301 | 3.373 | 4.27E-05 | 2.581 | 0.003403 | 3.312 | 0.000191 |
LOC104915275 | Versican core protein-like | 2.117 | 3.28E-07 | 2.508 | 9.4E-19 | 4.283 | 6.06E-45 | 3.322 | 3.36E-41 |
LOC104915303 | Structural maintenance of chromosomes protein 2-like | 2.372 | 1.45E-09 | 3.005 | 1.51E-47 | 3.395 | 5.32E-41 | 2.418 | 2.86E-19 |
LOC104915418 | Uncharacterized LOC104915418 (ncRNA) | 2.830 | 0.014401 | 2.592 | 0.013781 | 3.176 | 0.000745 | 2.861 | 0.004775 |
LOC104916051 | Uncharacterized LOC104916051 | 2.211 | 0.010683 | 4.739 | 1.72E-07 | 3.274 | 8.48E-08 | 5.184 | 2.35E-11 |
LOC104916792 | Uncharacterized LOC104916792 (ncRNA) | 2.430 | 0.004417 | 2.450 | 0.000331 | 3.118 | 8.43E-06 | 2.330 | 0.000822 |
LOC104916797 | Kinesin-like protein KIF20B | 5.546 | 0.012424 | 3.131 | 0.019325 | 5.632 | 0.003275 | 3.042 | 0.030237 |
LOC104916915 | ETS translocation variant 3-like protein | 2.198 | 7.61E-05 | 2.887 | 1.53E-07 | 2.179 | 6.09E-05 | 2.863 | 1.6E-07 |
LOC104916992 | Uncharacterized LOC104916992 (ncRNA) | −6.565 | 2.46E-78 | −6.353 | 4E-112 | −2.320 | 9.47E-22 | −2.022 | 2.86E-73 |
LOC104917145 | Uncharacterized LOC104917145 (ncRNA) | 2.147 | 5.47E-05 | 2.680 | 2.39E-09 | 2.443 | 1.16E-06 | 2.382 | 1.55E-10 |
LOC104917155 | Alpha-mannosidase 2-like | 3.184 | 8.61E-06 | 3.626 | 9.74E-23 | 4.006 | 1.55E-14 | 3.543 | 1.35E-21 |
LOC104917232 | Alpha-mannosidase 2-like | 3.317 | 1.09E-05 | 3.119 | 3.95E-11 | 3.585 | 5.11E-08 | 2.802 | 1.73E-07 |
LOC104917363 | Extended synaptotagmin-2-A-like | 2.174 | 0.01489 | 3.069 | 3.92E-12 | 2.452 | 0.000574 | 2.336 | 8.58E-05 |
LOC104917414 | Uncharacterized LOC104917414 (ncRNA) | 3.090 | 2.57E-06 | 3.525 | 2.34E-09 | 2.547 | 0.000728 | 2.029 | 0.020654 |
LOC104917580 | Sister chromatid cohesion protein PDS5 homolog B-like | 2.780 | 5.84E-05 | 2.052 | 2.19E-06 | 3.603 | 2.16E-11 | 2.307 | 2.47E-08 |
MKI67 | Marker of proliferation Ki-67 | 2.867 | 6.91E-09 | 3.127 | 1.48E-31 | 4.181 | 6.74E-22 | 2.829 | 1.9E-24 |
MSTN | Myostatin | −3.824 | 4.36E-08 | −2.382 | 1.94E-12 | −3.424 | 5.62E-07 | −2.480 | 5.71E-13 |
MTMR8 | Myotubularin related protein 8 | 2.148 | 1.6E-07 | 2.456 | 1.51E-14 | 2.262 | 3.14E-10 | 2.143 | 3.91E-11 |
MYL3 | Myosin, light chain 3, alkali; ventricular, skeletal, slow | −6.283 | 6.6E-130 | −7.377 | 0 | −3.687 | 3.26E-82 | −3.705 | 0 |
PTGFRN | Prostaglandin F2 receptor inhibitor | 3.291 | 4.18E-06 | 2.740 | 1.19E-06 | 3.536 | 1.27E-07 | 3.300 | 3.07E-11 |
REEP3 | Receptor accessory protein 3 | 2.363 | 5.13E-14 | 2.201 | 9.59E-31 | 2.646 | 6.37E-23 | 2.124 | 4.64E-30 |
SLN | Sarcolipin | −5.226 | 1.34E-54 | −6.440 | 7.03E-80 | −3.335 | 2.34E-30 | −2.338 | 1.23E-38 |
TEAD1 | TEA domain family member 1 (SV40 transcriptional enhancer factor) | 2.357 | 1.73E-12 | 2.259 | 9.1E-12 | 3.078 | 1.95E-19 | 2.654 | 5.9E-32 |
TNNC2 | Troponin C type 2 (fast) | −3.312 | 5.9E-126 | −3.349 | 0 | −4.650 | 7.4E-152 | −4.099 | 0 |
TPM2 | Tropomyosin 2 (beta) | −2.732 | 1.51E-36 | −2.541 | 1.5E-107 | −2.289 | 1.47E-22 | −2.163 | 9.04E-96 |
TSPAN10 | Tetraspanin 10 | −2.955 | 4.44E-18 | −2.432 | 3.11E-72 | −2.771 | 3.98E-16 | −3.485 | 3.72E-88 |
To further examine differences between the temperature treatments, the 2,926 significant DE genes (|Log2FC| > 1.0) shared among all treatment comparisons were further investigated. These represent 23.3% of the total number of DE genes identified across treatments. Of the 2,926 DE genes, 637 showed consistent directional expression change, with the 33 vs. 38°C comparisons and the 43 vs. 38°C comparisons having the same directional response in both lines. Up regulation across all treatment comparisons was observed for 407 genes whereas 141 were down regulated. Of the remaining 89 genes, 53 were down regulated by cold treatment in both lines and 36 were down regulated by heat treatment (Table
Common DE genes [FDR
ABLIM2 | Actin binding LIM protein family, member 2 | −2.054 | 0.001493 | −1.390 | 0.004024 | 1.353 | 0.001549 | 1.418 | 4.88E-07 |
LOC104912064 | Disintegrin and metalloproteinase domain-containing protein 12-like | −1.231 | 0.035684 | −1.447 | 0.000151 | 1.739 | 2.65E-07 | 1.573 | 9.79E-13 |
APBA2 | Amyloid beta (A4) precursor protein-binding, family A, member 2 | −1.319 | 0.001841 | −1.274 | 7.45E-09 | 2.041 | 1.52E-20 | 1.135 | 1.5E-14 |
ARHGAP28 | Rho GTPase activating protein 28 | −1.852 | 1.38E-05 | −1.690 | 2.9E-07 | 1.581 | 3.62E-06 | 1.145 | 6.23E-07 |
ASAP3 | ArfGAP with SH3 domain, ankyrin repeat and PH domain 3 | −1.356 | 0.001483 | −1.633 | 0.000236 | 1.193 | 0.000193 | 1.931 | 7.36E-16 |
BCL6 | B-cell CLL/lymphoma 6 | −1.024 | 1.31E-06 | −1.079 | 8.27E-30 | 1.171 | 1.37E-09 | 1.177 | 1.3E-65 |
C8H10orf71 | Chromosome 8 open reading frame, human C10orf71 | −2.257 | 2.07E-27 | −2.531 | 1.83E-83 | 1.786 | 3.24E-26 | 1.430 | 1.85E-50 |
CDK18 | Cyclin-dependent kinase 18 | −2.219 | 3.25E-10 | −1.751 | 3.86E-11 | 1.300 | 4.65E-08 | 1.053 | 6.42E-09 |
CHST1 | Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 | −3.057 | 3.89E-06 | −3.571 | 7.87E-08 | 1.440 | 1.37E-05 | 1.722 | 1.83E-08 |
COL3A1 | Collagen, type III, alpha 1 | −2.903 | 0 | −2.382 | 0 | 1.659 | 2.98E-12 | 1.923 | 1.18E-27 |
COL4A1 | Collagen, type IV, alpha 1 | −1.606 | 5.48E-14 | −1.224 | 0 | 1.217 | 1.83E-08 | 1.685 | 1.24E-40 |
DCX | Doublecortin | −2.199 | 4.6E-09 | −1.987 | 2.44E-19 | 1.815 | 3.08E-11 | 1.073 | 1.75E-14 |
LOC104911002 | Dickkopf-related protein 3-like | −2.513 | 4.9E-08 | −1.496 | 9.78E-05 | 1.626 | 6.64E-10 | 1.797 | 3.55E-19 |
LOC100540803 | Delta and Notch-like epidermal growth factor-related receptor | −2.602 | 0.003208 | −2.278 | 0.009568 | 1.750 | 6.59E-05 | 1.973 | 6.31E-07 |
LOC100545932 | Dystrobrevin alpha | −1.700 | 0.004161 | −2.497 | 4.6E-06 | 1.703 | 1.34E-06 | 1.535 | 5.69E-08 |
LOC104910320 | Dystrobrevin alpha-like | −2.452 | 0.000109 | −2.072 | 1.99E-05 | 1.320 | 0.00051 | 1.007 | 0.001196 |
LOC104910337 | Dystrobrevin alpha-like | −2.896 | 0.028765 | −2.277 | 0.010759 | 2.334 | 1.68E-05 | 1.380 | 0.001723 |
LOC100539830 | Dysferlin-like | −1.673 | 0.000158 | −1.487 | 0.001369 | 1.095 | 0.000961 | 1.553 | 3.96E-07 |
FRAS1 | Fraser extracellular matrix complex subunit 1 | −1.772 | 0.001852 | −2.548 | 1.79E-08 | 2.296 | 6.27E-12 | 1.137 | 7.91E-06 |
GNG2 | Guanine nucleotide binding protein (G protein), gamma 2 | −1.704 | 1.86E-06 | −1.474 | 2.75E-06 | 1.126 | 9.24E-06 | 1.158 | 4.56E-08 |
GRIN2A | Glutamate receptor, ionotropic, N-methyl D-aspartate 2A | −2.471 | 4.6E-12 | −1.899 | 1.35E-14 | 1.115 | 1.98E-07 | 1.354 | 4.38E-22 |
GUCA1B | Guanylate cyclase activator 1B (retina) | −3.228 | 2.28E-05 | −6.281 | 2.68E-05 | 2.990 | 1.44E-23 | 3.316 | 1.18E-25 |
LOC104909294 | Hypermethylated in cancer 1 protein | −2.281 | 2.93E-05 | −3.489 | 6.51E-10 | 1.876 | 2.64E-09 | 2.233 | 1.28E-29 |
LOC100541783 | Hypermethylated in cancer 1 protein-like | −1.977 | 0.027443 | −3.105 | 0.002499 | 2.380 | 3.83E-09 | 2.684 | 2.86E-15 |
INHA | Inhibin, alpha | −5.346 | 1.21E-15 | −6.115 | 4.21E-20 | 1.205 | 2.72E-05 | 1.180 | 2.92E-07 |
LOC100547979 | Junctophilin-1 | −4.175 | 1.97E-17 | −3.904 | 7.22E-23 | 2.198 | 2.21E-19 | 1.723 | 2.29E-23 |
LOC104917153 | Small conductance calcium-activated potassium channel protein 3 | −1.103 | 0.027791 | −1.368 | 0.000591 | 1.241 | 0.000199 | 1.234 | 1.4E-06 |
KLB | Klotho beta | −2.008 | 4.52E-14 | −2.114 | 1.07E-43 | 1.060 | 3.28E-07 | 1.049 | 1.72E-16 |
KLHL31 | Kelch-like family member 31 | −2.298 | 4.77E-08 | −3.752 | 1.06E-19 | 2.070 | 1.16E-12 | 1.284 | 5.06E-08 |
LEF1 | Lymphoid enhancer-binding factor 1 | −5.804 | 0.002693 | −4.107 | 0.00012 | 1.684 | 0.003038 | 1.957 | 1.61E-06 |
LHFPL3 | Lipoma HMGIC fusion partner-like 3 | −1.892 | 0.001752 | −1.144 | 0.005348 | 1.391 | 0.000141 | 1.788 | 6.76E-14 |
LOC104912085 | Uncharacterized LOC | −1.805 | 6.64E-11 | −1.791 | 3.01E-07 | 1.795 | 4.69E-15 | 1.270 | 5.13E-06 |
LOC104914708 | Uncharacterized LOC | −3.041 | 4.19E-08 | −2.491 | 1.47E-16 | 1.414 | 8.5E-05 | 1.543 | 3.96E-23 |
LTBP2 | Latent transforming growth factor beta binding protein 2 | −2.355 | 4.6E-44 | −2.465 | 4.81E-43 | 1.535 | 2.35E-24 | 1.854 | 5.97E-21 |
LOC104909252 | Myosin-3-like | −6.600 | 1.56E-05 | −6.693 | 6.28E-07 | 1.071 | 0.039632 | 1.195 | 0.003104 |
LOC100543020 | Myosin-7-like | −3.828 | 1.32E-05 | −3.409 | 0.000241 | 1.192 | 0.007426 | 1.221 | 0.004462 |
LOC100549331 | Myosin-7-like | −5.467 | 4.06E-71 | −6.412 | 0 | 1.275 | 3.75E-06 | 1.390 | 6.35E-31 |
LOC100544354 | Uncharacterized LOC | −5.397 | 0.014658 | −5.702 | 0.002833 | 1.954 | 0.002783 | 1.448 | 0.009527 |
LOC104914128 | Uncharacterized LOC | −2.861 | 0.00076 | −3.030 | 1.8E-05 | 1.456 | 0.001195 | 1.698 | 7.65E-09 |
NFATC2 | Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 | −2.476 | 0.020677 | −2.123 | 0.003688 | 1.779 | 0.000711 | 1.675 | 2.65E-05 |
OASL | 2′–5′-Oligoadenylate synthetase-like | −1.909 | 1.5E-08 | −1.554 | 4.62E-09 | 2.351 | 1.3E-29 | 2.333 | 7.81E-66 |
OPCML | Opioid binding protein/cell adhesion molecule-like | −2.642 | 1.09E-11 | −1.680 | 1.43E-21 | 1.270 | 2.36E-06 | 2.338 | 5.65E-21 |
P2RY1 | Purinergic receptor P2Y, G-protein coupled, 1 | −1.507 | 0.000988 | −1.140 | 0.013083 | 1.259 | 6.78E-05 | 1.932 | 6.03E-16 |
PALMD | Palmdelphin | −2.571 | 3.06E-05 | −4.140 | 5.6E-09 | 1.568 | 5.2E-06 | 1.613 | 2.51E-08 |
LOC104914006 | Receptor-type tyrosine-protein phosphatase T-like | −2.385 | 1.73E-12 | −2.643 | 2E-28 | 2.389 | 1.61E-33 | 1.880 | 5.84E-89 |
LOC104914794 | Iporin-like | −1.551 | 3.48E-05 | −1.618 | 2.51E-10 | 1.509 | 9.48E-07 | 1.310 | 1.39E-12 |
RYR1 | Ryanodine receptor 1 (skeletal) | −2.016 | 9.81E-05 | −2.523 | 0.000488 | 1.361 | 2.2E-05 | 1.894 | 1.47E-09 |
LOC104916851 | Ryanodine receptor 1-like | −1.297 | 0.01007 | −2.003 | 7.49E-06 | 1.469 | 9.89E-06 | 1.875 | 1.34E-17 |
LOC104917345 | Ryanodine receptor 1-like | −1.549 | 0.003033 | −1.727 | 0.00029 | 1.208 | 0.001046 | 1.777 | 3.21E-13 |
LOC104913331 | Putative E3 ubiquitin-protein ligase SH3RF2 | −5.398 | 0.013363 | −6.023 | 0.000157 | 2.091 | 0.000434 | 1.132 | 0.035376 |
SHROOM1 | Shroom family member 1 | −2.387 | 7.34E-07 | −2.096 | 3.88E-07 | 1.263 | 4.57E-05 | 1.482 | 7.73E-11 |
LOC100546217 | protein TENP | −5.045 | 0.038689 | −4.947 | 0.043268 | 1.767 | 0.021551 | 3.304 | 1.16E-12 |
TIMP3 | TIMP metallopeptidase inhibitor 3 | −1.582 | 0 | −1.683 | 0 | 1.690 | 5.37E-33 | 1.422 | 8.26E-31 |
LOC100542775 | Alpha-2-macroglobulin-like protein 1 | 2.407 | 7.18E-09 | 2.252 | 2.22E-11 | −1.990 | 0.040101 | −2.381 | 0.00191 |
C1QTNF4 | C1q and tumor necrosis factor related protein 4 | 1.106 | 1.2E-15 | 1.168 | 8.66E-38 | −1.107 | 2.2E-12 | −1.070 | 0 |
CD36 | CD36 molecule (thrombospondin receptor) | 2.300 | 2.05E-46 | 2.479 | 9.79E-46 | −2.606 | 3.59E-17 | −2.131 | 3.52E-27 |
CNDP1 | Carnosine dipeptidase 1 (metallopeptidase M20 family) | 1.117 | 0.007658 | 1.413 | 1.9E-05 | −3.750 | 1.16E-07 | −1.882 | 0.000797 |
LOC100550279 | HLA class II histocompatibility antigen, DM beta chain-like | 1.627 | 4.24E-29 | 1.928 | 6.54E-50 | −1.339 | 0 | −2.694 | 2.97E-91 |
ENTPD3 | Ectonucleoside triphosphate diphosphohydrolase 3 | 1.002 | 0.012146 | 1.079 | 3.4E-05 | −1.395 | 0.005746 | −1.241 | 0.001609 |
ESAM | Endothelial cell adhesion molecule | 1.958 | 1.42E-07 | 2.116 | 5.48E-25 | −1.339 | 0.012003 | −2.291 | 9.08E-08 |
ETV7 | ets variant 7 | 2.160 | 2.67E-06 | 1.664 | 5.7E-08 | −2.342 | 0.003252 | −2.101 | 0.000492 |
LOC104917139 | Germin-like protein subfamily 2 member 2 | 1.627 | 0.000198 | 1.141 | 6.34E-05 | −5.294 | 4.91E-09 | −3.698 | 1.06E-09 |
LOC100539100 | Guanylate cyclase soluble subunit beta-2-like | 2.063 | 6.72E-21 | 1.708 | 2.8E-25 | −1.209 | 0.000197 | −1.081 | 4.92E-05 |
LOC100543128 | Histone H3-like | 1.487 | 1.79E-07 | 1.169 | 1.34E-10 | −2.755 | 5.02E-09 | −2.480 | 2.18E-18 |
IL18BP | Interleukin 18 binding protein | 1.513 | 0.000535 | 1.325 | 4.57E-06 | −2.087 | 0.000679 | −2.409 | 3.58E-06 |
LOC104916207 | Kallikrein-8-like | 1.284 | 0.000119 | 1.215 | 6.35E-06 | −1.304 | 0.006723 | −1.537 | 0.000479 |
LRAT | Lecithin retinol acyltransferase (phosphatidylcholine–retinol O-acyltransferase) | 1.219 | 1.27E-05 | 1.178 | 4.02E-10 | −1.302 | 0.000548 | −1.028 | 0.000202 |
LOC100549167 | Mannose-binding protein A-like | 1.670 | 0.002909 | 1.232 | 0.010338 | −3.692 | 0.004571 | −3.695 | 0.00115 |
MTNR1A | Melatonin receptor 1A | 1.515 | 0.000877 | 1.153 | 0.0135 | −2.098 | 0.009624 | −1.611 | 0.023993 |
LOC104909506 | Uncharacterized LOC | 1.567 | 0.006758 | 1.695 | 5.46E-05 | −2.746 | 0.021177 | −2.932 | 0.002457 |
LOC104910409 | Uncharacterized LOC | 1.067 | 0.003554 | 1.193 | 7.65E-05 | −2.385 | 7.52E-06 | −1.160 | 0.010109 |
LOC104911590 | Uncharacterized LOC | 1.319 | 2.43E-06 | 1.103 | 9.54E-09 | −1.940 | 3.03E-07 | −2.072 | 8.46E-11 |
LOC104912270 | Uncharacterized LOC | 1.400 | 3.32E-11 | 1.230 | 2.85E-15 | −1.335 | 6.18E-05 | −1.116 | 4.73E-08 |
LOC104912544 | Uncharacterized LOC | 1.332 | 2.14E-05 | 1.304 | 8.93E-10 | −1.550 | 0.000542 | −1.963 | 2.5E-08 |
LOC104913567 | Uncharacterized LOC | 1.240 | 1.61E-14 | 1.029 | 6.62E-35 | −1.328 | 2.57E-11 | −1.137 | 3.08E-26 |
LOC104913826 | Uncharacterized LOC | 1.515 | 1.98E-22 | 1.564 | 4.58E-37 | −1.109 | 9.44E-07 | −1.104 | 5.72E-11 |
LOC104914493 | uncharacterized LOC | 1.418 | 0.001313 | 1.516 | 5E-05 | −1.869 | 0.007214 | −2.320 | 0.002089 |
NOXO1 | NADPH oxidase organizer 1 | 1.653 | 1.97E-10 | 1.357 | 7.28E-17 | −2.124 | 1.47E-09 | −1.591 | 6.04E-09 |
NTSR1 | Neurotensin receptor 1 (high affinity) | 1.209 | 0.029619 | 1.309 | 0.001145 | −2.517 | 0.002856 | −2.140 | 0.004664 |
PCBP3 | Poly(rC) binding protein 3 | 1.527 | 8.42E-09 | 1.406 | 8.33E-16 | −1.491 | 7.04E-05 | −1.521 | 4.59E-08 |
PIGM | Phosphatidylinositol glycan anchor biosynthesis, class M | 1.107 | 0.000363 | 1.095 | 4.43E-07 | −1.324 | 0.000604 | −1.023 | 0.000899 |
PLS1 | Plastin 1 | 1.251 | 2.14E-09 | 1.473 | 1.37E-32 | −2.179 | 3.22E-15 | −1.691 | 3.69E-20 |
PTGR1 | Prostaglandin reductase 1 | 2.046 | 7.33E-09 | 2.261 | 2.27E-14 | −1.937 | 0.001524 | −2.169 | 0.00077 |
RANBP17 | RAN binding protein 17 | 1.835 | 6.16E-08 | 1.038 | 5.8E-05 | −1.391 | 0.017844 | −2.861 | 8.69E-10 |
SERPINF2 | Serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 | 1.395 | 2.6E-16 | 1.163 | 1.15E-27 | −1.207 | 1.55E-08 | −1.096 | 1.28E-14 |
SH2D4A | SH2 domain containing 4A | 1.314 | 6.2E-07 | 1.371 | 8.49E-13 | −1.828 | 2.37E-07 | −2.367 | 1.25E-11 |
SH2D5 | SH2 domain containing 5 | 1.108 | 0.019073 | 1.063 | 0.00494 | −2.319 | 0.000812 | −1.171 | 0.030757 |
SH3GLB2 | SH3-domain GRB2-like endophilin B2 | 1.380 | 5.63E-21 | 1.065 | 1.78E-49 | −1.095 | 7.41E-11 | −1.345 | 3.32E-40 |
LOC104915044 | spErm-associated antigen 4 protein-like | 2.185 | 2.89E-06 | 1.644 | 1.1E–05 | −3.967 | 0.000862 | −2.010 | 0.007625 |
Comparisons between lines within temperature treatment found relatively few DE genes at the three incubation temperatures (Table
Distribution of differentially expressed genes between lines (F-line vs. RBC2) during p. major satellite cell differentiation. For each temperature comparison, the number of genes with FDR
In contrast to the control temperature, the numbers of genes where the cold (33°C) and hot (43°C) treatments significantly affected expression were considerably higher (Table
Significant DE genes in between-line comparisons [FDR
TECRL | Trans-2,3-enoyl-CoA reductase-like | −4.141 | 1E-07 | −10.569 | 0.0037668 | −6.170 | 6.44 |
CNGA3 | Cyclic nucleotide gated channel alpha 3 | 2.740 | 0.0030845 | 1.741 | 1.000 | 4.090 | 0.0007882 |
COL24A1 | Collagen, type XXIV, alpha 1 | −2.815 | 1.983E-06 | −2.710 | 1.000 | −2.851 | 8.81 |
LOC100539697 | Integrin beta-like protein 1 | 2.998 | 0.0318933 | 1.024 | 1.000 | 3.096 | 0.0088252 |
LOC104915513 | Histone deacetylase 7-like | −3.813 | 0.0051096 | −5.804 | 1.000 | −4.703 | 4.932 |
LOC104917072 | Zinc finger protein 502-like | −3.963 | 2.294E-06 | −2.846 | 1.000 | −5.982 | 0.0010235 |
MUC3A | Mucin 3A, cell surface associated | −6.218 | 0.0065859 | −4.082 | 1.000 | −6.338 | 1.051 |
ROBO2 | Roundabout, axon guidance receptor, homolog 2 (Drosophila) | −3.456 | 3.467E-05 | −1.795 | 1.000 | −2.654 | 0.0011681 |
Greater differential expression between the F and RBC2 cells was observed at 43°C (Table
Muscle satellite cells are self-renewing and give rise to differentiated cell types, thus being true stem cells. Satellite cells and myofibers are of the same origin, being derived from somites (Armand et al.,
The function of satellite cells is modulated by the cellular microenvironment and quiescent satellite cells are identifiable and being both PAX7 and MYF5 positive. The state of satellite cells is defined by antagonistic Notch and Wnt signaling, with Notch maintaining PAX7 and Wnt signaling driving
Thermal challenge of the turkey satellite cells had no significant effect on expression of
The gene
In addition to maintaining PAX7, Notch may also signal satellite cells to stop proliferating (Conboy and Rando,
The suggested upstream effects on AHR (Aryl Hydrocarbon Receptor) transcription factor, and the transforming growth factors TGFB1 and TGFB3 observed in comparison of the challenged cells are of interest because of the consistent directionality of expression changes and shared significance observed across treatment comparisons in the turkey satellite cells. Of particular interest is the effect of TGFB1 on genes like
In addition to TRPC channels, differentiating satellite cells beginning to express proteins responsible for regulation of Ca2+ homeostasis, including channel proteins, Ca2+-pumps, Ca2+-storage proteins, and proteins the regulate the activity of channels and pumps. These proteins are localized to the sarcoplasmic reticulum (SR) and the sarcolemma. The SR calcium release channels or ryanodine receptors RYR1 and RYR3 serve as conduits for Ca2+ to enter the sarcoplasm from the lumen of the sarcoplasmic reticulum (Rossi and Dirksen,
Interestingly, most of these genes associated with regulation of Ca2+ homeostasis were significantly affected by temperature treatment, particularly cold treatment. The
Among the most significantly altered gene pathways in the turkey satellite cells based on activation score were those corresponding to the eIF2, eIF4, and p70S6K, and mTOR signaling. The eIF2 imitation complex integrates a diverse array of stress-related signals to regulate mRNA translation, especially in response to stress. Likewise, eIF4 and p70S6K signaling play critical roles in translation regulation (Sonenberg and Hinnebusch,
The mTOR protein is central to two multi-protein complexes with distinct cellular functions that integrate cellular signals to regulate metabolism, proliferation and autophagy (Laplante and Sabatini,
Temperature can also stimulate the transdifferentiation of satellite cells to an adipogenic phenotype. Harding et al. (
Further support for the conversion of breast muscle satellite cells to an adipogenic cell fate, was the change in expression of
The post-hatch time-frame is critical and exposure of poultry to extreme temperatures, can seriously compromise the quality of meat. This study demonstrates significant alterations in gene expression and supports the hypothesis that satellite cell differentiation is directly altered in turkeys in response to ambient temperature. Numerous expression differences were observed between cells incubated at both lower (33°C) and higher (43°C) temperatures as compared to control (38°C). Greater expression differences were seen in the cold treatments where a greater number of the DE genes were down regulated. Fewer expression differences in the differentiating cells were observed between the genetic lines than observed for proliferating cells in the same experimental system (Reed et al.,
KR, SV, and GS conceived and designed the experiments. SV and KM performed the experiments. KR and KM analyzed the data. KR, KM, SV, and GS drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank Cindy Coy for technical assistance in culturing the satellite cells used in this study. Juan Abrahante, University of Minnesota Informatics Institute, assisted with data processing.
The Supplementary Material for this article can be found online at:
Hierarchical clustering of samples based on Euclidean distance reiterated relationships shown by PCA. Global gene expression differences among groups are illustrated in the heat map constructed from the co-expressed genes with the greatest experiment-wise differences in average normalized expression.
GO classification of genes expressed in cultured turkey
Volcano plot showing the relationship between the ANOVA
Distribution of differentially expressed genes for cold (33 vs. 38°C) and hot (43 vs. 38°C) comparisons of each line (RBC2 and F) during
Example gene networks identified from differentially expressed genes using QIAGEN's Ingenuity® Pathway Analysis (IPA®, QIAGEN Bioinformatics) software showing differential response of
Predicted upstream effects for three DE genes (