Differentially expressed (DE, P ≤ 0.05) genes in SL samples (NV, EV, AV and CV) relative to BL control samples
Relative to the BL sample, 395, 522, 524 and 526 DE genes were identified for NV, EV, AV and CV samples, respectively, which gave rise to 151, 213, 212 and 212 DE genes for Ingenuity Pathways Analysis (IPA) after deletion of genes without information, genes from cDNA libraries and clones and genes related to hypothetical proteins. The top 10% of up- and down-regulated DE genes for individual SLV states are summarized in Table .
Top 10% DE genesa in growing feathers from SL chickens relative to non-vitiliginous parental BL controls
The magnitude of the fold change of the top 10% up-regulated genes was lowest in NV samples, intermediate in EV and CV and highest in AV samples. The three up-regulated DE genes with the highest expression were immunoglobulin J chain (IGJ), acidic chitinase (CHIA) and T cell receptor (TCR) delta in NV; chemokine ligand 13 (CXCL13), guanylate binding protein (GBP) and lipopolysaccharide-induced TNF factor (LITAF) in EV; sodium channel alpha subunit, IGJ and CXCL13 in AV; and, IGJ, POU class 2 associating factor 1 (POU2AF1) and myeloid antimicrobial peptide 27 in CV. The magnitude of the fold change of down-regulated DE genes was similar in NV, EV, and AV samples, but higher in CV samples due to marked decreased expression of solute carrier family 24 member 5 (SLC24A5), tyrosinase (TYR), and matrix metalloproteinase 115 (MMP115).
Some of the top 10% DE genes were shared by more than two SL samples, although their expression-levels varied. Up-regulated DE genes shared by EV, AV and CV samples (SLV samples) included LITAF, tumor necrosis factor superfamily (TNFSF) 13B, interleukin 21 receptor (IL21R), complement component 3a receptor 1 (C3AR1) and sodium channel alpha subunit, with higher expression levels observed in AV than in EV and CV samples. Down-regulation was noticed in lipoprotein VSAF, matrix metallopeptidase 9 (MMP9), MMP13, growth hormone induced transmembrane protein (GHITM), transmembrane protein 22 (TMEM22), keratin, flavin-containing monooxygenase 6 (FMO6), and histone cluster 2H3C (HIST2H3C) in all SL samples (NV and SLV samples), with lipoprotein VSAF exhibiting the largest decrease in expression. Other down-regulated DE genes observed in SLV samples were parathyroid hormone (PTH) and tachykinin precursor 1 (TAC1), with larger decreases in PTH expression in AV and CV samples than in EV samples. Expression of SLC24A5, MMP115, and TYR, and genes for crystalline alphaB (CRYAB), Shikimate 5-dehydrogenase and amyotrophic lateral sclerosis 2 (ALS2) were commonly depressed in AV and CV samples relative to BL controls.
Within SL comparisons of DE genes in NV, EV, AV and CV samples
JMP genomics 4 analysis revealed 206 DE genes for within SL comparisons, which resulted in 88 DE genes after deletion of unknown genes (without information, from cDNA libraries and clones and related to hypothetical proteins). The 88 DE genes could be roughly divided into 4 functional groups which were immunity-, melanocyte-, stress-related and others (Table ) based on functions of their corresponding proteins. More than half of the DE genes were immune system related, including molecules of both innate and adaptive branches of the immune system with more DE genes relating to innate than adaptive immune response activities (Table ). The group of DE genes related to stress contained DE genes associated with cellular stress and apoptosis.
Comparison of DE genes between SL samples (within SL comparison)*
Compared to SLV samples, the majority of DE genes in NV samples had the lowest expression level except for all DE genes related to melanocyte functions (MMP115, TRP1, TYR and SLC24A5, SLC24A2, GRP143, V-ATPase C2 subunit and Shikimate 5-dehydrogenase). Additionally, DE genes related to stress (NPY and CRYAB) and intra/inter-cellular transport [gap junction protein alpha 5 (GJA5), transmembrane protein 9 (TMEM9) and synaptotagmin 12 (SYT12)], exhibited the highest expression in NV samples (Table ). For most DE genes, the expression levels in EV and CV samples were intermediate to their expression in NV and AV samples. All genes related to immune system activities had the highest expression levels in AV compared to NV, EV and CV samples (Table ). The expression levels of DE genes in CV samples tended to be comparable to EV and/or NV samples. Expression of melanocyte-related DE genes (i.e. MMP115, TYR, TRP1, SLC24A2, SLC24A5, V-ATPase C2 subunit), however, was decreased significantly in AV and reached the lowest expression in CV samples compared to those from NV and EV samples. DE genes in the others category, e.g. intra/inter-cellular transport (GJA5 and TMEM9), also exhibited the lowest expression levels in CV compared to NV, EV and AV samples (Table ).
Ingenuity Pathway Analysis (IPA) for SL samples
The three major output components provided by Ingenuity Pathway Analysis (IPA) are function, network and pathway interpretations (IPA, Version 9.0; Ingenuity Systems®
). The functional analysis identifies biological functions and/or diseases that are most closely related to the data set. Network interpretations are algorithmically generated by overlying genes from a data set onto a global molecular network developed from information contained in Ingenuity's Knowledge Base. Canonical pathway analysis identifies the pathways from the Ingenuity Pathways Analysis library of canonical pathways that most significantly fit the data set (IPA, Version 9.0; Ingenuity Systems®
IPA analysis provided similar composition and ranking of functions for all SL samples (Figure ). Common functions not only included those related to normal biological functions, such as cellular-function and -development and lipid metabolism, but also those associated with a variety of diseases, e.g. immunological disease, neurological disease, dermatological disease/condition and cancer. Among them, the inflammatory response was the most significant (lowest P values which were due to chance alone) functional interpretation and the first function identified for all SL groups. However, for any particular common function identified in the IPA analysis of SL samples, the associations were made with higher confidence (lower P values) for DE genes in SLV (EV, AV and CV) samples than in NV samples. Unique functional interpretations of NV DE genes included digestive system development and function as well as organismal survival. Based on function analysis of DE genes identified by within SL comparisons, amino acid/carbohydrate metabolism, energy production, protein folding and developmental disorder were uniquely demonstrated, however with very low confidence.
Figure 1 Graphical demonstration of associated functions from Ingenuity Pathway Analysis (IPA) of differentially expressed (P ≤ 0.05; DE) genes for NV, EV, AV, CV relative to BL samples. The functional analysis identifies biological functions and/or diseases (more ...)
Network output provided number wise more networks for DE genes in SLV samples than for DE genes in NV samples or DE genes identified by within SL comparison. Functional analysis of networks demonstrated that functions for the NV samples and the within SL comparison group were part of the functional networks identified for DE genes in SLV samples. In addition to shared functions between all groups, humoral immune response and immunological disease were only present in EV samples, and cell-mediated immune response was unique to CV samples. The 1st network for NV samples was associated with functions of cellular movement, immune cell trafficking and cell morphology; for EV samples, functions of inflammatory response, antimicrobial response and humoral immune response; and for AV and CV samples, inflammatory response, cellular growth and proliferation and hematological system development and function, and cellular development. These number one networks are illustrated in Figure .
Figure 2 Network #1 obtained by Ingenuity Pathway Analysis (IPA) of differentially expressed (P ≤ 0.05; DE) genes for NV, EV, AV, CV relative to BL samples. DE genes from the data sets were overlaid onto a global molecular network developed from information (more ...)
Canonical pathway analysis
Pathway composition was similar for DE genes in SL samples, with higher levels of similarity between DE genes for SLV samples. For a specific common pathway higher significance [suggested by a higher ratio (number of molecules from the data set divided by the total number of molecules in the pathway obtained from the Ingenuity's Knowledge Base) and a lower P value] was exhibited for SLV samples than for NV samples. Pathway analysis of the DE data-set generated by within SL comparison had numerically the most pathways which included all pathway components for all SL samples. The pathway of communication between innate and adaptive immune cells was most significant, constituting the number-one pathway for all SL samples and within SL comparison.
Microarray validation by qRT-PCR
Microarray data were validated by qRT-PCR based on comparable gene expression levels and similar expression trends of selected targets throughout SLV development (Table ). Fold changes in complement component receptor 2 (CR2) and POU2AF1 from qRT-PCR, however, were noticeable higher than those from microarray.
Relative expression of DE genes in SL samplesa from microarray vs. qRT-PCR analysisb