The Journal of Neuroscience, October 25, 2006, ():
Functional Genomic Analysis of Oligodendrocyte Differentiation
J. Neurosci. Dugas et al.
26: 10967
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Figure S1. Reproducibility and clustering of distinct temporal patterns of OL gene expression. A-E. In all graphs, expression levels are determined as fold changes relative average OPC levels and expressed on a log2 scale at the time points indicated; “AcOL” = acutely purified OL samples. A-C. The individual expression values for the four biologically independent repetitions of the time course are shown in colors, and the average expression values are shown in black. A. Examples of upregulated genes. OSP (rc_AA901342_at; squares) is reproducibly upregulated 3-4 days earlier than MOG (M99485_at; triangles). B. Examples of downregulated genes. CITED2 (rc_AI176963_at; CIT, circles) is repressed immediately after switch into differentiation medium, whereas cyclin B1 (X64589_at; CyB, X’s) peaks and is then downregulated. C. Example of a gene selected by solely by ≥4x changes (i.e. not statistically selected) that was reproducibly upregulated predominantly in acutely purified OLs (Carbonic Anhydrase 2; X58294_at, CA). D-E. Ad hoc clustering of upregulated genes from Table S2 with distinct time courses of induction. Probe sets were selected from Table S2 that are either robustly induced immediately after the initiation of OL differentiation (D), or are most strongly induced after three or more days in the presence of differentiation-promoting signals (E). Selection based on the time at which the greatest fold increase is seen between two subsequent time points in the in vitro time course, and also by comparing expression patterns to the observed early and late phases of myelin gene expression. Individual probe sets and values plotted are provided in Table S5. F-G. Functional category frequency of genes induced immediately (F) or after a three or more day delay (G) following initiation of OL differentiation. Multiple probe sets recognizing the same gene were categorized only once; some single genes are assigned to more than one functional group. Categories depicted are similar to those listed in Fig. 6.
- supplemental material
-
Figure S2. Expression of pancreatic lipase detected in CNS white matter. Verification of pancreatic lipase expression in mature optic nerves by western blot. Optic nerves were isolated from P3, P19, and adult (Ad) rats. Homogenate was tested for pancreatic lipase expression by probing with antibody to human pancreatic lipase (PL), which detects the protein at ~50kD and degradation products. The higher MW band detected at P3, P19, and adult is likely keratin (Mark Lowe, personal communication). hPL: sample of purified human pancreatic lipase. To confirm equal loading, the blot was stripped and probed for actin (Ac).
- supplemental material
-
Figure S3. Knock down of SOX10 and ZFP536 by siRNA. Semi-quantitative RT-PCR was performed on RNA collected from siRNA-transfected OLs. Cultured OPCs transfected with either a non-targeting siControl, siRNAs targeting SOX10 (siSOX10), or siRNAs targeting ZFP536 (siZFP536) were cultured in differentiation-promoting conditions (-PDGF, -NT-3, +T3) for 4 days in vitro before cells were lysed and total RNA purified. SuperScript III (Invitrogen) was used to reverse transcribe equivalent starting amounts of RNA, and then PCR reactions were performed to detect actin (first row; primer sequences 5’-CACTGGCATTGTGATGGACTCC-3’ and 5’-TGTTGGCATAGAGGTCTTTACGG-3’; 24 and 26 cycles), SOX10 (second row; primer sequences 5’-GCCACCCATCCCCAGAAATAAC-3’ and 5’-ATTGAGTGTCCCACCCTGCTCTTC-3’; 24 and 26 cycles, and ZFP536 (third row; primer sequences 5’-ACAGTTTGGCGTTTACCCCAC-3’ and 5’-GCTAAAGGTGAGACCAGTCCCTG-3’; 28 and 30 cycles). Strongest repression of SOX10 is seen with siSOX10 (second columns), and strongest repression of ZFP536 is seen with siZFP536 (third columns) compared to controls (first columns). Actin PCR demonstrates similar expression of non-targeted genes.
- supp
-
Table S1. Terminal OL differentiation time course data. Expression data for every Affymetrix probe set analyzed in the OL differentiation time course experiment. The identity of each probe set assayed from the complete Affymetrix RG-U34 A-C chip set is listed under Probe Set ID. Stat Rank: statistical relevance rankings (1 = best) as computed by a one-sample Hotelling T2 statistic. All expression values listed under OPC, d1-d9 correspond to in vitro time course points, acute OL corresponds to acutely purified OL samples; average raw expression values calculated as described in Experimental Procedures. All other columns are directly from Affymetrix-supplied RG-U34 annotations.
- supplemental material
-
Table S2. Probe sets highly regulated during terminal OL differentiation. 953 Affymetrix probe sets selected as most strongly and reproducibly regulated during OL differentiation. All numerical data is averaged from four independent biological replicates of the time course (Fig. 1), and four separate acute OL purifications (“AcOL”). Selection: the list is composed of the 824 probe sets that on average change ≥4x relative to average OPC expression levels (“4-fold”) combined with the top 824 statistically relevant probe sets, selected by a one-sample moderated Hotelling T2 statistic (“stat”); “both” refers to probe sets selected by both methods. Probe Set ID: Selected Affymetrix RG-U34A-C probe sets. Abs call: average absolute call values obtained from MAS 5.0 (Affymetrix) at indicated time points for probe sets – Present = 1, Marginal = 0.5, Absent = 0. Signal: average raw expression values calculated as described in Experimental Procedures. Norm: average fold expression change relative to OPC levels, expressed on a log2 scale. Fold Change: highest expression level / lowest expression level in time course; if highest expression level precedes lowest expression level, expressed as negative fold change. Pattern: description of expression patterns relative to OPC – most pattern notations based on in vitro time course, where acuteOL levels are similar to d9 levels; “-acute” denotes acute OL expression ≥2x higher than d9 in vitro expression; “type-2” denotes in vitro upregulation, then acute OL levels close to or lower than OPC levels. UniGene, Gene, Chromosome: NCBI UniGene and Gene accession numbers, and chromosomal locations are given for each listed Affymetrix rat probe set (r-), and also for mouse (m-) and human (h-) homologs where available (http://www.ncbi.nlm.nih.gov/). Category: classification of gene function, with the categories used listed at the top of the column; if multiple probe sets correspond to the same gene, only one is classified, with the rest listed as duplicates (“2”); putative categorizations are listed with a “!” after the chosen category. Name: gene name, which was occasionally determined from mouse or human homolog if rat gene is listed only as a transcribed locus. Affy Target Description, Gene Title, and Gene Symbol are from Affymetrix-supplied annotations.
- supplemental material
-
Table S3. Regulation of known CNS myelin genes. Genes from Table S2 that have been previously reported as enriched in CNS myelin sheaths are listed. Most columns are directly from Table S2. Symbol: gene symbols listed in the key of Fig. 3A-B. In cases where more than one probe set representing the same gene was strongly regulated and present on Table S2, all are listed, and the OPC-normalized log2 expression values are averaged (e.g. “MBP average” in Probe Set ID column). These average expression values are the ones plotted in Fig. 3.
- supplemental material
-
Table S4. Independent comparative RT-PCR verification of gene expression patterns. 55 genes from Table S2 that were selected for RT-PCR based independent verification of expression patterns. Probe Set ID, NAME, selection, pattern and fold change directly from Table S2. Peak Level: highest raw level of expression seen at any time point in the gene chip experiment. Verify? Indicates whether relative gene expression level in OPCs vs. OLs matches in both the gene chip data and the RT-PCR verifications (e.g. higher gene expression independently indicated in OLs by both methods).
- supplemental material
-
Table S5. Clustering of early vs. late induced OL genes. Probe sets plotted in Fig. S1D-E that are either induced immediately (EARLY) or only after a three or more day delay (LATE) following the initiation of OL differentiation. All depicted values directly from Table S2.
- supplemental material
-
Table S6. Regulation of transcription factor genes. Genes from Table S2 that are suspected transcription factors are listed. Columns shown are taken directly from Table S2. In cases where more than one probe set representing the same gene was strongly regulated and present on Table S2, all are listed, and the OPC-normalized log2 expression values are averaged (e.g. “average SOX11” in Probe Set ID column). These average expression values are the ones plotted in Fig. 4: “up” and “up-acute” patterns are plotted in Fig. 4A-B, “down” and “dip-down” patterns are plotted in Fig. 4C-D, and “peak-down” patterns are plotted in Fig. 4E. Symbol: gene symbols listed in the keys of Fig. 4 graphs. 37 of these genes were assayed and expression patterns independently confirmed by RT-PCR. Note that ATF5, BTBD3, and SHARP1 were not plotted as these genes failed independent verifications (Table S4).
- supplemental material
-
Table S7. Regulation of cell cycle control and DNA synthesis / repair genes. Genes from Table S2 that are either known regulators of the cell cycle or involved in DNA synthesis or repair are listed. Columns shown are taken from Table S2. In cases where more than one probe set representing the same gene was strongly regulated and present on Table S2, all are listed, and the OPC-normalized log2 expression values are averaged (e.g. “average Cyclin D1” in Probe Set ID column). These average expression values are the ones plotted in Fig. 3C-E: “dip-down” and “down” patterns are plotted in Fig. 3C, “peak-down” patterns are plotted in Fig. 3D, “up” and “up-acute” patterns are plotted in Fig. 3E.
