Extensive molecular differences between anterior- and posterior-half-sclerotomes underlie somite polarity and spinal nerve segmentation

Additional file 1:

Affymetrix identifiers and ranks for 11 known differentially-expressed sclerotome genes. Affymetrix identifiers of genes known to be differentially-expressed in A- or P-half-sclerotome. Multiple entries for the same gene reflect the transcripts represented on the Affymetrix GeneChip (26 transcripts corresponding to 11 genes, bold indicating the highest probability transcript for each gene). The probability of differential gene expression by Fisher's analysis, and the overall rank in array data are also listed.

Format: DOC Size: 61KB Download file

This file can be viewed with: Microsoft Word Viewer

Additional file 2:

Further clustering of individual array experiments. Dendrograms for agglomerative hierarchical clustering of individual array hybridization experiments showing that arrays do not separate into A- or P-clusters when analysed against: (i) the whole genome (45,101 GeneChip transcripts); (ii) sclerotome-expressed genes without differential A-P expression (pax1, pax9, scleraxis); (iii) general somite markers (cdx1, etv5, fgf6, foxc1, foxc2, pax1, pax3, pax7, pax9, myoD, myogenin, myf5, scleraxis, sfrp2); or (iv) house-keeping genes (β-actin and gapdh).

Format: PDF Size: 584KB Download file

This file can be viewed with: Adobe Acrobat Reader

Additional file 3:

Distribution of known transcripts amongst all rank-ordered transcripts. (i) The distribution of 11 transcripts, with known differential expression between A- and P- half-sclerotome, shows that while most are evenly distributed across the rank-ordered transcripts, more than one-third fall within the cell representing the highest statistical certainty of differential expression (see Additional File 1 for transcript identifiers). (ii) In contrast, the distribution of genes with no differential expression within the sclerotome shows no such peak. Histograms were generated using the hist function of R with a cell number of 50.

Format: PDF Size: 400KB Download file

This file can be viewed with: Adobe Acrobat Reader

Additional file 4:

Statistical significance of differential gene expression in the array data. The graph shows Fisher's exact score from Figure 4 for the range of 0–1500 rank-ordered transcripts. See legend to Figure 4 for further description. The blue line corresponds to p = 10^-7. Differential expression is highly significant for the top ~650 transcripts.

Format: PDF Size: 235KB Download file

This file can be viewed with: Adobe Acrobat Reader

Additional file 5:

Differentially-expressed genes between A- and P-half-sclerotome (genes 1–175). Table showing rank order of probability of differentially expression in A- or P-half-sclerotome for the top 175 transcripts as determined by t-test.

Format: XLS Size: 39KB Download file

This file can be viewed with: Microsoft Excel Viewer

Additional file 6:

Differentially-expressed genes between A- and P-half-sclerotome (genes 176–850). Table showing rank order of probability of differentially expression in A- or P-half-sclerotome for transcripts 176–850 as determined by t-test.

Format: XLS Size: 125KB Download file

This file can be viewed with: Microsoft Excel Viewer

Additional file 7:

Functional categorization of differentially-expressed genes. Table showing terms for biological processes that show a significant probability of enrichment relative to the genome for the first 175 genes as determined by DAVID.

Format: XLS Size: 32KB Download file

This file can be viewed with: Microsoft Excel Viewer

Additional file 8:

Whole-mount in situ hybridization of additional sclerotome differentially-expressed candidates. A further 44 genes in addition to those described in Figure 6 were typed by in situ hybridization for differential expression between A- and P-half-sclerotomes. Expression patterns were classified into 4 groups as for Figure 6 (see also Table 1). Group 1 (unambiguous differential expression, green text): flrt2, meox1, nmyc1, uncx4.1, igfbp5, lef1; Group 2 (very likely to be differentially-expressed, yellow text): efnb2, acpl2, mtdh, rbp1, rps6, sema3a, fap; Group 3 (likely to be differentially-expressed, blue text): anapc4, ets2, fgfr1op, gpr150, trappc6b, rab28, tpr, nm_172870, q8c5e4. Group 4 (non-staining or no differential expression, brown text): apg5l, btc, cnot, d17wsu104e, lxn, narg1, nm_028130, enh, nm_027740, q8cak8, st13, tbc1d24, tcfa2b, zc3h6, cd44, cdc42, col4a5, dcc, ddx3y, eif4e, galg1, hexb.

Format: PDF Size: 8.4MB Download file

This file can be viewed with: Adobe Acrobat Reader

Additional file 9:

Expression measures for qPCR analysis. Table showing the qPCR expression measures of a selection of candidate differentially-expressed genes between A- and P-half sclerotome. Genes in red are false-positive (FP) with no indication of differential expression. * Expression is highly dynamic within the dissected P-half-sclerotomes. While the average differences between A and P are <2, there is a greater than 2-fold difference between the highest levels of expression in P relative to A. # Genes showing small but distinct differences between A- and P-half-sclerotome. This may account for their initial categorization as non-differentially-expressed by whole-mount in situ hybridization.

Format: DOC Size: 47KB Download file

This file can be viewed with: Microsoft Word Viewer

Additional file 10:

Further qPCR expression analysis of differential expression. qPCR was used to show differential expression of 3 genes (mospd2, nedd4, apg5l) showing small but robust P-half-sclerotome enrichment (See Additional File 9). In contrast, 4 candidate differentially-expressed genes (fgf1rop, mtdh, st13, wnt5A) appear to be false-positives as qPCR provides no evidence for differential expression. Data are displayed as in Figure 7 legend.

Format: PDF Size: 992KB Download file

This file can be viewed with: Adobe Acrobat Reader

Additional file 11:

Primers used for qPCR analysis. Table indicating the primer sequences used for qPCR analysis of candidate differentially-expressed genes.

Format: DOC Size: 41KB Download file

This file can be viewed with: Microsoft Word Viewer