Pjeta R , Lindner H , Kremser L , Salvenmoser W , Sobral D , Ladurner P , Santos R .

P25404-B25 Austrian Science Fund, P30347 Austrian Science Fund, IF/00006/2015/CP1276/CT0001 Fundação para a Ciência e a Tecnologia, UID/Multi/00612/2019 Fundação para a Ciência e a Tecnologia, UID/MAR/04292/2013 Fundação para a Ciência e a Tecnologia

	Figure A1. In situ hybridization screen panel 1. (A–J) Ventral view onto the tube foot disc. The distribution of the in situ hybridization signal (blue) resembles the localization of the adhesive gland cells bodies. Therefore, these transcripts potentially constitute proteins involved in bioadhesion. Scale bar, 200 µm.
	Figure A2. In situ hybridization screen panel 2. (A–J) Ventral view onto the tube foot disc. The distribution of the in situ hybridization signal (blue) resembles the localization of the adhesive gland cells bodies plus additional cells of the adhesive disc. Therefore, these transcripts potentially constitute proteins involved in bioadhesion or other proteins of the disc cells. Scale bar, 200 µm.
	Figure A3. In situ hybridization screen panel 3. (A–H) Ventral view onto the tube foot disc. The distribution of the in situ hybridization signal (blue) is weak, but shows distinct expression in a subset of disc cells. Therefore, these transcripts represent proteins of unknown function. Scale bar, 200 µm.
	Figure A4. In situ hybridization screen panel 4. (A–K) Ventral view onto the tube foot disc. The distribution of the in situ hybridization signal (blue) is very weak and present only in a small subset of disc cells. Therefore, these transcripts represent proteins of unknown function. Scale bar, 200 µm, inset C, D and E, 25 µm.
	Figure A5. Spatial distribution of labeled cells within the tube foot disc. In situ hybridization signal (blue) in a ventral view onto the tube foot disc (A,D,G), in semi-thin sagittal section though the tube foot (B, E, H). The rectangle indicated in (B) is magnified in (C), the rectangle in (E) is magnified in (F). (I) shows the lateral part of the disc of a successive section of (H). s, stem; d, disc.
	Figure 1. Rock-boring sea urchin Paracentrotus lividus (A) has hundreds of oral tube feet specialized for locomotion and adhesion (B). Tube feet have a proximal cylindrical motile stem and a distal flattened disc with a duo-glandular adhesive epidermis with adhesive and de-adhesive secretory cells (C,E). After detachment, circles of adhesive secretion remain attached to the substrate and can be visualized after staining with an aqueous solution of Crystal Violet (D,F). Abbreviations: AC, adhesive secretory cell; AE, adhesive epidermis; CT, connective tissue; Cu, cuticle; D, disc; DC, de-adhesive secretory cell; L, lumen; M, myomesothelium; NE, non-adhesive epidermis; NP, nerve plexus; NR, nerve ring; S, stem; Sk, skeleton; TF, tube feet.
	Figure 2. Summary diagram of the integrative transcriptomic and proteomic analysis of the present study. 1 Raw data of Lebesgue et al., containing 10 P. lividus disc-, eight stem- and three adhesive secretion samples, were used for the present study. 2 P. lividus tube feet transcriptome was generated. 3 Disc and Stem specific differential RNAseq reads were generated. 4 Re-mapping of the Lebesgue et al. proteome data to the new P. lividus transcriptome. 5 Identification of adhesive disc specific transcripts using DESeq2 differential gene expression analysis. 6 Selection of candidate transcripts for in situ hybridization (ISH), based on the differential proteome and differential transcriptome. Only transcripts present in both datasets were considered for the ISH screen. 7 In order to ensure an encompassing dataset of disc-specific transcripts, a selection of differentially expressed transcripts, not present in the proteome (due to e.g., insolubility or post-translational modifications), was included. 8 ISH screen of the 59 selected disc-specific transcripts.
	Figure 3. RNAseq and differential gene expression. Scheme (A) representing the differential gene expression approach. (B) Differentially expressed transcripts with an adjusted p-value < 0.01 are indicated in grey and red. 2129 disc-specific transcripts with a log2 fold change ≤ 2 are highlighted in red (see Table S1).
	Figure 4. Illustration of biological samples used for differential proteomics and diagram indicating the number of transcripts allocated to disc-, stem- and adhesive secretion samples after re-mapping of the Lebesgue et al. proteome data to the P. lividus transcriptome. A total of 1324 transcripts (emphasized in bold) were used for downstream analysis.
	Figure 5. In situ hybridization candidate gene selection. (A) Candidate gene selection workflow. Initially, 121 transcripts were found in proteome data from disc and adhesive secretion samples as well as being over-expressed in the tube feet discs in the differential transcriptome. Exclusion of isoforms and similar transcripts, low expressed transcripts and exclusion of genes with non-adhesion related NCBI BLAST hit reduced in situ candidates to 49 transcripts. Additionally, 10 transcripts were selected for in situ hybridization that were differentially expressed but was not identified in the differential proteome dataset. (B) Differential gene expression analysis. Transcripts that were selected for in situ hybridization are highlighted in blue (differential proteome and differential transcriptome selection) and yellow (differential transcriptome only candidate selection).
	Figure 6. In situ hybridisation expression patterns of selected adhesion candidate genes previously identified in P. lividus (A) and in the sea star Asterias rubens (B–F). The respective BLAST hit for each transcript is shown on the bottom right of each image. Scale bar, 200 µm.
	Figure 7. Comparison of the differential proteome re-analysis with the initial dataset of Lebesgue et al. Mass spectrometry peptide mapping to the transcriptome resulted in a drastic increase of successfully mapped peptides and proteins compared to the mapping of the previous study, in which the peptides were mapped against UniProt database hits for sea urchins. Both protein mappings had an overlap of 877 proteins, 505 are uniquely found in the Lebesgue data, whereas 3926 proteins are newly identified in the present study.

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