Genes coding for extracellular matrix proteins are coordinately downregulated

GSEA analysis of Gene Sets enriched in the E10.5 and E11.5 UB and MM tissues from Robo2 and the Slit2 knockouts

GESA analysis of all gene expression data and comparisons at both E10.5 and E11.5 revealed that genes related to the extracellular matrix (ECM), collagen formation, ECM-receptor interaction, focal adhesions, actin cytoskeleton, axon guidance, and NCAM1 interactions were significantly coordinately downregulated in the E10.5 and E11.5 Robo2 and Slit2 knockout kidneys (Table 4.5). No gene-sets were coordinately upregulated at both E10.5 and E11.5 time points. At E10.5, many of the gene-sets most significantly upregulated were related to translation (e.g. ribosome) and metabolism (e.g.

mitochondria) (Table 4.6). These results suggest that loss of Robo2 or Slit2 during UB outgrowth may affect the gene expression of ECM compositions in the MM and around the UB.

Table 4.5: Sets of genes significantly coordinately down-regulated in Slit2 and Robo2 knockouts compared to the wild-type controls

Gene sets related to the extracellular matrix are highlighted in yellow.

Group Gene Set Name

KEGG: Kyoto Encyclopedia of Genes and Genomes, GO: Gene Ontology, NES:

Normalized Enrichment Score, p: nominal p-value, FDR q: FDR-corrected p value; FDR q < 0.25 is significant, WT- wildtype, combined KO: Slit2^-/- and Robo2^-/- knockout

Table 4.6: Sets of genes significantly coordinately up-regulated in E10.5 knockout

conversion from APCc CDC20 to APCc CDH1 in late

anaphase 16 2.00 0.00 0.02

GO mitochondrial ribosome 20 1.99 0.00 0.02

Reactome synthesis of glycosylphosphatidylinositol GPI 17 1.97 0.00 0.02 Reactome mitochondrial protein import 44 1.95 0.00 0.02

GO organellar ribosome 20 2.00 0.00 0.02

Reactome influenza viral rna transcription and replication 54 1.95 0.00 0.02

GO ribosomal subunit 18 2.01 0.00 0.02

Reactome

formation of the ternary complex and subsequently the

43s complex 29 1.89 0.00 0.04

GO Ribosome 29 1.88 0.00 0.04

Reactome 3 UTR mediated translational regulation 57 1.87 0.00 0.04 Reactome APC C CDC20 mediated degradation of cyclin B 16 1.85 0.00 0.05 Reactome

SRP dependent cotranslational protein targeting to

membrane 59 1.85 0.00 0.05

GO Translation 146 1.85 0.00 0.05

Reactome nucleotide like purinergic receptors 15 1.82 0.01 0.06 GO cellular protein catabolic process 55 1.76 0.00 0.12

inhibition of the proteolytic activity of APC c required for the onset of anaphase by mitotic spindle checkpoint

components 18 1.70 0.01 0.16

GO mitochondrial part 128 1.70 0.00 0.16

Reactome respiratory electron transport 57 1.68 0.00 0.17

Reactome

KEGG glycosylphosphatidylinositol GPI anchor biosynthesis 25 1.66 0.02 0.18

GO DNA dependent DNA replication 51 1.65 0.01 0.18

GO protein catabolic process 65 1.64 0.01 0.18 GO regulation of DNA metabolic process 44 1.64 0.00 0.18

GO mitochondrial membrane 77 1.62 0.00 0.20

GO mitochondrial membrane part 44 1.62 0.01 0.20

GO anion transport 29 1.63 0.01 0.20

GO inorganic anion transmembrane transporter activity 19 1.62 0.02 0.20

GO integral to organelle membrane 49 1.60 0.00 0.23

GO cytokine metabolic process 40 1.58 0.02 0.24

KEGG parkinsons disease 97 1.58 0.00 0.24

GO organelle inner membrane 66 1.59 0.01 0.24

GO hydrogen ion transmembrane transporter activity 25 1.58 0.02 0.24 KEGG: Kyoto Encyclopedia of Genes and Genomes, GO: Gene Ontology, NES:

Normalized Enrichment Score, p: nominal p-value, FDR q: FDR-corrected p value; FDR q < 0.25 is significant, WT- wildtype, combined KO: Slit2^-/- and Robo2^-/- knockout

Laminin and collagen coding genes are coordinately downregulated in E10.5 and E11.5 Slit2 and Robo2 knockouts versus wild type controls

I was specifically interested in the genes driving the association between the gene-sets and the differential expression in Slit2^-/- or Robo2^-/- knockout samples versus wildtype controls. Besides the “axon guidance” and the “actin cytoskeleton” gene-sets, all gene-sets differential expressed are related to the extracellular matrix (ECM). Analysis of the leading edges of these gene-sets revealed that genes encoded laminins and collagens are the genes driving the significant association between these gene-sets and differential expression in the dataset. Therefore I looked at the differential gene expression of genes encoded Laminins and collagens in the dataset (mean log2>8) in all the comparisons (Tables 4.7 and 4.8). Although the corrected significance has not been reached for any of these genes (FDR=1), many of the individual p-values based on the student t-test were lower than 0.05. To better visualize these results, heatmaps were

drawn using the mouse homologs of genes in the leading edges of the “GO cellular component- extracellular matrix” and the “reactome collagen formation” gene-sets at E10.5 (Figure 4.4). The intensities of the heatmaps revealed that most laminin and collagen coding genes were coordinately downregulated in E10.5 Robo2^-/- and the Slit2 -/-knockouts when compared to the wild-type controls (Figure 4.4).

Table 4.7: Most differential expressed Laminin coding genes at E10.5-E11.5 in the UBand MM (Mean log2 >8).

Individual p-values based on the student t-test were lower than 0.05 are highlighted in yellow. WT- wildtype, combined KO: Slit2^-/- and Robo2^-/- knockout

Table 4.8: Table 4.6: Most differential expressed Collagen coding genes at E10.5-E11.5 in the UB and MM (Mean log2 >8).

Individual p-values based on the student t-test were lower than 0.05 are highlighted in yellow. WT- wildtype, combined KO: Slit2^-/- and Robo2^-/- knockout

Figure 4.4: Heatmaps from leading edges for gene-sets downregulated in E10.5 Robo2 and Slit2 knockout kidneys

(A) The leading edge genes from “extracellular matrix” visualized in a heat map. The heatmap is color-coded when compared to average, Blue is below average (downregulate), white is at average (no change) and red is above average (upregulated). The darker the color is the bigger the difference is from average.

Each row represents a gene (labeled on the right). Each column represents a sample. Gray columns: wild-type, Orange columns: Robo2^-/-, Purple columns:

Slit2^-/-.

(B) The leading edge genes from “reactome collagen formation” visualized in a heat map. The heatmap is color-coded when compared to average, Blue is below average (downregulate), white is at average (no change) and red is above average (upregulated). The darker the color is the bigger the difference is from average.

Each row represents a gene (labeled on the right). Each column represents a sample. Gray columns: wild-type, Orange columns: Robo2^-/-, Purple columns:

Slit2^-/-.

4.5.3 Pathways related to the extracellular matrix proteins are downregulated in the