SUPPLEMENTAL MATERIAL

Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineages divergence

Elisa Rodriguez-Seguel1, Nancy Mah2, Heike Naumann1, Igor M. Pongrac1, Nuria Cerdá-

Esteban1, Jean-Fred Fontaine2, Yongbo Wang3, Wei Chen3, Miguel A. Andrade-

Navarro2, Francesca M. Spagnoli1*

LIST of SUPPLEMENTAL MATERIAL

- SUPPLEMENTAL FIGURES 1 to 8

- SUPPLEMENTAL TABLE S1, S2 AND S3 [uploaded separately as .xls files]

- SUPPLEMENTAL TABLE S4

- REFERENCES

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Supplemental Figure 1. Heat map-view of selected transcripts, which were previously shown to be expressed in liver and/or pancreas at E10.5 (Hou et al., 2007; Sherwood et al., 2009). Colors represent high (red), low (blue) or average (white) expression values based on Z-score normalized FPKM values for each gene. For example, Prox1 transcript is detected in the pancreatic populations [vpa (FPKM = 9.66275) and dpa (FPKM =

11.479)] as well as in the hepatic population [lv (FPKM = 30.3245)]. The white color in the heat map indicates a higher expression value of Prox1 in pancreas than in foregut [fg

2 and mfg (blue)], but lower value than in liver (red). This makes the vpa and dpa average

(white) respect to the values observed for Prox1 expression in the 5 samples.

Abbreviations, dpa, dorsal pancreas; fg, foregut; mfg, medial foregut; lv, liver; vpa, ventral pancreas.

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Supplemental Figure 2. Validation of candidate regulators of hepatic versus pancreatic fate decision. (A) Venn diagram showing the number of unique and common highly expressed transcripts between progenitor cells at different developmental stages.

The working dataset was divided into two groups based on a cutoff for high expression

(defined to be FPKM=10, at approximately the 50th percentile of RNA-Seq expression for each sample). For example, 5437 genes exhibited relative abundance values of > 10

FPKM in all samples, while 89 genes were highly expressed in foregut and liver, but not in ventral and dorsal pancreas (referred to as Group FL - Foregut Liver-). (B) Heat map- view of the FL Group transcripts that were differentially expressed between any of two samples [32 transcripts out of 89; Cufflink, p-value <0.05]. Colors represent high (red),

4 low (blue) or average (white) expression values based on Z-score normalized FPKM values for each gene. Group FL displayed moderate enrichment for GO “Molecular

Function” categories, such as TGF-β receptor binding [2 genes; p-value < 0.001] and

SMAD binding [2 genes; p-value < 0.00263] activities (see also Supplemental Table S1).

These include genes as Eng, Fst, Smad6. (C) RT-qPCR validation of a sub-set of differentially expressed genes of Group FL. Data were normalized to that of SDHA and represented as fold change compared to the E8.5 foregut sample (set to 1 as calibrator).

Error bars represent ± SEM.

5 Supplemental Figure 3. Heat map-view of E10.5 pancreatic and hepatic transcripts.

(A) Venn diagram showing the number of unique and common highly expressed

6 transcripts between progenitor cells at different developmental stages. The working dataset was divided into two groups based on a cutoff for high expression (FPKM = 10).

The diagram here shows transcripts with low expression in the foregut (FPKM < 10) and high expression (FPKM > 10) later in E10.5 hepatic or pancreatic progenitors. These transcripts represent cell type-specific markers of differentiation and, possibly, factors involved in late aspects of organogenesis. According to this cutoff, 256 transcripts were found enriched in the liver (referred to as Group L) and 274 in the pancreas (both vpa and dpa) (referred to as group P). (B) Heat map-view of pancreatic (left) and hepatic (right) transcripts that were differentially expressed between any of two samples [98 transcripts out of 274 (Group P); 123 transcripts out of 256 (Group L); Cufflink, p-value <0.05]. * indicate known pancreatic and hepatic marker genes.

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Supplemental Figure 4. Non-canonical Wnt ligands in mouse and Xenopus embryos.

(A) Gene expression levels of non-canonical Wnt ligands in the foregut (fg), liver (lv) and pancreas (vpa and dpa) progenitor datasets. FPKM values (y-axis) were plotted against the different progenitors cell types (x-axis). (B) RT-qPCR validation of Wnt5a and Wnt5b gene expression in foregut, dorsal pancreas and liver progenitor cells. Data were normalized to that of SDHA and represented as fold change compared to the E8.5

8 foregut sample (set to 1 as calibrator). Error bars represent ± SEM. (C) In situ hybridization analysis validated the expression of Wnt5a in the E8.5 foregut endoderm

(see red arrowheads) and adjacent lateral plate mesoderm (lpm). At E10.5, Wnt5a transcript was abundant in the limbs (Yamaguchi et al. 1999) and mesenchyme surrounding the midgut and pancreatic rudiments (demarcated by dotted yellow line), whereas its expression in pancreatic epithelial cells became weaker. Scale bars, 50 µm.

(D) RT-qPCR of Xenopus Wnt5a, Wnt5b and Wnt7b gene expression in endodermal explants. Both anterior (AE) and posterior endoderm (PE) explants were dissected at stage 10, cultured in isolation until stage 28 and assayed for expression of the indicated genes by RT-qPCR analysis. Data were normalized to that of ODC and represented as fold changes compared to AE sample (set to 1 as calibrator). Error bars represent ± SEM.

Wnt5a and Wnt5b were detected in both endoderm populations without any significant regionalized expression, whereas Pdx1 was expressed only in AE derivatives, including the pancreas. (E) Analysis of cell proliferation at early tadpole stages (stages 32-34) revealed no significant differences between control (CTRL) and Wnt5a-injected embryos.

Wnt5a mRNA was co-injected with β-gal mRNA into AE cells (dorsal vegetal cells) of 8- cell stage Xenopus embryos. Embryos were fixed at stages 32-34, stained for pHH3 and cleared with benzyl alcohol/benzyl benzoate. Subsequently, the number of pHH3+ cells relative to the anterior gut area (LacZ-staining positive area) was measured in transparent embryos. All results are expressed as means ± SEM. n=8 CTRL embryos; n=15 Wnt5a- injected embryos. Abbreviations: fg, foregut; dpa, dorsal pancreas; lpm, lateral plate mesoderm; lv, liver; vpa, ventral pancreas; AE, anterior endoderm; PE, posterior endoderm; ns, not significant.

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Supplemental Figure 5. Validation analysis of candidate regulators of pancreatic versus hepatic fate decision. (A) High magnification images of E10.5 mouse embryo cryosections immunostained for Celrs2, Fat1, Frizzled 2 (Fzd2) and Ror2 (see Fig. 3).

Pdx1 in blue marked pancreatic progenitors; HNF4α or Albumin in red stained hepatoblasts. Scale bars, 50 µm. (B) RT-qPCR validation of a sub-set of differentially expressed genes of the FP group in foregut (fg), liver (lv) and ventral pancreas (vpa) progenitor cells. Data were normalized to that of SDHA and represented as fold change compared to the E8.5 foregut sample (set to 1 as calibrator). Error bars represent ± SEM.

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Supplemental Figure 6. Gene models from UCSC are shown in panel a. Genomic coordinates from Cufflinks showed that the two genes XLOC_007215 (panel b, red) and

XLOC_007888 (panel c, blue) in the HoxB gene cluster loci constituted an overlapping transcript pair in opposite strands. Both are annotated to be HoxB gene members, but only XLOC_007215 represents HoxB genes. In one transcript model, Cufflinks assembles Hoxb7, Hoxb6 and Hoxb7 into one transcript, which is supported by a minority of reads that span exons between the three Hox genes (not shown).

XLOC_007888 represents an antisense gene (AK002860), which spans the genomic region encompassing the three HoxB genes, with some potential sense-antisense overlap with the 3'-terminal exon of Hoxb7. As seen in the raw reads (panel d), the three HoxB genes are highly expressed only in dorsal pancreas. Expression of the antisense transcript is lower than the HoxB genes, but still visible in the raw reads.

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Supplemental Figure 7. Validation of transcripts differentially expressed between

E10.5 ventral and dorsal pancreas. (A) Heat map-view of the transcripts that showed

12 significant differential expression between E10.5 vpa and dpa (192 transcripts; p-value <

0.05, absolute value of log2 FC > 3). On the left, transcripts upregulated in vpa. On the right, transcripts upregulated in dpa. Colors represent high (red), low (blue) or average

(white) expression values based on Z-score normalized FPKM values for each gene.

Gene Ontology analysis showed strong association with extracellular binding activities

(GO:0005488; 62 genes; p-value < 0.000129) in vpa group and DNA binding transcription factor function (GO:0003700; 25 genes; p-value < 9.41E-18) in dpa

(Supplemental Table S3). * indicate genes that were previously described to be enriched in either vpa or dpa. (B) RT-qPCR validation of a sub-set of transcripts identified as differentially expressed between vpa and dpa. Data were normalized to that of SDHA and represented as fold change compared to the E10.5 dpa sample (set to 1 as calibrator).

Error bars represent ± SEM. Additional differentially expressed genes, such as the Hox genes, were validated by in situ hybridization or immunofluorescence analysis, as shown in Fig. 6.

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Supplemental Figure S8. Novel divergent overlapping genes, XLOC_019721 and

XLOC_019656. The XLOC_019656 is supported by one EST, whereas the

XLOC_019271 is not supported by any spliced ESTs nor Genscan Predictions. By exon- junction analysis we predicted a gene model and, subsequently, validated the expression of this novel transcript in the fg and dorsal pancreas by RT-qPCR (see also Fig. 6).

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Supplemental Table S4.

Antibody Raised in Dilution Source Albumin Rabbit 1:100 Dako Celrs2 Rabbit undiluted gift from Dr. T. Uemura Claudin 4 Rabbit 1:250 Invitrogen Cytokeratin 19 Mouse 1:100 Hybridoma Babk EGFP Chick 1:200 Aves E-Cadherin Rat 1:500 Invitrogen Fat1 Rabbit 1:20 Novus Biologicals FoxA2 Rabbit 1:300 abcam FoxD3 Rabbit 1:300 Molecular Probes Fzd2 Goat 1:20 Santa Cruz HGF goat 1:10 Cell Signaling HNF4α Goat 1:100 Santa cruz Liv2 Rat 1:200 MBL Pdx1 Rabbit 1:2000 Abcam Pdx1 Mouse 1:100 Hybridoma Bank Prox1 Rabbit 1:200 Reliatech ROR2 Rabbit 1:200 gift from Prof. Minami Sox17 Goat 1:100 R&D system

List of applied primary antibodies, respective dilutions and manufacturers.

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REFERENCES

Hou J, Charters A, Lee S, Zhao Y, Wu M, Jones S, Marra M, Hoodless P. 2007. A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene

Expression (SAGE). BMC Dev. Biol. 7: 92.

Sherwood R, Chen T, Melton D. 2009. Transcriptional dynamics of endodermal organ formation. Dev. Dyn. 238: 29-42.

Yamaguchi TP, Bradley A, McMahon AP, Jones S. 1999. Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126: 1211-23.

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