VENOFT
2.00 Fold, p<0.001
B
R=0.94
R=0.93
R=0.91 R=0.93
R=0.94 R=0.90
52
Figure 6: Comparison of differential gene expression between E11.0 biological replicates. Plots mapping the fold (log base 2) difference (either (A)
>1.25-fold or (B) >2.00-fold) in expression between AVC/VEN, OFT/VEN or AVC/OFT in mouse biological replicates (X-axis: n=60, Y-axis: n=100) are shown. Genes that show agreement, defined as having >1.25 or >2.00-fold (p<0.001) increased or decreased expression in a specific comparison in both replicates, are mapped to quadrants I (upper right) or III (lower left) of a plot.
Genes that show disagreement, defined as having>1.25 or >2.00-fold (p<0.001) increased expression in a tissue in one replicate and decreased in another (or vis versa), are mapped to quadrants II (upper left) or IV (lower right). There was a high degree of agreement using either a >1.25-fold (R>0.91) or a >2.00-fold (R>0.90) across all comparisons.
53 qRT-PCR
First-strand synthesis was done with SuperScript III kit (Invitrogen). SYBR Fas Green (Biorad) was used for qRT-PCR. Primer sequences are in Table 2.
Table 2: Primers Used to Confirm Gene Knockdown
Gene Forward Primer Reverse Primer
FOXP2 5’(GTGCCACAGACAGATGCAACAACA)3’ 5’(TTTGACGAAGCTCATGCGGAGGAT)3’
MEIS2 5’(AAGGGAGCCTTATGGACCAAACCA)3’ 5’(AGCTTCAGCTTAAACACCCGACCT)3’
HAPLN1 5’(AGCACCCAGCTGTGGCTAATTACT)3’ 5’(TCTATGTGCAAATGTGCAAGGCCG)3’
ID1 5’(AGACAACAGGGCTCTCCTCAAACT)3’ 5’(ACTGCGAGACCCAAGTCAGCAATA)3’
TGFR3 5’(ACCAGAGTGGCGTGTTAAGAAGGT)3’ 5’(TGCACAGAAAGGACCCAAAGCAAC)3’
Collagen Gel Assay
Collagen gel assays (reviewed (Lencinas, Tavares et al. 2012)) with siRNA or small molecule addition were performed as described (Townsend, Wrana et al. 2008). AVC were excised from stage 16 chick embryos, transected, and explanted endocardial side down onto a collagen I gel and incubated at 37oC with siRNA constructs. Control for siRNA toxicity was a randomized GC matched construct that do not correspond to any sequence. The positive control was siRNA targeting Tgfbr3 (Townsend, Robinson et al. 2011). siRNA sequences are listed in Table 3. EMT was quantified by counting the number cells with mesenchymal morphology that invaded into the collagen gel (Table 4).
54
Table 3: siRNA Construct Sequences
Target: siRNA Construct Sequences:
TGFβR3 5’(UUCAUGUAGAUUUACUUCCtt)3’
FOXP2-A 5’(CGAAUUUUAUAAAAACGCAtt)3’
FOXP2-B 5’(GACAGGCAGUUAACACUUAtt)3’
MEIS2-A 5’(GUACAUUAGUUGUUUGAAAtt)3’
MEIS2-B 5’(GUAAACAACUGGUUUAUUAtt)3’
HAPLN1-A 5’(GGACAAGGAUAAAAGCAGAtt)3’
HAPLN1-B 5’(GCUACAAUUAACUUUCAtt)3’
ID1-A 5’(CCUCGUCUAUUGUUUAAAAtt)3’
ID1-B 5’(GUAUAUGCUUUAAUAGACUtt)3’
Table 4: Collagen Gel Assay Counting Data
Experiment Condition N
Total Number of Transformed
Cells
Normalized Mean Number of Transformed Cells +/- SEM Foxp2
Control 30 8886 100 +/- 4
TGFβR3 30 5349 60+/-3
FOXP2-A 31 5078 55+/-3
FOXP2-B 30 4785 53+/-2
Meis2 Control 30 8572 100+/-6
TGFβR3 29 4589 55+/-3
MEIS2-A 30 3413 39+/-3
MEIS2-B 30 3209 37+/-3
HAPLN1
Control 28 11592 100+/-7
TGFβR3 30 6127 49+/-3
HAPLN1-A 30 5500 44+/-3
HAPLN1-B 29 4884 40+/-3
ID1 Control 21 6867 100+/-9
TGFβR3 30 5971 60+/-5
ID1-A 30 5511 56+/-4
ID1-B 29 5755 60+/-5
55 In Situ Hybridization
Dioxygenin labeled probes were generated from cDNA derived from E13.5 whole heart tubes using primers in Table 5. In situ hybridization was performed as described (Wei, Manley et al. 2011) and embryos were cryosectioned.
Results
Defining a spatial transcriptional profile of the developing heart tube
Chick and mouse models were used for their relative strengths as model systems and the ability to use cross species comparison to identify key regulatory genes. Equivalent stages in the chick (HH18) and mouse (E11.0) were chosen when robust EMT occurs in both the OFT and AVC but not the VEN at this time (Camenisch, Molin et al. 2002). Spatial transcriptional profiles of chick and mouse heart tubes were generated as described in methods (Figure 4A-B).
The dynamic nature of gene expression profiles generated by RNA-seq identified approximately 8,000 genes expressed throughout either the chick or mouse heart tube. A smaller number of genes were also localized to specific regions such as the AVC, OFT, & VEN (Figure 4C). As a first step to enrich for Table 5: Primers Used to Generate ISH Probes
Gene Forward Primer Reverse Primer
Foxp2 5’(CGCGAGCCTCCGAGAAAGCG)3’ 5’(CATCATGGCCACCGACACGGG)3’
Meis2 5’(TGGCGGACAGGTTATGGACATTCA)3’ 5’(TGAAGAAGCCTTCGCTCTGTCTCT)3’
Hapln1 5’(TGGACCAGGACGACGCAGTGATT)3’ 5’(GCAGCGGTCATAGCCCAGAA)3’
Id1 5’(CCCCTCCGCCTGTTCTCAGGA)3’ 5’(TACCCACTGGACCGATCCGC)3’
56
genes mediating EMT, we subtracted genes found in the VEN sample from the AVC & OFT samples. By identifying genes upregulated in both cushions in the comparison we eliminated the influence of the epicardial cells (AVC) and neural crest cells (OFT) which are found only in a single cushion sample and therefore not in the shared gene list we identified (Figure 7A; see Wt1 (AVC, VEN), Sox10 (OFT), BMP10 (VEN)).
This approach yielded a robust list of genes upregulated in the cushions.
We mapped genes enriched >2-fold in each spatial region (Figure 4D) Genes associated with distinct regions of the heart tube in chick and mouse cushions, including the T-box family members, had spatial expression patterns consistent with the literature (Figure 7B). We observe a few hundred genes enriched in the cushions, consistent with the hypothesis that the AVC and OFT should share common genes involved in endothelial EMT (Figure 4D). Comparisons of the AVC & VEN or OFT & VEN did not reveal as many enriched genes since they do not share EMT as a common process. Therefore, genes with a significant p-value (p<.001) and >2-fold higher expression in the cushions (AVC & OFT) compared to VEN are considered to be enriched in these compartments and were potential candidates for involvement in EMT.
Overall, 198 genes were identified in the chick (Appendix A) and 105 in the mouse (Appendix B) that were >2-fold higher expressed in the cushions. A literature search was used to further characterize these gene lists. In total, 15 of the 198 genes in chick and 18 of the 105 genes in mouse have a previously described role in EMT (Table 6). Genes known to be expressed regionally in the
57
Figure 7: Spatial expression of select genes in the HH18 chick and E11.0 mouse heart tube. For each gene, expression was normalized to the region with the highest read count A, Regional expressed patterns in the mouse heart are consistent with previous observations at E10.5-E11.5. Bmp10 (VEN), Isl1 (OFT), Hand1 (VEN), Hand2 (endocardium throughout heart tube), Tbx18 and Wt1 (Epicardium attaching to AVC and migrating towards VEN), and Sox10 (neural crest in OFT and in OFT and AVC cushions). B, Expression of T-box genes in chick and mouse are consistent with previous reports (reviewed (Greulich, Rudat et al.)). n.d.- TBX1 has not been annotated in the chick genome and is not included in the analysis.
0 0.2 0.4 0.6 0.8 1
BMP10 ISL1 Hand1 Hand2 Tbx18 Wt1 Sox10
Normalized Expression
VEN OFT
AVC
0 0.2 0.4 0.6 0.8 1
TBX1 TBX2 TBX3 TBX5 TBX18 TBX20 TBX1 TBX2 TBX3 TBX5 TBX18 TBX20
HH18 Chick E11.0 Mouse
Normalized Expression
A
B
n.d.
Bmp10 Isl1 Hand1 Hand2 Tbx18 Wt1 Sox10
TBX1 TBX2 TBX3 TBX5 TBX18 TBX20 Tbx1 Tbx2 Tbx3 Tbx5 Tbx18 Tbx20
58
Table 6: Genes >2-fold Enriched in Chick or Mouse Cushions Previously Implicated in Cushion Development
Chick Genes Mouse Genes
BMP2 (Lyons, Pelton et al. 1990; Ma, Lu et al. 2005)
Col2a1 (Li, Prockop et al. 1995) CXCR7 (Yu, Crawford et al. 2011) Eln (Krishnamurthy, Opoka et al.
2012)
FBN1 (Ng, Cheng et al. 2004) Epha3 (Stephen, Fawkes et al. 2007) HAS2 (Camenisch, Spicer et al. 2000) Erbb3 (Erickson, O'Shea et al. 1997;
Camenisch, Schroeder et al. 2002) IGF2 (Hartnett, Glynn et al. 2010) Fzd1 (Yu, Smallwood et al. 2010) MSX2 (Chen, Ishii et al. 2008) Gpc1 (Niu, Bahl et al. 1998) SNAI2 (Niessen, Fu et al. 2008) Has2 (Camenisch, Spicer et al.
2000) SOX9 (Akiyama, Chaboissier et al. 2004;
Lincoln, Kist et al. 2007)
Msx1 (Chen, Ishii et al. 2008) TBX2 (Shirai, Imanaka-Yoshida et al.
2009; Singh, Hoogaars et al. 2012)
Msx2 (Chen, Ishii et al. 2008) TBX20 (Stennard, Costa et al. 2005) Pitx2 (Kitamura, Miura et al. 1999) TGFB2 (Sanford, Ormsby et al. 1997) Postn (Norris, Moreno-Rodriguez et
al. 2008)
WNT4 (Alfieri, Cheek et al. 2010) Rarg (Mendelsohn, Lohnes et al.
1994)
WNT6 (Alfieri, Cheek et al. 2010) Smad6 (Galvin, Donovan et al. 2000) WT1 (Zhou, Ma et al. 2009) Snai1 (Timmerman, Grego-Bessa et
al. 2004) ZFPM2 (Ackerman, Herron et al. 2005;
Zhou, Ma et al. 2009)
Sox9 (Akiyama, Chaboissier et al.
2004; Lincoln, Kist et al. 2007) Tbx2 (Shirai, Imanaka-Yoshida et al.
2009; Singh, Hoogaars et al. 2012) Tgfb2 (Sanford, Ormsby et al. 1997) Twist1(Chen and Behringer 1995;
Shelton and Yutzey 2008; Lee and Yutzey 2011; Vrljicak, Cullum et al.
2012)
59
myocardium (Bmp4 (Keyes, Logan et al. 2003), BMP2 (Keyes, Logan et al.
2003)) were identified as having cushion enriched expression in chick and mouse as expected. Of note, 31 of 105 identified in the mouse were found to be expressed in the endothelium or mesenchyme derived from endothelium. Thus, the RNA-seq methodology was sensitive enough to detect changes in gene expression in the endothelium and mesenchyme when the majority of the sample is derived from the myocardium. Relaxation of the stringency of gene enrichment to a level of >1.25-fold (p<0.001) (Figure 8) revealed 574 genes enriched in the chick (Appendix A) and 250 enriched in the mouse (Appendix B). Of these, 54 genes in the chick and 41 genes in the mouse were associated with abnormal heart development and function. Several genes known to play important roles in endocardial EMT were revealed in the >1.25-fold gene lists. Though there is an increased chance of including false positive genes by lowering the stringency, there may also be novel candidate genes in the 1.25 fold gene lists that would otherwise be missed with a 2 fold cutoff. In particular, small changes (<2-fold) in the expression levels of transcription factors may have a significant effect on cell behavior (Niwa, Miyazaki et al. 2000).
Gene Ontology (GO) Analysis of Cushion-Enriched Genes
To gain a better understanding of the genes in the >2-fold cushion enriched gene lists we examined the associated predicted protein location, predicted protein function and biological processes (Figure 9). Enriched genes encoded proteins with various predicted functions and cell locations, including
60
Figure 8: Spatial transcriptional profile of heart identifies cushion-enriched genes. A-B, AVC, OFT and VEN were dissected from equivalent stages of chick HH18 (A) and mouse E11.0 (B). C, RNA-seq analysis identified genes with significantly expressed (>10 reads) in each spatial region of the developing heart tube. D, Mapping genes with enriched expression (>1.25-fold) onto each spatial region identifies those genes with cushion-enriched expression (in red), 547 in the chick and 225 in the mouse.