Fig. S3

The lyc1 mutant lymphatic phenotype is enhanced with MO-pkd1b injection and targeting calcium signaling results in a lymphatic phenotype. Related to Figure 2.

(A) Quantitative real time PCR for kdrl, cdh5, prox1a, nfatc1, nrp2a, flt4 and lyve1 transcripts normalized expression at 3 dpf in sorted embryonic venous and lymphatic endothelial cells. Sorted cell populations display the predicted enrichment of marker genes.

(B) Quantitative real time PCR for pkd1b transcript normalized expression against ef1a and rpl13 at 3 dpf in WT and MO-pkd1a embryos at 24hpf. pkd1b is readily detectable in whole embryo cDNA but not altered by pkd1a knockdown.

(C) Quantification of parachordal lymphangioblasts in WT (n=18), lyc1 (n=9) WT/pkd1b (5ng MO) (n=21), lyc1/MO-pkd1b embryos (5ng MO) (n=23) at 56hpf.

(D-E) Quantification of thoracic duct extent in (D) WT (n=48), lyc1 (n=23), WT/MO-pkd1b (5ng MO) (n=24), lyc1/MO-pkd1b embryos (5 ng MO) (n=21), and (E) WT (n=50), MO-pkd2 embryos (7.5 ng MO)(n=136) at 4dpf.

(F) Quantitative real time PCR for cacna1s transcript normalized expression at 30 hpf in sorted embryonic venous and arterial endothelial cells. Endothelial expression of this calcium channel and Nifedipine target is confirmed.

(G-H) The vasculature of (G) DMSO 0.05% and (H) DMSO 0.05%/Nifedipine 25 μM treated embryos in Tg(fli1a:EGFP ^y1; kdrl:mcherry ^s916). The thoracic duct is markedly absent in the presence of a calcium signaling antagonist (Nifedipine).

(I) Quantification of parachordal lymphangioblasts in DMSO 0.2% (n=62) and DMSO 0.2%/Nifedipine 100μm treated embryos (n=101) at 56 hpf. PLs are unchanged in the presence of a calcium signaling antagonist (Nifedipine).

(J) Thoracic duct quantification in DMSO 0.05% (n=45) and DMSO/0.05%/Nifedipine 25 µM (n=68) at 5 dpf. Thoracic duct reduction similar to lyc1 mutants is observed.

(K) Quantification of thoracic duct extent in WT/MO-pkd1b/0.05% ethanol (5ng MO) (n=21), lyc1/MOpkd1b/ 0.05% ethanol (5ng MO) (n=21), MO-pkd1b/ethanol 0.05%/Bayk8644 (5ng MO) (n=28) and lyc1/MO-pkd1b /ethanol 0.05%/Bayk8644 (5ng MO)(n=15) embryos at 4dpf. A mildly penetrant lyc1 carrier was used. Remarkably, a phenotypic interaction with the calcium agonist is observed only in the mutant animals and not in the wildtype siblings. This suggests a sensitivity of mutant cells to further fluctuations in Ca²⁺ signalling.