Fig. S4

Mechanisms of liver size changes in cannabinoid receptor mutants

A) in situ hybridization shows reduction in the intestinal reporter fabp2 in cnr1^-/- (62.5% with phenotype, n=40) and cnr2^-/- mutants at 72hpf (57.1% with phenotype, n-56). Scale bar = 0.2 mm.

B) in situ hybridization images depict the size distribution of hhex expression after morpholino knockdown of cnr1 and cnr2. Morphant embryos do not exhibit differences in hhex liver progenitor expression. Scale bar = 0.2 mm.

C) FACS quantification of fabp10a:GFP WT embryos compared to cnr1^-/- and cnr2^-/- mutants at 96 and 120hpf. % of GFP+ cells were normalized to controls. Mean±s.e.m. n= 7-10 pooled samples of 10 embryos each, one-way ANOVA analysis, *p<0.05 fabp10a:GFP vs. cnr1^-/- and **p<0.01 fabp10a:GFP vs cnr2^-/-.

D - E) Scatter plot and representative in situ hybridization images showing liver size of cnr1^-/- and cnr2^-/- mutants at 96hpf (C) and 120hpf (D) based on liver morphometric measurements using ImageJ. At 96hpf, livers in mutants are still significantly smaller than controls, but this difference disappears by 120hpf. Data are represented as mean±s.e.m. with one-way ANOVA analysis, *p<0.05 for Tu compared to cnr1^-/- and cnr2^-/- at 96hpf only. Scale bar = 0.2 mm.

F) TUNEL staining in 96hpf embryos reveals no difference in cell death in the developing livers (outlined) of wild type (4.6% TUNEL positive cells, marked by white arrows) compared to cnr1- /- (3.9%) and cnr2^-/- mutants (3.0%), indicating that cell death does not substantially contribute to small liver size in cannabinoid receptor mutants. Percentages represent average of n=3, Scale bar = 0.2 mm.