Fig. S3

Heterotaxy Phenotype Is Not Due to Altered α₁-Adrenergic GPCR Signaling or Changed Expression of Key Components of Hh and RA Signaling, Related to Figure 4

(A) Overstimulation of α_1b-adrenergic receptors was shown to be causative for situs inversus in a rat model. As GRKs are considered the terminating enzymes in the signaling of GPCRs, loss of GRK activity could theoretically result in a situation similar to receptor overstimulation. However, as measured by lefty2 in situ hybridization at 22 ss, the phenotype upon loss of function of Grk5l is not caused by increased activity of the α_1b-adrenergic receptor. Subtype-specific inhibition of α_1b-adrenoceptors by treatment with WB-4101 from pregastrulation stages (5 hpf) on did not rescue the Grk5l depletion phenotype. Bar graph displays summary of three independent experiments. CTRL MO^DMSO versus Grk5l MO^DMSO: p < 0.0001; Grk5l MO^DMSO versus Grk5l MO^WB-4101: p = 0.6699; Fisher’s exact test.

(B–B2)2 Expression of the Hh target gene patched 1 (ptc1) remains unchanged after KD of Grk5l. B noninjected; (B2) injected with control MO; (B3) injected with Grk5l MO.

(C–C3) Expression pattern of cytochrome P450 family 26 subfamily A polypeptide 1 (cyp26a1), a key component of RA signaling, is unchanged after KD of Grk5l. (C) noninjected; (C2) injected with control MO; (C3) injected with Grk5l MO.)

(D–D3) Expression of fibroblast growth factor 8 (fgf8), a target of retinoic acid signaling, is not changed upon KD of Grk5l. (D, noninjected; (D2) injected with control MO; (D3) injected with Grk5l MO.

(E–E3) Similar to Grk5, Grk5l regulates canonical wnt signaling in a positive fashion. Loss of either zebrafish Grk5 variant causes downregulation of the wnt target gene axin2.