Fig. 4

Ela’s Cognate Receptor for Endoderm Differentiation Is Aplnr

(A) ELA and apelin are very basic hormones with isoelectric points exceeding 12.

(B) By qPCR, the onset of transcription of aplnra and aplnrb coincides with that of ela at the MBT; apln expression debuts 5 hr later during gastrulation (data are represented as mean ± SEM).

(C) Relative to control embryos, the expression of aplnra and aplnrb at 70% epiboly becomes stronger and confined to the most equatorial hypoblast in the ela mutant (white arrowheads: specific aplnra expression in the animal pole).

(D) WT embryo with a beating heart and blood circulation at 6 dpf, normal sox17 expression in definitive endoderm at 75% epiboly, no erythrocyte accumulation in the ICM, and cmlc1 expression in the heart-forming region at 30 hpf.

(E) aplnr morphants phenocopy ela mutant embryos, with no beating heart, loss of blood circulation at 6 dpf, reduced sox17 expression in definitive endoderm at 75% epiboly, accumulation of erythrocytes in the ICM, and loss of cmlc1 expression in the heart-forming region at 30 hpf.

(F) ela mutant embryos are nearly indistinguishable from Aplnr morphants and have no beating heart or blood circulation at 6 dpf, show reduced sox17 expression in definitive endoderm at 75% epiboly, have accumulated erythrocytes in the ICM, and no cmlc1 expression in the heart-forming region at 30 hpf.

(G) In 293T cells, overexpression of zebrafish aplnra or aplnrb, or human APLNR is sufficient to confer cell-surface binding to recombinant AP-ELA.

(H) In 293T cells, overexpression of zebrafish aplnrb, but not its mutant form (grinch) carrying the W90L missense mutation, or GPR15 (an orphan GPCR closely related to APLNR), is enough to afford binding to recombinant AP-ELA.