The orphan G protein-coupled receptor 161 is required for left-right patterning

Abstract

Gpr161 (also known as RE2) is an orphan G protein-coupled receptor (GPCR) that is expressed during embryonic development in zebrafish. Determining its biological function has proven difficult due to lack of knowledge regarding its natural or synthetic ligands. Here, we show that targeted knockdown of gpr161 disrupts asymmetric gene expression in the lateral plate mesoderm, resulting in aberrant looping of the heart tube. This is associated with elevated Ca(2+) levels in cells lining the Kupffer's vesicle and normalization of Ca(2+) levels, by over-expression of ncx1 or pmca-RNA, is able to partially rescue the cardiac looping defect in gpr161 knockdown embryos. Taken together, these data support a model in which gpr161 plays an essential role in left-right (L-R) patterning by modulating Ca(2+) levels in the cells surrounding the Kupffer's vesicle.

Fig. 1

Sequence comparison and in situ expression analysis of zebrafish gpr161. (A) Genbank accession no. of human GPR161: NM_007369 (Homo); zebrafish gpr161: EU090912 (Danio). 7TM (black), DRY (red) and PxxY (red) motifs were boxed. (B) gpr161 expression in the developing embryos by whole mount in situ hybridization. Inset showed the expression in developing somites and expression surrounding the Kupffer's vesicle (KV) near the tail bud region at 9 somite stage. All scale bars were 100μm.

Fig. 2

gpr161 knockdown disrupts cardiac looping morphogenesis. (A) Lateral view of zebrafish hearts at 4 dpf. Control and gpr161 knockdown in transgenic zebrafish embryos with cardiac specific GFP, TG[cmlc2:GFP], showing images of bright field (top panels) and fluorescent (bottom panels). A* labelled atrium, V* labelled ventricle. (B) Cardiac looping morphogenesis marked by cmlc2 expression in control (D-looping), gpr161 knockdown (unlooped and L-looping) and RNA rescued embryos. All scale bars were 100μm. (C) Graphical summary of gpr161 knockdown disrupted normal cardiac D-looping morphogenesis at 36 hpf. Calculation as % of embryos with D-looping in control (97.8±1.3%; n=223), embryos injected with gpr161 morpholino (MO#36, 32.4±4.1%; n=293) and RNA rescue (gpr161 RNA without 5'UTR; 52.2±3.4%; n=157), n was number of total embryos and results were from 4 injection experiments. Error bars were ± SEM. t-test * and ^# indicated statistical significance, p<0.01.

Fig. 3

gpr161 knockdown disrupts L-R identity in lateral plate mesoderm. (A) lefty2 expression in the left anterior lateral plate mesoderm (LPM) and lefty1 expression in the left diencephalon were disrupted in gpr161 knockdown embryos. (B) Graphical summary of control (99.3±1.1%; n=351), gpr161 morpholino (MO#24, 67.0±4.7%; n=348) and t-test (p<0.01) results were from 11 injection experiments. (C) spaw expression in the left anterior lateral plate mesoderm (LPM) at 19.5 hpf in control and gpr161 knockdown embryos. ntl expression marked the midline as a reference. (D) Graphical summary of spaw expression in control (98.7±1.4%; n=311), gpr161 morpholino (MO#24, 63.4±2.7%; n=306) and t-test (p<0.01) results were from 13 injection experiments. Error bars were ± SEM. All scale bars were 100μm.

Fig. 4

(A) bmp4 expression was predominantly on the left side of the cardiac cone in comparison to the uniform expression of cmlc2 (inset) was disrupted in gpr161 knockdown embryos. Graphical summary of control (85.8±3.7%; n=254), gpr161 knockdown (MO#24, 47.5±5.7%; n=193) and t-test (p<0.01) results were from 7 injection experiments. (B) bmp4 expression was predominantly on the left side of the heart tube in comparison to the uniform expression of cmlc2 (inset) was disrupted in gpr161 knockdown embryos. Graphical summary of control (99.5±1.3%; n=141), gpr161 knockdown (MO#24, 56.9±5.9%; n=102) and t-test (p<0.01) results were from 4 injection experiments. Dashed lines were drawn over the cardiac cone and heart tube and the midline of the structures. n was number of total embryos. Error bars were ± SEM. All scale bars were 100μm.

Fig. 5

L-R asymmetry of visceral organs in zebrafish embryos. (A) foxa3 expression marks the gut primordium at 36 hpf showing the liver bud (left) and pancreatic bud (right) in control embryo. (B) Graphical summary of normal visceral asymmetry in control (97.8±1.3%), gpr161 knockdown (32.4±4.1%) and RNA rescued (52.2±3.4%) embryos. t-test (p<0.01) results were from 6 injection experiments. n was number of total embryos. Error bars were ± SEM. (C) foxa3 expression of liver and pancreatic buds in normal L-R asymmetric position in control, symmetric or heterotaxia in gpr161 knockdown and normal L-R asymmetry in RNA rescued embryos. All scale bars were 100μm.

Fig. 6

gpr161 knockdown disrupts Ca²⁺ handling which is essential for establishment of L-R asymmetry. (A) Defective Ca²⁺ handling resulting in elevated intracellular Ca²⁺ in gpr161 knockdown embryos surrounding the Kupffer's vesicle. Pseudocolour scale for the intracellular Ca²⁺ signal as normal, intermediate and high intensity. The percentage of embryos exhibited such level of Ca²⁺ signal was listed in control, gpr161 knockdown, ncx1- and pmca-RNA rescue of the gpr161 knockdown embryos. (B) Removal of excessive Ca²⁺ by over-expression of 1pg of Ca²⁺ pump pmca or Na⁺/Ca²⁺ exchanger ncx1 can rescue gpr161 knockdown phenotype. Cardiac looping defect in gpr161-MO (MO#24, 54.1±4.0%; n=368), rescued by pmca-RNA (32.0±6.0%; n=238), rescued by ncx1-RNA (34.0±4.8%; n=144) and results were from 4 injection experiments. n was number of total embryos. Error bars were ± SEM. t-test * and ^# indicated statistical significance, p<0.01. All scale bars were 100μm. (C) A model of gpr161 involved in Ca²⁺ handling and essential for L-R asymmetry for cardiac morphogenesis and visceral asymmetry in developing zebrafish embryos. Blocking gpr161 function led to defect in Ca²⁺ handling and resulted in elevated free cytosolic Ca²⁺ and loss of cardiac looping and abnormal chamber morphogenesis. The gpr161 knockdown can be rescued by removing excessive cytosolic free Ca²⁺ using over-expression of Ca²⁺ pump pmca and Na⁺/Ca²⁺ exchanger ncx1.