Figure 1.
The downwards bending of the tail in gpr22 morphants is rescued by the co-injection of gpr22 mRNA.
(A–C) Lateral views with anterior to the left of zebrafish embryos at 24 hpf injected at one cell stage with (A) water, (B) 0.2 mM fluorescein tagged gpr22 MO, (C) 75 pg/nl gpr22 mRNA or (D) 0.2 mM fluorescein tagged gpr22 MO and 100 pg/nl gpr22 mRNA. (B, C) Deregulation of gpr22 results in a bending of the tail, a reduction in length of the anterior-posterior axis and heart edema (arrow). (E) Quantification of the tail phenotype. (D, E) The curly tail of embryos injected with fluorescein tagged gpr22 MO is rescued by the co-injection of gpr22 mRNA in a dose-dependent manner. MO = morpholino, hpf = hours post-fertilization, n = number of analyzed embryos, WT = wild type.
Figure 2.
gpr22 deregulation results in defective LR patterning.
(A) Ventral or (B, C) dorsal views with anterior to the top of embryos at (A, B) 4 dpf or (C) 20 somite stage. (A) Transgenic embryos expressing GFP under control of the cmlc2 promoter. In contrast to water controls, gpr22 morphants show (from top to bottom) normal, absent, reversed or bicardial cardiac looping. Co-injection of 0.2 mM gpr22 MO and 100 pg/nl gpr22 RNA partially rescues the randomized cardiac looping (last panel). (B) WISH for foxa3. The liver of water control embryos develops at the left side of the embryo. In contrast, the position of the liver in embryos injected with 0.2 mM gpr22 MO is randomized (straight line). (C) WISH for southpaw (spaw). gpr22 morphants show randomized expression of the early left-specific LPM marker southpaw (straight line). (D) Quantifications of the heart or (E) liver/gut phenotype. MO = morpholino, V = ventricle, A = atrium, dpf = days post-fertilization, n = number of analyzed embryos, GFP = green fluorescence protein, cmlc2 = cardiac myosin light chain type 2, LR = left-right, WISH = whole mount in situ hybridization, LPM = lateral plate mesoderm.
Table 1.
Randomized expression of the left-specific LPM markers in zebrafish with deregulated gpr22 expression.
Figure 3.
Gpr22 regulates LR asymmetry through its function in the KV.
(A) WISH on 1–5 somite stage zebrafish embryos using the gpr22 sense or gpr22 antisense probe. Shown are the dorsal views of the tail bud region with anterior to the top. gpr22 is expressed in the axial structures and in the forming KV (arrow). (B, C) Percentage of embryos with randomized southpaw (spaw) expression, injected with (B) water or 0.2 mM gpr22 MO into the yolk at one cell stage (knock down in the whole embryo), or with (C) 1 mM control MO or 1 mM gpr22 MO into the yolk at mid-blastula stage (DFC/KV specific knock down). A DFC/KV specific knock down of gpr22 results in a similar percentage of zebrafish embryos showing randomized southpaw expression as a knock down in the whole embryo. (D) Representative images of panel (C). MO = morpholino, n = number of analyzed embryos, yolksuperscript = injected at one cell stage, DFCsuperscript = injected at mid-blastula stage, WISH = whole-mount in situ hybridization, DFC = dorsal forerunner cell, LR = left-right, KV = Kupffer’s vesicle.
Figure 4.
Gpr22 is not involved in KV formation or morphology.
(A) WISH for the DFC marker casanova on embryos at 75% epiboly or (B) for the KV marker charon on 10 somite stage embryos. Shown is (A) the dorsal view (B) of the tailbud region with anterior to the bottom. gpr22 overexpression or knock down does not alter the expression of casanova or charon. MO = morpholino, KV = Kupffer’s vesicle, WISH = whole mount in situ hybridization, DFCs = dorsal forerunner cells.
Figure 5.
Gpr22 affects cilia length and structure in the KV.
(A–F) Immunostaining with anti-acetylated tubulin antibody on embryos at 10 somite stage injected at (A–D) one cell stage with (A) water, (B) 0.2 mM gpr22 MO, (C) 75 pg/nl gpr22 RNA, (D) 0.2 mM gpr22 MO and 100 pg/nl gpr22 RNA, or injected at (E, F) mid-blastula stage with (E) 1 mM control MO, (F) 1 mM gpr22 MO. Shown are the cilia of the KV. (G) Quantification of average cilia length ± s.d. (B, G) Whole embryo or (F, G) DFC-specific knock down of gpr22 significantly reduces cilia length. (C, G) In contrast, gpr22 overexpression results in significantly longer cilia. (D, G) Co-injection of 0.2 mM MO and 100 pg/nl RNA almost completely rescues cilia length. (H–J) TEM of cross sections of the KV cilia at 10 somite stage. (H) Structure of water control KV cilia showing 9 parallel outer microtubule doublets. (I) gpr22 knock down or (J) overexpression results in a disruption of the proper microtubuli arrangement or (I) even an absence of doublets. MO = morpholino, n = number of analyzed embryos, DFC = dorsal forerunner cells, DFCsuperscript = injected at mid-blastula stage, KV = Kupffer’s vesicle, TEM = transmission electron microscopy, * = P<0.01 (unpaired, two-tailed T-test).
Figure 6.
Gpr22 does not act upstream of Foxj1a/Rfx2 in KV ciliogenesis.
(A, B) WISH of (A) foxj1a or (B) rfx2 on embryos at bud stage. Shown are (A) the lateral view with anterior to the left or (B) dorsal view with anterior to the top. gpr22 knock down or overexpression does not alter the DFC expression of foxj1a or rfx2, two master regulators of ciliogenesis. (C) Confirmation by qPCR of the foxj1a, rfx2 and polaris (target gene) RNA levels in the whole embryo, normalized to beta-actin. MO = morpholino, WISH = whole-mount in situ hybridization, DFC = dorsal forerunner cell, KV = Kupffer’s vesicle.