Cxcl12b–Cxcr4a signaling axis is essential for L–R asymmetric development.

(A–D) WT embryos and cxcr4a^um20 mutants injected with or without 300 pg cxcr4a mRNA at the 256-cell stage were examined for cardiac looping and liver laterality at 48 hpf by WISH against cmlc2 (A) and hhex (C). Embryos with different phenotypes are shown in ventral (A) or dorsal view (C), and the ratios are shown in (B) and (D). Underlying data can be found in  S1 Data. (E–H) Embryo ratios with different expression patterns of cmlc2 and hhex at 48 hpf in WT embryos injected with 8 ng cxcr4a MO (4a MO) at the 256-cell stage (E and F) and cxcl12b mutants (G and H). Underlying data can be found in  S1 Data. (I–N) cxcr4a deficiency alters Nodal gene expression pattern. Representative images of spaw and pitx2c expression in cxcr4a mutants (I and M) and morphants (K). All embryos are shown in dorsal views with anterior on the top. Ratios of embryos are shown in (J), (L), and (N). Underlying data can be found in  S1 Data. cmlc2, cardiac myosin light chain 2; DFC, dorsal forerunner cell; hhex, hematopoietically expressed homeobox; hpf, hours postfertilization; L–R, left–right; MO, morpholino; pitx2c, paired-like homeodomain 2c; spaw, southpaw; WISH, whole-mount in situ hybridization; WT, wild-type.

cxcr4a is indispensable for KV formation and ciliogenesis.

(A–B) Light micrographs at the 10-somite stage showed smaller or even absent KVs in cxcr4a^um20 or cxcl12b^mu100 mutants. Scale bar, 200 μm. Embryo ratios with different KV sizes were shown in (B). Underlying data can be found in  S1 Data. (C) Time-lapse confocal images from the 1-somite stage to the 6-somite stage showed the dynamic formation of KV in WT and cxcr4a-deficient Tg(sox17:GFP) embryos. Scale bar, 20 μm. The ratios of affected embryos were indicated. (D–F) Fluorescent immunostaining of KV using anti-GFP and anti-α-Tubulin antibodies at the 10-somite stage in WT embryos and cxcr4a^um20 mutants. The boxed areas in the images were presented at higher magnification in the relevant insets. Scale bar, 20 μm. Cilia average number and length were quantified from three independent experiments, and the group values were expressed as the mean ± SD (E and F). Student t test, *P < 0.05, **P < 0.01. Underlying data can be found in  S1 Data. (G) Fluorescent bead tracking experiments showed that fluorescent beads moved in a persistent counterclockwise fashion in WT embryos but had no directional flow in cxcr4a^um20 mutants. White spots, yellow spots, and curves mark the start points, the end points, and the tracks of bead movements, respectively. GFP, green fluorescent protein; KV, Kupffer’s vesicle; sox, SRY-box transcription factor; Tg, transgene; WT, wild-type; α-Tubulin, acetylated tubulin. 

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Cxcr4 promotes Cyclin D1 expression through ERK signaling during DFC proliferation.

(A) ERK1/2 phosphorylation levels were dramatically decreased in cxcr4a^um20 mutants. WT and cxcr4a-deficient Tg(sox17:GFP) embryos were harvested at the 75% epiboly stage and subjected to immunostaining for p-ERK1/2 (red) and GFP (green). All embryos are shown in dorsal views with anterior to the top. Scale bar, 20 μm. (B–E) caMEK mRNA overexpression in DFCs rescued L–R patterning defects in cxcr4a^um20 mutants. Different types of heart looping and liver laterality at 48 hpf in cxcr4a^um20 mutants following midblastula injection of different caMEK mRNA doses were visualized by cmlc2 and hhex expression (B and C). The ratios are shown in (D) and (E). Underlying data can be found in  S1 Data. (F) Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with 60 pg caMEK mRNA at the 256-cell stage and then harvested at the 75% epiboly stage for fluorescence in situ hybridization experiments with cyclin D1 (red) and GFP (green) probes. Dorsal views with anterior to the left. Scale bar, 20 μm. (G–H) WT embryos were treated with 0.5 μM PD0332991 or 0.2 μM CY202 from the shield stage to bud stage and then analyzed for L–R patterning defects at 48 hpf by in situ hybridizations with cmlc2 and hhex probes. The proportion of treated embryos exhibiting each type of heart looping and liver laterality is shown in (G) and (H). Underlying data can be found in  S1 Data. (I–J) Reintroduction of cyclin D1 into DFCs relieves DFC proliferation defects in cxcr4a^um20 mutants. Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with or without 300 pg cyclin D1 mRNA at the 256-cell stage, followed by coimmunostaining with anti-BrdU (red) and anti-GFP (green) antibodies at the 75% epiboly stage. Representative images are shown in (I), and the percentage of BrdU-positive DFCs is indicated in (J). Scale bar, 20 μm. Student t test, ***P < 0.001. Underlying data can be found in  S1 Data. (K–M) The reduced ratios of embryos affected in KV size (K) and cmlc2 (L) or hhex (M) expression show that DFC-specific overexpression of cyclin D1 rescued the defects of KV formation (K) and L–R patterning (L and M) in cxcr4a mutants. Underlying data can be found in  S1 Data. BrdU, bromodeoxyuridine; caMEK, constitutively activated version of MEK; cmlc2, cardiac myosin light chain 2; DFC, dorsal forerunner cell; ERK, extracellular regulated MAP kinase; GFP, green fluorescent protein; hhex, hematopoietically expressed homeobox; hpf, hours postfertilization; KV, Kupffer’s vesicle; L–R, left–right; ns, no significant difference; p-ERK1/2, phosphorylated ERK1/2; sox, SRY-box transcription factor; Tg, transgene; WT, wild-type

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

foxj1a expression was examined by in situ hybridization at the 75% epiboly and bud stages in wild-type and  cxcr4a ^um20  mutant embryos. Foxj1a, forkhead box j1a.

(A)  cxcr4a expression during gastrulation. In situ hybridization of  cxcr4a in embryos at the 75% epiboly stage (dorsal view with animal pole to the top) and bud stage (lateral views with animal pole to the top). Black arrowhead indicates the DFCs. (B)  cxcr4a expression at the 6-somite stage. Lateral view was shown with animal pole to the top in the left panel, and dorsal view was shown in the right panel. Black arrowhead indicates the KV. (C) Confocal images depicting the formation of the lateral dorsal aorta in live  Tg(flk: GFP)embryos. Scale bar, 50 μm. DFC, dorsal forerunner cell; ep, epiboly;  flk,  fms-like tyrosine kinase; GFP, green fluorescent protein; KV, Kupffer’s vesicle; LDA, lateral dorsal aorta; PHBC, primordial hindbrain channel; Tg, transgene.

(A) Time-lapse confocal images showing DFC migration in wild-type and  cxcr4a ^um20 mutant embryos on a  Tg(sox17: GFP) background from 75%–90% epiboly stages. Scale bar, 50 μm. (B) Sox17 expression was examined by in situ hybridization in wild-type and  cxcr4a ^um20  mutants at the 75% epiboly stage. (C–D) Wild-type and  cxcr4a-deficient  Tg(sox17: GFP) embryos were harvested at the 10-somite stage for immunostaining. KV cells were labeled using an antibody against GFP. Expression of the basal–lateral marker E-cadherin (C) and the apical marker aPKC (D) were visualized using the indicated antibodies. Scale bar, 20 μm. aPKC, atypical protein kinase; DFC, dorsal forerunner cell; GFP, green fluorescent protein; KV, Kupffer’s vesicle;  sox, SRY-box transcription factor; Tg, transgene.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Tg(mKO2-zCdt1(1/190)) embryos were treated with 0.5 μM PD0332991 or 0.2 μM CYC202 from the shield stage to the 10-somite stage. Then, these embryos were harvested for in vivo confocal imaging. Note that both PD0332991 and CYC202 treatments induced a remarkable increase of the number of mKO2-zCdt1–positive cells. Scale bar, 200 μm. CDK, cyclin-dependent kinase; EF1α, eukaryotic translation elongation factor 1α; mKO2, monomeric Kusabira Orange2; Tg, transgene; zCdt1, zebrafish chromatin licensing and DNA replication factor 1.