FIGURE SUMMARY
Title

Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent

Authors
Mich, J.K., Blaser, H., Thomas, N.A., Firestone, A.J., Yelon, D., Raz, E., and Chen, J.K.
Source
Full text @ Dev. Biol.

Cyclopamine treatment induces zebrafish PGC mislocalization. (A, B) PGCs localize to the presumptive gonad in the ethanol-treated control embryos, as indicated by the distribution of vasa-expressing cells. In contrast, PGCs are widely dispersed in embryos treated with 60 μM cyclopamine. Cyclopamine and ethanol treatments were initiated at the one-cell stage and the resulting phenotypes at 24 hpf are shown. Scale bar: 200 μm. (C) Dose–response profile for cyclopamine-induced PGC mislocalization. At least 400 PGCs were scored at 24 hpf for each cyclopamine concentration. (D) Dose–response profile for cyclopamine inhibition of Shh signaling in Smo-/- mouse embryonic fibroblasts transfected with zebrafish smo. Data are the average of triplicate samples, with error bars representing the standard deviations. (E) Temporal window of cyclopamine action with respect to PGC mislocalization, as determined by a survey of treatment regimens. At least 500 PGCs were scored at 24 hpf for each time course of cyclopamine exposure.

Cyclopamine targets both PGCs and somatic cells to effect PGC mislocalization. (A) Schematic representation of the transplantation procedure used to differentiate cyclopamine action on PGCs and somatic tissues. Donor embryos were injected at the one-cell stage with EGFP-nanos1-3′UTR mRNA to selectively label PGCs and Alexa Fluor 594-dextran to label all donor cells, and one to three PGCs were transplanted from shield-stage (6 hpf) donors into unlabeled sphere-stage (4 hpf) hosts. Donor and host embryos were treated with 100 μM cyclopamine or an ethanol vehicle control prior to transplantation, and cultured until 24 hpf in cyclopamine-free medium. EGFP-expressing PGCs in the host embryos were then scored at 24 hpf for their localization to the presumptive gonad site. (B–E) Bright-field images, (B′–E′) EGFP fluorescence images, and (B″–E″) Alexa Fluor 594 fluorescence images of representative host embryos resulting from the following donor/host treatment regimens: (B) ethanol-treated donor and host; (C) cyclopamine-treated donor and host; (D) cyclopamine-treated donor and ethanol-treated host; and (E) ethanol-treated donor and cyclopamine-treated host. (F) Quantification of PGC mislocalization, scoring at least 100 transplanted PGCs for each donor/host condition per experiment. Data represent the average of three or more experiments, with error bars representing the standard error of the mean. (G–L) Distribution of sdf1a transcripts in ethanol- (G–I) and cyclopamine-treated (J–L) embryos from gastrulation to mid-somitogenesis. Embryos at the shield (G and J), 3-somite (H and K), and 15-somite (I and L) stages are shown. Scale bars: 200 μm.

Cyclopamine decreases PGC motility without disrupting chemotaxis. (A) Fifteen 100-min tracks of EGFP-expressing PGCs in Tg(kop:EGFP-F-nanos1-3′UTR) embryos treated with either ethanol or cyclopamine starting at the one-cell stage. PGC migration was followed during late gastrulation and early somitogenesis (8 to 12 hpf); the beginning of each track is set at the origin and the end of each track is indicated by a black-edged circle. (B) Representative migratory behavior of individual PGCs in ethanol- or cyclopamine-treated embryos over a similar time frame. Speed and vectoral changes are depicted by the blue and red lines, respectively. Time frames during which the PGCs exhibit an elongated morphology are indicated by the yellow bars, and run phases are indicated by the green bars. Note that run phases in embryos treated with the ethanol vehicle control usually coincide with periods of maximum speed and minimum angle changes. (C) Average speeds of PGC movement over the entire imaging period or during run phases. (D) Fraction of time, (E) average duration, and (F) frequency of PGC run phases or elongated morphological states in ethanol- or cyclopamine-treated embryos. Data in panels C–F are the averages of 35 ethanol-treated and 45 cyclopamine-treated PGCs, and error bars represent the standard error of the mean. (G) Chemotaxis of EGFP-expressing PGCs (green) toward transplanted Sdf1a-expressing cells (red) in ethanol- or cyclopamine-treated embryos. Scale bar: 50 μm.

Cyclopamine does not disrupt the subcellular organization or maturation of PGCs. (A, B) PGC-specific expression of nanos1, a maternally derived determinant of PGC specification, in ethanol- or cyclopamine-treated embryos. (C, D) Expression of Vasa protein, which is translated specifically in PGCs after 4 hpf. (E, F) Perinuclear granules containing Vasa-dsRedex protein (red) in EGFP-expressing PGCs (green). (G, H) PGC-specific expression of ziwi, which is transcribed in PGCs between 24 and 48 hpf. (I, J) Confocal immunofluorescence imaging of α-tubulin (red) and farnesylated EGFP (green) in the PGCs of Tg(kop:EGFP-F-nanos1–3′UTR) embryos. Developmental stages: A–F, 24 hpf; G–H, 48 hpf; I–J, 6 hpf. Scale bars: A–D and G, H, 100 μm; E–F, 20 μm; I, J, 10 μm.

PGC migration is not perturbed in zebrafish embryos lacking maternal and zygotic Smo but remains cyclopamine-sensitive. (A) Phenotypic distribution of progeny obtained from mating a chimeric female bearing smuhi1640 ova and either a smuhi160/+ male or a chimera bearing smuhi1640 sperm. (B, C) MZsmuhi1640 embryo treated with either ethanol or cyclopamine from the one-cell stage to 24 hpf and then stained for vasa expression. Scale bar: 200 μm.

Cyclopamine perturbs PGC migration in part through dysregulated cell adhesion. (A) Time-lapse imaging of PGC interactions in ethanol- or cyclopamine-treated Tg(kop:EGFP-F-nanos1-3′UTR) embryos. (B) Average duration of each PGC–PGC contact for both treatment conditions. Data are the averages of 35 ethanol-treated and 45 cyclopamine-treated PGCs, and error bars represent the standard error of the mean. (C, D) Distribution of vasa-positive PGCs in embryos injected with either water or an E-cadherin MO (300 pg/embryo) at the one-cell stage and then treated with 50 μM cyclopamine until 24 hpf. Scale bar: 200 μm. (E) Quantification of PGC mislocalization in embryos injected with varying doses of the E-cadherin MO and treated with cyclopamine or an ethanol vehicle control. At least 500 PGCs were scored for each experimental condition. (F) Western analysis of E-cadherin expression in embryos treated with ethanol or cyclopamine from the one-cell stage to the sphere stage (6 hpf). Quantification of E-cadherin expression normalized to a β-actin loading control is shown. (G, H) Confocal immunofluorescence imaging of E-cadherin expression and localization in identically treated embryos. Scale bars: A, 20 μm; C, D, 200 μm.

Cyclopamine induces PGC mislocalization during gastrulation and early somitogenesis. (A–B) Distribution of vasa-expressing PGCs and sdf1a transcripts in shield-stage (6 hpf) embryos treated with either ethanol or cyclopamine, starting from the one-cell stage. (C–D) Corresponding analysis of embryos at the 3-somite stage (11 hpf). Scale bars: 100 μm. (E) Quantification of PGC mislocalization in ethanol- or cyclopamine-treated embryos at various developmental time points. At least 400 PGCs were scored as either within or outside of the sdf1a expression domain for each experimental condition.

PGC migration is not perturbed in Hh pathway mutants or morphants. (A–E) PGCs are localized to the presumptive gonad in syutbx392, contm15a, smuhi1640, yotty119, and dtsts269 mutant zebrafish, as indicated by the distribution of vasa-expressing cells. (F) Embryos co-injected with smo MO (6 ng/embryo) and EGFP-nanos1-3′UTR RNA (100 pg/embryo) also exhibit properly localized PGCs (arrows). 24-hpf (A–D, F) and 6-dpf (E) zebrafish are shown. Scale bars: 200 μm.

PGC migration is not perturbed in MZsmub577 embryos but is cyclopamine-sensitive. (A–B) MZsmub577 embryo treated with either ethanol or cyclopamine from the one-cell stage to 1 day post fertilization (dpf) and then stained for vasa expression. Arrows indicate the locations of vasa-positive PGCs (blue) among the pigmented cells (brown). Scale bar: 200 μm.

Demonstration of the efficacy and specificity of the E-cadherin MO. (A) Embryos injected with 300 pg of E-cadherin MO exhibit decreased E-cadherin levels by 24 hpf, whereas wildtype embryos and those injected with 300 pg of a five-base mismatch E-cadherin MO do not. A representative immunoblot is shown, and quantitative data are the average E-cadherin/β-actin ratios of at least three independent samples ± s.e.m., normalized with respect to the wildtype condition. (B–C) Distribution of vasa-positive PGCs in embryos injected with either water or the mismatch E-cadherin MO (300 pg/embryo) at the one-cell stage and then treated with 50 μM cyclopamine until 24 hpf. Scale bar: 200 μm. (D) Quantification of PGC mislocalization in embryos injected with either water or the mismatch E-cadherin MO (300 pg/embryo) and treated with 50 μM cyclopamine or an ethanol vehicle control. At least 600 PGCs were scored for each experimental condition.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image.

Reprinted from Developmental Biology, 328(2), Mich, J.K., Blaser, H., Thomas, N.A., Firestone, A.J., Yelon, D., Raz, E., and Chen, J.K., Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent, 342-354, Copyright (2009) with permission from Elsevier. Full text @ Dev. Biol.