Stylianou P and Skourides PA (2009), Imaging morphogenesis, in Xenopus with Quantum ...

XB-ART-40181

Mech Dev 2009 Oct 01;12610:828-41. doi: 10.1016/j.mod.2009.07.008.

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Imaging morphogenesis, in Xenopus with Quantum Dot nanocrystals.

Stylianou P , Skourides PA .

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Mesoderm migration is a well studied morphogenetic movement that takes place during Xenopus gastrulation. The study of mesoderm migration and other morphogenetic movements has been primarily based on in vitro assays due to the inability to image deep tissue movements in the opaque embryo. We are the first to report the use of Near Infra Red Quantum Dots (NIR QD's) to image mesoderm migration in vivo with single cell resolution and provide quantitative in vivo data regarding migration rates. In addition we use QD's to address the function of the focal adhesion kinase (FAK) in this movement. Inhibition of FAK blocks mesoderm spreading and migration both in vitro and in vivo without affecting convergent extension highlighting the molecular differences between the two movements. These results provide new insights about the role of FAK and of focal adhesions during gastrulation and provide a new tool for the study of morphogenesis in vivo.

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Species referenced: Xenopus laevis
Genes referenced: actl6a bcr fn1 ptk2 pxn tbxt
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	Fig. 1. NIR QD’s can be used to label and image mesodermal cells during gastrulation. (A and B) NIR QD (800-nm peak emission) labelled animal pole cells in a living embryo viewed with two filter sets. (A) The embryo was imaged with 600 nm long pass emission filter allowing all wavelengths above 600 nm to reach the camera and in B the same area of the same embryo was imaged with an 800-nm long pass filter set allowing all wavelengths above 800 nm to reach the camera. Autofluorescence in the 600–750 nm range is so intense that the labelled cells (three indicated with red stars in A) cannot be differentiated from unlabelled neighboring cells. (C and D) NIR QD labelled cells break free from their neighbors and become interspersed within non labelled regions and can be visualized through the pigmented animal cap. (E–η) Four frames from a time-lapse movie of an embryo injected at the DMZ (yellow star) at the four-cell stage with NIR QD’s. The mesodermal front becomes clearly visible as it moves towards the top of the blastocoel (red star). (I–L) Four frames from a time-lapse movie of an embryo injected with cell tracker QD’s at early gastrula. The Qtracker QD’s were injected into the blastocoel cavity. Using this approach QD’s labelled vegetal pole cells (not visible) and the mesodermal belt (DMZ yellow star, top of the BCR red star, VMZ green star). (M–P) Injection of NIR QD’s at both the DMZ (yellow star) and VMZ (green star) enables visualization of the mesodermal mantle closure (DMZ yellow star, top of the BCR red star, VMZ green star).
	Fig. 2. Injection of non-targeted NIR QD’s at the 32 cell stage and Qtracker QD’s at mid gastrula enables visualization of the mesodermal mantle with single cell resolution. FRNK expressing cells fail to migrate directionally. (A–E) Five frames from a 4D time-lapse movie (Supplemental movie) of an embryo injected at the DMZ (yellow star) at the 32 cell stage. Frames are 2D maximum intensity projections of deconvolved z-stacks. Individual cells are clearly visible as they migrate towards the top of the BCR. Cells appear to have freedom of movement and display varying migration rates however they all display a high degree of directionality as indicated by their tracks (E). (F–J) Same as (A–E) but one out of two blastomeres was injected with GFP-FRNK (500 pg) at the four-cell stage followed by QD injections of two blastomeres (one on the left and one on the right hemisphere) with NIR QD’s at the 32 cell stage. (F) Shows the GFP and QD labelled regions around the dorsal lip of the blastopore. Cells from the GFP negative side of the embryo are seen migrating directionally on the BCR (see tracks in J) while the majority of the cells from the GFP positive site fail to do so and move randomly. (K–O) Five frames from a time-lapse movie of an embryo injected with Qtracker NIR QD’s inside the blastocoel. The embryo was injected with GFP-FRNK (500 pg) at the four-cell stage in one out of two dorsal blastomeres. FRNK expressing cells fail to migrate towards the BCR. (P–T) Same embryo as in (K) through (O) magnified and tracked (tracked region boxed in N). Tracking of cells at the border area between the FRNK expressing and non expressing regions of the embryo (FRNK expressing-red track, non expressing-green track). Initially the FRNK expressing (GFP positive) cells move towards the top of the BCR with the non expressing cells but then begin to slow down and move randomly. Final positions of tracked cells shown in (O) (green star GFP negative cell red star GFP positive cell). (U) Distribution of individual cell migration rates from in vivo tracking of NIR QD injected embryos as in A–E (10 embryos were used and a minimum of 10 cells were tracked from each embryo).
	Fig. 3. Migrating mesodermal cells form focal adhesion complexes and FRNK displaces FAK from its signaling complexes. (A–C) Colocalization of FAK PY861(red) with vinculin (green) at the focal adhesions formed by mesodermal cells plated on fibronectin. (D–F) Colocalization of paxillin (red) with FAK PY576 (green) (G) Staining with phalloidin (red) and vinculin (green) reveals that focal adhesions are formed at the end of actin filaments. (H) Higher magnification (of the boxed region of G). (I) Sagital section of 10½ stage embryo staining with anti-FAK PY576 (secondary antibody alexa633) reveals a concentration of phosphorylated FAK at the interface between mesoderm and deep ectoderm. (J–L) FRNK-HA (red) can compete FAK (green) from its signaling complexes. FRNK-HA (500 pg) was injected in one out of two blastomeres of two-cell stage embryos at the animal pole and then the embryos were allowed to develop to stage 11 fixed and processed for whole mount immunostaining. FRNK-HA was stained with an HA specific antibody while endogenous FAK (green) was visualized using a-FAK polyclonal antibody raised against the N-terminus of FAK that does not recognize FRNK. Note the diffuse signal of endogenous FAK (green) in the FRNK expressing cells (red) compared to the neighboring control cells.
	Fig. 4. FRNK can inhibit mesodermal cell spreading and migration but has no effect on convergent extension movements. (A–C) Small dorsal marginal zone explants and individual cells are inhibited from spreading by FRNK over-expression (note the visibility of the nuclei in the GFP negative cells indicating that these cells are spread). memGFP (100 pg) co-injected with FRNK (500 pg DMZ injection one out of two blastomeres) serves as linage tracer of FRNK expressing cells. (D–F) Co-injection of FAK (50 pg) can rescue the FRNK phenotype. The majority of cells expressing both FAK and FRNK together with memGFP as a marker spread and display a migratory morphology. (G–I) FRNK expressing regions of activin induced animal caps placed on fibronectin coated slides exhibit no intercalative behavior towards the substratum and remain multilayered appearing dark in transmitted light illumination (G). A total of 500 pg of FRNK was injected in two out of four animal pole blastomeres of four-cell stage embryos and the animal caps were dissected (stage 8) induced with activin and placed on FN coated wells. (J–L) Expression of FRNK (two blastomeres of two-cell stage embryos were injected at the animal pole with FRNK 500 pg per blastomere) does not affect convergent extension. FRNK was co-injected with memGFP and GFP positive explants elongate to the same extent as controls (20 injected embryos and 20 controls embryos were used). (M) Migration rates of FRNK expressing mesodermal cells (1) are similar to those of animal cap controls (5). FRNK was injected in both blastomeres (500 pg per blastomere) of two-cell stage embryos. Co injection of FAK (50 pg per blastomere) with FRNK raises the migration rates of these cells significantly (3). QD labelling does not affect migration rates of induced mesodermal cells (4). In vivo migration rates (6) are slightly higher than the rates of in vitro rates (1). (N) Merged still frame from a time-lapse movie used to calculate migration rates of FRNK expressing (GFP positive) and control mesodermal cells. The majority of FRNK expressing cells remain rounded up. 500 pg of FRNK was injected with 100 pg of GFP in one out of two blastomeres of two-cell stage embryos.
	Fig. 5. FRNK injection inhibits mesoderm migration and results in failure of blastopore closure which often prevents neural tube closure. FRNK injected embryos (two blastomeres of four-cell stage embryos were injected at the dorsal marginal region with FRNK 500 pg per blastomere) fail to close their blastopore (A) compared to uninjected sibling embryos (B). This often leads to failure of closure of the posterior regions of the neural tube. Co-injection of FAK (50 pg per blastomere) rescues the phenotype (inset of A) and the blastopore closes completely in the majority of embryos (white arrows). (C) As the injections become more lateral (away from the DMZ) the percentage of embryos failing to close their blastopore decreases dramatically. (D–E) Whole mount in situ hybridization for Xbra indicates that mesoderm specification is not affected in FRNK expressing embryos (two blastomeres of four-cell stage embryos were injected at the dorsal marginal region with FRNK 500 pg per blastomere). Injected embryos (E) appear to have a slightly shortened trunk compared to controls (D).