Laser-mediated cell ablation during post-implantation mouse development

Abstract

Background: Laser-mediated cell ablation is a powerful tool that has been used to understand cell fate in a variety of externally developing organisms but has not been used during mammalian post-implantation development.

Results: We describe a method pairing laser ablation with murine embryo culture and establish parameters that can be used to precisely ablate cells in the selected field with minimal disruption to adjacent cells or the underlying cell matrix. Ablation of a large domain of endoderm, followed by ~1 day of culture results in a phenotypically normal embryo and gut tube, indicating that laser ablation is compatible with normal development. We next focused on one of the three precursor populations that have been shown to produce the liver bud. Ablations of a single progenitor domain result in a unilateral delay in the liver bud while the contralateral side is unaffected.

Conclusions: We demonstrate that laser ablation is a specific and useful technique for studying cell fate in the mouse embryo. This method represents a powerful advance in developmental studies in the mouse and can be used to provide information on the specification of organs, differentiation, cell migration, and vital tissue interactions during development.

Keywords: embryo culture; endoderm; laser ablation; liver development; mouse.

Figure 1. Inverted microscope guided laser-ablation

A–C) The laser, conjugated to a 20X objective on an inverted microscope (A) is viewed from above (B) and below the tissue culture dish (C). D–E) Because precise and efficient ablation occurs at the inner surface of the tissue culture plastic, the buoyant embryo is positioned with a silicone rubber holder (D) and further stabilized with a silicone plug (small block in E). F) Correct positioning (foregut facing the bottom of the dish) is verified at higher magnification. G) The field of view during manipulation allows for clear identification of the notochord (No), somites (S) and ventral foregut lip (VF). The white asterisk is placed on the ectoplacental cone in D–F.

Figure 2. Laser-mediated ablation of surface cells

A) An illustrated transverse section of an 8.5 dpc embryo. Neural tube (blue), notochord (green), mesenchyme (orange) and endoderm (red) are drawn to scale. The predicted area of laser penetration (black ellipse) lies within the focal plane of the 20X objective. The light path from the objective is represented by grey triangles. The maximum temperature is generated at the center of the focal plane and decreases away from the focal plane as indicated (red-orange scale to the right). B) Similar sized areas were pulsed on the endoderm/visceral endoderm border of an 8.5 dpc embryo with durations ranging from 25 to 200 μs as indicated. After treatment the embryo was stained with both propidium iodide (PI), which produces a red fluorescence in the nucleus of cells with ruptured membranes, and the nuclear stain Hoechst. C–D′) Whole mount views of an embryo treated with 150 μs pulses targeted to the exposed endoderm before (C–C′) and after (D–D′) addition of trypan blue (TB). E–F) Transverse sections through an embryo treated as above (C–D) then sectioned and subjected to immunofluorescence, demonstrate that TB staining (E–F arrowheads) is limited to FOXA1 expressing cells (green, F) on the embryonic surface.

Figure 3. Optimized ablation parameters maintain embryo integrity

A–B, D–E and G–H) Whole mount views of individual pre- (A, D, G) and post-ablation (B, E, H) embryos. The indicated pulse durations were used on a similar sized area, on the left lateral endoderm of each embryo (between white arrows). With 300 μs pulses the ablated area bulges outward (compare G to H), indicating an immediate loss of tissue integrity. The red dot seen in some images is the laser-guide. C, F, I) Each ablated embryo was transversely sectioned and subjected to immunofluorescence analysis of CDH1 (red), a cell junction marker, and the basement membrane marker Laminin (green). The ablated area is located between the white arrowheads. The area outlined by each box is viewed at higher magnification (C′, F′, I′). The area between the green arrowheads demarcates the undisturbed contralateral side of the embryo. C–C′) Pulse durations of 140 μs result in loss of endoderm cell junctions and clustering of cell debris without significant disruption of the basement membrane. F–F′) 200 μs pulses produce gaps in the basement membrane and fragmentation of the underlying somites (yellow arrowhead). I–I′) 300 μs pulses extirpate the endoderm and underlying basement membrane and fragment the underlying somites (yellow arrowheads).

Figure 4. Ablation of putative liver precursors, but not adjacent endoderm, impairs liver bud development

A–E) The endoderm fated to contribute to the dorsal gut tube proximal to the liver and pancreas buds was ablated in this 6S embryo (between arrows in A). After culture through 25 S (B), section analysis reveals normal expression of the endoderm marker FOXA1, the liver bud (lb) differentiation marker HNF4α and the ventral (vp) and dorsal pancreas (dp) marker PDX1 (C–E, arrow in D indicates the region typically colonized by the ablated dorsal endoderm). F–G) Much of the left lateral liver progenitor domain of this 6 S embryo was ablated (between arrows in F) and the contralateral (right) side labeled with DiI. Whole mount views of the resultant 24 S embryo after culture reveals localization of the DiI labeled cells (G). Section analysis reveals that the HNF4α (green) expressing liver bud cells are confined to the DiI-positive (red) contralateral side of the embryo (H–I). J–O) Section analysis of a control (J–L) and a right liver progenitor ablated embryo (M–O) after culture. The control embryo expresses the liver bud markers HNF4α (grey, K) and AFP (red, L) throughout the liver bud. Although FOXA1 is found throughout the liver bud of ablated embryos (M) both HNF4α and AFP expression are confined to the prominent contralateral (left) side of the tissue (N–O). P) A size comparison of the ablated and contralateral side of liver buds from individual embryos manipulated at the somite stage indicated and cultured through the tissue budding stage. Q) A comparison of the average sizes of the right and left side of control (n=9) and that of the ablated and contralateral side of the liver precursor ablated embryos displayed in P.