Fate Mapping - New Techniques

New Techniques

More recently, scientists have developed new tools inspired by past approaches. The use of fluorescent peptide tracers can be helpful, but in order to extend fate mapping to later stages when cells are smaller and thus difficult to consistently and selectively inject (unlike the 0.5mm leech embryo), modifications were made. Chemically-"caged" fluorescent peptides, such as caged Rhodamine-dextran or caged Fluorescein-dextran (FITC), have been developed to be non-fluorescent until hit with an ultraviolet (450 nm) laser, which "uncages" the compound and causes it to fluoresce. This tool was particularly well received in the zebrafish community since it is ideal to inject caged compound into a freshly fertilized, single-cell embryo that will rapidly develop over 24 hours into a transparent swimming larvae with approximately 30,000 cells. Injection of the compound into the single-cell embryo allows uniform distribution throughout all the cells of the developing embryo, and the dextran carrier, developed by Jochen Braun and Bob Glimich prevents diffusion between cells through gap junctions, which are common during embryogenesis. At a given stage of development, one can use a UV laser to uncage the compound in a distinct cell or set of cells, effectively labeling them red (Rhodamine) or green (FITC). The embryos can be allowed to develop normally until a later time, at which point they can be imaged for red or green fluorescence in the progeny of the uncaged cells. However, it is important to note that properly focusing the UV laser beam on an individual cell deep within the embryo is difficult. Sub-optimal focusing can lead to unintentional uncaging of cells outside the focal plane of the target cell. Also, uncaged Rhodamine has a short half-life, and must be imaged within 48 hours or the signal may be difficult to see. Similarly uncaged FITC is sometimes difficult to image later in development, and thus detection by immunostaining is often performed. Injected embryos must also be kept in the dark to avoid non-specific uncaging from ambient light.

Aside from chemical tracers, we can also lineage trace with GFP mRNA injection, over-express a protein of interest by injecting mRNA, knockdown expression by shRNA injection or mutant protein construct injection to see what cell types and tissues are affected during embryogenesis. Particularly mRNAs encoding histone tagged with green, cyan, yellow or red fluorescent proteins co-injected with mRNA for a membrane-bound fusion protein conjugated to another fluorophore, greatly enhance our ability to obtain high-resolution images of individual cell movements over time. Such microinjection experiments allow highly specific and selective cell manipulations superior to gross ablation experiments. Thus, specificity will facilitate the effective observation of injected cells and their neighbors and resultant deviations from normal development.

Fate mapping is therefore an extremely powerful tool for biologists, with new and improved tools constantly evolving to allow great resolution of what goes on during embryogenesis in various model organisms. Many genetic and chemical tools have been generated that allow long-term cell lineage tracing, bringing insight into the longevity of embryonic stem cells for various tissues. The ability to over-express and knockdown putative cell-fate patterning molecules and fluorescently label them, will also enhance our understanding of the extrinsic and intrinsic molecular cues required by various cell types during embryogenesis.