gms | German Medical Science

Objectives/Interrogation: The processes of developmental patterning occurs in the embryo long before anatomical structures are visible. When these unseen developmental processes such as cell signalling and fate specification go wrong, however, congenital hand differences can occur. To understand where specific tissues are derived from and how they are patterned, developmental biologists undertake 'fate mapping' in model organisms, such as the chicken and mouse in order to establish the origins of the tissues that comprise the limb. We present the next generation of limb bud fate mapping using novel transgenic chicken embryos (Roslin Chameleon) that have a Cre-inducible, multi-fluorescent Cytbow construct. This method was used to determine the origins of the radius and ulna, structures that have been neglected in previous fate maps due to technical constraints.

Methods: Embryos were generated from a cross between heterozygous Chameleon males and stock hens generating eggs. These embryos allow implanted Cre protein within cells in the limb bud mesenchyme to exhibit fluorescence, even through cell multiplication, and therefore structures could be traced through their subsequent development. After 7 days, limb buds were fixed and processed for histology and immunohistochemistry, Optical Projection Tomography or Light Sheet Microscopy. In order to create accurate fate maps, the location of the bead for each fluorescent limb was plotted onto a stage specific outline of the wing bud, split into six segments - the stylopod, anterior and posterior zeugopod, anterior, medial and posterior autopod. These sections were imposed on the early limb bud, depending on the eventual location of the fluorescence induced by each bead.

Results and Conclusions: Application of an agarose bead soaked in TAT-Cre protein acted immediately and transiently to induce recombination in the transgene in a limited number of limb bud cells. Expression of fluorescent proteins was equal, stable and exclusive, occurring within 5 hours of TAT-Cre application and within a 380um distance from the bead. A total of 18 stage 20HH limbs buds were successfully implanted and had resulting fluorescence. After anatomical examination, 2 were found to have labelled cells in the radius and 2 in the ulna. Extrapolation from the placement of the bead at stage 20HH allowed us to hypothesise the orgin of the cells which give rise to the forearm. Such a fate map allows us to potentially decipher the origins of congenital hand differences such as radial or ulnar aplasia.