CHARACTERIZATION OF MIGRATING NEURAL CREST CELLS FROM HINDBRAIN EXPLANTS IN ZEBRAFISH

Abstract

Our lab was interested in creating a reproducible mechanism for obtaining neural crest cells in culture from cultured hindbrain explants of zebrafish. We have characterized the population of cells migrating from the explants as neural crest using a genetic variant of zebrafish with a GFP tag SOX-10, as well as immunostaining using HNK-1, a known neural marker for neural crest cells. We have also devised a modified protocol of in-situ hybridization that can be used for cells in culture for determining specific genes cells express in culture. Based on previous studies by Bingham et al., hindbrain explants have been extensively studied for preservation of their native (endogenous) cellular environments as well as the behavior of individual cell types. Biopidy ceramide labeling of live embryos has been used to extensively monitor and record cell shapes and organization in the developing zebrafish neural tube (Cooper et al). Studies by Bingham et al. have shown that morphogenesis of the neural tube in the zebrafish hindbrain is unaffected by the explantation of the hindbrain into a culture medium. The expression of krox20 and hoxb1a, which are expressed in the developing embryo in the hindbrain in rhombomeres 3 and 5 and rhombomere 4 respectively have been found to be unaffected when the hindbrain is explanted into a culture media. It has also been shown that valine expressing neural crest cells migrate normally out of r6. These results demonstrate that morphogenesis and patterning of the explanted hindbrain occur normally following the explanation of the hindbrain into a culture media (Bingham et al). Our lab has carried out dissections to remove the hindbrain from the developing embryo 15-18 hours post fertilization. We have found that it is possible to dissect the hindbrain out of a developing embryo and have cells migrate from these explants. We have also shown that these cells express Sox10 and are stained when using an antibody for HNK-1, which is known to be a neural crest cell marker. Our lab has also developed a working protocol of in-situ hybridization on cells in culture for Caco 2 and HT-29 using RNA probes for human beta actin and Hox A10.