ABSTRACT: We have developed a method for rapidly identifying early, regionally-expressed molecules: FACS-Assisted Microdissection of Photolabeled cells (FAM-P). For our first FAM-P project, we injected embryos with purified Kaede protein, a stony coral protein that fluoresces at 514 nm (green) prior to- and 582 nm (red) after photoconversion at 405 nm. We used the scanning laser of a confocal microscope to photolabel late blastula-stage embryos along 4-6 tiers of marginal cells, comprising the mesoderm and endoderm germ layer precursors. This procedure left the neurectoderm precursor cells marked by green fluorescence and the mesendoderm precursor cells marked by red fluorescence. Embryonic cells were dissociated and subjected to FACS, separating red mesendoderm precursors from green neurectoderm precursors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 30,000 oligos representing more than 20,000 unique zebrafish genes. In validation of our strategy, many genes known to have elevated expression in the late blastula margin (e.g., squint, fgf8, lim1 and gata5) and several genes known to be specific to the early neurectoderm (e.g., sox31 and otx2) were independently identified by this approach. In addition to known genes, our method has identified dozens of previously uncharacterized genes that are enriched in the pre-mesendoderm or pre-neurectoderm. Keywords: 40% epiboly-stage embryos, injected with purified Kaede protein, differentially photolabeled, disaggregated and FACS sorted 34k expression arrays were generated by printing Oligo nucleotides from three different sets (MWG, Compugen and Operon) onto epoxy slides. Total RNA from Kaede protein-injected and FAM-P treated zebrafish embryos was amplified using an Ambion kit (Cat#1753), labeled with Cy dyes and hybridized overnight to the oligo chip in a MAUI chamber. The experiment comparing mesendoderm was performed four times (twice with WIK/AB hybrid strain embryos, once with EK strain embryos and once with AB strain embryos) each time including a dye swap, for a total of eight (four forward and four dye-swap) hybridizations. Three control cRNA probes were also generated from AB-strain embryos, namely: (1) whole 40% epiboly stage embryos, (2) FACS-sorted green cells from dissociated 40% epiboly embryos that were Kaede-injected but NOT photolabeled and (3) FACS-sorted red cells from dissociated 40% epiboly embryos that were Kaede-injected AND photolabeled. These probes were each generated three times, each time including a dye swap and co-hybridized as follows: (1) whole embryo cRNA vs green cell cRNA (three forward and three dye-swap hybridiizations) and (2) green cell cRNA vs. red cell cRNA (three forward and three dye-swap hybridizations).
