Targeting the hemangioblast with a novel cell type-specific enhancer.

BACKGROUND: Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker. RESULTS: We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis. CONCLUSIONS: We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development.

Modified whole-mount in situ hybridization protocol for the detection of transgene expression in electroporated chick embryos.

BACKGROUND: In vivo electroporation has been extensively used as an effective means of DNA transfer for analyzing gene function as well as gene regulation in developmental systems. In any of these two types of studies, the correct spatial and temporal expression of the electroporated transgene can only be accurately assessed by in situ hybridization. METHODOLOGY/PRINCIPAL FINDINGS: While analyzing transgene expression in electroporated chicken embryos, we verified that transgene riboprobes cross-hybridized with the exogenous plasmid DNA when embryos were processed by conventional whole-mount in situ hybridization (WISH). CONCLUSIONS/SIGNIFICANCE: Here we describe a modification to the WISH protocol that is essential to prevent DNA cross-hybridization and to specifically detect transgene mRNA transcripts in electroporated embryos. Our optimized WISH procedure can be applied not only to electroporated chick embryos but also to other embryos or adult tissues that have been transfected with large amounts of reporter- or expression construct DNA.