Drosophila hematopoiesis: Markers and methods for molecular genetic analysis

Abstract

Analyses of the Drosophila hematopoietic system are becoming more and more prevalent as developmental and functional parallels with vertebrate blood cells become more evident. Investigative work on the fly blood system has, out of necessity, led to the identification of new molecular markers for blood cell types and lineages and to the refinement of useful molecular genetic tools and analytical methods. This review briefly describes the Drosophila hematopoietic system at different developmental stages, summarizes the major useful cell markers and tools for each stage, and provides basic protocols for practical analysis of circulating blood cells and of the lymph gland, the larval hematopoietic organ.

Keywords: Blood; Drosophila; Hematopoiesis; Hemocytes; Lymph gland; Protocol.

Figure 1. A new triple-fluorescence reporter line identifies mature, progenitor, and niche cells within the hematopoietic lymph gland

A) Differentiated blood cells in the cortical zone of a third-instar larval lymph gland are marked by DsRed protein (red) expression driven by the Hemolectin enhancer (Hml-DsRed). B) Blood progenitor cells in the medulary zone express EBFP2 protein (cyan) under the control of the domeless MESO enhancer (dome-MESO-EBFP2). C) PSC cells express EGFP protein (green) under the control of the hedgehog PSC enhancer (hh-EGFP). D) A combined image (Merge) showing differential marking of blood cell populations juxtaposed in the lymph gland primary lobe. DNA (blue) is labeled with TO-PRO-3 (Molecular Probes) in each panel. E) Overview of the generation of the dome-MESO-EGFP and dome-MESO-EBFP2 constructs. The 2.8 kb dome-MESO enhancer (1) was amplified by PCR from Drosophila genomic DNA, along with an added 5’ CACC nucleotide sequence for Gateway® Directional TOPO® cloning into the pENTR/D-TOPO® entry vector (2; Life Technologies). Upon directional cloning of the dome-MESO enhancer into the Gateway entry vector (3), the enhancer was recombined with the Ganesh-G2 Gateway destination vector (4), thereby creating the dome-MESO-EGFP Drosophila transformation vector (5). The DNA coding sequence for nuclear-localized Enhanced Blue Fluorescent Protein 2 (EBFP2-NLS) was PCR amplified from the expression vector pLV-EBFP2-nuc (Addgene) as an AgeI/MfeI fragment (6). The dome-MESO-EBFP2 expression vector (7) was subsequently created by removing the EGFP-NLS DNA sequence from the dome-MESO-EGFP vector by digesting with AgeI and MfeI restriction endonucleases and then ligating in the EBFP2-NLS DNA sequence into the vector using the same restriction sites.