piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome

<p>Abstract</p> <p>Background</p> <p>Much of the <it>Plasmodium falciparum </it>genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the <it>Plasmodium </it>genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the <it>Plasmodium </it>genome.</p> <p>Results</p> <p>In this study, we investigated the lepidopteran transposon, <it>piggyBac</it>, as a molecular genetic tool for functional characterization of the <it>Plasmodium falciparum </it>genome. Through multiple transfections, we generated 177 unique <it>P. falciparum </it>mutant clones with mostly single <it>piggyBac </it>insertions in their genomes. Analysis of <it>piggyBac </it>insertion sites revealed random insertions into the <it>P. falciparum </it>genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in <it>P. falciparum </it>with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.</p> <p>Conclusion</p> <p>Our results clearly demonstrate that <it>piggyBac </it>is a novel, indispensable tool for forward functional genomics in <it>P. falciparum </it>that will help better understand parasite biology and accelerate drug and vaccine development.</p

Gene Regulatory Networks Controlling Hematopoietic Progenitor Niche Cell Production and Differentiation in the <em>Drosophila</em> Lymph Gland

<div><p>Hematopoiesis occurs in two phases in <em>Drosophila</em>, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. In this report, we utilize a PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. We assessed the effect of abrogating the function of 820 genes as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, we characterized loss- and gain-of-function phenotypes. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the <em>hedgehog</em> (<em>hh</em>) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the <em>Pten</em>, <em>TSC1</em>, and <em>TSC2</em> tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland.</p> </div

Genetic interaction between the <i>osa</i> and <i>srp</i> genes.

<p>(A) <i>hhF4f-GFP</i> transgene activity in PSC cells (arrow) of a wild-type lymph gland. (B) Antp expression in PSC cells (arrow) of a wild-type lymph gland. (C) <i>osa³⁰⁸/+</i> heterozygous lymph glands contain a decreased number of <i>hhF4f-GFP</i>-positive PSC cells (arrowhead). (D) <i>osa³⁰⁸/+</i> heterozygous lymph glands contain a decreased number of Antp-positive PSC cells (arrowhead). (E) Normal <i>hhF4f-GFP</i> transgene activity in PSC cells (arrow) of a <i>srp⁰¹⁵⁴⁹/+</i> heterozygous lymph gland. (F) Normal Antp expression in PSC cells (arrow) of a <i>srp⁰¹⁵⁴⁹/+</i> heterozygous lymph gland. (G) Absence of <i>hhF4f-GFP</i>-positive PSC cells (arrowhead) in a <i>srp⁰¹⁵⁴⁹/osa³⁰⁸</i> double-heterozygous lymph gland. (H) <i>srp⁰¹⁵⁴⁹/osa³⁰⁸</i> double-heterozygous lymph glands contain a reduced population of Antp-positive PSC cells (arrowhead).</p

Quantification of hemolymph lamellocyte numbers.

<p>Quantification of hemolymph lamellocyte numbers.</p

Alteration of insulin-like growth factor signaling pathway gene functions.

<p>Expression of the PSC cell-specific markers <i>hhF4f-GFP</i> and Antp was assessed in lymph glands of the following loss- or gain-of-function genotypes: (A) wild-type, (B) <i>col>dAkt1</i> RNAi, (C) <i>col>dAkt1</i> cDNA, (D) <i>col>PDK1</i> RNAi, (E) <i>col>InR^DN</i> cDNA, (F) <i>col>InR</i> cDNA, (G) <i>col>Pi3K92E</i> RNAi, (H) <i>col>Pi3K92E^DN</i> cDNA, and (I) <i>col>Pi3K92E^CA</i> cDNA. Arrows in panels C, F, and I point out expanded populations of PSC niche cells, with an abnormal niche organization caused by PSC cell-specific expression of the <i>InR</i> cDNA. Arrowheads in panels B, D, E, G, and H point out reduced populations of PSC niche cells. Scale bar indicates 20 µm.</p

Alteration of TOR signaling pathway gene functions.

<p>Expression of the PSC cell-specific markers <i>hhF4f-GFP</i> and Antp was assessed in lymph glands of the following loss-of-function genotypes: (A) <i>col>Tor</i> RNAi, (B) <i>col>raptor</i> RNAi, (C) <i>col>rictor</i> RNAi, (D) <i>col>TSC1</i> RNAi, (G) <i>col>TSC2</i> RNAi, (J) <i>S6K^L-1/S6K^L-1</i> (Antp only), and (K) <i>Thor⁰⁶²⁷⁰/Thor⁰⁶²⁷⁰</i>. Arrows in panels G and K point out expanded populations and abnormal organization of PSC niche cells. Arrowheads in panels A, B, C, and J point out severely reduced populations of PSC niche cells. (E) Expanded expression of the <i>hhF4f-GFP</i> transgene in <i>TSC1^f01910/TSC1^f01910</i> lymph glands. (F) Increase in plasmatocyte number (detected by P1 antibody) and supernumerary lamellocyte production (detected by <i>MSNF9mCherry</i> activity) in <i>TSC1^f01910/TSC1^f01910</i> lymph glands. (H) Expanded expression of the <i>hhF4f-GFP</i> transgene in <i>TSC2¹⁰⁹/TSC2¹⁰⁹</i> lymph glands. (I) Increase in plasmatocyte number and supernumerary lamellocyte production in <i>TSC2¹⁰⁹/TSC2¹⁰⁹</i> lymph glands. Scale bar indicates 20 µm.</p

Lymph gland domains, cell markers, and strategy for a PSC-specific gene function knockdown analysis.

<p>(A) Dissected dorsal vessel and associated lymph glands assayed for DAPI (DNA), Antp protein (PSC cells), and <i>eater-GFP</i> transgene activity (plasmatocytes). Abbreviations: CZ, cortical zone; PSC, posterior signaling center. (B) Enlargement of a primary lymph gland lobe stained for Su(H) protein expressed in hematopoietic progenitors of the medullary zone (MZ) and crystal cells of the cortical zone (CZ). Also highlighted are <i>hhF4f-GFP</i>-positive niche cells of the PSC. (C) Focus on a lymph gland PSC niche assayed for nuclear Antp protein and membrane-associated GFP due to expression of the <i>col>UAS-gapGFP</i> combination. The arrow points out a filopodia extending from a niche cell. (D) Strategy for the PSC-specific gene function knockdown analysis undertaken in this study.</p

Quantification of PSC cell number.

<p>Quantification of PSC cell number.</p

Summary of findings of a PSC-specific gene function knockdown analysis of 820 <i>Drosophila</i> loci.

<p>Center: <i>hhF4f-GFP</i> transgene activity (hh in green circle) was the primary marker assessed in the RNAi-based gene function knockdown analysis, while expression of Antp protein (Antp in yellow circle) and filopodia formation based on <i>col>UAS-gapGFP</i> expression were monitored as secondary markers. Periphery: Grouping of genes (based on their encoded protein products), that when functionally altered by RNAi expression in PSC cells, resulted in abnormalities in <i>hh-GFP</i>, Antp, and/or <i>col>UAS-gapGFP</i> expression. Negative (blue circle) and positive (red circle) regulators are indicated. A negative regulator is defined as a gene whose loss-of-function condition leads to increased numbers of <i>hhF4f-GFP</i>-positive cells and/or enhanced transgene expression, while a positive regulator is defined as a gene whose loss-of-function condition leads to decreased numbers of <i>hhF4f-GFP</i>-positive cells and/or decreased transgene expression. Those loss-of-function conditions that resulted in lamellocyte induction (#) or absence of filopodia (*) are also indicated.</p