Argonaute2 Suppresses Drosophila Fragile X Expression Preventing Neurogenesis and Oogenesis Defects

Fragile X Syndrome is caused by the silencing of the Fragile X Mental Retardation gene (FMR1). Regulating dosage of FMR1 levels is critical for proper development and function of the nervous system and germ line, but the pathways responsible for maintaining normal expression levels are less clearly defined. Loss of Drosophila Fragile X protein (dFMR1) causes several behavioral and developmental defects in the fly, many of which are analogous to those seen in Fragile X patients. Over-expression of dFMR1 also causes specific neuronal and behavioral abnormalities. We have found that Argonaute2 (Ago2), the core component of the small interfering RNA (siRNA) pathway, regulates dfmr1 expression. Previously, the relationship between dFMR1 and Ago2 was defined by their physical interaction and co-regulation of downstream targets. We have found that Ago2 and dFMR1 are also connected through a regulatory relationship. Ago2 mediated repression of dFMR1 prevents axon growth and branching defects of the Drosophila neuromuscular junction (NMJ). Consequently, the neurogenesis defects in larvae mutant for both dfmr1 and Ago2 mirror those in dfmr1 null mutants. The Ago2 null phenotype at the NMJ is rescued in animals carrying an Ago2 genomic rescue construct. However, animals carrying a mutant Ago2 allele that produces Ago2 with significantly reduced endoribonuclease catalytic activity are normal with respect to the NMJ phenotypes examined. dFMR1 regulation by Ago2 is also observed in the germ line causing a multiple oocyte in a single egg chamber mutant phenotype. We have identified Ago2 as a regulator of dfmr1 expression and have clarified an important developmental role for Ago2 in the nervous system and germ line that requires dfmr1 function

Characterizing the regulation and activity of the Drosophila fragile X mental retardation gene, dfmr1

In order to better understand how a protein functions, it is also important to study the regulation of that protein, highlighting where and when its activity is important to promote or suppress. The studies described in this work aimed to investigate post-transcriptional pathways that regulate and interact with the Drosophila fragile X mental retardation protein, dFMR1, in order to better understand the function of dFMR1 in vivo. The loss of the human homologue FMRP, leads to fragile X syndrome, the most common form of inherited intellectual disability. Chapter Two examines the regulation of dfmr1 via one form of post-transcriptional regulation, alternative start codons. We identified a non-canonical start codon in the dfmr1 5\u27UTR that produces an additional isoform of dFMR1 with an amino-terminal (N-terminal) extension. We investigated the expression and activity of each isoform (with and without the N-terminal extension) individually in transgenic flies and found each isoform to be competent to rescue each aspect of dFMR1 activity that was tested. Interestingly, full expression of the N-terminal extended form requires the shorter dFMR1 isoform during specific developmental time points. Chapter Three provides an investigation into how dfmr1 and Ago2 interact in the circadian pathway (a pathway known to require dFMR1 function). We found that Ago2 is required for normal circadian locomotor activity under constant darkness. Secondly, Ago2 and dfmr1 interact genetically to regulate circadian behavior, and epistasis analyses showed that Ago2 acts downstream of dfmr1 in the circadian pathway in a sex-specific manner. Chapter Four characterizes how the small interfering RNA (siRNA) binding proteins, Argonaute2 (Ago2) and Dicer-2 (Dcr-2) regulate dfmr1 expression in the adult nervous system and female germ line. We tested the hypothesis that Ago2/and or Dcr-2 may directly regulate dfmr1 through a dsRNA intermediate containing the dfmr1 3\u27UTR and a downstream antisense transcript. We examined whether Dcr-2 regulates dfmr1 post-transcriptionally through the dfmr1 3\u27UTR using transgenic lines that express a GFP-dfmr1 3\u27UTR reporter. And finally we present evidence that Ago2 and Dcr-2 do not likely regulate dfmr1 through the canonical siRNA pathway

Characterizing the regulation and activity of the Drosophila fragile X mental retardation gene, dfmr1

In order to better understand how a protein functions, it is also important to study the regulation of that protein, highlighting where and when its activity is important to promote or suppress. The studies described in this work aimed to investigate post-transcriptional pathways that regulate and interact with the Drosophila fragile X mental retardation protein, dFMR1, in order to better understand the function of dFMR1 in vivo. The loss of the human homologue FMRP, leads to fragile X syndrome, the most common form of inherited intellectual disability. Chapter Two examines the regulation of dfmr1 via one form of post-transcriptional regulation, alternative start codons. We identified a non-canonical start codon in the dfmr1 5\u27UTR that produces an additional isoform of dFMR1 with an amino-terminal (N-terminal) extension. We investigated the expression and activity of each isoform (with and without the N-terminal extension) individually in transgenic flies and found each isoform to be competent to rescue each aspect of dFMR1 activity that was tested. Interestingly, full expression of the N-terminal extended form requires the shorter dFMR1 isoform during specific developmental time points. Chapter Three provides an investigation into how dfmr1 and Ago2 interact in the circadian pathway (a pathway known to require dFMR1 function). We found that Ago2 is required for normal circadian locomotor activity under constant darkness. Secondly, Ago2 and dfmr1 interact genetically to regulate circadian behavior, and epistasis analyses showed that Ago2 acts downstream of dfmr1 in the circadian pathway in a sex-specific manner. Chapter Four characterizes how the small interfering RNA (siRNA) binding proteins, Argonaute2 (Ago2) and Dicer-2 (Dcr-2) regulate dfmr1 expression in the adult nervous system and female germ line. We tested the hypothesis that Ago2/and or Dcr-2 may directly regulate dfmr1 through a dsRNA intermediate containing the dfmr1 3\u27UTR and a downstream antisense transcript. We examined whether Dcr-2 regulates dfmr1 post-transcriptionally through the dfmr1 3\u27UTR using transgenic lines that express a GFP-dfmr1 3\u27UTR reporter. And finally we present evidence that Ago2 and Dcr-2 do not likely regulate dfmr1 through the canonical siRNA pathway

The Macrophage-Specific Promoter <i>mfap4</i> Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish

<div><p>Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, <i>in vivo</i> analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the <i>mpeg1</i> promoter. However, <i>mpeg1</i> transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including <i>Salmonella typhimurium</i> and <i>Mycobacterium marinum</i>. Here, we describe <i>mfap4</i> as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of <i>mfap4</i>, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.</p></div

<i>mfap4</i>:<i>iCre</i>:<i>p2A-tdTomato</i> mediates macrophage-specific gene expression of loxP-containing transgenes.

<p><b>a-d.</b> Zebrafish larva at 3 dpf expressing both <i>Tg</i>(β-<i>actin2</i>:<i>loxP-DsRed-STOP-loxP-EGFP)</i>^<i>s928</i> and <i>Tg(mfap4</i>:<i>iCre</i>:<i>p2A-tdTomato)</i>^<i>xt8</i><i>;</i><b>a,b.</b> 50x magnification of GFP fluorescence and merge with brightfield, respectively. <b>c,d.</b> 200x magnification of the head, yolk sac, and anterior portion of the gut. <b>e-h.</b> Zebrafish larva at 3 dpf expressing <i>Tg(mfap4</i>:<i>dLanYFP-CAAX)</i>^<i>xt11</i> for comparison. <b>e,f.</b> 50x magnification of dLanYFP fluorescence and merge with brightfield, respectively. <b>g,h.</b> 200x magnification of the head, yolk sac, and anterior portion of the gut. Note that in both larvae, fluorescent protein expression is restricted to macrophages all along the body, with a particular enrichment in the head. Both larvae also exhibit expression within the caudal hematopoietic tissue (CHT). The larvae in <b>a-d</b> show a reduced number of fluorescent cells in the nascent macrophage population that predominates in the CHT region, and modestly reduced fluorescent cell population number in regions where mature macrophages have migrated throughout the body. Scale bars for 50x images = 500 μm; scale bars for 200x images = 100 μm.</p

Time-lapse imaging of a tail wound of an <i>mfap4</i>:<i>dLanYFP-CAAX</i> transgenic larva.

<p>Time-lapse imaging of macrophage recruitment to the site of a wound at the posterior end of the larval tail. Macrophage movements may be visualized in real-time via fluorescence imaging of <i>mfap4</i> transgenic larvae. Shown here are four representative frames of such an experiment, showing macrophage recruitment to the wound edge at 0, 1, 2, and 3 hours post-wounding. <b>a.</b> Brightfield detailing the extent and location of the wound. The wound edge is highlighted as a dashed white line. <b>b.</b> Macrophages visualized via dLanYFP fluorescence. The number of cells present proximal to and along the site of the wound can be seen increasing over time as macrophages track toward the damaged tissue. <b>c.</b> Merge of brightfield and fluorescence channels. Scale bars = 100 μm. Images representative of 12 animals from two independent biological replicates.</p

<i>mpeg1</i>-mediated fluorophore expression is attenuated in infected cells.

<p>Confocal image of a larval granuloma, 5 days post-infection. The region containing infected cells is indicated by the green dashed line. Within each cell, <i>M</i>. <i>marinum</i> expressing the Cerulean fluorescent protein (cyan signal) are clearly visible. In addition, faint tdTomato fluorescence is visible within the infected cells. White arrowheads indicate examples of bright, uninfected cells exhibiting normal levels of tdTomato fluorescence. <b>a.</b> Brightfield detailing the granuloma in which <i>mpeg1</i> fluorescent protein expression is attenuated. <b>b.</b> Fluorescent <i>M</i>. <i>marinum</i> in the same area <b>c.</b> Macrophages expressing the tdTomato fluorescent protein. <b>d.</b> Merged image of <b>a</b>-<b>c</b>. Scale bars = 50 μm.</p

<i>mfap4</i> transgene expression colocalizes with the expression of <i>mpeg1</i> transgenes.

<p>Representative image of the caudal hematopoietic tissue of a double transgenic larva, four days post-fertilization. <b>a.</b> Cells expressing dLanYFP via the <i>Tg(mfap4</i>:<i>dLanYFP-CAAX)</i>^<i>xt11</i> transgene. <b>b.</b> Cells expressing the tdTomato fluorescent protein via the <i>Tg(mpeg1</i>:<i>tdTomato)</i>^<i>xt3</i> transgene. <b>c.</b> Merged image of <b>a,b</b>. Scale bars = 50 μm. Images representative of at least 40 animals.</p

Differential regulation of <i>mfap4</i> and <i>mpeg1</i> transgenes during mycobacterial infection.

<p>Average fluorescence values of either <i>mpeg1</i>:<i>tdTomato-CAAX</i> or <i>mfap4</i>:<i>dLanYFP-CAAX</i> fluorescent proteins relative to uninfected, age-matched controls. Five randomly chosen infected macrophages per larva (“Infected”), or five randomly chosen macrophages within uninfected larvae (“Control”) were assessed. 20 larvae were analyzed for each of the infected and control groups at 1 dpi as well as for the Infected group at 5 dpi; 19 larvae were analyzed for the Control group at 5 dpi. Both tdTomato fluorescence (<b>a,b</b>) and dLanYFP fluorescence (<b>c,d</b>) were measured for the same pool of cells from each larva. <b>e.</b> Representative image of <i>mfap4</i> transgene expression (i) and <i>mpeg1</i> transgene expression (ii) within the same group of cells in an uninfected Control larva, 5 dpi. <b>f.</b> Representative image of <i>mfap4</i> transgene expression (i) and <i>mpeg1</i> transgene expression (iii) within the same group of cells in an Infected larva, 5 dpi; also shown are merged images of fluorescent <i>M</i>. <i>marinum</i> (cyan signal) with the YFP (ii) and tdTomato (iv) channels. Note the almost total loss of <i>mpeg1-</i>mediated fluorescence within the infected group of cells, while <i>mfap4</i> transgene fluorescence remains robust. Scale bars = 10 μm. * p < .05, ** p < .005, *** p = .0002. Student’s t-test with Welch’s correction for unequal variances. Error bars indicate +/- SD.</p