Steroid-triggered, cell-autonomous programmed cell death of identified Drosophila motoneurons during metamorphosis

x, 83 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.Programmed cell death (PCD) is a critical process during development and maturity of vertebrates and invertebrates. Aberrations in PCD are responsible for numerous developmental abnormalities and diseases in humans. Cell death pathways are surprisingly similar across species, so the study of PCD in simpler organisms such as insects provides important insight into the roles of cell death in higher animals including humans. Metamorphosis of the fruit fly, Drosophila melanogaster , provides an excellent model system in which to study PCD. During metamorphosis, many obsolete larval structures undergo PCD, largely in response to changes in circulating levels of steroid hormones known as ecdysteroids. These effects of ecdysteroids are particularly striking in the nervous system, where many larval neurons undergo PCD or functional remodeling during metamorphosis. One wave of neuronal PCD takes place during the first 24 hours of metamorphosis while a second follows adult emergence. Studies in another insect, Manduca sexta , suggested that the rise in ecdysteroids that initiates metamorphosis, the prepupal pulse, may trigger the first wave of neuronal PCD in Drosophila . This dissertation investigated steroid-regulated neuronal PCD in Drosophila by studying an individually-identified larval motoneuron, RP2. Using molecular genetics, ïmmunocytochemistry and primary cell culture, I showed that abdominal RP2s undergo PCD within the first 24 hours of Drosophila metamorphosis; identified a role for previously-identified PCD genes and ecdysteroid receptors in RP2's demise; and demonstrated that the prepupal pulse of ecdysteroids acts directly and cell-autonomously on RP2s to activate PCD. These experiments advance our understanding of hormonally-induced cell death and its regulation within the developing nervous system. This dissertation includes unpublished co-authored material.Adviser: Janis C. Week

Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors

<div><p>Food and feeding-state dependent changes in chemoreceptor gene expression may allow <i>Caenorhabditis elegans</i> to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, <i>srh-234</i>, in the ADL sensory neuron of <i>C</i>. <i>elegans</i> is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate <i>srh-234</i> expression as a function of feeding state. We identify a <i>cis</i>-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of <i>srh-234</i> expression, while an E-box site known to bind bHLH factors is required to drive <i>srh-234</i> expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate <i>srh-234</i> expression. We further demonstrate that the expression levels of <i>srh-234</i> in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of <i>srh-234</i> involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.</p></div

Mutagenesis of the <i>srh-234</i> regulatory region reveals an E-box and MEF2 site important for feeding-state dependent regulation of <i>srh-234</i>.

<p><b>(A)</b> The indicated lengths and positions of predicted regulatory elements relative to the translational start site of <i>srh-234</i> fused to <i>gfp</i> in expression vectors (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006237#sec015" target="_blank">Material and Methods</a>. Sequences in green and blue indicate the predicted MEF2 and E-box sequence motifs, respectively. Mutations generated in these sequences are indicated in red. <b>(B)</b> Representative expression of ADL sensory neurons driven by wild-type and mutated <i>srh-234</i> regulatory sequences in adult wild-type animals when either well-fed in the presence of <i>E</i>. <i>coli</i> OP50 food or starved in the absence of food for 12 hours. Confocal images are lateral views of ADL sensory neurons with the arrow pointing to the cell body; anterior is left. Images were acquired at the same exposure time at room temperature. <b>(C)</b> Relative expression of mutated <i>srh-234</i> regulatory sequences in the ADL cell body compared to wild-type adults when fed or starved. Data shown is the average of least three independent transgenic lines for each genotype with n>25 adult animals for each transgenic line. * indicates values that are different from that of wild-type adult animals at <i>P</i><0.001. Error bars denote the SEM.</p

An intestine-to-neuron model for regulation of <i>srh-234</i> expression in ADL as a function of feeding state.

<p>Changes in expression of <i>srh-234</i> are directed by multiple cell-autonomous and non-cell-autonomous transcriptional modules. In ADL neurons, one module consists of a MEF-2 transcription factor and its MEF2 binding site, and the other module consists of a bHLH heterodimer (e.g. HLH-2/3, and HLH-2/4) and its bi-partite E-box binding site. When fed <b>(A)</b>, MEF-2 activity is low and is prevented from repressing HLH-2/3/4 factors, and expression of <i>srh-234</i> in ADL is increased. When starved <b>(B)</b>, MEF-2 is no longer inhibited, allowing binding to the MEF2 site in <i>srh-234</i> (AGTTATATTTAA), which in turn represses HLH-2/3/4-mediated activation of <i>srh-234</i> at the E-box site (CACCTG), ultimately leading to the decrease in <i>srh-234</i> expression. In addition to cell-autonomous mechanisms, the bHLH factors MXL-3 and HLH-30 act in the intestine to remotely regulate changes in <i>srh-234</i> expression via the DAF-2/DAF-16 insulin pathway acting in ADL. When fed <b>(A)</b>, MXL-3 activity is high in the intestine (low HLH-30 activity) and increases <i>srh-234</i> expression, whereas when starved <b>(B)</b>, HLH-30 activity is high in the intestine (low MXL-3 activity) and represses the expression of <i>srh-234</i> in ADL. The combined action of ILPs from different tissues (the intestine) and other neurons (e.g. ASE) regulated by <i>mxl-3</i>-dependent (e.g. <i>ins-3</i>, <i>ins-26</i>) or independent mechanisms results in either high or low DAF-2 activity, which in turn modulates <i>srh-234</i> expression in ADL.</p

<i>hlh-2</i>, <i>hlh-3</i> and <i>hlh-4</i> act in ADL neurons to regulate <i>srh-234</i> expression, and <i>hlh-3</i> and <i>hlh-4</i> appear to be transiently expressed in ADL.

<p><b>(A)</b> Relative expression of <i>srh-234</i>p::<i>gfp</i> in adult <i>hlh-2</i> and <i>hlh-3</i> mutant animals compared to adult wild-type, or when fed <i>hlh-3(RNAi)</i> or <i>hlh-4(RNAi)</i> compared to control RNAi. Feeding RNAi was performed in <i>sid-1(pk3321)him-5(e1409)</i> mutants with the stably integrated <i>oyIs56</i> transgene carrying a <i>ADL</i>::<i>sid-1</i> extra chromosomal transgene to selectively enhance RNAi in ADL sensory neurons but not in other tissues (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006237#sec015" target="_blank">Material and Methods</a>). Data shown is the average of at least two independent transgenic lines with n>25 adult animals for each line. * indicates values that are different from that of wild-type adult animals at <i>P</i><0.001, and # between the genotypes compared by brackets at <i>P</i><0.001. Error bars denote the SEM. <b>(B)</b> Expression of <i>hlh-2</i> with the ADL-specific reporter <i>srh-234</i>p::<i>gfp</i> in late embryos and in adult animals. Animals carrying the <i>hlh-2p</i>::<i>mCherry</i>::<i>his-11</i> transgene expresses <i>hlh-2</i> in the nucleus of many cells, including ADL neurons. Co-expression in ADL is indicated with arrows, and ADL appears with a green cytoplasm and yellow nucleus in a merged fluorescent image. <b>(C)</b> Expression of <i>hlh-3p</i>::<i>hlh-3</i>::<i>SL2</i>::<i>gfp</i> or <i>hlh-4p</i>::<i>gfp</i> with the ADL-specific reporter <i>sri-51</i>p::<i>mCherry</i> in embryos or adult animals. Co-expression in ADL is indicated with arrows and yellow cell in a merged fluorescent image. <b>(D)</b> Overview of the temporal expression of HLH-2/3/4/10 in ADL sensory neurons.</p

Introduction of the <i>srh-234</i> MEF2 site into the ADL-expressed <i>sre-1</i> promoter confers regulation by starvation.

<p><b>(A)</b> The indicated lengths and positions of predicted regulatory elements relative to the translational start site of <i>sre-1</i> fused to <i>gfp</i> in expression vectors (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006237#sec015" target="_blank">Material and Methods</a>). Sequences in blue and green indicate the predicted E-box motif of <i>sre-1</i> with the inserted MEF2 site sequence of <i>srh-234</i>, respectively. <b>(B)</b> Relative expression of <i>sre-1</i> regulatory sequences in the ADL cell body without (<i>sre-1</i>p<i>(WT)</i>::<i>gfp</i>) or with the MEF2 motif of <i>srh-234</i> (<i>sre-1</i>p<i>(+MEF2)</i>::<i>gfp</i>) compared to wild-type adults when fed or starved. Data shown is the average of least two independent transgenic lines for each genotype with n>25 adult animals for each transgenic line. * indicates values that are different from that of wild-type adult animals at <i>P</i><0.001, and # between the genotypes compared by brackets at <i>P</i><0.001. Error bars denote the SEM. <b>(C)</b> Representative expression of ADL sensory neurons driven by wild-type <i>sre-1</i> regulatory sequences without (<i>sre-1</i>p<i>(WT)</i>::<i>gfp</i>) or with the MEF2 motif of <i>srh-234</i> (<i>sre-1</i>p<i>(+MEF2)</i>::<i>gfp</i>) in adult wild-type animals when either well-fed in the presence of <i>E</i>. <i>coli</i> OP50 food or starved in the absence of food for 12 hours. Confocal images are ventral views of ADL sensory neurons with the arrow pointing to the left and right cell body (anterior is left), and were acquired at the same exposure time at room temperature.</p

Mutations in <i>bHLH</i> genes and <i>mef-2</i> alter <i>srh-234</i> expression levels in ADL.

<p><b>(A, B)</b> Representative expression of ADL sensory neurons driven by wild-type <i>srh-234</i> regulatory sequences fused to <i>gfp</i> (<i>srh-234</i>p::<i>gfp</i>) in adult wild-type and mutant animals of the indicated <i>bHLH</i> genes when either well-fed <b>(A)</b> in the presence of <i>E</i>. <i>coli</i> OP50 food, or starved <b>(B)</b> in the absence of food for 12 hours. Confocal images are lateral views of ADL sensory neurons with the arrow pointing to the cell body; anterior is left. Images were acquired at the same exposure time at room temperature. <b>(C)</b> Relative expression of <i>srh-234</i>p::<i>gfp</i> in adult single and double mutants of the indicated genotypes in well-fed and starved conditions. Adult animals (n>20) for each genotype were examined at the same exposure time. * and ** indicates values that are different from that of wild type adult animals at <i>P</i><0.001 and <i>P</i><0.01, respectively. # and ## between the genotypes compared by brackets at <i>P</i><0.001 and <i>P</i><0.01, respectively. Error bars denote the SEM. ns indicates not significant. All strains contain stably integrated copies of a <i>srh-234</i>p::<i>gfp</i> transgene (<i>oyIs56</i>).</p