Transposition Driven Genomic Heterogeneity in the \u3cem\u3eDrosophila\u3c/em\u3e Brain: A Dissertation

In the Drosophila brain, memories are processed and stored in two mirrorsymmetrical structures composed of approximately 5,000 neurons called Mushroom Bodies (MB). Depending on their axonal extensions, neurons in the MB can be further classified into three different subgroups: αβ, α’β’ and γ. In addition to the morphological differences between these groups of neurons, there is evidence of functional differences too. For example, it has been previously shown that while neurotransmission from α’β’ neurons is required for consolidation of olfactory memory, output from αβ neurons is required for its later retrieval. To gain insight into the functional properties of these discrete neurons we analyzed whether they were different at the level of gene expression. We generated an intersectional genetic approach to exclusively label each population of neurons and permit their purification. Comparing expression profiles, revealed a large number of potentially interesting molecular differences between the populations. We focused on the finding that the MB αβ neurons, which are the presumed storage site for transcription-dependent long-term memory, express high levels of mRNA for transposable elements and histones suggesting that these neurons likely possess unique genomic characteristics. For decades, transposable elements (TE) were considered to be merely “selfish” DNA elements inserted at random in the genome and that they their sole function was to self-replicate. However, new studies have started to arise that indicate TE contribute more than just “junk” DNA to the genome. Although it is widely believed that mobilization of TE destabilize the genome by insertional mutagenesis, deletions and rearrangements of genes, some rearrangements might be advantageous for the organism. TE mobilization has recently been documented to occur in some somatic cells, including in neuronal precursor cells (NPCs). Moreover, mobilization in NPCs seems to favor insertions within neuronal expressed genes and in one case the insertion elevated the expression. During the last decade, the discovery of the small RNA pathways that suppress the expression and mobilization of TE throughout the animal have helped to uncover new functions that TE play. In this work, we demonstrate that proteins of the PIWI-associated RNA pathway that control TE expression in the germline are also required to suppress TE expression in the adult fly brain. Moreover, we find that they are differentially expressed in subsets of MB neurons, being under represented in the αβ neurons. This finding suggests that the αβ neurons tolerate TE mobilization. Lastly, we demonstrate by sequencing αβ neuron DNA that TE are mobile and we identify \u3e200 de novo insertions into neurally expressed genes. We conclude that this TE generated mosaicism, likely contributes a new level of neuronal diversity making, in theory, each αβ neuron genetically different. In principle the stochastic nature of this process could also render every fly an individual

Neuronal post-developmentally acting SAX-7S/L1CAM can function as cleaved fragments to maintain neuronal architecture in C. elegans [preprint]

Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal’s growth, maturation processes, the addition of new neurons, body movements, and aging. The C. elegans protein SAX-7, homologous to the vertebrate L1 protein family, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles, and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by post-developmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants’ neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance, and may help decipher processes that go awry in some neurodegenerative conditions

Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development

Memory-Relevant Mushroom Body Output Synapses Are Cholinergic

Memories are stored in the fan-out fan-in neural architectures of the mammalian cerebellum and hippocampus and the insect mushroom bodies. However, whereas key plasticity occurs at glutamatergic synapses in mammals, the neurochemistry of the memory-storing mushroom body Kenyon cell output synapses is unknown. Here we demonstrate a role for acetylcholine (ACh) in Drosophila. Kenyon cells express the ACh-processing proteins ChAT and VAChT, and reducing their expression impairs learned olfactory-driven behavior. Local ACh application, or direct Kenyon cell activation, evokes activity in mushroom body output neurons (MBONs). MBON activation depends on VAChT expression in Kenyon cells and is blocked by ACh receptor antagonism. Furthermore, reducing nicotinic ACh receptor subunit expression in MBONs compromises odor-evoked activation and redirects odor-driven behavior. Lastly, peptidergic corelease enhances ACh-evoked responses in MBONs, suggesting an interaction between the fast- and slow-acting transmitters. Therefore, olfactory memories in Drosophila are likely stored as plasticity of cholinergic synapses

Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11

Recurrent chromosomal rearrangements are associated with the development of acute myeloid leukemia (AML). The frequent inversion of chromosome 16 creates the CBFB-MYH11 fusion gene that encodes the fusion protein CBFbeta-SMMHC. This fusion protein inhibits the core-binding factor (CBF), resulting in a block of hematopoietic differentiation, and induces leukemia upon the acquisition of additional mutations. A recent genetic screen identified Plag1 and Plagl2 as CBF beta-SMMHC candidate cooperating proteins. In this study, we demonstrate that Plag1 and Plagl2 independently cooperate with CBF beta-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and Plagl2 increased proliferation by inducing G1 to S transition that resulted in the expansion of hematopoietic progenitors and increased cell renewal in vitro. Finally, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples. Interestingly, PLAGL2 was preferentially increased in samples with chromosome 16 inversion, suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Overall, this study shows that Plag1 and Plagl2 are novel leukemia oncogenes that act by expanding hematopoietic progenitors expressing CbF beta-SMMHC

Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration

There are many ways to train a fly

A biological understanding of memory remains one of the great quests of neuroscience. For over 30 years the fruit fly Drosophila melanogaster has primarily been viewed as an excellent vehicle to find \u27memory genes\u27. However, the recent advent of sophisticated genetic tools to manipulate neural activity has meant that these genes can now be viewed within the context of functioning neural circuits. A holistic understanding of memory in flies is therefore now a realistic goal. Larvae and adult flies exhibit remarkable behavioral complexity and they can both be trained in a number of ways. In this review, our intention is to summarize the many assays that have been developed to study plastic behaviors in flies. More specific and detailed reviews have been published by us and others, reviewed in references 1-6. While our bias for olfactory conditioning paradigms is obvious, our purpose here is not to pass judgment on each method. We would rather leave that to those readers who might be inspired to try each assay for themselves

<i>lon-2</i>/glypican functions in the attractive <i>unc-6</i>/netrin guidance pathway.

<p>(A) During the first larval stage of <i>C</i>. <i>elegans</i>, the pioneer neuron AVM extends ventrally along the body wall until it reaches the ventral nerve cord. Its migration results from the combined attractive response to UNC-6/netrin (secreted at the ventral midline) via the UNC-40/DCC receptor and the repulsive response to SLT-1/Slit (secreted by the dorsal muscles) via its SAX-3/Robo receptor. We visualized the morphology of the AVM axon using the transgene P<i>mec-4</i>::<i>gfp</i>. (B) The heparan sulfate proteoglycans <i>lon-2</i>/glypican and <i>sdn-1</i>/syndecan cooperate to guide the axon of AVM, as their simultaneous loss enhances guidance defects. The role of <i>lon-2</i>/glypican in axon guidance is specific, as the loss of <i>lon-2</i>/glypican, but not the loss of the other <i>C</i>. <i>elegans</i> glypican, <i>gpn-1</i>, enhances the defects of <i>sdn-1</i>/syndecan mutants. (C) Complete loss of <i>lon-2</i>/glypican enhances the axon guidance defects resulting from disrupted <i>slt-1</i>/Slit signaling in mutants for <i>slt-1</i>/Slit or its receptor <i>sax-3</i>/Robo, as well as in animals misexpressing <i>slt-1</i> in all body wall muscles (using a P<i>myo-3</i>::<i>slt-1</i> transgene). Data for wild type and <i>lon-2</i> are the same as in (B). (D) Complete loss of <i>lon-2</i>/glypican does not enhance the AVM guidance defects of <i>unc-6</i>/netrin mutants or of mutants for its receptor <i>unc-40</i>/DCC, suggesting that <i>lon-2</i>/glypican functions in the same genetic pathway as <i>unc-6</i>/netrin. Data for wild type and <i>lon-2</i> are the same as in (B). (E) Loss of <i>sdn-1</i>/syndecan function does not enhance the defects of <i>slt-1</i>/Slit or sax-3/Robo mutants but enhances the defects of <i>unc-</i>40/DCC mutants. Data for wild type, <i>sdn-1</i>, <i>slt-1</i>, <i>sax-3</i>, and <i>unc-40</i> are the same as in (B–D). Error bars are standard error of the proportion. Asterisks denote significant difference\: *** <i>p</i> ≤ 0.001,** <i>p</i> ≤ 0.01, and * <i>p</i> ≤ 0.05 (<i>z</i>-tests, <i>p</i>-values were corrected by multiplying by the number of comparisons). ns, not significant.</p

A secreted form of LON-2/glypican that lacks the heparan sulfate chain attachments is functional in axon guidance.

<p>(A) The HSPG LON-2/glypican is composed of a core protein and three HS chains. The core protein is predicted to fold into a globular domain on its N-terminal region and to be GPI-anchored. (B) Schematics of the engineered forms of LON-2 that we used: LON-2ΔGAG, in which the HS chain attachment sites are mutated; LON-2ΔGPI, in which the GPI anchor is deleted; N-LON-2, in which the C-terminus is deleted; and C-LON-2, in which the N-terminal globular domain is deleted. Western blot analysis of protein extracts of worms expressing LON-2::GFP or LON-2ΔGAG::GFP confirms that deleting the HS attachment sites on LON-2 affects HS addition on LON-2. Protein extracts from wild type (N2) and an unrelated GFP strain (<i>lqIs4</i>) are negative controls. (C) A form of LON-2/glypican lacking HS chain attachment sites (LON-2ΔGAG) functions in axon guidance. LON-2ΔGAG rescues the AVM guidance defects of double mutants <i>lon-2 slt-1</i> back to the level of <i>slt-1</i> single mutants. Secreted globular LON-2/glypican is functional in axon guidance. LON-2/glypican was engineered to be secreted by deleting its GPI anchor (LON-2ΔGPI) or by deleting the C-terminus, thus lacking the GPI anchor and the HS attachment sites (N-LON-2). Both LON-2ΔGPI and N-LON-2 function in axon guidance, as assayed by their ability to rescue axon guidance defects of <i>lon-2 slt-1</i> back down to the level of <i>slt-1</i> single mutants. In contrast, a form of LON-2/glypican containing its C-terminus including the three HS attachment sites, but lacking its N-terminal globular domain (C-LON-2), is not functional (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s013" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s014" target="_blank">S3</a> Tables), indicating that the N-terminal globular domain of the core protein is key to the function of LON-2/glypican in axon guidance. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s013" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s014" target="_blank">S3</a> Tables). Data for wild type, <i>lon-2</i>, <i>slt-1</i>, and <i>lon-2 slt-1</i> are the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.g001" target="_blank">Fig 1B and 1C</a>. (D) A form of LON-2/glypican lacking HS chain attachment sites is functional in DTC guidance. The DTC migration of <i>lon-2</i> mutants carrying the transgene P<i>lon-2</i>::LON-2ΔGAG is rescued back to wild-type levels. Secreted N-terminus globular LON-2/glypican (N-LON-2) is functional in DTC guidance, as DTC guidance defects of <i>lon-2</i> mutants are rescued by N-LON-2. Transgenic animals were compared to nontransgenic sibling controls (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.s018" target="_blank">S7 Table</a>). Data for wild type and <i>lon-2</i> are the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002183#pbio.1002183.g002" target="_blank">Fig 2B</a>. Error bars are standard error of the proportion. Asterisks denote significant difference: *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01 (<i>z</i>-tests, <i>p</i>-values were corrected by multiplying by the number of comparisons).</p

A model for the role of LON-2/glypican in UNC-6/netrin-UNC-40/DCC-mediated axon guidance.

<p>HSPG LON-2/glypican (red) is expressed from the hyp7 epidermal cells (pink) underlying the migrating growth cone of the AVM neuron (tan). LON-2/glypican is released from the hypodermal cell surface and may associate with the developing axon expressing the receptor UNC-40/DCC (blue), directly or indirectly interacting with UNC-40/DCC, to modulate UNC-6/netrin (green) signaling. A second HSPG, SDN-1/syndecan (black), acts in the SLT-1/Slit-SAX-3/Robo (grey) axon guidance pathway.</p