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.

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.

Cbf beta-SMMHC induces distinct abnormal myeloid progenitors able to develop acute myeloid leukemia.

The acute myeloid leukemia (AML)-associated CBF beta-SMMHC fusion protein impairs hematopoietic differentiation and predisposes to leukemic transformation. The mechanism of leukemia progression, however, is poorly understood. In this study, we report a conditional Cbfb-MYH11 knockin mouse model that develops AML with a median latency of 5 months. Cbf beta-SMMHC expression reduced the multilineage repopulation capacity of hematopoietic stem cells (HSCs) while maintaining their numbers under competitive conditions. The fusion protein induced abnormal myeloid progenitors (AMPs) with limited proliferative potential but leukemic predisposition similar to that of HSCs in transplanted mice. In addition, Cbf beta-SMMHC blocked megakaryocytic maturation at the CFU-Meg to megakaryocyte transition. These data show that a leukemia oncoprotein can inhibit differentiation and proliferation while not affecting the maintenance of long-term HSCs.

SAX-7/L1CAM acts with the adherens junction proteins MAGI-1, HMR-1/Cadherin, and AFD-1/Afadin to promote glial-mediated dendrite extension.

Adherens junctions (AJs) are a fundamental organizing structure for multicellular life. Although AJs are studied mainly in epithelia, their core function - stabilizing cell contacts by coupling adhesion molecules to the cytoskeleton - is important in diverse tissues. We find that two C. elegans sensory neurons, URX and BAG, require conserved AJ proteins for dendrite morphogenesis. We previously showed that URX and BAG dendrites attach to the embryonic nose via the adhesion molecule SAX-7/L1CAM, acting both in neurons and glia, and then extend by stretch during embryo elongation. Here, we find that a PDZ-binding motif (PB) in the SAX-7 cytoplasmic tail acts with other interaction motifs to promote dendrite extension. Using pull-down assays, we find that the SAX-7 PB binds the multi-PDZ scaffolding protein MAGI-1, which bridges it to the cadherin-catenin complex protein HMP-2/ß-catenin. Using cell-specific rescue and depletion, we find that both MAGI-1 and HMR-1/Cadherin act in glia to non-autonomously promote dendrite extension. Double mutant analysis indicates that each protein can act independently of SAX-7, suggesting a multivalent adhesion complex. The SAX-7 PB motif also binds AFD-1/Afadin, loss of which further enhances sax-7 BAG dendrite defects. As MAGI-1, HMR-1, and AFD-1 are all found in epithelial AJs, we propose that an AJ-like complex in glia promotes dendrite extension.

Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output.

Amnesiac mutant flies have an olfactory memory defect. The amn gene encodes a homolog of vertebrate pituitary adenylate cyclase-activating peptide (PACAP), and it is strongly expressed in dorsal paired medial (DPM) neurons. DPM neurons ramify throughout the mushroom bodies in the adult fly brain, and they are required for stable memory. Here, we show that DPM neuron output is only required during the consolidation phase for middle-term odor memory and is dispensable during acquisition and recall. However, we found that DPM neuron output is required during acquisition of a benzaldehyde odor memory. We show that flies sense benzaldehyde by the classical olfactory and a noncanonical route. These results suggest that DPM neurons are required to consolidate memory and are differently involved in memory of a volatile that requires multisensory integration.

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.

Transposition-driven genomic heterogeneity in the Drosophila brain.

Recent studies in mammals have documented the neural expression and mobility of retrotransposons and have suggested that neural genomes are diverse mosaics. We found that transposition occurs among memory-relevant neurons in the Drosophila brain. Cell type-specific gene expression profiling revealed that transposon expression is more abundant in mushroom body (MB) αß neurons than in neighboring MB neurons. The Piwi-interacting RNA (piRNA) proteins Aubergine and Argonaute 3, known to suppress transposons in the fly germline, are expressed in the brain and appear less abundant in αß MB neurons. Loss of piRNA proteins correlates with elevated transposon expression in the brain. Paired-end deep sequencing identified more than 200 de novo transposon insertions in αß neurons, including insertions into memory-relevant loci. Our observations indicate that genomic heterogeneity is a conserved feature of the brain.

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 'memory genes'. 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.

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.