Project description:Neurons are highly polarized cells with distinct protein compositions in axonal and dendritic compartments. Cellular mechanisms controlling polarized protein sorting have been described for mature nervous system but little is known about the segregation in newly differentiated neurons. In a forward genetic screen for regulators of Drosophila brain circuit development, we identified mutations in <span style="color: rgb(54, 54, 54); font-style: normal; font-weight: 400; background-color: rgb(245, 245, 245);">Serine Palmitoyltransferase </span>(SPT), an evolutionary conserved enzyme in sphingolipid biosynthesis. Here we show that reduced levels of sphingolipids in SPT mutants cause axonal morphology defects similar to loss of cell recognition molecule Dscam. Loss- and gain-of-function studies show that neuronal sphingolipids are critical to prevent aggregation of axonal and dendritic Dscam isoforms, thereby ensuring precise Dscam localization to support axon branch segregation. Furthermore, SPT mutations causing neurodegenerative HSAN-I disorder in humans also result in formation of stable Dscam aggregates and axonal branch phenotypes in Drosophila neurons, indicating a causal link between developmental protein sorting defects and neuronal dysfunction.
Project description:The Drosophila gene dLmo encodes a transcriptional regulator involved in wing development and behavioral responses to cocaine and ethanol. We were interested in discovering novel transcriptional targets of dLmo in the nervous system by examining gene expression changes in the heads of wild-type flies and flies carrying dLmo loss-of-function (EP1306) and gain-of-function mutants (BxJ).
Project description:The Drosophila gene dLmo encodes a transcriptional regulator involved in wing development and behavioral responses to cocaine and ethanol. We were interested in discovering novel transcriptional targets of dLmo in the nervous system by examining gene expression changes in the heads of wild-type flies and flies carrying dLmo loss-of-function (EP1306) and gain-of-function mutants (BxJ). RNA was isolated from 3 pooled groups of 200 fly heads from each genotype (w;iso control, EP1306, and BxJ) and hybridized to triplicate Affymetrix Drosophila 2.0 oligonucleotide microarray chips at the Partners HealthCare Center for Personalized Genetic Medicine microarray facility (Harvard University).
Project description:Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways. RNA-seq of wing imaginal discs expressing either H3.3WT-FLAG-HA or H3.3K27M-FLAG-HA.
Project description:Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways.
Project description:To show that FST cooperates with SHH to establish tonotopy by promoting apical cochlear characteristics responsible for low-frequency hearing in mammals, we analyzed cochlear gene expression, morphology, and auditory function of mouse mutants with loss or gain of SHH function in combination with loss or gain of follistatin (FST) function.