Project description:In order to knockdown bantam in the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs), we expressed the bantam microRNA-sponge (or the control scrambled-sponge) under the MB011B-split-Gal4 driver. Additionally, we expressed a membrane localized GFP transgene. At ZT12-13.5, MB011B>ban-spg, GFP and MB011B>scramble-spg, GFP female fly brains were diseccted, homogenized and FACS sorted using GFP flourescence to isolate the MBONs of interest. mRNA was extracted and used to construct Illumina RNAseq libraries
Project description:An animal’s skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues, however the mechanisms by which epidermal interactions shape processing of noxious inputs is still poorly understood. Here, we identify a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites, but not at other sites in the epidermis. From a genetic screen we identified miR-14 as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. We found that miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, we found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.
Project description:Class I and IV Drosophila dendritic arborization sensory neurons were isolated via magnetic bead sorting and total RNA isolated from the samples was used for gene expression profiling. Elucidating the molecular mechanisms controlling dendrite development is key to understanding the pivotal role these structures play in influencing synaptic integration and neural function. Despite significant advances in this field, genetic pleiotropy remains a significant impediment to investigating such complex developmental processes. To circumvent this problem, we have applied class specific neuron transcriptional expression profiling coupled to an in vivo RNAi functional validation screen in order to dissect the molecular bases of Drosophila class I and class IV dendritic arborization (da) neuron dendritogenesis.
