Journal: microPublication Biology

Article Title: Two distinct functions of Lim1 in the Drosophila antenna

doi: 10.17912/micropub.biology.001229

Figure Lengend Snippet: A) Third instar eye-antennal disk stained for Lim1 protein (magenta) shows expression in the second antennal segment. Counterstain: DAPI (blue). e = eye, a = antenna. B) Expression of Lim1 (red) depicted over Fly Cell Atlas antennal single nucleus RNA sequencing clusters (Li et al 2022) indicates that Lim1 is expressed primarily in JO neurons (circled). C) Sound-evoked potential (SEP) traces from a control animal ( elav-Gal4 alone) shows robust response to computer-generated pulse song (pulse stim). A representative Lim1 knockdown animal ( elav-Gal4; UAS-Lim1-RNAi ) shows reduced SEP, as do representative animals with Lim1 knockdown using 2xJO-AB driver or Lim1 E9 clones. D) Scatterplots of SEPs from control and Lim1 RNAi knockdown animals. Each dot represents the SEP from one antenna. Bars and whiskers represent mean ± standard error of the mean. Statistically significant differences from control are indicated at the top (Welch’s one-way ANOVA, p < 0.0001, Dunnett’s T3 post-hoc multiple comparisons: ns = not significant, * p < 0.05, ** p < 0.01, ***p < 0.001). E) (upper row) Third instar larval imaginal disks and brain of Lim1 E9 MARCM clone animal stained for the clone marker GFP (green), as well as anti-dLim1 (magenta) and the nuclear stain DAPI (blue). As ey -FLP does not express in the wing disks (asterisks), no GFP-positive clones are generated. (lower row) Magnified views of one eye-antennal imaginal disk, highlighted by the box in the upper row. In GFP-positive clones, dLim1 staining is absent. Scale bar in merged panel: 50 μm. F) Pupal antennae from control (upper row) and Lim1 E9 MARCM clone animals (lower row), stained for GFP (green), anti-dLim1 (magenta) as well as the neuronal marker Futsch (22C10, yellow), and actin (phalloidin, blue). Scale bars in merged images: 10 μm. G) Scanning electron micrographs of heads from a control animal and Lim1 E9 MARCM clone animals. Examples of defects in clone animals include deformed arista (blue arrow), reduced or deformed second antennal segment (orange arrows), excess tissue dorsal to the antenna (yellow arrows) and reduced eye size, usually on the ventral side (red arrows). Scale bars: 100 μm.

Article Snippet: The 22C10 monoclonal antibody developed by S. Benzer and N. Colley was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: Expressing, Staining, RNA Sequencing, Control, Generated, Knockdown, Clone Assay, Marker

Journal: eLife

Article Title: Cre-assisted fine-mapping of neural circuits using orthogonal split inteins

doi: 10.7554/eLife.53041

Figure Lengend Snippet: ( A ) Sequence of the chimeric neuroblast enhancer used in this study, which is composed of an enhancer for the gene encoding the transcriptional repressor, deadpan (blue) fused via a 10 bp linker (red) to an enhancer for the gene encoding nerfin-1 (black). Upper case letters signify the highly conserved sequences used to identify the enhancers using the Evoprinter. Underlined are conserved sequence blocks shared by the two enhancers that represent putative transcription factor binding sites. Yellow highlight indicates two nucleotide substitutions that expand the range of the nerfin-1 enhancer expression in NBs. For further details see Materials and Methods. ( B ) Cis-regulatory activity of the DNE. The DNE was used to drive Gal4 expression, which was monitored during embryonic development by mRNA in situ hybridization. Shown are filleted wholemount embryos, stages 8–13 (anterior up). Most, if not all, CNS NBs are labeled during early to late stages of lineage development. By early embryonic stage 15, only a small subset of late neuroblasts have detectable signal and most, if not all, neurons and glia of the ventral cord and cephalic lobes have no detectable expression (data not shown). Scale bar: 50 μM. ( C ) DNE-mediated expression of CreAB and CreC fragments leads to reconstitution of Cre activity, excision of Gal80, and activation of tdTomato expression (magenta) in the embryonic CNS beginning at stage 8. Dorsal view, s8; lateral view, s9-13. ( D ) DNE-dependent Cre reconstitution and tdTomato expression in the NB 3–5 lineage of VNC hemisegments in a late-stage embryo. The previously described NB 3–5-specific Gal4 line, R59E09-Gal4 , was used to drive expression of UAS-Cre C in flies bearing the DNE-Cre B , DNE-Cre A and CreStop (i.e. actin^STOP^tdTomato) constructs. Excision of Gal80 specifically in NB 3–5 leads to tdTomato expression (red). Blue: 22C10 (anti- futsch) immunostaining. An enlarged image of the outlined region (left) is shown on the right. Scale bar: 50 μM.

Article Snippet: Antibody , Mouse monoclonal Anti-futsch Antibody , Developmental Studies Hybridoma Bank , RRID: AB_528403 , 1:50 dilution.

Techniques: Sequencing, Binding Assay, Expressing, Activity Assay, In Situ Hybridization, Labeling, Activation Assay, Construct, Immunostaining

Journal: eLife

Article Title: Cre-assisted fine-mapping of neural circuits using orthogonal split inteins

doi: 10.7554/eLife.53041

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal Anti-futsch Antibody , Developmental Studies Hybridoma Bank , RRID: AB_528403 , 1:50 dilution.

Techniques: Recombinant, Cloning, TA Cloning, Ligation