Journal: eLife

Article Title: BDNF-TrkB signaling in oxytocin neurons contributes to maternal behavior

doi: 10.7554/eLife.33676

Figure Lengend Snippet: ( a ) qPCR analysis validating significant enrichment ( Myo5a, Ank2, Peg3, and Kmt2a) and de-enrichment ( Agrp, Cartpt ) of select transcripts in IP compared to Input fractions. ( b–d ) Representative confocal z-projections of Oxt ( b, c, d ), Kmt2a ( b’ ), Peg3 ( c’ ), and Ank2 ( d’ ) transcripts in brain sections containing the PVN from an adult WT virgin female mouse visualized with RNAscope in situ hybridization. In the merged images ( b”, c”, d” ), Kmt2a , Peg3 , and Ank2 transcripts (white) are enriched in Oxt -expressing neurons (red) independently corroborating RNA-seq and qPCR findings.

Article Snippet: Taqman probes were commercially available from Life Technologies (Mm01329577_g1, Mm01182684_m1, 4352932E, Mm00487823_m1, Mm00618325_m1, Mm01337379_m1, Mm01179235_m1, Mm00475829_g1, Mm04210469_m1).

Techniques: RNAscope, In Situ Hybridization, Expressing, RNA Sequencing

Journal: eLife

Article Title: BDNF-TrkB signaling in oxytocin neurons contributes to maternal behavior

doi: 10.7554/eLife.33676

Figure Lengend Snippet:

Article Snippet: Taqman probes were commercially available from Life Technologies (Mm01329577_g1, Mm01182684_m1, 4352932E, Mm00487823_m1, Mm00618325_m1, Mm01337379_m1, Mm01179235_m1, Mm00475829_g1, Mm04210469_m1).

Techniques: Virus, Library Quantification, RNAscope, Multiplex Assay, Magnetic Beads, Sequencing

Journal: Molecular neurobiology

Article Title: Expression of a fragment of Ankyrin 2 disrupts the structure of the Axon Initial Segment and causes axonal degeneration in Drosophila

doi: 10.1007/s12035-019-1477-6

Figure Lengend Snippet: Z-projected confocal images of late third-instar larval brains stained with anti-Fas2. (a-d) Pink arrow indicates a stereotypical nerve that crosses over in front of the MB peduncle, and is used as a fiducial landmark to measure position of AIS boundary. The proximal border of the axonal Fas2 accumulation in the MB peduncle is indicated by the light blue arrow; the mean position of this proximal border in 201Y control samples is defined as zero. All scale bars are equal to 10μm. MB-specific GAL4 driver 201Y is present in all samples. (a) control, (b) UAS-VENUS- Ank2-L4 (U-A2L4), (c) UAS-VENUS-Ank2-L8 (U-A2L8), and (d) UAS-VENUS-Ank2-S (U-A2S). (e) Quantification of the distance of the proximal edge of the Fas2-positive boundary from the average position of the control boundary. Bars are presented as mean+SEM; statistical significance is relative to the 201Y-GAL4 control (****=p<0.0001). For each genotype, the number of MBs analyzed is presented at the bottom of the bar

Article Snippet: Z-stacks were collected from individual brain hemispheres and were analyzed using IMARIS and its ‘Add spots’ function to automatically count the labeled neurons. qRT-PCR RNA was isolated from 25 Drosophila heads using TRI reagent, and 1000ng of RNA was used for synthesis of cDNA using High Capacity cDNA Reverse Transcription Kit (both reagents from ThermoFisher Scientific, used following manufacturer’s protocol). qRT-CR reactions were prepared using the Affymetrix VeriQuest Probe qPCR Master Mix (Santa Clara, CA) with specific TaqMan gene primers: Ank (Probe# Dm01835806_g1 Cat# 4351372), and control: Rpl32 (Probe# Dm02151827_g1 Cat# 4331182).

Techniques: Staining, Control

Journal: Molecular neurobiology

Article Title: Expression of a fragment of Ankyrin 2 disrupts the structure of the Axon Initial Segment and causes axonal degeneration in Drosophila

doi: 10.1007/s12035-019-1477-6

Figure Lengend Snippet: Projected confocal images of late third-instar larval brains expressing markers that delineate the AIS. (a-c, g-i) 201Y-GAL4 controls; (d-f, j-l) 201Y-driven expression of Ank2-L4 (U-A2L4); (a) and (d) show Actin-RFP expression, and (g) and (j) outline the AIS using Synaptotagmin-HA; the same samples were co-stained with anti-Fas2 (b, e, h, and k), with panels (c, f, i, and l) showing the resulting merged images. In control samples, the somatodendritic boundary is marked by the green arrow; in Ank2-L4 samples, this boundary is indistinguishable from the proximal border of the axonal signal (indicated by the blue arrow). The pink arrow indicates a stereotypical nerve that crosses over in front of the MB peduncle, and is used as a landmark to quantify position of AIS boundary. All scale bars are equal to 10μm

Article Snippet: Z-stacks were collected from individual brain hemispheres and were analyzed using IMARIS and its ‘Add spots’ function to automatically count the labeled neurons. qRT-PCR RNA was isolated from 25 Drosophila heads using TRI reagent, and 1000ng of RNA was used for synthesis of cDNA using High Capacity cDNA Reverse Transcription Kit (both reagents from ThermoFisher Scientific, used following manufacturer’s protocol). qRT-CR reactions were prepared using the Affymetrix VeriQuest Probe qPCR Master Mix (Santa Clara, CA) with specific TaqMan gene primers: Ank (Probe# Dm01835806_g1 Cat# 4351372), and control: Rpl32 (Probe# Dm02151827_g1 Cat# 4331182).

Techniques: Expressing, Staining, Control

Journal: Molecular neurobiology

Article Title: Expression of a fragment of Ankyrin 2 disrupts the structure of the Axon Initial Segment and causes axonal degeneration in Drosophila

doi: 10.1007/s12035-019-1477-6

Figure Lengend Snippet: Z-projected confocal images of late third-instar larval brains stained with anti-Fas2. (a-f) Pink arrow indicates a stereotypical nerve that crosses over in front of the MB peduncle, and is used as a fiducial landmark to measure position of AIS boundary. The proximal border of the axonal Fas2 accumulation in the MB peduncle is indicated by the light blue arrow. All scale bars are equal to 10μm. (a) w1118 control samples were compared to (b) samples homozygous for the Ank22001 mutation and thus null for Ank2-L. (c-f) MB-specific GAL4 driver 201Y is present in all samples. (c) control, (d) UAS-VENUS-Ank2-L4 (U-A2L4), (e) UAS-Ank2-RNAi (U-A2RNAi), or (f) both UAS-VENUS-Ank2-L4 and UAS-Ank2-RNAi. (g) Quantification of the shift in Fas2 accumulation was performed as in Fig. 1e. Bars are presented as mean±SEM; statistical significance is relative to the w1118 or 201Y-GAL4 control (**=p<0.01, ***=p<0.001, ****=p<0.0001). For each genotype, the number of MBs analyzed is presented at the bottom of the bar

Article Snippet: Z-stacks were collected from individual brain hemispheres and were analyzed using IMARIS and its ‘Add spots’ function to automatically count the labeled neurons. qRT-PCR RNA was isolated from 25 Drosophila heads using TRI reagent, and 1000ng of RNA was used for synthesis of cDNA using High Capacity cDNA Reverse Transcription Kit (both reagents from ThermoFisher Scientific, used following manufacturer’s protocol). qRT-CR reactions were prepared using the Affymetrix VeriQuest Probe qPCR Master Mix (Santa Clara, CA) with specific TaqMan gene primers: Ank (Probe# Dm01835806_g1 Cat# 4351372), and control: Rpl32 (Probe# Dm02151827_g1 Cat# 4331182).

Techniques: Staining, Control, Mutagenesis

Journal: Molecular neurobiology

Article Title: Expression of a fragment of Ankyrin 2 disrupts the structure of the Axon Initial Segment and causes axonal degeneration in Drosophila

doi: 10.1007/s12035-019-1477-6

Figure Lengend Snippet: Ank2-L4 overexpression does not cause lethality. Results from a genetic lethality screen to assess viability upon pan-neuronal expression of UAS- Ank2-RNAi (U-A2RNAi) or UAS-VENUS-Ank2-L4 (U-A2L4). Virgins heterozygous for the pan- neuronal driver ELAV-GAL4 and a balancer chromosome ( CyO – curly wing) were crossed with males carrying the respective UAS construct. The ratio of balancer to non-balancer offspring was quantified; significant differences in ratios were assessed by chi-square analysis

Article Snippet: Z-stacks were collected from individual brain hemispheres and were analyzed using IMARIS and its ‘Add spots’ function to automatically count the labeled neurons. qRT-PCR RNA was isolated from 25 Drosophila heads using TRI reagent, and 1000ng of RNA was used for synthesis of cDNA using High Capacity cDNA Reverse Transcription Kit (both reagents from ThermoFisher Scientific, used following manufacturer’s protocol). qRT-CR reactions were prepared using the Affymetrix VeriQuest Probe qPCR Master Mix (Santa Clara, CA) with specific TaqMan gene primers: Ank (Probe# Dm01835806_g1 Cat# 4351372), and control: Rpl32 (Probe# Dm02151827_g1 Cat# 4331182).

Techniques: Over Expression, Expressing, Construct

Journal: Molecular neurobiology

Article Title: Expression of a fragment of Ankyrin 2 disrupts the structure of the Axon Initial Segment and causes axonal degeneration in Drosophila

doi: 10.1007/s12035-019-1477-6

Figure Lengend Snippet: Projected confocal images of late third-instar larval brains stained with anti-Fas2. (A-D) Pink arrow indicates a stereotypical nerve that crosses over in front of the MB peduncle, and is used as a fiducial landmark to measure position of AIS boundary. The proximal border of the axonal Fas2 accumulation in the MB peduncle is indicated by the light blue arrow; the mean position of this proximal border in 201Y control samples is defined as zero. All scale bars are equal to 10μm. MB-specific GAL4 driver 201Y is present in all samples. (a) control, (b) UAS-Cdk5α (U- Cdk5α), (c) UAS-VENUS-Ank2-L4 (U-A2L4), and (d) both UAS-VENUS-Ank2-L4 and UAS- Cdk5α. (e) Quantification of the observed shift in Fas2 accumulation was completed as in Fig. 1e. Bars are presented as mean±SEM; statistical significance is relative to the 201Y-GAL4 control (*=p<0.05, ****=p<0.0001). For each genotype, the number of MBs analyzed is presented at the bottom of the bar

Article Snippet: Z-stacks were collected from individual brain hemispheres and were analyzed using IMARIS and its ‘Add spots’ function to automatically count the labeled neurons. qRT-PCR RNA was isolated from 25 Drosophila heads using TRI reagent, and 1000ng of RNA was used for synthesis of cDNA using High Capacity cDNA Reverse Transcription Kit (both reagents from ThermoFisher Scientific, used following manufacturer’s protocol). qRT-CR reactions were prepared using the Affymetrix VeriQuest Probe qPCR Master Mix (Santa Clara, CA) with specific TaqMan gene primers: Ank (Probe# Dm01835806_g1 Cat# 4351372), and control: Rpl32 (Probe# Dm02151827_g1 Cat# 4331182).

Techniques: Staining, Control

Journal: Molecular neurobiology

Article Title: Expression of a fragment of Ankyrin 2 disrupts the structure of the Axon Initial Segment and causes axonal degeneration in Drosophila

doi: 10.1007/s12035-019-1477-6

Figure Lengend Snippet: (a-f) Confocal images of single MARCM clones from 30-day old brains. MB-specific GAL4 driver 201Y is present in all samples. All scale bars are equal to 10μm. (a,d) control, (b-c) UAS-VENUS-Ank2-L4, and (e-f) UAS-Cdk5α. Representative images show (b, e) swelling or (c, f) a “beads-on-a-string” phenotype in the proximal axon. (g) Quantification of the prevalence of degenerative morphologies; significance assessed by χ2 test, with Yates’ correction (**p<0.01). For each genotype, the total number of clones analyzed is presented at the bottom of the bar. (h) Quantification of the number of MB neurons per brain hemisphere in aged flies with MB-specific expression of UAS-nls-mCherry alone, UAS-nls-mCherry plus UAS-GFP, or UAS-nls-mCherry plus UAS-VENUS-Ank2-L4. The number of 201Y>nls-mCherry positive MB neurons per hemisphere is presented as mean±SEM. Significant differences were assessed by two-way ANOVA; differences labeled with asterisks (*) are relative to the Day 3 UAS-nls-mCherry alone control; differences marked with “#” are relative to the Day 3 sample co-expressing UAS-GFP and UAS-nls-mCherry. *p<0.05; ***p<0.001; #p<0.05

Article Snippet: Z-stacks were collected from individual brain hemispheres and were analyzed using IMARIS and its ‘Add spots’ function to automatically count the labeled neurons. qRT-PCR RNA was isolated from 25 Drosophila heads using TRI reagent, and 1000ng of RNA was used for synthesis of cDNA using High Capacity cDNA Reverse Transcription Kit (both reagents from ThermoFisher Scientific, used following manufacturer’s protocol). qRT-CR reactions were prepared using the Affymetrix VeriQuest Probe qPCR Master Mix (Santa Clara, CA) with specific TaqMan gene primers: Ank (Probe# Dm01835806_g1 Cat# 4351372), and control: Rpl32 (Probe# Dm02151827_g1 Cat# 4331182).

Techniques: Clone Assay, Control, Expressing, Labeling