Dataset DOI: 10.5061/dryad.z612jm6r9

Description of the data and file structure

Original data used in the study entitled: "The IKC-leptin axis functions as a molecular bridge between neurodevelopment and endocrine signaling” Elena Forzisi, Anika Gaur, Aniket Gaur, Maria del Carmen Medranda, Rachel Philip, Srishti Anand, Clotilde Impaloni, Kiarah Leonard, Srinidi Venkateswaran, Federico Sesti.

Data are organized in folders named after each figure of the related paper. For each figure of the paper, the corresponding original data are in folders named after the figure's experimental procedures. For example the folder FIGURE 1 contains the zipped sub-folders Behaviors, and zip sub-sub-folders for marble burying, nesting behavior, open field, y maze. Folders can contain sub-folders, sub-sub-folders, etc. corresponding to different experimental procedures. Please note:

1.        The genotype "Kcnb1Null" is often called "Null" or "KO".

2.        The genotype Wild type is often called "WT"

Usage notes for file formats:

Confocal images in Nikon .ND2 format or Olympus .iob or Leica .lif format can be imported in ImageJ/Fiji or read with the freely available software NIS Elements.

Confocal images in .XCF format can be imported into ImageJ or read with the Olympus Fluoview software

Images are typically in JPEG format but some can be in other readable formats such as, PNG and TIFF.

Files in HEIF format (Apple high efficiency image file) can be read using Apple OS or ImageJ.

Data files are in Excel format .xlsx and/or .csv (comma separated values) format or more commonly in Prism format. These data can be read using Microsoft office or numbers or Prism software.

For behavioral analysis, ANY maze files in .szd can only be opened with the ANY maze software.

Files and variables

FIGURE 1

File: Figure_1.zip

The folder contains original data from behavioral experiments, including marble burying, nesting behavior, open field, and Y maze. The folder “behaviors” contains four subfolders: “marble burying,” “Nesting behavior,” “open field,” and “Y maze,” as shown in Figure 1. Videos are in the original ANY format and have also been converted to .avi. Videos named numerically correspond to the animal performing the behavioral test indicated in the corresponding folder. 

The “Marble burying” subfolder contains an Excel file ("MB data.xlsx/.csv") used for data collection and analysis. This file reports counts of buried, partially buried, and unburied marbles, per mouse/trial. Columns F and M report the percent of marbles buried; this is reported in Figure 1D in the associated manuscript.

The “Nesting behavior” subfolder contains sub-subfolders labeled according to the date of the experiment in the format MMDDYY. These sub-subfolders contain JPEG photos of the nesting material along with the mouse cage card for reference. The file "Nesting behavior data.xlsx/.csv" reports nesting behavior for each mouse, with a score from 0 to 5 (see Methods in associated manuscript; reported in Figure 1C).

The “open field” subfolder contains ANY-maze .sdz, .szp and .xlsx files used for behavioral data analysis, as well as a sub-subfolder named “open field videos.” This folder contains sub-sub-subfolders of open field behavioral experiments divided by date in the format MMDDYY. These sub-sub-subfolders contain videos of the experiments, named appropriately with genotype and conditions, in .avi format. Heatmap files contain trajectory plots depicting the paths traveled by the animals in the open-field arena. The file "Open field data.xlsx/.csv" reports behavioral data per mouse, with distance in meters and time in seconds; shown in Figure 1A.

The “Y maze” subfolder contains an .xlsx file for data collection and analysis, and a sub-subfolder named “Y maze plots and videos,” which contains videos and plots in .mpa, .avi, or .szv (ANY-maze compatible) formats, divided by experiment date in the format MMDDYY. The file "Y maze data.xlsx/.csv" reports counts of correct and incorrect alternations per mouse, shown in Figure 1E.

If a subfolder is not present, the genotype is indicated in the title of the image or video. Images and videos were analyzed by multiple observers using the methodologies described in the Methods section of the related paper.

Figure descriptions and units for related Excel files:

Panel B Distance traveled in meters.

Panel C Nesting score (dimensionless).

Panel D Percent of marbles buried.

Panel E Percent of incorrect alternations.

FIGURE 2_S5

File: Figure_2_S5.zip

The folder contains raw images and analysis data for immunofluorescence staining of MAP2 (magenta, far-red channel), L1CAM (red, red channel), NFL (green, green channel), and DAPI (blue, violet channel), as shown in Figure 2 and Supplementary Figure 5.

The folder contains a subfolder named “MAP2 NFL L1CAM staining,” in which images are divided into sub-subfolders named according to genotype and condition. For example, the sub-subfolder “WT leptin” contains .lif files for each biological and technical replicate, divided by number and date of experiment execution.

Images can be opened using ImageJ. For analysis, pictures were converted to grayscale, and the thickness and fluorescence intensity of specific brain regions were calculated. Background values were measured and subtracted for each replicate. Data were collected and analyzed in Excel, and all data are stored in the “Measurements anatomy.xlsx” file (also available as CSV exports, named according to each tab of the original Excel). Column A of each sheet indicates which .lif file contains the raw data reported in each section of the sheet. Each column is labeled with the relevant anatomical structure. "Thick" or "Thickness" in variable names denotes thickness measurements (in µm or normalized). "Int" denotes intensity measurements.

Figure descriptions and units for related Excel files:

Figure 2

Panel C Normalized layer thickness (dimensionless).

Panel E Thickness in µm.

Figure S5

Panels A-F Fluorescence intensities in arbitrary units.

FIGURE 3

File: Figure_3.zip

The folder contains raw images and analysis data for BrdU migration experiments, as shown in Figure 3. It contains a folder named “BRDU stainings,” which includes all raw images in .lif format, divided into subfolders based on genotype and condition. For example, the “WT leptin” folder contains .lif files named with the genotype, animal number, and the date of the experimental procedure in the format MMDDYY.

The data analysis is presented in the “BRDU Analysis_final.xlsx” file (also available as CSV exports, named according to each tab of the original Excel). Raw imaging data can be used to reproduce analyses; the spreadsheets are included to preserve a record of the original analytic workflow.

Figure descriptions and units for related Excel files:

Panel B Number of cells.

Panel D Thickness in µm.

FIGURE 4

File: Figure_4.zip

The folder contains raw images and analysis data for Golgi staining experiments, as shown in Figure 4. The folder contains Excel files used for analysis and a subfolder named “Cortex all photos.”

This subfolder contains multiple sub-subfolders divided by genotype and treatment, such as “KO control.” The sub-subfolders contain sub-sub-subfolders with images divided by the date of the experiment, such as “KO Cont #2 2 072524.” Images are in JPEG format and date format is MMDDAA.

For analysis, images were opened in ImageJ and converted to 8-bit format. The threshold was adjusted, and Sholl analysis or dendritic analysis was performed. Sholl analysis data are stored in the “Layer_2_3 normalized interactions.xlsx” and “Layer_5_6 normalized interactions.xlsx” files. Dendritic density analysis data are stored in the “Layer_2_3 dendrite density.xlsx” and “Layer_5_6 dendrite density.xlsx” files. Column descriptions are included in the Excel spreadsheets.

Sholl analyses were performed using a Fiji plug-in. Sholl analysis quantifies dendritic complexity by counting the number of dendritic intersections with concentric circles of progressively increasing radius centered on the neuronal soma. It is graphed as number of dendritic intersections vs. radial distance from the soma.

Figure descriptions and units for related Excel files:

Panel C Normalized Sholl interactions (dimensionless).

Panels E-F Cell density in µm^-1.

FIGURE 5_S6

File: Figure_5_S6.zip

The folder contains raw images and data analysis for spine morphology experiments, as shown in Figure 5 and Supplementary Figure 6. The folder contains a subfolder named “Golgi spines ctx,” where “ctx” stands for cortex.

This subfolder is divided into sub-subfolders based on genotype and condition. For example, the “WT leptin” folder contains .lif files with raw images from three biological replicates, named with identification numbers and the date of the experiment in the format MMDDYY.

Analysis was performed using ImageJ on 15–25 z-stack images. The data analysis is presented in the “analysis spine CTX.xlsx” file. Raw imaging data can be used to reproduce analyses; the spreadsheets are included to preserve a record of the original analytic workflow.

Figure descriptions and units for related Excel files:

Panels B-C Number of spines.

Panel D % of spines.

Figure S6  Number of spines.

FIGURE 6_S7

File: Figure_6_S7.zip

The folder contains raw data and analysis for Fluorescence Recovery After Photobleaching (FRAP), neuronal staining for the leptin receptor (LepR) and actin, and Sholl and cytoskeletal experiments, as shown in Figure 6 and Supplementary Figure 7, under control and leptin treatment conditions. The folder contains three subfolders: “FRAP,” “LepR-Actin staining,” and “Neurons for sholl, dendritic length and actin analysis.”

The “FRAP” subfolder contains sub-subfolders divided by genotype (WT and Null) and the data analysis file “Analysis CRTX.xlsx.” The sub-subfolders contain sub-sub-subfolders of FRAP experiments divided by condition: “CTRL,” which stands for control, and “LEPTIN.” These sub-sub-subfolders contain additional sub-sub-sub-subfolders with raw images divided by experiment date in the format MMDDYY. Raw images are in .oib format and can be opened in ImageJ for analysis. Fluorescence is shown in the green channel, and intensity is measured in grayscale for 24 frames using the grayscale measurement tool.

The “LepR-Actin staining” subfolder contains raw images for WT and Null co-staining of LepR (red channel), MAP2 (far-red channel), and actin (green channel) in .lif format. The analysis is presented in the “colocalization analysis.xlsx” file. Colocalization analysis was performed by measuring the number of overlapping particles using an “AND” and “OR” ratio equation expressed as a percentage.

The “Neurons for sholl, dendritic length and actin analysis” subfolder contains sub-subfolders named according to genotype (WT and Null). These sub-subfolders contain sub-sub-subfolders named according to condition, and within them are sub-sub-sub-subfolders containing images divided into technical replicates named “disk #number.” Image files are in .nd2 format and can be opened in ImageJ, where they were morphologically analyzed using the Sholl analysis neuronal anatomy plugin, the segmented line tool for dendritic length, and the particle analysis tool for actin aggregates. Sholl analyses were performed using a Fiji plug-in. Sholl analysis quantifies dendritic complexity by counting the number of dendritic intersections with concentric circles of progressively increasing radius centered on the neuronal soma. It is graphed as number of dendritic intersections vs. radial distance from the soma.

Excel files named “Analysis” in the FRAP folders contain the original FRAP measurements. The first column indicates the frame number, acquired at 0.5-s intervals, for a total of 24 time points corresponding to 12 s. The remaining columns report fluorescence measurements, in arbitrary units, with individual cells distinguished by alternating column colors. Below these raw measurements are the averaged data for each cell, normalized to the pre-bleach fluorescence. Each worksheet corresponds to the indicated genotype or experimental condition.

Figure descriptions and units for related Excel files:

Figure 6

Panels B-D Number of Sholl interactions.

Panels F-G Length in µm.

Panel H Fluorescence recovery in percent (dimensionless).

Panel I Rate constants in s^-1.

Panels J-K Steady-state fluorescence recovery as percent of total recovery (dimensionless).

Figure S7

Panel B GFP fluorescence in arbitrary units.

Panels C-E Fluorescence recovery in percent (dimensionless).

Panels F-G Rate constants in s^-1.

FIGURE 7

File: Figure_7.zip

The folder contains raw data and analysis for co-immunoprecipitation, neuronal staining, and RNAscope experiments, as shown in Figure 7. The folder contains the subfolders “COIP,” “Neuronal Stainings,” and “RNA SCOPE.”

The “COIP” subfolder contains sub-subfolders of Western blot (WB) data for co-immunoprecipitation experiments, divided by date in the format MMDDYY. WB images are in JPEG format and are named according to the antibody used for band identification. TL stands for Total Lysate, IP stands for Immunoprecipitate, and COIP stands for Co-immunoprecipitate. Intβ1 stands for integrin beta 1, and LepR stands for leptin receptor. The order of samples is described in the image name. For example, the image named “Lepr TL wt e17 hypo wt e17 ctx wt p1 wt p13 wt ctrl ko ctrl” indicates a Western blot for leptin receptor protein total lysate from wild-type (WT) embryonic day 17 hypothalamus and cortex, WT postnatal day 1, postnatal day 13, and adult WT and adult null samples, respectively.

The “Neuronal Stainings” subfolder contains .lif files of raw images for WT neuronal staining of KCNB1 (green channel), integrin beta 1 (Intβ1, far-red channel), and leptin receptor (LepR, red channel). The analysis is presented in the “colocalization analysis.xlsx” file. Colocalization analysis was performed in ImageJ using the colocalization plugin. Raw imaging data can be used to reproduce analyses; the spreadsheets are included to preserve a record of the original analytic workflow.

The “RNA SCOPE” subfolder contains .lif files of raw images from WT and Null technical and biological replicates for RNAscope analysis. The corresponding data analysis is presented in the “RNA scope analysis.xlsx” file. Analysis was performed in ImageJ using the particle analysis tool. Raw data can be used to reproduce analyses; the spreadsheets are included to preserve a record of the original analytic workflow.

Figure descriptions and units for related Excel files:

Panel C mRNA density in mm^-1.

Panel F Manders coefficients expressed in percent (dimensionless).

Panel G Protein molecular weights in kDa.

FIGURE 8_S7

File: Figure_8_S7.zip

The folder contains raw images and data for FRAP experiments and Sholl and cytoskeletal analyses, as shown in Figure 8 and Supplementary Figure 7, under Allo-Aca or Allo-Aca plus leptin treatment conditions. The folder contains two subfolders: “FRAP” and “Neurons for sholl, dendritic length and actin analysis.”

The “FRAP” subfolder contains sub-subfolders divided by genotype (WT and Null) and the data analysis file “Analysis CRTX.xlsx.” These sub-subfolders contain sub-sub-subfolders of FRAP experiments divided by condition: “allo-aca” and “allo-aca+lep.” The sub-sub-subfolders contain additional sub-sub-sub-subfolders with raw images divided by experiment date in the format MMDDYY.

The “Neurons for sholl, dendritic length and actin analysis” subfolder contains sub-subfolders named according to genotype (WT and Null). These sub-subfolders contain sub-sub-subfolders named according to condition, and within them are sub-sub-sub-subfolders containing images divided into technical replicates named “disk #number.” Image files are in .nd2 format and can be opened in ImageJ, where they were morphologically analyzed using the Sholl analysis neuronal anatomy plugin, the segmented line tool for dendritic length, and the particle analysis tool for actin aggregates. Sholl analyses were performed using a Fiji plug-in. Sholl analysis quantifies dendritic complexity by counting the number of dendritic intersections with concentric circles of progressively increasing radius centered on the neuronal soma. It is graphed as number of dendritic intersections vs. radial distance from the soma.

Excel files named “Analysis” in the FRAP folder contain the original FRAP measurements. The first column indicates the frame number, acquired at 0.5-s intervals, for a total of 24 time points corresponding to 12 s. The remaining columns report fluorescence measurements, in arbitrary units, with individual cells distinguished by alternating column colors. Below these raw measurements are the averaged data for each cell, normalized to the pre-bleach fluorescence. Each worksheet corresponds to the indicated genotype or experimental condition.

Figure descriptions and units for related Excel files:

Figure 8

Panels A and C Number of Sholl interactions and thicknesses in µm.

Panels B and D Rate constants in s^-1 and steady-state fluorescence recovery as percent of total recovery (dimensionless).

Figure S7

Panel B GFP fluorescence in arbitrary units.

Panels C-E Fluorescence recovery in percent (dimensionless).

Panels F-G Rate constants in s^-1.

FIGURE 9_S7

File: Figure_9_S7.zip

The folder contains raw images and data for FRAP experiments and Sholl and cytoskeletal analyses, as shown in Figure 8 and Supplementary Figure 7, under cilengitide, fibronectin, or fibronectin plus leptin treatment conditions. The folder contains two subfolders: “FRAP” and “Neurons for sholl, dendritic length and actin analysis.”

The “FRAP” subfolder contains sub-subfolders divided by genotype (WT and Null) and the data analysis file “Analysis CRTX.xlsx.” These sub-subfolders contain sub-sub-subfolders of FRAP experiments divided by condition: “Cilen” (cilengitide), “Cilen+Lep,” “Fibro” (fibronectin), and “Fibro+Lep.” The sub-sub-subfolders contain additional sub-sub-sub-subfolders with raw images divided by experiment date in the format MMDDYY.

The “Neurons for sholl, dendritic length and actin analysis” subfolder contains sub-subfolders named according to genotype (WT and Null). These sub-subfolders contain sub-sub-subfolders named according to condition, and within them are sub-sub-sub-subfolders containing images divided into technical replicates named “disk #number.” Image files are in .nd2 format and can be opened in ImageJ, where they were morphologically analyzed using the Sholl analysis neuronal anatomy plugin, the segmented line tool for dendritic length, and the particle analysis tool for actin aggregates. Sholl analyses were performed using a Fiji plug-in. Sholl analysis quantifies dendritic complexity by counting the number of dendritic intersections with concentric circles of progressively increasing radius centered on the neuronal soma. It is graphed as number of dendritic intersections vs. radial distance from the soma.

Excel files named “Analysis” in the FRAP folder contain the original FRAP measurements. The first column indicates the frame number, acquired at 0.5-s intervals, for a total of 24 time points corresponding to 12 s. The remaining columns report fluorescence measurements, in arbitrary units, with individual cells distinguished by alternating column colors. Below these raw measurements are the averaged data for each cell, normalized to the pre-bleach fluorescence. Each worksheet corresponds to the indicated genotype or experimental condition.

Figure descriptions and units for related Excel files:

Figure 9

Panel B Number of Sholl interactions.

Panels C-D thicknesses in µm.

Panel E Steady-state fluorescence recovery as percent of total recovery (dimensionless).

Figure S7

Panel B GFP fluorescence in arbitrary units.

Panels C-E Fluorescence recovery in percent (dimensionless).

Panels F-G Rate constants in s^-1.

FIGURE S1

File: Figure_S1.zip

The folder contains data for leptin ELISA and mouse weight measurements, as shown in Supplementary Figure 1. The folder contains the subfolders “Leptin ELISA” and “Weight.”

The “Leptin ELISA” subfolder contains Excel files for ELISA experiment data analysis from 1-month- and 3-month-old mice. The “Weight” subfolder contains an Excel file with mouse weight data at 1 month of age.

Figure descriptions and units for related Excel files:

Panel A Weight in gr.

Panels B-D Serum leptin in pg/mL.

FIGURE S2 

File: Figure_S2.zip

The folder contains behavioral data for the Object Recognition Test, as shown in Supplementary Figure S2. It contains two ANY-maze files in .sdz format, which can be opened using ANY-maze software. The data are organized by genotype and treatment condition. Data were collected, analyzed, and stored in the Excel file “Behaviors mice log ORT.” Distances reported in cm.

Figure descriptions and units for related Excel files:

Panel B Exploration time in secs.

Panel C Discrimination index (dimensionless).

FIGURE S3

File: Figure_S3.zip

The folder contains behavioral data for the Barnes Maze test, as shown in Supplementary Figure S3. It includes two ANY-maze files in .sdz format, which store the data for the probe test and can only be opened using ANY-maze software. All data are organized by genotype and treatment condition. The folder also contains subfolders with videos in .mp4 or .szv format (the latter can only be opened in ANY-maze). Data were collected, analyzed, and stored in the Excel file “Behaviors mice log BM.”

Figure descriptions and units for related Excel files:

Panels A-F Times in platform in secs.

Panel H Number of entries (dimensionless).

Panels I-J Time in quadrants in secs.

FIGURE S4

File: Figure_S4.zip

The folder contains raw data for the PSD95 and Synapsin staining colocalization experiment, as shown in Supplementary Figure S4. It includes a subfolder named “PSD95 Syn staining,” which contains sub-subfolders organized by genotype and treatment condition. Each sub-subfolder contains .lif files from three biological replicates, named using the format “condition #animal date fluorophores,” with the date in MMDDYY format (for example, “ko control #2 62625 syn 594 psd 488.lif”). The .lif files can be opened in ImageJ.

The analysis is presented in the Excel file “colocalization analysis syn psd95.xlsx.” Colocalization analysis was performed by measuring the number of overlapping particles using an “AND” and “OR” ratio equation, expressed as a percentage. Raw data can be used to reproduce analyses; the spreadsheet is included to preserve a record of the original analytic workflow.

Figure descriptions and units for related Excel file:

Panel B-C Percent of protein colocalization.

Additional data for figure 6,8,9

File: Addittional_data_for_figure_6_8_9.zip

The folder contains data from FRAP experiments and neuronal in vitro experiments, as shown in Figures 6, 8, 9, and Supplementary Figure S7. It contains two subfolders: “FRAP” and “Neurons for sholl, actin aggregates, dendrites.”

The “FRAP” subfolder contains data from FRAP experiments under Allo-Aca, Fibronectin (Fibro), or Cilengitide (Cil or Cilen) treatments. This subfolder includes the Excel data analysis file “Analysis CRTX.xlsx” and two sub-subfolders named according to genotype: “Null” and “WT.” These sub-subfolders contain sub-sub-subfolders named according to the treatment used in the experiment, such as “allo+cilen,” where “allo” stands for Allo-Aca. Inside these sub-sub-subfolders are sub-sub-sub-subfolders containing images divided by experiment date, for example “FRAP_120825_allo+cil_1,” with dates in the format MMDDYY.

The “Neurons for sholl, actin aggregates, dendrites” subfolder contains four Excel files for data analysis: actin analysis (“Actin Analysis 081625 CTX”), Sholl analysis (“CTX NULL sholl single cell final” and “CTX WT sholl single cell final”), and dendritic length analysis (“DL Analysis CTX”) for all in vitro neuronal experiments shown in Figures 6, 8, and 9. This subfolder also contains two sub-subfolders named by genotype: “Null” and “WT.” Each of these contains sub-sub-subfolders named according to treatment conditions, such as “Cil+Allo.” Within the sub-sub-subfolders are .lif and .lifext files containing the raw images for the experiments.

The analysis is organized within sub-sub-sub-subfolders named “disc 1, 2, 3.” For each disc, 15 images were acquired, and 2 cells were analyzed. The data for each cell are organized into sub-sub-sub-sub-subfolders. Raw imaging data can be used to reproduce analyses; the spreadsheets are included to preserve a record of the original analytic workflow.

Figure descriptions and units for related Excel files:

Figure 6

Panels B-D Number of Sholl interactions.

Panels F-G Length/size in µm.

Panel H Fluorescence recovery in percent (dimensionless).

Panel I Rate constants in s^-1.

Panels J-K Steady-state fluorescence recovery as percent of total recovery (dimensionless).

Figure 8

Panels A and C Number of Sholl interactions and thicknesses in µm.

Panels B and D Rate constants in s^-1 and steady-state fluorescence recovery as percent of total recovery (dimensionless).

Figure 9

Panel B Number of Sholl interactions.

Panels C-D thicknesses in µm.

Panel E Steady-state fluorescence recovery as percent of total recovery (dimensionless).

Crosslink_COIP_.zip 

File: Crosslink_COIP_.zip

The folder contains raw data used in Fig. S8. These are JPEG and TIFF files showing total Western blots from the same membrane stripped and reloaded with different antibodies. Each file is named with the antibody used for the co-Ip or total lysate or, in the case of the LepR antibody for the IP and total lysate. 

Units:

Protein molecular weights in kDa.

Code/software

no code software was used/generated.