Retinal calcium waves coordinate uniform tissue patterning of the Drosophila eye
Data files
Abstract
Dataset DOI: 10.5061/dryad.1g1jwsv9d
Description of the data and file structure
Raw data deposit from manuscript "Retinal Calcium Waves Coordinate Uniform Tissue Patterning of the Drosophila Eye."
Files and variables
File: F1.xlsx
Description: Raw data for main Fig. 1:
1C: Retinal calcium activity across pupal retinal development.
Each rows indicate individual time point.
Column labelings are following.
Time point : Each time points are 2 min intervals
Avg : Average of deltaF of whole retinal calcium signal.
SEM : Standard Error of the Mean of deltaF of whole retinal calcium signal.
n : Number of retina
Dev Stage (hAPF) : Developmental stages by hour from pupation
Normalized Ca activity : Normalized Avg values by average basal line activity. Depicted in Fig. 1C
Normalized SEM : Normalized Standard Error of the Mean values by average basal line activity. Depicted in Fig. 1C
1H. Retinal calcium activity of per individual retina during retinal waves.
Each rows indicate individual time point.
Column labelings are following.
T(sec) : Time points in seconds.
T(min) :Time points in minutes.
retina 1: Normalized Avg values of calcium activity of retina 1 by average basal line. Depicted in Fig. 1H
retina 2: Normalized Avg values of calcium activity of retina 2 by average basal line. Depicted in Fig. 1H
retina 3: Normalized Avg values of calcium activity of retina 3 by average basal line. Depicted in Fig. 1H
retina 4: Normalized Avg values of calcium activity of retina 4 by average basal line. Depicted in Fig. 1H
avg : Average of retina1-4. Depicted in Fig. 1H
1I. Quantification of the timing of the onset of retinal waves.
Each rows indicate individual retina.
Column labelings are following.
Stage1: Dev (developmental) timing in hAPF (hours after pupation) when stage1 begins.
Stage2: Dev timing in hAPF when stage2 begins.
Stage3: Dev timing in hAPF when stage3 begins.
1J. Quantification of the duration of retinal waves.
Each rows indicate individual retina.
Column labelings are following.
Stage1: Duration for stage1 in minutes.
Stage2: Duration for stage2 in minutes.
Stage3: Duration for stage3 in minutes.
1KtoP. Ommatidial-level retinal wave-induced integrative calcium activity between 37hAPF and 41hAPF. Each bin represents a 30-minute time period, and each hexagon represents a single ommatidium. The level of integrative calcium signal for individual ommatidium during each 30-minute time window is color-coded. All images came from the same retina as in Fig. 1C.
Each rows indicate ROI for individual ommatidia.
Column labelings are following.
roi_id : ROI identifier
ON_count_Bin1 : Wave counts during Bin1 (0-30minutes)
ON_count_Bin2 : Wave counts during Bin2
ON_count_Bin3 : Wave counts during Bin3
ON_count_Bin4 : Wave counts during Bin4
ON_count_Bin5 : Wave counts during Bin5
ON_count_Bin6 : Wave counts during Bin6
ON_count_Bin7 : Wave counts during Bin7
ON_count_Bin8 : Wave counts during Bin8
ON_count_Bin9 : Wave counts during Bin9
ON_count_Bin10 : Wave counts during Bin10
1Q. Ommatidial configuration grid of integrative calcium activity. Each circle represents a group of ommatidia in an 8×8 grid along dorsoventral (DV) and anteroposterior (AP) axes. Color indicates average level of integrative calcium activity during the wave period. Data from 6 control retinas.
Each rows indicate each grid points in 8x8 map.
Column labelings are following.
8x8 Grid : Grid identifier
4Q : Positioning identifier as quadrants. Va = ventral-anterior, vp = ventral-posterior, da = dorsal-anterior, dp = dorsal-posterior,
Normalized integrative calcium activity : Normalized integrative calcium activity per grid points by group average calcium activity,
1R. Quantification of ommatidial-level integrative calcium activity between different retinal quadrants shown in Fig. 1Q. n ≥ 432 ommatidia from 6 retinas.
Each value from normalized calcium activity from individual grid points.
Column labelings are following.
Va = ventral-anterior,
vp = ventral-posterior,
da = dorsal-anterior,
dp = dorsal-posterior
Variables include retina identifier, imaging time (hAPF or hours after puparium formation), wave duration (seconds or minutes), ΔF/F₀ amplitude (ratio), and region identity (VA, VP, DA, DP).
Methods: Retinal calcium activity was recorded using longGMR-Gal4 driven GCaMP6s under a Leica SP8 confocal microscope (dry-lens configuration). Fiji, Python, and Imaris were used for ΔF/F₀ normalization, wave detection, and quantification.
File: F2.xlsx
Description: Raw data for Main Fig2.
2Q. This file contains wave event logs during early developmental stages (stage 1 and early stage 2).
Each rows indicate time points
Column labelings are following.
time 1min : each time points
CC firing : individual CC-firing counts
cc-cc : CC-CC propagation counts
cc-cc-1p: CC-CC-1PC propagation counts
cc-cc-1p-ioc: CC-CC-1PC-IOC propagation counts
cc-cc-1p-ioc-ioc: CC-CC-1PC-IOC-IOC propagation counts
Variables include elapsed time (minutes), event frequency (waves per minute), propagation pathway category, and retina identifier. See also B. Cell type code notation, C. Propagation code notation in the last session.
Methods: Wave events were manually with Fiji-processed time-lapse movies.
File: F3.xlsx
Description: Raw data for main Fig. 3. Sheets include:
3G. Quantification of the calcium activity of individual ommatidia made by WT or Mut clones from IP3R mosaic retinas. n ≥ 8 ommatidia from 3 retinas.
Each rows indicate individual ommatidia.
Column labelings indicate genotypes.
See also A. Genotype Abbreviation Key in the last session.
Each values indicate retinal calcium activity per individual ommatidia normalized to average of control groups.
3H. Quantification of retinal wave calcium activity from 3E. n ≥ 10 retinas.
Each rows indicate individual retinas.
Column labelings indicate genotypes. See also A. Genotype Abbreviation Key in the last session.
Each values indicate retinal calcium activity per individual retinas normalized to average of control groups.
Variables include sample identifier, genotype (wild-type or mutant), ΔF/F₀ amplitude (percent), and sample number.
Methods: IP₃R mosaic retinas were generated using the MARCM technique. Calcium imaging and quantification followed the same procedure as above.
File: F4.xlsx
Description: Raw data for main Fig. 4.
4O. Quantification of the frequency of vertical pathway in inx mutant retinas during stage 2.
Each rows indicate genotypes.
Column labelings indicate following wave events
cc>cc nor avg : Normalized average ratio of CC to CC events to control (all event collectively)
cc>cc nor sd : Normalized SD (standard deviation) of CC to CC events to control (all event collectively)
cc>1PC nor avg: Normalized average ratio of CC to 1PC events to control (all event collectively)
cc>1PC nor sd: Normalized SD of CC to 1PC events to control (all event collectively)
1PC>IOC nor avg: Normalized average ratio of 1PC to IOC events to control (all event collectively)
1PC>IOC nor sd: Normalized SD of 1PC to IOC events to control (all event collectively)
1PC>CC nor avg: Normalized average ratio of 1PC to CC events to control (all event collectively)
1PC>CC nor sd: Normalized SD of 1PC to CC events to control (all event collectively)
See also A. Genotype Abbreviation Key, B. Cell type code notation in the last session.
4P. Quantification of the frequency of lateral pathway in inx mutant retinas during stage 2.
Each rows indicate individual retinas.
Column labelings indicate genotypes.
All values are normalized to control average.
See also A. Genotype Abbreviation Key in the last session.
4Q. Quantification of the frequency of spontaneously emerging cell-autonomous activity from each cell type in inx mutant retinas during stage 1. n ≥ 5 retinas.
Each rows indicate genotypes.
Column labelings indicate following wave events
CC firing nor avg : Normalized average frequncy of CC firing to control (all event collectively)
CC firing nor sd: Normalized SD of CC firing to control (all event collectively)
1PC firing nor avg: Normalized average frequncy of 1pc firing to control (all event collectively)
1PC firing nor sd: Normalized SD of 1pc firing to control (all event collectively)
IOC firing nor avg: Normalized average frequncy of ion firing to control (all event collectively)
IOC firing nor sd: Normalized SD of ion firing to control (all event collectively)
See also A. Genotype Abbreviation Key, B. Cell type code notation in the last session.
Variables include genotype (e.g., WT, inx1-sKO), propagation type (vertical or lateral), frequency (events per minute), and number of retinas analyzed.
Methods: Stage 2 retinal waves were recorded using GCaMP6s and analyzed manually in Fiji to classify propagation patterns.
File: F5n6.xlsx
Description: Raw data for main Fig. 5 and 6.
5A-E.
Each rows indicate individual grid points.
Column labelings indicate following.
Bin_ID : Grid point identifier.
4Q (manual) : Retinal quadrant indicator like Fig.1
omm size : Average ommatidial size per grid points for indicated genotype to avg control.
RW : Average**** normalized integrative calcium activity**** per grid points for indicated genotype to avg control.
See also A. Genotype Abbreviation Key in the last session.
5O.
Each rows indicate individual ommatidia
Column labelings indicate following.
36 size : Normalized average ommatidial size per individual ommatidia at 36hAPF. (Normalized to 36 size)
36 Gap: Normalized average interommadidial gap per individual ommatidia at 36hAPF. (Normalized to 36 gap)
46 size: Normalized average ommatidial size per individual ommatidia at 46hAPF. (Normalized to 36 size)
46 Gap: Normalized average interommadidial gap per individual ommatidia at 46hAPF. (Normalized to 36 gap)
5P. Quantification of the gap contraction ratio between 36hAPF and 46hAPF for small (magenta) and large (green) size groups of ommatidia using Dlg1 immunostaining.
Each rows indicate avg interommadidial gap from individual ommatidia
Column labelings indicate following.
Small 36: Normalized average interommadidial gap per individual ommatidia of small size at 36hAPF. (Normalized to small 36)
Large 36: Normalized average interommadidial gap per individual ommatidia of large size at 36hAPF. (Normalized to small 36)
Small 46: Normalized average interommadidial gap per individual ommatidia of small size at 46hAPF. (Normalized to small 36)
Large 46: Normalized average interommadidial gap per individual ommatidia of large size at 46hAPF. (Normalized to small 36)
5Q.
Each rows indicate avg interommadidial gap from individual ommatidia
Column labelings indicate following.
Small : Ratio of average interommadidial gap per individual ommatidia of small size between 36hAPF to 46hAPF for indicated genotype.
Large : Ratio of average interommadidial gap per individual ommatidia of large size between 36hAPF to 46hAPF for indicated genotype.
See also A. Genotype Abbreviation Key in the last session.
6J.
Each rows indicate coefficient of variation of Sqh-GFP within individual interommatidial boundaries.
Column labelings indicate following.
Small 36: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from small ommatidia at 36hAPF to control. (Control is Small 36)
Small 38.5: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from small ommatidia at 38.5hAPF to control. (Control is Small 36)
Small 41: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from small ommatidia at 41hAPF to control. (Control is Small 36)
Small 43.5: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from small ommatidia at 43.5hAPF to control. (Control is Small 36)
Small 46: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from small ommatidia at 46hAPF to control. (Control is Small 36)
Large 36: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from large ommatidia at 36hAPF to control. (Control is large 36)
Large 38.5: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from large ommatidia at 38.5hAPF to control. (Control is large 36)
Large 41: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from large ommatidia at 41hAPF to control. (Control is large 36)
Large 43.5: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from large ommatidia at 43.5hAPF to control. (Control is large 36)
Large 46: Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from large ommatidia at 46hAPF to control. (Control is large 36)
6K.
Each rows indicate coefficient of variation of Sqh-GFP within individual interommatidial boundaries.
Column labelings indicate following.
Small : Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from small ommatidia for indicated genotype at 46hAPF to control. (Control is Small control)
Large : Normalized coefficient of variation of Sqh-GFP within interommatidial boundaries from large ommatidia for indicated genotype at 46hAPF to control. (Control is Small control)
See also A. Genotype Abbreviation Key in the last session.
Variables include ommatidium identifier, normalized size, integrated calcium signal (ΔF/F₀), inter-ommatidial gap distance (normalized to indicated average), developmental stage (36 h or 46 h APF), and ommatidial size group.
Methods: Dlg1 immunostaining was used to visualize ommatidial boundaries. Images were analyzed using Fiji, Python, and Imaris 3D reconstrcution to extract size and gap parameters from apical side.
File: F7.xlsx
Description: Raw data for main Fig. 7.
7M. Quantification of pseudocone size
7N. Variability of pseudocone size
Each row represents one individual pseudocone measured from an adult retina.
Column headers indicate genotype or condition. Definitions are as follows:
Small : Normalized pseudocone size of indicated genotype. (Control is Small control)
Large : Normalized pseudocone size of indicated genotype. (Control is Small control)
Con: Control retina.
sl-sKO: Retina-specific somatic knockout of sl using somatic CRISPR.
inx1-sKO: Retina-specific somatic knockout of inx1 using somatic CRISPR.
Weak R: Weak rescue condition of calcium activity in sl-sKO background using TrpA activation under weak heat-shock.
Strong R: Weak rescue condition of calcium activity in sl-sKO background using TrpA activation under strong heat-shock.
See also A. Genotype Abbreviation Key in the last session.
Variables include retina identifier, genotype, pseudocone size (normalized to the mean value of control group), and sample number.
Methods: Adult eyes were imaged under two photon microscopy. Pseudocone areas were measured in Fiji, Imaris.
File: S1.xlsx
Description: Raw data for main Fig. S1.
S1M, N, O
Each rows indicate individual retina showing developmental stage (hAPF or hours after pupal formation) of the onset of stage 2 retinal waves
Column labelings indicated genotypes and experimental conditions.
S1M
lGMRG4>uG6s: LongGMR-Gal4 driven GCamP6s
GMR-G6s: GMR-Gal4 driven GCamP6s
S1N
G6S: LongGMR-Gal4 driven GCamP6s
G8S: LongGMR-Gal4 driven GCamP8s
JRgeco1a: LongGMR-Gal4 driven JRgeco1a
S1O
488nm: Imaging 488nm confocal laser with LongGMR-Gal4 driven GCamP6s
920nm: Imaging 920nm two-photon laser with LongGMR-Gal4 driven GCamP6s
1knm: Imaging 1000nm two-photon laser with LongGMR-Gal4 driven JRgeco1a
S1P
Each rows indicate individual ommatidial grid points.
Column labelings indicate followings
Each values are initial ommatidial counts per each grid points
8x8 Grid : Grid point identifier.
4Q : Quadrant identifier (same with Fig.1)
1-10 : individual retina identifier
Initial omm : total (1-10 retinas) initial ommatidial counts per each grid points
S1Q
Each rows indicate individual retina.
Column labelings indicate followings
Retina : individual retina identifier
DA, DP, VA, VP : Quadrant label
Column labelings are following.
Va = ventral-anterior,
vp = ventral-posterior,
da = dorsal-anterior,
dp = dorsal-posterior
Each values are probability of initial ommatidial firing per each quadrant.
Variables include retina identifier, genotype or condition, calcium sensor type, imaging time, wave onset time, DV–AP coordinates, probability of activity initiation, and quadrant identity.
Methods: Calcium imaging was performed using longGMR-GAL4 driving UAS-GCaMP6s or other UAS-transgenes as indicated. Activity onset and regional distributions were quantified using Fiji, Imaris and Python scripts.
File: S2.xlsx
Description: Raw data for main Fig. S2.
S2B. Quantification of initial calcium activity probability by CC (cone-cell) orientation
Each rows indicate individual retina.
Column labelings indicate followings
Retina : individual retina identifier
D, V, A, P : CC orientation.
D: Dorsal
V: Ventral
A: Anterior
P: Posterior
Each values are probability of initial firing per each CC.
Variables include ommatidium identifier, cone-cell orientation, and activation probability.
Methods: Ommatidial positions were mapped manually, and propagation paths were manually verified by cross-referencing Fiji processed time-lapse imaging frames.
File: S3.xlsx
Description: Raw data for main Fig. S3.
S3C. Quantification of the level of retinal calcium signal
Each rows indicate individual retina.
Column labelings indicate genotype.
Con : LongGMR-Gal4 driven GCamP6s
PR-G4: Chaoptin-Gal4 driven GCamP6s
Each values are normalized retinal calcium activity during retinal waves to control.
S3H. Quantification of adult eye size.
Each rows indicate individual retina.
Column labelings indicate genotype.
See also A. Genotype Abbreviation Key in the last session.
Each values are normalized adult eye size to control.
S3L. Quantification of the fraction of *cad96Ca-GAL4 *expressing cell-types.
Each rows indicate individual retina.
Column labelings indicate cell types.
See also B. Cell type code notation in the last session.
Each values are fraction of cell types expressing Cad96Ca-Gal4.
Variables include genotype, GAL4 driver type, signal intensity (ΔF/F₀), eye area (normalized to control), and cell-type fraction.
Methods: Calcium imaging and adult eye imaging were performed under the same setup as the main figures. Cell-type identification was confirmed using Cad96Ca-GAL4 expression in Fiji-processed time-lape imaging.
File: S4.xlsx
Description: Raw data for main Fig. S4.
S4G. Quantification of the level of retinal calcium signal
Each rows indicate individual retina.
Column labelings indicate genotype.
Con : Control
PR-G4: ShakB[2] mutants
Each values are normalized retinal calcium activity during retinal waves to control.
S4W, X, Y. Quantification of Inx1 (W), Inx2 (X), and Inx3 (Y) immunostaining signals in inxs-sKO mutant retinas.
Each rows indicate individual retina.
Column labelings indicate genotype.
See also A. Genotype Abbreviation Key in the last session.
Each values are normalized retinal innexin immunostaining signal to control.
S4Z. Quantification of vertical pathway frequency for each step indicated above in inx mutant retinas during late stage 1.
Each rows indicate retinas.
Column labelings indicate following wave events
CC firing: Normalized average frequency of CC firing to control of indicated genotype.
CC>CC: Normalized average frequency of CC>CC propagation to control of indicated genotype.
CC>CC>1PC: Normalized average frequency of CC>CC>1PC propagation to control of indicated genotype.
CC>CC>1PC>IOCs: Normalized average frequency of CC>CC>1PC propagation to control of indicated genotype.
Also see A. Genotype Abbreviation Key, B. Cell type code notation in the last session.
Variables include genotype, innexin type, calcium signal level, normalized fluorescence intensity, pathway frequency, and number of samples.
Methods: Immunostaining and live imaging were used to measure Innexin expression and wave propagation. All signal values were normalized to control means.
File: S5.xlsx
Description: Raw data for main Fig. S5.
S5A, B, C. Ommatidial configuration grid of size distribution in control retinas.
Each rows indicate individual grid points from 8x8 map.
Column labelings indicate following
Bin_ID : Grid identifier
Quadrant : Quadrant identifier from Fig.1
p24 : 24hAPF retina
p36: 36hAPF retina
p46: 46hAPF retina
Each values are normalized average ommatidial size per grid points to control (average of p36)
S5O, P. Quantification of apical contraction of gap-width in 2PCs as related to control size (5O) and 3PC area (5P).
Each rows indicate individual 2PC/3PCs
Column labelings indicate following
X 36 : Apical gap-width (5O) or area (5P) of indicated genotype (X) at 36hAPF. Normalized to control (con 36).
X 46 : Apical gap-width (5O) or area (5P) of indicated genotype (X) at 46hAPF. Normalized to control (con 36).
See also A. Genotype Abbreviation Key in the last session.
Variables include developmental stage, ommatidial size, inter-ommatidial gap width, contraction ratio, and genotype.
Methods: Dlg1 immunostaining was used to define ommatidial boundaries, and apical gap width and contraction were quantified using Fiji and Imaris same with Fig. 5.
A. Genotype Abbreviation Key
In all figures
Con: Control
IP3R MUT: IP3 receptor mutant (Retinal clones)
sl-sKO: Retina-specific somatic knockout of sl using somatic CRISPR.
cad96Ca-sKO: Retina-specific somatic knockout of cad96Ca using somatic CRISPR.
inx1-sKO: Retina-specific somatic knockout of inx1 using somatic CRISPR.
inx2-sKO: Retina-specific somatic knockout of inx2 using somatic CRISPR.
inx3-sKO: Retina-specific somatic knockout of inx3 using somatic CRISPR.
inx1+3-DsKO: Retina-specific somatic knockout of inx3 using somatic CRISPR.
Weak R: Weak rescue condition of calcium activity in sl-sKO background using TrpA activation under weak heat-shock.
Strong R: Weak rescue condition of calcium activity in sl-sKO background using TrpA activation under strong heat-shock.
B. Cell type code notation
In all figures
CC: Cone cell
1PC or 1PC: Primary pigement cell
2PC: Secondary pigement cell
3PC: Tertiary pigement cell
IOC: Interommatidial cell
PR: Photoreceptor neuron
C. Propagation code notation
In F2 and F4:
CC firing: Spontaeous firing from Cone cell, cc-cc : Propagation from Cone cell to Cone cell, cc-cc-1p : Propagation from Cone cell to Cone cell to Primary pigement cell, cc-cc-1p-ioc : Propagation from Cone cell to Cone cell to Primary pigement cell to Interommatidial cell
Code/software
https://doi.org/10.5281/zenodo.16416573.
Quantification of Retinal Wave-Induced Calcium Activity: Calcium imaging data from SP8 confocal microscopy of whole retinas was quantified using a hybrid model integrating event detection and intensity measurement for improved sensitivity and accuracy. Time-lapse images were pre-processed in Fiji to obtain corrected F/F0 values. Photobleaching was corrected by exponential decay fitting in Fiji under consistent exposure settings. ROIs for individual ommatidia were defined in a semi-automatic manner for further analysis in R. A two-component Gaussian mixture model (GMM) was applied to classify each ROI in each frame as “On” or “Off”, with thresholds semi-manually optimized to enhance detection accuracy. Total calcium activity was calculated by summing ΔF/F0 values exclusively during “On” states, reducing background noise and ensuring meaningful activity quantification. This approach improves sensitivity and accuracy by combining event frequency and intensity.
Access information
Other publicly accessible locations of the data:
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Data was derived from the following sources:
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