DOI: 10.5061/dryad.0k6djhb8d
title: Scaling between cell cycle duration and wing growth is regulated by Fat-Dachsous signaling in Drosophila

description: The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo
 pathway to regulate growth of numerous organs, including
 the Drosophila wing. Here, we find that Ds-Fat signaling
 tunes a unique feature of cell proliferation found to control the rate of
 wing growth. The duration of the cell cycle increases in direct proportion
 to the size of the wing, leading to linear rather than exponential growth.
 Ds-Fat signaling enhances the rate at which the cell cycle lengthens with
 wing size, thus diminishing the linear rate of wing growth. We show that
 this results in a complex but stereotyped relative scaling of wing growth
 with body growth in Drosophila. Finally, we examine the dynamics
 of Fat and Ds protein distribution in the wing, observing graded
 distributions that change during growth. However, the significance of
 these dynamics is unclear since perturbations in expression have
 negligible impact on wing growth.
description: Data accompanying a published eLife paper titled "Scaling
 between cell cycle duration and wing growth is regulated by Fat-Dachsous
 signaling in Drosophila."  
 Imaging data were acquired on confocal micrscope and processed.
 Please refer to the Methods section of the accompanying eLife paper for
 detailed methodology of how images were collected. How the data was
 processed are descripted below. Code is available at
 https://github.com/andrewliu321/LarvaSeg and
 https://github.com/andrewliu321/ProteinIntensity.
   <strong>Image
 processing</strong>  
 Raw images were processed using a custom-built Matlab pipeline
 with no prior preprocessing. The pipeline consists of several modules: 1)
 surface detection, 2) wing disc, pouch, and midline segmentation, 3)
 volume measurement, 4) fluorescence intensity measurement, 5) cell
 segmentation, 6) mitotic index measurement.
   <em>Surface
 detection</em>  
 Fat-GFP and Ds-GFP proteins localize to the apical region of
 cells in the wing disc proper. These proteins are also localized in cells
 of the peripodial membrane, which is positioned near the apical surface of
 the disc proper. In order to get a 2D projection of the signal in the wing
 disc proper excluding the peripodial membrane signal, we used an
 open-source software package called ImSAnE – Image Surface Analysis
 Environment. The detailed parameters we used have been previously
 described. Briefly, we used the MIPDetector module to find the brightest
 z-position of every xy pixel followed by tpsFitter to fit a single layer
 surface through these identified z-positions. Using the onionOpts function
 in ImSAnE, we output a 9-layer z-stack, 4 layers above and below the
 computed surface that capture the entire signal from the wing disc proper.
 However, this operation still sometimes includes fluorescence signals from
 the peripodial membrane. Therefore, we manually masked the residual
 peripodial signal using FIJI 1.53t. The resulting z-stack was
 sum-projected to form a 2D surface projection of the wing disc
 proper.   <em>Wing
 disc, pouch, and midline segmentation</em>
   Wing discs were counterstained for
 both Wg and En proteins, which mark the wing pouch dorsal-ventral (DV)
 midline and anterior-posterior (AP) midline, respectively. Although both
 Wg and En proteins were stained with the same Alexa 546 fluorescent
 antibody, the two signals were readily distinguished by their distinct
 separation in z space. Wg is apically localized in cells of the disc
 proper and En is nuclear localized more basally in the disc proper.
 Moreover, the Wg signal was far stronger than En, allowing for detection
 of its stripe in the posterior compartment even with a max
 projection.   We built a
 semi-automated Matlab script that computationally segments the wing disc
 into discrete objects. i) <em>Wing disc segmentation</em>.
 Endogenous Fat-GFP or Ds-GFP signal was used to segment the wing disc from
 surrounding media. ii) <em>Wing pouch segmentation</em>. The
 3D morphology of the wing disc creates narrow and deep tissue folds that
 surround the wing pouch. Using these morphological landmarks that were
 visualized by the Fat-GFP or Ds-GFP signals, the Matlab script recorded
 user-derived mouse-clicks that defined the wing pouch boundary. This
 method was validated to be 95.5% accurate when compared to a wing pouch
 boundary that was defined by the expression boundary of a reporter for the
 vestigial quadrant enhancer, 5x-QE-DsRed. iii) <em>DV midline
 segmentation</em>. The Wg signal was used to segment the DV midline
 running through the segmented wing pouch. Since the Wg and En signals are
 distinguishable in z space, the upper third of the z-stack was max
 projected to segment Wg. An adaptive threshold of 0.6 was used to binarize
 the image into Wg-positive pixels. The binarized and raw images were used
 to inform manual input of the DV midline. iv) <em>AP midine
 segmentation</em>. The En signal was used to segment the AP midline
 running through the segmented wing pouch. The lower two-thirds of the
 z-stack was max projected and binarized in En+ pixels. The binarized and
 raw images were used to inform manual input of the AP midline.
   <em>Volume
 measurement</em>  
 Areas of each of the segmented objects were calculated by
 summing the number of pixels in each object and multiplying by the pixel
 dimensions in xy physical space. Notum-hinge area was calculated by
 subtracting the segmented pouch area from total segmented wing disc area.
 The thickness of the wing pouch was measured at the intersection of the AP
 and DV midlines using the orthogonal views tool in FIJI. The first layer
 is defined by the initial signal of Fat-GFP or Ds-GFP at this xy position.
 The last layer is defined by the first appearance of background signal in
 the composite image. Thickness of the object was calculated by multiplying
 the sum of z-slices by the z-separation. To calculate the volume of
 segmented objects, we multiplied the thickness of the object (in µm) by
 the object’s surface area (in µm<sup>2</sup>). Conversion from
 µm<sup>3</sup> to nL units was performed.
   <em>Fluorescence intensity
 measurement of Fat-GFP and Ds-GFP</em>
   Fluorescence intensity values were
 averaged across a vector of 50 pixels length that was orthogonal to the
 segmented boundary of interest and having 25 pixels residing on each side
 of the segmented line. These values were then averaged in a sliding window
 of 100 pixels length that moved along the segmented boundary of interest.
 Physical distance along the boundaries were measured using ImSAnE function
 Proper_Dist to account for the curvature of the segmented objects. The
 intersection of the segmented DV and AP midlines was defined as the center
 (0,0 µm) of the wing pouch, with the anterior/dorsal annotated in units of
 negative µm and the posterior/ventral annotated in units of positive µm. A
 minimum of three wing discs of the same age and genotype were aligned by
 their (0,0) centers and their fluorescent measurements were averaged along
 the AP and DV midlines.  
 Older third instar, WPP, and BPP wing discs begin to evert such
 that the ventral compartment is partially folded underneath the dorsal
 compartment. However, the ventral compartment can still be accurately
 segmented to measure area and thickness even under these conditions. For
 GFP measurements, the folded specimen resulted in dimmer fluorescent
 signals from the compartment farthest from the objective due to tissue
 thickness and light scattering. Thus, measurements were limited to the
 compartment closest to the objective.  
 <em>Cell boundary segmentation</em>
   To count cell numbers and cell sizes
 in the wing pouch, we analyzed wing discs imaged from
 <em>E-cadherin</em>-<em>GFP</em> or
 <em>E-cadherin-mCherry</em> larvae. We used a machine learning
 pixel-classification model based on a convolutional neural net to segment
 cell boundaries in the surface projections. This model was trained on a
 broad range of image data derived from Cadherin-GFP labeled<em>
 Drosophila</em> imaginal discs. The model is &gt; 99.5% accurate
 at segmenting cells when compared to ground truth. Cell size (surface
 area) and number were computed for specific compartments in the wing
 pouch.   <em>Mitotic
 index measurement</em>  
 Phospho-histone H3 (PHH3) has been used to estimate mitotic
 index previously. Wing discs were immunostained for PHH3, which labels
 nuclei undergoing mitosis. These nuclei were manually recorded by
 user-defined mouse clicks at or near the center of each nucleus. Their
 Euclidean distances relative to the segmented AP and DV midlines were
 calculated as was the number of PHH3+ cells. To estimate the total cell
 number in a wing pouch, we used E-cadherin to computationally segment
 cells as described above. Each imaged wing pouch had a subset of cell
 boundaries segmented in a subdomain of the pouch. This was then used to
 calculate cell density: number of segmented cells divided by subdomain
 area. The density value was multiplied by total wing pouch area to
 estimate the total number of wing pouch cells for that sample. We then
 derived an averaged conversion factor to apply to each volume measurement
 in order to estimate total cell number. This was done by plotting the
 estimated total cell number versus wing pouch volume for all discs of a
 given genotype. Linear regression of the data produced an equation to
 convert pouch volume to cell number.
description: # Scaling between cell cycle duration and wing growth is regulated by
 Fat-Dachsous signaling in Drosophila
 [https://elifesciences.org/reviewed-preprints/91572](https://elifesciences.org/reviewed-preprints/91572) ## Summary of the data and file structure Excel files with fluorescent intensity measured along midlines of wing discs. File name represent genotypes while individual tabs within each excel file specify whether the DV or AP midline was measured. Pay attention to the title row for further specification of age or genotype.  Excel file titled Animal Properties include various measurements of larvae sizes as well as wing disc measurements, including area, mitotic cells, and cell cycle times. ## Detailed description of each dataset **Actin_trol_RNAi.xlsx -** all data supplementing the accompanied paper Fig. 3A-E. WPP staged animals ubiquitously express RNAi against *trol* under the control of Actin5c promoter using the Gal4/UAS system. The "Dimensions" sheet: * *Genotype*: actin5c gal4 denotes the control group, actin5c&gt;trol RNAi denotes the experimental group * *Pouch Area*: the wing pouch area of each individual sample with units of 10^3 um^2. * *Pouch Thickness*: the wing pouch thickness of each individual sample with units in um.   * *Pouch Volume:* the wing pouch total volume (area multiplied by thickness) of each individual sample with units of 10^3 um^3. The "Fat GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'actin5c Gal4/ actin5c&gt;trol RNAi'*: Each column denotes one sample of the respective genotype, with Fat-GFP protein intensity measured at each relative distance.  The "Ds GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'actin5c Gal4/ actin5c&gt;trol RNAi'*: Each column denotes one sample of the respective genotype, with Ds-GFP protein intensity measured at each relative distance. **Animal Properties.xlsx -** all data supplementing the accompanied paper Fig. 1, 4, 5, 6. The "Pouch Measurements" sheet: * *Genotype*: genotype of the sample * *Stage*: L3 is the third instar larval stage, WPP is the white prepupae stage, WPP+1 is one hour after WPP stage. * *Age*: hours after larval hatching, only applicable for L3 stage. n/a indicates that L3 samples were not collected at specific times *i.e. ds-Gal4* and *ds-Gal4, uas-ds.* * *Pouch Area*: the wing pouch area of each individual sample with units in um^2. n/a indicates that pouch area was not measured or recorded. * *Pouch Thickness*: the wing pouch thickness of each individual sample with units in um. n/a indicates that pouch thickness was not measured or recorded.  * *Pouch Vol:* the wing pouch total volume (area multiplied by thickness) of each individual sample with units in um^3. * *number PHH3 cells*: raw counts of nuclei stained with PHH3 which marks mitotic cells. This was only applicable to L3 stage. n/a indicates that number of PHH3 positive cells was not measured.  * *cell density*: cell density is measured in a subset of samples in L3 stage, measured as number of cells per um^2. n/a indicates that cell density was not measured.  * *TotalCellFitPerGeno*: Total cell number was calculated using a regression analysis after multiplying pouch area and cell density. This was done for each genotype. This was only applicable to L3 stage. n/a indicates that the regression analysis was not performed. * *MitoticIndex*: number of mitotic cells divided by total cells for each sample. This was only applicable to L3 stage. n/a indicates that mitotic index was not calculated. * *Cell Cycle Time*: Duration of cell cycle is calculated by duration of M phase / MitoticIndex. The duration of mitosis is measured to be 0.34 hr. This was only applicable to L3 stage. n/a indicates that cell cycle time was not calculated. The "Animal Dimensions" sheet: * *Genotype*: genotype of the sample * *Stage*: L3 is the third instar larval stage, WPP is the white prepupae stage, WPP+1 is one hour after WPP stage. * *Age*: hours after larval hatching, only applicable for L3 stage. Some L3 samples were not collected at specific times *i.e. ds-Gal4* and *ds-Gal4, uas-ds.* * *LarvaLength*: length of the larva for each sample, measured in pixels. 125 pixels is the same as 1 mm. * *LarvaWidth*: width of the larva for each sample, measured in pixels. 125 pixels is the same as 1 mm. * *LarvaVol*: volume of the larva for each sample is calculated by approximating a larva as a cylinder. * *LarvaArea*: area of the larva for each sample.  The "Animal Weight" sheet: * *Genotype*: genotype of the sample * *Stage*: L3 is the third instar larval stage, WPP is the white prepupae stage, WPP+1 is one hour after WPP stage. * *Age*: hours after larval hatching, only applicable for L3 stage. * *Larva Weight*: weight of the larva for each sample, measured in mg. The "Total Wing Disc Area" sheet: * *Genotype*: genotype of the sample * *Stage*: L3 is the third instar larval stage, WPP is the white prepupae stage, WPP+1 is one hour after WPP stage. * *Age*: hours after larval hatching, only applicable for L3 stage. * *Wing Disc Area*: total area of the entire wing disc, measured in um^2. * *Wing Pouch Area*: total area of the wing pouch, measured in um^2. Note: wing pouch and wing disc areas on the same row are not from the same animal/sample. For example, for one genotype at one time point, 10 wing discs were measured but only 5 wing pouches were measured. Therefore, n/a indicates no sample was measured. Furthermore, notum area can be calculated by subtracting the average wing pouch area from the average wing disc area. The "En&gt;RNAi Pouch Measurement" sheet: * *Relative Pouch Area To En Gal4 Controls*: wing pouch area of each individual sample normalized to the average of En Gal4 control samples. The genotype is listed on the 2nd row. 'En Gal4' is the control. 'En&gt;fat RNAi' is *fat* knockdown specifically in the posterior compartment (En expression region).  'En&gt;ds RNAi' is *ds* knockdown specifically in the posterior compartment. 'En&gt;fat, ds RNAi' is both *fat* and *ds* knockdown specifically in the posterior compartment. n/a indicates no sample was measured. * *Posterior/Anterior Compartment Area Ratio:* posterior compartment area divided by anterior compartment area of each individual sample. The genotype is listed on the 2nd row. This measures how much overgrowth occurred in the posterior compartment relative to the wildtype anterior compartment. n/a indicates no sample was measured. The "Ap&gt; ds RNAi" sheet: * *Genotype*: Ap Gal4 denotes the control group, Ap&gt;ds RNAi denotes the experimental group, where the dorsal compartment driver, Ap Gal4 is expressing *ds* RNAi. * *Relative Cell Number*: Number of cells in the dorsal compartment normalized by the average in the control group. Each column is a separate sample. * *Relative Cell Area*: Average area of cells in the dorsal compartment normalized by the average in the control group. Each column is a separate sample. n/a indicate no sample was measured. * *Dorsal/Ventral Area Ratio*: dorsal compartment area divided by ventral compartment area of each individual sample. This measures how much overgrowth occurred in the dorsal compartment relative to the wildtype ventral compartment. n/a indicate no sample was measured. * *Relative Pouch Area*: wing pouch area of each individual sample normalized to the average of control samples. n/a indicate no sample was measured. **Da_Fat_HA_and Baz_mCherry.xlsx -** all data supplementing the accompanied paper Fig. 2L. WPP staged animals ubiquitously express Fat-HA and Bazooka-mCherry under the control of Daughterless(Da) promoter using the Gal4/UAS system. The "AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'Baz mCherry WPP/ Fat HA WPP'*: Each column denotes the intensity of either Bazooka-mCherry or Fat-HA measured at each relative distance for one sample.   **Ds_Gal4_Uas_Ds_Fat_GFP.xlsx -** all data supplementing the accompanied paper Fig. 7D-F . Time-staged animals overexpressing Ds under the control of Ds promoter using the Gal4/UAS system. Endogenously tagged Fat-GFP is also measured. The "4.5 Day Anti Ds" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. All animals are 4.5 days old after larvae hatching.  * *'Ds Gal4/ Ds Gal4, UAS ds'*: each column denotes the intensity of anti-ds antibody from either the control Ds Gal4 sample or Ds overexpression sample.   The "Ds Gal4, Fat GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1.  * *'4.5 Days/ 5 Days/ WPP'*: each column denotes the age of the control, Ds Gal4 sample, with endogenous Fat-GFP protein intensity measured at each relative distance. The "Ds&gt;ds, Fat GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1.  * *'4.5 Days/ 5 Days/ WPP'*: each column denotes the age of the Ds overexpression sample, with endogenous Fat-GFP protein intensity measured at each relative distance. **En_RBF.xlsx -** all data supplementing the accompanied paper Fig. 3F-I. WPP staged animals express RBF the control of En promoter using the Gal4/UAS system. The "Ratio" sheet: * *Genotype*: en gal4 denotes the control group, en&gt;RBF denotes the experimental group * *Posterior/Anterior Ratio*: posterior compartment area divided by anterior compartment area to measure change in posterior compartment caused by RBF overexpression relative to the wildtype anterior compartment. The "Fat GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'en Gal4/ en&gt;RBF'*: Each column denotes one sample of the respective genotype, with Fat-GFP protein intensity measured at each relative distance.  The "Ds GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'en Gal4/ en&gt;RBF'*: Each column denotes one sample of the respective genotype, with Ds-GFP protein intensity measured at each relative distance. **Mutant_background_Fat-GFP.xlsx -** all data supplementing the accompanied paper Fig. 2M and Fig 2 Supplement 3. Time-staged animals expressing endogenously tagged Fat-GFP in mutant backgrounds. The "ds null AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *''4 Days/ 4.5 Days/ 5 Days/ WPP/ WPP+1'*: Each column denotes one sample of the respective age of the ds null animals, with Fat-GFP protein intensity measured at each relative distance.  The "fj null AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days'*: Each column denotes one sample of the respective age of the fj null animals, with Fat-GFP protein intensity measured at each relative distance. **Nub_Ds.xlsx -** all data supplementing the accompanied paper Fig. 7B-C and Fig 7 Supplement 1B. Time-staged animals expressing endogenously tagged Fat-GFP, anti-Ds, or GFP under the expression of the nubbin promoter. The "nub&gt;GFP NLS AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *''L3/ WPP/ WPP+1'*: Each column denotes one sample of the respective stage, with GFP protein intensity measured at each relative distance. Animals are expressing GFP-NLS under the control of the nubbin promoter using the Gal4/UAS system. Nubbin is expressed in the wing pouch.  The "anti Ds AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ wpp'*: Each column denotes one sample of the respective age of animals expressing Ds under the nubbin promoter, with anti-Ds protein intensity measured at each relative distance. The "anti Ds DV Normalized" sheet: * *Normalized Distance*: Distance measured along the DV midline within the wing pouch (Engrailed staining). The most dorsal is marked at -1, the center is at 0, while the most ventral is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ wpp'*: Each column denotes one sample of the respective age of animals expressing Ds under the nubbin promoter, with anti-Ds protein intensity measured at each relative distance. The "Fat GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ wpp'*: Each column denotes one sample of the respective stage of animals expressing Ds under the nubbin promoter, with endogenously tagged Fat-GFP protein intensity measured at each relative distance. **Nub_Fat_HA.xlsx -** all data supplementing the accompanied paper Fig. 7H-I. Staged animals express Fat-HA under the control of Nub promoter using the Gal4/UAS system as well as endogenously tagged Fat-GFP. The "Pouch Vol" sheet: * *Genotype*: Nub gal4 denotes the control group, nub&gt; Fat HA denotes the experimental group * *Pouch Vol at WPP (nL)*: wing pouch volume measured at wpp stage in nL. The "Fat HA AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ wpp/ wpp+1'*: Each column denotes one sample of the respective genotype, with Fat-HA protein intensity measured at each relative distance.  The "Fat GFP AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ wpp/ wpp+1'*: Each column denotes one sample of the respective genotype, with Fat-GFP protein intensity measured at each relative distance.  **WT_Ds-GFP.xlsx -** all data supplementing the accompanied paper Fig. 2C-F. Time-staged animals expressing endogenously tagged Ds-GFP. The "AP Axis" sheet: * Distance (um)*: Distance measured in um along the AP midline within the wing pouch (Wingless staining). The anterior is marked with negative integers, the center is at 0, while the posterior is marked at positive integers. n/a indicates wings are smaller and not measured at the specific distance. * *''4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Ds-GFP protein intensity measured at each distance. The "DV Axis" sheet: * Distance (um)*: Distance measured in um along the DV midline within the wing pouch (Engrailed staining). The dorsal is marked with negative integers, the center is at 0, while the ventral is marked at positive integers. n/a indicates wings are smaller and not measured at the specific distance. * *''4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Ds-GFP protein intensity measured at each distance. The "AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Ds-GFP protein intensity measured at each relative distance. The "DV Normalized" sheet: * *Normalized Distance*: Distance measured along the DV midline within the wing pouch (Engrailed staining). The most dorsal is marked at -1, the center is at 0, while the most ventral is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Ds-GFP protein intensity measured at each relative distance. **WT_Fat-GFP.xlsx -** all data supplementing the accompanied paper Fig. 2G-J. Time-staged animals expressing endogenously tagged Fat-GFP. The "AP Axis" sheet: * Distance (um)*: Distance measured in um along the AP midline within the wing pouch (Wingless staining). The anterior is marked with negative integers, the center is at 0, while the posterior is marked at positive integers. * *''4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Fat-GFP protein intensity measured at each distance. n/a indicates wings are smaller and not measured at the specific distance. The "DV Axis" sheet: * Distance (um)*: Distance measured in um along the DV midline within the wing pouch (Engrailed staining). The dorsal is marked with negative integers, the center is at 0, while the ventral is marked at positive integers. * *''4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Fat-GFP protein intensity measured at each distance.n/a indicates wings are smaller and not measured at the specific distance. The "AP Normalized" sheet: * *Normalized Distance*: Distance measured along the AP midline within the wing pouch (Wingless staining). The most anterior is marked at -1, the center is at 0, while the most posterior is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Fat-GFP protein intensity measured at each relative distance. The "DV Normalized" sheet: * *Normalized Distance*: Distance measured along the DV midline within the wing pouch (Engrailed staining). The most dorsal is marked at -1, the center is at 0, while the most ventral is marked at +1. * *'4 Days/ 4.5 Days/ 5 Days/ WPP'*: Each column denotes one sample of the respective age, with Fat-GFP protein intensity measured at each relative distance. ## Code/Software MatLab codes and detailed protocols are available at: [https://github.com/andrewliu321/ProteinIntensity](https://github.com/andrewliu321/ProteinIntensity) [https://github.com/andrewliu321/LarvaSeg](https://github.com/andrewliu321/LarvaSeg)