Allometric growth of the wing pouch in Drosophila.

(A) Schematic depicting relative size of the wing imaginal discs inside a larva starting from 3 days after egg laying (AEL). The wing pouch (blue) begins everting during larva-pupa molt and eventually becomes the adult wing blade. The notum and hinge (red) surrounding the wing pouch becomes the wing hinge and notum of the adult.

(B) Wet-weight of third instar larvae as a function of age measured every 12 hours. Lines connect average weight measurements, and the shaded region denotes the standard error of the mean.

(C) At 5 days AEL, the larva-pupa molt begins. Wet-weight of wildtype animals at early pupariation stages. Lines connect average weight measurements, and the shaded region denotes the standard error of the mean.

(D) Volume of the wing pouch as a function of age. Lines connect average volume measurements and the shaded region denotes the standard error of the mean.

(E) Volume of wing pouch at early pupariation stages. Lines connect average volume measurements and the shaded region denotes the standard error of the mean.

(F) Schematic depicting isometric growth (green arrow) where growth rates of the organ (wing) and the body are the same, positive allometric growth (orange arrow) where the organ is growing faster than the body, and negative allometric growth (magenta arrow) where the organ is growing slower than the body.

(G) Allometric growth relationship of the wing pouch versus body weight. Dashed line depicts the trajectory for an isometric growth curve. Error bars denote standard error of the mean.

Dynamics of Ds and Fat protein distributions across the wing pouch.

(A) Schematic representation of E-cadherin, Fat and Ds protein structures, which are endogenously tagged with GFP at the C-terminus. Adapted from Tanoue and Takeichi, 2005.

(B) Schematic of the wing disc depicting the anterior-posterior (AP, blue) and dorsal-ventral (DV, red) axes of symmetry.

(C) Moving line average of Ds-GFP fluorescence as a function of position along the AP axis. Shown are profiles from wing pouches of different ages, as indicated. Shaded regions for each profile represent the standard error of the mean.

(D) Moving line average of Ds-GFP fluorescence as a function of position along the DV axis. Shown are profiles from wing pouches of different ages, as indicated. Shaded regions for each profile represent the standard error of the mean. In the WPP, the pouch begins everting and only a portion of the ventral compartment is visible.

(E) Moving line average of Ds-GFP fluorescence as a function of position along the AP axis normalized to the total distance of the axis. Shown are profiles from wing pouches of different ages, each normalized independently.

(F) Moving line average of Ds-GFP fluorescence as a function of position along the DV axis normalized to the total distance of the axis. Shown are profiles from wing pouches of different ages, each normalized independently.

(G) Moving line average of Fat-GFP fluorescence as a function of position along the AP axis. Shown are profiles from wing pouches of different ages, as indicated at right. Shaded regions for each profile represent the standard error of the mean.

(H) Moving line average of Fat-GFP fluorescence as a function of position along the DV axis. Shown are profiles from wing pouches of different ages, as indicated at right. Shaded regions for each profile represent the standard error of the mean.

(I) Moving line average of Fat-GFP fluorescence as a function of position along the AP axis normalized to the total distance of the axis. Shown are profiles from wing pouches of different ages, each normalized independently.

(J) Moving line average of Fat-GFP fluorescence as a function of position along the DV axis normalized to the total distance of the axis. Shown are profiles from wing pouches of different ages, each normalized independently.

(K) Schematic of the da-Gal4 driver, active everywhere in the wing disc, expressing UAS-fat-HA and UAS-bazooka-mCherry.

(L) Moving line averages of Fat-HA (red) and Bazooka-mCherry (brown) fluorescence along the normalized AP axis in third instar larval wing pouches. Shaded regions for each profile represent the standard error of the mean.

(M) Moving line average of Fat-GFP fluorescence along the normalized AP axis of wildtype and ds33k/UAO71 mutant wing pouches from 4-day old larvae. Shaded regions for each profile represent the standard error of the mean.

Ds and Fat expression dynamics correlate with wing pouch volume.

(A) Wing pouch area of nub-Gal4 control and nub>trol(RNAi) discs from the WPP stage. Shown are replicates and the mean.

(B) Wing pouch thickness of nub-Gal4 control and nub>trol(RNAi) discs from the WPP stage. Shown are replicates and the mean.

(C) Wing pouch volume of nub-Gal4 control and nub>trol(RNAi) discs from the WPP stage. Shown are replicates and the mean.

(D) Moving line average of Ds-GFP fluorescence as a function of position along the AP axis normalized to the total distance of the axis in nub-Gal4 control and nub>trol(RNAi) WPP wing pouches. Shaded regions for each profile represent the standard error of the mean.

(E) Moving line average of Fat-GFP fluorescence as a function of position along the AP axis normalized to the total distance of the axis in nub-Gal4 control and nub>trol(RNAi) WPP wing pouches. Shaded regions for each profile represent the standard error of the mean.

(F) Confocal image of DAPI-stained nuclei in an en>RBF wing pouch. Note the lower density of nuclei in the P compartment (to the right of the dashed red line). This is due to the enlarged size of cells in this compartment. Scale bar is 30 micrometers.

(G) The area ratio of P compartment to A compartment in en-Gal4 control and en>RBF wing pouches from WPP animals. Shown are replicates and the mean.

(H) Moving line average of Ds-GFP fluorescence as a function of position along the AP axis normalized to the total distance of the axis in en-Gal4 control and en>RBF WPP wing pouches. Shaded regions for each profile represent the standard error of the mean.

(I) Moving line average of Fat-GFP fluorescence as a function of position along the AP axis normalized to the total distance of the axis in en-Gal4 control and en>RBF WPP wing pouches. Shaded regions for each profile represent the standard error of the mean.

Allometric growth of the wing pouch is altered in ds and fat mutants.

(A) Wet-weight of wildtype, ds33k/UAO71, and fatG-rv/8 mutant third instar larvae as a function of age. Shaded regions represent standard error of the mean in this and the other panels.

(B) Wet-weight of wildtype and mutant animals during early pupariation.

(C) Volume of wildtype and mutant wing pouches as a function of larval age.

(D) Volume of wildtype and mutant wing pouches during early pupariation.

(E) Allometric growth relationship of the wing pouch versus body weight in wildtype and mutants.

Autonomous requirements for Fat and Ds in wing pouch growth.

(A) Schematic of the nub-Gal4 driver inducing RNAi of fat or ds in the wing pouch by shRNA expression.

(B) Wet-weight of nub-Gal4 control, nub>ds(RNAi), nub>fat(RNAi), and nub>ds fat(RNAi) third instar larvae as a function of age. Shaded regions represent standard error of the mean.

(C) Wet-weight of control and RNAi-treated animals during early pupariation. Shaded regions represent standard error of the mean.

(D) Volume of control and RNAi-treated wing pouches as a function of larval age. Shaded regions represent standard error of the mean.

(E) Volume of control and RNAi-treated wing pouches during early pupariation. Shaded regions represent standard error of the mean.

(F) Allometric growth relationship of the wing pouch versus body weight in control and RNAi-treated animals.

(G) Schematic of the ap-Gal4 driver inducing RNAi of ds in the D compartment by shRNA expression.

(H) Ratio of D compartment area to V compartment area in ap-Gal4 control and ap>ds(RNAi) wing pouches from WPP animals. Shown are replicate measurements and their mean.

(I) Wing pouch area of ap-Gal4 control and ap>ds(RNAi) WPP animals. Shown are replicate measurements and their mean. The area is normalized to the average of ap-Gal4 controls.

(J) Cell number in the D compartment of the wing pouch of ap-Gal4 control and ap>ds(RNAi) WPP animals. Shown are replicate measurements and their mean. The cell number is normalized to the average of ap-Gal4 controls.

(K) Average cell size (apical area) in the D compartment of the wing pouch of ap-Gal4 control and ap>ds(RNAi) WPP animals. Shown are replicate measurements and their mean. The cell size is normalized to the average of ap-Gal4 controls.

(L) Schematic of the en-Gal4 driver inducing RNAi of ds and fat in the P compartment by shRNA expression.

(M) Ratio of P compartment area to A compartment area in en-Gal4 control, en>fat(RNAi), en>ds(RNAi), and en>fat ds(RNAi) wing pouches from WPP animals. Shown are replicate measurements and their mean.

(N) Wing pouch area of en-Gal4 control and RNAi knockdown WPP animals. Shown are replicate measurements and their mean. The area is normalized to the average of en-Gal4 controls.

Samples that were significantly different are marked with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Scaling of cell cycle duration with wing size is regulated by Ds and Fat.

The average cell cycle time for wing pouch cells is plotted against wing pouch volume for wing discs sampled over 1.5 days of larval growth. Solid lines show the linear regression model, and dotted lines show the 95% confidence intervals of the fit.

(A) nub-gal4 control.

(B) nub>ds(RNAi) and nub-gal4 control.

(C) nub>fat(RNAi) and nub-gal4 control.

(D) nub>ds fat(RNAi) and nub-gal4 control.

(E) nub>ds(RNAi) and nub>ds fat(RNAi).

The endogenous graded distribution of Fat and Ds is not essential for controlling growth rate of the wing pouch.>

(A) nub-Gal4 driving expression of UAS-GFP-NLS along the AP axis of symmetry in the wing pouch. This demonstrates the graded expression of genes transcribed by nub-Gal4. Shown are moving line averages for larval and pupal wing pouches. Shaded regions represent standard error of the mean.

(B) Schematic of the nub-Gal4 driver expressing ds under the UAS promoter.

(C) Moving line average of Ds protein stained with anti-Ds as a function of position along the AP axis and normalized to the total distance of the axis. Shown are staged larval wing pouches from nub>ds animals. This measurement also detects expression from the endogenous ds gene. Shaded regions for each profile represent the standard error of the mean.

(D) Moving line average of Ds protein stained with anti-Ds as a function of position along the DV axis and normalized to the total distance of the axis. Shown are staged larval wing pouches from nub>ds animals. Shaded regions for each profile represent the standard error of the mean.

(E) Moving line average of Fat-GFP fluorescence as a function of position along the AP axis and normalized to the total distance of the axis. Shown are staged larval and pupal wing pouches from nub>ds animals. Shaded regions for each profile represent the standard error of the mean.

(F) Schematic of the nub-Gal4 driver expressing fat-HA under the UAS promoter.

(G) Moving line average of transgenic Fat-HA protein stained with anti-HA as a function of position along the AP axis and normalized to the total distance of the axis. Shown are staged larval and pupal wing pouches from nub>fat-HA; fat-GFP animals. This measurement does not detect expression from the endogenous fat-GFP gene. Shaded regions for each profile represent the standard error of the mean.

(H) Moving line average of endogenous Fat-GFP fluorescence as a function of position along the AP axis and normalized to the total distance of the axis. Shown are staged larval and pupal wing pouches from nub>fat-HA; fat-GFP animals. Shaded regions for each profile represent the standard error of the mean.

(I) The average cell cycle time for wing pouch cells plotted against wing pouch volume for wing discs from nub-Gal4 control animals and nub>fat-HA; fat-GFP animals. Solid lines show the linear regression model, and dotted lines show the 95% confidence intervals for the fit. There is no significant difference between the slopes (p = 0.65).

Summary statistics of linear regressions

Measured volume of individuals correlates with measured weight throughout the third instar larval and WPP stages.

Since the weight of 1 µL water is 1 mg, the wet weight predicted by volume measurements is close to the measured weight. The strong correlation is independent of age and genotype of measured individuals. Genotypes listed are described later in the Results.

Validation of method in defining the wing pouch boundary.

(A) Confocal image of E-cadherin-GFP in a wing disc, which displays folds in the epithelium as morphological landmarks that define the wing pouch. In magenta is the marked wing pouch boundary.

(B) Confocal image of wing disc expression of the vestigial gene reporter 5x-QE-dsRed, which is specifically expressed in the wing pouch. In magenta is the marked wing pouch boundary.

(C) Comparison of wing pouch area measured in third instar larval wing discs using the two methods. Dotted line shows outcome if both methods were in perfect agreement (slope = 1.0000). Linear regression of the measurement data shows the two methods are in strong agreement (slope = 0.9554).

Allometric growth of the notum-hinge in Drosophila.

(A) Area of the larval notum-hinge as a function of age. Lines connect average area measurements, and the shaded region denotes the standard error of the mean.

(B) Area of the notum-hinge at early pupariation stages. Lines connect average area measurements, and the shaded region denotes the standard error of the mean.

(C) Allometric growth relationship of the notum-hinge versus body weight. Dashed line depicts the trajectory for an isometric growth curve. Error bars denote standard error of the mean.

Pattern of cell proliferation in the wing pouch as it ages.

(A) Confocal image of a 4-day old larval wing pouch stained with anti-En and anti-Wg on the left and anti-PHH3 on the right. Wg and En label the AP and DV axes, respectively. The pouch boundary is highlighted with a magenta dashed line while the AP axis is illustrated with cyan dashed line. Scale bar is 50 micrometers.

(B) Centroid positions of PHH3-positive nuclei in the 4-day old larval wing pouch are plotted to show the distribution of cell divisions in the wing pouch. Three wing replicates are shown in different colors. Magenta dashed line illustrates a typical wing pouch.

(C) Confocal image of a WPP wing pouch stained with anti-En and anti-Wg on the left and anti-PHH3 on the right. The pouch boundary is highlighted with a magenta dashed line while the AP axis is illustrated with cyan dashed line. Scale bar is 50 micrometers.

(D) Centroid positions of PHH3-positive nuclei in the WPP wing pouch are plotted to show the distribution of cell divisions in the wing pouch. Three wing replicates are shown in different colors. Magenta dashed line illustrates a typical wing pouch. Note the concentration of dividing cells along the AP axis of symmetry, where sensory organ precursor cells are dividing.

(E) Confocal images of a wing pouch from a WPP stage animal stained for anti-PHH3 (left), sfGFP-Senseless (Sens) (center), and the merge (right). Sens is expressed in cells fated to become sensory organ precursors, each of which undergo two rounds of cell division to form a sensory bristle. At the WPP stage, the only cells actively dividing are those with high Sens expression. Scale bar is 50 micrometers.

smFISH images of fat and ds expression in the wing pouch.

(A) Confocal images of a ds-GFP larval wing pouch stained for GFP mRNAs (left) and DAPI (right). Scale bar is 50 micrometers.

(B) Confocal images of a fat-GFP larval wing pouch stained for GFP mRNAs (left) and DAPI (right). Scale bar is 50 micrometers.

Methods to measure distributions of Ds-GFP and Fat-GFP.

(A) Confocal images of a ds-GFP larval wing pouch showing GFP fluorescence (left) and staining of En and Wg proteins (right). Blue and red lines are the AP and DV axes of symmetry, respectively. Scale bar is 50 micrometers.

(B) Schematic of the wing disc depicting the AP (blue) and DV (red) axes of symmetry.

(C) Representative Ds-GFP fluorescence image in which fluorescence from cells in the disc proper has been computationally segregated from signal from the peripodial membrane. Left is a max projection of all sections. Middle is the surface projected signal from the disc proper. Right is the projected signal from the peripodial membrane.

(D) Confocal images of a fat-GFP larval wing pouch showing GFP fluorescence (left) and staining of En and Wg proteins (right). Blue and red lines are the AP and DV axes of symmetry, respectively. Scale bar is 50 micrometers.

Fat expression in ds and fj mutants.

(A) Moving line average of Fat-GFP fluorescence along the normalized AP axis. Shown are profiles from different ages (as indicated) of ds33k/UAO71 mutants. Shaded regions for each profile represent the standard error of the mean.

(B) Moving line average of Fat-GFP fluorescence along the normalized AP axis of ds33k/UAO71mutant wing pouches from WPP and BPP animals. Shaded regions for each profile represent the standard error of the mean.

(C) Moving line average of Fat-GFP fluorescence along the normalized AP axis of fj d1/p1 mutant wing pouches from 4-day old larvae. Shaded regions for each profile represent the standard error of the mean.

Allometric growth of the notum-hinge is altered in ds and fat mutants.

(A) Area of the wildtype, ds33k/UAO71, and fatG-rv/8 mutant notum-hinge as a function of larval age. Shaded regions represent standard error of the mean in this and the other panels.

(B) Area of the wildtype and mutant notum-hinge during early pupariation.

(C) Allometric growth relationship of the notum-hinge versus body weight in wildtype and mutants.

RNAi effectively knocks down fat and ds expression.

All panels show confocal images of third instar larval wing discs. Scale bars are 50 micrometers.

(A) Endogenously expressed Ds-GFP.

(B) Anti-Ds staining of nub>ds(RNAi) disc. Ds expression in the peripodial membrane and notum remains unchanged.

(C) Endogenously expressed Fat-GFP.

(D) Fat-GFP expression in a nub>fat(RNAi) disc. Fat expression in the peripodial membrane and notum remains unchanged.

(E) Ds-GFP expression in an ap>ds(RNAi) disc (left). Ds expression in the ventral compartment remains unchanged. E-cadherin-mCherry is used for cell segmentation (right). The compartment boundary is shown in yellow.

(F) Ds-GFP expression in an en>ds(RNAi) disc. Ds expression in the anterior compartment remains unchanged. The compartment boundary is shown in yellow.

(G) Fat-GFP expression in an en>fat(RNAi) disc. Fat expression in the anterior compartment remains unchanged. The compartment boundary is shown in yellow.

Growth of the notum-hinge when fat and ds are knocked down in the wing pouch.

(A) Notum-hinge area of nub-Gal4 control, nub>ds(RNAi), nub>fat(RNAi), and nub>ds fat(RNAi) as a function of larval age. Shaded regions represent standard error of the mean in this and the other panels.

(B) Notum-hinge area of control and RNAi-treated animals during early pupariation.

(C) Allometric growth relationship of the notum-hinge versus body weight in control and RNAi-treated animals.

Measurement of length of M phase in wing pouch cells.

Third instar wing discs expressing E-cadherin-GFP were dissected and cultured ex vivo as described by Gallagher et al. (2022). Each disc was successively imaged by microscopy over a 2-hr period, with an image frame time interval of 5 min.

(A) A cell undergoing mitosis starting at t = 5 minutes. Cytokinesis is completed at t = 25 minutes. The beginning of mitosis is marked by the first detectable increase in a cell’s apical area. The cell expands and becomes circular, after which mitosis completes and the cell contracts in area as it divides. The end of cytokinesis is marked by the last detectable decrease in daughter cell apical area.

(B) Histogram of M phase times for 33 wing pouch cells. The mean time is 20.5 min.

Summary statistics of linear regression analysis.

Reversal of the Ds expression gradient does not affect growth.

(A) Confocal images of a nub>ds; fat-GFP third instar larval wing disc showing Fat-GFP fluorescence (left), anti-Ds fluorescence (center), and anti-Wg / anti-En fluorescence (right). The AP (blue) and DV (red) axes are shown. Scale bar is 50 micrometers.

(B) Wet-weight of nub-Gal4 control and nub>ds third instar larvae as a function of age. Shaded regions represent standard error of the mean.

(C) Wet-weight of nub-Gal4 control and nub>ds animals during early pupariation. Shaded regions represent standard error of the mean.

(D) Volume of nub-Gal4 control and nub>ds larval wing pouches as a function of age. Shaded regions represent standard error of the mean.

(E) Volume of nub-Gal4 control and nub>ds wing pouches during early pupariation. Shaded regions represent standard error of the mean.

Disruption of Fat gradient dynamics has no detectable effect on wing pouch growth.

(A) Confocal images of a nub>fat-HA; fat-GFP third instar larval wing disc showing Fat-GFP fluorescence (left), anti-HA fluorescence (center), and anti-Wg / anti-En fluorescence (right). The AP (blue) and DV (red) axes are shown. Scale bar is 50 micrometers.

(B) Volume of WPP wing pouches from nub-Gal4 control and nub>fat-HA; fat-GFP animals. There is no significant difference between the two groups as determined by a t-test.