(A) Differentiation is initiated in the eye disc by the MF, which moves across the eye epithelium. As the MF transits the eye, cells on either side proliferate, which generates disc growth. Shown …
(A) A Z-stack of optical sections are collected at each time point. This stack is centered around the adherens junction plane of the eye disc epithelium in the z dimension. (B) Since the adherens …
For panels A to D, all images are oriented with anterior to the left and dorsal is top. For each panel, the time course shown is arbitrarily set to begin at 0 and does not represent the actual time …
(A) Velocities for all cells in the MF at the indicated times. Arrow length is proportional to the magnitude of velocity. Clusters of cells with higher anterior velocity alternate with clusters of …
Position of the MF along the anterior-posterior axis of the field of view for two replicate eye discs. The data strongly fits a linear model with a slope of 1.44 μm/hr for both replicates.
(A) Tracking over time the positions of all presumptive R cells binned by their ommatidia column. Each bin is color coded. The moving line averages follow relative position along the …
Time frames of a wildtype disc captured every 2 hr. A group of seven presumptive R8 cells spanning five neighboring columns are connected to one another by purple lines. All cells are labeled with …
(A-D’’) Analysis of scabrousBP2 eye disc with severe patterning defects. (A) Time point showing presumptive and fated R cells colored - R8 (purple), R2 and R5 (orange), R3 and R4 (cyan). (B) Cell …
(A) Cell division and delamination events as a function of their distance from the MF (vertical dashed line) along the anterior-posterior axis of scabrous mutant discs. Shown are all cell division …
(A) The rate of area change for all cells in the field of view at a randomly chosen time point. A stripe of cells anterior to the MF shows rapid area contraction, while a stripe of cells posterior …
(A) Hypothetical mechanism for how an inferred pressure gradient generates periodic flows in the MF. There are periodic domains of cell dilation (red) in the source of cell flows, causing periodic …
Each unique tracked cell is labeled a specific color. Note that cells gain and lose color when they enter and exit the field of view, which marks the beginning and end, respectively, of those cells’ …
All R cells belonging to the same column are labeled with the same color. Cells shaded gray represent a strip of cells residing within the MF.
A group of seven presumptive R8 cells spanning five neighboring columns are connected to one another by purple lines. All cells are labeled with their respective velocity vectors. Note how the …
R8 (purple), R2 and R5 (orange), R3 and R4 (cyan). Cells shaded gray represent a strip of cells residing within the MF. The disc has a more severe spacing phenotype.
Each cell at each time-frame was fitted with an ellipse, and the dorsal-ventral vs. anterior-posterior span of the ellipse was used to calculate the Shape Index of that cell. The Shape Index was …
Cells retain that color identity throughout the movie no matter where they move. Note that stripes of color within the anterior side of the MF retain their integrity, meaning that there are few T1 …
We performed non-parametric Mann-Whitney U tests since most distributions are not Gaussian. The large sample sizes going into all our measurements resulted in extremely low p-values for many. …
Compared populations | -log10(Mann Whitney p-value) |
---|---|
Figure 3D & E - R8s vs. non-R cell flow in the MF | |
3D - R8 vs. non-R cells in high flow regions of the MF | 1.1467 |
3E - R8 vs. non-R cells in low flow regions of the MF | 24.8128 |
Figure 3F - velocity distributions of R8s to non-R cells | |
Dark blue (far posterior) | 0.5838 |
Orange (PTZ) | 1.6628 |
Yellow (posterior side MF) | 8.9340 |
Purple (anterior side MF) | 15.5146 |
Figure 4D–D’’ velocity distributions of strong sca vs. WT | |
4D top - R8s vs. non-R cells | 72.4072 |
4D bottom - R8s vs non-R cells | 23.9553 |
4D’ top - Strong sca vs. WT | Infinity* |
4D’ bottom - Strong sca vs. WT | Infinity* |
4D’’ top - Strong sca vs. WT | 68.3809 |
4D’’ bottom - Strong sca vs. WT | 22.5819 |
Figure 4H–H’’ velocity distributions of weak sca vs. WT | |
4 H top - R8s vs. non-R cells | 12.7047 |
4 H bottom - R8s vs non-R cells | 20.9813 |
4 H’ top - Weak sca vs. WT | 447.4232 |
4 H’ bottom - Weak sca vs. WT | 24.8584 |
4 H’’ top - Weak sca vs. WT | 17.4521 |
4 H’’ bottom - Weak sca vs. WT | 0.3525 |
Figure 6B - velocity distributions of R8s to non-R cells | |
Far posterior | 0.5838 |
Posterior Transition Zone (PTZ) | 1.6628 |
Morphogenetic furrow (MF) | 8.9340 |
p Value reported in the test lower than the computational limit of the program.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Drosophila melanogaster) | DE-Cadherin::GFP | Huang et al., 2009 | Flybase: FBal0247908 | Knock-in in-frame fusion of GFP into endogenous shotgun (shg) gene. Gift from Suzanne Eaton |
Gene (Drosophila melanogaster) | scabrousBP2 | Bloomington Drosophila Stock Center | FlyBase IDFBal0032653BDSC ID 7320 | ~ 2 kb deletion of 5’ region of sca, including first exon and start site. Protein null allele. |
Other | Grace’s Insect Medium | Sigma Aldrich | G9771 | |
Chemical compound, drug | PenStrep Stock Solution | Gibco | 15140–122 | |
Chemical compound, drug | BIS-TRIS | Sigma Aldrich | B4429 | |
Chemical compound, drug | Fetal Bovine Serum (FBS) | Thermo Fisher Scientific | 10270098 | |
Chemical compound, drug | Insulin solution, human | Sigma Aldrich | 19,278 | |
Other | Dumont forceps (0208–5-PO) | Fine Science Tools | 11252–00 | |
Other | 26G × 5/8 Syringe | BD | 305,115 | |
Other | 35 mm dish, No. 1.5 coverslip | MatTek | P35G-1.5–14 C | |
Chemical compound, drug | 0.01% (w/v) Poly-L-Lysine | Sigma Aldrich | P4707 | |
Other | Tesa double sided sticky tape | Tesa | 5,338 | |
Other | 0.25 in (6 mm) hole puncher | Staples | 10,573 CC | |
Other | Whatman polycarbonate membrane | Sigma Aldrich | WHA70602513 | |
Chemical compound, drug | SeaKem Gold Agarose | Lonza Rockland | 50,150 | |
Software, algorithm | ImSAnE MATLAB software | Heemskerk and Streichan, 2015 | https://github.com/idse/imsane | |
Software, algorithm | Linear stack alignment with SIFT ImageJ plugin | Lowe, 2004 | https://imagej.net/plugins/linear-stack-alignment-with-sift | |
Software, algorithm | MATLAB implementation of Hungarian algorithm | Cao, 2021 | https://www.mathworks.com/matlabcentral/fileexchange/20328-munkres-assignment-algorithm | |
Software, algorithm | Convolutional neural network used for pixel classification | This paper | https://github.com/K-D-Gallagher/CNN-pixel-classification | |
Software, algorithm | Trained CNN model for pixel classification of epithelial fluorescence confocal data | This paper | https://drive.google.com/drive/folders/1I-nRpn1esRzs5t4ztgbNvkBQuTN2vT7L?usp=sharing | |
Software, algorithm | MATLAB pipeline for segmenting cells from pixel classified images and tracking them | This paper | https://github.com/K-D-Gallagher/eye-patterning | |
Software, algorithm | MATLAB GUI for manually correcting segmentation errors | This paper | https://github.com/K-D-Gallagher/eye-patterning | |
Software, algorithm | MATLAB datasets for Wildtype 1, Wildtype 2, Strong scabrous mutant, and Weak scabrous mutant | This paper | https://drive.google.com/drive/folders/1I-nRpn1esRzs5t4ztgbNvkBQuTN2vT7L?usp=sharing |
Reference* | Medium | Serum (v/v) | Antibiotic | Fly extract (v/v) | Insulin | Ecdysone | Sample type | Duration |
---|---|---|---|---|---|---|---|---|
Robb, 1969 | R-14 | --- | 1X | --- | --- | --- | wing disc | --- |
Davis and Shearn, 1977 | X (XCS) | --- | --- | --- | 0.4 mU/ml | 1 ng/ml | discs | --- |
Wyss, 1982 | ZW | ??? FBS | --- | 22.5% | 10 mg/ml | 10 ng/ml | disc cells | --- |
Currie et al., 1988 | Shields and Sang M3 | 2% FBS | --- | 5% | 125 mU/ml | 1 ng/ml | disc cells | --- |
Schubiger and Truman, 2000 | Shields and Sang M3 D22 | 7.5% FCS | --- | --- | --- | 1 mg/ml | wing disc | 24 hr |
Gibson et al., 2006 | Shields and Sang M3 | 10% FBS | 1X | --- | 0.01 mU/ml | --- | 1.5–2 hr | |
Cafferty et al., 2009 | Schneider’s | 1% FBS | 10 X | --- | 200 mg/ml | --- | 4 hr | |
Landsberg et al., 2009 | Shields and Sang M3 | 2% FCS | 1X | 2.5% | 0.125 iu/ml | --- | wing disc cells | |
Aldaz et al., 2010 | Shields and Sang M3 | 2% FCS | 5X | --- | --- | 100–500 ng/ml | disc eversion | 10 hr |
Mao et al., 2011 | Shields and Sang M3 | 2% FBS | 1X | --- | 0.01 mU/ml | --- | wing disc | 5 hr |
Ohsawa et al., 2012 | Schneider’s | 10% FBS | --- | --- | --- | --- | 3 hr | |
Zartman et al., 2013 | Schneider’s | variable | 4X | 5% | 6.2 µg/ml | --- | wing disc | 5 hr |
Legoff et al., 2013 | Shields and Sang M3 | 2% FCS | --- | 2.5% | 125 mU/ml | --- | wing disc | 8 hr |
Handke et al., 2014 | Shields and Sang M3 | 2% FBS | 10 X | 5% | 5 µg/ml | 1 ng/ml | wing disc | 7 hr |
Tsao et al., 2016 | Schneider’s | 2% FBS | 4X | --- | 1250 µg/ml | --- | multiple discs | 18 hr |
Dye et al., 2017 | Grace’s Insect Medium | 5% FBS | 1X | --- | --- | 1 ng/ml | wing discs | 12+ hr |
This study | Grace’s Insect Medium | 2% FBS | 1X | --- | 625 µg/ml | --- | eye disc | 12–16 hr |