Emergence of a geometric pattern of cell fates from tissue-scale mechanics in the Drosophila eye

  1. Kevin D Gallagher
  2. Madhav Mani  Is a corresponding author
  3. Richard W Carthew  Is a corresponding author
  1. Department of Molecular Biosciences, Northwestern University, United States
  2. NSF Simons Center for Quantitative Biology, Northwestern University, United States
  3. Department of Engineering Sciences and Applied Mathematics, Northwestern University, United States
7 figures, 7 videos, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Living eye discs imaged by time-lapse microscopy.

(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 …

Figure 1—figure supplement 1
Pipeline for image capture and processing.

(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 …

Figure 1—figure supplement 2
General features of ex vivo eye development.

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 …

Figure 2 with 1 supplement
A periodic cell flow exists in the MF.

(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 …

Figure 2—figure supplement 1
Position of the MF as it changes over time.

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.

Figure 3 with 1 supplement
Complex cell flows within the eye disc.

(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 …

Figure 3—figure supplement 1
Triangular grid formation.

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 …

Figure 4 with 1 supplement
Scabrous is required for robust periodic cell flow in the MF.

(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 …

Figure 4—figure supplement 1
Macroscopic features of scabrous mutant discs.

(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 …

Scabrous is required for normal cell dilation and cell flow.

(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 …

Relationship between cell flow and cell dilation/contraction.

(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 …

Self-organized triangular lattice formation.

(A) Summary of newly observed phenomena described in this study. Arrows show the cell flows across the eye disc. (B) Prior model for self-organized lattice formation based on a standard …

Videos

Video 1
A wildtype disc after segmentation and tracking.

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’ …

Video 2
A wildtype disc with the presumptive R cells labeled a specific color.

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.

Video 3
A wildtype disc with all cells labeled with their respective velocity vectors, in which arrow length is proportional to the magnitude, and direction is marked by arrow orientation. Presumptive R8 cell velocities are labeled red.
Video 4
Emergence of the triangular lattice as a consequence of cell flow.

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 …

Video 5
A scabrous mutant disc with the presumptive R cells labeled with a specific color.

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.

Video 6
A wildtype disc with all cells colored according to their Shape Index.

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 …

Video 7
A wildtype disc with cells initially colored in arbitrary stripes of one to two cells thickness and that are aligned with the dorsal-ventral axis (vertical).

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 …

Tables

Table 1
Summary statistics of populations under comparison.

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 MF1.1467
3E - R8 vs. non-R cells in low flow regions of the MF24.8128
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 cells72.4072
4D bottom - R8s vs non-R cells23.9553
4D’ top - Strong sca vs. WTInfinity*
4D’ bottom - Strong sca vs. WTInfinity*
4D’’ top - Strong sca vs. WT68.3809
4D’’ bottom - Strong sca vs. WT22.5819
Figure 4H–H’’ velocity distributions of weak sca vs. WT
4 H top - R8s vs. non-R cells12.7047
4 H bottom - R8s vs non-R cells20.9813
4 H’ top - Weak sca vs. WT447.4232
4 H’ bottom - Weak sca vs. WT24.8584
4 H’’ top - Weak sca vs. WT17.4521
4 H’’ bottom - Weak sca vs. WT0.3525
Figure 6B - velocity distributions of R8s to non-R cells
Far posterior0.5838
Posterior Transition Zone (PTZ)1.6628
Morphogenetic furrow (MF)8.9340
  1. *

    p Value reported in the test lower than the computational limit of the program.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Drosophila melanogaster)DE-Cadherin::GFPHuang et al., 2009Flybase: FBal0247908Knock-in in-frame fusion of GFP into endogenous shotgun (shg) gene. Gift from Suzanne Eaton
Gene (Drosophila melanogaster)scabrousBP2Bloomington Drosophila Stock CenterFlyBase IDFBal0032653BDSC ID 7320~ 2 kb deletion of 5’ region of sca, including first exon and start site. Protein null allele.
OtherGrace’s Insect MediumSigma AldrichG9771
Chemical compound, drugPenStrep Stock SolutionGibco15140–122
Chemical compound, drugBIS-TRISSigma AldrichB4429
Chemical compound, drugFetal Bovine Serum (FBS)Thermo Fisher Scientific10270098
Chemical compound, drugInsulin solution, humanSigma Aldrich19,278
OtherDumont forceps (0208–5-PO)Fine Science Tools11252–00
Other26G × 5/8 SyringeBD305,115
Other35 mm dish, No. 1.5 coverslipMatTekP35G-1.5–14 C
Chemical compound, drug0.01% (w/v) Poly-L-LysineSigma AldrichP4707
OtherTesa double sided sticky tapeTesa5,338
Other0.25 in (6 mm) hole puncherStaples10,573 CC
OtherWhatman polycarbonate membraneSigma AldrichWHA70602513
Chemical compound, drugSeaKem Gold AgaroseLonza Rockland50,150
Software, algorithmImSAnE MATLAB softwareHeemskerk and Streichan, 2015https://github.com/idse/imsane
Software, algorithmLinear stack alignment with SIFT ImageJ pluginLowe, 2004https://imagej.net/plugins/linear-stack-alignment-with-sift
Software, algorithmMATLAB implementation of Hungarian algorithmCao, 2021https://www.mathworks.com/matlabcentral/fileexchange/20328-munkres-assignment-algorithm
Software, algorithmConvolutional neural network used for pixel classificationThis paperhttps://github.com/K-D-Gallagher/CNN-pixel-classification
Software, algorithmTrained CNN model for pixel classification of epithelial fluorescence confocal dataThis paperhttps://drive.google.com/drive/folders/1I-nRpn1esRzs5t4ztgbNvkBQuTN2vT7L?usp=sharing
Software, algorithmMATLAB pipeline for segmenting cells from pixel classified images and tracking themThis paperhttps://github.com/K-D-Gallagher/eye-patterning
Software, algorithmMATLAB GUI for manually correcting segmentation errorsThis paperhttps://github.com/K-D-Gallagher/eye-patterning
Software, algorithmMATLAB datasets for Wildtype 1, Wildtype 2, Strong scabrous mutant, and Weak scabrous mutantThis paperhttps://drive.google.com/drive/folders/1I-nRpn1esRzs5t4ztgbNvkBQuTN2vT7L?usp=sharing
Table 2
Summary of published conditions to culture Drosophila imaginal discs ex vivo. Adapted from Tsao et al., 2016.
Reference*MediumSerum (v/v)AntibioticFly extract (v/v)InsulinEcdysoneSample typeDuration
Robb, 1969R-14---1X---------wing disc---
Davis and Shearn, 1977X (XCS)---------0.4 mU/ml1 ng/mldiscs---
Wyss, 1982ZW??? FBS---22.5%10 mg/ml10 ng/mldisc cells---
Currie et al., 1988Shields and Sang M32% FBS---5%125 mU/ml1 ng/mldisc cells---
Schubiger and Truman, 2000Shields and Sang M3 D227.5% FCS---------1 mg/mlwing disc24 hr
Gibson et al., 2006Shields and Sang M310% FBS1X---0.01 mU/ml---1.5–2 hr
Cafferty et al., 2009Schneider’s1% FBS10 X---200 mg/ml---4 hr
Landsberg et al., 2009Shields and Sang M32% FCS1X2.5%0.125 iu/ml---wing disc cells
Aldaz et al., 2010Shields and Sang M32% FCS5X------100–500 ng/mldisc eversion10 hr
Mao et al., 2011Shields and Sang M32% FBS1X---0.01 mU/ml---wing disc5 hr
Ohsawa et al., 2012Schneider’s10% FBS------------3 hr
Zartman et al., 2013Schneider’svariable4X5%6.2 µg/ml---wing disc5 hr
Legoff et al., 2013Shields and Sang M32% FCS---2.5%125 mU/ml---wing disc8 hr
Handke et al., 2014Shields and Sang M32% FBS10 X5%5 µg/ml1 ng/mlwing disc7 hr
Tsao et al., 2016Schneider’s2% FBS4X---1250 µg/ml---multiple discs18 hr
Dye et al., 2017Grace’s Insect Medium5% FBS1X------1 ng/mlwing discs12+ hr
This studyGrace’s Insect Medium2% FBS1X---625 µg/ml---eye disc12–16 hr

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