Kinetics of blood cell differentiation during hematopoiesis revealed by quantitative long-term live imaging
- Department of Cellular and Physiological Sciences, University of British Columbia, Canada
- School of Biomedical Engineering, University of British Columbia, Canada
- British Columbia Children’s Hospital, Canada
Figures
Figure 1 with 3 supplements
Long-term ex vivo culture system for extended imaging of developing Lgs.
(A) Quantification of the percentage of videos where proliferation was observed versus videos where no proliferation was observed under four different conditions: co-culture with or without the …
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Figure 1—source data 1
Raw data of Figure 1A, B, E, F, I.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
A LG stays integrated during long-term culture and imaging.
Time-lapse DIC images (0, 4, 8, 10, 12 hr) from a representative video (of Figure 1D) of an ex vivo cultured LG during long-term live imaging period. Scale bar in (A) represents 50 μm.
Figure 1—figure supplement 2
Long-term ex vivo cultured LGs exhibit low oxidative stress and high cell viability.
(A–B) Representative time-lapse images (0, 4, 8, 12 hr) and quantification showing the oxidative stress level over the course of 12 hr in whole LGs cultured in SM with fat bodies (A) and in SM …
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Figure 1—figure supplement 2—source data 1
Raw data of Figure 1—figure supplement 2A, B, C, E.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Ex vivo cultured LGs demonstrate comparable cell cycle, proliferation, and differentiation profiles to in vivo LGs.
(A–C) Quantification of cell cycle profile (shown as the percentage of cells staying in G1, S, G2 phases) in ex vivo cultured LGs that were uncompressed (A, n=4 videos [each 12 hrs]), ex vivo …
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Figure 1—figure supplement 3—source data 1
Raw data of Figure 1—figure supplement 3A, B, C, D, E, F, G.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig1-figsupp3-data1-v2.xlsx
Figure 2 with 2 supplements
Blood progenitors undergo symmetric divisions in the LG.
(A, B) Time-lapse images from representative videos showing a blood progenitor (labelled as “mother cell”) undergoing symmetric division over the course of 40 min (progenies labelled as ‘daughter …
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Figure 2—source data 1
Raw data of Figure 2D and E.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Genetic tools and markers to study blood progenitor cell fate transition during hematopoiesis.
(A) LG schematic and genetic tools including Gal4 and reporter lines that were used in different combinations in the current study to reveal different cell types and cell states in the LG. Cell …
Figure 2—figure supplement 2
Long-term tracking of dome-MESO-GFP intensities in daughter progenitor cells.
Example showing real-time tracking of dome-MESO-GFP fluorescent intensities of two daughter cells over 8 hr. Each dot represents the level of dome-MESO-GFP intensity at a single time point.
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Figure 2—figure supplement 2—source data 1
Raw data of Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig2-figsupp2-data1-v2.xlsx
Figure 3 with 1 supplement
Kinetics of blood progenitor mitosis, progeny growth, and mitotic axes in the LG.
(A) Top panel: time-lapse images from a representative video showing multiple dividing blood progenitors over 2.4 hr. Blood progenitors labelled with dome-Gal4 driven membranous GFP (of genotype dome…
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Figure 3—source data 1
Raw data of Figure 3B, C, D, I, J.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Critical cell size and spatial distribution of blood progenitor divisions.
(A-A’’’) Representative images showing core and distal progenitors are mitotically active revealed by pH3 staining (n=8 lobes analyzed). Core progenitors labelled by both Tep4-Cherry and …
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Figure 3—figure supplement 1—source data 1
Raw data of Figure 3—figure supplement 1B, C, D, E, F, G.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 2 supplements
Reduction of blood progenitor proliferation upon infection.
(A–E) Representative images (A–B) and quantification of EdU incorporation in blood progenitors (C, dome+ Hml-, n=13 lobes in wild-type control, n=17 lobes in infection group, P-value = 0.032), …
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Figure 4—source data 1
Raw data of Figure 4C, D, E, F, G, H, I, L, M.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Reduction of blood progenitor divisions upon infection.
(A–D) Representative images (A–B) and quantification of the number of mitotically active blood progenitors (C, dome+ eater-, p-value = 0.023) and mitotically active mature hemocytes (D, dome- eater+,…
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Figure 4—figure supplement 1—source data 1
Raw data of Figure 4—figure supplement 1C, D.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Workflow of spatial analysis on cell divisions and differentiations.
A step-by-step schematic introducing the methodology applied to extract spatial information of cellular events (including mitosis and differentiation) from long-term LG live imaging videos (detailed …
Figure 5 with 1 supplement
Long-term imaging identifies distinct dynamics of blood progenitor differentiation.
(A) Time-lapse images of blood progenitor differentiation over the course of 7.5 hrs. Blood progenitors labelled with dome-MESO-GFP (green in top panel, white in below panel). Mature hemocytes …
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Figure 5—source data 1
Raw data of Figure 5B, C, D, E, F and H.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Linear and sigmoid type differentiation can occur in parallel or at distinct time points.
(A) Information derived from cells collected in regions containing cells undergoing sigmoid or linear type of differentiation in wild-type LGs. (B) Information derived from cells of collected in …
Figure 6 with 1 supplement
Spatial distribution of sigmoid and linear type differentiations upon immune activation.
(A) Quantification of the number of blood progenitor differentiation events recorded in LGs from wild-type control (n=8 videos that capture 11 differentiation events in total) and E. coli infected …
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Figure 6—source data 1
Raw data of Figure 6A and B.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Ex vivo cultured LGs demonstrate comparable differentiation trends to in vivo LGs following infection.
Quantification of differentiation index (shown as a ratio of the number of eater+ cells/ the number of dome+ cells) following infection in ex vivo cultured LGs and in vivo LGs that were all matched …
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Figure 6—figure supplement 1—source data 1
Raw data of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 2 supplements
Temporal control of sigmoid and linear type differentiations upon bacterial infection.
(A–B) Representative sigmoid differentiation trajectories in blood progenitors from LGs of wild-type control (A-A’’’) or E. coli infected larvae (B-B’’’) over the course of 5–6 hr. (C–D) …
Figure 7—figure supplement 1
Tracking differentiation events at single cell resolution in real time following infection.
(A–B) Time-lapse images of a blood progenitor from a wild-type LG undergoing sigmoid (A) or linear type differentiation (B). (C–D) Time-lapse images of a blood progenitor from a LG upon infection …
Figure 7—figure supplement 2
The normalization method preserves the original trend of cell fate markers during differentiation in wild-type condition and upon infection.
(A–D) Quantification of the raw intensities and normalized intensities of eater-dsRed and dome-MESO-GFP at the start and end of linear differentiation (A–B) and sigmoid differentiation (C–D) in LGs …
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Figure 7—figure supplement 2—source data 1
Raw data of Figure 7—figure supplement 1A, B, C, D, E, F, G, H.
- https://cdn.elifesciences.org/articles/84085/elife-84085-fig7-figsupp2-data1-v2.xlsx
Author response image 1
Author response image 2
Videos
Video 1
Long-term imaging of an ex vivo wild-type LG at single cell resolution.
Representative long-term live imaging video of a primary lobe from an ex vivo LG showing blood progenitor divisions (highlighted by yellow ROIs in the video) over a cultured period of 13 hr. Blood …
Video 2
Long-term monitoring of blood progenitor viability during overnight ex vivo culture.
Representative video showing only three blood progenitors undergoing cell death in a live LG cultured ex vivo over a period of 12 hr. The blood progenitors were marked by Tep4-Gal4-driven dsRed …
Video 3
Long-term tracking of cell cycle progression of blood progenitors in a wild-type LG.
Representative video showing cell cycle progression of a blood progenitor (highlighted in a yellow ROI) in wild-type LG over a period of 1 hr. The cell cycle indicator FUCCI construct was expressed …
Video 4
Blood progenitors divide symmetrically in a wild-type LG.
Representative video showing an example of a blood progenitor undergoing symmetric cell division over a period of 50 min (see also Video 1). The blood progenitors were marked by JAK-STAT signaling …
Video 5
Long-term tracking of blood progenitor differentiation in a wild-type LG.
Representative video of a differentiating blood progenitor (the cell was green at the beginning) turning into a differentiated mature blood cell (the cell became red in the end) in a live intact LG. …
Video 6
Dynamics of sigmoid type differentiation in a wild-type blood progenitor.
Real-time tracking of dome-MESO-GFP and eater-dsRed intensities in a wild-type blood progenitor undergoing sigmoid type differentiation over the course of 5~6 hr. Each dot represents a single time …
Video 7
Dynamics of linear type differentiation in a wild-type blood progenitor.
Real-time tracking of dome-MESO-GFP and eater-dsRed intensities in a wild-type blood progenitor undergoing linear type differentiation over the course of 7~8 hr. Each dot represents a single time …
Video 8
Dynamics of sigmoid type differentiation in a blood progenitor upon E. coli infection.
Real-time tracking of dome-MESO-GFP and eater-dsRed intensities in a blood progenitor undergoing sigmoid type differentiation in the LG derived from an E. coli infected larva over the course of 5~6 …
Video 9
Dynamics of linear type differentiation in a blood progenitor upon E. coli infection.
Real-time tracking of dome-MESO-GFP and eater-dsRed intensity in a blood progenitor undergoing linear type differentiation in the LG derived from an E. coli infected larva over the course of 7~8 hr. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | Tep4-Gal4 | Avet-Rochex et al., 2010 | Flybase ID: FBti0037434 | Gift from Dr. Lucas Waltzer, Université Clermont Auvergne, France |
| Genetic reagent (Drosophila melanogaster) | dome-MESO-Gal4 | Hombría et al., 2005 | Flybase ID: FBtp0146166 | Gift from Dr. Lucas Waltzer, Université Clermont Auvergne, France |
| Genetic reagent (Drosophila melanogaster) | eater-dsRed | Kroeger et al., 2012 Tokusumi et al., 2009 | Flybase ID: FBtp0084524 | Gift from Dr. Elio Sucena, Instituto Gulbenkian de Ciência, Portugal |
| Genetic reagent (Drosophila melanogaster) | dome-MESO-GFP.nls | Oyallon et al., 2016 | Flybase ID: FBtp0142446 | Gift from Dr. Michele Crozatier, Université de Toulouse, France |
| Genetic reagent (Drosophila melanogaster) | gstD-GFP | Sykiotis and Bohmann, 2008 | Flybase ID: FBtp0069371 | Gift from Dr. Dirk Bohmann, University of Rochester Medical Center, USA |
| Genetic reagent (Drosophila melanogaster) | dome-MESO-LacZ | Hombría et al., 2005 | Flybase ID: FBtp0022619 | Gift from Dr. Nancy Fossett, University of Maryland, Baltimore, USA |
| Genetic reagent (Drosophila melanogaster) | HmlΔ-dsRed.nls | Makhijani et al., 2011 | Flybase ID: FBtp0150011 | Gift from Dr. Katja Brüeckner, University of California, San Francisco, USA |
| Genetic reagent (Drosophila melanogaster) | Tep4-QF>QUAS-mCherry | Girard et al., 2021 | N/A | Gift from Dr. Utpal Banerjee, University of California, Los Angeles, USA |
| Genetic reagent (Drosophila melanogaster) | Ubi-FUCCI | Bloomington Drosophila Stock Center | RRID: BDSC_55124 | |
| Genetic reagent (Drosophila melanogaster) | UAS-FUCCI | Bloomington Drosophila Stock Center | RRID: BDSC_55117 | |
| Genetic reagent (Drosophila melanogaster) | w1118 | Bloomington Drosophila Stock Center | RRID: BDSC_3605 | |
| Antibody | Mouse monoclonal anti-phospho-Histone H3 | Invitrogen | Cat# MA3-064, RRID: AB_2633021 | Used in 1:1000 |
| Antibody | Mouse monoclonal anti-LacZ | Developmental State Hybridoma Bank | Cat# 40–1 a, RRID: AB_2314509 | Used in 1:100 |
| Antibody | Donkey polyclonal anti-mouse Cy5 | Jackson Immunoresearch laboratories Inc | Code: 715-175-151, RRID: AB_2340820 | Used in 1:400 |
| Chemical compound, drug | VECTASHIELD with DAPI | Vector Laboratories | Cat# H-1200, RRID:AB_2336790 | |
| Chemical compound, drug | 16% Paraformaldehyde | ThermoFisher Scientific | Cat#28908 | Used in 4% |
| Chemical compound, drug | Triton X | ThermoFisher Scientific | Cat#BP151100 | Used in 0.1% |
| Chemical compound, drug | Normal Goat Serum | Abcam | Cat# ab7481; RRID:AB_2716553 | Used in 16% |
| Chemical compound, drug | Schneider’s Drosophila medium | ThermoFisher Scientific | Cat# 21720001 | |
| Chemical compound, drug | Fetal Bovine Serum | ThermoFisher Scientific | Cat# 12483–020 | Used in 15% |
| Chemical compound, drug | Insulin solution from bovine pancreas | Sigma Aldrich | Cat# I0516 | Used in 0.2 mg/mL |
| Chemical compound, drug | Sytox Green | ThermoFisher Scientific | Cat# S7020 | Used in 2 μM |
| Commercial assay or kit | Click-iT EdU kit | Life technologies | Cat# C10337 | See detail protocol in the Methods |
| Software, algorithm | MATLAB | Commercial | https://www.mathworks.com/products/matlab.html | |
| Software, algorithm | FIJI | Source of the software Schindelin et al., 2012 | https://fiji.sc/ | |
| Software, algorithm | MATLAB script used to create heat maps | Codes deposited in the Tanentzapf lab GitHub (https://github.com/Tanentzapf-Lab/LiveImaging_HematopiesisKinetics_Infection_Ho_Carr; Ho et al., 2023) | This study | See the Tanentzapf lab GitHub |
| Software, algorithm | MATLAB scripts used to calculate the number of progenitors, plasmatocyte differentiation, and total number of cells in a LG | Scripts deposited in the study Khadilkar et al., 2017 | N/A | Khadilkar et al., 2017 |
| Other | Glass bottom mounting dishes | MatTek Corporation | Cat# P35G-0–14 C | See Immunohistochemistry and antibodies section in the Materials and methods. |
| Other | Incubation system | TOKAI HIT | Cat# INU-ONICS F1 | Temperature set at 25οC. See Long-term ex vivo organ culture and confocal imaging section in the Methods. |
