Long-term, in toto live imaging of the developing mouse heart

doi:10.21203/rs.2.21499/v1

Method Article

Long-term, in toto live imaging of the developing mouse heart

https://doi.org/10.21203/rs.2.21499/v1

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Mapping holistic cell behaviors sculpting mammalian heart has been a goal, but so far only successes in transparent invertebrates and lower vertebrates. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a culture module, a heartbeat-gated imaging strategy, and a digital image processing framework, we realized imaging of developing mouse hearts with uninterrupted cell lineages for up to 1.5 days. Four-dimensional landscapes of cell behaviors revealed a blueprint for ventricle chamber formation in which biased outward migration of outermost cardiomyocytes coupled with cell intercalation and horizontal division. The trabeculae, an inner muscle architecture, was developed through early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction affords a transformative means for deciphering mammalian organogenesis.

Developmental biology

Continuous live imaging

light-sheet microscopy

heart development

lineage tracing

cell behaviors

A central theme in organogenesis is how cell lineages of divergent sources are spatiotemporally deployed to assemble complex organ structures in mammals^1,2. In particular, the formation of mammalian heart as the first functional organ reflects an exquisite choreography of massive numbers of cells dividing and migrating through the processes of heart tube looping, ballooning, and formation and coalescence of the inner trabecular layer, ultimately creating a four-chambered muscular pump ^1-4. Many congenital malformations result from abnormal cardiac growth⁵. However, cellular mechanisms of heart chamber formation remain controversial because an effective means offering spatiotemporal, holistic information of cell lineages is still lacking.

Ideally, to precisely map the developmental processes of embryogenesis and organogenesis, all single cells should be followed noninvasively through live imaging for a certain period of time. Recent breakthroughs in live imaging coupled with digital reconstructions open a new avenue to decipher morphogenetic rules through precisely tracking cell fate decisions and depicting a holistic lineage landscape^6-9. Not only it has brought new fundamental understandings of the development of Caenorhabditis elegans and Drosophila embryos^10,11, great strides have also been made on mouse embryo imaging using light-sheet microscopy, enabling single-cell imaging of yolk sac expansion¹² and gastrulation⁹. Because of technical challenges including rigorous culture conditions to support ex vivo development, depth of penetration to image solid organs ^9,15,16, and spontaneous heartbeats onset around E8.0 ¹⁷, its implementation to mammalian hearts has not been possible, until now.

Here, we overcame the technical challenges as previously encountered by optimizing mouse embryo culture and mounting methods, developing a vertical, dual-side illumination light-sheet microscope, equipping it with an integrated embryo culture module, together with a heartbeat-gated imaging module. With this integrative approach, we realized a 36-h, all cell-resolved imaging of developing mouse heart at 3-min intervals. Further aided with innovative digital image processing pipelines, Grapebio, we generated, for the first time, all-cell landscapes and uninterrupted cell lineages for the developing mouse heart, through robustly segmenting and accurately locating ~20,000,000 cells of different shapes and intensities in ~7,200 volumetric stacks (obtained over 1.5 days at 3 min intervals). Taking these digital reconstructions, we reveal the cellular basis for the heart chamber ballooning and the cells of origin for the heart trabeculation.

NotI (NEB, R3189V)

BamHI (NEB, R3136V)

DMEM/F12 (Sigma, D2906)

Fetal bovine serum (Gibco, 16000-044)

Pen-Strep solution (Hyclone, SV30010)

HEPES (Sigma, H4034-500G)

Sodium bicarbonate (Sigma, S5761)

Glucose (Sigma, G7021)

Low-melt agarose (Sigma, A9414)

OCT (Leica, 14020108926)

Doxycycline (RPI, P6148-500G)

BrdU (Sigma, B5002)

anti-BrdU antibody (Abcam, Ab6326)

EdU (Life Technologies, C10638)

Click-it EdU imaging Kit (Life Technologies, C10638)

TNNI3 antibody (Abcam, Ab56357)

GATA4 antibody (Santa, Sc-25310)

Alexa Fluor 488 donkey anti rabbit (Invitrogen, A11078)

Alexa Fluor 488 donkey anti rat (Invitrogen, A21208)

Alexa Fluor 555 donkey anti goat (Invitrogen, A21432)

Alexa Fluor 647 donkey anti goat (Invitrogen , A21447)

T-A clone vector EZ-T (Vazyme, T168-101)

DIG RNA labeling kit (Roche, 11277073910)

anti-DIG-AP antibody (Roch, 11093274910)

BM Purple AP Substrate (Roche, 11442074001)

1.5 ml tube (Axygen, MCT-150-C)

0.2 ml 8-strip PCR tube (Axygen, PCR-0208-C)

Confocal microscope (Zeiss LSM 710)

Nikon SMZ18 microscope

Zeiss Axio Vert.A1 microscope

Zeiss Lumar V12

Centrifuge (Eppendorf)

Thermal cycler (Eppendorf)

ThermoMixer

Reagents Setup

• Dissection medium

88% DMEM/F12 (PH 7.2-7.4)

10% fetal bovine serum

2% Pen-Strep solution

25 mM HEPES

• Basic Culture medium-V1

98% DMEM/F12 (PH 7.2-7.4)

2% Pen-Strep solution

15 mM HEPES

• Culture medium-V2

50% basic culture medium

50% rat serum

• Culture medium-V3

83% dissection medium

17% rat serum

Dissection medium and culture medium pre-heated and balanced in an incubator (37°C, 5% CO₂, and 95% air) for 1 h before using.

Detailed Experiment Procedure for embryo preparation

Embryo collection

1. Pregnant mice are euthanized with 4% chloral hydrate.

2. Soak abdomen with 70% ethanol, pinch the skin and make an incision at the midline of the abdomen with surgical scissors, then pull the skin apart.

3. Cut the peritoneum to expose abdominal cavity.

4. Cut the uterus and place it into the dissection medium.

5. Separate each embryo with forceps between implantation sites.

6. Peel back the surrounding muscle layer and expose decidua tissue.

7. Clip off 1/5 portion of the decidua, and expose the embryo;

8. Dissect out the enveloped decidua, Reichert’s membrane with fine forceps.

9. Remove ectoplacental cone, yolk sac and amnion with super fine forceps (when embryos are later than E8.25).

Embryo culture

10 Transfer the embryos to a Petri dish/agarose tube with culture medium-V2, put the Petri dish/agarose tube in an incubator (37°C, 5% CO₂, and 95% air).

Embryo mounting

11 Make hollow agarose cylinders: Component B inserted with component A is filled with 2% low melting-temperature agarose. Triangular hollow is formed after removing component A (Figure 5).

12 Suck the mouse embryo with culture medium-V2 to the hollow in the agarose holder.

13 Combine the agarose holder with a base support, and mount the agarose holder and base on the chamber, full the chamber with culture medium-V3.

Live imaging of the mouse embryos

14 Set up the environment on the microscopy: Open and adjust gas mixer to 5% CO₂, 5% O₂and 90% N₂, pre-heat the chamber to 37°C using water bath which is rolling surrounding the chamber.

15 Image the embryos: Generally, 2.5 μm step size, up to total of ~400 μm, 3 min intervals, lower laser power and exposure time are used to obtain better results.

Step: 9.

Problem: The state of mouse embryo is not well when dissecting.

Possible reason: It takes too much time to dissect mouse embryos or destroys the embryo.

Solution: Practice more times and decrease the operating time.

Step: 15.

Problem: The state of mouse embryo is not well when imaging .

Possible reason: The culture condition may be changed during long-term imaging.

Solution: Check the imaging system, containing gas mixture (O₂, N₂, and CO₂) and temperature control.

Day 1

• Dissect mouse embryos

• Mount embryos to V-slot

• Place the V-slot with embryo onto the vLSFM

• Adjust the imaging conditions

• Live imaging

Day 2-3

• Live imaging

• Obtain and analyze the imaging data

1 The mouse embryos exhibit good state before imaging.

2 The imaging mouse embryo shows normal heart development.

3 Imaging results show positive signals at single-cell resolution.

1 Meilhac, S. M., Lescroart, F., Blanpain, C. & Buckingham, M. E. Cardiac cell lineages that form the heart. Cold Spring Harb Perspect Med 4, a013888, doi:10.1101/cshperspect.a013888 (2014).

2 Kelly, R. G., Buckingham, M. E. & Moorman, A. F. Heart fields and cardiac morphogenesis. Cold Spring Harb Perspect Med 4, doi:10.1101/cshperspect.a015750 (2014).

3 Vincent, S. D. & Buckingham, M. E. How to make a heart: the origin and regulation of cardiac progenitor cells. Curr Top Dev Biol 90, 1-41, doi:10.1016/S0070-2153(10)90001-X (2010).

4 Sedmera, D., Pexieder, T., Vuillemin, M., Thompson, R. P. & Anderson, R. H. Developmental patterning of the myocardium. Anat Rec 258, 319-337 (2000).

5 Hoffman, J. I. E., Kaplan, S. & Liberthson, R. R. Prevalence of congenital heart disease. Am Heart J 147, 425-439, doi:10.1016/j.ahj.2003.05.003 (2004).

6 Keller, P. J., Schmidt, A. D., Wittbrodt, J. & Stelzer, E. H. K. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065-1069, doi:10.1126/science.1162493 (2008).

7 Ichikawa, T. et al. Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools. Nat Protoc 9, 575-585, doi:10.1038/nprot.2014.035 (2014).

8 Amat, F. et al. Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data. Nat Methods 11, 951-958, doi:10.1038/nmeth.3036 (2014).

9 McDole, K. et al. In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level. Cell 175, 859-876 e833, doi:10.1016/j.cell.2018.09.031 (2018).

10 Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998, doi:10.1126/science.1257998 (2014).

11 Royer, L. A. et al. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat Biotechnol 34, 1267-1278, doi:10.1038/nbt.3708 (2016).

12 Udan, R. S., Piazza, V. G., Hsu, C. W., Hadjantonakis, A. K. & Dickinson, M. E. Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy. Development 141, 4406-4414, doi:10.1242/dev.111021 (2014).

13 Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007-1009, doi:DOI 10.1126/science.1100035 (2004).

14 Mickoleit, M. et al. High-resolution reconstruction of the beating zebrafish heart. Nat Methods 11, 919-922, doi:10.1038/nmeth.3037 (2014).

15 Skylaki, S., Hilsenbeck, O. & Schroeder, T. Challenges in long-term imaging and quantification of single-cell dynamics. Nat Biotechnol 34, 1137-1144, doi:10.1038/nbt.3713 (2016).

16 Massarwa, R. & Niswander, L. In toto live imaging of mouse morphogenesis and new insights into neural tube closure. Development 140, 226-236, doi:10.1242/dev.085001 (2013).

17 Tyser, R. C. et al. Calcium handling precedes cardiac differentiation to initiate the first heartbeat. Elife 5, doi:10.7554/eLife.17113 (2016).

We thank R.H. Harvey for reviewing this manuscript, I.C. Bruce for manuscript editing, and Yangyang Xu, Lufeng Yuan, and Erlin Yao for technical assistance. A.H. was supported by grants from the National Basic Research Program of China (2017YFA0103402 and 2019YFA0801802), the National Natural Science Foundation of China (31571487, 31771607 and 31327901), the Peking-Tsinghua Center for Life Sciences, and the 1000 Youth Talents Program of China. H.C. was supported by grants from the National Key Technology R&D Program (SQ2011SF11B01041), the National Key Basic Research Program of China (2016YFA0500403), and the National Science Foundation of China (31521062). W.Z. was supported by grants from the National Key Technology R&D Program (2018YFA0109600). R.W. was supported by grants from the National Postdoctoral Program for Innovative Talents (8206200030).

The authors declare no financial interests.

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Long-term, in toto live imaging of the developing mouse heart

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Long-term, in toto live imaging of the developing mouse heart

Status:

Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Troubleshooting

Time Taken

Anticipated Results

References

Acknowledgements

Additional Declarations

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

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