Cell
Volume 186, Issue 3, 2 February 2023, Pages 479-496.e23
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Article
Graded mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart

https://doi.org/10.1016/j.cell.2023.01.001Get rights and content

Highlights

  • Improved workflow for in toto reconstruction of mouse embryogenesis, single-cell tracking

  • Patterned filling of cardiac mesoderm without linear paths, but with marked cell mixing

  • Cardiac fields exhibit surprising collective and individual spatiotemporal behaviors

  • Mesp1 mutant mesoderm progenitors maintain motility but lose directionality

Summary

Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development at single-cell resolution. Nascent mesoderm progenitors form opposing density and motility gradients, converting the temporal birth sequence of gastrulation into a spatial anterolateral-to-posteromedial arrangement. Migrating precardiac mesoderm does not strictly preserve cellular neighbor relationships, and spatial patterns only become solidified as the cardiac crescent emerges. Progenitors undergo a mesenchymal-to-epithelial transition, with a first heart field (FHF) ridge apposing a motile juxta-cardiac field (JCF). Anchored along the ridge, the FHF epithelium rotates the JCF forward to form the initial heart tube, along with push-pull morphodynamics of the second heart field. In Mesp1 mutants that fail to make a cardiac crescent, mesoderm remains highly motile but directionally incoherent, resulting in density gradient inversion. Our practicable live embryo imaging approach defines spatial origins and behaviors of cardiac progenitors and identifies their unanticipated morphological transitions.

Introduction

The emergence and allocation of the progenitors of organs offers insights into the events that ensure robust morphogenesis. The developing heart is particularly sensitive to disturbed morphogenesis, as congenital heart defects (CHDs) occur in over 1% of live births. Understanding the stepwise allocation and assembly of cardiac precursors will provide insights into heart development and disease. Cell labeling and histological studies have shown how the heart forms from its earliest discernible stages,1,2,3,4,5 but individual cellular events following gastrulation remain mostly uncharacterized.

Cardiovascular progenitors emerge during gastrulation as a subset of the Mesp1+ nascent mesoderm population and migrate to lateral regions that will become the cardiac crescent (CC).6,7,8 Early cardiac progenitors comprise multipotent progenitor pools, the first and second heart fields (FHF and SHF), as well as a newly classified juxta-cardiac field (JCF). The JCF contributes to epicardium and left ventricle (LV).9,10 Partially overlapping the JCF, the FHF contributes to atria, atroventricular canal (AVC), and LV.6,11 SHF cells contribute to the atria, right ventricle (RV), and outflow tract (OFT).12,13

Lineage and clone labeling strategies have revealed that Mesp1+ progenitors have rudimentary assignments to final cardiac structures (even before formation of the heart fields), with either temporal or spatial restriction.6,7,14 However, unifying evidence between temporal and spatial domains is incomplete, and concretely linking early progenitors to their progeny structures requires examination at a greater temporal resolution than lineage tracing alone can afford.

Live imaging of avian cardiogenesis has been insightful, by exploiting the relative accessibility of such embryos for visualization and micro-manipulation.2,15,16,17 Imaging studies of early mouse development, however, have grown at a relatively slower pace, owing to the fragility and limited longevity of ex vivo embryo culture.18,19,20,21 Recent studies have examined gastrulation22 and cardiogenesis23 in the mouse but were limited to examining only a few cells at a time with confocal microscopy.

Light sheet fluorescence microscopy (LSFM) is well suited to the morphogenetic studies of mouse development,24,25,26,27 although most in toto embryo imaging has been performed on highly specialized, custom-build instruments. Although computational analysis of large-scale LSFM data is now possible,27,28,29,30 many existing software applications were designed with the same specialization as the custom microscopes with which they are paired.

Overcoming these roadblocks, we performed comprehensive whole-embryo analyses to examine early cardiac progenitors and their emergence from Mesp1+ mesoderm. We combined a widely available LSFM setup and murine ex vivo embryo culture (Figure 1A), integrating data from fluorescent reporters for both Mesp1 lineage and the Smarcd3 “F6” enhancer, the latter being the earliest known cardiac-specific identifier.6 Furthermore, we improved computational tools to enhance data collection, image processing, and analysis of such large-scale data, as well as to help democratize the use of live embryo imaging.

By tracking cardiogenesis at single-cell resolution with retrospective in silico labeling, our work reveals how cardiac regional fate is intimately tied to the temporal birth and migration sequence of cardiac progenitors. Additionally, we highlight the morphological formation of cardiac epithelium, uncovering region-specific migration and movement behaviors that ultimately shape and sculpt the early heart.

Section snippets

An improved computational workflow for in toto mouse embryogenesis by multi-view LSFM

McDole et al. described a comprehensive, whole-embryo imaging workflow of mouse post-implantation development.29 The powerful LSFM microscope utilized in that study provides unparalleled imaging, but its complex assembly and upkeep requires dedicated specialists. Alternatively, advanced commercial LSFM setups (now widely available), such as the Zeiss Lightsheet Z.1, performed excellently in auditions with our embryos. However, long imaging runs required minor accommodations, including the

Discussion

Our improved comprehensive workflow was an important step in overcoming the big data intimidation of LSFM and aims to simplify and democratize the complexities of live LSFM. Its software components are open source, portable, easier to use than ever before, and the requisite hardware is broadly accessible. Armed with fluorescent reporter mice, a widely available LSFM instrument, and this computational toolbox, we examined a short yet critical and highly dynamic window in mouse development from

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIER
Antibodies
tdTomato (rabbit polyclonal)RocklandCat# 600-401-379; RRID:AB_2209751
multi-RFP 5F8 (rat monoclonal)Allele BiotechnologyCat# ACT-CM-MRRFP10; RRID:AB_2336064
Cre (rabbit polyclonal)MilliporeCat# 69050; RRID:AB_10806983
GFP (chicken polyclonal)AvesCat# GFP-1020; RRID:AB_10000240
Foxc2 (sheep polyclonal)R&DCat# AF6989; RRID:AB_10973139
Nkx2.5 (goat polyclonal)Santa CruzCat# sc-8697X; RRID:AB_650280
Isl1 (rabbit polyclonal)AbcamCat# ab-109517; RRID:AB_10866454

Acknowledgments

We thank Blaise Ndjamen for help with microscopy, Kathryn Claiborn for editorial guidance, Mark Kahn for workspace and use of equipment, Yumiko Saga for her gift of Mesp1-Cre mice, as well as W. Patrick Devine and Junli Zhang for design and creation of Smarcd3 reporter mice. This work was funded by a grant from the NHLBI (R01 HL114948) and The Younger Family Fund. M.H.D. was supported by NIH T32 training grants 2T32-HL007731-26 and T32-HL007843-24, as well as funding from UCSF Department of

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