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Single-cell analysis of mammalian cardiogenesis elucidating an essential role of outflow tract progenitors

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posted on 2024-09-02, 22:59 authored by Chikai Zhou

The heart is the first organ to form and start to function during mammalian embryogenesis. This complex organ system is constructed by a diverse set of cell types, involving mesodermal precursors and heart progenitors, at the earliest embryonic stages. These progenitor cells contribute to the formation of the distinct heart regions, such as atria, right and left ventricle, and outflow tract (OFT). However, it is still controversial and undefined which specific progenitors and paracrine molecular cues are responsible to form each of the distinct heart regions (e.g., OFT), requiring the rigorous analysis at higher resolution to identify the detailed cellular and molecular pathways on developing hearts. To tackle this problem, we applied a wide variety of the state-of-the-art biotechnologies and assays, including the in vitro cardiac differentiation system of human embryonic stem cells (hESCs), handling of the murine and human embryonic hearts, CRISPR/Cas9 gene editing, single-cell RNA sequencing (RNA-seq), and a mouse lineage tracing approach. We provide a comprehensive gene expression resource, characterizing the transcriptional dynamics of cardiac lineage specification and identifying novel markers of developing cardiac derivatives from multipotent progenitors to mature cardiac cells. Importantly, we have discovered the uniquely stage- and region-specific mesodermal precursors and/or heart progenitors that are essential on mammalian cardiogenesis.

In Paper I, to determine the key regulators for cardiac linage specification and commitment, we first established a genome-wide CRISPR/Cas9 knockout screen platform using the in vitro hESC differentiation where we monitored the two distinct stage markers, an early cardiac mesodermal marker MESP1 and a heart progenitor marker ISL1. From the screen output, we compiled a list of 15 candidate genes and finally identified ZIC2 as an essential gene for early cardiac mesoderm formation. Interestingly, RNA-seq profiles of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single-cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps during the in vitro hESC cardiogenesis. In Paper II, through population and single-cell analysis of the in vitro hESC cardiac differentiation and the in vivo human embryonic/fetal hearts, we chart the developmental landscape of human cardiac formation at the cellular and molecular basis. Importantly, we have discovered a uniquely human subset of the OFT region-specific heart progenitors, marked by LGR5. The LGR5+ progenitors emerge specifically in the proximal OFT of human embryonic hearts (4 to 5 weeks of fetal age) and likely contribute to the OFT formation and alignment. Our results provide a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations. In Paper III, to obtain a whole picture of transcriptional and epigenetic regulation in the mesoderm lineage on the developing hearts, we first established Mesp1+ mesodermal lineage tracing mice. Mesp1 gene encodes a transcription factor of the b-HLH family, which is expressed broadly in the mesodermal cells and critical for the cardiovascular development in mammals. Using the CRISPR/Cas9 system and an IRES2-Cre cassette, we generated a Mesp1-IRES2-Cre knock-in mouse line and cross-bred them with reporter mice (Rosa26-tdTomato). On the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), we observed that more than 95% of the atria and ventricular cells in the hearts on an embryonic day 10.5 are the Mesp1+ mesodermal lineage. Interestingly, less percentage (<90%) of the OFT cells are positive for Mesp1, while the rest OFT cells (»10%) are positive for a neural crest marker, SOX10. We showed developmental dynamics of the Mesp1+ mesodermal lineage on murine embryonic hearts using the advanced light sheet microscopic images. Further, we showcased the single-cell RNA and chromatin sequencing analysis data of the Mesp1+ lineage on murine cardiogenesis.

In summary, our studies in this thesis provide a comprehensive gene expression resource of developing cardiac derivatives including novel mesodermal precursors and heart progenitors. Therefore, the current works contribute to a better understanding of mammalian heart development.

List of scientific papers

I. Jiejia Xu, Chikai Zhou, Kylie S. Foo, Ran Yang, Yao Xiao, Kristine Bylund, Makoto Sahara, Kenneth R. Chien#. Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors. Stem Cells. 38, no. 6 (2020):741-755. #Corresponding author.
https://doi.org/10.1002/stem.3168

II. Sahara, Makoto*#, Federica Santoro*, Jesper Sohlmér, Chikai Zhou, Nevin Witman, Chuen Yan Leung, Mimmi Mononen, Kristine Bylund, Peter Gruber, Kenneth R. Chien#. Population and Single-Cell analysis of human cardiogenesis reveals unique LGR5 ventricular progenitors in embryonic outflow tract. Developmental Cell. 48, no. 4 (2019):475-490. *These authors contributed equally to this work. #Corresponding author.
https://doi.org/10.1016/j.devcel.2019.01.005

III. Chikai Zhou, Ran Yang, Kenneth R Chien, Makoto Sahara#. Single-cell analysis of Mesp1 mesodermal precursors for embryonic outflow tract formation. #Corresponding author. [Manuscript]

History

Defence date

2021-10-04

Department

  • Department of Cell and Molecular Biology

Publisher/Institution

Karolinska Institutet

Main supervisor

Chien, Kenneth

Co-supervisors

Bergmann, Olaf; Sahara, Makoto; Hansson, Emil

Publication year

2021

Thesis type

  • Doctoral thesis

ISBN

978-91-8016-296-8

Number of supporting papers

3

Language

  • eng

Original publication date

2021-09-14

Author name in thesis

Zhou, Chikai

Original department name

Department of Cell and Molecular Biology

Place of publication

Stockholm

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