Dataset Information

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

ABSTRACT: A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. To address this, we developed a Cre-loxP based method to mosaically label precursor cells, called dorsal forerunner cells, that give rise to the zebrafish LRO known as Kupffer's vesicle. This methodology allows lineage tracing, 3-dimensional (3D) reconstruction and morphometric analysis of single LRO cells in living embryos. The ability to visualize and quantify individual LRO cell dynamics provides an opportunity to advance our understanding of LRO development, and in a broader sense, investigate the interplay between intrinsic biochemical mechanisms and extrinsic mechanical forces that drive morphogenesis of epithelial tissues.

SUBMITTER: Dasgupta A

PROVIDER: S-EPMC6319891 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Publications

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

Dasgupta Agnik A Jacob Andrew E AE Amack Jeffrey D JD

Bio-protocol 20181101 22

A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. To address this, we developed a Cre-<i>loxP</i> based method to mosaically label precursor cells, called dorsal forerunner cells, th ...[more]

PMID: 30613762

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Project description:Several of our internal organs, including heart, lungs, stomach, and spleen, develop asymmetrically along the left-right (LR) body axis. Errors in establishing LR asymmetry, or laterality, of internal organs during early embryonic development can result in birth defects. In several vertebrates-including humans, mice, frogs, and fish-cilia play a central role in establishing organ laterality. Motile cilia in a transient embryonic structure called the "left-right organizer" (LRO) generate a directional fluid flow that has been proposed to be detected by mechanosensory cilia to trigger asymmetric signaling pathways that orient the LR axis. However, the mechanisms that control the form and function of the ciliated LRO remain poorly understood. In the zebrafish embryo, precursor cells called dorsal forerunner cells (DFCs) develop into a transient ciliated structure called Kupffer's vesicle (KV) that functions as the LRO. DFCs can be visualized and tracked in the embryo, thereby providing an opportunity to investigate mechanisms that control LRO development. Previous work revealed that proliferation of DFCs via mitosis is a critical step for developing a functional KV. Here, we conducted a targeted pharmacological screen to identify mechanisms that control DFC proliferation. Small molecule inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) were found to reduce DFC mitosis. The SERCA pump is involved in regulating intracellular calcium ion (Ca2+) concentration. To visualize Ca2+ in living embryos, we generated transgenic zebrafish using the fluorescent Ca2+ biosensor GCaMP6f. Live imaging identified dynamic cytoplasmic Ca2+ transients ("flux") that occur unambiguously in DFCs. In addition, we report Ca2+ flux events that occur in the nucleus of DFCs. Nuclear Ca2+ flux occurred in DFCs that were about to undergo mitosis. We find that SERCA inhibitor treatments during DFC proliferation stages alters Ca2+ dynamics, reduces the number of ciliated cells in KV, and alters embryo laterality. Mechanistically, SERCA inhibitor treatments eliminated both cytoplasmic and nuclear Ca2+ flux events, and reduced progression of DFCs through the S/G2 phases of the cell cycle. These results identify SERCA-mediated Ca2+ signaling as a mitotic regulator of the precursor cells that give rise to the ciliated LRO.

Dataset Information

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

Publications

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

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