Culture of and experiments with sea urchin embryo primary mesenchyme cells

Bradley Moreno, Allessandra DiCorato, Alexander Park, Kellen Mobilia, Regina Knapp, Reiner Bleher, Charlene Wilke, Keith Alvares, Derk Joester*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Skeletogenesis in the sea urchin embryo gives rise to a pair of intricate endoskeletal spicules. Deposition of these skeletal elements in the early larva is the outcome of a morphogenetic program that begins with maternal inputs in the early zygote and results in the specification of the large micromere-primary mesenchyme cell (PMC) lineage. PMCs are of considerable interest as a model system, not only to dissect the mechanism of specific developmental processes, but also to investigate their evolution and the unrivaled level of control over the formation of a graded, mechanically robust, yet single crystalline biomineral. The ability to study gene regulatory circuits, cellular behavior, signaling pathways, and molecular players involved in biomineralization is significantly boosted by the high level of autonomy of PMCs. In fact, in the presence of horse serum, micromeres differentiate into PMCs and produce spicules in vitro, separated from the embryonic milieu. PMC culture eliminates indirect effects that can complicate the interpretation of experiments in vivo, offers superior spatiotemporal control, enables PMC-specific readouts, and is compatible with most imaging and characterization techniques. In this chapter, we provide an updated protocol, based on the pioneering work by Okazaki and Wilt, for the isolation of micromeres and subsequent culture of PMCs, as well as protocols for fixation and staining for fluorescent microscopy, preparation of cell cultures for electron microscopy, and the isolation of RNA.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc
Pages293-330
Number of pages38
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Cell Biology
Volume150
ISSN (Print)0091-679X

Fingerprint

Sea Urchins
Mesoderm
Embryonic Structures
Primary Cell Culture
Zygote
Gene Regulatory Networks
Cell Lineage
Horses
Larva
Microscopy
Electron Microscopy
Cell Culture Techniques
Mothers
RNA
Staining and Labeling
Serum

Keywords

  • Cell culture
  • Development
  • Electron microscopy
  • Fluorescence microscopy
  • Primary mesenchyme cells
  • Sea urchin embryo
  • Skeletogenesis
  • Spiculogenesis
  • Transcriptomics

ASJC Scopus subject areas

  • Cell Biology

Cite this

Moreno, B., DiCorato, A., Park, A., Mobilia, K., Knapp, R., Bleher, R., ... Joester, D. (2019). Culture of and experiments with sea urchin embryo primary mesenchyme cells. In Methods in Cell Biology (pp. 293-330). (Methods in Cell Biology; Vol. 150). Academic Press Inc. https://doi.org/10.1016/bs.mcb.2019.01.002
Moreno, Bradley ; DiCorato, Allessandra ; Park, Alexander ; Mobilia, Kellen ; Knapp, Regina ; Bleher, Reiner ; Wilke, Charlene ; Alvares, Keith ; Joester, Derk. / Culture of and experiments with sea urchin embryo primary mesenchyme cells. Methods in Cell Biology. Academic Press Inc, 2019. pp. 293-330 (Methods in Cell Biology).
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Moreno, B, DiCorato, A, Park, A, Mobilia, K, Knapp, R, Bleher, R, Wilke, C, Alvares, K & Joester, D 2019, Culture of and experiments with sea urchin embryo primary mesenchyme cells. in Methods in Cell Biology. Methods in Cell Biology, vol. 150, Academic Press Inc, pp. 293-330. https://doi.org/10.1016/bs.mcb.2019.01.002

Culture of and experiments with sea urchin embryo primary mesenchyme cells. / Moreno, Bradley; DiCorato, Allessandra; Park, Alexander; Mobilia, Kellen; Knapp, Regina; Bleher, Reiner; Wilke, Charlene; Alvares, Keith; Joester, Derk.

Methods in Cell Biology. Academic Press Inc, 2019. p. 293-330 (Methods in Cell Biology; Vol. 150).

Research output: Chapter in Book/Report/Conference proceedingChapter

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AU - DiCorato, Allessandra

AU - Park, Alexander

AU - Mobilia, Kellen

AU - Knapp, Regina

AU - Bleher, Reiner

AU - Wilke, Charlene

AU - Alvares, Keith

AU - Joester, Derk

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KW - Fluorescence microscopy

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KW - Transcriptomics

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Moreno B, DiCorato A, Park A, Mobilia K, Knapp R, Bleher R et al. Culture of and experiments with sea urchin embryo primary mesenchyme cells. In Methods in Cell Biology. Academic Press Inc. 2019. p. 293-330. (Methods in Cell Biology). https://doi.org/10.1016/bs.mcb.2019.01.002