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Echinobase
ECB-ART-46970
Methods Cell Biol 2019 Jan 01;150:269-292. doi: 10.1016/bs.mcb.2018.11.007.
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Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs).

Campanale JP , Hamdoun A , Wessel GM , Su YH , Oulhen N .


Abstract
Small micromeres of the sea urchin are believed to be primordial germ cells (PGCs), fated to give rise to sperm or eggs in the adult. Sea urchin PGCs are formed at the fifth cleavage, undergo one additional division during blastulation, and migrate to the coelomic pouches of the pluteus larva. The goal of this chapter is to detail classical and modern techniques used to analyze primordial germ cell specification, gene expression programs, and cell behaviors in fixed and live embryos. The transparency of the sea urchin embryo enables both live imaging techniques and in situ RNA hybridization and immunolabeling for a detailed molecular characterization of these cells. Four approaches are presented to highlight small micromeres with fluorescent molecules for analysis by live and fixed cell microscopy: (1) small molecule dye accumulation during cleavage and blastula stages, (2) primordial germ cell targeted RNA expression using the Nanos untranslated regions, (3) fusing genes of interest with a Nanos2 targeting peptide, and (4) EdU and BrdU labeling. Applications of the live labeling techniques are discussed, including sorting by fluorescence-activated cell sorting for transcriptomic analysis, and, methods to image small micromere behavior in whole and dissociated embryos by live confocal microscopy. Finally, summary table of antibody and RNA probes as well as small molecule dyes to label small micromeres at a variety of developmental stages is provided.

PubMed ID: 30777180
PMC ID: PMC6487853
Article link: Methods Cell Biol
Grant support: [+]

Genes referenced: LOC100887844 LOC115919910 nanos2l
Antibodies: ddx4 Ab6 ddx4 Ab7 ddx4 Ab8 methyl-LOC754126 Ab1

References [+] :
Campanale, Programmed reduction of ABC transporter activity in sea urchin germline progenitors. 2012, Pubmed, Echinobase