Abstract
AbstractBlastomeres of early vertebrate embryos undergo numerous fate choices for division, motility, pluripotency maintenance and restriction culminating in various cell lineages. Tracing blastomere fate choices at the single cell level in vitro has not been possible because of the inability to isolate and cultivate early blastomeres as single cells. Here we report the establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell embryos for fate tracing at the single cell level in vitro. Interestingly, these blastomeres immediately upon isolation exhibit motility, lose synchronous divisions and even stop dividing in &8805;50% cases, suggesting that the widely accepted nucleocytoplasmic ratio controlling synchronous divisions in entire embryos does not operate on individual blastomeres. We even observed abortive division, endomitosis and cell fusion. Strikingly, ~5% of blastomeres in single cell culture generated extraembryonic yolk syncytial cells, embryonic stem cells and neural crest-derived pigment cells with timings mimicking their appearance in embryos. We revealed the maternal inheritance of key lineage regulators and their differential expression in cleavage embryos. Therefore, medaka blastomeres possess the accessibility for single cell culture, previously unidentified heterogeneity in motility, division, gene expression and intrinsic ability to generate major extraembryonic and embryonic lineages without positioning cues. Our data demonstrate the fidelity and potential of the single cell culture system for tracking blastomere fate decisions under defined conditions in vitro.
Highlights
Blastomeres of early vertebrate embryos undergo numerous fate choices 2 for division, motility, pluripotency maintenance and restriction culminating in various cell lineages
The establishment of a single blastomere culture system and its 14 exploitation in tracing cell fate choices lead to twelve important findings that are novel or different from previous results obtained in whole embryos
In situ hybridization reveals that maternal RNAs often do not completely enter the cellular blastodisc but leave a substantial level in the yolk to form an area beneath the blastodisc, indicating the presence of a barrier 2 between the yolk and blastodisc as early as the 2-cell stage
Summary
24 Key words: blastomere, cleavage, fate tracing, neural crest, pigment, single cell culture, yolk syncytium. We report the 6 establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell 8 embryos for fate tracing at the single cell level in vitro. We began with the 16-cell stage (Figure 1e) to examine cell divisions as 2 the first fate choice of blastomeres In many animals, both invertebrates and vertebrates including Xenopus[16] and zebrafish[14], all blastomeres until the 4 midblastula transition undergo 10~12 abbreviated cycles of rapid synchronous cell divisions without G1 and G2 phases and motility[16]. Blastomeres have the heterogeneity in division, 18 suggesting again the presence of novel mechanisms in medaka other than the nucleocytoplasmic ratio
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