Illuminating cell-cycle progression in the developing zebrafish embryo

Abstract

By exploiting the cell-cycle-dependent proteolysis of two ubiquitination oscillators, human Cdt1 and geminin, which are the direct substrates of SCF Skp2 and APC Cdh1 complexes, respectively, Fucci technique labels mammalian cell nuclei in G 1 and S/G2/M phases with different colors. Transgenic mice expressing these G 1 and S/G2/M markers offer a powerful means to investigate the coordination of the cell cycle with morphogenetic processes. We attempted to introduce these markers into zebrafish embryos to take advantage of their favorable optical properties. However, although the fundamental mechanisms for cell-cycle control appear to be well conserved among species, the G 1 marker based on the SCF Skp2 -mediated degradation of human Cdt1 did not work in fish cells, probably because the marker was not ubiquitinated properly by a fish E3 ligase complex. We describe here the generation of a Fucci derivative using zebrafish homologs of Cdt1 and geminin, which provides sweeping views of cell proliferation in whole fish embryos. Remarkably, we discovered two anterior-toposterior waves of cell-cycle transitions, G 1/S and M/G1, in the differentiating notochord. Our study demonstrates the effectiveness of using the Cul4 Ddb1 -mediated Cdt1 degradation pathway common to all metazoans for the development of a G 1 marker that works in the nonmammalian animal model. cell cycle ͉ fluorescent protein ͉ imaging ͉ ubiquitination E ukaryotic cells ensure tight regulation of cell division by maintaining close control over the levels of cell-cycle proteins. For example, Cdt1 and geminin have opposite effects on DNA replication during S phase, and their levels fluctuate accordingly throughout the cell cycle (1, 2). Cdt1 levels are highest in G 1 phase just before DNA replication and decrease as cells transition into S phase, whereas geminin levels rise during S phase and fall during G 1 phase. Cells control Cdt1 and geminin activity at the protein level by ubiquitination, which precisely targets unwanted proteins for destruction.We harnessed the regulation of cell-cycle-dependent ubiquitination to develop a genetically encoded indicator for cell-cycle progression: Fucci ( fluorescent ubiquitination-based cell cycle indicator) (3). The original Fucci probe was generated by fusing mKO2 (monomeric Kusabira Orange2) and mAG (monomeric Azami Green) to the ubiquitination domains of human Cdt1 (hCdt1) and human geminin (hGem): hCdt1(30/120) and hGem(1/110), respectively. These two chimeric proteins, mKO2-hCdt1(30/120) and mAG-hGem(1/110) ( Fig. 1 A and B), accumulate reciprocally in the nuclei of transfected mammalian cells during the cell cycle, labeling nuclei of G 1 phase cells orange and those in S/G 2 /M phase green. Thus, these proteins function as effective G 1 and S/G 2 /M markers. We also developed a S/G 2 /M marker, mAG-hGem(1/60), which accumulates in both the nucleus and cytoplasm (4) and reveals the morphology of individual cells that have undergone DNA replication. This permits cell proliferation to be monitored along with t...

Protein tyrosine phosphatase PTPN9 regulates erythroid cell development through STAT3 dephosphorylation in zebrafish

“…The Tg(ef1a:mAG-zGem) transgene has been shown to label S, G2 and M cells with green fluorescence (Sugiyama et al, 2009). We found comparable numbers of cells that were positive for green fluorescence in the ICM region in ptpn9a-MO-injected and wild-type Tg(ef1a:mAG-zGem) transgenic embryos at 24 hpf (Fig.…”

“…Tg(ef1a:mAG-zGem) transgenic reporter zebrafish for the S, G2 and M phases of the cell cycle were generated, as described previously (Sugiyama et al, 2009). Tg(gata1:DsRed) were kindly provided by Shuo Lin (University of California Los Angeles, Los Angeles, CA).…”

Cdc25 and the importance of G2control

“…41 For example, the Fucci approach reveals that retinal cells of the early embryo are all in the S/G 2 /M state (Fig. 5B), 49 and it will be worthwhile to examine the role of Cdc25 in regulating the cell cycle state of these cells.…”

“…42,49 One note of caution, it is important not to use promoters that express the Fucci at high levels, or cells will not degrade the fluorescent indicators or faithfully report the state of the cell cycle. Even with this caveat, Fucci promises the ability to identify new instances where cells are held in G 2 in different animal systems at all stages of development and in all tissues.…”

“…Fucci uses fluorescently labeled protein fragments that are differentially degraded as a live cell moves through the cell cycle. 49 In the latest incarnation, called Dual Fucci, a red G 1 /G 0 indicator and a blue G 2 /S/M indicator are expressed from a single transcript in transgenic zebrafish embryos and adults using a ubiquitous promoter (Fig. 5A).…”