Temporal remodeling of the cell cycle accompanies differentiation in the Drosophila germline

Abstract

Development of multicellular organisms relies upon the coordinated regulation of cellular differentiation and proliferation. Growing evidence suggests that some molecular regulatory pathways associated with the cell cycle machinery also dictate cell fate; however, it remains largely unclear how the cell cycle is remodeled in concert with cell differentiation. During Drosophila oogenesis, mature oocytes are created through a series of precisely controlled division and differentiation steps, originating from a single tissue-specific stem cell. Further, germline stem cells (GSCs) and their differentiating progeny remain in a predominantly linear arrangement as oogenesis proceeds. The ability to visualize the stepwise events of differentiation within the context of a single tissue make the Drosophila ovary an exceptional model for study of cell cycle remodeling. To describe how the cell cycle is remodeled in germ cells as they differentiate in situ, we used the Drosophila Fluorescence Ubiquitin-based Cell Cycle Indicator (Fly-FUCCI) system, in which degradable versions of GFP::E2f1 and RFP::CycB fluorescently label cells in each phase of the cell cycle. We found that the lengths of the G1, S, and G2 phases of the cell cycle change dramatically over the course of differentiation, and identified the 4/8-cell cyst as a key developmental transition state in which cells prepare for specialized cell cycles. Our data suggest that the transcriptional activator E2f1, which controls the transition from G1 to S phase, is a key regulator of mitotic divisions in the early germline. Our data support the model that E2f1 is necessary for proper GSC proliferation, self-renewal, and daughter cell development. In contrast, while E2f1 degradation by the Cullin 4 (Cul4)-containing ubiquitin E3 ligase (CRL4) is essential for developmental transitions in the early germline, our data do not support a role for E2f1 degradation as a mechanism to limit GSC proliferation or self-renewal. Taken together, these findings provide further insight into the regulation of cell proliferation and the acquisition of differentiated cell fate, with broad implications across developing tissues.

A demographic model for estimating the inter-division lifespans of stem cells and the subsequent transit amplifying stages

“…To understand whether there is any correlation between the cell cycle phases and the rates of TA divisions, we scored the number and relative proportion of cysts marked for different cell cycle stage markers, spanning from G1 to M phases in wild-type testes ( Figure 1D, E, F). A previous study in Drosophila ovary had reported a progressive shrinkage of the Manuscript text, Tables and Figures Gadre et al, 2019 G2 phases after the second TA division, and an increase in the length of M and S phases (Hinnant et al 2017). However, these results were based on Fly FUCCI reporter genes driven by nosGAl4vp16 (nos>).…”

“…However, these results were based on Fly FUCCI reporter genes driven by nosGAl4vp16 (nos>). nos> driver leads to a non-uniform expression of the downstream UAS transgene (Hinnant et al 2017), which can skew the cell cycle phase distributions. Such false negative results were apparent in cases where the germ cells were positive for 5-ethynyl-2′-deoxyuridine (EdU) or Phospho-histone3 (pH3) but expressed no FUCCI marker genes (Hinnant et al 2017).…”

“…nos> driver leads to a non-uniform expression of the downstream UAS transgene (Hinnant et al 2017), which can skew the cell cycle phase distributions. Such false negative results were apparent in cases where the germ cells were positive for 5-ethynyl-2′-deoxyuridine (EdU) or Phospho-histone3 (pH3) but expressed no FUCCI marker genes (Hinnant et al 2017). In contrast to their observations, we observed that at the 8-cell and 16cell stage, the percentage of cysts marked by Cyclin E decreased, and the percentage of cysts expressing Cyclin B increased ( Figure 1E and F).…”

“…Finally, a visible increase in the nuclear size during the time-lapse series was identified as the end of telophase (Figure 3A-g). Accordingly, the Mphase period was estimated to take an average of 70 minutes for GSCs.The previous study in Drosophila ovary reported that the M-phase period varies significantly across TA stages based on the mitotic indices of different TA stages(Hinnant et al, 2017). Estimation of M-phase periods using time-lapsed imaging (Fig.…”

Ecdysone signaling promotes expression of multifunctional RNA binding proteins essential for ovarian germline stem cell self-renewal inDrosophila

“…Other genetic tools are described in FlyBase. Genetic mosaics were generated using FLP/FRT-mediated recombination in 1-3 day old females carrying a mutant allele in trans to a wild-type allele (linked to a Ubi-GFP or NLS-RFP marker) on homologous FRT arms with a hs-FLP transgene, as previously described (Hinnant et al, 2017;Laws and Drummond-Barbosa, 2015). Flies were heat shocked at 37°C twice per day 6-8 hours apart for 3 days, then incubated at 25°C on standard media supplemented first with dry yeast, then with wet yeast 3 days prior to dissection.…”