Background

While permitting the visualization of various tissue-resident stem cells in several model systems, recent advances in intravital imaging often rely on invasive surgery or sophisticated and expensive imaging modalities ( Yoshida et al., 2007; Rompolas et al., 2012; Ritsma et al., 2014; Barbosa et al., 2015; Park et al., 2016; Martin et al., 2018; Nguyen and Currie, 2018). C. elegans GSCs are an established stem cell model that has yielded generalizable insights into many aspects of stem cell biology (Kimble and White, 1981; Baugh and Sternberg, 2006; Fielenbach and Antebi, 2008; Angelo and Van Gilst, 2009). In addition, C. elegans GSCs can be imaged in living animals using standard fluorescence microscopy techniques and without surgical manipulation. In particular, live imaging of GSC mitosis provides an opportunity to investigate the dynamics of cell division and how they are influenced by in vivo factors such as tissue organization, niche signaling, and organismal physiology. Here we describe a simple, fast, and reproducible method to immobilize C. elegans and to image and analyze GSC mitosis, while preserving animal viability, fertility, and seemingly normal GSC divisions.

Adult C. elegans hermaphrodites house two populations of GSCs located at the distal ends of the two tube-shaped gonad arms (Figure 1A). Like other germlines, the C. elegans gonad is organized as a syncytium. GSCs form a rough circumferential monolayer around a shared inner core of cytoplasm named the rachis (Hirsh et al., 1976) (Figure 1A and 1C). Each GSC is connected to the rachis by a stable actomyosin ring, which forms a cytoplasmic bridge (Maddox et al., 2005; Zhou et al., 2013; Amini et al., 2014; Priti et al., 2018). Like other stem cells, C. elegans GSCs are kept in a stem-like state by signaling from a somatic niche (the distal tip cell, Kimble and White, 1981; Figure 1A and 1E). Like several types of mammalian stem cells, the size of the C. elegans GSC pool is maintained according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions to maintain a relatively constant number of stem cells. according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions, thus maintaining a relatively constant number of stem cells (Morrison and Kimble, 2006; Joshi et al., 2010).

The majority of studies in C. elegans GSCs rely on dissected, fixed, and stained gonads, or single-timepoint imaging of gonads bearing fluorescently tagged proteins in living worms. Long-term imaging of GSCs has been accomplished using “catch and release” approaches that permit periodic visualization of GSCs over hours or days (Wong et al., 2013; Rosu and Cohen-Fix, 2017). Moreover, a recently developed microfluidics device may permit continuous observation on a similar time scale (Berger et al., 2018, Berger and Spiri, 2021). Fewer studies have reported live cell imaging of GSCs under conditions suitable for documenting dynamic subcellular events such as mitosis (Gerhold et al., 2015; Gordon et al., 2020; Zellag et al., 2021).

To achieve high temporal and spatial resolution during live imaging, animals must be immobilized and the overall impact of mounting on animal and GSC physiology must be considered. Typical mounting methods immobilize animals using a combination of paralytic drugs and physical compression (Sulston and Horvitz, 1977; Chai et al., 2012; Kim et al., 2013; Hwang et al., 2014; Luke et al., 2014; Burnett et al., 2018; Fabig et al., 2020a and 2020b; Gordon et al., 2020). We use an etched silicon wafer to pattern grooves on an agarose pad, which constrains the animals in a straight position (Figure 1B); this prevents the sinusoidal movements required for worm locomotion (Gray and Lissmann, 1964) and reduces the requirement for physical compression and anesthetics.

Figure 1. A mounting method allowing for live-imaging of dividing GSCs in intact C. elegans. A. Schematic showing a top view of a late L4 (top) and an adult (bottom) hermaphrodite worm mounted in grooves. The position and overall organization of the two gonad arms are shown in white, and key germline features are highlighted, such as the rachis (green), and the distal tip cell (DTC) or niche (magenta). The position of the GSCs is indicated by purple circles. B. Schematic showing a cross-sectional view of animals mounted in v-shaped (top) and square-shaped (bottom) grooves that match the width of the animals. (C-E) Maximum intensity projections of the distal gonad from adult animals expressing (C) mNG::ANI-1 (green) and mCH::β-tubulin (magenta; strain = UM679), D. GFP::β-tubulin (green) and mCH::Histone H2B (magenta; strain = JDU19), and (E) LAG-2::GFP (green) and mCH::β-tubulin (magenta; strain = UM211). A dividing GSC in each gonad is boxed in yellow and an enlarged image is shown below. Fluorescently labelled β-tubulin marks the mitotic spindle in all three strains, with mNG::ANI-1 marking the rachis, mCH::Histone H2B marking nuclei and LAG-2::GFP marking the DTC/niche. Scale bar = 10 µm (top, gonad view) and 5 µm (bottom, single cell view).

We have used this mounting method to provide the first characterization of GSC mitosis by live imaging (Gerhold et al., 2015). Recently, we investigated the technical factors that impact GSC mitosis during live imaging, which allowed us to define optimal mounting and imaging conditions, and to determine that animal starvation during mounting/imaging has the most deleterious effect on GSC mitosis (Zellag et al., 2021). Our data suggest that GSC mitosis can be imaged under near physiological conditions within an approximately 40 min window, which starts from the moment worms are removed from food. Although some GSCs enter mitosis after 40 min, we recommend caution when interpreting their behavior. In addition, under optimal conditions, the number of mitoses per germline and the duration of these mitoses in wild-type worms are relatively constant, and offer a reproducible baseline by which others may benchmark their results (see Videos 1 and 2 and Figure 4I-4K).

A parallel challenge that arises when using live imaging to study GSC mitosis is how to monitor mitotic progression. GSCs divide within the tube-shaped gonad and can divide in any orientation relative to the imaging plane, and at a range of depths relative to the gonad surface. Therefore, accurate monitoring of GSC mitosis requires a tracking method that accounts for the three-dimensional (3D) nature of their division. A good method will need to be sufficiently high-throughput to generate large data sets with minimal user time. In addition, fluorescently labelled proteins are often expressed at low levels in the germline (Merritt and Seydoux, 2010), which limits the availability of suitable markers.

We have shown that fluorescently tagged β-tubulin provides a robust marker for GSC mitotic centrosomes and that we can use centrosome-to-centrosome distance as a reliable readout for mitotic progression by tracking centrosome pairs in 3D (Gerhold et al., 2015; Zellag et al., 2021). In addition, tracking centrosome pairs can provide information on mitotic features, such as spindle dynamics and orientation (Zellag et al., 2021). To make this approach amenable to large-scale studies, we recently developed CentTracker, a largely automated image analysis pipeline that allows for fast extraction of mitotic parameters in any genetic background and in other cell types and organisms, provided that centrosomes are trackable (Zellag et al., 2021). Here, we describe the basic imaging processing steps required for using CentTracker. The relevant code and detailed instructions are freely available for download.