Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.


Introduction
The periodic formation of segments along the vertebrate body axis, or somitogenesis, is evidence of a spatial and temporal oscillator in the developing embryo. The favored mechanism controlling somitogenesis is conceptually described by a "clock and wavefront" model 1 , wherein the "clock" consisting of cellular oscillators, now thought to be intracellularly driven by the rhythmic expression of a set of cyclic genes 2 , ticks off the formation of somites from the presomitic mesoderm (PSM). As the embryo develops, a maturation "wavefront" in the PSM moves in concert with the regressing tissue towards the posterior, slowing and arresting cellular oscillators as it passes 3 . Together, this spatiotemporally dynamic system is termed the segmentation clock. Current approaches to study the segmentation clock span three increasing levels of organization from the genetic oscillator in single cells to local coupling that occurs between cells and finally to global regulation of positional information in the collective PSM tissue 4 .
Previous studies suggest the cell-autonomous segmentation oscillator in zebrafish consists of genes and protein products from the her/hes transcription factorfamily, which are thought to form a negative feedback loop through transcriptional repression [5][6][7] . The Delta/Notch signaling pathway synchronizes oscillations between neighboring cells and regulates the collective period of the population [8][9][10] . Fgf signaling molecules produced in the tailbud appear to build a gradient across the zebrafish PSM, and are thereby hypothesized to contribute to slowing and arresting oscillating cells in the anterior 11 . Until now, the functional roles of each of these molecules in somitogenesis have been investigated by genetic mutation, morpholino injection, heat-shock over-expression, and antagonist drug treatment of clock components and signaling between cells 5,7,10,12 . Using these perturbations, segmentation clock function has been inferred from tissue-level descriptions of somite defects and the loss of uniform oscillations in expression of cyclic genes like her1, her7, and deltaC. However, nearly all of these data are from fixed embryos and fail to accurately capture changes that are inherent to the dynamic function of the segmentation clock. More recently, multiple-embryo timelapse imaging has revealed the first mutants with altered oscillator period, but these observations were also made at the tissue level 7,13 . Thus, behavior of the hypothesized cell-autonomous oscillator during somitogenesis has not been observed.
Static snapshots of somitogenesis give an incomplete picture because, inherently, the process is driven by an oscillatory system. Earlier work in mouse and chick cells showed that levels of transcript and protein rise and fall, but sampling an approximately 2 hr oscillation every 30 or 45 min necessarily restricts the data collected and therefore the conclusions that can be drawn 14,15 . Study of other biological oscillators, most notably, circadian clocks, has moved away from staged measurements of gene and protein expression to real-time monitoring using fluorescent and bioluminescent reporters maintain their phase relations. Additionally, the effects of signaling molecules found in the embryo on the cell-autonomous clock have not been directly tested. Consequently, these fundamental properties of the single cell oscillator remain completely unknown.
We have recently developed transgenic fish lines using BAC recombineering 20 to drive Venus (YFP) fluorescence reporters of her1 expression 30 . Such lines exploit the regulatory cues of the intact chromosomal locus in which they are embedded and recapitulate the temporal dynamics and spatial pattern of her1. This breakthrough allows for real-time monitoring of gene expression in the developing zebrafish embryo in vivo. To study the fundamental properties of cell-autonomous oscillations and how such expression is regulated over time, we recently developed a reliable method to isolate and record from PSM cells in vitro. This protocol describes how we have utilized our transgenic reporter lines to generate dispersed cell cultures, from which we can characterize the oscillations of the zebrafish segmentation clock in single cells. We can thereby tackle outstanding questions in the field that were not accessible with static or tissue-level analysis, as well as directly manipulate the segmentation clock at the single cell level with signaling molecules and inhibitors. Sub-optimal cultures will contain cells that do not remain rounded and healthy throughout the recording. We hypothesized that our ECM substrate, a fragment of zebrafish fibronectin1 keeps cells in an undifferentiated PSM-like state, in comparison to other commonly used substrates like poly-lysine or laminin. Other substrates we tested caused cells to flatten on the glass and the loss of oscillating fluorescent signal over the course of the recording. We also found that the addition of serum to the medium during dissection, dispersal, and recording was not only important to quench the trypsin used for dissociation, but also increased fluorescence intensity over background, likely due to improved cell viability. To ensure optimal signal capture from single cells we used a 40x objective specifically designed for fluorescence imaging (Zeiss Plan NeoFluor series) with a high NA. We also found that a solid-state light source provided more stable illumination than traditional mercury lamps, which is critical to minimize background fluctuations that contribute to noisy images. These modifications are important to ensure robust results.
Using this protocol we expect cultures in which the majority of cells in a given field are fluorescent at some point during the recording. We find that fluorescent cells typically remain rounded and are sometimes quite motile during recordings. A few cells, including fluorescent cells, may become apoptotic during the recording. These cells are excluded from any analysis. We also exclude cells that come into contact with other cells or move outside of the field of view. On average, we see a 12% reduction in cell number per field by the end of the 10 hr recording as measured by counting the number of healthy cells in the transmitted light channel. This loss includes both cell death and cells that have moved out of the field. Given these caveats we can, on average, track 5 fluorescent cells per field that remain viable and visible, and typically record 6 fields per condition. For example, an experiment with 4 conditions will have 24 total fields acquired and typically about 30 tracked cells per condition. Under standard imaging conditions with L15 medium containing 10% fetal bovine serum, we find that PSM cells (n=101 cells from 4 experimental replicates) can produce between 2 and 7 peaks, with the mean and standard deviation of 3±1 peaks (2-3 cycles). The median peak number, as well as the 25% percentile, is 2 peaks and the 75% percentile is 3 peaks. Using our semi-automated tracking and analysis, we can quickly generate fluorescence intensity over time traces for individual PSM cells, with a representative cell trace shown in Figure 2C. These raw traces can then be used to make quantitative measurements of properties of the oscillating PSM cells, such as frequency, amplitude, number of cycles, and timing of peaks.

Discussion
To study a cellular process that takes place over the course of embryonic development, biologists typically utilize an approach in the context of the entire embryo. However, to fully understand how a single cell behaves within a developmental time frame, a method to examine and perturb individual cells in isolation is also highly beneficial. By generating dispersed PSM cell cultures using transgenic zebrafish embryos we now have a tool to directly study the cell autonomous nature of genetic oscillations in segmentation clock in a quantitative way. It is possible to measure dynamics of the oscillations in our fluorescent reporter in hundreds of cells under a variety of conditions. The observed period of single cells in culture is longer than the somitogenesis period in the intact embryo. We observe that an intact PSM explant in culture also exhibits slower oscillations than the intact embryo (data not shown), suggesting that the longer period observed in isolated cells is not simply due to damage from dispersal. A variable period and amplitude is observed in most of our single-cell time series. The source of this variability is unknown, but should reveal important details about the segmentation clock's pace-making circuits.
With this method we can study the genetic components of segmentation at the cellular level, and address questions that are challenging to examine in the whole embryo. For example, by controlled addition of known signaling molecules found in the developing embryo to our cultures, we can examine their effects on the single PSM cellular oscillator in a robust and reproducible assay. Our PSM culture system opens the door to a rigorous evaluation of what factors, alone, or in combination promote oscillations in these cells, what factors inhibit these oscillations, and to test the interplay between such molecules. With these tools in hand, we aim to evaluate existing models of somitogenesis that are based on published tissue-level data, as well as use results in single cells to generate predictions that can be tested in the whole embryo.
To our knowledge, this is the first zebrafish primary cell culture protocol for acute time-lapse recording of fluorescence in single cells; further development and refinement of this protocol is no doubt possible. Other protocols often use zebrafish embryos to generate stable cell lines that can be transfected with reporters and used for long-term imaging [27][28][29] . While stable lines are useful for imaging a process that is not tied to development timing, such as the circadian clock, the question of embryonic segmentation necessitates immediate imaging while cells are still in their oscillatory, progenitor state. Once cells stop oscillating, they assume a differentiated cell fate, and when in the tissue, would be incorporated