Imaging Promoter Assay of Adenylyl Cyclase A Gene in Dictyostelium discoideum during Fruiting Body Formation by Dual-Color Bioluminescence Microscopy

1. Introduction

Gene expression and regulation are essential processes in cellular proliferation and differentiation and are involved in morphogenesis and embryogenesis. The social amoebae Dictyostelium discoideum is known to have a simple life cycle, short generation time, and small genome size. Thus, it is a model organism that is often used in research on morphogenesis, especially the formation of fruiting bodies from amoeba cell aggregation. This cell aggregation is mediated by extracellular cyclic adenosine monophosphate (cAMP) [1]. Then, cAMP is secreted from the tip of the cell mound for prestalk and prespore cell migration [2]. High concentrations of extracellular cAMP are required for prestalk and prespore cell differentiation [3, 4] and spore formation [5]. Thus, cAMP plays an important role in Dictyostelium discoideum development. Its synthesis is catalyzed by adenylyl cyclases A, B, and G, which are encoded by the genes acaA, acrA, and acgA, respectively [6, 7].

Adenylyl cyclase A is considered a development-specific enzyme [8, 9]. Galardi-Castilla et al. [10] characterized the promoter region of the acaA gene in Dictyostelium discoideum Ax4 cells by histochemistry using a lacZ/X-Gal staining system, β-galactosidase reporter system, quantitative RT-PCR, and in situ hybridization. The acaA gene is transcribed under the control of three different alternative promoters: promoter 1 (distal region), promoter 2 (intermediate region), and promoter 3 (proximal region). Promoter 1 is active during the cell aggregation stage, and promoters 2 and 3 are active in the mound, slug, and fruiting body stages [10].

Promoter assays using histochemical techniques are quite cumbersome, as the samples have to be fixed, stained, and observed over time sequentially for each promoter. For this purpose, many samples must be prepared. On the other hand, a promoter assay using bioluminescence microscopy can be used to obtain time-lapse image data from a single experiment using one sample. This method is often used in the study of clock genes [11, 12, 13] and developmental biology [14, 15, 16]. In this chapter, we applied bioluminescence microscopy and used two luciferases in the development of an acaA promoter assay that can monitor acaA promoters 1 and 3 simultaneously during Dictyostelium discoideum development, and compared the result with those of histochemistry and β-galactosidase reporter system [10]. We also demonstrated the advantages of this promoter assay and discussed the perspectives that need further consideration.

2. Materials and methods

3. Results and discussion

Bright-field and bioluminescence images of the activities of promoters 1 and 3 are shown in Figure 1. According to the bright-field image, the amoeba cells began to aggregate after 10 h of seeding (Figure 1A) and formed the mound (Figure 1D), slug, and fruiting body (Figure 1F) after 16, 18, and 20 h, respectively. The image of the slug is not shown in Figure 1, since the moving slug disappeared from the field of view. In the case of Figure 1, the slug stage was extremely short and the fruiting body formation occurred immediately from the mound.

According to the bioluminescence image, promoter 1 activity was observed in single amoeba cells. It increased gradually (Figure 1A–C) and peaked at the mound stage after 16 h (Figure 1D). Then, it decreased during fruiting body formation (Figure 1E–G) and eventually disappeared (Figure 1H). On the other hand, promoter 3 activity increased during the cell aggregation stage (Figure 1C), peaked during the fruiting body stage (Figure 1F), and then decreased (Figure 1G and H). Thereafter, the activity of the stalk cells disappeared. Histochemical detection of promoter activity using a lacZ/X-Gal staining system [10] showed that promoter 1 activity was detected in the cell aggregation and early mound stages, but was not detected after the slug stage. On the other hand, promoter 3 activity was detected in the early mound, slug, and spore stages. This activity was particularly strong in the upper cup of the spores. The two methods yielded almost the same results, but promoter 3 activity was detected earlier by bioluminescence imaging than by histochemical detection at the cell aggregation stage after 14 h (Figure 1C). However, the resolution of the images obtained by histochemical detection was superior to that by bioluminescence imaging.

To show the time course of the promoter activities, two regions of interest (ROI) (ROI-1 and ROI-2) were assigned to cover the process from cell aggregation to fruiting body formation, as shown in Figure 1. Figure 2 shows the time course of luminescence intensity reflecting the activities of promoters 1 and 3 in ROI-1 (Figure 2A) and ROI-2 (Figure 2B) using 961 time-lapse images captured at 90s intervals for 24 h. The intensity of promoter 1 activity increased after 13 h, peaked after 16 h, decreased, and disappeared after 24 h in ROI-1 and ROI-2. On the other hand, the intensity of promoter 3 activity increased to the same timing as that of promoter 1 after 13 h, but peaked after around 20–22 h. Then, the intensity decreased gradually in ROI-2, but rapidly decreased and recovered in ROI-1. Since the measurement of the intensities involves live imaging, the discrepancy may be caused by the movement of the spores in the ROI during fruiting body formation. The measurement of the time courses of the activities of promoters 1 and 3 by bioluminescence imaging and by a β-galactosidase reporter system [10] showed similar results.

The results of the promoter assay using bioluminescence microscopy were the same as those of the promoter assays using histochemistry and β-galactosidase, confirming the convenience of this imaging promoter assay for Dictyostelium studies. Moreover, the imaging promoter assay enabled the spatiotemporal information of promoter activity to be obtained sequentially in a single experiment. Based on the result, detailed analysis of promoter activity can be performed efficiently by histochemical or immunofluorescence microscopy. However, several experiments are required for each measurement in histochemical and β-galactosidase promoter assays. Multiple promoters can also be analyzed using multicolor luciferases, but the number of promoters evaluated is limited to the number of luciferases of different colors. Moreover, there are some concerns regarding the use of multicolor luciferases in bioluminescence microscopy as follows. Figure 3 shows the normalized luminescence spectra of the luciferases (variant 1 of Luci sp1 and Psa) in the transparent range of the emission filter for each luciferase channel. The luciferases were expressed in HeLa cells [17, 18]. In the transparent range of the 610ALP filter, cross talk between the two spectra was observed between 610 and 700 nm. To prevent spectral cross talk, a spectral unmixing operation must be done, as is performed in fluorescence microscopy [22]. Nakajima et al. [23] demonstrated the unmixing of tri-colored bioluminescence for a luciferase promoter assay using one color to normalize the activity of two genes. In addition to the number of promoters to evaluate, we need one more color luciferase to normalize different promoter activities.

One of the advantages of bioluminescence microscopy is that it is not affected by autofluorescence background. Figure 4 shows the bright-field and autofluorescence images of the mound, slug, and fruiting body stages of Dictyostelium discoideum development captured by fluorescence microscopy with a mirror unit for green fluorescent protein (GFP). Autofluorescence from the upper tip of the mound, the periphery of the slug body, and the spore and stalk of the fruiting body was observed. Therefore, the imaging conditions used GFP as a reporter require optimization of the excitation intensity, etc.

4. Conclusion

The imaging promoter assay of the acaA promoters 1 and 3 by bioluminescence microscopy and the histochemical and β-galactosidase promoter assays yielded similar results in the evaluation acaA promoter activity in the different stages of Dictyostelium discoideum development. Moreover, we found that obtaining an overall picture of promoter activity spatiotemporally during the entire developmental process is possible with the bioluminescence imaging promoter assay. However, spectral unmixing is required to effectively normalize the different promoter activities. We also found that the imaging promoter assay is not significantly affected by autofluorescence.

Acknowledgments

We thank Prof. H. Kuwayama (Tsukuba University, Japan) for providing the materials needed in the Dictyostelium discoideum experiments, offering valuable advice, and allotting time for discussion. We also thank our colleagues at Olympus Corporation for providing technical assistance during the development of the LV200 bioluminescence microscope.

This work was conducted in association with Nimura Genetic Solutions and Perak State Development Corporation for luciferase gene cloning under a contract with the Convention on Biological Diversity in Malaysia. This research did not receive grants from funding agencies in the public, commercial, or non-profit sectors.

Conflict of interest

The authors T. Hayashi, K. Ogoh, and H. Suzuki are employees of Olympus Corporation (Tokyo, Japan).