**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 [11–13] and developmental biology [14–16]. 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.
