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Whole-mount in situ hybridization of cell-type specific mRNAs in DictyosteliumDev Biol 171:262-6Contributed by Ricardo Escalante and William F. Loomis*
Center for Molecular Genetics, Department of Biology, University
of California, San Diego, La Jolla CA 92093
AbstractWe have been able to hybridize non-radioactive probes from cell-type specific genes to fixed whole-mounts prepared at the mound, slug and culminant stages of Dictyostelium development. The cellular patterns of labelling with probes from the prespore gene, cotB, and the prestalk genes, ecmA and ecmB, confirmed the patterns seen in strains carrying reporter constructs in which the regulatory regions of these genes drive b-galactosidase. This technique permits the direct observation of protein synthetic capacity from characterized genes without the need of generating transformed lines carrying specific reporter constructs. Moreover, the pattern is not complicated by previous developmental history of gene expression.IntroductionThe advent of cell-type specific molecular markers has facilitated the description and analysis of multicellular morphogenesis in many systems. Hybridization of probes prepared from specific cloned genes to whole-mounts can provide three dimensional information on cells that have accumulated the respective mRNA. This in situ technique has been developed and widely used in several developmental systems including Drosophila (Tautz and Pfeifle, 1989), Xenopus (Hemmati-Brivanlou et al., 1990), and quail (Coutinho et al., 1992) but has not been used previously in Dictyostelium. Another approach to recognizing specific cell-types is to transform with constructs in which the control regions associated with specific genes are used to drive reporter genes such as bacterial b-galactosidase. Cells carrying the prespore specific reporter construct cotB::lacZ can first be seen to stain 8 hours after the initiation of development (Fosnaugh and Loomis, 1993). Stained cells are initially found throughout the loose aggregates and subsequently found in the posterior of the slug shaped structures that form. At culmination, the stained cells are found in the rising sorus and can be seen to form spores. Cells carrying either of the prestalk specific reporter constructs ecmA::lacZ or ecmB::lacZ can first be seen to stain a few hours after the prespore cells (Williams et al., 1989). Like the prespore cells, the prestalk cells are initially found scattered throughout the mound. A little later the ecmA::lacZ positive cells sort to the tip and the ecmB::lacZ positive cells sort to the base (Williams et al., 1989). The ecmA::lacZ positive cells remain in the tip as it elongates to form a slug and enter the stalk tube during culmination (Jermyn and Williams, 1991; Early et al., 1993). About an equal number of ecmA::lacZ positive cells are found in the posterior of the slug where they are referred to as anterior-like cells (Early et al., 1993). Cells in the central core of the slug tip express ecmB::lacZ and are the first to enter the stalk. During culmination ecmB::lacZ is expressed in the upper and lower cups generated by anterior-like cells (Jermyn and Williams, 1991; Early et al., 1993).We have found that hybridization of probes prepared from cotB, ecmA and ecmB to cells fixed at the mound, slug or culmination stages gives the identical spatial and temporal patterns seen by staining the reporter strain with X-gal. The congruence of the data indicates that expression of the reporter genes accurately mimics the presence of mRNA from these genes throughout development. Our results show that whole-mount in situ hybridization can be usefully applied to Dictyostelium. Materials and MethodsGrowth and developmentDictyostelium discoideum strain AX4 was grown in HL5 medium and developed at high density on nitrocellulose filters saturated with buffered salts in a humid chamber (Sussman, 1987). Probe labelingWe used a 2.3 kb fragment from the clone gDd63 and a 2.4 kb fragment from the clone gDd56 (Jermyn et al., 1987; Williams et al., 1987) as the ecmA and ecmB probes respectivelly and a 0.7 kb fragment from the clone pSP70 as a cotB probe (Fosnaugh and Loomis, 1989). The DNA probes (50-100 ng) were labeled for 16 hours by random priming and incorporation of dUTP-digoxigenin using the DIG labeling kit from Boheringer Mannheim (cat. no. 1175033) according to the manufacturer's instructions. Probes were purified by ethanol precipitation. In situ hybridization
Samples collected at different times of development were transferred
to sterilized glass test tubes by washing with buffered salt solution
(PBS: 133 mM NaCl, 10 mM sodium potassium phosphate pH 7.4); all
subsequent steps were carried out in these tubes. The structures
were washed twice with methanol for 5 minutes and gently dispersed
before replacing it with 4% paraformaldehide in PBS. After fixation
for 2.5 hours at room temperature, the preparations were washed
3 times for 5 min each in PBS and incubated with 20 mg/ml Proteinase
K (Sigma) in PBS for 20 to 60 minutes, washed in PBS for 5 min,
and further fixed in 4% paraformaldehyde solution at room temperature
for 20 min. After three washes with PBS for 5 min each, the structures
were prehybridized at 42°C for 3 hours in 4X SSC (1X SSC:
150 mM NaCl, 15 mM sodium citrate pH 7), 1X Denhardt's, 0.5 mg/ml
sonicated salmon sperm DNA, 0.25 mg/ml yeast RNA, 60% formamide.
Hybridization was carried out at 42°C in 4X SSC, 0.5 mg/ml
denatured salmon sperm DNA, 0.25 mg/ml yeast RNA, 60% formamide
and 50 ng/ml of heat denaturalized probe.
Results and DiscussionSpatial localization of Prespore mRNA (Figure 1)Structures collected at different times of development were fixed and hybridized with digoxigenin labeled cotB probe and the hybrids detected with antibody to digoxigenin coupled to alkaline phosphatase. Some mounds which had developed for 12 hours were seen to have stained cells scattered about with no evidence of spatial localization (Fig. 1A) while others showed localization of the cotB reactive cells at the base and unstained cells at the tip (Fig. 1B, 1C). At the slug stage (15 hours of development) cells that had accumulated cotB mRNA were seen only in the posterior (Fig.1D, 1E). During culmination (20 hours of development) stained cells were restricted to the prespore cells in the sorus (Fig. 1F, 1G). This pattern of spatial and temporal localization of cotB mRNA is in agreement with those seen with antibodies to SP70, the product of cotB (Gomer et al., 1986), as well as those obtained in cells carrying cotB::lacZ (Fosnaugh and Loomis, 1993). Staining was reduced or absent after the prespore cells had encapsulated, indicating that permeabilization may not have been sufficient to open spores (data not shown). Spatial localization of Prestalk mRNAs (Figure 2)
Cells which had accumulated ecmA and ecmB mRNAs
were initially distributed randomly in the loose aggregates (Fig.
2A and 2H). ecmA positive cells were subsequently localized
to the tip (Fig. 2B,2C) while ecmB positive cells were
found in the base (Fig 2I, 2J). At the slug stage ecmA
expressing cells were seen in the prestalk region at the anterior
as well as scattered throughout the back where anterior-like cells
are found (Fig 2D, 2E). Cells expressing ecmB were localized
to a central core in the prestalk region as well as scattered
throughout the posterior (Fig 2K,2L). During culmination cells
expressing ecmA could be seen in the tip, in the stalk
and in the basal disk (Fig. 2F, 2G). We have observed that the
level of ecmA expression is higher in the most anterior
portion of the tip than in the region below where PST-O cells
are found during culmination in agreement with the previous observations
of Early et al. (1993) using reporter constructs. Cells
expressing ecmB during culmination were seen in the upper
and lower cups, the basal disk and the stalk (Fig. 2M, 2N, 2O).
References
FiguresFigure 1. Localization of cotB mRNA during Dictyostelium development by in situ hybridization. Structures were collected at diferent times of development and hybridized with digoxigenin labeled cotB probe. A, B and C are tight aggregates fixed after 12 hours of development; D and E are from the slug stage at 15 hours of development; F and G are culminants collected after 20 hours of development. Bar: 0.2 mm. 
Figure 2. Localization of ecmA and ecmB mRNAs. Structures were collected at diferent times of development and hybridized with digoxigenin labeled ecmA probe (A-G) or ecmB probe (H-O). A, B, C, H, I, J were collected at the tight aggregate stage after 12 hours of development; D, E, K, L were collected at the slug stage after 15 hours of development; and F, G, M, N, O were collected during culmination after 20 hours of development. Bar: 0.2 mm. 
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