Expression patterns of prespore genes. RNAs prepared at 2-h intervals throughout development on filters were analyzed on microarrays. Values were normalized to 1 at the time of initiation of development, and the fold changes are presented for the 54 prespore genes analyzed in this study. Values are the averages of four independent determinations. Genes showing the greatest changes are indicated by name.

Prespore regions in slugs of wild-type and DIF-less slugs. Expression of three prespore genes was analyzed by in situ hybridization on more than 20 slugs prepared from wild-type Ax2 and dmtA mutant strains. Proportional lengths were measured, and the ratio and standard deviations were calculated for each gene by NIH image.

Cell type conversion in isolated prestalk fragments. The anterior 20% of wild-type Ax2 slugs and the anterior 10% of dmtA mutant slugs were dissected and incubated in isolation. At the indicated times (in hours) after removal of the posterior tissue, the prespore marker SSJ770 (pspA) was used for in situ hybridization. Cell type conversion was apparent in the back of the fragments within a few hours.

Prestalk regions in wild-type and DIF-less slugs. Expression of three PST-O genes and two PST-O genes was analyzed by in situ hybridization of 20 slugs prepared from wild-type Ax2 and dmtA mutant strains. Proportional lengths were measured, and the ratio and standard deviations were calculated for each gene. The PST-O genes (SSH630 and SSM184) are not expressed in the most anterior cells. The anterior as well as the posterior borders were measured for these genes.

In situ hybridization with prespore genes. The pattern of staining of posterior cells by SLB126 (cotC) for mRNA (a to d) or antisense RNA (a′ to d′) is representative of almost all of the prespore genes. Patterns for each of the 54 prespore genes can be inspected at http://www.csm.biol.tsukuba.ac.jp/∼tools/bin/ISH/index.html. Only SSH707 and two other cDNAs derived from the same gene, which encodes a protein with no significant homologs, gave a significantly different pattern (e to h′). Up to the culmination stage its mRNA accumulated in posterior cells just like that of other prespore genes; however, its antisense RNA was seen in anterior cells (f′). During culmination, SSH704 mRNA disappeared from the prespore cells and both mRNA and antisense RNA accumulated in cells at the back of the prestalk domain.

Double-staining in situ hybridization. (A) Slugs formed from wild-type Ax2 and dmtA mutant strains were hybridized with prestalk probes to generate a green signal and were hybridized simultaneously with prespore probe SSJ770 (pspA) to generate a red signal. (a to d′) SLE474 (ecmA); (e to h′) SSA854; (i to l′) SSM184. (a to l) wild-type Ax2; (a′ to l′) dmtA mutant strain. (a, a′, e, e′, i, and i′) Prestalk gene expression revealed by the Alexa 488 signal. (b, b′, f, f′, j, j′) Expression of SSJ770 (pspA) revealed by the rhodamine signal. (c, c′, g, g′, k, k′) Merged pictures. (d, d′, h, h′, l, l′) Enlarged slug anteriors. The stained regions did not overlap and were separated by unstained cells in the case of SSA854. (B) Simultaneous analysis of spatial expression patterns of PST-AO and PST-O genes. Slugs formed from wild-type Ax2 (a to c) and dmtA mutant strain (a′ to c′). Expression of SSM184 (a PST-O gene) was detected with rhodamine (a), while expression of SLE474 (ecmA, a PST-AO gene) in Ax2 slugs was detected with Alexa 488 (b). (c) Merged picture of the images shown in panels a and b. In dmtA mutant slugs, SSM184 was detected with Alexa 488 (a′), while SLE474 was detected with rhodamine (b′). (c′) Merged picture of the images shown in panels a′ and b′. Regions with cells expressing both the PST-O and the PST-AO genes appear yellow in the merged pictures.