Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses

Abstract

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

FIG. 1.

Prestalk cell types. (A) Anatomy of a slug. PstA cells are at the very front, and PstO cells are immediately behind them. PstAB cells form a central core at the anterior. Prespore cells are found in the posterior. (B) Representative in situ hybridization patterns: mRNA recognized by cDNA clone SLF308 is found only in PstA cells, mRNA recognized by clone SLA128 is found only in PstAB cells, mRNA recognized by cDNA SSM184 is found only in PstO cells, and mRNA recognized by clone SSJ314 is found in both PstA and PstO cells.

FIG. 2.

Developmental expression patterns of PstA genes. Spatial expression patterns of the three PstA genes that we identified in this study were analyzed by in situ hybridization. Expression patterns at the slug (b, g, and l), Mexican hat (c, h, and m), early culmination (d, i, and n), and late culmination (e, j, and n) stages are shown. SLF308 was expressed at high levels in the most anterior cells throughout development (a to e); expression of SSK861 decreased in PstA cells as culmination proceeded but transiently appeared in cells as they entered the stalk tube during early culmination (f to j). SLI271 recognized mRNA that became abundant in all prestalk cells during culmination (k to n).

FIG. 3.

Expression of representative PstAB genes. In situ hybridization analysis of 10 PstAB genes identified in this study revealed that these genes are regulated in four distinct patterns, exemplified by SLA128 (a to d), SLB233 (e to h), SLC388 (i to l), SSK348 (m to p), SSB312 (q to t), or SLK182 (u to x). (a, e, i, and u) Tipped aggregate stage; (b, f, j, m, q, and v) slug stage; (c, g, k, n, r, and w) Mexican hat stage; (o, s, and x) early culmination stage; (d, h, l, p, and t) late culmination stage. The insets in panels j and q show a higher magnification of the anterior portion of a slug in a rectangle in each panel.

FIG. 4.

Expression of representative PstO genes. We found 30 genes that are expressed in PstO cells but not in PstA cells. These genes can be grouped into three classes according to their expression patterns during culmination. SSM184 is a representative of six genes that are continuously expressed in PstO cells but at progressively reduced levels (a to d). SLG775 is a representative of six genes that become more strongly expressed in PstO cells during culmination and are also expressed in cells at the top of the stalk tube (e to h). SLH659 is a representative of the remainder of the PstO genes that continue to be strongly expressed in PstO cells throughout development (i to l). (a, e, and i) Slug stage; (b, f, and j) Mexican hat stage; (c, g, and k) early culmination stage; (d, h, and l) late culmination stage.

FIG. 5.

Genes expressed in PstA and PstO cells. We identified 38 PstAO genes that are expressed in both PstA and PstO cells in the slug stage. There were 26 genes that became preferentially restricted to PstO cells during culmination, as exemplified by SSH475 (a to f). SLE474 is a representative of six PstAO genes whose expression was maintained in all prestalk cells by early culmination (g to k). Their mRNAs could be seen in the vacuolized cells within the stalk tube (j). SSA854 is a representative of six PstAO genes that were expressed in both PstA and PstO cells up through early culmination and were subsequently repressed (l to p). (a) Mound stage; (b, g, and l) tipped aggregate stage; (c, h, and m) slug stage; (d, i, and n) Mexican hat stage; (e, j, and o) early culmination stage; (f, k, and p) late culmination stage.

FIG. 6.

Genes expressed in prestalk cells only during culmination. We found 23 genes which gave very little in situ signal at the slug stage. SSE634 is a representative of 14 of these genes that were subsequently expressed at high levels in PstO cells (a to d). Another nine genes, exemplified by SSG721, failed to show cell type specificity at the slug stage but were expressed in cells within the top of the stalk tube during culmination. Their mRNAs could be seen in vacuolized cells near the top of the stalk tube (e to h). (a and e) Slug stage; (b and f) Mexican hat stage; (c and g) early culmination stage; (d and h) late culmination stage.

FIG. 7.

Expression patterns of representative prestalk genes. RNAs prepared at 2-h intervals throughout development were analyzed on microarrays. The fold increase relative to time-averaged RNAs prepared by pooling samples from different stages was normalized at the time of initiation of development. (A) Representative prestalk genes are indicated by their cDNA number (Table 1). (B) Developmental expression of 65 prestalk genes, including those marked in panel A. Five clusters were generated by K-means in the Genespring program and color coded. Values are the averages from four independent determinations.

FIG. 8.

Expression of PstO genes in Ax2 and the dmtA⁻ mutant lacking DIF. Thirty PstO genes were analyzed by in situ hybridization in both Ax2 and the dmtA⁻ mutant lacking DIF at the slug stage. Among those, 18 genes were down-regulated in the mutant, as exemplified by SSD764 (a and b) and SSM184 (c and d). Although residual expression was detectable in SSM184, SSD764 was totally extinguished. In contrast to these genes, 12 genes were not affected, as exemplified by SLF774 (e and f) and SLA769 (g and h). (a, c, e, and g) Ax2; (b, d, f, and h) dmtA⁻ mutant.