A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium.

Abstract

Previously, we identified a class of genes in Dictyostelium that are prespore cell-type specific in their expression in the multicellular aggregate and are inducible by cAMP acting through cell-surface cAMP receptors. In this paper, we report the cloning and analysis of the regulatory regions controlling the expression of one such gene that encodes a spore coat protein, SP60. By use of a fusion of the firefly luciferase gene and Escherichia coli lacZ [expresses [3-galactosidase (~-gal)], we have identified cis-acting regions required for proper spatial and temporal expression in multicellular aggregates and for cAMP induction in shaking cell culture. Deletion analysis suggests that a CA-rich element (CAE) and surrounding sequences present three times within the 5'-flanking sequence are required for proper regulation. SP60-IacZ fusions that include all three of these regions express lacZ only in the posterior -85% of migrating slugs (prespore zone). Studies show that SP60 is expressed during mid to late aggregation, and SP60-lacZ-positive cells are spatially localized as a doughnut-shaped ring within the forming aggregate. Cells within the skirt that surrounds the aggregate and that are still migrating into the aggregate do not stain. Sequential 5' deletions of CAEs and surrounding regions affect the expression level of SP60-luciferase in response to developmental signals and cAMP, as well as the spatial pattern of SP60-IacZ. Deletion of the first (most 5') of these regions restricts the spatial expression of SP60-IacZ fusions to the anterior of the prespore zone. When both the first and second regions are removed, the expression level drops, and the staining is restricted to the prespore/prestalk boundary. Furthermore, the staining pattern that is seen with these two deletions is present as a gradient from anterior to posterior within the prespore zone. Deletion of all three regions results in a loss of both cAMP and developmentally induced expression. These results suggest the presence of a gradient within the prespore zone that differentially affects the activity of promoters containing different numbers of response elements.

Tissue-specific G1-phase cell-cycle arrest prior to terminal differentiation inDictyostelium

“…It will be important to determine whether the late mitotic events and G1-phase arrest are required for spore cell differentiation or stalk formation. Certainly, prespore-specific genes and genes that become prespore enriched are expressed in some cells well before mitosis begins so it is unlikely that G1 arrest is required for all aspects of prespore differentiation (Fosnaugh and Loomis, 1993;Good et al, 2003;Haberstroh and Firtel, 1990;Iranfar et al, 2001;VanDriessche et al, 2002;Williams et al, 1989). Microtubule destabilizing drugs that block mitosis do not block development suggesting that prespore cells need not divide prior to spore differentiation (Cappuccinelli and Ashworth, 1976;Cappuccinelli et al, 1979).…”

A bZIP/bRLZ transcription factor required for DIF signaling inDictyostelium

“…Prestalk cells occupy the anterior quarter and are of two major types: the prestalk-A (pstA) cells, which are at the very front, and the prestalk-O (pstO) cells just behind them (Early et al, 1993;Jermyn et al, 1989). The posterior three-quarters of the slug comprise the prespore zone, and there is some evidence for the subdivision of that region as well (Haberstroh and Firtel, 1990;Kibler et al, 2003). The question of how pattern arises is fundamental to our understanding of Dictyostelium development.…”

Developmental and cAMP-mediated regulation of glycogen phosphorylase 1 in Dictyostelium discoideum

“…The promoter inserts were cut out of pBluescript with ClaI/BssHII double digests and were blunt-ended by filling in overhangs with the Klenow enzyme. The vector SP60-luciferase-A20 (SP60-luc ; see Haberstroh & Firtel, 1990) was the generous gift of Richard A. Firtel from the University of California, San Diego, USA. The SP60 promoter was excised from this vector with a NdeI/ClaI double digest, and the overhangs were filled in.…”