DIF-1 induces the basal disc of the Dictyostelium fruiting body - PubMed (original) (raw)

Comparative Study

DIF-1 induces the basal disc of the Dictyostelium fruiting body

Tamao Saito et al. Dev Biol. 2008.

Abstract

The polyketide DIF-1 induces Dictyostelium amoebae to form stalk cells in culture. To better define its role in normal development, we examined the phenotype of a mutant blocking the first step of DIF-1 synthesis, which lacks both DIF-1 and its biosynthetic intermediate, dM-DIF-1 (des-methyl-DIF-1). Slugs of this polyketide synthase mutant (stlB(-)) are long and thin and rapidly break up, leaving an immotile prespore mass. They have approximately 30% fewer prestalk cells than their wild-type parent and lack a subset of anterior-like cells, which later form the outer basal disc. This structure is missing from the fruiting body, which perhaps in consequence initiates culmination along the substratum. The lower cup is rudimentary at best and the spore mass, lacking support, slips down the stalk. The dmtA(-) methyltransferase mutant, blocked in the last step of DIF-1 synthesis, resembles the stlB(-) mutant but has delayed tip formation and fewer prestalk-O cells. This difference may be due to accumulation of dM-DIF-1 in the dmtA(-) mutant, since dM-DIF-1 inhibits prestalk-O differentiation. Thus, DIF-1 is required for slug migration and specifies the anterior-like cells forming the basal disc and much of the lower cup; significantly the DIF-1 biosynthetic pathway may supply a second signal - dM-DIF-1.

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Figures

Fig. 1

Fig. 1

DIF-1 biosynthetic pathway. The polyketide skeleton of DIF-1 is assembled by the StlB, ‘steely’, polyketide synthase, and then decorated by two successive chlorinations (where the chloroperoxidase is only known as an enzymatic activity) and a final methylation by the DmtA methyltransferase (Kay, 1998; Thompson and Kay, 2000b; Austin et al., 2006). DIF-1 is further metabolized by mono-dechlorination to DIF-3 and then by successive hydroxylations/oxidations to yield around 10 further metabolites (Traynor and Kay, 1991; Morandini et al., 1995). These metabolites are all much less active than DIF-1 in the stalk cell bioassay, but may conceivably have other activities in vivo.

Fig. 2

Fig. 2

Slugs formed without DIF-1 break up and regulate. Slugs of the PKS null strain were observed from soon after alighting on the agar surface. Note that though the posterior part of the slug does not move, it still regulates and makes a fruiting body. Scale bar indicates 1 mm.

Fig. 3

Fig. 3

Prestalk subtypes in DIF-1 biosynthetic mutants. (A) ecmAO reporter expressed in the PKS null strain; (B) ecmAO reporter expressed in the PKS null strain with 100 nM DIF-1 added to the agar; (C) ecmAO reporter expressed in the wild-type strain; (D) ecmO reporter expressed in the PKS null strain; (E) ecmO reporter expressed in the PKS/methyltransferase double null strain; (F) ecmO reporter expressed in the wild-type strain; (G) ecmO reporter expressed in the methyltransferase null mutant. Reporters are as described (Jermyn et al., 1989; Early et al., 1993).

Fig. 4

Fig. 4

Des-methyl-DIF-1 represses prestalk-O cell differentiation. Low magnification images of ecmO-lacZ staining of double null mutant cells developed to the early slug stage with additions to the support filter as follows: (A) control, no additions; (B) 10 nM dM-DIF-1; (C) 50 nM dM-DIF-1; (D) 100 nM dM-DIF-1; (E) 200 nM dM-DIF-1; (F) 100 nM DIF-1. Approximately 50 nM dM-DIF-1 is required to suppress ecmO expression.

Fig. 5

Fig. 5

A class of anterior-like cells is lacking in the PKS null mutant. In the wild-type slugs, an aggregate of neutral red stained cells (indicated with black arrow) is located at the base of the prespore zone adjacent to the substratum (A and B). This aggregate of stained cells is absent in the PKS null mutant (C and D), but can be restored by development with DIF-1 (F). The dorsal side of the mutant slug is often bent, as in this case (indicated with red arrow in panels C and D).

Fig. 6

Fig. 6

The fruiting bodies of the PKS null and methyltransferase null mutants lack a basal disc. Whole fruiting body structure of (A) wild-type strain; (B) PKS null mutant; (C) methyltransferase null mutant. The stalk of the PKS null mutant lies partially on the substratum and the spores slip down the stalk. Higher power detail of the basal discs of (D) wild-type strain; (E) PKS null mutant. The wild-type strain has both inner and outer basal discs, while the PKS null mutant has only the inner basal disc, which derives from the stalk proper.

Fig. 7

Fig. 7

The lower cup is frequently missing in PKS null mutant fruiting bodies. Typical staining pattern of _ecmB_-lacZ in culminants: (A) PKS null mutant developed without DIF-1; (B) PKS null mutant developed with 100 nM DIF-1 added to the support filter (note the staining of the lower cup indicated by an arrow); (C) wild-type parental strain; (D) methyltransferase null mutant (note that there is some lower cup staining, but much less than the wild-type strain). Arrows indicate the lower cups.

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