Mound Stage Research Articles

CAR2, a prestalk cAMP receptor required for normal tip formation and late development of Dictyostelium discoideum.

Extracellular cAMP serves as a primary signaling molecule to regulate the development of Dictyostelium discoideum. It is required for chemotaxis, aggregation, cytodifferentiation, and morphogenetic movement. The receptors for cAMP are members of the family of cell-surface receptors that are linked to G proteins and characterized by seven putative transmembrane domains. Previously, we have isolated the gene for the cAMP receptor subtype 1 (CAR1) from Dictyostelium and suggested that several genes related to CAR1 were present in the genome. Here, we describe a family of cAMP receptor genes of Dictyostelium and the isolation and function of the gene for the cAMP receptor subtype 2, CAR2. CAR2 is structurally similar to CAR1. Overall, their transmembrane and loop domains are approximately 75% identical in amino acid sequence; however, their carboxyl termini are quite dissimilar; CAR2 possesses homopolymeric runs of histidines and asparagines that are absent from the corresponding region in CAR1. Although CAR1 is maximally expressed during the early stages of development, CAR2 is expressed only after cells have aggregated and, then, preferentially in prestalk cells. Transgenic Dictyostelium that have had their wild-type CAR2 gene replaced by a defective copy using homologous recombination proceed through early development but are detained at the tight mound stage. CAR2 may be required for cAMP-directed sorting of prestalk cells during pattern formation within the aggregation mound. Furthermore, although prestalk genes are expressed normally in aggregates that lack CAR2, they exhibit an enhanced expression of prespore-specific mRNA. Previously, we had shown that there was a requirement for CAR1 during early development. The present results demonstrate that the multiple responses of Dictyostelium to cAMP are regulated by distinct cAMP receptors that are encoded by unique genes.

The DIF-1 signaling system in Dictyostelium. Metabolism of the signal.

DIF-1 is a novel, chlorinated alkyl phenone from Dictyostelium which, at very low concentrations, induces amoebae to differentiate into stalk cells and may act as a morphogen in the formation of the prestalkprespore pattern during development. We report here the existence of a developmentally regulated metabolic pathway which inactivates DIF-1. Radioisotopically labeled DIF-1 was synthesized, incubated with developing cells, the metabolites recovered, and then analyzed by high pressure liquid chromatography and TLC. At least 12 metabolites are produced and the early steps of a complex metabolic pathway have been deduced by following the flow of counts from one metabolite to another and by determining the fate of purified metabolites when they are incubated with cells. The first metabolite, DM1, is largely cell-associated whereas the more distal ones are found mainly in the medium. Metabolism inactivates DIF-1, since DM1 retains only 7% of the specific activity of DIF-1 in the stalk cell differentiation bioassay and later metabolites possess even less activity. Metabolism is developmentally regulated, increasing toward the end of aggregation to reach maximal levels at the tipped mound stage, as endogenous DIF-1 levels are themselves rising. Cells at this stage of development possess the capacity to metabolize their endogenous DIF-1 with a half-life of a few minutes. We suggest that DIF-1 metabolism is important to prevent the DIF-1 receptor system from becoming saturated by excess ligand, thus allowing cells to respond to changes in DIF-1 production. Metabolism may also produce other effector molecules from DIF-1 or produce DIF-1 gradients in the aggregate by the localized destruction of DIF-1.

Origins of the prestalk-prespore pattern in Dictyostelium development

Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line ( mhcA ) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain ( mhcA ) the light chains are unstable.

Changes during differentiation in requirements for cAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum

A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not to be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development.

Sedimentology and budget of a Recent carbonate mound, Florida Keys

ABSTRACTThe sedimentology of a Recent carbonate mound is investigated to further our understanding of mound building communities, surface and subsurface mound sediments, and the overall sediment budget of mounds. Nine sedimentary facies of the surface of Tavernier mound, Florida Keys are described. These sediments are composed of Neogoniolithon, Halimeda, Porites, mollusc and foraminiferal grains, and lime mud. Muds rich in aragonite and high magnesian calcite show little mineralogical variation over the mound surface. Geochemical evidence suggests that the mud is mainly formed from breakdown of codiacean algae and Thalassia blade epibionts. Production rates of the facies are established from in situ growth rate experiments and standing‐crop surveys. Annual calcium carbonate production is c. 500gm‐2, intermediate between reef and other bay and lagoonal environment production rates in the Caribbean. The internal structure of the mound, studied from piston cores and sediment probes, indicates that seven facies can be identified. Five of these can be related to the present‐day facies, and occur in the upper part of the mound (gravel‐mound stage). The remaining two facies, characterized by molluscs and aragonite‐rich muds, occur in the lower part of the mound (mud‐mound stage), and are most similar to facies from typical Florida Bay mud mounds. Mangrove peats within the mound indicate former intertidal areas and C14 dates from these peats provide a time framework for mound sedimentation. The mound appears to have formed because of an initial valley in the Pleistocene surface which accumulated mud in a shallow embayment during the Holocene transgression.A sediment budget for the mound is presented which compares production rates from present‐day facies with subsurface sediment masses. During the mud mound stage production rates were similar to accumulation rates and the mound was similar to the present‐day mounds of Florida Bay. During the gravel mound stage (3400 yr BP‐present day), conditions were more normal marine and the establishment of Porites and Neogoniolithon on the mound increased production rates 10% over accumulation rates. This excess sediment is thought to be transported off the mound to the surrounding seabed. Models are proposed which divide carbonate mounds on the basis of internal versus external sediment supply. Comparisons are made with other Recent and ancient mounds. Similarities exist between the roles of the biotic components of late Palaeozoic mounds but major differences are found when structures and early diagenesis are compared.

Regulation of discoidin I gene expression in dictyostelium discoideum by cell-cell contact and cAMP.

We have previously presented evidence that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion-defective mutant, strain EB-21, which enters the developmental program but is blocked at the loose mound stage. When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB-21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB-21 to form normal cell-cell contacts and its deficiency in inactivating discoidin I gene expression. Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact-regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several-fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state. Furthermore, overexpression of discoidin I mRNA late in development in EB-21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact-mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion-defective EB-21 strain.

The timing of cell-type-specific differentiation in Dictyostelium discoideum

We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10–12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.

Novel morphogenesis in Ax-3, a mutant strain of the cellular slime mould Dictyostelium discoideum.

The cellular slime mould Dictyostelium discoideum constructs a succession of characteristic structures (grexes) in the multicellular phase of its life cycle. Compared with the wild-type strain, NC-4, grexes of a mutant strain, Ax-3, exhibited a profound prolongation of the early mound stage of development and a premature and exaggerated tendency to construct short, broad and bulbous forms. The aberrant Ax-3 phenotype was partially corrected by increasing the temperature of development, decreasing the plating density, perfusing with fresh air, or by introducing 0.1 nm-formycin B or 3-deazaadenosine into the agar. Mixtures of NC-4 and Ax-3 formed hybrid grexes which often became transformed into toruses by spiral centrifugal movement of the cells. Under other conditions, hybrid mounds developed into binary grex structures, cylindrical columns on top of hemispherical mounds, each component of which completed the morphogenetic sequence independently. The narrow upper structure appeared to be composed primarily of NC-4 cells. These novel patterns of morphogenesis support our hypothesis that the shape of the organism is controlled by the specification of the circumference of loops of cells within the grex.

Biosynthesis and self-assembly of protein S, a development-specific protein of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that has a complex life cycle including a temporal sequence of cellular aggregation, mound formation, and myxosporulation. During development, protein S (molecuar weight 23,000) is induced and accumulates in very large amounts. Protein S was found in the soluble fraction of early developmental extracts and in the insoluble fraction in later extracts. This insoluble form of protein S can be solubilized by the addition of 1 M NaCl at 0 degrees C to extracts from aggregated cells (mound stage) or by the addition of 1 M NaCl at 30 degrees C to mature spores. Salt extraction (1 M NaCl) of protein S from mature spores was partially inhibited by the addition of Mg(2+) and almost completely inhibited by the addition of Ca(2+). The viability of spores was not changed by a salt extraction that removed their protein S. Examination of thin sections of mature spores and extracted spores by electron microscopy suggested that the protein S-deficient spores lacked a spore surface coat about 300 A thick. Purified protein S will spontaneously self-assemble onto protein S-deficient spores after removal of the NaCl by dialysis or by addition of 10 mM Ca(2+) to undialyzed samples. Glycerol-induced spores did not contain protein S and did not serve as primers for assembly of protein S. Quantitation of the self-assembly process showed almost stoichiometric binding of protein S to the protein S-deficient spores until saturation at 3.3 x 10(6) molecules per spore, a value 1.35 times higher than the normal level of proteins S found in mature spores. Protein S in the "reconstituted" spores was as protease resistant and sonication resistant as the protein S of native spores. Electron microscopy of the reconstituted spores revealed the assembly of new material on the spore surface. Adjacent spores were sometimes observed to be fused to each other through a common protein S layer. These results suggest that protein S serves a function in spore-spore interaction in the fruiting body.

Axillary buds in normal and irradiated apple and pear

Dormant buds of apple and pear are convenient material to irradiate for the production of mutations or chimeral rearrangements. The sites of action of ionizing radiation are the apical meristem and the secondary bud primordia of the dormant bud. Such buds and growing shoot tips of normal and irradiated McIntosh (2×) and Northern Spy (2×) apples, their cytochimeras Kimball McIntosh (2-4-4-4) and Loop Spy (2-2-4-4), and Bartlett pear (2×) were sectioned or dissected. Apple scions received 0 to 2850 rad of X-rays and pear scions were exposed to 0, 2000, 4000 or 6000 rad of γ-rays. Dormant pear buds had seven bud primordia which would normally continue growth while those of apple had only three. No bud primordium in a dormant bud had reached the mound stage. After irradiation of apple and pear radiation-damaged cells were found in the subsurface central region of both apical and axillary meristems. Greatest damage occurred in layers II and III. Cells adjacent to the damage, notably those surrounding the damaged area of the shoot between the youngest three leaves and those adaxial to the older bud primordia, became meristematic. One or more secondary buds developed from the axils of pre-irradiation leaves 1–5 in apple and 1–10 in pear. In older recovering shoots an adventitious bud might arise on the stem, as suggested by one cauline bud.