A new measure for assessing the physical activity behaviors of persons with disabilities and chronic health conditions: the Physical Activity and Disability Survey.

PURPOSE: Assess the psychometric properties of the Physical Activity and Disability Survey (PADS), a new physical activity measure for persons with disabilities and chronic health conditions. DESIGN: Cross-sectional and pre-post designs were employed. SETTING: A Midwestern university fitness center. SUBJECTS: Participants were 103 individuals with disabilities and/or chronic health conditions. MEASURES: The Physical Activity and Disability Survey (PADS), peak oxygen uptake (peak VO2), maximum workload (MW), and time to exhaustion (TE) during exercise. RESULTS: Factor analysis revealed a four-factor model that generally corresponded to PADS subscales. Cronbach alpha coefficients ranged from .67 (Exercise) to .77 (Time Indoors). Test-retest reliability (1-week interval) ranged from .78 (Time Indoors) to .95 (Leisure Time Physical Activity). Interrater reliability ranged from .92 (Household Activities) to .99 (Exercise, Leisure Time Physical Activity, Total Activity). Significant (p < .05) correlations were found between PADS subscales and absolute peak VO2 (Leisure Time Physical Activity, Household Activity, Total Activity), relative peak VO2 (Exercise, Time Indoors), MW (Time Indoors, Household Activity), and TE (Household Activity, Total Activity). Analyses of variance revealed that, unlike controls, health promotion program participants evidenced significant pre-post gains as measured by the Exercise subscale and Total Activity score. CONCLUSIONS: The findings lend support for the reliability and validity of the PADS as a measure of physical activity of groups who are sedentary and disabled.

Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction.

Members of the fibroblast growth factor (FGF) family of peptide ligands have been implicated in otic placode induction in several vertebrate species. Here, we have functionally analyzed the roles of fgf3 and fgf8 in zebrafish otic development. The role of fgf8 was assessed by analyzing acerebellar (ace) mutants. fgf3 function was disrupted by injecting embryos with antisense morpholino oligomers (MO) specifically designed to block translation of fgf3 transcripts. Disruption of either fgf3 or fgf8 causes moderate reduction in the size of the otic vesicle. Injection of fgf3-MO into ace/ace mutants causes much more severe reduction or complete loss of otic tissue. Moreover, preplacode cells fail to express pax8 and pax2.1, indicating disruption of early stages of otic induction in fgf3-depleted ace/ace mutants. Both fgf3 and fgf8 are normally expressed in the germring by 50% epiboly and are induced in the primordium of rhombomere 4 by 80% epibloy. In addition, fgf3 is expressed during the latter half of gastrulation in the prechordal plate and paraxial cephalic mesendoderm, tissues that either pass beneath or persist near the prospective otic ectoderm. Conditions that alter the pattern of expression of fgf3 and/or fgf8 cause corresponding changes in otic induction. Loss of maternal and zygotic one-eyed pinhead (oep) does not alter expression of fgf3 or fgf8 in the hindbrain, but ablates mesendodermal sources of fgf signaling and delays otic induction by several hours. Conversely, treatment of wild-type embryos with retinoic acid greatly expands the periotic domains of expression of fgf3, fgf8, and pax8 and leads to formation of supernumerary and ectopic otic vesicles. These data support the hypothesis that fgf3 and fgf8 cooperate during the latter half of gastrulation to induce differentiation of otic placodes.

Development of utricular otoliths, but not saccular otoliths, is necessary for vestibular function and survival in zebrafish.

We have been studying the consequences of embryonic vestibular dysfunction caused by the monolith (mnl) mutation in zebrafish. mnl is a dominant mutation that specifically inhibits formation of utricular otoliths. However, briefly immobilizing mnl/mnl embryos in agarose with the otic vesicle orientated at certain angles selectively induces or prevents formation of utricular and/or saccular otoliths. With this noninvasive technique, we generated six phenotypic classes of mnl/mnl mutants, designated S-S, U-U, U-S, S-US, U-US, and US-US, depending on which otoliths are present on each side (U, utricular otolith; S, saccular otolith). All mnl/mnl larvae survived through day 10 of development. Thereafter, S-S larvae showed a rapid decline, probably because of starvation, and none survived to adulthood. Survival rates in all other classes of mnl/mnl larvae (those having at least one utricular otolith) were close to normal. The presence or absence of utricular otoliths also correlated with vestibular function during early larval development, as measured by three criteria: First, unlike wild-type larvae, S-S mutant larvae showed almost no detectable counter-rotation of the eyes when tilted tail up or tail down. Second, 95% of S-S mutant larvae never acquired the ability to maintain a balanced dorsal-up posture. Third, although most wild-type larvae responded to gentle prodding by swimming in a straight line, S-S larvae responded by swimming in rapid circles, showing sudden and frequent changes in direction ("zigzagging"), and/or rolling and spiraling. All other phenotypic classes of mnl/mnl larvae behaved normally in these assays. These data demonstrate that bilateral loss of utricular otoliths disrupts the ability to sense gravity, severely impairs balance and motor coordination, and is invariably lethal. The presence of a utricular otolith in at least one inner ear is necessary and sufficient for vestibular function and survival. In contrast, saccular otoliths are dispensable for these functions.

The deltaA gene of zebrafish mediates lateral inhibition of hair cells in the inner ear and is regulated by pax2.1.

Recent studies of inner ear development suggest that hair cells and support cells arise within a common equivalence group by cell-cell interactions mediated by Delta and Notch proteins. We have extended these studies by analyzing the effects of a mutant allele of the zebrafish deltaA gene, deltaA(dx2), which encodes a dominant-negative protein. deltaA(dx2/dx2 )homozygous mutants develop with a 5- to 6-fold excess of hair cells and a severe deficiency of support cells. In addition, deltaA(dx2/dx2) mutants show an increased number of cells expressing pax2.1 in regions where hair cells are normally produced. Immunohistological analysis of wild-type and deltaA(dx2/dx2) mutant embryos confirmed that pax2.1 is expressed during the initial stages of hair cell differentiation and is later maintained at high levels in mature hair cells. In contrast, pax2.1 is not expressed in support cells. To address the function of pax2.1, we analyzed hair cell differentiation in no isthmus mutant embryos, which are deficient for pax2.1 function. no isthmus mutant embryos develop with approximately twice the normal number of hair cells. This neurogenic defect correlates with reduced levels of expression of deltaA and deltaD in the hair cells in no isthmus mutants. Analysis of deltaA(dx2/dx2); no isthmus double mutants showed that no isthmus suppresses the deltaA(dx2) phenotype, probably by reducing levels of the dominant-negative mutant protein. This interpretation was supported by analysis of T(msxB)(b220), a deletion that removes the deltaA locus. Reducing the dose of deltaA(dx2) by generating deltaA(dx2)/T(msxB)(b220 )trans-heterozygotes weakens the neurogenic effects of deltaA(dx2), whereas T(msxB)(b220) enhances the neurogenic defects of no isthmus. mind bomb, another strong neurogenic mutation that may disrupt reception of Delta signals, causes a 10-fold increase in hair cell production and is epistatic to both no isthmus and deltaA(dx2). These data indicate that deltaA expressed by hair cells normally prevents adjacent cells from adopting the same cell fate, and that pax2.1 is required for normal levels of Delta-mediated lateral inhibition.

Delta-mediated specification of midline cell fates in zebrafish embryos.

BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.

Genetic analysis of tissue interactions required for otic placode induction in the zebrafish.

Development of the vertebrate inner ear begins during gastrulation with induction of the otic placode. Several embryonic tissues, including cephalic mesendoderm, notochord, and hindbrain, have been implicated as potential sources of otic-inducing signals. However, the relative contributions of these tissues have not been determined, nor have any genes affecting placode induction been identified. To address these issues, we analyzed otic placode induction in zebrafish mutants that are deficient in prospective otic-inducing tissues. Otic development was monitored by examining mutant embryos for morphological changes and, in some cases, by visualizing expression patterns of dlx-3 or pax-2.1 in preotic cells several hours before otic placode formation. In cyclops (cyc-) mutants, which develop with a partial deficiency of prechordal mesendoderm, otic induction is delayed by up to 1 h. In one-eyed pinhead (oep-) mutants, which are more completely deficient in prechordal mesendoderm, otic induction is delayed by 1.5 h, and morphology of the otic vesicles is abnormal. Expression of marker genes in other regions of the neural plate is normal, suggesting that ablation of prechordal mesendoderm selectively inhibits otic induction. In contrast, the timing and morphology of otic development is not affected by mutations in no tail (ntl) or floating head (flh), which prevent notochord differentiation. Similarly, a mutation in valentino (val), which blocks early differentiation of rhombomeres 5 and 6 in the hindbrain, does not delay otic induction, although subsequent patterning of the otic vesicle is impaired. To test whether inductive signals from one tissue can compensate for loss of another, we generated double or triple mutants with various combinations of the above mutations. In none of the multiple mutants do the flh or val mutations exacerbate delays in placode induction, although val does contribute additively to defects in subsequent patterning of the otic vesicle. In contrast, mutants homozygous for both oep and ntl, which interact synergistically to disrupt differentiation of cephalic and axial mesendoderm, show a delay in otic development of about 3 h. These data suggest that cephalic mesendoderm, including prechordal mesendoderm and anterior paraxial mesendoderm, provides the first otic-inducing signals during gastrulation, whereas chordamesoderm plays no discernible role in this process. Because val- mutants are deficient for only a portion of the hindbrain, we cannot rule out a role for that tissue in otic placode induction. However, if the hindbrain does provide otic-inducing signals, they apparently differ quantitatively or qualitatively from the signals required for vesicle patterning, as val disrupts only the latter.

Types of spiritual well-being among persons with chronic illness: their relation to various forms of quality of life.

OBJECTIVES: Derive a spiritual well-being classification and thereby enhance understanding of the relation between spiritual well-being, quality of life (QOL), and health among persons with chronic illness or disability. DESIGN: Cluster analyses were performed to develop a spiritual well-being classification. Analysis of variance was used to compare cluster groups on various dimensions of QOL. SETTING: Part of a larger QOL study conducted at a midwestern medical center. PATIENTS: A convenience sample of 216 inpatients: amputation (n = 74), postpolio (n = 37), spinal cord injury (n = 34), breast cancer (n = 36), and prostate cancer (n = 35). Minors were excluded from the study. MAIN OUTCOME MEASURES: Spiritual Well-Being Scale (SWBS), Functional Assessment of Cancer Therapy (FACT), Functional Living Index-Cancer (FLIC), Sickness Impact Profile (SIP), Medical Outcome Survey-Short Form (SF-36), and the Satisfaction With Life Scale (SWLS). RESULTS: Three types of spiritual well-being were identified: religious (n = 146), existential (n = 37), and nonspiritual (n = 30). Significant cluster differences (p < .03 to p < .001) were observed across all QOL domains and life satisfaction. Compared with the other cluster groups, the nonspiritual group reported significantly lower levels of QOL and life satisfaction and the highest proportion of health status change with respect to both improvement and decline in health. CONCLUSIONS: Three types of spiritual well-being were empirically identified in this sample. Subtypes differed significantly with respect to various aspects of QOL. Further research is needed to validate this classification and to determine if type of spiritual well-being has a causal effect on treatment outcome or on the recovery process.

A critical period of ear development controlled by distinct populations of ciliated cells in the zebrafish.

The zebrafish (Danio rerio) is a useful model system for analyzing development of the inner ear. A number of mutations affecting the inner ear have been identified. Here we investigate the initial stages of otolith morphogenesis in wild-type embryos as well as in monolith (mnl) mutant embryos, which fail to form anterior otoliths but otherwise appear normal. Otolith growth is initiated at 18-18.5 h by localized accretion of free-moving precursor particles. This process, referred to as otolith seeding, is regulated by two classes of cilia: First, kinocilia of precociously forming hair cells (tether cells) bind seeding particles, thereby localizing otolith formation. Tether cells usually occur in pairs at the anterior and posterior ends of the ear. Despite the presence of functional kinocilia, tether cells initially appear immature and do not acquire the characteristics of mature hair cells until approximately 21.5 h. Second, beating cilia distributed throughout the ear agitate seeding particles, thereby inhibiting premature agglutination. Constraining particles with laser tweezers caused them to fuse into large untethered masses. Bringing such masses into contact with tethered otoliths caused them to fuse, greatly enhancing otolith growth. Selectively enhancing one otolith greatly inhibited growth of the second, creating an imbalance that persisted for many days. Seeding particles and beating cilia disappear soon after 24 h, and the rate of otolith growth decreases by nearly 90%. In mnl mutant embryos, tethers and beating cilia are distributed normally, but anterior otoliths fail to form in 80-85% of mutant ears. The binding properties of seeding particles appear normal, as shown by their ability to fuse when entrapped by laser tweezers and their binding to posterior tethers. We infer that anterior tethers have a weakened ability to bind seeding particles in mnl embryos. Immobilizing mnl embryos with the anterior end of the ear oriented downward effectively concentrated the dense seeding particles near the anterior tethers and permitted all to form anterior otoliths. However, immobilizing mnl embryos after 24 h when seeding particles were depleted did not facilitate anterior otolith formation. Together, these data demonstrate that the ability to initiate otolith formation is limited to a critical period, from 18.5 to 24 h, and that interfering with the functions of tether cell kinocilia or beating cilia impairs otolith seeding and subsequent otolith morphogenesis.

Quality of life issues among women with physical disabilities or breast cancer.

OBJECTIVE: The purpose of this study was to assess quality of life (QOL) and life satisfaction among women with physical disabilities or breast cancer, and to identify factors predictive of QOL and life satisfaction for women and men. QOL and life satisfaction differences were examined between women and men with physical disabilities and cancer, and between women with traumatic and chronic physical conditions. DESIGN: A cross-sectional design employing several QOL and life satisfaction measures was used. SAMPLE: Two hundred sixteen outpatient subjects (99 women, 117 men) with physical disabilities or cancer were studied. INSTRUMENTS: The Health Status Questionnaire-Short-Form 36 (SF-36), Functional Assessment of Cancer Therapy (FACT), Functional Living Index-Cancer (FLIC), and the Satisfaction With Life Scale (SWLS). RESULTS: Women with traumatic conditions (amputation, spinal cord injury) reported poorer physical functioning and well-being, whereas women in the chronic (postpolio, breast cancer) group reported poorer health status. No significant gender differences were found with respect to QOL or life satisfaction. Whereas functional and emotional well-being were the strongest predictors of overall QOL for both men and women, self-perceived general health significantly predicted QOL for women (p < .05) and social well-being significantly predicted QOL for men (p < .01). Among men, life satisfaction was best predicted by marital status (p < .05), general health (p < .05), and social well-being (p < .01). The resulting QOL models had adjusted R2 values of .77 and .76 for women and men, respectively. Among women with traumatic conditions, functional well-being best predicted QOL (p < .01). Life satisfaction for women with chronic conditions was best predicted by age, education, and spiritual well-being. CONCLUSION: QOL as measured by the impact of illness on an individual is best predicted by physical and functional well-being. Satisfaction with one's life was best predicted by functional ability. Although functional and physical ability were the best predictors for both QOL and life satisfaction, social functioning made significant and substantive contributions to these constructs. Spinal cord injury had the most impact on physical functioning, whereas prostate cancer had the least. Psychosocial functioning was most affected by amputation and least affected by prostate cancer.

A mutation in zebrafish affecting a localized cellular function required for normal ear development.

Zebrafish holds great potential as a model system for studying inner ear development because genetic techniques are highly efficient and inner ear development is a conspicuous and manipulable feature of zebrafish embryogenesis. Here we describe analysis of a semilethal dominant mutation, termed monolith (mnl), that specifically perturbs formation of the anterior (utricular) otolith in the developing ear. Other than the utricular otolith deficiency, all structures in the ear appear morphologically normal in mutant embryos, including posterior otoliths and all sensory epithelia. Expression patterns of several ear marker genes (msxC, msxD, and dlx3) also appear normal in the mutant. To identify the cell type(s) affected by the mnl mutation, chimeras were generated by transplanting dye-labeled +/+ cells into unlabeled mnl/mnl host embryos. Roughly half of such chimeras formed utricular otoliths normally, indicating that the transplanted wild-type cells rescued their mutant hosts. Detailed analysis of +/+ cell fates revealed that virtually all chimeras in which +/+ cells formed support cells in the utricular sensory epithelium were rescued. In contrast, wild-type cells forming other cell types (such as hair cells) or colonizing other regions of the host were not sufficient to facilitate rescue. These data indicate that support cells are required for normal otolith formation, providing the first experimentally established role for support cells in vertebrate sensory epithelia. The data also provide the first clear indication that otolith formation is controlled independently in different regions of the ear by localized cellular functions.

Efficient induction of point mutations allowing recovery of specific locus mutations in zebrafish.

A technique is described that greatly increases the efficiency of recovering specific locus point mutations in zebrafish (Danio rerio). Founder individuals that were mosaic for point mutations were produced by mutagenizing postmeiotic gametes with the alkylating agent N-ethyl-N-nitrosourea. Under optimal conditions, each founder carried an average of 10 mutations affecting genes required for embryogenesis. Moreover, approximately 2% of these founders transmitted new mutations at any prespecified pigmentation locus. Analyses of new pigmentation mutations confirmed that most were likely to be point mutations. Thus, mutagenesis of postmeiotic gametes with N-ethyl-N-nitrosourea yielded frequencies of point mutations at specific loci that were 10- to 15-fold higher than previously achieved in zebrafish. Our procedure should, therefore, greatly facilitate recovery of multiple mutant alleles at any locus of interest.

Distribution of FGF-2 suggests it has a role in chick limb bud growth.

We developed and characterized antibodies specific for FGF-2 and used them to locate FGF-2 during chick embryo development. A series of micrographs demonstrated the progression of FGF-2 staining during development of the different tissues and organs. FGF-2 was present in the ectoderm covering the entire embryo, muscle cells, nervous system, neural crest cells, and mesonephros. FGF-2 was also present in the limb from initiation of budding through differentiation. The limb ectoderm and subjacent mesoderm showed the strongest immunostaining, with lower levels in the center of the bud. However, the distribution of FGF-2 positive cells in the mesoderm was not homogeneous. This heterogeneity was not due to cell cycle specific distribution of FGF-2 protein, as flow cytometric analysis showed that FGF-2-positive cells were distributed throughout the cell cycle. However, the amount of anti-FGF-2 fluorescence varied most during G1, consistent with the possibility that FGF-2 is low after M phase and increases during G1. A bioassay was used to demonstrate FGF-2 levels in the wing ectoderm were approximately 2.7-fold greater than in the mesoderm. We propose that the location of FGF-2 in the embryo is consistent with a role in epithelial-mesenchymal interactions; in the limb bud it may prevent differentiation and permit limb outgrowth and subsequent expression of patterning events.

Retroviral expression of FGF-2 (bFGF) affects patterning in chick limb bud.

To investigate the role of fibroblast growth factor-2 (basic fibroblast growth factor) in chick limb development, we constructed a replication-defective spleen necrosis virus to ectopically express fibroblast growth factor-2 in stage 20-22 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, infected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of host wings receiving infected grafts at their anterior borders had duplications of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressively more posterior sites of host limb buds, and grafting to the posterior border had no effect at all. Several techniques were used to determine the role of infected tissue in forming skeletal duplications. First, staining with an fibroblast growth factor-2 specific monoclonal antibody showed higher than endogenous levels of fibroblast growth factor-2 expression associated with extra elements. Second, the host/donor composition of duplicated elements was determined by simultaneously infecting donor cells with viruses encoding fibroblast growth factor-2 or beta-galactosidase; donor tissue was then visualized by X-gal staining. Patterns of ectopic fibroblast growth factor-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicated structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications caused by infected quail wing bud cells grafted to the chick wing bud. These observations suggest that fibroblast growth factor-2-expressing donor tissue induced host tissue to form normally patterned extra elements. In support of this conclusion, implanting beads containing fibroblast growth factor-2 caused partial duplications of digit 2. These data provide the first direct evidence that fibroblast growth factor-2 plays a role in patterning in the limb bud.

Ammonia promotes accumulation of intracellular cAMP in differentiating amoebae of Dictyostelium discoideum.

We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous NH4Cl and high ambient pH promote spore formation during in vitro differentiation. The level of NH4Cl required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH4+ is the active species. The spore-promoting activity of high ambient pH (without exogenous NH4Cl) was eliminated by the addition of an NH3-scavenging cocktail, suggesting that high pH promotes spore differentiation by increasing the ratio of NH3:NH4+ secreted into the medium by developing cells. High ammonia levels and high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells. In both treatments, peak cAMP levels equaled or exceeded control levels and were maintained for longer periods than in control cells. In contrast, ammonia strongly inhibited accumulation of extracellular cAMP without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous observations that factors that raise intracellular cAMP levels increase spore formation. Lowering intracellular cAMP levels with caffeine or progesterone inhibited spore formation, but simultaneous exposure to these drugs and optimal concentrations of NH4Cl restored both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high intracellular cAMP levels.

Conditions that alter intracellular cAMP levels affect expression of the cAMP phosphodiesterase gene in Dictyostelium.

We examined expression of the Dictyostelium cAMP phosphodiesterase (PDE) gene under conditions that alter intracellular cAMP levels during in vitro differentiation of wild-type strain V12M2 and a sporogenous derivative, HB200. In control cultures, cellular PDE activity peaked at 6 hr and declined by 8 hr, while secreted PDE activity continued to increase through 8 hr. Lowering intracellular cAMP levels with caffeine or progesterone increased cellular and secreted PDE activities 2-fold, increased stalk cell differentiation, and inhibited spore differentiation. In contrast, exposure to 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP; a membrane-permeable cAMP analog) or ammonia (which promotes intracellular cAMP accumulation in V12M2 and HB200 cells) lowered PDE activities by as much as 45%, decreased stalk cell differentiation, and increased spore differentiation. Simultaneous exposure to 8-Br-cAMP and caffeine gave intermediate PDE activities as would be expected if 8-Br-cAMP entered the cell and bypassed the caffeine-mediated block to adenylate cyclase activation. In all cases, we observed commensurate changes in developmental PDE transcript levels. The developmental time course of expression was not significantly altered by these treatments. These results suggest that the magnitude of PDE gene expression is negatively regulated by intracellular cAMP levels and provide evidence for one of the earliest changes in gene expression that is consistent with cell-type specificity. These results are discussed in terms of a bistable switch employing intracellular cAMP as a regulator of cell fate.

Conditions that elevate intracellular cyclic AMP levels promote spore formation in Dictyostelium.

We have been using sporogenous mutants of Dictyostelium discoideum strain V12M2 to study regulation of cell fate during terminal differentiation of spores and stalk cells. Analyses of intracellular cAMP accumulation, cAMP secretion, cAMP binding to cell surface receptors, and chemotactic sensitivity to exogenous cAMP during aggregation showed that all of these functions were identical in V12M2 and HB200, a sporogenous mutant. We used several methods of altering intracellular cAMP levels in HB200 cells to test the hypothesis that intracellular cAMP levels affect cell fate. First, HB200 amoebae were treated with 5 mM caffeine for 4 h during growth, washed, and allowed to develop in the absence of caffeine. Treated cells had normal levels of intracellular cAMP and adenylate cyclase activities at the beginning of differentiation; by 6 h development, they contained two to three times more intracellular cAMP and two times more GTP-dependent adenylate cyclase activity than untreated cells. However, their level of basal Mn++-dependent adenylate cyclase activity was the same as untreated controls. Thus, treatment of growing HB200 amoebae with caffeine for only 4 h leads to hyperinduction of a GTP-dependent regulator (or inhibition of a negative regulator) of adenylate cyclase during subsequent differentiation, without induction of basal activity. The fraction of amoebae forming spores increased twofold when HB200 amoebae were treated with caffeine during growth. Spore (but not stalk cell) differentiation by such treated cells was blocked by inhibitors of cAMP accumulation. Second, cells grown on nutrient agar accumulated higher levels of intracellular cAMP and formed more spores in vitro than cells grown in shaken suspension.(ABSTRACT TRUNCATED AT 250 WORDS)