The effects of organic environmental toxicants on hard tissue formation in developing tooth : An in vitro study in mice

Dioxins are organic toxicants that are known to impair tooth development, especially dental hard tissue formation. The most toxic dioxin congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further, clinical studies suggest that maternal smoking during pregnancy can affect child s tooth development. One of the main components of tobacco smoke is the group of non-halogenated polycyclic aromatic hydrocarbons (PAHs), a representative of which is 7,12-dimethylbenz[a]anthracene (DMBA). Tributyltin (TBT), an organic tin compound, has been shown to impair bone mineralization in experimental animals. In addition to exposure to organic toxicants, a well-established cause for enamel hypomineralization is excess fluoride intake. The principal aim of this thesis project was to examine in vitro if, in addition to dioxins, other organic environmental toxicants, like PAHs and organic tin compounds, have adverse effects on tooth development, specifically on formation and mineralization of the major dental hard tissues, the dentin and the enamel. The second aim was to investigate in vitro if fluoride could intensify the manifestation of the detrimental developmental dental effects elicited by TCDD. The study was conducted by culturing mandibular first and second molar tooth germs of E18 NMRI mouse embryos in a Trowell-type organ culture and exposing them to DMBA, TBT, and sodium fluoride (NaF) and/or TCDD at various concentrations during the secretory and mineralization stages of development. Specific methods used were HE-staining for studying cell and tissue morphology, BrdU-staining for cell proliferation, TUNEL-staining for apoptosis, and QPCR, in situ hybridization and immunohistochemistry for the expressions of selected genes associated with mineralization. This thesis work showed that DMBA, TBT, TCDD and NaF interfere with dentin and enamel formation of embryonic mouse tooth in vitro, and that fluoride can potentiate the harmful effect of TCDD. The results suggested that adverse effects of TBT involve altered expression of genes associated with mineralization, and that DMBA and TBT as well as NaF and TCDD together primarily affect dentin mineralization. Since amelogenesis does not start until mineralization of dentin begins, impaired enamel matrix secretion could be a secondary effect. Dioxins, PAHs and organotins are all liposoluble and can be transferred to the infant by breast-feeding. Since doses are usually very low, developmental toxicity on most of the organs is difficult to indentify clinically. However, tooth may act as an indicator of exposure, since the major dental hard tissues, the dentin and the enamel, are not replaced once they have been formed. Thus, disturbed dental hard tissue formation raises the question of more extensive developmental toxicity.

Miniature Compliant Grippers With Vision-Based Force Sensing

This paper is concerned with grasping biological cells in aqueous medium with miniature grippers that can also help estimate forces using vision-based displacement measurement and computation. We present the design, fabrication, and testing of three single-piece, compliant miniature grippers with parallel and angular jaw motions. Two grippers were designed using experience and intuition, while the third one was designed using topology optimization with implicit manufacturing constraints. These grippers were fabricated using different manufacturing techniques using spring steel and polydimethylsiloxane ( PDMS). The grippers also serve the purpose of a force sensor. Toward this, we present a vision-based force-sensing technique by solving Cauchy's problem in elasticity using an improved algorithm. We validated this technique at the macroscale, where there was an independent method to estimate the force. In this study, the gripper was used to hold a yeast ball and a zebrafish egg cell of less than 1 mm in diameter. The forces involved were estimated to be about 30 and 10 mN for the yeast ball and the zebrafish egg cell, respectively.

Evolution of polyembryony: Consequences to the fitness of mother and offspring

Polyembryony, referring here to situations where a nucellar embryo is formed along with the zygotic embryo, has different consequences for the fitness of the maternal parent and offspring. We have developed genetic and inclusive fitness models to derive the conditions that permit the evolution of polyembryony under maternal and offspring control. We have also derived expressions for the optimal allocation (evolutionarily stable strategy, ESS) of resources between zygotic and nucellar embryos. It is seen that (i) Polyembryony can evolve more easily under maternal control than under that of either the offspring or the ‘selfish’ endosperm. Under maternal regulation, evolution of polyembryony can occur for any clutch size. Under offspring control polyembryony is more likely to evolve for high clutch sizes, and is unlikely for low clutch sizes (<3). This conflict between mother and offspring decreases with increase in clutch size and favours the evolution of polyembryony at high clutch sizes, (ii) Polyembryony can evolve for values of “x” (the power of the function relating fitness to seed resource) greater than 0.5758; the possibility of its occurrence increases with “x”, indicating that a more efficient conversion of resource into fitness favours polyembryony. (iii) Under both maternal parent and offspring control, the evolution of polyembryony becomes increasingly unlikely as the level of inbreeding increases, (iv) The proportion of resources allocated to the nucellar embryo at ESS is always higher than that which maximizes the rate of spread of the allele against a non-polyembryonic allele.Finally we argue that polyembryony is a maternal counter strategy to compensate for the loss in her fitness due to brood reduction caused by sibling rivalry. We support this assertion by two empirical evidences: (a) the extent of polyembryony is positively correlated with brood reduction inCitrus, and (b) species exhibiting polyembryony are more often those that frequently exhibit brood reduction.

How do heterogametic females survive without gene dosage compensation?

When the male is the heterogametic sex (XX♀-XY♂ or XX♀-XO♂), as inDrosophila, orthopteran insects, mammals andCaenorhabditis elegans, X-linked genes are subject to dosage compensation: the single X in the male is functionally equivalent to the two Xs in the female. However, when the female is heterogametic (ZZ♂-ZW♀), as in birds, butterflies and moths, Z-linked genes are apparently not dosage-compensated. This difference between X-linked and Z-linked genes raises fundamental questions about the role of dosage compensation. It is argued that (i) genes which require dosage compensation are primarily those that control morphogenesis and the prospective body plan; (ii) the products of these genes are required in disomic doses especially during oogenesis and early embryonic development; (iii) heterogametic females synthesize and store during oogenesis itself morphogenetically essential gene products - including those encoded by Z-linked genes — in large quantities; (iv) the abundance of these gene products in the egg and their persistence relatively late into embryogenesis enables heterogametic females to overcome the monosomic state of the Z chromosome in ZW embryos. Female heterogamety is predominant in birds, reptiles and amphibians, all of which have megalecithal eggs containing several thousand times more maternal RNA and other maternal messages than eggs of mammals,Caenorhabditis elegans, orDrosophila. This increase in egg size, yolk content and, concomitantly, the size of the maternal legacy to the embryo, may have facilitated female heterogamety and the absence of dosage compensation.

Functional expression and subcellular localization of the Cl- cotransporters KCC2 and NKCC1 in rodent hippocampal and neocortical neurons

The work presented here has focused on the role of cation-chloride cotransporters (CCCs) in (1) the regulation of intracellular chloride concentration within postsynaptic neurons and (2) on the consequent effects on the actions of the neurotransmitter gamma-aminobutyric acid (GABA) mediated by GABAA receptors (GABAARs) during development and in pathophysiological conditions such as epilepsy. In addition, (3) we found that a member of the CCC family, the K-Cl cotransporter isoform 2 (KCC2), has a structural role in the development of dendritic spines during the differentiation of pyramidal neurons. Despite the large number of publications dedicated to regulation of intracellular Cl-, our understanding of the underlying mechanisms is not complete. Experiments on GABA actions under resting steady-state have shown that the effect of GABA shifts from depolarizing to hyperpolarizing during maturation of cortical neurons. However, it remains unclear, whether conclusions from these steady-state measurements can be extrapolated to the highly dynamic situation within an intact and active neuronal network. Indeed, GABAergic signaling in active neuronal networks results in a continuous Cl- load, which must be constantly removed by efficient Cl- extrusion mechanisms. Therefore, it seems plausible to suggest that key parameters are the efficacy and subcellular distribution of Cl- transporters rather than the polarity of steady-state GABA actions. A further related question is: what are the mechanisms of Cl- regulation and homeostasis during pathophysiological conditions such as epilepsy in adults and neonates? Here I present results that were obtained by means of a newly developed method of measurements of the efficacy of a K-Cl cotransport. In Study I, the developmental profile of KCC2 functionality during development was analyzed both in dissociated neuronal cultures and in acute hippocampal slices. A novel method of photolysis of caged GABA in combination with Cl- loading to the somata was used in this study to assess the extrusion efficacy of KCC2. We demonstrated that these two preparations exhibit a different temporal profile of functional KCC2 upregulation. In Study II, we reported an observation of highly distorted dendritic spines in neurons cultured from KCC2-/- embryos. During their development in the culture dish, KCC2-lacking neurons failed to develop mature, mushroom-shaped dendritic spines but instead maintained an immature phenotype of long, branching and extremely motile protrusions. It was shown that the role of KCC2 in spine maturation is not based on its transport activity, but is mediated by interactions with cytoskeletal proteins. Another important player in Cl- regulation, NKCC1 and its role in the induction and maintenance of native Cl- gradients between the axon initial segment (AIS) and soma was the subject of Study III. There we demonstrated that this transporter mediates accumulation of Cl- in the axon initial segment of neocortical and hippocampal principal neurons. The results suggest that the reversal potential of the GABAA response triggered by distinct populations of interneurons show large subcellular variations. Finally, a novel mechanism of fast post-translational upregulation of the membrane-inserted, functionally active KCC2 pool during in-vivo neonatal seizures and epileptiform-like activity in vitro was identified and characterized in Study IV. The seizure-induced KCC2 upregulation may act as an intrinsic antiepileptogenic mechanism.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.

GATA Factors Regulate Inner Ear Development and Midbrain Neurogenesis

The zinc-finger transcription factors GATA2 and GATA3 in vertebrates belong to the six-member family that are essential regulators in the development of various organs. The aim of this study was to gain new information of the roles of GATA2 and GATA3 in inner ear morphogenesis and of the function of GATA2 in neuronal fate specification in the midbrain using genetically modified mouse and chicken embryos as models. A century ago the stepwise process of inner ear epithelial morphogenesis was described, but the molecular players regulating the cellular differentiation of the otic epithelium are still not fully resolved. This study provided novel data on GATA factor roles in several developmental processes during otic development. The expression analysis in chicken suggested that GATA2 and GATA3 possess redundant roles during otic cup and vesicle formation, but complementary cell-type specific functions during vestibular and cochlear morphogenesis. The comparative analysis between mouse and chicken Gata2 and Gata3 expression revealed many conserved aspects, especially during later stages of inner ear development, while the expression was more divergent at early stages. Namely, expression of both Gata genes was initiated earlier in chicken than mouse otic epithelium relative to the morphogenetic stages. Likewise, important differences concerning Gata3 expression in the otic cup epithelium were detected between mouse and chicken, suggesting that distinct molecular mechanisms regulate otic vesicle closure in different vertebrate species. Temporally distinct Gata2 and Gata3 expression was also found during otic ganglion formation in mouse and chicken. Targeted inactivation of Gata3 in mouse embryos caused aberrant morphology of the otic vesicle that in severe cases was disrupted into two parts, a dorsal and a ventral vesicle. Detailed analyses of Gata3 mutant embryos unveiled a crucial role for GATA3 in the initial inner ear morphogenetic event, the invagination of the otic placode. A large-scale comparative expression analysis suggested that GATA3 could control cell adhesion and motility in otic epithelium, which could be important for early morphogenesis. GATA3 was also identified as the first factor to directly regulate Fgf10 expression in the otic epithelium and could thus influence the development of the semicircular ducts. Despite the serious problems in the early inner ear development, the otic sensory fate establishment and some vestibular hair cell differentiation was observable in pharmacologically rescued Gata3-/- embryos. Cochlear sensory differentiation was, however, completely blocked so that no auditory hair cells were detected. In contrast to the early morphogenetic phenotype in Gata3-/- mutants, conditional inactivation of Gata2 in mouse embryos resulted in a relatively late growth defect of the three semicircular ducts. GATA2 was required for the proliferation of the vestibular nonsensory epithelium to support growing of the three ducts. Concurrently, with the role in epithelial semicircular ducts, GATA2 was also required for the mesenchymal cell clearance from the vestibular perilymphatic region between the membranous labyrinth and bony capsule. The gamma-aminobutyric acid-secreting (GABAergic) neurons in the midbrain are clinically relevant since they contribute to fear, anxiety, and addiction regulation. The molecular mechanisms regulating the GABAergic neuronal development, however, are largely unknown. Using tissue-specific mutagenesis in mice, GATA2 was characterized as a critical determinant of the GABAergic neuronal fate in the midbrain. In Gata2-deficient mouse midbrain, GABAergic neurons were not produced, instead the Gata2-mutant cells acquired a glutamatergic neuronal phenotype. Gain-of-function experiments in chicken also revealed that GATA2 was sufficient to induce GABAergic differentiation in the midbrain.

The evolution of genomic imprinting

We explore three possible pathways for the evolution of genomic imprinting. (1) Imprinting may be advantageous in itself when imprinted and unimprinted alleles of a locus confer different phenotypes. If a segment of DNA is imprinted in the gametes of one sex but not in those of the other, it might lead to effects correlated with sexual dimorphism. More fundamentally, in certain organisms, sex determination might have evolved because of imprinting. When imprinting leads to chromosome elimination or inactivation and occurs in some embryos but not in others, two classes of embryos, differing in the number of functional gene copies, would result. A model for sex determination based on inequality in the actual or effective copy-number of particular noncoding, regulatory sequences of DNA has been proposed (Chandra, Proc. natn. Acad. Sci. U.S.A. 82. 1165–1169 and 6947–6949, 1985). Maternal control of offspring sex is another possible consequence of imprinting; this would indicate a potential role for imprinting in sex ratio evolution. (2) Genes responsible for imprinting may have pleiotropic effects and they may have been selected for reasons other than their imprinting ability. Lack of evidence precludes further consideration of this possibility. (3) Imprinting could have co-evolved with other traits. For instance, gamete-specific imprinting could lead to a lowered fitness of androgenetic or gynogenetic diploids relative to the fitness of ‘normal’ diploids. This in turn would reinforce the evolution of anisogamy. The reversibility of imprinting raises the possibility of occasional incomplete or improper erasure. If the site of imprinting is the egg – as appears to be the case with the human X (Chandra and Brown, Nature 253. 165–168, 1975) – either improper imprinting or improper erasure could lead to unusual patterns of inheritance (as in the fragile-X syndrome) or fitness effects skipping generations.

Cloning and sequence of a cDNA encoding a novel hybrid prolme-rich protein associated with cytokinin-induced haustoria formation in Cuscuta reflexa

A complete cDNA encoding a novel hybrid Pro-rich protein (HyPRP) was identified by differentially screening 3x10(4) recombinant plaques of a Cuscuta reflexa cytokinin-induced haustorial cDNA library constructed in lambda gt10. The nucleotide (nt) sequence consists of: (i) a 424-bp 5'-non coding region having five start codons (ATGs) and three upstream open reading frames (uORFs); (ii) an ORF of 987 bp with coding potential for a 329-amino-acid (aa) protein of M(r), 35203 with a hydrophobic N-terminal region including a stretch of nine consecutive Phe followed by a Pro-rich sequence and a Cys-rich hydrophobic C terminus; and (iii) a 178-bp 3'-UTR (untranslated region). Comparison of the predicted aa sequence with the NBRF and SWISSPROT databases and with a recent report of an embryo-specific protein of maize [Jose-Estanyol et al., Plant Cell 4 (1992) 413-423] showed it to be similar to the class of HyPRPs encoded by genes preferentially expressed in young tomato fruits, maize embryos and in vitro-cultured carrot embryos. Northern analysis revealed an approx. 1.8-kb mRNA of this gene expressed in the subapical region of the C. reflexa vine which exhibited maximum sensitivity to cytokinin in haustorial induction.

Proposed role of w chromosome inactivation and the absence of dosage compensation in avian sex determination

Three features of avian sex chromosomes - female heterogamety (ZZ male, ZW female), the apparently inactive state of the W chromosome, and dose-dependent expression of Z-linked genes - are examined in regard to their possible relation to sex determination. It is proposed that the W chromosome is facultatively heterochromatic and that the Z and W chromosomes carry one or more homologous sex-determination genes. The absence of dosage compensation in ZZ embryos, and W inactivation in ZW embryos, would then bring about a 2n(ZZ)-n(ZW) inequality in the effective copy number of such genes. The absence of dosage compensation of Z-linked genes in ZZ embryos is viewed as a means by which two copies of Z-W homologous sex determination genes are kept active to meet the requirements of testis determination. W inactivation may promote ovarian development by reducing the effective copy number of these genes from 2n to n. If there is a W-specific gene for femaleness, spread of heterochromatization to this gene in cells forming the right gonadal primordium may explain the latter's normally undifferentiated state; reversal of heterochromatization may similarly explain the development of the right gonad into a testis following left ovariectomy.

Synergistic interaction between particular X-chromosome deletions andSex-lethal causes female lethality inDrosophila melanogaster

We studied the effect on female viability of trans-heterozygous combinations of X-chromosome deficiencies and Sxt-(fl), a null allele of Sex-lethal. Twentyfive deficiencies, which together covered 80% of the X chromosome, were tested. Seven of these trans-heterozygous combinations caused significant levels of female lethality. Two of the seven interacting deficiencies include the previously known sex determination genes sans fille and sisterless-a. Four of the remaining uncover X-chromosomal regions that were not hitherto known to contain sex determination genes. These newly identified regions are defined by deficiencies Df(1)RA2 (7D10; 8A4-5), Df(1)KA14 (7F1-2; 8C6), Df(1)C52 (8E; 9C-D) and Df(1)N19 (17A1; 18A2). These four deficiencies were characterized further to determine whether it was the maternal or zygotic dosage that was primarily responsible for the observed lethality of female embryos, daughterless and extra macrochaetae, two known regulators of Sxl, influence the interaction of these deficiencies with Sxl.

Synthesis of Trilaurin by Developing Pisa Seeds (Actinodaphne hookeri)

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C-16:0, C-18:0, C-18:2, and C-18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C-12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% FL. At this stage the TAG contained exclusively C-12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C-12:0. Experiments on [1-C-14]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in FL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into FL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into FL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C-12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering, Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C-12:0 and 61% of C-14:0, but not C-16:0, C-18:2, and C-18:3, into TAG. In contrast the LCFA were incorporated preferentially into FL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C-12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C-12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering. (C) 1994 Academic Press, Inc.

Social selection and the evolution of cooperative groups: The example of the cellular slime moulds

In social selection the phenotype of an individual depends on its own genotype as well as on the phenotypes, and so genotypes, of other individuals. This makes it impossible to associate an invariant phenotype with a genotype: the social context is crucial. Descriptions of metazoan development, which often is viewed as the acme of cooperative social behaviour, ignore or downplay this fact. The implicit justification for doing so is based on a group-selectionist point of view. Namely, embryos are clones, therefore all cells have the same evolutionary interest, and the visible differences between cells result from a common strategy. The reasoning is flawed, because phenotypic heterogeneity within groups can result from contingent choices made by cells from a flexible repertoire as in multicellular development. What makes that possible is phenotypic plasticity, namely the ability of a genotype to exhibit different phenotypes. However, co-operative social behaviour with division of labour requires that different phenotypes interact appropriately, not that they belong to the same genotype, or have overlapping genetic interests. We sketch a possible route to the evolution of social groups that involves many steps: (a) individuals that happen to be in spatial proximity benefit simply by virtue of their number; (b) traits that are already present act as preadaptations and improve the efficiency of the group; and (c) new adaptations evolve under selection in the social context-that is, via interactions between individuals-and further strengthen group behaviour. The Dictyostelid or cellular slime mould amoebae (CSMs) become multicellular in an unusual way, by the aggregation of free-living cells. In nature the resulting group can be genetically homogeneous (clonal) or heterogeneous (polyclonal); in either case its development, which displays strong cooperation between cells (to the extent of so-called altruism) is not affected. This makes the CSMs exemplars for the study of social behaviour.

Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug, Planococcus lilacinus

A clone showing female-specific expression was identified from an embryonic cDNA library of a mealybug, Planococcus lilacinus, In Southern blots this clone (P7) showed hybridization to genomic DNA of females, but not to that of males, However, P7 showed no hybridization to nuclei of either sex, raising the possibility that it was extrachromosomal in origin, In sectioned adult females P7 hybridized to an abdominal organ called the mycetome. The mycetome is formed by mycetocytes, which are polyploid cells originating from the polar bodies and cleavage nuclei that harbour maternally transmitted, intracellular symbionts. Electron microscopy confirmed the presence of symbionts within the mycetocytes, Sequence analysis showed that P7 is a 16S rRNA gene, confirming its prokaryotic origin, P7 transcripts are localized to one pole in young embryos but are found in the pole as well as in the germ band during later stages of development, P7 expression is detectable in young embryos of both sexes but the absence of P7 in third instar and adult males suggests that this gene, and hence the endosymbionts, are subject to sex-specific elimination. Copyright (C) 1997 Elsevier Science Ltd.

The determination of spatial pattern in Dictyostelium discoideum

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other, and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ.