RESUMEN
FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.
Asunto(s)
Dictyostelium/genética , Proteínas F-Box , Proteínas Protozoarias/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cloruro de Amonio/farmacología , Animales , Western Blotting , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Dictyostelium/citología , Dictyostelium/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Mutagénesis Insercional , Fenotipo , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismoRESUMEN
Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.
Asunto(s)
Dictyostelium/citología , Dictyostelium/fisiología , Proteínas Protozoarias/fisiología , Proteínas Represoras/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , ADN Complementario/genética , ADN Protozoario/genética , Dictyostelium/genética , Genes Protozoarios , Genes Reporteros , Marcadores Genéticos , Operón Lac , Microscopía por Video , Mutagénesis Insercional , Mutación , Proteínas Protozoarias/genética , Proteínas Represoras/genéticaRESUMEN
The TTGA-binding factor is a transcriptional regulator activated by DIF, the chlorinated hexaphenone that induces prestalk cell differentiation in Dictyostelium. The same activity also functions as a repressor, controlling stalk cell differentiation. We show that the TTGA-binding factor is a STAT protein. Like the metazoan STATs, it functions via the reciprocal interaction of a phosphotyrosine residue on one molecule with an SH2 domain on a dimerizing partner. Furthermore, it will bind specifically to a mammalian interferon-stimulated response element. In Saccharomyces cerevisiae, where the entire genomic sequence is known, SH2 domains have not been identified. It would seem, therefore, that SH2 signaling pathways arose very early in the evolution of multicellular organisms, perhaps to facilitate intercellular comunication.
Asunto(s)
Dictyostelium/fisiología , Proteínas de la Leche , Transducción de Señal/fisiología , Dominios Homologos src/fisiología , Animales , Diferenciación Celular/fisiología , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Janus Quinasa 1 , Mamíferos , Espectrometría de Masas , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT5 , Homología de Secuencia de Aminoácido , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
DIF-1 [Differentiation-Inducing Factor 1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one] is a novel chlorinated signal molecule that induces stalk-cell differentiation during development of Dictyostelium discoideum. Here we introduce the use of the radioisotope 36Cl to label DIF-1 and other low-Mr chlorinated compounds produced during development. H.p.l.c. and t.l.c. were used to resolve the labelled compounds. We find the following. (1) At least 14 dialysable 36Cl-labelled compounds are released into the medium by cells labelled continuously through development with Na36Cl. (2) The compounds can be classified into two major groups according to their times of accumulation in development. The early group of compounds starts accumulating at the end of aggregation, co-ordinately with DIF-1; the late group is only made at the end of development, by mature fruiting bodies. There may also be an intermediate group made during culmination. (3) The early group of compounds has been identified as comprising DIF-1 and seven of its metabolites by co-chromatography with the authentic compounds. These metabolites had previously only been recognized in suspensions of living cells incubated with exogenous DIF-1. Their detection here, from cells undergoing normal development, suggests that endogenous DIF-1 is metabolized in normal development in much the same way as is DIF-1 added to cells in suspension. (4) The intermediate and late groups of compounds are not obvious DIF-1 metabolites. They may have some role unconnected with DIF signalling. (5) A group of 36Cl-labelled late compounds remain cell-associated after washing of the fruiting bodies, and these are greatly enriched in stalk, compared with spore, cells. (6) Other slime-mould species were labelled with 36Cl. All three tested, namely D. mucoroides, D. vinaceo-fuscum and P. violaceum, also produced chloro compounds. D. mucoroides produced DIF-1 by the criterion of h.p.l.c. co-elution with authentic DIF-1. A developmentally regulated metabolism of chlorinated compounds may therefore be widespread amongst slime moulds. To our knowledge, labelling with 36Cl in vivo has not been reported before and provides a powerful general method for investigating chlorinated compounds in diverse organisms.
Asunto(s)
Cloruros/metabolismo , Dictyostelium/metabolismo , Animales , Cloruros/aislamiento & purificación , Cloro , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Dictyostelium/crecimiento & desarrollo , Técnica de Dilución de Radioisótopos , RadioisótoposRESUMEN
The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devl Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst 'O' region is a preparatory step in the process.
Asunto(s)
Dictyostelium/genética , Proteínas de la Matriz Extracelular/genética , Expresión Génica/fisiología , Movimiento Celular/fisiología , Dictyostelium/citología , Dictyostelium/ultraestructura , Marcadores Genéticos , Luz , Microscopía Fluorescente , Morfogénesis/genética , Proteínas Recombinantes de FusiónRESUMEN
We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the prestalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.
Asunto(s)
Dictyostelium/fisiología , ARN Mensajero/análisis , Northern Blotting , Movimiento Celular , Clonación Molecular , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Regulación Fúngica de la Expresión Génica , Sondas de Oligonucleótidos , ARN Mensajero/genética , Esporas Fúngicas/fisiologíaRESUMEN
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.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Antígenos Transformadores de Poliomavirus/genética , Diferenciación Celular , Dictyostelium/citología , Dictyostelium/genética , Expresión Génica , Genes Bacterianos , Fusión de Membrana , Morfogénesis , Esporas Fúngicas/fisiología , Transformación Genética , beta-Galactosidasa/genéticaRESUMEN
The characteristic structure of the mature Dictyostelium culminant is created by the regionalized cellular differentiation and directed movement of prestalk cells. The front prestalk zone of the migratory slug has previously been considered to be a homogeneous tissue. Here we demonstrate, however, the existence of multiple classes of prestalk cells located in different parts or the slug anterior. The pDd56 and pDd63 genes encoding closely related extracellular matrix proteins are dependent for their expression upon DIF-1, the specific stalk-cell inducer. We have fused the promoters of the two genes to a modified chloramphenicol acetyltransferase (cat) gene to produce immunologically detectable proteins which localize to the cell nucleus. These two markers define three distinct kinds of 'prestalk' cells. One class, which we term 'prestalk A' cells, expressed the pDd63 gene. 'Prestalk B' cells express pDd56 and may also express the pDd63 gene. A third class, which we term 'prestalk 0' cells, expresses neither marker.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Diferenciación Celular , Clonación Molecular , Dictyostelium/genética , Dictyostelium/fisiología , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
The expression of ras-related protein in the cellular slime mold Dictyostelium discoideum is developmentally regulated. It was previously reported that Dictyostelium possesses a single ras gene (Ddras) that is maximally expressed during the pseudoplasmodial stage of development. We have isolated a series of cDNA clones derived from a second ras gene, DdrasG. It encodes a protein that is very similar to the protein encoded by Ddras, but in contrast to Ddras, DdrasG is only expressed during growth and early development. Although other eukaryotic organisms possess more than one ras gene, Dictyostelium is thus far unique in expressing different ras genes at different stages of development. In Dictyostelium the two ras proteins may fulfill different functions, with the DdrasG protein playing a role during cell growth and the Ddras protein playing a role in signal transduction during multicellular development.
Asunto(s)
Dictyostelium/genética , Proteínas Fúngicas/genética , Genes ras , Proteínas ras , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Dictyostelium/crecimiento & desarrollo , Genes , Genes Fúngicos , Datos de Secuencia Molecular , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
The pDd63 and pDd56 genes encode extracellular matrix proteins which, respectively, surround the migratory slug and mature stalk cells. Both genes are dependent for their expression upon, and rapidly induced by, DIF, the stalk cell inducer. Using these genes as cell-autonomous markers, we have defined three distinct kinds of 'prestalk' cells localized to different parts of the anterior region of the slug. At least one, and probably both, prestalk cell types initially differentiates at the base of the aggregate. The most abundant of the two prestalk cell types then migrates into the tip, the precursor of the prestalk zone which arises at the apex of the aggregate. Thus we believe that morphogenesis of the prestalk zone, the primary pattern-forming event in Dictyostelium development, involves a combination of positionally localized differentiation and directed cell migration. To account for the positionally localized differentiation of prestalk cells, we invoke the existence of gradients of the known antagonists of DIF-cAMP and NH3. We further suggest that differences in the motility of pstA and pstB cells might result from differences in their chemotactic responsiveness to cAMP signals propagated from the tip.
Asunto(s)
Diferenciación Celular , Dictyostelium/crecimiento & desarrollo , Transducción de Señal , MorfogénesisRESUMEN
The migratory slug of Dictyostelium discoideum is surrounded by, and continuously synthesizes, an extracellular protein-cellulose matrix known as the slime sheath which is deposited on the substratum as a trail marking the slug's progress. We show that the stalk-specific proteins, ST310 and ST430, are exclusively located in the slime sheath and trail and that fusion genes, containing upstream sequences from the cognate genes, direct correct mRNA accumulation during development and correct localization of the fusion protein. Immunoelectron microscopy shows the ST310 and ST430 proteins to be present throughout the entire thickness of the slime sheath and almost totally absent from the cells of the slug. The genes that encode the ST310 and ST430 polypeptides are inducible by DIF, a stalk-specific inducing agent, and the mRNAs are highly enriched in prestalk over prespore cells. The production of these extracellular proteins by prestalk cells suggests that, in a manner somewhat analogous to that of extracellular matrix proteins of higher eukaryotes, the anterior region of the slug may be responsible for the continuous deposition of a track, along which the slug cells migrate. In the mature culminant, the ST310, and possibly the ST430, protein form part of the stalk tube and stalk cell wall. Therefore, the results also show that there are proteins common to both slime trial and stalk tube, indicating a possible precursor-product relationship between these chemically similar integuments.
Asunto(s)
Dictyostelium/metabolismo , Matriz Extracelular/análisis , Proteínas Fúngicas/análisis , ARN Mensajero/metabolismo , Immunoblotting , Microscopía Electrónica , Microscopía Fluorescente , Péptidos/análisisRESUMEN
Two endogenous differentiation-inducing factors (DIF-2 and DIF-3), which induce stalk-cell differentiation in the cellular slime mould Dictyostelium discoideum, have been identified as the pentan-1-one and monochloro analogues respectively of (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one). These compounds represent a new chemical class of effector molecules.
Asunto(s)
Dictyostelium/análisis , Hexanonas , Cetonas , Diferenciación Celular , Fenómenos Químicos , Química , Espectrometría de MasasRESUMEN
The nucleotide sequence of D19, a Dictyostelium gene that encodes a prespore-specific mRNA sequence shows it to encode PsA, the cell surface protein detected by the MUD 1 monoclonal antibody. The predicted sequence of the protein reveals a largely hydrophobic C terminus, with chemical similarity to proteins known to be attached to the plasma membrane via a phosphatidylinositol link. The C-terminal region has direct sequence homology to the contact sites A protein and to the phosphatidylinositol-linked form of a chicken N-CAM, suggesting that it might play a role in cell adhesion. Expression of the D19 gene is known to be induced by cAMP and repressed by adenosine. The accumulation of the D19 mRNA is also repressed by DIF, the putative stalk-specific morphogen, and this effect is mediated at the transcriptional level. The pDd56 and pDd63 genes are induced by DIF, and they are specific markers of prestalk and stalk cells. They encode, respectively, ST310 and ST430, two proteins that were first identified by two-dimensional gel electrophoresis. Both proteins are predominantly composed of a highly conserved, 24-amino acid repeat. The two proteins are localized in the slime sheath of the migratory slug and in the stalk tube and stalk cell wall of the mature culminant, where they presumably function as structural components of the extracellular matrix. We have constructed marked derivatives of the pDd56, pDd63, and D19 genes, and these are correctly regulated after transformation into Dictyostelium cells. Thus we have determined the structure, and elucidated possible functions, for one prespore and two prestalk genes. These sequences should be of value, both as markers of the earliest events in cellular differentiation and in identifying the regulatory sequences controlling cell type-specific gene expression.
Asunto(s)
Dictyostelium/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Dictyostelium/fisiología , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Esporas Fúngicas/fisiologíaRESUMEN
The pDd56 mRNA sequence is highly enriched in prestalk over prespore cells and is inducible by DIF, the putative Dictyostelium stalk-specific morphogen. We show that the pDd56 gene is composed of forty one copies of a twenty four amino acid, cysteine rich repeat. This is highly homologus to a repeat which we have previously shown to compose the major fraction of the pDd63 mRNA, another DIF inducible, prestalk-enriched sequence. The predicted pDd56 protein contains a putative signal peptide but does not appear to contain a transmembrane segment. In combination these features suggest it to be an extrinsic protein and we confirm this elsewhere by showing that the pDd56 gene encodes a known, extracellular protein of the stalk. The pDd56 mRNA is dependent upon exogenous DIF for its accumulation. We show that this control is exerted at the transcriptional level and that a restriction fragment containing 1.7Kb of upstream sequence directs temporally-regulated expression of the gene.
Asunto(s)
Dictyostelium/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Hexanonas/farmacología , Cetonas/farmacología , ARN de Hongos/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , ADN Recombinante , Dictyostelium/crecimiento & desarrollo , Regulación de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
Morphogens are signal molecules presumed to exist in embryos and to be involved in establishing the spatial pattern of cells during development. Differentiation inducing factor (DIF) has the properties of a morphogen required for producing the prestalk/prespore pattern in the aggregate formed by cells of the slime mould Dictyostelium in response to starvation. DIF-1, the major bioactive species after purification, has now been identified using a combined microchemical, spectroscopic and synthetic approach. The structure is defined as 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone, and represents a new class of effector molecule. The availability of relatively large quantities of synthetic and isotopically labelled materials should now allow progress towards a detailed understanding of the pattern-forming processes in Dictyostelium development.
Asunto(s)
Dictyostelium/análisis , Hexanonas , Cetonas , Diferenciación Celular , Fenómenos Químicos , Química , Dictyostelium/citología , Hexanonas/aislamiento & purificación , Cetonas/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Morfogénesis , Espectrofotometría UltravioletaRESUMEN
We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , ARN Mensajero/clasificación , Secuencia de Bases , Clonación Molecular , Dictyostelium/efectos de los fármacos , Electroforesis en Gel de Agar , Hexanonas/farmacología , Cinética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
The Dictyostelium slug contains a simple anterior-posterior pattern of prestalk and prespore cells. It is likely that DIF, the morphogen which induces stalk cells, is involved in establishing this pattern. Previous work has shown that a number of distinct species of DIF are released by developing cells and that cell-associated DIF activity increases rapidly during the slug stage of development. In this paper we describe a comparison of the DIF extracted from slugs with the DIF released into the medium. Analysis by high-pressure liquid chromatography (HPLC) using different solvent systems shows that the major species of DIF activity extracted from slugs coelutes with DIF-1, the major species of released DIF and is similarly sensitive to sodium borohydride reduction. Since DIF specifically induces the differentiation of prestalk cells, the anterior cells of the slug, it could be anticipated that DIF is localized in the prestalk region. We have therefore determined the distribution of DIF within the slug. Migrating slugs from strain V12M2 were manually dissected into anterior one-third and posterior two-third fragments and the DIF activity extracted. Surprisingly, we found that DIF was not restricted to the prestalk fragment. Instead there appears to be a reverse gradient of DIF in the slug with at least twice the specific activity of total DIF in the prespore region than in the prestalk region.
Asunto(s)
Dictyostelium/análisis , Hexanonas/análisis , Cetonas/análisis , Animales , Bioensayo , Cromatografía Líquida de Alta Presión , MorfogénesisRESUMEN
We have isolated a gene that is very rapidly induced at the transcriptional level by DIF--a low molecular weight, diffusible factor necessary for stalk cell differentiation in Dictyostelium cells developing in vitro. The gene encodes a protein containing an N-terminal signal peptide preceding approximately 70 tandem repeats of a highly conserved 24 amino acid sequence with a high cysteine content. These features suggest it is an extracellular structural protein. During normal development, the gene is maximally expressed in the slug, in which the mRNA is very highly enriched in prestalk over prespore cells. The gene is not detectably expressed until the tipped aggregate stage, several hours later than prespore genes, suggesting that prespore cell differentiation precedes prestalk cell differentiation. The demonstration that DIF induces a gene normally only expressed in the prestalk zone of the slug provides strong evidence that DIF is a Dictyostelium morphogen.
Asunto(s)
Dictyostelium/genética , Proteínas Fúngicas/fisiología , Secuencia de Aminoácidos , Diferenciación Celular , Dictyostelium/citología , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Genes Fúngicos , Morfogénesis , ARN de Hongos/genética , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low-Mr factors, which can induce isolated cells to differentiate into stalk cells in the presence of cyclic AMP [Town, C. D., Gross, J. D. and Kay, R. R. (1976) Nature (Lond.) 262, 717-719; Town, C. D. and Stanford, E. (1979) Proc. Natl Acad. Sci. USA, 76, 308-312]. These differentiation-inducing factors (DIF) have now been purified by a procedure involving binding to and elution from XAD-2 resin, extraction into hexane and two steps of reverse-phase high-pressure liquid chromatography (HPLC). Our results show the following. HPLC resolves a major stalk-cell-inducing activity (DIF-1) and at least four minor and more polar activities (DIFs 2-5). DIF-1 has been purified at least 3000-fold over the starting dialysed medium with a recovery of about 2%. This low recovery of DIF-1 can be explained in part by the loss of non-specific stimulatory ('helper') factors during the purification. A few micrograms purified DIF-1 were obtained from 10(12) cells. This material could induce stalk cell differentiation in the standard assay at less than 0.2 nM. The biological activity of DIFs 1, 2 and 3 was sensitive to borohydride reduction, suggesting the presence of an essential carbonyl group. DIF-5 was partially sensitive and DIF-4 resistant. Other properties of DIF-1 suggest that it is a non-polar molecule of Mr less than 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties. The purification of DIF should make possible its eventual identification by sensitive physical techniques, such as mass spectroscopy, and will allow further investigation of its biological effects.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Bioensayo , Diferenciación Celular , Fenómenos Químicos , Química , Cromatografía Líquida de Alta Presión , Dictyostelium/análisis , MorfogénesisRESUMEN
The complex morphology of a higher organism is generated partly by such developmental processes as cell movement and cohesion but also by a social interaction between cells in small areas of embryonic tissue known as morphogenetic fields. The initially similar cells within such a field organize themselves and differentiate, forming a discrete spatial pattern which is remarkably independent of field size and which can regenerate after some part is removed. Although it is believed that a cell signalling system must underlie this behaviour, the putative signals--or morphogens--have so far proved elusive. Perhaps the simplest known morphogenetic field arises within the multicellular aggregate formed by developing cells of the slime mould Dictyostelium discoideum. As the amorphous aggregate transforms into a cylindrical slug, a simple pattern emerges, with prestalk cells differentiating in the anterior and prespores in the posterior. One great difficulty in identifying any morphogen has been to predict properties that could form the basis of a bioassay. However, in Dictyostelium it is almost essential that the morphogens should dictate to cells their choice of differentiation pathway. We have described previously a crude factor termed DIF which stimulates the differentiation of isolated amoebae into stalk cells. We now show that purified DIF also inhibits spore formation and so switches cells to stalk cell formation. Thus, we believe that DIF is a morphogen which regulates the choice of differentiation pathway of cells in the Dictyostelium slug.