A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium

The cellular slime mold, Dictyostelium discoideum is a non-metazoan organism, yet we now demonstrate that a disintegrin domain-containing protein, the product of the ampA gene, plays a role in cell type specification. Disintegrin domain-containing proteins are involved in Notch signaling in Drosophila and C. elegans via an ectodomain shedding mechanism that depends on a metalloprotease domain. The Dictyostelium protein lacks a metalloprotease domain. Nonetheless, analysis of cell type specific reporter gene expression during development of the ampA null strain identifies patterning defects that define two distinct roles for the AmpA protein in specifying cell fate. In the absence of a functional ampA gene, cells prematurely specify as prespore cells. Prestalk cell differentiation and migration are delayed. Both of these defects can be rescued by the inclusion of 10% wild-type cells in the developing null mutant aggregates, indicating that the defect is non-cell autonomous. The ampA gene is also demonstrated to be necessary in a cell-autonomous manner for the correct localization of anterior-like cells to the upper cup of the fruiting body. When derived from ampA null cells, the anterior-like cells are unable to localize to positions in the interior of the developing mounds. Wild-type cells can rescue defects in morphogenesis by substituting for null cells when they differentiate as anterior-like cells, but they cannot rescue the ability of ampA null cells to fill this role. Thus, in spite of its simpler structure, the Dictyostelium ampA protein carries out the same diversity of functions that have been observed for the ADAM and ADAMTS families in metazoans.     

Expression pattern of alkaline phosphatase in Dictyostelium

We used two different methods to study the expression pattern of alkaline phosphatase (alp) in Dictyostelium. In situ staining of the endogenous enzyme activity at different stages of development showed that the enzyme was active early in the aggregation stage and localized to the area where the tip of the first finger was initiated. The activity was localized to the anterior region of developing slugs, then became restricted to the region between the prestalk and prespore cells at the culmination stage. In the complete fruiting body, the activity was confined to the lower and upper cup. A second method to study alp expression utilized a beta-galactosidase reporter gene under the control of the alp promoter. A low level of beta-galactosidase activity was observed in vegetative cells, then increased during development. Reporter gene activity was restricted to PstO cells at the slug stage. At the culmination stage, the expression was restricted to prestalk cells at the interface between the prestalk and prespore cells. In the completed fruiting body, the expression was observed in the upper and lower cup.     

The trishanku gene and terminal morphogenesis in Dictyostelium discoideum

SUMMARY Multicellular development in the social amoeba Dictyostelium discoideum is triggered by starvation. It involves a series of morphogenetic movements, among them being the rising of the spore mass to the tip of the stalk. The process requires precise coordination between two distinct cell types—presumptive (pre-) spore cells and presumptive (pre-) stalk cells. Trishanku ( triA ) is a gene expressed in prespore cells that is required for normal morphogenesis. The triA⁻ mutant shows pleiotropic effects that include an inability of the spore mass to go all the way to the top. We have examined the cellular behavior required for the normal ascent of the spore mass. Grafting and mixing experiments carried out with tissue fragments and cells show that the upper cup, a tissue that derives from prestalk cells and anterior-like cells (ALCs), does not develop properly in a triA⁻ background. A mutant upper cup is unable to lift the spore mass to the top of the fruiting body, likely due to defective intercellular adhesion. If wild-type upper cup function is provided by prestalk and ALCs, trishanku spores ascend all the way. Conversely, Ax2 spores fail to do so in chimeras in which the upper cup is largely made up of mutant cells. Besides proving that under these conditions the wild-type phenotype of the upper cup is necessary and sufficient for terminal morphogenesis in D. discoideum , this study provides novel insights into developmental and evolutionary aspects of morphogenesis in general. Genes that are active exclusively in one cell type can elicit behavior in a second cell type that enhances the reproductive fitness of the first cell type, thereby showing that morphogenesis is a cooperative process.     

Origins of the prestalk-prespore pattern in Dictyostelium development

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

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12.

Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal beta-lactamasee. From E. coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.     

Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms.

Factors governing the morphogenesis of Bacillus subtilis colonies as well as the spatial-temporal pattern of expression of a reporter gene during colony development were examined by systematically varying the initial nutrient levels and agar concentrations (wetness), the relative humidity throughout incubation, and the genotype of the inoculum. A relationship between colony form and reporter gene expression pattern was found, indicating that cells respond to local signals during colony development as well as global conditions. The most complex colony forms were produced by motile strains grown under specific conditions such that cells could swim within the colony but not swarm outward uniformly from the colony periphery. The wetness of the growth environment was found to be a critical factor. Complex colonies consisted of structures produced by growth of finger-like projections that expanded outward a finite distance before giving rise to a successive round of fingers that behaved in a similar fashion. Finger tip expansion occurred when groups of cells penetrated the peripheral boundary. Although surfactin production was found to influence similar colony forms in other B. subtilis strains, the strains used here to study reporter gene expression do not produce it. The temporal expression of a reporter gene during morphogenesis of complex colonies by motile strains such as M18 was investigated. Expression arose first in cells located at the tips of fingers that were no longer expanding. The final expression pattern obtained reflects the developmental history of the colony.     

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPAR??, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.     

Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.     

A demonstration of pattern formation without positional information in Dictyostelium

Although positional information, conveyed by morphogen gradients, is a widely accepted way of forming patterns during development, an alternative method is conceivable, based on the intermingled differentiation of cells with different fates, followed by their sorting into discrete pattern elements. It has been proposed that Dictyostelium prestalk and prespore cells behave in this way at the mound stage of development. However, it has been difficult to conclusively demonstrate that they initially differentiate intermingled, because rapid cell movement within the mound makes it impossible to be sure where prestalk and prespore cells originate. We have taken a novel approach to address this problem by blocking cell movement at different stages in development, using the actin-depolymerizing drug, latrunculin-A. Prestalk and prespore cells differentiate with essentially normal efficiency and timing in such paralyzed structures. When movement is blocked sufficiently early, the major cell types all subsequently differentiate at scattered positions throughout the aggregate, and even in the streams leading into it. Our work strongly supports the idea that the prestalk/prespore pattern in Dictyostelium forms without positional information and demonstrate that latrunculin-A may provide a useful tool for the investigation of patterning in other organisms.     

Fate determination of fetal Leydig cells

Leydig cell (LC) is one of the most important somatic cell types in testis, which localized in the interstitium between seminiferous tubules. The major function of Leydig cells is to produce steroid hormone, androgens. LC differentiation exhibits a biphasic pattern in rodent testes, which are divided into two different temporal mature populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are transiently present in fetal testes and undergo involution or degeneration after birth. FLCs are completely devoid and replaced by ALCs in adult testes. Comparing to ALCs, FLCs display unique morphology, ultrastructure and functions. The origin of FLCs has been debated for many years, but it is still a mystery. Many factors have been reported regulating the specification, proliferation and differentiation of FLCs. FLCs degenerate in a few weeks postnatally, however, the underlying mechanism is still unknown. In this review, we will focus on the fate determination of FLCs, and summarize the resent progress on the morphology, ultrastructure, function, origin and involution of FLCs.     

Expression of type-1 cannabinoid receptor during rat postnatal testicular development: possible involvement in adult leydig cell differentiation

Endocannabinoids are lipidic modulators able to bind cannabinoid receptors (CNRs). Two types of CNRs have been cloned, CNR1 (central) and CNR2 (peripheral). The objectives of the present study were to investigate the expression pattern of CNR1 in the rat testis during prepubertal development and to define the CNR1 spatiotemporal pattern. From 31 to 60 days of age, CNR1 was immunolocalized in round elongating spermatids and spermatozoa, suggesting an important role for this receptor in spermatogenesis. From 14 to 60 days of age, adult Leydig cells (ALCs) at different developmental stages were positive for CNR1. In particular, CNR1 expression in differentiating ALCs was negatively correlated to cell division. Bromodeoxyuridine uptake experiments on serial sections showed that immature Leydig cells in mitosis were negative for CNR1; in contrast, immature nonmitotic Leydig cells were positive for CNR1. A further observation of few ALCs in CNR1KO mice validates the role of CNR1 during proliferative activity involved in ALC differentiation. In addition, starting from 41 days of age, a faint CNR1 signal was also observed in Sertoli cells. Taken together, these results demonstrate the first clear evidence (to our knowledge) of CNR1 in mammalian germinal epithelium, ALCs, and Sertoli cells and indicate that differentiation of ALCs may depend on the endocannabinoid system.     

gp150蛋白在盘基网柄菌发育中的作用及粘附分子间关系的分析

饥饿的盘基网柄菌进入多细胞发育期 ,在发育早期 ,AK12 7细胞 (gp150突变细胞 )能表达DdCAD 1和 gp80两种粘附分子 ,但它们不足以促进细胞继续发育 ,发育停留在细胞疏松结合阶段。粘附分子 gp150调节的细胞与细胞间的粘着影响了细胞丘“突出”的形成 ,由此影响了盘基网柄菌多细胞发育的形态发生。TL93细胞 (DdCAD 1和gp80突变细胞 )能完成发育。主要原因是在细胞流发育阶段就表达了gp150分子 ,在细胞粘着的功能上有替代DdCAD 1和 gp80的作用。因此 gp150蛋白对盘基网柄菌多细胞发育有着不可或缺的作用