Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

Spatially restricted expression of candidate taste receptors in the Drosophila gustatory system

BACKGROUND: Taste is an important sensory modality in most animals. In Drosophila, taste is perceived by gustatory neurons located in sensilla distributed on several different appendages throughout the body of the animal. Here we show that the gustatory receptors are encoded by a family of at least 54 genes (Gr genes), most of which are expressed exclusively in a small subset of taste sensilla located in narrowly defined regions of the fly's body. RESULTS: BLAST searches with the predicted amino acid sequences of 6 7-transmembrane-receptor genes of unknown function and 20 previously identified, putative gustatory receptor genes led to the identification of a large gene family comprising at least 54 genes. We investigated the expression of eight genes by using a Gal4 reporter gene assay and found that five of them were expressed in the gustatory system of the fly. Four genes were expressed in 1%-4% of taste sensilla, located in well-defined regions of the proboscis, the legs, or both. The fifth gene was expressed in about 20% of taste sensilla in all major gustatory organs, including the taste bristles on the anterior wing margin. Axon-tracing experiments demonstrated that neurons expressing a given Gr gene project their axons to a spatially restricted domain of the subesophageal ganglion in the fly brain. CONCLUSIONS: Our findings suggest that each taste sensillum represents a discrete, functional unit expressing at least one Gr receptor and that most Gr genes are expressed in spatially restricted domains of the gustatory system. These observations imply the potential for high taste discrimination of the Drosophila brain.     

Spatially restricted actions of BDNF

In this issue of Neuron, Zhang and Poo present evidence for localized BDNF-induced synaptic potentiation that is accompanied by spatially restricted calcium influx and requires local axonal protein synthesis. These results are consistent with a synapse-specific role for BDNF and provide a potentially novel way to think about cellular mechanisms for potentiation of neurotransmitter release.     

Hedgehog does not guide migrating Drosophila germ cells

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.     

Identification of a mouse germ cell-less homologue with conserved activity in Drosophila

Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed.     

The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell-cell adhesion

BACKGROUND: Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive. RESULTS: Via bead implantations, we show that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca(2+)/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction. CONCLUSIONS: We propose that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula.     

Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. We now show that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. We find that the integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs we show that wun function in the trachea is distinct from its role in germ cell migration.     

Mutagenicity of sumithion tested in Drosophila somatic and germ cells

Sumithion, a broad-spectrum insecticide, was tested for its mutagenicity in the Drosophila wing-spot test and sex-linked recessive lethal test. Strains carrying the recessive mutant markers mwh and flr3 in their third chromosomes, expressed phenotypically as multiple trichomes or thickened and misshapen wing hairs in the adult wings, were used in the wing-spot test. Larvae transheterozygous for these markers were exposed to the insecticide in instant food and the sex-linked recessive lethal test was performed by the standard technique using the Basc strain. The compound is mutagenic in the wing primordial cells and induces recombination at high doses. Further, the frequency of induction of sex-linked recessive lethals is significant only at high treatment doses.     

Evidence for the absence of mutagenic activity of furfuryl alcohol in tests of germ cells in Drosophila melanogaster

Furfuryl alcohol was evaluated for mutagenic activity in D. melanogaster by means of the sex-linked recessive lethal test and the sex-chromosome loss test. Brooding was employed in order to test different stages of spermatogenesis. No evidence was found of a mutagenic effect after adult injection and larval feeding.     

Recombination and disjunction in female germ cells of Drosophila depend on the germline activity of the gene sex-lethal

Gametogenesis in males and females differs in many ways. An important difference in Drosophila is that recombination between homologous chromosomes occurs only in female meiosis. Here, we report that this process relies on the correct functioning of Sex-lethal (Sxl) which is primarily known as the master gene in somatic sex determination. Certain alleles of this gene (Sxl(fs)) disrupt the germline, but not the somatic function of Sxl and cause an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxl(fs) mutant females, we discovered additional requirements of Sxl for normal meiotic differentiation of the oocyte. Females mutant for Sxl(fs) and carrying a suppressor become fertile, but pairing of homologous chromosomes and formation of chiasmata is severely perturbed, resulting in an almost complete lack of recombinants and a high incidence of non-disjunction events. Similar results were obtained when germline expression of wild-type Sxl was compromised by mutations in virilizer (vir), a positive regulator of Sxl. Ectopic expression of a Sxl transgene in premeiotic stages of male germline development, on the other hand, is not sufficient to allow recombination to take place, which suggests that Sxl does not have a discriminatory role in this female-specific process. We propose that Sxl performs at least two tasks in oogenesis: an 'early' function in formation of the egg chamber, and a 'late' function in progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism.     

Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor

Protein kinases and phosphatases are organized into complex intracellular signaling networks designed to coordinate their activities in both space and time. In order to better understand the molecular mechanisms underlying the regulation of signal transduction networks, it is important to define the spatiotemporal dynamics of both protein kinases and phosphatases within their endogenous environment. Herein, we report the development of a genetically-encoded protein biosensor designed to specifically probe the activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. Our reporter design utilizes a phosphatase activity-dependent molecular switch based on the N-terminal regulatory domain of the nuclear factor of activated T-cells as a specific substrate of calcineurin, sandwiched between cyan fluorescent protein and yellow fluorescent protein. Using this reporter, calcineurin activity can be monitored as dephosphorylation-induced increases in fluorescence resonance energy transfer and can be simultaneously imaged with intracellular calcium dynamics. The successful design of a prototype phosphatase activity sensor lays a foundation for studying targeting and compartmentation of phosphatases.     

Endocytic activity of male germ cells during puberty

It is well known that numerous germ cells degenerate during the first meiotic division and spermatid elongation is due mostly to an adverse physiological microenvironment in relation to hormonal deficiency. The present study is aimed at investigating the endocytic activity of germ cells, during the first wave of mouse spermatogenesis, using transferrin coupled to gold particles, in order to study the efficiency of this possible pathway of communication between Sertoli cells and germ cells. Labelling experiments in control animals confirmed a receptor-mediated pathway in all germ cells during puberty. Furthermore, our morphological and quantitative data revealed that during spermatid elongation, degenerating germ cells possessed a highly developed endocytic apparatus which contained twice as many transferrin gold particles than normal adult cells. The fact that endocytosis of transferrin was increased in degenerating germ cells indicates that, most probably, germ cell degeneration during the first wave of spermatogenesis did not result from a deficiency in iron transport. The higher endocytic activity of degenerating germ cells, compared to adult control cells, could not only be the result of a simple process of plasma membrane internalization but also a complex mechanism which could be involved in the degradation of the cells.