Sxl in the germline of Drosophila: A target for somatic late induction

In Drosophila, the sex of germ cells is determined by autonomous and inductive signals. Somatic inductive signals can drive XX germ cells into oogenesis or into spermatogenesis. An autonomous signal makes XY germ cells male and unresponsive to sex determination by induction. The elements forming the X:A ratio in the soma and the genes tra, tra2, dsx, and ix that determine the sex of somatic cells have no similar role in the germline. The gene Sxl, however, is required for female differentiation of somatic and germ cells. Inductive signals that are dependent on somatic tra and dsx expression already affect the sex-specific development of germ cells of first instar larvae. At this early stage, however, germline expression of Sxl does not appear to affect the sexual characteristics of germ cells. Since inductive signals dependent on tra and dsx nevertheless influence the choice of sex-specific splicing of Sxl, it can be concluded that Sxl is a target of the inductive signal, but that its product is required late for oogenesis. Other genes must therefore control the early sexual dimorphism of larval germ cells. © 1994 Wiley-Liss, Inc.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

 In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA. Received:23 May 1996 Accepted:3 July 1996     

Maternal Nanos‐Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos

Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3′ UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3′ UTR of CG32425 mRNA mediates Nos‐dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3′ UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3′ UTR, we identified the region required for mRNA stabilization, which includes Nos‐binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development.     

Perturbation of Notch/Suppressor of Hairless pathway disturbs migration of primordial germ cells in Xenopus embryo

Primordial germ cells (PGCs) in Xenopus embryo are specified in the endodermal cell mass and migrate dorsally toward the future gonads. The role of the signal mediated by Notch and Suppressor of Hairless [Su(H)] was analyzed on the migrating PGCs at the tailbud stage. X‐Notch‐1 and X‐Delta‐1 are expressed in the migrating PGCs and surrounding endodermal cells, whereas X‐Delta‐2 and X‐Serrate‐1 are expressed preferentially in the PGCs. Suppression and constitutive activation of the Notch/Su(H) signaling in the whole endoderm region or selectively in the PGCs resulted in an increase in ectopic PGCs located in lateral or ventral regions. Knocking down of the Notch ligands by morpholino oligonucleotides revealed that X‐Delta‐2 was indispensable for the correct PGC migration. The ectopic PGCs seemed to have lost their motility in the Notch/Su(H) signal‐manipulated embryos. Our results suggest that a cell‐to‐cell interaction via the Notch/Su(H) pathway has a significant role in the PGC migration by regulating cell motility.     

A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

Sex determination in Drosophila melanogaster: X-linked genes involved in the initial step of Sex-lethal activation

Sex determination is the commitment of an embryo to either the female or the male developmental pathway. The ratio of X chromosomes to sets of autosomes is the primary genetic signal that determines sex in Drosophila, by triggering the functional state of the gene Sex-lethal: in females (2X;2A) Sxl will be ON, whereas in males (X;2A) Sxl will be OFF. Genetic and molecuar studies have defined a set of genes involved in the formation of the X:A signal, as well as other genes, with either maternal or zygotic effects, which are also involved in regulating the initial step of Sex-lethal activation. We review these data and present new data on two more regions of the X chromosome that define other genes needed for Sxl activation. In addition, we report on the interaction between some of the genes regulating Sxl activation. © 1994 Wiley-Liss, Inc.     

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.     

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

Nanos3 of the frog Rana rugosa: Molecular cloning and characterization

Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3‐deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT‐PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3‐knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild‐type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.     

Sexual back talk with evolutionary implications: stimulation of the Drosophila sex-determination gene sex-lethal by its target transformer

We describe a surprising new regulatory relationship between two key genes of the Drosophila sex-determination gene hierarchy, Sex-lethal (Sxl) and transformer (tra). A positive autoregulatory feedback loop for Sxl was known to maintain somatic cell female identity by producing SXL-F protein to continually instruct the target gene transformer (tra) to make its feminizing product, TRA-F. We discovered the reciprocal regulatory effect by studying genetically sensitized females: TRA-F from either maternal or zygotic tra expression stimulates Sxl-positive autoregulation. We found female-specific tra mRNA in eggs as predicted by this tra maternal effect, but not predicted by the prevailing view that tra has no germline function. TRA-F stimulation of Sxl seems to be direct at some point, since Sxl harbors highly conserved predicted TRA-F binding sites. Nevertheless, TRA-F stimulation of Sxl autoregulation in the gonadal soma also appears to have a cell-nonautonomous aspect, unprecedented for somatic Sxl regulation. This tra-Sxl retrograde regulatory circuit has evolutionary implications. In some Diptera, tra occupies Sxl's position as the gene that epigenetically maintains female identity through direct positive feedback on pre-mRNA splicing. The tra-mediated Sxl feedback in Drosophila may be a vestige of regulatory redundancy that facilitated the evolutionary transition from tra to Sxl as the master sex switch.     

Developmental regulation of locomotive activity in Xenopus primordial germ cells

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species‐specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re‐examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz‐like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re‐aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re‐aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F‐actin, myosin II activity and RhoA/Rho‐associated protein kinase (ROCK) signaling.