scholarly journals Efficient expression of genes in the Drosophila germline using a UAS-promoter free of interference by Hsp70 piRNAs

2018 ◽  
Author(s):  
Steven Z. DeLuca ◽  
Allan C. Spradling
Keyword(s):  
Germ Cells ◽  
Somatic Cells ◽  
Developmental Genetics ◽  
Technical Limitation ◽  
Promoter Sequences ◽  
Expression Of Genes ◽  
Gal4 Expression ◽  
Efficient Expression ◽  
Germline Cells ◽  
Female Germline

ABSTRACTControlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of Drosophila melanogaster developmental genetics for twenty-five years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special UAS promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a significant technical limitation. Here we show that the expression problem of UASt and many other Drosophila molecular tools in germline cells is caused by their core Hsp70 promoter sequences, which are targeted in female germ cells by Hsp70-directed piRNAs generated from endogenous Hsp70 gene sequences. In a genetic background lacking genomic Hsp70 genes and associated piRNAs, UASt-based constructs function effectively during oogenesis. By reducing Hsp70 sequences targeted by piRNAs, we created UASz, which functions better than UASp in the germline and like UASt in somatic cells.

scholarly journals Collective cell sorting requires contractile cortical waves in germline cells

2020 ◽  
Author(s):  
Soline Chanet ◽  
Jean-René Huynh
Keyword(s):  
Germ Cells ◽  
Somatic Cells ◽  
Active Role ◽  
Basic Unit ◽  
Female Reproduction ◽  
Primordial Follicles ◽  
Genetic Perturbations ◽  
Egg Chambers ◽  
Germline Cells ◽  
Cortical Contractility

ABSTRACTEncapsulation of germline cells by layers of somatic cells forms the basic unit of female reproduction called primordial follicles in mammals and egg chambers in Drosophila. How germline and somatic tissues are coordinated for the morphogenesis of each separated unit remains poorly understood. Here, using improved live-imaging of Drosophila ovaries, we uncovered periodic actomyosin waves at the cortex of germ cells. These contractile waves are associated with pressure release blebs, which project from germ cells into somatic cells. We demonstrate that these cortical activities, together with cadherin-based adhesion, are required to sort each germline cyst as one collective unit. Genetic perturbations of cortical contractility, blebs protrusion or adhesion between germline and somatic cells induced failures to encapsulate any germ cells or the inclusion of too many germ cells or even the mechanical split of germline cysts. Our results reveal that germ cells play an active role in the physical coupling with somatic cells to produce the female gamete.

scholarly journals Male-biased protein expression in primordial germ cells, identified through a comparative study of UAS vectors in Drosophila

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masaki Masukawa ◽  
Yuki Ishizaki ◽  
Hiroki Miura ◽  
Makoto Hayashi ◽  
Ryoma Ota ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Translational Activity ◽  
Male Germline ◽  
Germline Cells ◽  
Female Germline ◽  
Go Terms ◽  
Mrna Expression Levels

AbstractIn Drosophila, three types of UAS vectors (UASt, UASp, and UASz) are currently available for use with the Gal4-UAS system. They have been used successfully in somatic cells and germline cells from ovaries. However, it remains unclear whether they are functional in the germline cells of embryos, larvae, and adult testes. In this study, we found that all three types of UAS vectors were functional in the germline cells of embryos and larvae and that the UASt and UASz vectors were active in the germline of the distal tip region in adult testes. Moreover, we observed that protein expression from the UAS vectors was male-biased in germline cells of late embryos, whereas their respective mRNA expression levels were not. Furthermore, O-propargyl-puromycin (OPP) staining revealed that protein synthesis was male-biased in these germline cells. In addition, GO terms related to translation and ribosomal maturation were significantly enriched in the male germline. These observations show that translational activity is higher in male than in female germline cells. Therefore, we propose that male-biased protein synthesis may be responsible for the sex differences observed in the early germline.

scholarly journals Frequent gain- and loss-of-function mutations of the BjMYB113 gene accounted for leaf color variation in Brassica juncea

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Guanghui An ◽  
Jiongjiong Chen
Keyword(s):  
Brassica Juncea ◽  
Anthocyanin Biosynthesis ◽  
Color Variation ◽  
Nucleotide Polymorphisms ◽  
Loss Of Function ◽  
High Accumulation ◽  
Single Nucleotide ◽  
Breeding Programs ◽  
Promoter Sequences ◽  
Expression Of Genes

Abstract Background Mustard (Brassica juncea) is an important economic vegetable, and some cultivars have purple leaves and accumulate more anthocyanins than the green. The genetic and evolution of purple trait in mustard has not been well studied. Result In this study, free-hand sections and metabolomics showed that the purple leaves of mustard accumulated more anthocyanins than green ones. The gene controlling purple leaves in mustard, Mustard Purple Leaves (MPL), was genetically mapped and a MYB113-like homolog was identified as the candidate gene. We identified three alleles of the MYB113-like gene, BjMYB113a from a purple cultivar, BjMYB113b and BjMYB113c from green cultivars. A total of 45 single nucleotide polymorphisms (SNPs) and 8 InDels were found between the promoter sequences of the purple allele BjMYB113a and the green allele BjMYB113b. On the other hand, the only sequence variation between the purple allele BjMYB113a and the green allele BjMYB113c is an insertion of 1,033-bp fragment in the 3’region of BjMYB113c. Transgenic assay and promoter activity studies showed that the polymorphism in the promoter region was responsible for the up-regulation of the purple allele BjMYB113a and high accumulation of anthocyanin in the purple cultivar. The up-regulation of BjMYB113a increased the expression of genes in the anthocyanin biosynthesis pathway including BjCHS, BjF3H, BjF3’H, BjDFR, BjANS and BjUGFT, and consequently led to high accumulation of anthocyanin. However, the up-regulation of BjMYB113 was compromised by the insertion of 1,033-bp in 3’region of the allele BjMYB113c. Conclusions Our results contribute to a better understanding of the genetics and evolution of the BjMYB113 gene controlling purple leaves and provide useful information for further breeding programs of mustard.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

Neonatal administration of diethylstilbestrol has adverse effects on somatic cells rather than germ cells

2006 ◽  
Vol 22 (4) ◽  
pp. 746-753 ◽  
Author(s):  
Kyu-Bom Koh ◽  
Yoshiro Toyama ◽  
Masatoshi Komiyama ◽  
Tetsuya Adachi ◽  
Hideki Fukata ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Germ Cells ◽  
Somatic Cells ◽  
Neonatal Administration

scholarly journals The Mammalian Ovary from Genesis to Revelation

2009 ◽  
Vol 30 (6) ◽  
pp. 624-712 ◽  
Author(s):  
Mark A. Edson ◽  
Ankur K. Nagaraja ◽  
Martin M. Matzuk
Keyword(s):  
Germ Cells ◽  
Ovarian Function ◽  
Primordial Germ Cells ◽  
Bioactive Molecules ◽  
Sex Characteristics ◽  
Germline Development ◽  
Peptide Growth Factors ◽  
Development And Differentiation ◽  
Common Destiny ◽  
Female Germline

Abstract Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago.

scholarly journals Two Alternative Mechanisms That Regulate the Presentation of Apoptotic Cell Engulfment Signal in Caenorhabditis elegans

2007 ◽  
Vol 18 (8) ◽  
pp. 3180-3192 ◽  
Author(s):  
Victor Venegas ◽  
Zheng Zhou
Keyword(s):  
Caenorhabditis Elegans ◽  
Abc Transporter ◽  
Germ Cells ◽  
Mammalian Cells ◽  
Developmental Stages ◽  
Apoptotic Cell ◽  
Somatic Cells ◽  
Apoptotic Cells ◽  
Phospholipid Scramblase ◽  
Cell Engulfment

Phosphatidylserine exposed on the surface of apoptotic mammalian cells is considered an “eat-me” signal that attracts phagocytes. The generality of using phosphatidylserine as a clearance signal for apoptotic cells in animals and the regulation of this event remain uncertain. Using ectopically expressed mouse MFG-E8, a secreted phosphatidylserine-binding protein, we detected specific exposure of phosphatidylserine on the surface of apoptotic cells in Caenorhabditis elegans. Masking the surface phosphatidylserine inhibits apoptotic cell engulfment. CED-7, an ATP-binding cassette (ABC) transporter, is necessary for the efficient exposure of phosphatidylserine on apoptotic somatic cells, and for the recognition of these cells by phagocytic receptor CED-1. Alternatively, phosphatidylserine exposure on apoptotic germ cells is not CED-7 dependent, but instead requires phospholipid scramblase PLSC-1, a homologue of mammalian phospholipid scramblases. Moreover, deleting plsc-1 results in the accumulation of apoptotic germ cells but not apoptotic somatic cells. These observations suggest that phosphatidylserine might be recognized by CED-1 and act as a conserved eat-me signal from nematodes to mammals. Furthermore, the two different biochemical activities used in somatic cells (ABC transporter) and germ cells (phospholipid scramblase) suggest an increased complexity in the regulation of phosphatidylserine presentation in response to apoptotic signals in different tissues and during different developmental stages.

Evidence of male germ cell redifferentiation into female germ cells in planarian regeneration

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 29-36
Author(s):  
V. Gremigni ◽  
M. Nigro ◽  
I. Puccinelli
Keyword(s):  
Germ Cells ◽  
Somatic Cells ◽  
The Body ◽  
Diploid Male ◽  
Female Gonad ◽  
Anterior Portion ◽  
Male Germ Cells ◽  
Blastema Formation ◽  
Planarian Regeneration ◽  
Undifferentiated Cells

The source and fate of blastema cells are important and still unresolved problems in planarian regeneration. In the present investigation we have attempted to obtain new evidence of cell dedifferentiation-redifferentiation by using a polyploid biotype of Dugesia lugubris s.1. This biotype is provided with a natural karyological marker which allows the discrimination of triploid embryonic and somatic cells from diploid male germ cells and from hexaploid female germ cells. Thanks to this cell mosaic we previously demonstrated that male germ cells take part in blastema formation and are then capable of redifferentiating into somatic cells. In the present investigation sexually mature specimens were transected behind the ovaries and the posterior stumps containing testes were allowed to regenerate the anterior portion of the body. Along with the usual hexaploid oocytes, a small percentage (3.2%) of tetraploid oocytes were produced from regenerated specimens provided with new ovaries. By contrast only hexaploid oocytes were produced from control untransected specimens. The tetraploid oocytes are interpreted as original diploid male germ cells which following the transection take part in blastema formation and then during regeneration redifferentiate into female germ cells thus doubling their chromosome number as usual for undifferentiated cells entering the female gonad in this biotype.

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