Germ Cell-less Research Articles

Identification and functional analysis of foxl2 and nodal in sea cucumber, Apostichopus japonicus

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation.

Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation.

In germ cell transplantation experiments, the use of sterile recipients that do not produce their own gametes is an important prerequisite. Triploidization and dnd gene knockdown (KD) methods have been widely used to produce sterile fish. However, triploidization does not produce complete sterility in some fish species, and gene KD is labor and time intensive since it requires microinjection into individual fertilized eggs. To overcome these problems, in this study, we generated homozygous mutants of the dead end (dnd) gene in rainbow trout (Oncorhynchus mykiss) using the clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, analyzed their reproductive capacity, and evaluated their suitability as recipients for germ cell transplantation. By crossing F1 heterozygous mutants produced from founders subjected to genome editing, an F2 generation consisting of approximately 1/4 homozygous knockout mutants (dnd KO) was obtained. The dnd KO hatchlings retained the same number of primordial germ cells (PGCs) as the wild-type (WT) individuals, after which the number gradually decreased. At 1year of age, germ cells were completely absent in all analyzed individuals. To evaluate the dnd KO individuals as recipients for germ cell transplantation, germ cells prepared from donor individuals were transplanted into the abdominal cavity of dnd KO hatchlings. These cells migrated to the recipient gonads, where they initiated gametogenesis. The mature recipient individuals produced only donor-derived sperm and eggs in equivalent numbers to WT rainbow trout. These results indicate that dnd KO rainbow trout are suitable recipient candidates possessing a high capacity to nurse donor-derived germ cells.

Identification and functional characterization of piwi1 gene in sea cucumber, Apostichopus japonicas.

The sea cucumber (Apostichopus japonicus) is an economically important mariculture species in Asia. However, the genetic breeding of sea cucumbers is difficult because the sexes cannot be identified by appearance. Therefore, studies on sex-related genes are helpful in revealing the mechanisms of sex determination and differentiation in sea cucumbers. P-element induced wimpy testis (piwi) is a germ cell marker involved in gametogenesis in vertebrates; however, the expression pattern and function during gametogenesis remain unclear in sea cucumbers. In this study, we identified a piwi homolog gene in A. japonicus (Ajpiwi1) and investigated its expression pattern, and function. Ajpiwi1 is a maternal factor and is ubiquitously expressed in adult tissues, including the ovary and testis. Ajpiwi1 expression is strong in early oocytes, spermatocytes, and spermatogonia; weak in mature oocytes; and undetected in spermatids and intra-gonadal somatic cells. The knockdown of Ajpiwi1 by RNA interference (RNAi) led to the downregulation of other conserved sex-related genes such as dmrt1, foxl2, and germ cell-less. Therefore, Ajpiwi1 might play a critical role during gametogenesis in A. japonicus. This study creates new possibilities for studying sex-related gene functions in the sea cucumber and builds a gene function research platform based on RNAi for the first time.

Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction.

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. Here we demonstrate that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torsoDeg) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torsoDeg in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction.

Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish†.

In animals, spermatogonial transplantation in sterile adult males is widely developed; however, despite its utility, ovarian germ cell transplantation is not well developed. We previously showed that the interspecific hybrid offspring of sciaenid was a suitable model for germ cell transplantation studies as they have germ cell-less gonads. However, all these gonads have testis-like characteristics. Here, we tested whether triploidization in hybrid embryos could result in germ cell-less ovary development. Gonadal structure dimorphism and sex-specific gene expression patterns were examined in 6-month-old triploid hybrids (3nHybs). Thirty-one percent of 3nHybs had germ cell-less gonads with an ovarian cavity. cyp19a1a and foxl2, ovarian differentiation-related genes, were expressed in these gonads, whereas dmrt1 and vasa were not expressed, suggesting ovary-like germ cell-less gonad development. Some (26%) 3nHybs had testis-like germ cell-less gonads. Ovarian germ cells collected from homozygous green fluorescent protein (GFP) transgenic blue drum (BD) (Nibea mitsukurii) were transplanted into 6-month-old 3nHybs gonads via the urogenital papilla or oviduct. After 9months, the recipients were crossed with wild type BD. Among the six 3nHyb recipients that survived, one female and one male produced fertile eggs and motile sperm carrying gfp-specific DNA sequences. Progeny tests revealed that all F1 offspring possessed gfp-specific DNA sequences, suggesting that these recipients produced only donor-derived eggs or sperm. Histological observation confirmed donor-derived gametogenesis in the 3nHyb recipients' gonads. Overall, triploidization reduces male-biased sex differentiation in germ cell-less gonads. We report, for the first time, donor-derived egg production in an animal via direct ovarian germ cell transplantation into a germ cell-less ovary.

Distinct cis-acting elements mediate targeting and clustering of Drosophila polar granule mRNAs.

Specification and development of Drosophila germ cells depend on molecular determinants within the germ plasm, a specialized cytoplasmic domain at the posterior of the embryo. Localization of numerous mRNAs to the germ plasm occurs by their incorporation, as single-transcript ribonucleoprotein (RNP) particles, into complex RNP granules called polar granules. Incorporation of mRNAs into polar granules is followed by recruitment of additional like transcripts to form discrete homotypic clusters. The cis-acting localization signals that target mRNAs to polar granules and promote homotypic clustering remain largely uncharacterized. Here, we show that the polar granule component (pgc) and germ cell-less (gcl) 3' untranslated regions contain complex localization signals comprising multiple, independently weak and partially functionally redundant localization elements (LEs). We demonstrate that targeting of pgc to polar granules and self-assembly into homotypic clusters are functionally separable processes mediated by distinct classes of LEs. We identify a sequence motif shared by other polar granule mRNAs that contributes to homotypic clustering. Our results suggest that mRNA localization signal complexity may be a feature required by the targeting and self-recruitment mechanism that drives germ plasm mRNA localization.

GCL and CUL3 Control the Switch between Cell Lineages by Mediating Localized Degradation of an RTK

The separation of germline from somatic lineages is fundamental to reproduction and species preservation. Here, we show that Drosophila Germ cell-less (GCL) is a critical component in this process by acting as a switch that turns off a somatic lineage pathway. GCL, a conserved BTB (Broad-complex, Tramtrack, and Bric-a-brac) protein, is a substrate-specific adaptor for Cullin3-RING ubiquitin ligase complex (CRL3GCL). We show that CRL3GCL promotes PGC fate by mediating degradation of Torso, a receptor tyrosine kinase (RTK) and major determinant of somatic cell fate. This mode of RTK degradation does not depend upon receptor activation but isprompted by release of GCL from the nuclear envelope during mitosis. The cell-cycle-dependent change in GCL localization provides spatiotemporal specificity for RTK degradation and sequesters CRL3GCL to prevent it from participating in excessive activities. This precisely orchestrated mechanism of CRL3GCL function and regulation defines cell fate at the single-cell level.

How Primordial Germ Cells Destroy Somatic Signals

The Germ Cell-Less (GCL) protein is a key regulator of primordial germ cell (PGC) formation in Drosophila embryos. Reporting in this issue of Developmental Cell, Pae etal. (2017) show that GCL blocks somatic cell fate by specifically destroying the Torso Receptor Tyrosine Kinase.

Germ Cell-less Promotes Centrosome Segregation to Induce Germ Cell Formation

The primordial germ cells (PGCs) specified during embryogenesis serve as progenitors to the adult germline stem cells. In Drosophila, the proper specification and formation of PGCs require both centrosomes and germ plasm, which contains the germline determinants. Centrosomes are microtubule (MT)-organizing centers that ensure the faithful segregation of germ plasm into PGCs. To date, mechanisms that modulate centrosome behavior to engineer PGC development have remained elusive. Only one germ plasm component, Germ cell-less (Gcl), is known to play a role in PGC formation. Here, we show that Gcl engineers PGC formation by regulating centrosome dynamics. Loss of gcl leads to aberrant centrosome separation and elaboration of the astral MT network, resulting in inefficient germ plasm segregation and aborted PGC cellularization. Importantly, compromising centrosome separation alone is sufficient to mimic the gcl loss-of-function phenotypes. We conclude Gcl functions as a key regulator of centrosome separation required for proper PGC development.

Germ Cell Segregation from the Drosophila Soma Is Controlled by an Inhibitory Threshold Set by the Arf-GEF Steppke.

Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1-actomyosin activity. Recently, we found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. Here, we report that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo . These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses we found that Step normally activates Arf small G proteins and antagonizes Rho1-actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, we examined the effect of Step on PGC formation at the posterior pole. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP-Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. We propose that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.

Transcriptome profiles of Penaeus (Marsupenaeus) japonicus animal and vegetal half-embryos: identification of sex determination, germ line, mesoderm, and other developmental genes.

There is virtually no knowledge of the molecular events controlling early embryogenesis in Penaeid shrimp. A combination of controlled spawning environment, shrimp embryo micro-dissection techniques, and next-generation sequencing was used to produce transcriptome EST datasets of Penaeus japonicus animal and vegetal half-embryos. Embryos were collected immediately after spawning, and then blastomeres were separated at the two-cell stage and allowed to develop to late gastrulation, then pooled for RNA isolation and cDNA synthesis. Ion Torrent sequencing of cDNA from approximately 500 pooled animal and vegetal half-embryos from multiple spawnings resulted in 560,516 and 493,703 reads, respectively. Reads from each library were assembled and Gene Ontogeny analysis produced 3479 annotated animal contigs and 4173 annotated vegetal contigs, with 159/139 hits for developmental processes in the animal/vegetal contigs, respectively. Contigs were subject to BLAST for selected developmental toolbox genes. Some of the genes found included the sex determination genes sex-lethal and transformer; the germ line genes argonaute 1, boule, germ cell-less, gustavus, maelstrom, mex-3, par-1, pumilio, SmB, staufen, and tudor; the mesoderm genes brachyury, mef2, snail, and twist; the axis determination/segmentation genes β-catenin, deformed, distal-less, engrailed, giant, hairy, hunchback, kruppel, orthodenticle, patched, tailless, and wingless/wnt-8c; and a number of cell-cycle regulators. Animal and vegetal contigs were computationally subtracted from each other to produce sets unique to either half-embryo library. Genes expressed only in the animal half included bmp1, kruppel, maelstrom, and orthodenticle. Genes expressed only in the vegetal half included boule, brachyury, deformed, dorsal, engrailed, hunchback, spalt, twist, and wingless/wnt-8c.

Gene expression during ovarian differentiation in parasitic and non-parasitic lampreys: Implications for fecundity and life history types

Lampreys diverged from the jawed vertebrate lineage approximately 500million years ago. Lampreys undergo sex differentiation much later than most other vertebrates, and ovarian differentiation occurs several years before testicular differentiation. The genetic basis of lamprey sex differentiation is of particular interest both because of the phylogenetic importance of lampreys and because of their unusual pattern of sex differentiation. As well, differences between parasitic and non-parasitic lampreys may first become evident at ovarian differentiation. However, nothing is known about the genetic basis of ovarian differentiation in lampreys. This study examined potential differences in gene expression before, during, and after ovarian differentiation in parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzonfossor. Eight target genes (17β-hydroxysteroid dehydrogenase, germ cell-less, estrogen receptor β, insulin-like growth factor 1 receptor, daz-associated protein 1, cytochrome c oxidase subunit III, Wilms’ tumour suppressor protein 1, and dehydrocholesterol reductase 7) were examined. Northern brook lamprey displayed higher expression of cytochrome c oxidase subunit III, whereas chestnut lamprey displayed higher expression of insulin-like growth factor 1 receptor; these genes may be involved in apoptosis and oocyte growth, respectively. Presumptive male larvae had higher expression of Wilms’ tumour suppressor protein 1, which may be involved in the undifferentiated gonad and/or later testicular development. Differentiated females had higher expression of 17β hydroxysteroid dehydrogenase and daz-associated protein 1, which may be involved in female development. This study is the first to identify genes that may be involved in ovarian differentiation and fecundity in lampreys.

A spindle-independent cleavage pathway controls germ cell formation in Drosophila.

The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. While the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using 4D multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to utilizing core regulators of cleavage, including the small GTPase RhoA (Drosophila Rho) and the Rho associated kinase, ROCK (Drosophila Rok), we show that this pathway requires Germ cell-less (Gcl), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternate form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage.

GAGE Cancer-Germline Antigens Are Recruited to the Nuclear Envelope by Germ Cell-Less (GCL)

GAGE proteins are highly similar, primate-specific molecules with unique primary structure and undefined cellular roles. They are restricted to cells of the germ line in adult healthy individuals, but are broadly expressed in a wide range of cancers. In a yeast two-hybrid screen we identified the metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2β, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two-hybrid analysis and pull-down experiments of GCL polypeptides, GCL residues 209–320 (which includes the BACK domain) were deduced sufficient for association with GAGE proteins. GAGE mRNAs and GCL mRNA were demonstrated in human testis and most types of cancers, and at the protein level GAGE members and GCL were co-expressed in cancer cell lines. Structural studies of GAGE proteins revealed no distinct secondary or tertiary structure, suggesting they are intrinsically disordered. Interestingly GAGE proteins formed stable complexes with dsDNA in vitro at physiological concentrations, and GAGE12I bound several different dsDNA fragments, suggesting sequence-nonspecific binding. Dual association of GAGE family members with GCL at the nuclear envelope inner membrane in cells, and with dsDNA in vitro, implicate GAGE proteins in chromatin regulation in germ cells and cancer cells.

The LINC complex and human disease

The LINC (linker of nucleoskeleton and cytoskeleton) complex is a proposed mechanical link tethering the nucleo- and cyto-skeleton via the NE (nuclear envelope). The LINC components emerin, lamin A/C, SUN1, SUN2, nesprin-1 and nesprin-2 interact with each other at the NE and also with other binding partners including actin filaments and B-type lamins. Besides the mechanostructural functions, the LINC complex is also involved in signalling pathways and gene regulation. Emerin was the first LINC component associated with a human disease, namely EDMD (Emery-Dreifuss muscular dystrophy). Later on, other components of the LINC complex, such as lamins A/C and small isoforms of nesprin-1 and nesprin-2, were found to be associated with EDMD, reflecting a genetic heterogeneity that has not been resolved so far. Only approximately 46% of the EDMD patients can be linked to genes of LINC and non-LINC components, pointing to further genes involved in the pathology of EDMD. Obvious candidates are the LINC proteins SUN1 and SUN2. Recently, screening of binding partners of LINC components as candidates identified LUMA (TMEM43), encoding a binding partner of emerin and lamins, as a gene involved in atypical EDMD. Nevertheless, such mutations contribute only to a very small fraction of EDMD patients. EDMD-causing mutations in STA/EMD (encoding emerin) that disrupt emerin binding to Btf (Bcl-2-associated transcription factor), GCL (germ cell-less) and BAF (barrier to autointegration factor) provide the first glimpses into LINC being involved in gene regulation and thus opening new avenues for functional studies. Thus the association of LINC with human disease provides tools for understanding its functions within the cell.

Bruno negatively regulates germ cell-less expression in a BRE-independent manner

Mechanisms of post-transcriptional control are essential during Drosophila oogenesis and embryogenesis to sequester gene products in discrete regions and ultimately achieve embryonic asymmetry. Maternal germ cell-less ( gcl) mRNA accumulates in the pole plasm of the embryo before Gcl protein is detectable. gcl mRNA, but not Gcl protein, can also be detected in somatic regions of the embryo, suggesting that gcl RNA is subject to translational control. We find that Gcl is expressed during oogenesis, and that it is regulated by the translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru level is reduced, and overexpression of Bru reduces Gcl expression. Consistently, reduction of the maternal dosage of Bruno leads to ectopic Gcl expression in the embryo, which, in turn, represses anterior hückebein ( hkb) expression. Bru binds directly to the gcl 3’UTR in vitro, but, surprisingly, this binding is independent of a BRE (Bruno response element)-like motif. This motif is also not required for in vivo repression of Gcl expression during oogenesis or early embryogenesis. Bru binds the gcl 3’UTR via its C-terminal domain, which includes RNA recognition motif 3 (RRM3), with little or no contribution from the remainder of the protein. We conclude that repression by Bruno during oogenesis is required to restrict Gcl expression in the early embryo and that Bru represses gcl expression in a manner that involves RRM3 and a sequence unrelated to the BRE.

Cloning and expression of BTB/POZ-domain from zebrafish gcl (germ cell-less), and appraisement of its polyclonal-antibody

In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl (germ cell-less) into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21(DE3) pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.

Germ cell‐less expression in zebrafish embryos

The gene of germ cell-less (gcl) has been shown to be important in early differentiation of germ cells in Drosophila. Although the gcl homologue genes have been identified in some organisms, there is little data on the expression pattern and functional analysis of the gcl gene in zebrafish. In this research, real-time quantitative reverse transcription-polymerase chain reaction showed that the level of gcl mRNA expression rapidly decreases from the 4-cell stage to the sphere stage at which it reaches a minimum, gradually increases from the 50%-epiboly stage, and then remains stable during the posterior stages. Results of in situ hybridization indicated that the transcripts of zebrafish gcl are evenly distributed in all blastomeres from the 2-cell stage to the blastula period, different from that of vasa, nonas1 and dead end mRNA, and condense into some clusters of cells located along the blastoderm margin from the gastrulation period. During subsequent development, the transcripts are segregated as subcellular clumps to a small number of cells that would migrate to the position of the gonad in the dorsal side. In the adult, gcl mRNA was widely expressed in developing germ cells of both ovary and testis. These data suggest that zebrafish gcl have potentially important roles in the formation of primordial germ cells.

Role of mitochondrial ribosome-dependent translation in germline formation in Drosophila embryos

In Drosophila, mitochondrially encoded ribosomal RNAs (mtrRNAs) form mitochondrial-type ribosomes on the polar granules, distinctive organelles of the germ plasm. Since a reduction in the amount of mtrRNA results in the failure of embryos to produce germline progenitors, or pole cells, it has been proposed that translation by mitochondrial-type ribosomes is required for germline formation. Here, we report that injection of kasugamycin (KA) and chloramphenicol (CH), inhibitors for prokaryotic-type translation, disrupted pole cell formation in early embryos. The number of mitochondrial-type ribosomes on polar granules was significantly decreased by KA treatment, as shown by electron microscopy. In contrast, ribosomes in the mitochondria and mitochondrial activity were unaffected by KA and CH. We further found that injection of KA and CH impairs production of Germ cell-less (Gcl) protein, which is required for pole cell formation. The above observations suggest that mitochondrial-type translation is required for pole cell formation, and Gcl is a probable candidate for the protein produced by this translation system.

Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.