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functional antagonism between the pgc germline repressor and torso in the development of somatic cells.
The main questions of research in this paper that the authors wish to address are: what is the status of pgc and torso in Drosophila development? What are the mechanisms involved in transcriptional quiescence of somatic cells and germ cells located at the posterior pole of embryos? Does pgc of germ cells have an independent and direct role of inhibiting tor transcription in somatic cells to prevent differentiation into a somatic cell fate instead of a germline fate? The aim of the experiment was to record the effects of transcriptional inhibition by pgc interactions in embryonic development by raising the expression levels of pgc in six different genes of Drosophila wild-type using a probe of pgc RNA in situ hybridization. Progeny of these flies; 6x[pgc], resulted in a greater amount of pgc expression and a pole-hole phenotype. This is analogous to the cellularization phenotype of tor mutants indicating that both excessive amounts of pgc and limited tor is likely to cause transcription of genes within somatic cells to be less efficient than normal. In order for the germline to be precisely developed, tor signalling repression is required which directs faults in morphological cellularization producing a pole-hole phenotype. This phenotype depends on pgc and other components of the germ-plasm which may or may not be transcriptionally-dependent. The results agree with the hypothesis that the tor gene is specific to somatic cells whilst pgc carries out a similar function in germ cells of Drosophila. There are many key conclusions which include tor signalling antagonising pgc activity in germ cells meaning there is a similar function in development of tor and the pgc repressor in Drosophila. Germ cells have a transcriptive silencing mechanism to prevent them from differentiating like their surrounding somatic cells. This allows the functioning of germline and somatic cells to be located at the posterior pole of the embryo. The maternal cytoplasm of the egg containing transcription factors determines the gradients of gene expression after the mitotic cleavage stages of development and tor is located in somatic cells abundant in the terminal parts of the embryo whilst pgc is present in germ cells at the posterior. On the whole, this paper focuses on the germline and somatic identities whilst demonstrating an illustrative model for the presence of pgc and other factors on cell fate, along with the development in Drosophila melanogaster. The specificity of germ cells is highlighted in the paper as fundamental for germline fate and the meiotic reproductive potential that promotes genetic diversity between species. Tor and pgc of Drosophila are recognised as vital for the protective mechanisms of both somatic and germ cell fate positioned posteriorly in the embryo. This allows further research to be undertaken on the functional antagonism of tor signalling in germline cells and pgc activity in somatic cells as well as germline biology in general. A potential comparison with other invertebrates such as C.elegans as well as other eukaryotic organisms like mice and frogs could be preformed. The developmental patterns and mechanisms of both invertebrates and vertebrates can then be evaluated as a whole. The evolution of germ biology is also important in understanding adaptation and phylogeny between organisms.
The development of Drosophila melanogaster begins with formation of a zygote via the fusion of eggs and sperm. Embryonic development begins with multiple syncytial nuclear divisions exclusive of cytokinesis. This process is followed by blastoderm cellularization which involves cleavage of the fertilized egg to form a morula stage. Pole-hole phenotype is characteristic of flaws in the cellularization process. There are three separate germ layers: endoderm, mesoderm and ectoderm formed at gastrulation before the embryo finally progresses into an adult fruit fly.
The configuration of germ cells in Drosophila depends on the production of pgc expression at the posterior pole during embryogenesis which initiates the congregation of germplasm (a specialised form of cytoplasm) via the expression of specific genes. Expression of genes typically involves the transcription of DNA to produce mRNA followed by translation to form a protein. Germ cells are fundamental in all organisms in the respect of totipotency as they have reproductive potential, allowing germlines to be passed from one generation to another. They are different from somatic cells in the respect of having the potential to fuse and reproduce to produce either reproductive cells that can undergo sexual meiosis (gametes) or body cells which are diploid (somatic).
Torso (tor) is a transmembrane protein with the receptor tyrosine kinase (RTK) and is vital for the anterior and posterior identity of the embryo. Expression of tor is at the somatic cells situated at the terminal poles of the embryo and depends on the activity of several genes including torso-like (tsl) and trunk; a ligand of tor. The tor RTK pathway initiates two other somatic genes indirectly; the expression of zygotic terminal gap genes tailless (tll) and huckebein (hkb) at the embryonic poles by inhibiting capicua. The expressional effects of tor activity determine the specificity of cell fate; somatic differentiation occurs when tor is expressed and germline differentiation in low activity of tor.
Germ cell fate can be determined by germ granules. These polar granule components (pgcâ€™s) otherwise known as primordial germ cells are present in the germplasm of germ cells to delay zygotic gene expression until this stage of development. The polar granules are located posteriorly in Drosophila embryos and participate in transcriptional silencing of somatic differentiation; vital for the specification of germ cells. The maternal germplasm components are passed on to the germ cells of their progeny and transcription factors provide a variety of concentration gradients of genes in all post-cleavage cells.
Transcriptional silencing occurs in cells such as germ cell-less, nanos and pumilo. Nanos is expressed in the posterior part of a Drosophila embryo and inhibits the expression of the anterior regulated gene hunchback via the expression of its binding partner pumilo. Germ cell-less is a gene necessary for the formation of polar granules in germ cells whilst nanos and pumilo vital for pole cell migration. Germ cell specification occurs during early embryogenesis where pgc inhibits somatic synthesis via deactivation of the RNA Polymerase II (Pol II) which is phosphorylated as mRNA levels increase with its interacting kinase complex P-TEFb. The pgc regulator of germline varies between species; pgc in Drosophila; PIE1 in C.elegans; and Blimp1 in mammals. The effects of pgc on embryonic development of Drosophila were experimented.
The methods included the use of six Drosophila transgenic embryos (6x[pgc]); progeny of wild-type flies; having a pole-hole phenotype similar to the results of embryos from tor-mutant mothers. In germline cells there is the potential to reproduce which involves meiotic divisions from haploid cells to a diploid zygote. Somatic cells are diploid cells which are only able to mitotically divide to renew cells during an adultâ€™s lifetime. Germ cell formation depends on the assembly of the constituents of the germplasm as a result of the maternally mRNA which was deposited in its oocyte. These can then be activated when they have reached the posterior pole of the embryo. Since germ cells and somatic cells are both located towards the posterior poles of a Drosophila embryo their usual expression of specific genes at specific times may affect each other and obstruct proper functioning of these cells. As a result it is found that an alternative mechanism of particularly pgc and tor genes coincided; they are able to function alongside each other positional within the embryo by transcriptional silencing. Tor signalling is thought by the authors to shield somatic cells from deletion during germ cell expression of the gene interaction mechanisms of pgc. The specificity of cells: somatic and germline allows cell fate to be determined at early stages of development. Germ cell fates are not only specified by transcriptional silencing but also by the control of translation, unique to Drosophila. This indicates that there may be other proteins of similar role in other species differing in the mechanisms used to determine cell fate.
Germline cells differ from somatic cells by the presence of alternative transcription factors inherited maternally in the egg, before the egg and sperm even fuse. These transcription factors are gene specific and either directly activate or ultimately switch off genes through down-regulation. Another factor which distinguishes germline cells in Drosophila is the presence of polar granules in their cytoplasm as when cells divide mitotically a differential gradient of constituents occurs in the post-cleavage cells which are formed in the process of cellularization. The authors suggest that transcriptional silencing is fundamental for the segregation of germline and somatic cells. The process of transcriptional silencing must have evolved as it efficiently avoids the production of transcription factors which determine a somatic fate. There is a disadvantage in the formation of germline cells of Drosophila which is their preventive effect on somatic cell development by inhibiting some of their genes. However, this is the cost of initiating the very specific cell fate of germ cells.
Posterior nuclei are found to fall into the yolk during cellularization in both of these types of embryo. In situ hybridization of the entire Drosophila embryo tissue requires the use of a probe to localise the pgc RNA sequence. The outcomes of increased pgc expression were recorded to allow the monitoring of pgc behaviour in somatic cells and a comparison to the germline whilst a relative understanding of the interactions of this gene with other genes to be produced. The hypothesis suggested was that tor is specific to somatic cells and therefore will not be expressed in germ cells of Drosophila. The hypothesis was proven as the results showed that tor was down-regulated in germ cells to permit development. A major finding that backs up this suggestion is that the results implemented the fact that tor in somatic cells and pgc in germ cells are similar in function and phenotypically. Pgc is present in germ cells whereas tor is located in somatic; both thought to have an antagonistic behaviour as they are involved in similar developmental mechanisms.
The expression of tll in fliesâ€™ mutant for pgc (pgc mutant) was compared to flies mutant to tor and pgc (tor, pgc mutants). In embryos of pgc mutant, expression of tll was found to be in germ cells in both pgc and tor, pgc mutants; however tll was not expressed in posterior somatic cells of tor, pgc mutants and is usually expressed in posterior blastoderm cells of pgc mutants. This indicates that tll is independent of tor signalling. Posterior somatic cells regulate the expression of genes such as tll, zen and slam which are found to also be expressed via germ cells lacking pgc and Ser2. This indicates that transcription is inhibited by pgc. A decrease in transcription was recorded for the gene needed for the formation of somatic cells: slam. There was a much higher expression rate of slam in the posterior soma of six wild-type copies of pgc compared to the decrease of tor activity. Wild-type posterior somatic cells contained less RNA Pol II than 6x[pgc] embryos. The pole-hole phenotype was found to be promoted by pgc in tor mutant progeny indicating the likelihood that pgc activity unsuitably affects this phenotype. Inhibition of pole-hole phenotype was evidently stronger in vas, tor mutants compared to tor, pgc mutants. Defects in cellularization of tor mutant progeny only occurs at the posterior pole of the embryo indicating that tor may inhibit the transcription of pgc contained within germ cells. Pole-hole phenotype was found to be dependent on the presence of the germline cells and its components, mainly pgc in tor mutant progeny. Overall, these results suggest a role for pgc in transcriptional repression and in 6x[pgc] embryos the pole-hole phenotype is due to the inhibition of gene expression in blastoderm nuclei of posterior origin. Also, there may be other germ-plasm components that are not necessarily transcriptionally-dependent acting alongside pgc to cause the pole-hole phenotype in tor mutant progeny and tor signalling enhances the deleterious defects of somatic development. As a result of these conclusions the author proposes the idea that this cellularization defect must occur from specific irregularities such as impairment of slam expression up-regulated by pgc.
Interestingly, low levels of pgc and germline components were observed in posterior somatic cells of wild type embryos. Also, the greatest amount of pgc RNA expression was found in the posterior somatic cells of 6x[pgc] embryos as oppose to tor-mutant progeny and wild-type embryos. Over-expression of pgc causes a decrease in RNA Pol II activity and slam expression in wild-type embryos affecting the activity of tor. Since tor signalling inactivates transcriptional repressor activity in the mechanism that mediates the phosphorylation of the Cic transcription factor by MAPK it is likely to have more than one transcriptional repressor inactivations. This evidence was used to conjure information that proved tor inactivates pgc-dependant activity in somatic cells indirectly.
Developmental genes are controlled by transcription factors which allow the stages of development such as patterned migrations, the formation of germ layers in gastrulation and gamete formation to be regulated at specific time periods. The efficient and complex developmental system enables genes to be switched on at specific times in development which increases in perfection via adaptation and natural selection. The survival of a species in terms of evolution by natural selection is largely dependent on the differences between germ cell development lineages. This is due to germ cells playing a major role in the propagation of genes from generation to generation, particularly in higher organisms.
The major findings in this paper include; similar functioning of tor and pgc in development of Drosophila; transcriptional silencing occurs at the posterior pole of the embryos as the result of the location of both germ and somatic cells in this region and prevents them from hindering each otherâ€™s functioning; the pole-hole phenotype is expressed in both 6x[pgc] embryos and those from tor-mutant mothers; increased pgc activity or tor mutants causes a pole-hole phenotype; transcription factors are maternally inherited in the cytoplasm, having a positive or negative effect on transcription of RNA from the DNA of target genes; mitotic division during cleavage produces gradients in the cytoplasm determining the fate of the cell as either soma or germline; and germ cells require high levels of pgc and lack of tor to function efficiently.
A comparison between vertebrates and invertebrates enables their phylogenetic lineage to be calculated whilst highlighting their developmental differences. The recognition of homologous genes among species enables the functional differences of development and their origins by considering the degree of conservation between amino acid sequences. The understanding of germ biology on both a cellular and evolutionary level is important for; finding potential cancer remedies by recognising the immune response mechanisms; propagation; and the survival of organisms. More research on germline biology is required to have a deeper understanding on many topics of development including; the mechanisms involved in transcriptional silencing of somatic and germline cells; the functional antagonism of tor signalling and pgc activity; as well as the identification of RNA Pol II zygotic genes in fruit flies, nematodes, mice and a variety of other organisms.