nanos
Early in oogenesis (stages 4-5), NOS RNA synthesized in nurse cells accumulates in the oocyte, uniformly distributed until stage 9 when it accumulates in the oocyte's anterior margin. By stage 10, marking mid-oogenesis, the RNA becomes uniformly distributed again, and there is a high level of expression in nurse cells. This newly transcribed RNA is deposited in the oocyte following stage 10, and becomes posteriorly localized (Wang, 1994).
nos transcript is concentrated at the posterior pole in cleavage embryos and freshly laid eggs. At the pole bud stage (stage 3 early in zygotic development), the transcript segregates into the nascent pole cells. The transcript remains concentrated in the pole cells during blastoderm stages. During gastrulation and germ band extension, when the pole cells are carried dorsally and internalized into the embryo, staining for NOS mRNA is visible in the pole cells, but staining is not detected once pole cells have left the pocket formed by the posterior midgut invagination. After fertilization NOS protein is transcribed during the syncytial stage, emanating from the posterior pole and extending a short distance anteriorly (Lehmann, 1991 and Smith, 1992).
Translational repression of maternal Nanos (NOS) mRNA by a cis-acting Translational Control Element (TCE) in the NOS 3'UTR is critical for anterior-posterior patterning of the Drosophila embryo. It has been shown,
through ectopic expression experiments, that the NOS TCE is capable of repressing gene expression at
later stages of development in neuronal cells that regulate the molting cycle. These results predict additional
targets of TCE-mediated repression within the nervous system. They also suggest that mechanisms that
regulate maternal mRNAs, like TCE-mediated repression, may function more widely during development to spatially or temporally control gene expression (Clark, 2002).
To explore the possibility that other mRNAs may be regulated by the TCE or a TCE-like motif at different developmental stages,
whether the TCE could repress translation in sites other than the Drosophila oocyte and early embryo was investigated. Evidence is provided that TCE-mediated repression can occur in a subset of cells in the
central nervous system, by using the GAL4/UAS system to ectopically express either a regulatable NOS mRNA, which bears the TCE, or an unregulatable NOS mRNA, which lacks the TCE,
in a variety of temporal and spatial patterns during development. Production of Nos from the unregulatable NOS mRNA in these cells produces a characteristic adult phenotype. Strikingly, this
phenotype is suppressed when NOS RNA bearing the TCE is expressed in the identical cells. The phenotype caused by ectopic Nos resembles one
caused by ablation of neurosecretory cells that produce eclosion hormone (EH). However, TCE-mediated regulation does not occur in EH
cells themselves, but in other cells that act within this complex neuroendocrine signaling pathway. The ability of the TCE to repress Nos activity in the
nervous system implies that TCE-mediated repression may be a more general mechanism for both spatial and temporal control during development (Clark, 2002).
Embryos derived from nanos mutant females lack all abdominal segments while the regions anterior (the head and thorax) and posterior (the telson) to the abdomen appear normal (Lehmann, 1988).
Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the Hunchback (HB) morphogen. Nanos determines the distribution of the maternal HB protein by regulating its translation. To identify further components of this pathway, suppressors of nanos have been isolated. In the absence of nanos, high levels of HB protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high HB levels. The suppressors are alleles of Enhancer of zeste (E[z]) a member of the Polycomb group (Pc-G) of genes. E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal HB protein gradient. (Pelegri, 1994).
In a variety of tissues in eukaryotes, multipotential stem cells are responsible for maintaining a germinal population and generating a
differentiated progeny. The Drosophila germline is one such tissue where a continuous supply of both eggs and sperm relies on the
normal functioning of stem cells. Recent studies have implicated a possible role for the posterior determinant gene nanos (nos) in
stem cells. nanos is required in the Drosophila female germline as well as in the male germline. In the female,
nos is required for the functioning of stem cells. In nos mutants, while the stem cells are specified, these cells divide only a few
times at the most and then degenerate. The loss of germline stem cells in nos mutant mothers appears to be due to a progressive degeneration of the plasma
membrane. Furthermore, following germ cell loss, the germaria in the nos mutant mothers appear to carry on massive mitochondrial biogenesis activity. Thus, the
syncytia of such germaria are filled with mitochondria. In the male germline, the male fertility assay indicates that nos appears to be also required for the maintenance
of stem cells. In these mutant males, spermatogenesis is progressively affected and these males eventually become sterile. These results indicate novel requirements
for nos in the Drosophila germline (Bhata, 1999).
To determine the ovarian phenotypes in nos mutant females, ovaries from nos mutant mothers were stained with Hoechst, a DNA stain, and then examined under
a fluorescent microscope. In wild type, each ovariole contains six to seven developing egg chambers at different stages and generally one, but occasionally two,
mature eggs. In nos mutant mothers, the ovaries contain ovarioles with either no egg chambers or a
single to a maximum of seven egg chambers at different stages. Some of the
ovarioles in nos mutants produce only one egg. In addition, the Hoechst staining of mutant ovaries indicates that ovaries with no developing germ cells can also be observed
among nos mutant females. These results are consistent with the observation that ovarioles in nos ovaries produce a variable number of eggs and argue that the egg-laying defect in nos mothers is due to an interruption in the oogenesis process itself (Bhata, 1999).
Because the strong loss-of-function alleles of nos produce only a few eggs, it is possible that the germline stem cells are not specified in nos mutants and that the
few eggs deposited by the mutant females are due to a subset of prestem cells that differentiate directly without going through a stem cell stage. Alternatively, one can also suppose that the loss of nos function affects the functioning but not the specification of stem cells and that
the few eggs generated are due to both direct differentiation of prestem cells and to stem cells asymmetrically dividing a few times to produce a limited number of eggs. To distinguish between these two possibilities, first the egg-laying pattern of nos mutant females was determined. Because each asymmetric division of a stem cell
yields one egg, determining the pattern and the number of eggs deposited over a period of time in a given mutation indicates the functional history of stem
cells in that mutation. In wild type, a stem cell divides once in 12-22 hr to self-renew and to produce a cystoblast. A
cystoblast undergoes 6-7 days of development before being deposited as an egg. A female Drosophila begins to lay eggs by its second day of emergence, indicating
that parent cystoblasts for these eggs enter the differentiation pathway in the early pupal stage (the pupal stage extends for ~5 days at room temperature). Generally,
a wild-type female deposits an average of 30-40 eggs per day for ~4 wk, after which the number becomes progressively reduced. nos
females show three different types of disrupted egg-producing patterns. In Type I, nos females begin to deposit their eggs by the second day of emergence as in
wild type, indicating that the process of egg development is not delayed in the mutants. However, these nos females stop laying eggs altogether by their fifth to
ninth day of emergence. In Type II, nos mothers begin to deposit their eggs by their second day of emergence and cease egg laying between the fifth to ninth
day, as in Type I; however, after an interval of 2 to 12 days of no egg deposition, these females lay a few more eggs. In Type III, nos mutant females do
not lay any eggs. Phenotypic analysis of these mutant ovaries indicates that the ovarioles in Type III individuals are rudimentary and devoid of any developing egg
chambers or other developing germ cells (Bhata, 1999).
The results presented in this article indicate that nos is required for the survival of stem cells in the female germline but not for their formation or specification. At least two different lines of evidence indicate that
stem cells are indeed specified in nos mutant ovaries. First is the Type II egg-laying pattern displayed by nos
mutant females; while the first batch of eggs in Type II may also contain eggs originating from a direct differentiation of prestem cell, the second peak of eggs (which are laid as late as 12 days after eclosion) are likely to have been generated from the
asymmetric division of stem cells because all the prestem cells would have been differentiated and laid as eggs within the first 2-3 days of eclosion. Thus, the Type II
egg-laying pattern indicates that stem cells are specified in nos mutant females and also that stem cells divide transiently to generate a few cystoblasts. The second line of evidence comes from the staining pattern of the mutant germaria with several cell-type specific markers: in freshly eclosed females, the germaria in
a large number of ovarioles in nos mutants appear to be wild type, with cells that would account for stem cells, cystoblasts, and two-cell cystocytes in region 1. In
older females, however, only the stem cells can be observed at the tip of the germarium, not the cystoblasts or the cystocyte clusters. Additional indication that the large
cells at the tip of the germarium are indeed stem cells is provided due to their lack of Bag of marbles mRNA, a cystoblast specific marker. In contrast, ovarioles of 4- to 5-day-old mutants containing only stem cells in the germarium and a few egg chambers
in the vitellarium indicate that stem cells have divided earlier to generate a few cystoblasts. Moreover, the presence of empty agametic
regions between stem cells and cystocyte clusters (as revealed by Orb staining) within the germarium of 2- to 3-day-old nos mutants argues that stem
cells are, indeed, specified in nos mutants; however, they fail to generate cystoblasts in an uninterrupted manner (Bhata, 1999).
The requirements for the multi sex combs (mxc) gene during development have been examined to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila. Although mxc has not yet been cloned, it is known to be allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm [l(1)mbn]. The mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc also has
a strong maternal effect. Hypomorphic mxc mutations are found to enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. The mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression known to involve some other PcG genes and suggests that the mechanism
of this early PcG function could be different from the PcG-mediated regulation of homeotic selector
genes later in development (Saget, 1998).
Induction of
uncontrolled growth and deregulation of Hox genes are linked in mammals, where Hox products can
induce leukemia. In Drosophila,
modification of homeotic gene expression causes homeosis, sometimes associated with increased
proliferation but not with uncontrolled tumorous growth, possibly because
the identity of each segment is specified by a combination of HOM products. Loss or gain of one
HOM gene will likely lead to a new combination that is found elsewhere in wild type, and cells
expressing this combination could be expected to follow the corresponding developmental pathway and give rise to homeotic
transformations. However, because each cellular identity apparently corresponds to a given
proliferation rate, loss or ambiguity of identity due to deregulation of several selector genes in a single
cell, such as mxc mutations apparently induce, could lead to loss of proliferation control. Identification
of mxc partners and targets, as well as of the molecular nature of the mxc product, may help throw
light on the genes and mechanisms involved in this process (Saget, 1998).
It has been proposed that certain PcG genes are required for the
maintenance of the expression domains of knirps and giant, through a mechanism similar to the
regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid
(bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in
the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and
giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal
segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also
be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress
kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products
of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine
if mutation of mxc also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This
genetic background was used because a decrease in the amount of maternal hb product can partially rescue the
nos phenotype in F1 embryos. Such embryos can differentiate a few
abdominal denticle belts and form an adequate background to evaluate increased rescue of
nos. Thus loss-of-function PcG mutations should have a strong effect on rescue,
and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background
should permit increased rescue of the nos phenotype (Saget, 1998).
Any of three E(z)son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2
complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed
with the gain-of-function E(z)son alleles. The
EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc does not. Some rescue with the
Psc/Su(z)2 complex deletion Df(2)vgB is also observed and
strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as
amorphic. This suggests that phob and mxcG48 are probably not
amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with
a non-null mutation (Saget, 1998).
Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb
nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap
gene regulation by these PcG mutations may be, it does not function like the PcG-mediated
maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by
several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that
modified PcG proteins are poisoning a normal process. How this process depends on wild-type
regulation by PcG products has yet to be established (Saget, 1998).
The zinc-finger protein Nanos functions during development (to promote germ cell migration) and during oogenesis (during germ line stem cell development). In a third role, early in development, Nanos and the RNA-binding protein Pumilio act together to repress the translation of maternal hunchback RNA in the posterior of the Drosophila embryo, thereby allowing abdomen formation. Nanos RNA is localized to the posterior pole during oogenesis; the posteriorly synthesized Nanos protein is sequestered into the germ cells as they form in the embryo. This maternally provided Nanos protein is present in germ cells throughout embryogenesis. Maternally deposited Nanos protein is essential for germ cell migration. In embryos lacking maternal Nos, defects in germ cell migration are seen from stage 10 onward. Following the exit of germ cells from the posterior midgut pocket, the germ cells fail to migrate over the surface of the gut and instead cluster tightly together on the outer gut surface. In many embryos most of the germ cells remain in a large cluster associated with the distal tip of the posterior midgut as it extends anteriorly during embryonic development. Mutant germ cells seem to cluster very tightly together as soon as they exit the midgut whereas, in wild type embryos, this tight association between germ cells is only seen following their association with the gonadal mesoderm at a later stage in embryogenesis. Zygotic nos expression cannot compensate for the loss of maternal Nos (Forbes, 1998).
Lack of zygotic nanos and pumilio activity in adults has a dramatic effect on germline development of homozygous females. Given the coordinate function of nanos and pumilio in embryonic patterning early in development, an analysis was made of the roles of these genes in oogenesis. Both genes act in the germline. Although the nanos and pumilio ovarian phenotypes have similarities and both genes ultimately affect germline stem cell development, the focus of these phenotypes appears to be different. While pumilio mutant ovaries fail to maintain stem cells and all germline cells differentiate into egg chambers, the focus of nanos function seems to lie in the differentiation of the stem cell progeny, the cystoblast. Thus, in egg chambers pum acts early in the developmental hierarchy in the maintenance of stem cells and nos functions later in stem cell progeny (Forbes, 1998).
When comparing pum and nos mutant germaria, and consistent with differences in their effects on early oogenesis, differences in the distribution of Spectrin are also seen. In nos mutants, very small spectrosomes are seen in the germline stem cells closely associated with the cell membrane adjacent to the cap cells. The amount of Spectrin associated with the fusome in the dividing germline cysts is greatly reduced in nos mutants. This phenotype suggests that while stem cells are established in nos mutants, they are not entirely normal. In pum mutant germaria, Spectrin-staining dots, which are almost as large as in wild type but more irregularly shaped, are seen in the most anterior germline cells. However, in contrast to wild-type stem cells, these spectrosome-containing cells are not associated with the basal cells of terminal filaments or cap cells. This is consistent with the failure to maintain germline stem cells at the germarium tip in pum mutants. Consistent with the model that nanos and pumilio have different phenotypic foci during oogenesis, high levels of Pumilio protein are detected in the germline stem cells and high levels of Nanos in the dividing cystoblasts. Therefore, it is suggested that in contrast to early embryonic patterning, Nanos and Pumilio may interact with different partners in the germline (Forbes, 1998).
The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) act together to specify the abdomen in
Drosophila embryos. Both proteins later accumulate in pole cells, the germline progenitors. Nos is required
for pole cells to differentiate into functional germline. Pum is also essential for germline
development in embryos. A mutation in pum causes a defect in pole-cell migration into the gonads.
In such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely.
pum mutation causes premature mitosis in the migrating pole cells. Pum is found to inhibit
pole-cell division by repressing translation of cyclin B messenger RNA. Since these phenotypes are
indistinguishable from those produced by nos mutation, it is concluded that Pum acts together with Nos to
regulate these germline-specific events (Asaoka-Taguchi, 1999).
The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos
protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells.
During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of
oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of Hunchback RNA.
Nanos' targets for germ cell migration and development are not known. A selective genetic screen was carried out aimed at
isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed the identification of amino acids critical for nanos function. This analysis shows
that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers
seems to constitute a novel functional domain within the Nanos protein. This 'tail region' of Nanos is required for abdomen formation and germ cell migration, but not
for oogenesis (Arrizabalaga, 1999).
The last 87 amino acids of Nanos contain two metal-binding domains of the CCHC type. In addition to mutations that alter the CCHC motif and thereby affect the ability of the mutant protein to chelate Zn, mutations in 9 amino acids located within the Zn finger domain that completely abolish nanos function were identified. While these mutations could affect the structure or stability of the protein, some of these mutations may identify amino acids important for the ability of Nanos protein to interact with RNA or protein targets. Zinc fingers of the CCHC type are not commonly found. The spacing between the Cys and His residues in Nanos are unique to this protein and its homologs. Other proteins, such as the HIV nucleocapsid protein, Xenopus CNBP, and Drosophila Clipper, have multiple copies of CCHC zinc fingers, but the ligand spacing is different. All of these proteins have been implicated in binding to single-stranded RNA. For instance, Clipper (Clp) is a Drosophila endoribonuclease that cleaves RNA hairpins. This protein contains five CCCH fingers that confer the endonucleolytic function and two CCHC fingers implicated in specific RNA binding. In addition to the CCHC motif, the HIV-I nucleocapsid protein and Nanos share a seven-amino-acid spacing between the zinc fingers. Of particular interest is the fact that the fourth amino acid in this spacer is an Arg in both proteins. The mutational analysis has identified this Arg351 as important for nanos function. This Arg is conserved among HIV nucleocapsid proteins and has been shown to be required for viral genomic packaging. In addition, crystallography studies of the nucleocapsid protein bound to its RNA target shows that this Arg makes direct contact with nucleic acids. Thus, it is an intriguing possibility that this Arg plays a similar role in Nanos (Arrizabalaga, 1999 and references therein).
The specific function of the zinc fingers in Nanos is not known. However, it has been shown that Nanos can bind to RNA with high affinity and that the ability of Nanos to bind RNA resides in the C terminus. While a specific, high-affinity interaction between Nanos and the NREs has not been established, Pumilio protein has been shown to bind with high affinity and specificity to the NREs. Point mutations in the NREs that affect Pumilio binding do not affect the affinity of Nanos for the RNA. Nevertheless, a small number of nucleotides outside the conserved NRE motif have been shown to affect translational regulation of hunchback binding, but not Pumilio binding. Further experiments are required to determine whether Nanos indeed binds to the NREs or other RNA targets with sequence specificity and whether Arg351 plays a role in such an interaction (Arrizabalaga, 1999 and references therein).
Mutations in a region C terminal to the zinc fingers of Nanos cause abdominal and germ cell migration defects without affecting the function of nanos in oogenesis. Since increasing the dosage of Nanos protein containing a mutation in the tail domain does not alter the abdominal phenotype of mutant embryos, the hypothesis is favored that the tail region of Nanos constitutes a separate functional domain. Mutations in the tail domain affect germ cell migration differently from nanos null mutations. Null mutations or mutations in the zinc finger region of Nanos have been shown to have a dramatic effect on germ cell migration. Primordial germ cells devoid of Nanos have altered morphology, fail to leave the gut toward the mesoderm, and tightly associate with each other in clusters. Furthermore, enhancer trap lines that are normally expressed in germ cells late during embryogenesis are expressed earlier in nanos mutant germ cells. This has led to the hypothesis that some of the phenotypes displayed by nanos mutant germ cells may be caused by the precocious expression of genes normally expressed at a later stage. Mutants in the tail domain affect germ cell migration to a lesser extent than null mutations. nosL7 germ cells, like germ cells lacking Nanos, form clusters and fail to leave the gut. However, aberrant germ cell clustering is not as extreme as that seen for the null mutant, and germ cell morphology seems normal. Furthermore, premature gene expression has not been observed in nosL7 mutants. Finally, many nosL7 germ cells reach the embryonic gonad and the embryos develop into fertile adults (Arrizabalaga, 1999 and references therein).
These differences in phenotypes might indicate that nosL7 is a weak allele with respect to germ cell migration. Contrary to what is observed in hunchback regulation, nosL7 may retain some function in germ cell migration. Alternatively, the tail domain may just affect a subset of the processes disrupted in the null mutants. Contrary to nosL7 germ cells, nanos null germ cells show aberrant morphology and even when some cells reach the gonad, the resulting adults are often sterile. Consequently, nanos might be required in the germ cells for two independent functions: migration, which requires the tail domain, and germ-line stem cell identity, which does not require the tail domain. Problems in germ cell identity might exacerbate the migration defect, hence the greater loss of germ cells in the null mutants. Clearly, the identification of Nanos germ-line targets is required to further address the function of its different domains in germ cell migration (Arrizabalaga, 1999).
The Nanos CCHC motifs show significant homology with sequences from other insects, Xenopus, leech, and C. elegans. The Xenopus nanos homolog Xcat-2 and the leech homolog are expressed in the developing oocyte. Xcat-2 RNA is localized to the vegetal pole of the oocyte during oogenesis. In the embryo, Xcat-2 RNA is initially taken up into the vegetal blastomeres and its expression becomes restricted to the primordial germ cells later in embryogenesis. The function of the Nanos homolog in Xenopus is not known. However, the similarity in localization pattern suggests a role for the protein in establishing embryonic polarity or germ-line development. Most amino acids mutated in this screen are conserved in these divergent species. Only two mutations, alleles nos538/549 and nos512, lead to changes in nonconserved amino acids (V354M and S337L). Given that these amino acids are next to His and Cys residues of the CCHC motif, it is possible that these mutations may affect the metal coordination within their respective zinc fingers. Despite the high degree of conservation between the two zinc finger domains of Drosophila Nanos and Xenopus Xcat-2, the two protein regions are functionally not interchangeable.
. An RNA in which the Nanos zinc fingers were replaced in frame by those of Xcat-2 was shown to be unable to rescue the nanos mutant abdominal phenotype (Arrizabalaga, 1999 and references therein).
Three family members of Nanos have been identified in C. elegans, and a role for these three proteins in germ-line development has been suggested. Among the three C. elegans Nanos homologs, the characteristic CCHC motif with the exact spacing within and between the two fingers is only conserved in Nos-3. Like Xcat-2, the homology between Drosophila Nanos and the C. elegans homolog includes only the zinc finger region, while there is no homology with the tail domain. The results presented in this paper suggest that the tail domain of Nanos is specifically required for the regulation of hunchback and bicoid and certain aspects of germ cell migration. One explanation for the functions of the tail domain and the zinc finger is that the tail domain carries out functions that are unique to insect development, while the zinc finger domain plays a conserved role in germ-line development. In support of a conserved role for Nanos, homologs of Pumilio, the functional partner of Nanos, have been found in many species, including C. elegans. There are at least eight pumilio homologs in C. elegans, and the role of two functionally redundant homologs, FBF1 and FBF2, has been reported. Fem-3 binding factor (FBF) proteins, like their Drosophila counterpart, are specific RNA-binding proteins and act as translational repressors. One of the identified FBF targets is the fem-3 gene, which is involved in germ-line sex determination. Fem-3 directs spermatogenesis in the hermaphrodite, and its translation must be suppressed to allow the switch to oogenesis to occur. These observations suggest a role for FBF in translational regulation similar to that performed by Pumilio and Nanos in Drosophila. Furthermore, the fact that the FBFs like Pumilio and Nanos regulate germ-line cell fate may suggest a conserved role for Nanos and Pumilio in germ-line development (Arrizabalaga, 1999 and references therein).
In summary, the analysis of a large number of nanos mutants has led to the following model for Nanos protein function: the C terminus of Nanos plays a crucial role during three developmental stages of Drosophila development: embryogenesis, primordial germ cell migration, and oogenesis. Nanos' role during embryogenesis is to silence the translation of maternal Hunchback RNA. This function requires the Nanos zinc finger and tail region as well as the RNA-binding protein Pumilio. During primordial germ cell development, Pumilio and Nanos are required for migratory behavior, the temporal control of gene expression, and the differentiation of germ cells into germ-line stem cells. These processes require Pumilio and the zinc finger region of Nanos. The function of Nanos' tail region seems to be restricted to certain aspects of germ cell migration. During oogenesis, only the zinc finger region is necessary for Nanos' function, which has aspects both overlapping with and separate from Pumilio. Nanos homologs in other organisms suggest a conserved role in germ cell development. In these organisms, the region of homology is restricted to the zinc finger motif and does not span the tail domain, suggesting that the tail domain may have been recruited later in evolution and may fulfill a more specialized roles (Arrizabalaga, 1999).
Germ cell identity and development are controlled by autonomous cues in the germ plasm as well as by interactions between germ cells and somatic cells. This study investigated the formation of a germ cell-specific organelle, the spectrosome. Spectrosome formation is independent of germ cell-soma interactions and is autonomous to the germ cells. Furthermore, the germ plasm component nanos (nos) is essential for spectrosome formation. The role of nos in spectrosome formation is independent of its role in germ cell survival; nos mutant germ cells that are prevented from undergoing programmed cell death still fail to form spectrosomes. Thus, nos is required to regulate the formation of this germ cell-specific organelle, further supporting a role for nos in promoting germ cell identity (Wawersik, 2005).
Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation has been unknown. nos and pum are shown in this study to control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis (Ye, 2004).
Early in Drosophila embryogenesis, Nos protein is first detected in the posterior end of the embryo and then in the pole cells, whereas Pum protein is uniformly distributed. Characterization of later expression has been limited to the ovary for Nos and to the adult head for Pum. Several recent findings implicate Nos and Pum in eye development, optic nerve development, neuronal excitability, and long-term memory. To determine whether Nos and Pum regulate dendrite morphogenesis, their expression and function were examined in the dendritic arborization neurons in the Drosophila peripheral nervous system (PNS) (Ye, 2004).
The da neurons have proven to be useful for studies of dendrite development. The 15 da neurons in each hemisegment of larvae fall into four classes (class I, II, III, and IV) with increasing complexity of dendritic morphology. The highest order dendrites of class I and II da neurons are fourth-order dendrites. Most of the terminal branches of class III neurons are fifth-order dendrites with a distinctive structure referred to as dendritic spikes. The class IV neurons have highly branched dendrites with terminal branches typically above the fifth-order (Ye, 2004 and references therein).
Nos and Pum were expressed in all da neurons, as revealed by immunocytochemistry with antibodies against Nos or Pum in third-instar larvae from a transgenic line carrying GFP marker 80G2, which marks all da neurons. Neuronal expression of nos was further confirmed with two independent GAL4 drivers under the control of the nos promoter, P{GAL4-nos.NGT}40. mCD8-GFP immunoreactivity is observed in da neurons of larvae that carry both P{GAL4-nos.NGT}40 and a reporter gene, UAS-mCD8-GFP, suggesting that the nos promoter is active in da neurons. Similar neuron-specific expression was also observed with nos-GAL4::VP16, which was inserted into a different chromosome and yielded some segment-to-segment variations in the expression pattern (Ye, 2004).
Overexpression of nos-tub3'UTR in class III and class IV neurons, but not class I neurons, dramatically changes dendrite morphology. In both class III and class IV neurons, the number of high-order dendritic branches was significantly reduced while the morphology of the major branches was not affected. Overexpressing pum causes a similar change specific to dendrites of class III and IV neurons. Neither the dendrites of bipolar neurons nor those of chordotonal neurons were affected by overexpressing nos or pum (Ye, 2004).
The loss of function phenotype of nos and pum in dendrite morphogenesis was assessed via mosaic analysis with a repressible cell marker (MARCM). The MARCM system provides an effective way to study every type of PNS neuron, including da neurons, chordotonal neurons, bipolar neurons, and external sensory (es) neurons, with single cell resolution. It was therefore determined which of these neurons is affected by nos or pum mutation. In addition, by specifically eliminating nos or pum function in da neurons, whether these genes act cell-autonomously in dendritic morphogenesis was determined. As a control, MARCM analysis was performed with a chromosome carrying an unrelated transgene (Ye, 2004).
Loss of nos or pum in class I or II da neurons did not alter dendrite morphology. In contrast, in class III neurons lacking nos or pum function, the characteristic dendritic spikes are significantly elongated, but the order of dendrites and the length of major dendritic branches (all dendrites except dendritic spikes) are indistinguishable from those of wild-type neurons. Whereas around 2%-10% of dendritic spikes of wild-type ddaA neurons are longer than 10 μm, loss of nos or pum function causes about 10%-30% of spikes to be longer than 10 μm in about 50% of ddaA neurons (Ye, 2004).
Class IV neurons deficient for nos or pum function also exhibit abnormality in their dendrites. The dendrites of wild-type class IV neurons cover the epidermis in a complete but nonoverlapping fashion and thereby 'tile' the body wall. Incomplete coverage of the epidermis was observed in 20% of neurons mutant for nos (3 in 15) as a result of the reduction of higher-order branches. Therefore, both nos and pum are required for the proper morphogenesis of dendrites, especially the high-order dendritic branches, in a cell type-specific manner (Ye, 2004).
Given the similar dendrite phenotypes of nos and pum mutants, it was wondered whether there is a mutual requirement of nos and pum for dendrite morphogenesis, as in embryogenesis. First it was tested whether pum function is required for nos overexpression to eliminate high-order dendritic branches in class IV neurons. Indeed, when nos is overexpressed in a pum null background, the high-order dendritic branches are not as drastically reduced as those in the case of nos overexpression in a wild-type background. It was then reasoned that, if nos and pum require each other in regulating dendrite morphogenesis, the dendrite phenotypes of pum, nos double mutants should resemble those of single mutants of nos or pum. MARCM analysis was employed to examine the dendrites of da neurons mutant for both nos and pum. Eliminating both nos and pum functions in class I da neurons does not result in any defect in dendrite morphology. The number of long dendritic spikes in class III neurons is increased to a similar extent as in the nos and pum single mutants. Moreover, incomplete innervation of the territory was observed in 18% of neurons mutant for both nos and pum (5 in 28 clones), an extent similar to that in the single mutant of either nos (20%) or pum (15%), as a result of reduced numbers of high-order branches in class IV neurons. Taken together, these data indicate that nos and pum require each other to regulate dendrite morphology, possibly by forming a protein complex as they do in embryogenesis (Ye, 2004).
The only domain structure identified so far in Pum protein is the so-called 'Pumilio-homology domain' (Pum-HD), which consists of eight repeats of 36 amino acids and is conserved in various species, including humans. Pum-HD is responsible for binding to the nos response elements (NRE's) of hb mRNA, and is sufficient for Pum function in embryogenesis. To investigate whether this RNA binding domain is sufficient for dendrite morphogenesis, Pum-HD was overexpressed in class IV da neurons. Overexpression of Pum-HD virtually replicates the dendrite phenotype that was produced by overexpression of full-length Pum. Therefore, it is likely that the Nos/Pum complex regulates the downstream molecules through the RNA binding domain of Pum (Ye, 2004).
If the roles of Nos and Pum in dendrite morphogenesis are to be fully understood, it is crucial to identify the RNA targets of this complex in dendrites. The dendrite phenotypes described here provide a guide for searching for the RNA targets of the Nos/Pum complex in an ongoing genetic screen for dendrite development. Moreover, the epitope-tagged Pum RNA binding domain, which is sufficient for Pum function in dendrites, will be a useful tool for biochemically identifying the RNA targets (Ye, 2004).
To elucidate the possible site of Nos/Pum action, the subcellular distribution of Nos was studied. Because the anti-Nos antibody also stains muscle in larvae, it was difficult to examine Nos distribution in neuronal processes situated near muscle. To circumvent this technical problem, a transgene of Nos fused to an HA-epitope tag at the N terminus was generated and it was expressed in da neurons, but not muscles, by using the GAL4/UAS binary system. HA-Nos is localized to distinctively punctuate structures in both soma and dendrites. These structures are round and uniform in size, with a diameter of around 0.3 μm; this is reminiscent of the RNA granules ranging from 0.175-0.6 μm in diameter in mammalian cortical neurons. Then the larval preparations were double stained with both anti-HA antibody and Syto 14, a nucleic acid dye that preferentially labels RNA; Nos was found to colocalize with RNA granules (Ye, 2004).
Essential for the posteriorly localized translation repression of hb by Nos/Pum complex, translation of Nos itself is repressed in the anterior of the embryo via a 90 nucleotide translational control element (TCE) located in the 3' untranslated region (3'UTR) of nos mRNA. In a subset of Drosophila central neurons, ectopic expression of nos causes the wing expansion phenotype only upon replacement of the TCE-bearing 3'UTR with α-tubulin (tub) 3'UTR (nos-tub3'UTR), thereby removing the TCE-dependent translational suppression of the nos transgene. To examine whether a mechanism analogous to that for the translational repression of nos in the embryo exists in da neurons, a GAL4 driver (GAL48-123) was used to ectopically express nos-tub3'UTR mRNA inserted with the nos TCE (nos-tub:nos+2). The nos-tub3'UTR and nos-tub:nos+2 transgenes have been shown to have little position effect in expression. Overexpression of nos-tub3'UTR resulted in reduction of the amount of high-order dendritic branches, a dendrite phenotype similar to that produced by GAL44-77. Overexpression of nos-tub:nos+2 with GAL48-123 significantly reduces the severity of the phenotype, thereby suggesting the presence of a mechanism for translational repression of nos in class IV da neurons (Ye, 2004).
Both nos and pum genes are conserved in various species, including mammals. Two nos genes have been identified in humans. The zygotic nos1 is highly expressed in the nervous system but not in developing germ cells. It is unclear whether nos2 and 3 are expressed in the nervous system. There have been no gross anatomical defects observed in mice deficient for nos1. In light of this study, it would be of interest to conduct a detailed investigation on neuron morphology with single-cell resolution to ascertain whether these mice exhibit any defects in dendrite morphology, especially of high-order dendritic branches. It is also important to determine whether nos2 and nos3 are expressed in the nervous system and if their functions are redundant to those of nos1. Two pum genes, pum1 and pum2, have been cloned in both mice and humans; both genes are expressed in the brain. It will be interesting to see whether these genes take on separate or redundant roles in neurodevelopment and long-term memory, both functions of the pum gene in Drosophila (Ye, 2004).
In summary, nos and pum have been shown to be essential for proper dendrite morphogenesis in subsets of Drosophila PNS neurons: evidence is provided suggesting that they act by forming a translation control complex, possibly in dendrites. This study could serve as a starting point for future identification and characterization of molecules regulating local translation in both Drosophila and mammalian dendrites (Ye, 2004).
Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. The translational repressor Pumilio (Pum) is a regulator of sodium current [I(Na)] and excitability in Drosophila motoneurons. This study shows that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel Paralytic (Para). A putative binding site for Pum was identified in the 3' end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. Pum is able to reduce the level of nanos mRNA and as such a potential negative-feedback mechanism has been identified that protects neurons from overactivity of Pum. Finally, coupling was shown between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3'-untranslated region of target transcripts (Muraro, 2008).
Identification of the molecular components that underlie homeostasis of membrane excitability in neurons remains a key challenge. This study shows that the translational repressor Pum binds para mRNA, which encodes the Drosophila voltage-gated Na+ channel. This observation provides a mechanistic understanding for the previously documented ability of Pum to regulate INa and membrane excitability in Drosophila motoneurons (Mee, 2004). Thus, alteration in activity of Pum, in response to changing exposure to synaptic excitation, enables neurons to continually reset membrane excitability through the translational control of a voltage-gated Na+ channel (Muraro, 2008).
Previous studies report several mRNAs subject to direct Pum regulation including hb, bicoid (bcd), CycB, eIF4E, and possibly the transcript destabilization factor smaug (smg). The majority of these identified transcripts concentrate the roles of Pum to the establishment of the embryonic anterior-posterior axis (hb and bcd) and germ-line function/oogenesis (CycB). However, in the last few years, new findings have expanded the role of Pum to encompass predicted roles in memory formation, neuron dendrite morphology, and glutamate receptor expression in muscle. Indeed, the role of Pum is likely to be very much more widespread given that Pum pull-down assays followed by microarray analysis of bound mRNAs have now identified a plethora of possible additional targets of translational regulation (Gerber, 2006). The ~1000 or so genes identified are implicated to be involved in various cellular functions, suggesting that Pum-dependent translational repression might be a mechanism used in different stages of development and in diverse tissue function. To date, para is the first confirmed Pum target encoding a voltage-gated ion channel (Muraro, 2008).
Pum-binding motifs have been identified in the 3'-UTRs of many mRNAs known to bind to this protein. Analysis of 113 such genes expressed in adult Drosophila ovaries has identified a consensus 8 nt binding motif [UGUAHAUA]. This sequence contains the UGUA tetranucleotide that is a defining characteristic of the NRE-like motif described in the 3'-UTR of hb mRNA. Such an 8 nt motif has been identified within the ORF of para at the 3' end of the transcript. The biochemical binding data support the notion that this motif is indeed sufficient to bind Pum and as such represents the first such site to be localized to an ORF of any transcript. However, to translationally repress para mRNA, the data also show a requirement for regions of Pum in addition to the RBD. Interestingly, this kind of requirement has also been shown for another Pum target, eIF4E. The translational silencing of mRNAs is a complex mechanism on which only little information is available. It could involve deadenylation and degradation of the mRNA and/or the circularization of the mRNA and the recruitment of factors that would preclude translation. The fact that different Pum targets may require only the RBD (hb) or the full-length protein (eIF4E and para) suggests that Pum-mediated translational repression may follow complex target mRNA-specific mechanisms, most probably involving the interaction of other domains of Pum with additional, so far unknown, factors. In this regard, it is interesting to note that the N terminus of Pum has regions of low complexity including prion-like domains rich in Q/R. These domains may provide a platform for other proteins that influence the fate of Pum targets (Muraro, 2008).
The putative Pum binding motif lies within an exon that is common to all para splice variants identified (at least in the embryo) but is possibly subject to editing by adenosine deamination. Thus, in an analysis of splicing of para, a number of individual cDNA clones were sequenced and one splice variant was recovered that shows A-to-I editing in this motif. Together with a differential requirement for specific cofactors, editing of this motif might serve to influence how para is affected by Pum and, as such, further increase diversity in level of expression of INa in differing neurons or disease states (Muraro, 2008).
The known mechanism of action of Pum-dependent translational repression is absolutely dependent on additional cofactors. The most studied example, that of hb mRNA during early embryogenesis, requires the presence of both Nanos and Brat. However, the requirement for these two cofactors is seemingly transcript dependent. Thus, Pum-mediated repression of CycB mRNA requires Nanos but not Brat. However, Pum-dependent repression of bcd is apparently Nanos independent, because levels of Nanos in the anterior of the early embryo are undetectable. Although it was clearly shown that Pum-dependent repression of para mRNA in the Drosophila CNS requires Nanos, the requirement for Brat is less clear and seems to be neuronal cell type specific. A requirement for a different combination of cofactors for Pum-dependent translational regulation of a single gene transcript has not been reported previously, but clearly might represent an additional level of regulation. Such differential regulation might be required to spatially restrict the effect of Pum to certain cell types within the CNS. Voltage-gated Na+ currents are responsible for the initiation and propagation of the action potential and determine, together with other voltage-gated ion conductances, the membrane excitability of a neuron. Despite para being the sole voltage-gated sodium channel gene in Drosophila [compared with at least nine different genes in mammals, neuronal subpopulations nevertheless exhibit distinctive INa characteristics. To achieve this, para is known to undergo extensive alternative splicing and, additionally, RNA editing. It is highly likely that both alternative splicing and RNA editing generate mRNAs that encode channels with differing electrophysiological properties. It is also conceivable that these mechanisms might yield para transcripts that contain differing arrangements of Pum/Nanos binding sites, which may, or may not, recruit Brat. Indeed, it has been proposed that variations of the NRE consensus sequence may result in Pum-NRE-Nanos complexes with different topographies, resulting in altered recruitment abilities for additional cofactors such as Brat. Additional work is necessary to clarify where, in para mRNA, the binding sites for the Pum/Nanos complex are localized and how the recruitment of Brat is facilitated in only some neurons. In the hb repression complex, Brat has been shown to interact with the cap-binding protein d4EHP. Therefore, additional cofactors might be necessary for Pum-dependent para repression in the Brat-independent neuronal cell subtypes (Muraro, 2008).
In contrast to translational repression of hb, the data show that Nanos is unlikely to be a limiting factor of Pum-dependent repression of para translation. Consistent with this finding is the observation that overexpression of pum is sufficient to downregulate (and probably translationally repress) nanos mRNA. However, the opposite is not true; overexpression of nanos does not affect levels of pum mRNA. These data suggest that Pum is at least a principal orchestrating factor (if not the prime factor) in regulation of para translation. Moreover, the demonstration that overexpression of pum is sufficient to greatly downregulate nanos mRNA (relative to para mRNA), together with a requirement of Nanos for Pum-dependent para mRNA repression, implicates the existence of a protective negative-feedback mechanism that prevents overrepression of para mRNA. In the absence of such feedback, it is conceivable that excessive overrepression of para mRNA might lead to neurons falling silent as their membrane excitability drops below a critical threshold. Were this to happen, then signaling in the affected neuronal circuit would be severely compromised (Muraro, 2008).
Overexpression of full-length Pum in aCC/RP2 motoneurons not only causes a decrease in INa but also a significant decrease in IKfast. Additionally, pan-neuronal overexpression of Pum causes a significant decrease in Shal mRNA, a gene encoding a potassium channel known to contribute to IKfast. This result was surprising given that Shal was not identified as a Pum target from microarray analysis. That this mechanism might, therefore, be indirect is corroborated by the finding that IKfast and Shal mRNA remain at wild-type levels when Pum is overexpressed in a para-null background. It is, perhaps, counterintuitive that a reduction in INa, to achieve a reduction in membrane excitability, should be accompanied by a similar decrease in outward IKfast. However, changes in ionic conductances should not be considered in isolation and such a relationship might serve to maintain action potential kinetics within physiological constraints. Covariation of INa and IK as a mechanism for changing neuronal excitability has been described in these motoneurons previously. Moreover, there is precedent for coupling between transcripts: injection of Shal mRNA into lobster PD (pyloric dilator) neurons results in an expected increase in IA but also an unexpected linearly correlated increase in Ih, an effect that acts to preserve membrane excitability. Injection of a mutated, nonfunctional, Shal mRNA is also sufficient to increase Ih indicative that this coregulation is activity independent (MacLean, 2003). It remains to be shown whether genetic manipulation of para mRNA levels in Drosophila motoneurons will similarly evoke compensatory changes in Shal expression (Muraro, 2008).
In a previous study, it was shown that blockade of synaptic release, through pan-neuronal expression of tetanus toxin light chain, is sufficient to evoke a compensatory increase in membrane excitability in aCC/RP2 that was accompanied by increases in INa, IKfast, and also IKslow (Baines, 2001). In contrast, the current study showed that overexpression of pum is sufficient to decrease INa and IKfast but does not significantly affect IKslow (although there is a small nonsignificant reduction in this current). Clearly, the complete absence of synaptic input is a more severe change that likely elicits a greater compensatory change in these neurons than when Pum is overexpressed. However, whether removal of synaptic excitation also invokes additional compensatory mechanisms that act preferentially on IKslow remains to be determined. What is consistent, however, is that change in synaptic excitation of these motoneurons is countered by Pum-dependent regulation of both para mRNA translation and magnitude of INa (Muraro, 2008).
A key question remains as to what the mechanism is that transduces changes in synaptic excitation to altered Pum activity. Perhaps the most parsimonious mechanism will be one linked to influx of extracellular Ca2+. Indeed, experimental evidence supports a role for Ca2+, because blocking its entry can preclude changes in neuronal excitability observed as a result of activity manipulation. In addition, changes of gene expression resulting from activity-mediated Ca2+ entry have been described both in vitro and in vivo after plasticity changes such as long-term potentiation. Whether Ca2+ influx influences translation and/or transcription of Pum remains to be shown. Stimulation of mammalian neurons in culture with glutamate, after a preconditioning period of forced quiescence, results in an increase of Pum2 protein levels after just 10 min. The rapidity of this response suggests that it is mediated by a posttranscriptional mechanism. This study examined the role of Pum on Ca2+ channel activity. Neither IBa(Ca) nor levels of the voltage-gated calcium channel coded by Dmca1A (cacophony, Calcium channel α1 subunit, type A) are affected in aCC/RP2 motoneurons in which pum [full length (FL)] is overexpressed. The fact that Pum does not affect Ca2+channel activity directly could reinforce the idea of its serving as a primary sensor of activity changes (Muraro, 2008).
In summary, this study has shown that Pum is able to bind to para mRNA, an effect that is sufficient to regulate both INa and membrane excitability in Drosophila motoneurons. This mechanism requires the cofactor Nanos but does not obligatorily require Brat. Given that mammals express two Pum genes, Pum1 and Pum2, it will be of importance to determine whether this protein is also able to regulate sodium channel translation in the mammalian CNS (Muraro, 2008).
Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. This study shows that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. These results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species (Janic, 2010).
The Drosophila tumor-suppressor gene l(3)mbt was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain. Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype. L(3)mbt's closest homologs, Drosophila Scm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins. L3MBTL1, the human homolog of Drosophila L(3)MBT, is a transcriptional repressor that is found in a complex with core histones, heterochromatin protein 1γ (HP1γ), and RB (Retinoblastoma protein) and can compact nucleosomes. Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex. Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression. These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (Janic, 2010).
To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), genome-wide gene expression profiling was carried out of l(3)mbtE2 and l(3)mbtts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). l(3)mbtts1 tumors were also analyzed at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white1118 (w1118), l(3)mbtE2, or l(3)mbtts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins), were also profiled (Janic, 2010).
Hierarchical clustering plots of these data reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbtts1 tumors, respectively. From these data, 151 genes were identified that were either overexpressed or underexpressed in all three larval mbt tumor types compared to all three controls. From this list, those genes were removed that were also up- or down-regulated) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. The expression levels of the remaining 102 up-regulated genes are referred to as as the mbt signature (MBTS). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat. Individual MBTS genes, however, are also up-regulated in some of these tumors (Janic, 2010).
The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line. For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation. The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific γTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development. Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses. Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality), and nos is required during nervous system development (Janic, 2010).
Driven by the high percentage of MBTS genes that have germline functions, other germline-related genes were sought that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors. Among these, the genes were found that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively. The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (Janic, 2010).
To determine whether the mRNAs found ectopically expressed in mbt tumors are translated, protein expression was examined with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development, it was included in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, it was confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors. Immunofluorescence studies also revealed the ectopic expression in l(3)mbtts1 brains raised at 29°C of C(3)G, SQU, and VASA. These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, it has not been possible to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated (Janic, 2010).
Prompted by the expression in l(3)mbtts1 brains of several genes involved in the biogenesis and regulation of piRNAs, 23- to 30-nucleotide RNAs were sequenced from l(3)mbtts1 larval brain tumors and from wild-type brains and ovaries. 117 known piRNAs and microRNAs (miRNAs) were detected in l(3)mbtts1 larval brain tumor samples. Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors (Janic, 2010).
It is not known which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component, has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells. In addition, there is no estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (Janic, 2010).
The extent to which ectopic expression of germline genes contributes to mbt tumor growth was determined. To this end, larval brain growth was quantified in individuals that were mutant for l(3)mbtts1 alone, or double mutant for l(3)mbtts1 and one of several of the germline genes that are ectopically expressed in mbt tumors. Measured as the total amount of protein, the average brain size in l(3)mbtts1 is about seven times as large as that in control w1118 larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbtts1 larvae that are also mutant for either piwi, vasa, aub, or nos. The loss of piwi does not prevent brain overgrowth in brat k06028 mutant larvae. Then tumor growth was quantified after allograft in adult flies. The frequency with which l(3)mbtts1 homozygous larval brain tissue develops tumors in this assay is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt, but it is markedly reduced by the additional loss of piwi, vasa, aub), or nos. The frequency of brat k06028 tumor formation is not affected by the loss of piwi or nos. These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth (Janic, 2010).
A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains, has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells. Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas. These are known as cancer-testis (CT) genes or cancer-germline (CG) genes. A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (Janic, 2010 and references therein).
Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation, but their actual role in cancer remains unknown. The current results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi, NANOS1/nanos, and SYCP1 /c(3)G. The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer (Janic, 2010).
nanos:
Biological Overview
| Evolutionary Homologs
| Regulation
| mRNA localization and post-transcriptional regulation
| Developmental Biology
| References
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