pumilio

DEVELOPMENTAL BIOLOGY

Embryonic

PUM mRNA first appears in the most mature portions of the germarium. It is here that a cystoblast divides four times to yield 15 nurse cells and the oocyte(collectively referred to as an egg chamber). Early PUM mRNA is present throughout the egg chamber, but during stage 9, PUM mRNA is restricted to nurse cells with little or no detectable mRNA in the oocyte (Macdonald, 1992).

PUM protein is cytoplasmic, concentrated in a subcortical region of the embryo. The distribution of PUM protein exhibits no asymmetry along the anterior-posterior axis of the embryo (Macdonald, 1992). PUM mRNA is enriched at the posterior pole of early embryos. In addition, unlocalized PUM mRNA is also present (Barker, 1992).

Effects of Mutation or Deletion

Embryos derived from mutant females develop very few or no abdominal segments [Images]. The expression and distribution of Nanos mRNA and protein in embryos derived from pumilio mutant females are indistinguishable from wild type. Abdomen formation depends both on Nanos activity, spreading from the localized posterior source, and on Pumilio activity, present throughout the embryo. In embryos from mutant females, the domain of Hunchback protein expands in the posterior domain In strong pumilio mutants, the HB protein is expressed at high levels throughout the embryo (Barker, 1992).

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 pumilio (pum) gene plays an essential role in embryonic patterning and germline stem cell (GSC) maintenance during oogenesis in Drosophila. Using pum^ovarette mutations, a phenotypic analysis was carried out; it revealed multiple functions for pum: in primordial germ cell proliferation, larval ovary formation, germ-line stem cell (GSC) division, involvement in subsequent oogenic processes, as well as oviposition. Specifically, by inducing pum^- GSC clones at the onset of oogenesis, pum has been shown to be directly involved in GSC division, a function that is distinct from its requirement in primordial germ cells. Furthermore, pum encodes 156- and 130-kD proteins, both of which are functional isoforms. Among pum^ovarette mutations, pum¹⁶⁸⁸ specifically eliminates the 156-kD isoform but not the 130-kD isoform, while pum²⁰⁰³ and pum⁴²⁷⁷ specifically affect the 130-kD isoform but not the 156-kD isoform. Normal doses of both isoforms are required for the zygotic function of pum, yet either isoform alone at a normal dose is sufficient for the maternal effect function of pum. A pum cDNA transgene that contains the known open reading frame encodes only the 156-kD isoform and rescues the phenotype of both pum¹⁶⁸⁸ and pum²⁰⁰³ mutants. These observations suggest that the 156- and 130-kD isoforms can compensate for each other's function in a dosage-dependent manner. Finally, evidence is presented suggesting that the two Pum isoforms share some of their primary structures (Parisi, 1999).

pum mutations cause failure of germline stem cell maintenance during oogenesis. To investigate whether pum is also required for germline development before oogenesis, the phenotypes of the ovarette class of pum mutants (pum^ovt) were examined for potential defects in primordial germ cell development. Unlike 'classical' maternal effect pum mutants that can undergo oogenesis to produce embryos defective in posterior patterning, pum^ovt mutants show severe oogenic defects and fail to produce any eggs. Third instar larval ovaries from homozygous pum^ovt mutant and wild-type larvae were stained with anti-VASA antibodies to specifically label germline cells. Because pre-germline stem cells in the third instar larval ovaries reflect the final stage of primordial germ cell proliferation and migration, any abnormality in number or morphology reflects a defect in primordial germ cell proliferation and development. Wild-type third instar larval ovaries typically contain approximately 54.6 germ cells located in the medial region of the ovary. However, ovaries from pum^ovt mutants contain either significantly reduced or increased numbers of germ cells. The requirement of pum during primordial germ cell development is further confirmed by dramatic overproliferation of primordial germ cells seen in pum⁴²⁷⁷ and pum¹⁶⁸⁸ mutant ovaries. These observations suggest that pum function is required for the normal proliferation of primordial germ cells before oogenesis (Parisi, 1999).

In addition to abnormal numbers of germ cells, the mutant germ cells also exhibit various morphological defects. The abnormal size and morphology of pre-germline stem cells suggest that the development of primordial germ cells is highly aberrant in pum mutants even though certain germline characteristics, such as Vasa expression, are still maintained. The germline defects are often accompanied by a drastic increase in the size of the ovary, with an increased number of somatic cells. These results suggest that pum is also required for the proper formation of the larval ovary (Parisi, 1999).

The proliferation defects of the primordial germ cells and the abnormal morphology of the resulting pre-germline stem cells in the pum^ovt larval ovary suggest that these cells may not be able to function normally as germline stem cells during subseqeunt oogenesis. Examined was the hypothesis that germline defects at the larval stage correlate to subsequent ovarian defects in pupal and adult ovaries. The viability of pum^ovt mutants was examined at the pupal stage. The viability difference between the pum^ovt mutants and their heterozygous siblings is within 25%. This rules out any potentially significant skew in the observed pupal or adult defects caused by selective lethality against pupae with a particular type of oogenic defect. The pum⁶⁸⁹⁷ mutant was examined because a high proportion (77%) of pum⁶⁸⁹⁷ larval ovaries contain either underproliferated (54%) or overproliferated (23%) primordial germ cells. Ovaries were isolated from homozygous pum⁶⁸⁹⁷ females at ~48 hr after pupation. They were double stained with anti-Vasa antisera to label germline cells and anti-1B1 antibody to outline somatic cells and label spectrosomes and fusomes, two special structures in the early germline cells. By this stage, pupal ovaries from wild-type siblings have differentiated into an average of 16.7 ovarioles per ovary. In mutant ovaries, 1B1 staining of somatic cells shows that a similar number of germaria have also formed. In contrast to the control ovarioles that always contain a full complement of germline cells, however, 63% of the mutant ovarioles are germlineless. The remaining mutant ovarioles contain a small number of germline cells. By the adult stage, 86% of the pum⁶⁸⁹⁷ ovaries lack germline. These observations indicate that the pum⁶⁸⁹⁷ mutant is severely defective in oogenesis but not in ovariole formation (Parisi, 1999).

Parallel analyses on six other pum^ovt mutants reveal similar oogenic defects, with the proportion of germlineless ovaries varying among the mutants. The remaining ovaries contain either developing egg chambers or undifferentiated germ cell clusters. This confirms the previous conclusion that the self-renewing asymmetric division of germline stem cells is disrupted in the pum²⁰⁰³ mutant (Parisi, 1999).

pum^ovt mutations also affect subsequent oogenic events. Developing mutant egg chambers sometimes contain few nurse cells but no oocytes. This defect also exists in the maternal effect lethal class of pum mutants. Staining of these mutant egg chambers with rhodamine-conjugated phalloidin reveals a reduced number of ring canals, indicating that the reduced number of nurse cells results from reduced divisions of cystoblasts. Furthermore, these mutant egg chambers show a pronounced unequal ploidy among these nurse cells, indicating that the endoreplication mechanism in these egg chambers has been affected severely. These defects suggest that pum is required for the proper division of cystoblasts and differentiation of germline cysts. In addition to germline defects, somatic defects have also been detected at a low frequency in pum^ovt mutants, such as long interfollicular stalks and the disruption of single-stack cells in the terminal filament. Together, these data reveal that multiple germline and somatic processes of oogenesis are disrupted by pum^ovt mutations (Parisi, 1999).

The severe defects observed in pum mutant priomordial germ cells suggest that these cells are unlikely to undergo normal oogenesis, which precludes the opportunity to analyze whether pum also plays a direct role in germline stem cell division and other oogenic processes. To overcome this problem, germ cells were allowed develop normally before oogenesis and then the pum activity in germline stem cells was removed at the onset of oogenesis by inducing homozygous pum^- germline clones using the FLP-DFS technique. Pole cell transplantation have shown that pum functions cell autonomously in the germline. Thus, by removing pum from germ cells at the onset of oogenesis, whether or not pum is required during oogenesis could be examined directly. The homozygous pum mutant germline was induced in pum²⁰⁰³, pum¹⁶⁸⁸, and a maternal effect mutation (pum^ET1), because these are all strong mutations, representing three types of molecular lesions. Homozygous pum^- germline clones were generated in the ovo^D1 background. ovo^D1 dominantly blocks oogenesis in a cell-autonomous manner, arresting egg chambers uniformly at stage 3. This defect is distinctively different from either the germlineless or differentiating phenotype of pum^ovt or the wild-type ovariole. The developmental fate of homozygous pum^- germline stem cell clones depends on whether pum is required during oogenesis. If pum is required during oogenesis, pum^- clones should show corresponding pum^- oogenic defects. Alternatively, if pum is not required during oogenesis, the pum^- clones should undergo oogenesis normally (Parisi, 1999).

For all three pum alleles tested, pum^- germline clones exhibited typical pum^- defects. For example, many pum²⁰⁰³ germline clones produce ovarioles that contain only one to three mature egg chambers but no other germ cells. These typical differentiated pum²⁰⁰³ ovarioles are distinctively different from the ovo^D1 ovarioles. The differentiated ovarioles are also seen in pum¹⁶⁸⁸ and pum^ET1 germline clones. Sometimes, differentiated ovarioles contain four to five egg chambers, indicating that these pum^- germline stem cells have divided once before entering oogenesis. Other defects, such as germlineless germaria and ovarioles with undifferentiated germ cell clusters, though more difficult to quantify, also exist in the clone-induced ovaries. These defects indicate that pum activity is directly required in the germline stem cells during oogenesis (Parisi, 1999).

A striking difference between the pum germline clonal females and their corresponding nonclonal homozygous females is that the eggs produced by the clonal females are often laid, yet eggs produced by their nonclonal counterparts are never laid. This suggests that pum may be required in somatic cells for oviposition. To further test this possibility, the germline clonal females were allowed to lay eggs for 9 days and then the females were dissected to count the number of eggs that were still held in the ovary to measure the efficiency of oviposition. Among three alleles tested, the pum²⁰⁰³, pum¹⁶⁸⁸, and pum^ET1 clonal females achieved oviposition efficiency of 56%, 85%, and 78%, respectively. This egg-laying ability is never observed in homozygous pum²⁰⁰³ or pum¹⁶⁸⁸ females. The restoration of the egg-laying ability in females whose soma is no longer deficient in pum suggests that the pum gene is required in somatic cells for oviposition. This function is affected by pum²⁰⁰³ and pum¹⁶⁸⁸ mutations as well as by the maternal effect class of mutations such as pum^ET1 (Parisi, 1999).

A more surprising observation is that, despite pum²⁰⁰³ or pum¹⁶⁸⁸ mutations causing both germline and somatic defects in oogenesis and oviposition, eggs laid by females containing the homozygous pum²⁰⁰³ or pum¹⁶⁸⁸ germline were sometimes capable of developing into adulthood. This is in contrast to eggs laid by females containing the homozygous pum^ET1 germline; these eggs show typical posterior patterning defects and fail to hatch, as has been reported previously for maternal effect alleles. The ability of the pum²⁰⁰³ or pum¹⁶⁸⁸ eggs to develop further suggests that pum²⁰⁰³ or pum¹⁶⁸⁸ mutations do not abolish the maternally provided pum activity required for embryogenesis. The pum^- embryos were produced by mating virgin mutant pum²⁰⁰³ or pum¹⁶⁸⁸ clonal females to homozygous pum²⁰⁰³ or pum¹⁶⁸⁸ males, respectively. This rules out any possible paternal contribution to the embryonic pum function (Parisi, 1999).

Both pum²⁰⁰³ and pum¹⁶⁸⁸ mutations lead to more defects than maternal effect mutations during oogenesis, yet they do not completely affect embryonic development. Conversely, maternal effect mutations have fewer pleiotropic effects during oogenesis, yet they completely block embryogenesis. These differences could suggest that maternal effect mutations are stronger mutations. Alternatively, it is possible that pum is a complex locus encoding several discrete and complementable functions. To test these possibilities, inter se genetic complementation tests were conducted among pum^ovt and maternal effect alleles of pum. Previous complementation analyses have shown that pum²⁰⁰³ and pum³²⁰³ fail to complement several of the maternal effect lethal pum alleles, yet pum¹⁶⁸⁸ partially complements the maternal effect alleles. pum¹⁶⁸⁸ partially complements the embryonic lethality of maternal effect alleles pum^ET1, pum^MSC, and pum^ET7; of these, pum^MSC and pum^ET7 are known to disrupt the RNA-binding domain at the C terminus of the PUM protein. This complementation leads to the production of viable yet sterile progeny (Parisi, 1999).

The partial complementation between different P alleles suggests that they may differentially affect the pum gene product. Although two splicing variants of the pum transcript have been characterized, they differ only in the 5'-untranslated region but share the same predicted ORF. However, previous analysis of the Pum protein from embryonic extracts detected two bands positioned at ~156 kD among several bands of smaller molecular weight. It was known only that the 156-kD doublet represents the Pum protein. All antisera tested showed two major bands, the 156- and the 130-kD bands, in wild-type flies. The 156-kD band can be resolved as a doublet upon shorter exposure. Interestingly, the pum¹⁶⁸⁸ mutation completely eliminates the 156-kD doublet but does not affect the 130-kD band. Conversely, pum²⁰⁰³ and pum⁴²⁷⁷ mutations diminish the 130-kD band but do not appear to affect the 156-kD doublet. These results suggest that both the 156-kD doublet and the 130-kD band are functionally important protein isoforms of the pum gene. Affecting either one of the isoforms leads to defective pum function during preoogenic germline development and oogenesis, but one of the isoforms is sufficient for embryogenesis. The heteroallelic pum^MSC/pum^ET9 and pum^MSC/pum^ET1 combinations eliminate both the 156-kD doublet and the 130-kD isoform, suggesting that these three maternal effect mutations are null mutations (Parisi, 1999).

The 156- and 130-kD isoforms of Pum could both derive from the known ORF of pum by posttranslational processing. Alternatively, one of them could be encoded by a novel species of alternatively spliced pum mRNA that is yet to be identified. Exeriments indicate that the known pum ORF encodes only the 156-kD isoform. Although a P[nos-pum] transgene encodes only the 156-kD isoform, it rescues the preoogenic and oogenic defects of both pum²⁰⁰³ and pum¹⁶⁸⁸ mutants. In both homozygous pum²⁰⁰³ and pum¹⁶⁸⁸ mutants carrying the transgene, most ovarioles contain actively dividing germline stem cells that support normal oogenesis, even in 8-day-old mutant females, as is evident by the presence of a progression of wild-type egg chambers in most of their ovarioles. This indicates the complete rescue of germline and oogenic defects of both pum²⁰⁰³ and pum¹⁶⁸⁸ mutants by the P[nos-pum] transgene. Thus, even though the lack of either the 156- or 130-kD isoform leads to severe defects during zygotic germline development and oogenesis, increasing the expression of the 156-kD isoform alone can compensate for the lack of the 130-kD isoform and rescue the germline and oogenic defects. In addition to the complete oogenic rescue, 8 out of 15 transgene-carrying pum²⁰⁰³ females produced progeny; 3 out of 11 transgene-carrying pum¹⁶⁸⁸ females also produced progeny. These observations further support the conclusion that the 156- and the 130-kD PUM isoforms are not functionally distinct. Either isoform is sufficient for the maternal effect function of pum. At the present time, it is not known how the 130-kD protein is produced. The results suggest that this protein is either translated from an alternatively spliced PUM mRNA or derived from the 156-kD protein by posttranslational cleavage. An additional intricacy of Pum production is that the 156-kD protein itself consists of two isoforms of similar molecular weight. Given the identical behavior of the 156-kD doublet in this analysis, it is likely that these doublet isoforms are generated by posttranslational modification. This study has thus revealed a surprising complexity in the regulation of pum expression (Parisi, 1999).

Despite the rescue of the female sterility, P[nos-pum] does not rescue the semilethality of pum²⁰⁰³. The viability of homozygous P[nos-pum]; pum²⁰⁰³/pum²⁰⁰³ flies is 14% that of P[nos-pum]; pum²⁰⁰³/+ flies, which is similar to the viability of pum²⁰⁰³/pum²⁰⁰³ flies. Thus, as expected, the somatic function of pum in supporting viability is not rescued by the germline-specific expression of the P[nos-pum] transgene (Parisi, 1999).

Phenotypic analysis reveals a somatic function for pum during oogenesis, yet previous RNA and protein in situ analyses have shown only the germline expression of pum. To detect the possible somatic expression of pum, the enhancer trap staining pattern of most pum^ovt mutations was examined. This examination revealed that they are specifically expressed in the terminal filament cells. PUMmRNA is easily detectable in the terminal filament cells and epithelial sheath cells in the interfollicular stack region but is barely detectable in the follicle cells. In the germline, PUM mRNA is present in the germarium and later stages of oogenesis. The terminal filament and interfollicular cell expression may reflect the involvement of pum in ovary differentiation and oviposition (Parisi, 1999).

Immunostaining using different anti-Pum antibodies consistently reveals that the Pum protein is present in several somatic and germline cell types in the ovaries, including the terminal filament and the invaginating follicle cells in germarial regions IIb and III, as well as postgermarial follicle cells. In the germline, Pum is present at the highest level in germline stem cells and at lower levels in other germarial germline cells. The follicle cell expression of Pum may be related to the aging property of follicle cells (Parisi, 1999).

The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Zpg is required during oogenesis for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. Germ-line stem cells (GSCs) lacking Zpg can divide, but the daughter cell destined to differentiate dies. These results suggest that zpg may be necessary for the differentiation process itself, as well as for survival of differentiated germ cells, and that zpg probably acts in parallel to bam and bgcn. The differentiation of the GSC to a cystoblast is gradual, and it is suggested many of the germ cells in 'stem cell tumors' caused either by strong mutations in bam or by overexpression of Dpp may be at an intermediate state between GSCs and cystoblasts. These findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual. (Gilboa, 2003).

To further explore the role of Zpg in early germ cell differentiation and survival, zpg alleles were recombined with mutant alleles of the gene pumilio (pum). pum mutant ovaries exhibit a compound phenotype. Many ovarioles lack germ line completely, a defect that may be attributed to preoogenic defects. Ovarioles with germ line have a germ line-depleted germarium connected to a few defective egg chambers. This phenotype suggests that Pum has roles in GSC maintenance. In ovaries from zpg, pum double-mutant females, many ovarioles were empty. This is consistent with the embryonic and larval requirement for pum. Ovarioles occupied by germ line exhibited a phenotype more similar to zpg than to pum mutants: few germ cells at the tip of the ovariole. Thus Zpg function is required for the differentiation of pum mutant germ cells. The apparent difference between the zpg, pum and hs-Bam; zpg phenotypes may reflect the different roles of Pum and Bam in GSC differentiation. Pum, as a translational repressor may permit GSC maintenance by repressing differentiation, which requires Zpg. By contrast, Bam may have a more instructive role in GSC differentiation, such that its overexpression can overrule GSC-maintenance cues emanating from the niche, independent of zpg (Gilboa, 2003).

In addition to defects in GSC maintenance, pum mutants also show defects in cyst development. This function is also evident in the zpg, pum phenotype. Although zpg, pum ovarioles occupied by germ line mostly resemble the zpg phenotype, they contain more germ cells, and have more dividing cysts and differentiating egg chambers, than those of the single zpg mutant. The double mutant had an average of 0.23 egg chambers per ovariole (n=290), compared with 0.02 (n=500) in flies homozygous for zpg and heterozygous for pum. The higher number of single cells and egg chambers in zpg, pum double mutants may indicate that Zpg function is less essential when cyst development is abrogated, as is the case in pum mutants (Gilboa, 2003).

Maintenance of proper neuronal excitability is vital to nervous system function and normal behavior. A subset of Drosophila mutants that exhibit altered behavior also exhibit defective motor neuron excitability, which can be monitored with electrophysiological methods. One such mutant is the P-element insertion mutant bemused (bem). The bem mutant exhibits female sterility, sluggishness, and increased motor neuron excitability. The bem P element is located in the large intron of the previously characterized translational repressor gene pumilio (pum). Pum protein has been shown to bind directly to specific sequences in the 3' untranslated region (UTR) of maternally supplied hunchback (hb) mRNA (known as nanos-response elements or NREs) and then recruits at least two other proteins, Nanos (Nos) and Brain Tumor (Brat), to the mRNA. The resulting complex results in repression of hb translation via deadenylation of the hb message (Schweers, 2002)

This study shows by several criteria that bem is a new allele of pum: (1) ovary-specific expression of pum partially rescues bem female sterility; (2) pum null mutations fail to complement bem female sterility, behavioral defects, and neuronal hyperexcitability; (3) heads from bem mutant flies exhibit greatly reduced levels of Pum protein and the absence of two pum transcripts; (4) two previously identified pum mutants exhibit neuronal hyperexcitability; (5) overexpression of pum in the nervous system reduces neuronal excitability, which is the opposite phenotype of pum loss of function. Collectively, these findings describe a new role of pum in the regulation of neuronal excitability and may afford the opportunity to study the role of translational regulation in the maintenance of proper neuronal excitability (Schweers, 2002).

These studies suggest that Pum might regulate translation in the cell body, dendrites, or axons and that this translational regulation is important in maintaining proper neuronal excitability. For example, maintenance of proper neuronal excitability may be achieved by translational regulation of ion channel mRNAs directly or through regulation of an upstream ion channel regulator. Therefore, isolation of the pum^bem mutation allows the opportunity to study with genetic methods the role played by translational regulation in maintaining proper neuronal excitability (Schweers, 2002).

The staufen/pumilio pathway is involved in Drosophila long-term memory

Memory formation after olfactory learning in Drosophila displays behavioral and molecular properties similar to those of other species. Particularly, long-term memory requires CREB-dependent transcription, suggesting the regulation of 'downstream' genes. At the cellular level, long-lasting synaptic plasticity in many species also appears to depend on CREB-mediated gene transcription and subsequent structural and functional modification of relevant synapses. To date, little is known about the molecular-genetic mechanisms that contribute to this process during memory formation. Two complementary strategies were used to identify these genes. From DNA microarrays, 42 candidate memory genes were identified that appear to be transcriptionally regulated in normal flies during memory formation. Via mutagenesis, 60 mutants with defective long-term memory have been independently identified and molecular lesions have been identified for 58 of these. The pumilio translational repressor was found from both approaches, along with six additional genes with established roles in local control of mRNA translation. In vivo disruptions of four genes, staufen, pumilio, oskar, and eIF-5C, yield defective memory. It is concluded that convergent findings from the behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio/staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification (Daubnau, 2003).

The 60 memory mutants were generated with enhancer-trap transposons, which often drive reporter genes (lacZ or GFP) in patterns of expression similar to those of the endogenous genes they disrupt. Thus, reporter gene expression patterns were examined for milord-1 and -2 (pum), norka (oskar), and krasavietz (eIF-5C) in the adult CNS. Each of these enhancer-trap memory mutants shows common reporter gene expression in the mushroom body, an anatomical focus with a demonstrated role for olfactory memory. The norka and krasavietz strains carry a PGAL4 transposon that can drive expression of GFP in neuronal somata and processes. These data clearly reveal a common site of expression in a subset of intrinsic mushroom body neurons (Kenyon cells) that comprise the α and β lobes. The milord-1 and milord-2 strains, in contrast, carry a PlacZ transposon that expresses β galactosidase only in somata and, thus, only around the calyx region of mushroom bodies (Daubnau, 2003).

An existing mouse polyclonal antibody was to determine the expression pattern of Pum protein in the adult CNS. Consistent with the pattern of enhancer-trap expression for milord-1 and -2, Pum immunoreactivity is detected broadly in the CNS but appears to be expressed at high levels in mushroom body neurons. Strong immunoreactivity appears to be perinuclear in Kenyon cells, whereas weaker, punctate expression is detected in mushroom body neuropil (calyx). This antibody shows appreciable specificity on Western blots of embryonic extracts. It was not possible to use pumilio null mutants to establish antibody specificity for adult brain tissue, however, because the null mutants are not viable as adults. Coexpression in mushroom bodies of the reporter genes for oskar, pum, and eIF-5C and anti-Pum immunostaining are consistent with the notion that these genes function together in the CNS during long-term memory formation (Daubnau, 2003).

STAU already has been implicated in mRNA localization in mammalian CNS. In cultured hippocampal neurons, for instance, STAU has a punctate, somato-dendritic distribution and is a component of large RNP-containing neural granules, which themselves are associated with microtubules. These neural granules seem to play an analogous role in targeting mRNA translation to subcellular (synaptic) compartments in neurons, as do STAU-containing RNP particles (polar granules) in Drosophila oocytes. In cultured hippocampal neurons, neural granules are located near dendritic spines, appear to dissociate in response to local synaptic activity, and thereby release translationally repressed mRNAs. This process has been proposed as a mechanism for synapse-specific modification via local protein synthesis in response to neural activity in vitro. The data indicate that this staufen-dependent pathway underlies memory formation per se. Moreover, the further identification of oskar as a memory mutant and of moesin and orb as confirmed candidate memory genes suggests that additional genetic components of the machinery used for mRNA translocation and translation in oocytes also may function in neurons (Daubnau, 2003).

Combined with these observations from the literature, the data suggest a molecular mechanism for synapse-specific delivery of gene products during long-term memory formation. (1) Behavioral training results in the activation of CREB-mediated transcription, and nascent mRNAs are packaged into an RNP complex, a neural granule. In addition to staufen, oskar, and moesin, these granules may well include other components of polar granules such as mago and faf. (2) These neural granules then are transported into dendritic shafts along an organized microtubule network, as proposed above for vertebrate neurons. These activity-induced transcripts may be delivered nonselectively throughout the neuron or selectively to sites of recent synaptic activity. In either case, packaged mRNAs probably are translationally quiescent while in transport, thereby preventing ubiquitous expression of protein products. The data implicate pumilio as part of this translational repression complex. Finally, synapse-specific modification may result from the depolarization-dependent release of neural granule-associated mRNAs and a concomittant translational derepression (Daubnau, 2003 and references therein).

Local derepression of translation, in part, may involve phosphorylation of CPEB (orb) by aurora kinase, resulting in cytoplasmic polyadenylation and the dissociation of MASKIN from eIF-4E, which then allows interaction between eIF-4E and eIF-4G. Release of eIF-4E via phosphorylation of other 4E binding proteins also may promote assembly of the rest of the translation initiation complex. The presence during synaptic or behavioral plasticity of several other persistently active kinases also might contribute to such phosphorylation. CPEB-mediated translational activation in Xenopus oocytes, for instance, is associated with phosphorylation of ORB by CDC2 kinase (which is a dimer of CycB and CDC2) and ubiquitin-mediated degradation of Orb, perhaps modulated by faf or another ubiquitin hydrolase. Here again, DNA chip and memory mutant experiments have identified several of these additional components (Daubnau, 2003 and references therein).

nanos and pumilio are essential for dendrite morphogenesis in Drosophila peripheral neurons

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 (GAL4^8-123) 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 GAL4^4-77. Overexpression of nos-tub:nos+2 with GAL4^8-123 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).

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