InteractiveFly: GeneBrief

Zinc finger protein RP-8: Biological Overview | References


Gene name - Zinc finger protein RP-8

Synonyms -

Cytological map position - 60B11-60B12

Function - Zinc finger protein

Keywords - control of heterochromatin silencing, interacts with the piRNA pathway that is essential for the maintenance of germline stem cells, a component of the small ribosomal subunit (40S), controls RNA processing, centrosome function, regulates cell proliferation, hematopoesis, lymph gland new germline stem cells heterochromatin silencing piRNA pathway

Symbol - Zfrp8

FlyBase ID: FBgn0021875

Genetic map position - chr2R:24,133,989-24,135,405

Classification - MYND finger, Programmed cell death protein 2, C-terminal putative domain

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Minakhina, S., Naryshkina, T., Changela, N., Tan, W. and Steward, R. (2016). Zfrp8/PDCD2 interacts with RpS2 connecting ribosome maturation and gene-specific translation. PLoS One 11: e0147631. PubMed ID: 26807849
Summary:
Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). This study shows that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. Zfrp8/PDCD2 was also shown to interact with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins, it was demonstrated that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. These results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation.
BIOLOGICAL OVERVIEW

Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. This study identifies FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. It is proposed that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus (Tan, 2016).

Stem cell maintenance is essential for the generation of cells with high rates of renewal, such as blood and intestinal cells, and for the regeneration of many organs such as the brain and skin. Previous work has shown that Zfrp8 is essential for maintaining hematopoietic, follicle, and germline stem cells (GSCs) in Drosophila melanogaster (Minakhina, 2014). Knockdown (KD) of Zfrp8 in GSCs results in the loss of stem cell self-renewal, followed by the eventual loss of all germline cells (Minakhina, 2014). Similarly in vertebrates, the Zfrp8 homolog, Pdcd2, is essential for embryonic stem cell maintenance and the growth of mouse embryonic fibroblasts; Pdcd2 mouse embryos die before implantation (Granier, 2014 and Mu, 2010). PDCD2 is abundantly expressed and essential in highly proliferative cells including cultured cells and clinical isolates obtained from patients with hematologic malignancies (Barboza, 2013). The function of Zfrp8 and PDCD2 is highly conserved, as expression of transgenic PDCD2 is sufficient to rescue Zfrp8 phenotypes (Minakhina, 2014). Zfrp8 directly binds to Ribosomal Protein 2 (RpS2), a component of the small ribosomal subunit (40S), controls its stability and localization, and hence RNA processing. Zfrp8 also interacts with the piRNA pathway, which is conserved throughout all metazoans and is also essential for the maintenance of GSCs (Tan, 2016).

The piRNA pathway functions in maintaining heterochromatin stability and regulating the expression levels of retrotransposons. Both processes are thought to occur through piRNA targeting of chromatin modifying factors to the DNA. Guided by piRNAs, the piRNA pathway protein Piwi and associated proteins can set repressive epigenetic modifications to block transcription of nearby genes. Levels of transposon transcripts are also controlled by cytoplasmic PIWI-piRNA complexes, which can bind complementary mRNAs and mark them for translational repression and degradation (Tan, 2016).

Fragile-X Mental Retardation Protein (FMRP) functions as a translational repressor involved in RNA silencing. FMRP is a Piwi interactor and part of the piRNA pathway. FMRP-deficient animals display phenotypes similar to piRNA pathway mutants including genomic instability and de-repression of retrotransposons. While FMRP is predominantly localized within the cytoplasm, FMRP complexes have also been demonstrated within the nucleus. In Xenopus, FMRP has been shown to bind target mRNAs co-transcriptionally in the nucleus. Like Zfrp8, FMRP has been shown to bind ribosomal proteins prior to nuclear export (Chen, 2014; Taha, 2014). In the cytoplasm, the FMRP-containing RNP complex controls mRNAs stability, localization, and miRNA-dependent repression. FMRP mRNA targets are not well defined, as different studies show low overlap of putative targets in neuronal tissues (Tan, 2016).

In Drosophila, FMRP is required to maintain GSCs, and loss of Fmr1 is associated with infertility and developmental defects in oogenesis and neural development. Fmr1, the gene encoding FMRP, is essential in both vertebrates and Drosophila for the maintenance of neural stem cells (NSCs). In humans, loss of FMRP is associated with Fragile X-associated disorders, which cover a spectrum of mental, motor, and reproductive disabilities. Fragile X-associated disorders are the most commonly inherited cause of mental disabilities and autism. In vertebrates, FMRP physically interacts in the nucleus with NUFIP1 (Nuclear FMRP-Interacting Protein 1), a nucleo-cytoplasmic shuttling protein involved in ribonucleoprotein (RNP) complex formation. NUFIP1 is found in the nucleus in proximity to nascent RNA, and in the cytoplasm associated with ribosomes. In the cytoplasm, FMRP co-localizes and associates with Trailer Hitch (Tral) to form a translational repressor complex. The Tral complex contains a number of translational repressor proteins, which together control the initiation of translation and the stability of mRNAs, such as gurken (grk). In Drosophila, loss of Tral causes ovary phenotypes similar to piRNA pathway mutants, including oocyte polarity defects and transposon activation (Tan, 2016).

This study has identified Zfrp8 interactors by performing a yeast-two hybrid screen, and also by analyzing the components of the Zfrp8 complex by mass spectrometry. The nature of the proteins in the Zfrp8 complex indicates that it is involved in mRNA metabolism and translational regulation. Zfrp8, Nufip, FMRP, and Tral are all part of the complex, and Zfrp8 interacts antagonistically with Fmr1 and tral, suppressing their oogenesis defects. Furthermore, it was determined that Zfrp8 is required within the nucleus, and controls FMRP localization within the cytoplasm. It was further confirmed that FMRP functions in heterochromatin silencing and that Zfrp8 is required in the same process, but has an opposite function of FMRP. It is proposed that Zfrp8 functions as a chaperone of the FMRP-containing RNP translational repression complex and controls the temporal and spatial activity of this complex (Tan, 2016).

Zfrp8 is essential for stem cell maintenance, but its molecular functions have not yet been clearly defined (Minakhina, 2014). Two distinct approaches were taken to address this question. A yeast-two hybrid screen was performed to identify direct interactors of Zfrp8, and the components of the Zfrp8 complex were characterized by mass spectrometry (Tan, 2016).

Because of the high sequence and functional conservation of Zfrp8 (flies) and PDCD2 (mammals) (Minakhina, 2014), and because no stem cell-derived cDNA library exists in Drosophila, a mouse embryonic stem cell cDNA library was screened using mammalian PDCD2 as bait. Forty-six initial positives were isolated, and 19 potential interactors were identified after re-testing of the positives (Tan, 2016).

In order to purify the Zfrp8 protein complex a transgenic line was established expressing NTAP-tagged Zfrp8 under the control of the general da-Gal4 (daughterless) driver. Two-step tandem affinity purification was performed on embryonic extracts and the purified proteins were separated by SDS-PAGE electrophoresis. The proteins were eluted and analyzed by mass spectrometry. Thirty proteins were identified as part of the Zfrp8 complex. The threshold for interactors was set to at least 5x peptide enrichment in Zfrp8 over vector control fractions. Eighteen of the proteins are predicted to function in ribosomal assembly or translational regulation, strongly suggestive of a function of Zfrp8 in mRNA processing (i.e., translation, localization, and stability). In the complex six ribosomal subunits were found (five 40S subunits and one 60S subunit); EF2 and eIF-4a, which are required for translation initiation and elongation; and FMRP, Tral and Glorund which function in mRNA transport and translational repression. While Zfrp8 interacts with several ribosomal proteins it does not appear to be part of the ribosome itself (Tan, 2016).

No overlapping interactors were found in the yeast-two hybrid screen and mass spectrometry assay. But interestingly, FMRP was identified as part of the Zfrp8 complex by mass spectrometry and NUFIP1 in a yeast-two hybrid assay. Most likely Nufip (estimated 57 kD) was not identified as part of the Zfrp8 complex in the TAP-purification approach, because proteins with similar size to tagged Zfrp8 (~55 kD) were excluded from the mass spectrometry analysis. To investigate whether these proteins could work together in the same molecular process, the interaction of both Zfrp8 and PDCD2 with Nufip (flies) and NUFIP1 (mammals) was confirmed in tissue culture cells. Immunoprecipitation of human HEK293 cell extracts expressing FLAG-tagged NUFIP1 pulled down endogenous PDCD2. Next whether this protein interaction also exists in Drosophila was examined. It was possible to co-purify endogenous Zfrp8 with NTAP-tagged Nufip from transfected S2 cells. An additional Western blot was performed on the purified NTAP-Nufip isolate and it was shown that FMRP is present in the protein complex, indicating that Nufip physically interacts with both Zfrp8 and FMRP. These results suggest that all three proteins function together in a molecular complex which regulates RNP processing/assembly and translation. Based on these results, and the requirement of both Zfrp8 and Fmr1 in stem cell maintenance, it was decided to characterize the genetic interaction between these genes (Tan, 2016).

To further characterize the connection between the two genes, whether the loss of Zfrp8 can modify oogenesis defects reported for Fmr1 females. Similar to what was previously reported, 100% of Fmr1Δ50M/Df(3 R)Exel6265 and 80% of Fmr1Δ50M/Fmr13 ovaries displayed developmental defects. The ovarioles contained fused egg chambers, aberrant nurse cell numbers. Occasionally, egg chambers with oocyte misspecification/multiple oocytes were also observed. Interestingly, the loss of one copy of Zfrp8 suppressed the majority of Fmr1 ovary defects, restoring cell division in the germline, as well as egg chamber morphology and separation. In Zfrp8/+; Fmr1Δ50M/Df(3R)6265, fusion of the first egg chamber is still observed in most germaria, but despite this, oogenesis appears to proceed normally resulting in normal looking ovarioles. Zfrp8/+; Fmr1Δ50M/Fmr13 ovaries appear almost completely normal even though these ovarioles contain no FMRP (Tan, 2016).

The loss of Fmr1 has also been associated with a strong reduction in egg production. This study found that similar to previous reports, Fmr1Δ50M/Df(3R)Exel6265 and Fmr1Δ50M/Fmr13 mutants display a strong reduction in fertility; females laid on average of 1 and 6 eggs/day, respectively, as compared to 18 eggs/day for wild-type flies. The removal of one copy of Zfrp8 partially suppressed Fmr1 infertility and resulted in 8 eggs/day from Fmr1Δ50M/Df(3R)Exel6265 and 15 eggs/day from Fmr1Δ50M/Fmr13 females. These results demonstrate that Zfrp8 and Fmr1 affect the same process and that even though they are found in the same complex, have opposing functions (Tan, 2016).

To investigate the nature of the Zfrp8 interaction with FMRP, the localization of the proteins within the ovary was examined. Zfrp8 displays ubiquitous distribution in all cells and cell compartments of the wild type ovary. No significant changes in Zfrp8 localization or levels are visible in Fmr1 ovaries. FMRP has a more varied distribution pattern, present in strong, cytoplasmic puncta in the cytoplasm of nurse cells and follicle cells, and also in high levels in the cytoplasm of the maturing oocyte. FMRP is also detectable in low levels in nurse cell nuclei at stage 8 egg chambers at an average of 9.76 puncta per nucleus. As expected, Fmr1 ovaries display no FMRP staining in either the cytoplasm or nucleus (Tan, 2016).

To determine whether Zfrp8 functions in FMRP regulation, Zfrp8 was depleted in the germline by expressing Zfrp8 RNAi under the control of the nos-Gal4 driver (Minakhina, 2014), and changes in FMRP expression were assessed. In control nos-Gal4 ovaries, FMRP levels and distribution were similar to that in wild-type ovaries. However, in Zfrp8 KD ovaries, aberrant FMRP localization is observed in the germline; FMRP is more uniformly distributed throughout the cytoplasm and puncta are strongly diminished. Remaining FMRP puncta appear fragmented, reduced in intensity, size and number (~10% of wild-type). These results indicate a Zfrp8 requirement for proper FMRP localization to the cytoplasm. FMRP normally functions by shuttling mRNA cargo from the nucleus to the cytoplasm, where it represses the translation of bound mRNA. The observed change of FMRP localization in Zfrp8 KD ovaries therefore may indicate a regulatory function for Zfrp8 in the nuclear export and localization of FMRP (Tan, 2016).

Zfrp8 protein is present in both the cytoplasm and nucleus (Minakhina, 2014) and, as demonstrated above, controls the distribution of FMRP in the cytoplasm. It was decided to investigate the cell compartment in which Zfrp8 is required, in order to elucidate how Zfrp8 regulates FMRP. To do so, the capability of Zfrp8 deletion constructs to rescue mutant lethality was examined. Expression of human PDCD2 cDNAs driven by the general driver da-Gal4 is fully capable of rescuing Zfrp8 lethality (Barboza, 2013 and Minakhina, 2014). Mutated Zfrp8 constructs were created, removing either the two putative NLSs or the putative NES domains. These proteins were expressed under the da-Gal4 driver, and while clearly overexpressed on Western blots, failed to rescue mutant lethality, suggesting that the three domains are essential for the function of the protein (Tan, 2016).

In an alternative approach, the function of Zfrp8 proteins targeted to a distinct cell compartment was examined. Four N-terminal GFP-tagged transgenic proteins were expressed, encoding a wild-type Zfrp8, nuclear-localized NLS-Zfrp8, cytoplasmic-localized NES-Zfrp8, and cell membrane-localized CD8-GFP-Zfrp8. Transgenic Zfrp8 subcellular localization is visible when the proteins are strongly overexpressed. When the transgenes were expressed at lower levels, similar to the endogenous levels, with the hsp70-Gal4 driver at 25°C, both wild-type and nuclear-localized Zfrp8 were able to rescue mutant lethality at similar rates, whereas the cytoplasmic- and membrane-localized proteins did not show rescue. These results show that Zfrp8 is required in the nucleus and suggest that like FMRP, Zfrp8 may function by shuttling between nuclear and cytoplasmic compartments (Tan, 2016).

This study has shown that FMRP and Zfrp8 are present in the same protein complex. In addition to FMRP, the mass spectrometry results have also identified other translational regulators, such as Tral. Tral has previously been shown to function in conjunction with FMRP to control the translation of mRNAs (Tan, 2016).

To determine whether Zfrp8 functions in Tral/FMRP-associated translational regulation, the genetic interaction between Zfrp8 and tral was investigated. Tral regulates dorsal-ventral (D/V) patterning through the localization and translational control of gurken (grk) mRNA. Eggs laid by tral females display ventralized chorion phenotypes, due to the aberrant Gurken morphogen gradient. If Zfrp8 functions to regulate the translational activity of FMRP/Tral, a suppression of the tral ventralized phenotypes should be apparent when Zfrp8 is reduced. Tral was depleted in the germline by expressing a TRiP RNAi lineunder the control of the nos-Gal4 driver. Tral KD resulted in similar ventralized egg phenotypes as previously observed in eggs laid by tral1 females: 1% of eggs displayed two normal dorsal appendages (Wt), 36% had fused appendages, and 63% had no dorsal appendages. Removing one copy of Zfrp8 in the tral KD background suppressed the tral phenotypes. This genetic interaction suggests that in addition to controlling the localization of FMRP in the cytoplasm, Zfrp8 also influences the translational control by Tral, essential for formation of dorsal-ventral polarity in the egg (Tan, 2016).

Whether Zfrp8 regulates Tral localization as it does FMRP was investigated by examining the distribution of GFP-fusion Tral protein trap line. Tral protein was uniformly present in cytoplasmic compartments of germline and somatic cells, with stronger granules surrounding nuclei, and was highly enriched within the oocyte. Zfrp8 KD results in loss of oocyte identity (Minakhina, 2014), and the distribution of Tral was significantly altered in those cells. But in all other germline cells Tral distribution remained unaffected. Tral and its orthologs are cytoplasmic proteins and examination of the Tral protein sequence identifies no NLSs. Zfrp8 may therefore interact only indirectly with Tral and not regulate its localization (Tan, 2016).

Zfrp8 and Fmr1 control position effect variegation piRNA pathway genes have been shown to be essential for heterochromatin packaging in position effect variegation (PEV) experiments. PEV measures expression of endogenous or reporter genes inserted within or adjacent to heterochromatin. Fmr1 is specifically required for chromatin packaging as loss of a single copy of Fmr1 is sufficient to inhibit heterochromatin silencing of a white reporter inserted into the pericentric heterochromatin region 118E10 on the 4th chromosome (Tan, 2016).

PEV of Zfrp8 heterozygotes, Fmr1 heterozygotes and Fmr1, Zfrp8 transheterozygotes were examined using 118E10 (4th chromosome centromeric) and an additional white reporter, inserted into heterochromatin region 118E15 (4th chromosome telomeric). While the white+ reporters in Zfrp8null/+ eyes were expressed at levels comparable to those in wild-type controls, expression in Fmr1Δ50M/+ of both white reporters was strongly enhanced. But, the removal of one copy of both Zfrp8 and Fmr1 decreased expression of the reporters back to the Zfrp8/+, near wild-type levels, indicating restored heterochromatin silencing of both 4th chromosomal insertions. These findings suggest that in normal eyes, Zfrp8 functions upstream of Fmr1 and controls Fmr1 effects on heterochromatin packaging (Tan, 2016).

A connection between regulation of heterochromatin silencing and Piwi has clearly been established and the current results show that Zfrp8 and FMRP are part of the mechanism that controls heterochromatin silencing. Heterochromatin is established at the blastoderm stage in Drosophila embryos and is subsequently maintained throughout development. Thus, FMRP and Zfrp8 function together in heterochromatin packaging in the early embryo in the same way as they do during oogenesis (Tan, 2016).

This study has shown that Zfrp8 is part of a complex that is involved in RNA processing, i.e. translation, localization, and stability. It is proposed that Zfrp8 likely forms a ribonucleoprotein complex with Nufip, FMRP and select mRNAs in the nucleus, and is required for localization of this complex in the cytoplasm. After nuclear export, mRNAs within the complex are targeted for translational control and repression by FMRP and Tral. The suppression of the Fmr1 and tral phenotypes in a Zfrp8 heterozygous background, occurs in the absence of Fmr1 and the strong reduction of tral. This suggests that Zfrp8 function is not protein specific, but rather that it controls the FMRP and Tral-associated complex, even in the absence of each of the two proteins. This hypothesis is consistent with Zfrp8 actively controlling the localization of FMRP to cytoplasmic foci, as this localization is affected in Zfrp8 germ cells (Tan, 2016).

Previous studies identified a piRNA pathway protein, Maelstrom (Mael), that is controlled by Zfrp8 in a similar manner as FMRP. Zfrp8 forms a protein complex with Mael, genetically suppresses the loss of mael, and controls Mael localization to the nuage, a perinuclear structure (Minakhina, 2014). But the Zfrp8 phenotype is stronger and appears earlier than that of mael, tral, Fmr1, or other piRNA pathway regulatory genes studied so far. Zfrp8 may therefore control a central step in the regulation of specific RNPs. Consistent with this hypothesis, the TAP purification and mass spectrometry analysis identified a number of Zfrp8-associated proteins, the majority of which function in ribosomal assembly or translational regulation, such as the ribosomal protein RpS2. And Zfrp8 KD in the germ line and partial loss of rps2 result in a similar 'string of pearls phenotype', caused by developmental arrest in early stages of oogenesis. In addition, a recent study has shown that Zfrp8 and PDCD2 contain a TYPP (TSR4 in yeast, YwqG in E. coli, PDCD2 and PDCD2L in vertebrates and flies) domain, which has been suggested to perform a chaperone-like function in facilitating protein-protein interactions during RNA processing (Burroughs, 2014). These observations lead to a hypothesis that Zfrp8 functions as a chaperone essential for the assembly of ribosomes and the early recruitment and localization of ribosomal-associated regulatory proteins, such as FMRP, Tral and Mael (Tan, 2016).

Zfrp8 negatively controls the functions of Fmr1 and tral. In the absence of FMRP and Tral the temporal and spatial control of translation of their associated RNPs is lost. It is proposed that reducing the level of Zfrp8 diminishes the availability of these RNP-complexes in the cytoplasm resulting in suppression of the Fmr1 and tral phenotypes (Tan, 2016).

Zfrp8, Fmr1 and tral have all been shown to genetically and physically interact with components of the piRNA pathway, and to regulate the expression levels of select transposable elements. Transposon de-repression is often associated with the loss of heterochromatin silencing. The molecular mechanisms underlying heterochromatin formation appear to involve maternally contributed piRNAs and piRNA pathway proteins that control the setting of epigenetic marks in the form of histone modifications, maintained throughout development. But transposon expression can also be controlled post-transcriptionally by cytoplasmic PIWI-piRNA complexes, suggesting that transposon deregulation and heterochromatin silencing phenotypes seen in FMRP and Zfrp8 may be linked to translational de-repression. It is proposed that by facilitating the early assembly of ribosomes with specific translational repressors, Zfrp8 regulates several developmental processes during oogenesis and early embryogenesis including dorsal-ventral signaling, transposon de-repression, and position effect variegation (Tan, 2016).

Zfrp8/PDCD2 interacts with RpS2 connecting ribosome maturation and gene-specific translation

Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). This study shows that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins this study demonstrates that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. These results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation (Minakhina, 2016).

RpS2 is a component of the small ribosomal subunit and may bind Zfrp8/PDCD2 as an individual protein or as part of the subunit. Previous work has identified several 40S ribosomal proteins (RpS2 (uS5), RpS3 (uS3), RpS4 (eS4), RpS5a and RpS7 (eS7) as part of the Zfrp8 complex (Tan, 2016). Knock down of Zfrp8 also affects the cytoplasmic levels of several 40S components (RpS2 (uS5), RpS11 (uS17), and RpS13 (uS15)). Based on these data, it is proposed that Zfrp8/PDCD2 interacts with the small ribosomal subunit rather than with free RpS2. However, it is not clear if Zfrp8 interacts with the partially assembled or the mature 40S subunit. Interestingly, Zfrp8 KD does not affect the stability of all RpS proteins, for instance RpS18 (uS13) and RpS15 (uS19) remain unaffected by loss of Zfrp8, but then these ribosomal proteins may be more stable than others when not assembled into the subunit (Minakhina, 2016).

Like most ribosomal proteins, RpS2 is synthesized in the cytoplasm, transported into the nucleolus, where it is required for several steps of ribosome maturation: assembly of ribosomal proteins on pre-rRNA, pre-rRNA cleavage, nuclear export of the competent pre-40S subunit, its cytoplasmic maturation, interaction of the small subunit with mRNA and assembly into the ribosome. At each of these steps the stability of individual ribosomal proteins greatly increases, and Zfrp8/PDCD2 may control one or more of these steps. In normal ovaries, tagged RpS2 was undetectable in nuclei and nucleoli, but it showed visible nuclear accumulation in Zfrp8 KD(s) follicle cells. Furthermore, the relatively high nuclear and nucleolar levels of two other ribosomal proteins, RpS11 and RpS13, were not changed in Zfrp8 KD(s) cells, but the cytoplasmic levels of all three proteins were dramatically reduced. This suggests that Zfrp8/PDCD2 does not affect the synthesis of ribosomal proteins nor their nuclear import, but that it functions later, in ribosome assembly in the nucleolus, or nuclear export of the 40S subunit, or it may stabilize the small ribosomal subunit in the cytoplasm (Minakhina, 2016).

Because pre-rRNA cleavage and 18S rRNA trimming are essential steps in 40S maturation, whether Zfrp8 has an effect on rRNA processing was tested. No significant accumulation was observed of pre-rRNA precursors in Zfrp8 KD ovaries. This result is similar to what was observed in PDCD2 KO mouse embryonic fibroblasts, where pre-rRNAs were not increased. These results indicate that Zfrp8/PDCD2 functions after pre-40S assembly and pre-rRNA cleavage, possibly at the level of 40S subunit nuclear export or its final assembly into the mature ribosome in the cytoplasm (Minakhina, 2016).

The apparent reduction of 40S subunit components in the cytoplasm of Zfrp8 KD cells does not lead to a universal block of translation as many proteins are being produced at relatively normal levels. Further, Zfrp8 mutant and KD phenotypes as well as PDCD2 KO and KD in mouse and human cells show that Zfrp8/PDCD2 is essential in stem cells and highly proliferative cells, but has little or no function in differentiated cells and cells with low proliferative activity. Several explanations for these cell- or tissue-specific phenotypes are possible. First, it is argued that highly proliferative and stem cells, requiring high levels of protein synthesis, might be especially sensitive to levels of ribosomes. For instance, the decrease in ribosomal biogenesis may trigger premature stem cell differentiation. This phenotype, was observed in wcd mutants (wicked encodes U3 snoRNP associated protein) and may result not only from general abnormalities in rRNA maturation, but also from defects in asymmetric segregation of ribosomal biogenesis factors. Second, it may be that the lack of Zfrp8/PDCD2 may cause an imbalance of ribosomal proteins that are not assembled into subunits and this is detrimental to stem- and highly proliferative cells. In this context it is interesting to note that select unbound ribosomal proteins inhibit MDM2 E3 ligase activity, cause p53 stabilization, and cell cycle arrest. Therefore, imbalance of ribosomal proteins may explain the cell cycle arrest and marked increase of nuclear p53 observed in PDCD2 KO MEFs, ESCs, and embryonic blastocysts (Minakhina, 2016).

A third explanation could be that, Zfrp8/PDCD2 functions in regulating select transcripts essential in stem- and proliferating cells. Several TE transcripts and protein coding mRNAs were identified that are selectively regulated by Zfrp8 in the germ line (Minakhina, 2014). The subcellular localization of one of the TEs (TAHRE) was tested that is de-repressed in Zfrp8 KD ovaries, and TAHRE transcript levels were not only increased, but also showed uniform nuclear-cytoplasmic distribution in Zfrp8 KD ovaries. This effect is different from what is observed in the majority of piRNA pathway mutants, where TE (e.g. TAHRE) transcripts are predominantly seen in the cytoplasm. Therefore, it is proposed that Zfrp8 may facilitate export of TAHRE RNA from the nucleus as well as its targeting to the cytoplasmic sites of RNA processing and degradation. Similarly, Zfrp8 affects the transport of select protein coding mRNAs from the nuclei to proper cytoplasmic locations. The mRNA of two genes Pino and RpL36, showed visible nuclear accumulation in Zfrp8 KD ovaries. Pino also showed some increase in transcript level while RpL36 mRNA levels were unchanged. Tests were performed to see if nuclear accumulation of Pino and RpL36 transcripts was associated with inefficient splicing. No accumulation of non-spliced RNAs was detected, and it is therefore proposed that Zfrp8/PDCD2 regulates nuclear export and post-export localization of mature transcripts, steps occurring post splicing. Therefore, Zfrp8 may influence not only degradation but also the efficiency and the spatial control of their translation (Minakhina, 2016).

Zfrp8/PDCD2 is distributed in the cytoplasm and nuclei of most cells and while required in nuclei, may have an important function in both cellular compartments (Mu, 2010; Minakhina, 2014; Tan, 2016). Zfrp8/PDCD2 could control the formation of specialized RNA binding complexes or RNPs co-transcriptionally by recruiting different RNA binding proteins (NUFIP/FMRP, MAEL/PIWI, Hrb27C) and thereby regulate the fate of various transcripts (Minakhina, 2016).

In Zfrp8 KD cells, both mis-regulation of select transcripts and ribosomal abnormalities were observed. While it is possible that Zfrp8/PDCD2 influences assembly of the ribosome and transcript-specific RNPs independently, the hypothesis is favored that Zfrp8/PDCD2 provides a functional link between the transcripts and ribosomes. For instance, by assisting the formation of competent transcript-specific-RNPs in the nucleus, Zfrp8 may prevent their premature binding to pre-40S and translation. Recent studies suggest that the pre-40S/40S subunit may be able to bind mRNA and initiate translation in the nucleus. However, the vast majority of protein synthesis happens in the cytoplasm, and eukaryotic cells utilize check point mechanisms to prevent binding of immature subunits to mRNA to avoid unnecessary nuclear translation. If Zfrp8/PDCD2 is part of such a check point mechanism, lack of Zfrp8 in the nucleus may cause improper binding between pre-40S and transcript-specific RNPs or mRNAs, and therefore disrupt their nuclear export. In addition, in the cytoplasm, Zfrp8/PDCD2 may stimulate the binding between RNPs and 40S subunits, facilitating final ribosome assembly, and thereby, stabilize ribosomal proteins, and ensure translation of select transcripts. This step in the control of gene expression is not well studied. Because Zfrp8 is specifically required in stem and rapidly dividing cells, such as cancer cells, these results further confirm the cell type specificity of RNA processing and ribosomal biogenesis. Much additional work will be necessary to understand how these specific mechanisms are linked to cell fate (Minakhina, 2016).

Zfrp8/PDCD2 is required in ovarian stem cells and interacts with the piRNA pathway machinery

The maintenance of stem cells is central to generating diverse cell populations in many tissues throughout the life of an animal. Elucidating the mechanisms involved in how stem cells are formed and maintained is crucial to understanding both normal developmental processes and the growth of many cancers. Previous studies have shown that Zfrp8/PDCD2 is essential for the maintenance of Drosophila hematopoietic stem cells. This study shows that Zfrp8/PDCD2 is also required in both germline and follicle stem cells in the Drosophila ovary. Expression of human PDCD2 fully rescues the Zfrp8 phenotype, underlining the functional conservation of Zfrp8/PDCD2. The piRNA pathway is essential in early oogenesis, and this study found that nuclear localization of Zfrp8 in germline stem cells and their offspring is regulated by some piRNA pathway genes. Zfrp8 forms a complex with the piRNA pathway protein Maelstrom and controls the accumulation of Maelstrom in the nuage. Furthermore, Zfrp8 regulates the activity of specific transposable elements also controlled by Maelstrom and Piwi. These results suggest that Zfrp8/PDCD2 is not an integral member of the piRNA pathway, but has an overlapping function, possibly competing with Maelstrom and Piwi (Minakhina, 2014).

Studies on Zfrp8 requirement in the Drosophila ovary show that the gene is essential in stem cells. The results suggest that Zfrp8 is not required in cells with limited developmental potential, as transient wild-type and mutant clones were similar in number and size. No difference was found in Zfrp8 and wild-type escort cell clones, indicating that Zfrp8 is not required in these cells that multiply by self-duplication. Furthermore, Zfrp8 and wild-type MARCM clones induced in third instar larvae were indistinguishable in the adult antenna and legs 20 days ACI. These results support the conclusion that Zfrp8 function is primarily required in stem cells (Minakhina, 2014).

Despite this functional requirement, Zfrp8 protein was not enriched in Drosophila GSCs and FSCs. This is surprising, because in mice Zfrp8/PDCD2 is enriched in several types of stem cell. Zfrp8/PDCD2 is also highly expressed in human bone marrow and cord blood stem and precursor cells with protein levels decreasing significantly as these cells differentiate (Minakhina, 2014).

This study observed that loss of Zfrp8 in the Drosophila germline did not affect signaling from the niche to the stem cells. But the stem cells themselves are highly sensitive to loss of Zfrp8. In both Zfrp8 germline stem cell clones and Zfr8 KD germaria abnormal spectrosomes were observed reminiscent of fusomes. These phenotypes suggest that these germline stem cells are losing stem identity and show features of a stem cell and a more advanced cystocyte. Germline and somatic stem cells and their daughter cells ultimately stop dividing when depleted of Zfrp8 but continue to survive for several days, as evident from the phenotype of the persistent stem cell clones. Similarly, in leukemia and in cancer cell lines that initially have high levels of the protein, reduction of Zfrp8/PDCD2 correlates with delay or arrest of the cell cycle rather than cell death (Minakhina, 2014).

The most severe abnormalities were observed 10-20 days ACI in Zfrp8 GSC clones induced in larvae and adults. The phenotype of Zfrp8 KD ovarioles also became more pronounced with age, starting from a relatively normal-looking germarium and a few egg chambers in young flies, to ovarioles made up of disorganized cysts, and finally, to ovarioles in which germ cells were almost entirely absent. The temporal change in phenotype can be explained in two ways. First, it is possible that Zfrp8 levels are initially high enough in mutant and KD stem cells to support a few divisions and the formation of mutant cysts. However, as Zfrp8 is gradually depleted the cells stop dividing and are eventually lost. Alternatively, lack of Zfrp8 may induce changes in parental cells that affect the developmental potential of the daughter cells. For instance, chromatin modifications could be affected in the absence of Zfrp8, but it could take several cell generations for these changes to have a phenotypic effect. In both these scenarios, loss of Zfrp8 would predominantly affect cells undergoing constant or rapid divisions, such as stem cells and cancer cells (Minakhina, 2014).

The loss of asymmetry in the stem cells, the mislocalization of BicD and Orb proteins to and within the oocyte, the mislocalization of Zfrp8 protein in GSCs of several piRNA pathway mutants, and the genetic interaction of Zfrp8 with piRNA pathway genes suggested a connection between Zfrp8 and the piRNA pathway. The de-repression of the subset of transposons in Zfrp8 KD ovaries further links the gene with the piRNA pathway (Minakhina, 2014).

This study tested several LTR and non-LTR retroelements that represent three major TE classes based on their tissue-specific activity in the germline, soma or in both tissues (intermediate). When Zfrp8 is depleted in the germline, two out of seven intermediate and germline elements tested, HeT-A and TART, show significant de-repression. These elements are different from the majority of Drosophila TEs. The HeT-A, TART and the TAHRE elements are integral components of fly telomere. Their activity is tightly regulated and is required to protect chromosome ends. These elements, like other TEs, are controlled by the piRNA machinery, but their primary piRNAs are likely to be derived from the same telomeric loci that are also their targets for repression. By contrast, the majority of primary piRNAs are derived from piRNA clusters and target TEs dispersed throughout the genome. Furthermore, the repression of TART and HeT-A in the germline involves an unusual combination of piRNA factors. We found that at early stages of oogenesis they appear to be regulated by piwi and mael, but not by the germline-specific Piwi family member Aub. This result is in agreement with recent studies on piwi function in the soma and germline that showed that HeT-A and TART elements are among the TEs most strongly regulated by Piwi in the germline. Thus, Zfrp8 may target the same TEs as Piwi and Mael but not those regulated by Aub (Minakhina, 2014).

De-repression of TEs caused by Zfrp8 KD could be responsible for the enhancement of developmental defects seen in piRNA pathway mutants. For instance, the increase of TE transcripts may enhance dorsoventral patterning defects in armi, AGO3, aub, spnE and vas because of the competition between TE transcripts and oocyte polarity factors for the same RNA transport machinery. However, the interaction of Zfrp8 with the piRNA pathway machinery seems to be more complex. Zfrp8 enhanced the egg phenotype of only three mutants, spnE, AGO3 and vas, and in these mutants the nuclear localization of Zfrp8 protein was also affected. These results suggest that Zfrp8 functions downstream of the three factors (Minakhina, 2014).

Both piwi and mael are dominantly suppressed by Zfrp8. Both these factors have important nuclear functions, regulating chromatin modifications and controlling TEs at the transcriptional level, and both are required to repress HeT-A and TART-A elements. Zfrp8 could suppress piwi or mael by inducing a competing chromatin modification at the genomic loci targeted by Piwi or Mael. Chromatin modifications are generally stable through several cell generations. Such a function would therefore be consistent with the temporal changes of phenotypes in Zfrp8 ovarian clones and KD ovarioles (Minakhina, 2014).

Although Piwi and Mael target the same genomic loci, no interaction between the two proteins have been detected. This study's co-immunoprecipitation experiments suggest that Zfrp8 complexes with Mael but not with Piwi, indicating that the observed genetic interaction between Zfrp8 and piwi may be mediated by mael. Mael is one of the most enigmatic proteins in the piRNA pathway. It is found in the cytoplasm, nuage and nucleus, and has been implicated in diverse cellular processes including the ping-pong piRNA amplification cycle in the germline, MTOC assembley in the oocyte and Piwi-dependent chromatin modification in somatic cells. Zfrp8/PDCD2 is also required in the soma and germline and may function both in the cytoplasm and in nuclei. However, in contrast to mael, Zfrp8 homozygous mutants are lethal and Zfrp8 ovaries show a stronger phenotype. Based on the observation that Mael and Zfrp8 are found in the same complex and that Zfrp8 dominantly suppresses Mael, we propose that they act in opposite fashion on a common target, whether during piRNA biogenesis or chromatin modifications (Minakhina, 2014).

Hematopoietic stem cells in Drosophila

The Drosophila lymph gland, the source of adult hemocytes, flanks the dorsal vessel and is established by mid-embryogenesis. During larval stages, a pool of pluripotent hemocyte precursors differentiate into hemocytes that are released into circulation upon metamorphosis or in respond to immune challenge. This process is controlled by the posterior signaling center (PSC), which is reminiscent of the vertebrate hematopoietic stem cell niche. Using lineage analysis, bona fide hematopoietic stem cells (HSCs) were identified in the lymph glands of embryos and young larvae, which give rise to a hematopoietic lineage. These lymph glands also contain pluripotent precursor cells that undergo a limited number of mitotic divisions and differentiate. It was further found that the conserved factor Zfrp8/PDCD2 (Minakhina, 2007) is essential for the maintenance of the HSCs, but dispensable for their daughter cells, the pluripotent precursors. Zfrp8/PDCD2 is likely to have similar functions in hematopoietic stem cell maintenance in vertebrates (Minakhina, 2010).

Drosophila blood cell development occurs in two phases. In the first, 'primitive' phase, hemocytes develop from the early embryo head mesoderm and supply the pool of circulating blood cells. The second phase gives rise to adult hemocytes, produced in a small organ, the lymph gland. The larval lymph gland and the differentiation of hemocytes have been studied using a range of cell-specific markers. The primary, largest lobe of the larval lymph gland is sub-divided into the posterior signaling center (PSC), the medullary zone (MZ) and the cortical zone (CZ) (see illustration in Jung, 2005). The MZ has been thought to contain a relatively uniform population of pluripotent prohemocytes (PH), sometimes called stem-like cells. These cells migrate into the CZ as they differentiate into plasmatocytes (PM), crystal cells (CC) and lamellocytes (LM). The homeostasis between prohemocytes and differentiated blood cells is maintained by the PSC (Minakhina, 2010 and references therein).

To investigate the existence of stem cells in the Drosophila lymph gland, clones were induced in embryos and first instar larvae, using the MARCM technique combined with UAS-GFP reporters. This technique results in marking a single cell and its progeny, and revealed that in wild-type lymph glands both persistent and transient clones are induced, indicating the presence of hematopoietic stem cells (Minakhina, 2010).

Because stem cells usually represent only a small fraction of the cells in an organ, they are difficult to identify and study. The MARCM technique was chosen because it marks cells undergoing mitosis, such as stem cells, which are particularly active in young animals. Clones were produced at four embryonic stages, 2-6 hours, 6-12 hours, 12-18 hours and 18-24 hours, and in first instar larvae, by exposing animals to 38oC for one hour to activate the heat-inducible FLP-recombinase. 2-6 hour embryos contain about nine precursor cells that will form one lymph gland lobe and the cardioblasts. In 6-12 hour embryos, a lymph gland lobe contains about 12 cells, and at stage 16, 20-25 cells. By late first instar larval stage cells have undergone, on average, one additional division. In the absence of infection, hemocytes remain in the lymph gland until metamorphosis when they are released into circulation. This aspect of hemocyte development allowed wild-type and Zfrp8 clones to be followed from the embryo to the third instar larval stage by noting the distribution of marked cells in the lymph glands. As expected, wild-type and mutant PSC clones were obtained with similar frequency (about 6%-9%) after induction between 6-18 hours. They had comparable phenotypes and did not mix with non-PSC hemocytes (Minakhina, 2010).

Except for the cells in the PSC, the hemocyte precursors within the embryonic lymph glands appear identical and were therefore expected to have similar lineage potential, and to produce clones of comparable size and appearance. However, a large variety of non-PSC clones were recovered that were subdivided into four types according to their size, shape and location. Type 1 are large clones encompassing 10-30% of all of the lymph gland cells that form cohesive clusters. They occupy a large part of the medulla and extend into the cortex, where they scatter into secondary small clusters. All ten type 1 clones that were stained with the PSC marker Antp contained cells, probably the founder cells, and were in immediate contact with the PSC. The frequency of type 1 clones remained about the same (18-29%) independent of when they were induced during embryogenesis. But their frequency was strongly reduced when the clones were induced in first instar larvae (Minakhina, 2010).

Type 1 clones showed the characteristics of 'persistent; clones that are expected when the clone is induced in HSCs or their precursors (primordial cells). Founder cells in these clones were in contact with the PSC hematopoietic niche, they could self-renew and were pluripotent, meaning that they could differentiate into plasmatocytes, crystal cells and probably lamellocytes (there are too few lamellocytes in a normal lymph gland to establish this positively). By contrast, type 3 and 4 clones clearly arose from cells that have no self-renewal properties, cells that divide, migrate into the cortex, and differentiate. Because these cells are gradually removed from the medulla, type 3 and 4 clones are considered to be 'transient'. The types of clones obtained are consistent with the existence of stem cells that can self-renew and replenish the population of pluripotent hemocyte precursors, while their daughter cells divide several times and commit to differentiation. The four types of clones also indicate that the hematopoietic lineage contains at least three developmental stages in addition to the stem cells. All persistent and most transient clones consisted of one or several contiguous patches and scattered cells, indicating that cell mixing was prevalent, especially when cells moved into the cortex (Minakhina, 2010).

All four types of clones were observed in wild-type glands, independently of when the clones were induced, suggesting that already at the earliest embryonic stage the lymph gland cells have different developmental potentials, and that all cell types persist at least through the first larval instar. These observations suggest that some of the primordial cells do not form stem cells but undergo differentiation similar to what is observed in the ovary, where some prestem cells fail to form stem cells and instead undergo differentiation. The proportion of type 1 clones was significantly lower in first instar larvae than in early embyos, indicating that the number of stem cells stays relatively constant while their daughter cells multiply. Stem cells are likely to be present still in later larval stages, but they would be difficult to detect because of their relatively low numbers and because their mitotic activity may be reduced. Furthermore, if clones were induced in second and third instar larvae, the short time between the induction of the clones and their analysis would not be sufficient to see a clear difference beween persistent and transient clones (Minakhina, 2010).

The results show that embryonic and first instar larval lymph glands contained HSCs (type 1 clones), transient pluripotent progenitors (type 2 and 3 clones), and cells with limited mitotic potential (type 4). The presence of HSCs in wild-type glands was further validated by the fact that these cells were lost in the absence of Zfrp8 (Minakhina, 2010).

Zfrp8 (Minakhina, 2007), also called PDCD2, is highly conserved from flies to humans, and its molecular and physiological function is generally not well understood. Loss of Zfrp8 causes a unique phenotype in Drosophila. The lymph gland is enlarged already in mid-embryogenesis and by the late third instar larval stage, the lymph gland size is increased 10 to 50 times, accompanied by lamellocyte overproliferation (Minakhina, 2010).

To study the function of Zfrp8 throughout hematopoiesis, GFP-labeled homozygous mutant Zfrp8 clones were induced in Zfrp8 heterozygous animals. Analysis of the Zfrp8 mutant lymph gland clones showed that their occurrence differed remarkably from that of wild type. The most striking result was that no type 1 (HSC) clones were detected. The percentage of type 2 clones was reduced, whereas that of type 3 and 4 clones was increased, especially when induced in young embryos. The percentage of mosaic animals with no lymph gland clones was double that of wild type (Minakhina, 2010).

In spite of this shift, the phenotypes of type 2, 3 and 4 clones were indistinguishable from that of wild type. Lack of Zfrp8 did not result in hemocyte or lamellocyte overproliferation within the clone. The pluripotency of Zfrp8 mutant prohemocytes was the same as that of wild-type cells, indicating that Zfrp8 is not required in cells that give rise to transient clones. A similar result was found in other tissues where the clonal loss of Zfrp8 resulted in cells that looked indistinguishable from their wild-type neighbors. Cell proliferation, viability or differentiation was not affected (Minakhina, 2010).

The absence of persistent clones, the decrease of animals with clones in the lymph gland, the increase of type 3 and 4 clones in young animals, and the absence of a phenotype within the clones, all suggest that Zfrp8 is required specifically in stem cells. Stem cells lacking Zfrp8 loose their ability to self-renew and instead behave like more mature prohemocytes (Minakhina, 2010).

To ascertain whether the Zfrp8 mutant phenotype was consistent with the loss of HSCs, mutant lymph gland growth and hemocyte differentiation were examined during several stages of larval development. Peroxidasin (Pxn) is an early cortex marker expressed in cells committed to differentiation (see Jung, 2005). As in wild type, in second instar mutant glands Pxn-negative cells were detected in the medulla and positive cells in the cortex, indicating that these Zfrp8 mutant glands contain hemocyte precursors and prohemocytes. But in early third instar mutant larvae, all lymph gland cells had become Pxn-positive, indicating that all hemocyte precursor cells, normally present in the medulla, had matured. The absence of hemocyte precursors is consistent with the finding that HSCs, which would replenish this hemocyte population throughout development, were missing in Zfrp8 mutant lymph glands. Thus, the lack of Zfrp8 explains the absence of HSCs and the subsequent loss of the medulla. Larvae without a PSC also lack medulla. The overlap of these two phenotypes is consistent with the PSC controlling the development of the HSCs. Conversely, the massive Zfrp8 mutant hemocyte overgrowth was not seen in animals without a PSC, which indicates the existence of an additional signal, possibly also originating in the PSC, that controls hemocyte proliferation and differentiation (Minakhina, 2010).

This study has found evidence for a Drosophila hematopoietic lineage established by a stem cell and, further, that the identity of the HCS is dependent on the function of Zfrp8. It is possible that the Zfrp8 human homolog, the PDCD2 protein, has a similar function. PDCD2 is more highly expressed in a CD34+ bone marrow fraction, enriched in HSCs, than in a sample of total bone marrow cells. Consistent with this observation, transcriptional profiling of mouse embryonic, neural and hematopoietic stem cells showed an enrichment of PDCD2 mRNA in all three stem cells (Ramalho-Santos, 2002; Minakhina, 2010).

Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation

This study has identified a new gene, Zfrp8, as being essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of gamma-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Gamma-tubulin ring protein 91 (Dgrip91), which is involved in anchoring gamma-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, it is proposed that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation (Minakhina, 2007).

An early study found that the Zfrp8 vertebrate ortholog, PDCD2, is routinely referred to as an apoptotic gene solely because it was upregulated during steroid-induced programmed cell death in rat thymocytes. Subsequent studies, using different cells and assay conditions, found no connection between PDCD2 expression and programmed cell death. It is unlikely that a reduction in cell death is the cause of the lymph gland overgrowth observed in Zfrp8 mutant larvae. Very few or no apoptotic cells are detected in wild-type larval lymph glands. A statistically insignificant increase was found in the number of apoptotic cells in Zfrp8 mutants. No other evidence was found of change in programmed cell death in Zfrp8 mutant animals, there was no increase in apoptotic gene expression, no change in caspase cleavage and no genetic interaction of Zfrp8 with known apoptotic genes (Minakhina, 2007).

The results are consistent with an increase in cell division in Zfrp8 mutants throughout development. This conclusion is supported by the observation that Zfrp8 lymph glands are already twice the size of their normal counterparts in late-stage embryos, and that the number of cells in mitosis is about 30% higher in the mutant glands than in wild type (Minakhina, 2007).

Detailed analysis of Zfrp8 lymph glands shows that its phenotype is different from that of Drosophila hematopoietic/immunity mutants. Unlike hematopoietic/immunity mutants, the increase in lymph gland cell numbers is much larger than the increase in circulating hemocytes. Furthermore, the blood cell overproliferation in Zfrp8-null mutants is not accompanied by constitutive activation of immunity. Zfrp8 larvae show normal induction of immune peptide genes in response to bacterial challenge and normal wound clogging and wound melanization. That the requirements are different for Zfrp8 and known hematopoiesis and immunity genes is underlined by the absence of their genetic interaction (and see Table S1 in Minakhina, 2007).

In normal lymph glands, plasmatocytes are found mostly in the cortical region and very few lamellocytes are detected. The posterior signaling center (PSC) is formed at the base of each primary lobe. The presence of additional PSCs in mutant lymph glands might indicate that additional primary lobes are formed by the large number of cells (Minakhina, 2007).

Two recent papers report that the PSCs are essential for maintaining the undifferentiated hemocyte population in the medullary zone and that they control lamellocyte differentiation during parasitic infection (Krzemien, 2007; Mandal, 2007). Lack of the transcription factor collier, essential for PSC maintenance, leads to a decrease in the pro-hemocyte population and abolishes lamellocyte differentiation. Loss of Zfrp8 leads to the opposite phenotype: an increase in pro-hemocyte proliferation, beginning during embryogenesis, and an increased number of cells acquiring the lamellocyte fate. Expansion of the PSCs alone does not account for this phenotype. Ectopic expression of the homeotic gene Antennapedia results in expansion of the PSCs, and a concomitant increase of the medullar zone, but not the gland overgrowth (Mandal, 2007). Therefore, it is unlikely that Zfrp8 is directly involved in the establishment of PSCs (Minakhina, 2007).

The results link the Zfrp8 overgrowth phenotype to a defect in normal cell proliferation. In mutant lymph glands, the cell-cycle markers γ-Tubulin and CycB are misregulated. Zfrp8 genetically interacts with at least two genes functioning in the cell cycle, Cdc27 encoding a subunit of the anaphase-promoting complex (APC), and l(1)dd4 encoding the Drosophila gamma-ring protein Dgrip91 (Minakhina, 2007).

Dgrip91 and γ-Tubulin are components of the γ-TuRC microtubule-nucleating complex anchored to centrosomes. Beyond the conventional role in microtubule organization, centrosomes also serve as a scaffold for anchoring a number of cell-cycle regulators. For instance, centrosome-association of Cdc27 and CycB proteins plays an important role in CycB activation, degradation and entrance into mitosis (Minakhina, 2007).

The link between the phenotypes described above and Zfrp8 function became clear when it was discovered that a proportion of Zfrp8 protein localizes adjacent to the centrosome in wild-type tissue. This subcellular localization is consistent with a function of Zfrp8 in centrosome organization and in the anchoring of proteins such as γ-Tubulin and CycB to this organelle (Minakhina, 2007).

Zfrp8 might also affect the expression of bona fide cell-cycle regulators. The protein contains a zinc-finger domain, MYND, present in a number of transcriptional regulators, that fosters protein-protein interactions and recruits co-repressors. PDCD2/Zfrp8 is known to interact with the HCF-1 transcriptional regulator, which suggests that PDCD2/Zfrp8 might be involved in regulating the cell cycle at the transcriptional level (Minakhina, 2007).

Zfrp8 might have a dual function, through its association with the centrosome and as a transcriptional regulator of the cell cycle. Several transcriptional regulators have been found to localize to the centrosome, but their centrosomal function has not been documented (Minakhina, 2007).

Zfrp8 function is essential for the control of cell proliferation already in the embryo. With this being the case, it functions upstream from most of the conserved signaling pathways involved in fly hematopoiesis and immunity. Because of the similarity of the protein in flies and vertebrates, it is possible that PDCD2 has a similar function in vertebrate hematopoiesis (Minakhina, 2007).


REFERENCES

Search PubMed for articles about Drosophila Zfrp8

Barboza, N., Minakhina, S., Medina, D. J., Balsara, B., Greenwood, S., Huzzy, L., Rabson, A. B., Steward, R. and Schaar, D. G. (2013). PDCD2 functions in cancer cell proliferation and predicts relapsed leukemia. Cancer Biol Ther 14: 546-555. PubMed ID: 23760497

Burroughs, A. M. and Aravind, L. (2014). Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis. Front Genet 5: 424. PubMed ID: 25566315

Chen, E., Sharma, M. R., Shi, X., Agrawal, R. K. and Joseph, S. (2014). Fragile X mental retardation protein regulates translation by binding directly to the ribosome. Mol Cell 54: 407-417. PubMed ID: 24746697

Granier, C. J., Wang, W., Tsang, T., Steward, R., Sabaawy, H. E., Bhaumik, M. and Rabson, A. B. (2014). Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest. Biol Open 3: 821-831. PubMed ID: 25150276

Krzemien, J., Dubois, L., Makki, R., Meister, M., Vincent, A. and Crozatier, M. (2007). Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre. Nature 446: 325-328. PubMed ID: 17361184

Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V. and Banerjee, U. (2007). A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446: 320-324. PubMed ID: 17361183

Minakhina, S., Druzhinina, M. and Steward, R. (2007). Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation. Development 134: 2387-2396. PubMed ID: 17522156

Minakhina, S., Changela, N. and Steward, R. (2014). Zfrp8/PDCD2 is required in ovarian stem cells and interacts with the piRNA pathway machinery. Development 141: 259-268. PubMed ID: 24381196

Minakhina, S., Naryshkina, T., Changela, N., Tan, W. and Steward, R. (2016). Zfrp8/PDCD2 Interacts with RpS2 Connecting Ribosome Maturation and Gene-Specific Translation. PLoS One 11: e0147631. PubMed ID: 26807849

Mu, W., Munroe, R. J., Barker, A. K. and Schimenti, J. C. (2010). PDCD2 is essential for inner cell mass development and embryonic stem cell maintenance. Dev Biol 347: 279-288. PubMed ID: 20813103

Ramalho-Santos M., Yoon S., Matsuzaki Y., Mulligan R. C. and Melton D. A. (2002). 'Stemness': transcriptional profiling of embryonic and adult stem cells. Science 298: 597-600. PubMed ID: 12228720

Taha, M. S., Nouri, K., Milroy, L. G., Moll, J. M., Herrmann, C., Brunsveld, L., Piekorz, R. P. and Ahmadian, M. R. (2014). Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP) with nucleolin. PLoS One 9: e91465. PubMed ID: 24658146

Tan, W., Schauder, C., Naryshkina, T., Minakhina, S. and Steward, R. (2016). Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function. Dev Biol. 410(2):202-12. PubMed ID: 26772998


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date revised: 3 April 2016

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