Drosophila Importin Alpha 1 (Dα1) Is Required to Maintain Germline Stem Cells in the Testis Niche.

Stem cell maintenance and differentiation can be regulated via the differential activity of transcription factors within stem cells and their progeny. For these factors to be active, they need to be transported from their site of synthesis in the cytoplasm into the nucleus. A tissue-specific requirement for factors involved in nuclear importation is a potential mechanism to regulate stem cell differentiation. We have undertaken a characterization of male sterile importin alpha 1 (Dα1) null alleles in Drosophila and found that Dα1 is required for maintaining germline stem cells (GSCs) in the testis niche. The loss of GSCs can be rescued by ectopic expression of Dα1 within the germline but the animals are still infertile, indicating a second role for Dα1 in spermatogenesis. Expression of a Dα1 dominant negative transgene in GSCs confirmed a functional requirement for Dα1 in GSC maintenance but expression of the transgene in differentiating spermatogonia did not exhibit a phenotype indicating a specific role for Dα1 within GSCs. Our data indicate that Dα1 is utilized as a regulatory protein within GSCs to facilitate nuclear importation of proteins that maintain the stem cell pool.

Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects.

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Deficiency of the mitochondrial ribosomal subunit, MRPL50, causes autosomal recessive syndromic premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.

MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function.

The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, we show that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.

RNA binding protein Musashi-2 regulates PIWIL1 and TBX1 in mouse spermatogenesis.

RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi-2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis-expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post-transcriptional regulation during male gamete development.

Rbf Regulates Drosophila Spermatogenesis via Control of Somatic Stem and Progenitor Cell Fate in the Larval Testis.

The Drosophila testis has been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. Here, we report the identification of two independent alleles for the highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third-instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. We demonstrate that Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together our observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage.

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis.

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development.

RNA binding proteins in spermatogenesis: an in depth focus on the Musashi family.

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the complex process of mammalian spermatogenesis, male germ cells experience extended periods of the inactive transcription despite heavy translational requirements for continued growth and differentiation. Hence, spermatogenesis is highly reliant on mechanisms of posttranscriptional regulation of gene expression, facilitated by RNA binding proteins (RBPs), which remain abundantly expressed throughout this process. One such group of proteins is the Musashi family, previously identified as critical regulators of testis germ cell development and meiosis in Drosophila, and also shown to be vital to sperm development and reproductive potential in the mouse. This review describes the role and function of RBPs within the scope of male germ cell development, focusing on our recent knowledge of the Musashi proteins in spermatogenesis. The functional mechanisms utilized by RBPs within the cell are outlined in depth, and the significance of sub-cellular localization and stage-specific expression in relation to the mode and impact of posttranscriptional regulation is also highlighted. We emphasize the historical role of the Musashi family of RBPs in stem cell function and cell fate determination, as originally characterized in Drosophila and Xenopus, and conclude with our current understanding of the differential roles and functions of the mammalian Musashi proteins, Musashi-1 and Musashi-2, with a primary focus on our findings in spermatogenesis. This review highlights both the essential contribution of RBPs to posttranscriptional regulation and the importance of the Musashi family as master regulators of male gamete development.

RNA binding protein Musashi-1 directly targets Msi2 and Erh during early testis germ cell development and interacts with IPO5 upon translocation to the nucleus.

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in Drosophila and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic Msi1 testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, Msi2 and Erh, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function.

Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster.

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, we show that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. We propose that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.

Developmental expression of Musashi-1 and Musashi-2 RNA-binding proteins during spermatogenesis: analysis of the deleterious effects of dysregulated expression.

Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

The Musashi family of RNA binding proteins: master regulators of multiple stem cell populations.

In order to maintain their unlimited capacity to divide, stem cells require controlled temporal and spatial protein expression. The Musashi family of RNA-binding proteins have been shown to exhibit this necessary translational control through both repression and activation in order to regulate multiple stem cell populations. This chapter looks in depth at the initial discovery and characterisation of Musashi in the model organism Drosophila, and its subsequent emergence as a master regulator in a number of stem cell populations. Furthermore the unique roles for mammalian Musashi-1 and Musashi-2 in different stem cell types are correlated with the perceived diagnostic power of Musashi expression in specific stem cell derived oncologies. In particular the potential role for Musashi in the identification and treatment of human cancer is considered, with a focus on the role of Musashi-2 in leukaemia. Finally, the manipulation of Musashi expression is proposed as a potential avenue towards the targeted treatment of specific aggressive stem cell cancers.

Drosophila Rbp6 is an orthologue of vertebrate Msi-1 and Msi-2, but does not function redundantly with dMsi to regulate germline stem cell behaviour.

The vertebrate RNA-binding proteins, Musashi-1 (Msi-1) and Musashi-2 (Msi-2) are expressed in multiple stem cell populations. A role for Musashi proteins in preventing stem cell differentiation has been suggested from genetic analysis of the Drosophila family member, dMsi, and both vertebrate Msi proteins function co-operatively to regulate neural stem cell behaviour. Here we have identified a second Drosophila Msi family member, Rbp6, which shares more amino acid identity with vertebrate Msi-1 and Msi-2 than dMsi. We generated an antibody that detects most Rbp6 splice isoforms and show that Rbp6 is expressed in multiple tissues throughout development. However, Rbp6 deletion mutants generated in this study are viable and fertile, and show only minor defects. We used Drosophila spermatogonial germline stem cells (GSC's) as a model to test whether Drosophila Msi proteins function redundantly to regulate stem cell behaviour. However, like vertebrate Msi-1 and Msi-2, Rbp6 and Msi do not appear to be co-expressed in spermatogenic GSC's and do not function co-operatively in the regulation of GSC maintenance. Thus while two Msi family members are present in Drosophila, the function of the family members have diverged.

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes.

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4-encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline-specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis.

Akap200 suppresses the effects of Dv-cbl expression in the Drosophila eye.

The Drosophila melanogaster orthologue of the c-Cbl proto-oncogene acts to downregulate signalling from receptor tyrosine kinases by enhancing endocytosis of activated receptors. Expression of an analogue of the C-terminally truncated v-Cbl oncogene, Dv-cbl, in the developing Drosophila eye conversely leads to excess signalling and disruption to the well-ordered adult compound eye. Co-expression of activated Ras with Dv-cbl leads to a severe disruption of eye development. We have used a transposon-based inducible expression system to screen for molecules that can suppress the Dv-cbl phenotype and have identified an allele that upregulates the A-kinase anchoring protein, Akap200. Overexpression of Akap200 not only suppresses the phenotype caused by Dv-cbl expression, but also the severe disruption to eye development caused by the combined expression of Dv-cbl and activated Ras. Akap200 is also endogenously expressed in the developing Drosophila eye at a level that modulates the effects of excessive signalling caused by expression of Dv-cbl.

Differential roles of HOW in male and female Drosophila germline differentiation.

The adult gonads in both male and female Drosophila melanogaster produce gametes that originate from a regenerative pool of germline stem cells (GSCs). The differentiation programme that produces gametes must be co-ordinated with GSC maintenance and proliferation in order to regulate tissue regeneration. The HOW RNA-binding protein has been shown to maintain mitotic progression of male GSCs and their daughters by maintenance of Cyclin B expression as well as suppressing accumulation of the differentiation factor Bam. Loss of HOW function in the male germline results in loss of GSCs due to a delay in G2 and subsequent apoptosis. Here we show that female how mutant GSCs do not have any cell cycle defects although HOW continues to bind bam mRNA and suppress Bam expression. The role of HOW in suppressing germ cell Bam expression appears to be conserved between sexes, leading to different cellular outcomes in how mutants due to the different functions of Bam. In addition the role in maintaining Cyclin B expression has not been conserved so female how GSCs differentiate rather than arrest.

HOW is required for stem cell maintenance in the Drosophila testis and for the onset of transit-amplifying divisions.

The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric division to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic divisions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial divisions. HOW is normally expressed in a complementary pattern to Bam in the germline and bam mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Bam to the level required for differentiation, resulting in extra mitotic divisions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit-amplifying divisions.

Ttk69-dependent repression of lozenge prevents the ectopic development of R7 cells in the Drosophila larval eye disc.

BACKGROUND: During the development of the Drosophila eye, specific cell types differentiate from an initially equipotent group of uncommitted precursor cells. The lozenge (lz) gene, which is a member of the Runt family of transcriptional regulators, plays a pivotal role in mediating this process through regulating the expression of several fate-specifying transcription factors. However, the regulation of lz, and the control of lz expression levels in different cell types is not fully understood. RESULTS: Here, we show a genetic interaction between Tramtrack69 (Ttk69) a key transcriptional repressor and an inhibitor of neuronal fate specification, and lz, the master patterning gene of cells posterior to the morphogenetic furrow in the Drosophila eye disc. Loss of Ttk69 expression causes the development of ectopic R7 cells in the third instar eye disc, with these cells being dependent upon Lz for their development. Using the binary UAS Gal4 system, we show that overexpression of Ttk69 causes the loss of lz-dependent differentiating cells, and a down-regulation of Lz expression in the developing eye. The loss of lz-dependent cells can be rescued by overexpressing lz via a GMR-lz transgene. We provide additional data showing that factors functioning upstream of Ttk69 in eye development regulate lz in a Ttk69-dependent manner. CONCLUSIONS: Our results lead us to conclude that Ttk69 can either directly or indirectly repress lz gene expression to prevent the premature development of R7 precursor cells in the developing eye of Drosophila. We therefore define a mechanism for the tight regulatory control of the master pre-patterning gene, lz, in early Drosophila eye development and provide insight into how differential levels of lz expression can be achieved to effect specific cell fate outcomes.

The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity.

A key goal of regenerative medicine is an understanding of the genetic factors that define the properties of stem cells. However, stem cell research in mammalian tissue has been hampered by a paucity of stem cell-specific markers. Although increasing evidence suggests that members of the Musashi (Msi) family of RNA-binding proteins play important functions in progenitor cells, it remains unclear whether there is a stem cell-autonomous requirement for Msi because of an inability to distinguish stem cells from early-lineage cells in mammalian tissues. Here, using the Drosophila testis as a model system for the study of stem cell regulation, we show specific evidence for a cell-autonomous requirement for Msi family proteins in regulating stem cell differentiation, leading to the identification of an RNA-binding protein required for spermatogonial stem cell maintenance. We found that loss of Msi function disrupts the balance between germ-line stem cell renewal and differentiation, resulting in the premature differentiation of germ-line stem cells. Moreover, we found that, although Msi is expressed in both somatic and germ cells, Msi function is required intrinsically in stem cells for maintenance of stem cell identity. We also discovered a requirement for Msi function in male meiosis, revealing that Msi has distinct roles at different stages of germ cell differentiation. We describe the complementary expression patterns of the murine Msi paralogues Msi1 and Msi2 during spermatogenesis, which support the idea of distinct, evolutionarily conserved roles of Msi.