SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear ...targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.
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.
•The EcR is expressed in the somatic cyst cells of Drosophila testes.•EcR signaling restores gametogenesis in EGF mutants.•EcR signaling acts antagonistic to EGF signaling.•EcR signaling may promote the undifferentiated state in cyst cells.
Abstract
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.
Male subfertility or infertility is a common condition often characterized by men producing a low number of sperm with poor quality. To gain insight into this condition, we performed a quantitative ...proteomic analysis of semen samples obtained from infertile and fertile men. At least 6 proteins showed significant differences in regulation of alternatively spliced isoforms. To investigate this link between aberrant alternative splicing and production of poor-quality spermatozoa, we overexpressed the hnrnpH/F-orthologue Glorund (Glo) in Drosophila, which was also found to be abundant in poor quality human sperm. Transgenic animals produced low numbers of morphologically defective spermatozoa and aberrant formation of the “dense body,” an organelle akin to the mammalian manchette. Furthermore, fertility trials demonstrated that transgenic flies were either completely infertile or highly subfertile. These findings suggest that dysregulation of hnrnpH/F is likely to result in the production of low-quality semen, leading to subfertility or infertility in men.
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•The RNA splicing factor HnrnpF/H is more abundant in infertile human spermatozoa•Infertile human spermatozoa possess aberrant alternative splicing events•Overexpressing the Drosophila HnrnpF/h orthologue Glo causes poor quality sperm•Overexpression of Glo causes infertility or severe male subfertility in Drosophila
Reproductive medicine; Cell biology.
Transmembrane protein 161b (Tmem161b) was recently identified in multiple high-through-put phenotypic screens, including in fly, zebrafish, and mouse. In zebrafish, Tmem161b was identified as an ...essential regulator of cardiac rhythm. In mouse, Tmem161b shows conserved function in regulating cardiac rhythm but has also been shown to impact cardiac morphology. Homozygous or heterozygous missense mutations have also recently been reported for TMEM161B in patients with structural brain malformations, although its significance in the human heart remains to be determined. Across the three model organisms studied to date (fly, fish, and mouse), Tmem161b loss of function is implicated in intracellular calcium ion handling, which may explain the diverse phenotypes observed. This review summarises the current knowledge of this conserved and functionally essential protein in the context of cardiac biology.
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.
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•Rbf null testes exhibit failure of germline stem cells to differentiate•Rbf expression in somatic cells of L3 testes rescues the GSC differentiation defect•Somatic Rbf RNAi disrupts cyst stem cell and germline stem cell differentiation•Somatic depletion of E2f1 rescues Rbf germline proliferation and differentiation
The Drosophila testis contains germline and somatic cyst stem cell populations. Progeny from these populations cross regulate the differentiation of each lineage. In this article, Hime, Quinn, and colleagues show that the Drosophila Retinoblastoma ortholog, Rbf, is cell autonomously required by somatic cells of the testis to facilitate cyst stem cell differentiation and subsequent germline stem cell differentiation.
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