Search

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.07.031 Byline: X. Rebecca Sheng (a), Trevor Posenau (b), Juliann J. Gumulak-Smith (b), Erika Matunis (a), Mark Van Doren (c), Matthew Wawersik (b)(c) Keywords: Primordial germ cell; Germline; Spermatogonia; Niche; Jak-STAT; Gonad; Stem cell Abstract: Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes. Author Affiliation: (a) Department of Cell Biology, 725 N. Wolfe Street, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA (b) Department of Biology, Integrated Science Center, Rm 2139, The College of William and Mary, Williamsburg, VA 23185, USA (c) Department of Biology, Mudd Hall 305, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA Article History: Received 6 May 2009; Revised 9 July 2009; Accepted 10 July 2009

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF, and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Owing to the important role that the human leukocyte antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk., Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising 5159 cases and 27 459 controls, including a prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold of p<4.5×10 -4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report., Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies., Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF., Competing Interests: Conflict of interest: L.J. Donoghue, A. Stockwell, M. Neighbours, X.R. Sheng and B.L. Yaspan are full-time employees of Genentech; A. Stockwell, M. Neighbours, X.R. Sheng and B.L. Yaspan hold stock options in Roche. J.M. Oldham reports personal fees from Boehringer Ingelheim, Genentech, United Therapeutics, AmMax Bio and Lupin Pharmaceuticals outside the submitted work. D.A. Schwartz is the founder and chief scientific officer of Eleven P15, a company focused on the early detection and treatment of pulmonary fibrosis. R.G. Jenkins reports honoraria from Chiesi, Roche, PatientMPower, AstraZeneca, GlaxoSmithKline and Boehringer Ingelheim, and consulting fees from Bristol Myers Squibb, Daewoong, Veracyte, Resolution Therapeutics, RedX, Pliant and Chiesi. L.V. Wain reports research funding from GlaxoSmithKline, Roche and Orion Pharma, and consultancy for GlaxoSmithKline and Galapagos, outside of the submitted work. S.P. Hart and P.L. Molyneaux are associate editors of this journal. The other authors declare no competing interests., (Copyright ©The authors 2024.)

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.

The reversal of cellular differentiation, or dedifferentiation, has fascinated biologists for many decades. While cells can be re-programmed extensively in culture, examples of in vivo dedifferentiation have recently emerged in both vertebrate and invertebrate systems, allowing for analysis of this intriguing process under more physiologically relevant conditions. Studies suggest that dedifferentiation occurs not only during large-scale cellular regeneration, but also at low levels to replenish stem cells lost due to normal turnover. Our recent paper demonstrates a novel method to induce the dedifferentiation of lineage-committed stem cell daughters back into germline stem cells (GSCs) in the Drosophila testis. We also show a requirement for activation of Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling in this process, and suggest that normally non-motile germline cells gain mobility and out-compete resident somatic cells for occupancy in the stem cell-maintaining microenvironment (niche). Here, we discuss what our findings reveal about stem cell competition and the capacity of various cell types to dedifferentiate.

The mouse mu-opioid receptor gene, Oprm1, currently contains 18 recognized alternatively spliced exons [Doyle, G.A., Sheng, X.R., Lin, S.S.J., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007. Identification of three mouse mu-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 388 (1-2) 135-147, in press (doi:10.1016/j.gene.2006.10.017). Electronic publication 2006 November 1] that generate 27 splice variants encoding at least 11 morphine-binding isoforms of the receptor. Here, we identify five MOR variants that contain an as yet undescribed exon (exon 19) of the gene, and we provide evidence that these MOR splice variants are expressed in the C57BL/6 and DBA/2 mouse strains. Three splice variants, MOR-1Eii, MOR-1Eiii and MOR-1Eiv, encode the MOR-1E isoform and contain the newly identified exon 19 in their 3' untranslated regions. The fourth splice variant encodes a novel mu-opioid receptor isoform, MOR-1U, and contains exon 19 in its coding region. The cytoplasmic tail of the putative MOR-1U isoform contains a putative nuclear localization signal encoded by the sequence of exon 19. Exon 19 appears to be conserved in the rat, but not in humans. In mouse and rat Oprm1, exon 19 is located between described exons 7 and 8. We also report the cloning of the "full-length" MOR-1T splice variant [Kvam, T.-M., Baar, C., Rakvag, T.T., Kaasa, S., Krokan, H.E., Skorpen, F., 2004. Genetic analysis of the murine mu-opioid receptor: increased complexity of Oprm1 gene splicing, J. Mol. Med. 82 (4) 250-255] that encodes MOR-1 and contains the newly identified exon in its 3' UTR. RT-PCR analysis suggests that splice variants MOR-1Eii, MOR-1Eiii, MOR-1Eiv, MOR-1T and MOR-1U are expressed in all brain regions analyzed (cortex, cerebellum, hypothalamus, thalamus and striatum). These exon 19-containing splice variants add to the growing complexity of the mouse Oprm1 gene.

C57BL/6J and DBA/2J mice demonstrate differences in morphine preference when tested in a two-bottle choice paradigm. Quantitative trait loci (QTL) mapping suggested the proximal region of chromosome 10 was responsible for 41% of the observed genetic variance. The mu-opioid receptor (MOR) gene (Oprm) maps to this region and is a prime candidate for explaining the QTL. We hypothesized that variations in Oprm between these strains are responsible for differences in morphine preference. We identify five single nucleotide polymorphisms (SNPs) in the Oprm promoter; three within or near putative transcription factor binding sites. Promoter fragments were amplified from genomic DNA by polymerase chain reaction (PCR) and subcloned into luciferase reporter vectors. A significant difference in basal Oprm promoter activity was seen with C57BL/6 and DBA/2 approximately 1675 constructs in MOR-positive BE(2)-C cells, but not in MOR-negative Neuro-2a cells. In BE(2)-C cells, average DBA/2 approximately 1675 construct activity was 1.3-2.0x greater than average C57BL/6 activity suggesting that the SNPs might alter MOR expression in these two mouse strains. Significant differences in promoter activities between the two cell lines suggest that cell-type-specific transcription factors are involved. No significant differences in construct activity were found between untreated and morphine-treated BE(2)-C or Neuro-2a cells, suggesting that morphine does not regulate transcription of Oprm.