Regulation of spermatogenic stem cell homeostasis by mitogen competition in an open niche microenvironment.

Continuity of spermatogenesis in mammals is underpinned by spermatogenic (also called spermatogonial) stem cells (SSCs) that self-renew and differentiate into sperm that pass on genetic information to the next generation. Despite the fundamental role of SSCs, the mechanisms underlying SSC homeostasis are only partly understood. During homeostasis, the stem cell pool remains constant while differentiating cells are continually produced to replenish the lost differentiated cells. One of the outstanding questions here is how self-renewal and differentiation of SSCs are balanced to achieve a constant self-renewing pool. In this review, we shed light on the regulatory mechanism of SSC homeostasis, with focus on the recently proposed mitogen competition model in a facultative (or open) niche microenvironment.

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche.

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice.

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/ß-Catenin Signaling in Mouse Spermatogenic Stem Cells.

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/ß-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/ß-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

Notch and Egfr signaling act antagonistically to regulate germ-line stem cell niche formation in Drosophila male embryonic gonads.

Germ-line stem cells (GSCs) are maintained by the somatic microenvironment, or GSC niche, which ensures that GSCs can both self-renew and produce functional gametes. However, it remains unclear how the proper niche size and location are regulated within the developing gonads. In the Drosophila testis, the hub cells that form the GSC niche are derived from a subset of somatic gonadal precursors (SGPs) in the anterior portion of the embryonic gonad. Here we show that Notch signaling induces hub differentiation. Notch is activated in almost all SGPs in the male embryonic gonad, but Epidermal growth factor receptor (Egfr) is activated in posterior SGPs to repress hub differentiation, thereby restricting the expansion of hub differentiation in the embryonic gonad. We further show that Egfr is activated in posterior SGPs by Spitz ligand secreted from primordial germ cells (PGCs), whereas the Notch ligand Serrate is expressed in SGPs. This suggests that varying the number of PGCs alters niche size. Indeed, a decrease in the number of PGCs causes ectopic hub differentiation, which consequently increases their opportunity to recruit PGCs as GSCs. When ectopic hub differentiation is repressed, the decreased number of PGCs fails to become GSCs. Thus, we propose that SGPs sense PGC number via signals from PGCs to SGPs that modulate niche size, and that this serves as a mechanism for securing GSCs.

Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads.

Drosophila germline stem cells are regulated by the somatic microenvironment, or "niche," which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. Here, we demonstrate that a receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, we propose that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads.

MAMO, a maternal BTB/POZ-Zn-finger protein enriched in germline progenitors is required for the production of functional eggs in Drosophila.

A hallmark of germline cells throughout the animal kingdom is their ability to execute meiosis. However, despite its prime importance, little is known about how germline progenitors acquire this ability. In Drosophila, the primordial germ cells (PGCs) are characterized by the inheritance of germ plasm, which contains maternal factors that have sufficient ability to direct germline development. Here, we show that a novel maternal factor, MAMO, is autonomously required in PGCs to produce functional gametes. MAMO protein which contains both a BTB/POZ (Broad Complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) domain and C(2)H(2) zinc finger motifs is enriched in PGCs during embryogenesis. The PGCs with reduced maternal MAMO activity are able to undergo oogenesis, but fail to execute meiosis properly. In the resulting oocytes, meiosis-specific chromosomal configurations are impaired. We additionally show that the decondensation of fertilized sperm nuclei is also affected in the eggs. We propose that maternal MAMO activates downstream genes to promote specialized morphological changes of both female meiotic chromosomes and the sperm nucleus, which are critical in zygote formation.

Molecular characterization of embryonic gonads by gene expression profiling in Drosophila melanogaster.

In many animal species, germ-line progenitors associate with gonadal somatic cells to form the embryonic gonads (EGs) that later develop into functional organ producing gametes. To explore the genetic regulation of the germ-line development, we initiated a comprehensive identification and functional analysis of the genes expressed within the EGs. First, we generated a cDNA library from gonads purified from Drosophila embryos by FACS. Using this library, we catalogued the genes expressed in the gonad by EST analysis. A total of 17,218 high-quality ESTs representing 3,051 genes were obtained, corresponding to 20% of the predicted genes in the genome. The EG transcriptome is unexpectedly distinct from that of adult gonads and includes an extremely high proportion of retrotransposon-derived transcripts. We verified 101 genes preferentially expressed in the EGs by whole-mount in situ hybridization. Within this subset, 39 and 58 genes were expressed predominantly in germ-line and somatic cells, respectively, whereas four genes were expressed in the both cell lineages. The gonad-enriched genes encompassed a variety of predicted functions. However, genes implicated in SUMOylation and protein translation, including germ-line-specific ribosomal proteins, are preferentially expressed in the germ line, whereas the expression of various retrotransposons and RNAi-related genes are more prominent in the gonadal soma. These transcriptome data are a resource for understanding the mechanism of various cellular events during germ-line development.

Isolation of germline cells from Drosophila embryos by flow cytometry.

Primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. To understand the molecular basis of germline development, isolation of pure PGC is required. We report here the use of fluorescence-activated cell sorting (FACS) to isolate pure populations of Drosophila pole cells, which are the presumptive primordial germ cells in flies. In order to fluorescently mark pole cells, we used an EGFP-vasa transgenic line that expresses green fluorescent protein (GFP) specifically and continuously in the germ line throughout the life cycle. The purity of FACS-sorted pole cells from embryos was confirmed by microscopic inspection and quantitative polymerase chain reaction. Moreover, by optimizing the sample preparation and the sorting protocol, embryonic gonads could also be isolated. This technique opens the way for genome-wide transcriptome analysis of germline cells. In a pilot experiment, we generated a cDNA library from purified embryonic gonad and identified a novel germline-specific gene, RpL22-like.

Expression of meiotic genes in the germline progenitors of Drosophila embryos.

Meiosis is one of the fundamental characteristics of germ cells. In Drosophila, genetic screens have identified many genes required for meiotic division. However, it remains elusive as to when and how these meiotic genes are activated during germline development. To obtain insights into their regulatory mechanisms, we examined the expression of 38 meiotic genes in the germline progenitors, pole cells, during embryogenesis. We found that the transcripts of 12 meiotic genes were enriched in pole cells within the embryonic gonads. Among them, bag of marbles (bam), benign gonial cell neoplasia (bgcn), deadhead (dhd), matotopetli (topi) and twine (twe) were activated only in pole cells within the gonads, whereas the transcripts from grapes (grp), Kinesin-like protein at 3A (Klp3A), pavarotti (pav), lesswright (lwr), mei-P26, Topoisomerase 2 (Top2) and out at first (oaf) were distributed ubiquitously in early embryos and then became restricted to pole cells and to a subset of somatic tissues at later embryonic stages. The remaining meiotic genes were either expressed ubiquitously in the embryos (15 genes) or were undetectable in pole cells within the gonads (11 genes). These observations suggest that pole cells have already acquired the potential to express several meiotic genes. Our data will thus provide a useful basis for analyzing how the germline acquires a potential to execute meiosis.