Argonaute 1 regulates the fate of germline stem cells in Drosophila

The Argonaute-family proteins play crucial roles in small-RNA-mediated gene regulation. In Drosophila, previous studies have demonstrated that Piwi, one member of the PIWI subfamily of Argonaute proteins, plays an essential role in regulating the fate of germline stem cells (GSCs). However, whether other Argonaute proteins also play similar roles remains elusive. Here, we show that overexpression of Argonaute 1 (AGO1) protein, another subfamily (AGO) of the Argonaute proteins, leads to GSC overproliferation, whereas loss of Ago1 results in the loss of GSCs. Combined with germline clonal analyses of Ago1, these findings strongly support the argument that Ago1 plays an essential and intrinsic role in the maintenance of GSCs. In contrast to previous observations of Piwi function in the maintenance of GSCs, we show that AGO1 is not required for bag of marbles (bam) silencing and probably acts downstream or parallel of bam in the regulation of GSC fate. Given that AGO1 serves as a key component of the miRNA pathway, we propose that an AGO1-dependent miRNA pathway probably plays an instructive role in repressing GSC/cystoblast differentiation.     

Hsp83 regulates the fate of germline stem cells in Drosophila ovary

正In adult tissues,stem cells are defined by their unique capacity to self-renew and produce differentiated cells to maintain tissue homeostasis.Drosophila ovarian germline stem cells(GSCs)provide a powerful model for investigating the regulatory mechanisms underlying stem cell fate determination in vivo(Chen and Mckearin,     

CRIM1 regulates the rate of processing and delivery of bone morphogenetic proteins to the cell surface

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of co-expression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface.     

Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub–GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.     

Heterodimerization-dependent secretion of bone morphogenetic proteins in Drosophila

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Cross talk between hedgehog and bone morphogenetic proteins occurs during cardiomyogenesis in P19 cells

Hedgehog (Hh) signaling plays a role in heart morphogenesis and can initiate cardiomyogenesis in P19 cells. To determine if Hh signaling is essential for P19 cell cardiomyogenesis, we determined which Hh factors are expressed and the effect of Hh signal transduction inhibitors. Here, we find that the Hh gene family and their downstream mediators are expressed during cardiomyogenesis but an active Hh signaling pathway is not essential. However, loss of Hh signaling resulted in a delay of BMP-4, GATA-4, Gli2, and Meox1 expression during cardiomyogenesis. By using Noggin-overexpressing P19 cells, we determined that Hh signaling was not active during Noggin-mediated inhibition of cardiomyogenesis. Thus, there is cross talk between the Hh and BMP signaling pathways and the Hh pathway appears important for timely cardiomyogenesis.     

HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis

In humans and mice, loss of HOXA13 function causes defects in the growth and patterning of the digits and interdigital tissues. Analysis of Hoxa13 expression reveals a pattern of localization overlapping with sites of reduced Bmp2 and Bmp7 expression in Hoxa13 mutant limbs. Biochemical analyses identified a novel series of Bmp2 and Bmp7 enhancer regions that directly interact with the HOXA13 DNA-binding domain and activate gene expression in the presence of HOXA13. Immunoprecipitation of HOXA13-Bmp2 and HOXA13-Bmp7 enhancer complexes from the developing autopod confirm that endogenous HOXA13 associates with these regions. Exogenous application of BMP2 or BMP7 partially rescues the Hoxa13 mutant limb phenotype, suggesting that decreased BMP signaling contributes to the malformations present in these tissues. Together, these results provide conclusive evidence that HOXA13 regulates Bmp2 and Bmp7 expression, providing a mechanistic link between HOXA13, its target genes and the specific developmental processes affected by loss of HOXA13 function.     

The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche

The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. Here we show that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions.     

Bone morphogenetic proteins secreted by breast cancer cells upregulate bone sialoprotein expression in preosteoblast cells

It is well established that bone metastases comprise bone; however, the exact factors/mechanisms involved remain unknown. We hypothesized that tumor cells secreted factors capable of altering normal bone metabolism. The aims of the present study were to (1) determine the effects of secretory products isolated from HT-39 cells, a human breast cancer cell line, on osteoprogenitor cell (MC3T3-E1 cells) behavior, and (2) identify tumor-derived factor(s) that alters osteoblast activities. Conditioned media (CM) from HT-39 cells were collected following a 24-h serum-free culture. The ability of CM to alter gene expression in MC3T3-E1 cells was determined by Northern analysis. CM effects on cell proliferation and mineralization ability were determined using a Coulter counter and von Kossa stain, respectively. MC3T3-E1 cells were treated with CM plus noggin, a factor known to block bone morphogenic proteins (BMPs), to determine whether BMPs, shown to be present in CM, were linked with CM effects on MC3T3-E1 cell activity. In addition, inhibitors of MAP kinase kinase (MEK), protein kinase C (PKC), and protein kinase A were used to identify the intracellular signaling pathway(s) by which the active factors in CM regulated osteoblast behavior. CM treatment significantly enhanced BSP mRNA (2.5-fold over control), but had no effect on cell proliferation. Mineralization assay showed that CM enhanced mineral nodule formation compared to controls. Noggin inhibited CM-induced upregulation of BSP mRNA, suggesting that BMPs were responsible for upregulating BSP gene expression in MC3T3-E1 cells. The PKC inhibitor blocked CM-mediated upregulation of BSP, suggesting involvement of the PKC pathway in regulating BSP expression. BMPs secreted by HT-39 cells may be responsible for enhancing BSP expression in MC3T3-E1 cells. Continued studies targeted at determining the role of BMPs in regulating bone metabolism are important for understanding the pathogenesis of bone diseases.     

Clonal expansion of ovarian germline stem cells during niche formation in Drosophila

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. We report that signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Our lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic (dpp), the Drosophila homolog of human bone morphogenetic protein 2/4, is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential dpp receptor, loses the ability to clonally populate a niche. Therefore, dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches.     

Cross-talk between fibroblast growth factor and bone morphogenetic proteins regulates gap junction-mediated intercellular communication in lens cells

Homeostasis in the lens is dependent on an extensive network of cell-to-cell gap junctional channels. Gap junction-mediated intercellular coupling (GJIC) is higher in the equatorial region of the lens than at either pole, an asymmetry believed essential for lens transparency. Primary cultures of embryonic chick lens epithelial cells up-regulate GJIC in response to purified fibroblast growth factor (FGF)1/2 or to medium conditioned by vitreous bodies, the major reservoir of factors (including FGF) for the lens equator. We show that purified bone morphogenetic protein (BMP)2, -4, and -7 also up-regulate GJIC in these cultures. BMP2, -4, or both are present in vitreous body conditioned medium, and BMP4 and -7 are endogenously expressed by lens cells. Remarkably, lens-derived BMP signaling is required for up-regulation of GJIC by purified FGF, and sufficient for up-regulation by vitreous humor. This is the first demonstration of an obligatory interaction between FGF and BMPs in postplacode lens cells, and of a role for FGF/BMP cross-talk in regulating GJIC in any cell type. Our results support a model in which the angular gradient in GJIC in the lens, and thus proper lens function, is dependent on signaling between the FGF and BMP pathways.