Wnt Signaling in Stem Cell Maintenance and Differentiation in the Drosophila Germarium

Wnt signaling is a conserved regulator of stem cell behaviors, and the Drosophila germarium has been an important model tissue for the study of stem cell maintenance, differentiation, and proliferation. Here we review Wnt signaling in the germarium, which houses two distinct types of ovarian stem cells: the anteriorly located germline stem cells (GSCs), which give rise to oocytes; and the mid-posteriorly located follicle stem cells (FSCs), which give rise to the somatic follicle cells that cover a developing oocyte. The maintenance and proliferation of GSCs and FSCs is regulated by the stem cell niches, whereas differentiation of the germline is regulated by the differentiation niche. Four distinct Wnt ligands are localized in the germarium, and we focus review on how these Wnt ligands and Wnt signaling affects maintenance and differentiation of both germline and follicle stem cells in their respective niches

Drosophila Ninjurin A induces nonapoptotic cell death.

Ninjurins are conserved transmembrane proteins that are upregulated across species in response to injury and stress. Their biological functions are not understood, in part because there have been few in vivo studies of their function. We analyzed the expression and function of one of three Drosophila Ninjurins, NijA. We found that NijA protein is redistributed to the cell surface in larval immune tissues after septic injury and is upregulated by the Toll pathway. We generated a null mutant of NijA, which displayed no detectable phenotype. In ectopic expression studies, NijA induced cell death, as evidenced by cell loss and acridine orange staining. These dying cells did not display hallmarks of apoptotic cells including TUNEL staining and inhibition by p35, indicating that NijA induced nonapoptotic cell death. In cell culture, NijA also induced cell death, which appeared to be cell autonomous. These in vivo studies identify a new role for the Ninjurin family in inducing nonapoptotic cell death

Distinct functions for the catalytic and hemopexin domains of a Drosophila matrix metalloproteinase

Human matrix metalloproteinases (MMPs) are believed to contribute to tumor progression. Therapies based on inhibiting the catalytic domain of MMPs have been unsuccessful, but these studies raise the question of whether other MMP domains might be appropriate targets. The genetic dissection of domain function has been stymied in mouse because there are 24 related and partially redundant MMP genes in the mouse genome. Here, we present a genetic dissection of the functions of the hemopexin and catalytic domains of a canonical MMP in Drosophila melanogaster, an organism with only 2 MMPs that function nonredundantly. We compare the phenotypes of Mmp1 null alleles with alleles that have specific hemopexin domain lesions, and we also examine phenotypes of dominant-negative mutants. We find that, although the catalytic domain appears to be required for all MMP functions including extracellular matrix remodeling of the tracheal system, the hemopexin domain is required specifically for tissue invasion events later in metamorphosis but not for tracheal remodeling. Thus, we find that this MMP hemopexin domain has an apparent specialization for tissue invasion events, a finding with potential implications for inhibitor therapies

<i>NijA</i> mutants behave <i>in vivo</i> as they do in cell culture.

<p>(<b>A</b>) Third instar <i>hml>GFP,NijA^152156A</i> larvae were devoid of GFP-positive hemocytes, indicating that these mutants were capable of inducing cell death and that polar amino acids 152 and 156 were not required for cell death. (<b>B</b>) <i>hml>NijA^ect,GFP</i> larvae displayed visible GFP-positive hemocytes on the body wall, suggesting that NijA^ect was not sufficient to induce cell death.</p

Ninjurin A protein response to septic wounding.

<p>(<b>A</b>) Western blot of whole adult male lysates probed with anti-NijA. NijA increases expression two hours after infection in adults. <i>NijA^D3</i> null lysates demonstrate antibody specificity. Black lines indicate regions of the blot that were omitted for clarity. (<b>B</b>) Graph representing three replicates of the western blot pictured in (A). NijA levels increase significantly in adults after septic injury (p = 0.003). (<b>C</b>) Western blot of whole male larval lysates probed with anti-NijA. NijA levels do not change 2 h after septic injury in third instar larvae; in contrast larval <i>Toll^10b</i> gain-of-function mutant larvae have increased levels of NijA protein. (<b>D</b>) Graph representing five replicates of the western blot pictured in (C). NijA levels increase significantly in constitutively activate <i>Toll^10b</i> mutant larvae (p<0.0001). (<b>E–M</b>) Anti-NijA (red) and DAPI (blue) labeling nuclei. All scale bars are 10 µm. (<b>E–G</b>) Anti-NijA stained non-permeabilized fat bodies of male third instar larvae show an increase in NijA at the cell surface 2 h after septic injury (compare E and F). (<b>G</b>) <i>NijA^D3</i> larvae demonstrate the NijA antibody specificity. (<b>H,I</b>) Anti-NijA stained non-permeabilized hemocytes of third instar larvae <i>ex-vivo</i> show an increase in NijA at the cell surface 2 h after septic injury. (<b>K–M</b>) Anti-NijA stained permeabilized fat bodies of male third instar larvae show increased NijA expression in gain-of-function <i>Toll^10b</i> mutants. Error bars in (B,D) represent standard error of the mean.</p

NijA induces cell death in <i>Drosophila</i> S2 cell culture.

<p>(<b>A</b>) <i>NijA</i> expression kills S2 cells. Cells were transiently transfected with <i>pRmHa3</i> empty vector or <i>pRmHa3-NijA</i> and induced with copper for 48 h. The percentage of dead cells was determined by dividing the number of trypan blue positive cells by that of total cells counted for each sample. Data from 8 experiments are shown. Error bars indicate S.E.M, and Student's T test was used to calculate p value. (<b>B</b>) Mmp1 activity is not required for <i>NijA</i>-induced cell death. Cells were transiently transfected and induced for 48 h. <i>Mmp1^E255A</i> is a dominant-negative catalytically inactive mutant of <i>Mmp1</i>. Data from 4 experiments are shown. (<b>C</b>) <i>NijA</i> is not required for actinomycin D-induced apoptosis. Cells were treated with <i>NijA</i> dsRNA or no dsRNA (mock) for 48 h, then incubated with 100 nM actinomycin D for 6 h. Trypan blue staining was used to determine cell survival, which was normalized to the untreated (DMSO), wild-type (mock) sample. Data from 4 experiments are shown. (<b>D</b>) Western blot showing the NijA protein levels in mock and <i>NijA</i> dsRNA-treated cells. Actin was used as the loading control.</p

<i>NijA^D3</i> mutants do not express mRNA from the <i>NijA</i> genomic locus.

<p>(<b>A</b>) Schematic of the <i>NijA</i> locus showing all four exons. Gray indicates untranslated regions and white indicates open reading frame. Three excision alleles (<i>D3, E1</i>, and <i>F9</i>) were generated from imprecise excisions of <i>EP G4196</i>. (<b>B</b>) qPCR data from primers specific to exon 3 (a negative control, as it is deleted in the <i>D3</i> allele) or exon 4 of <i>NijA</i>. The <i>NijA^D3</i> mutant did not produce any detectable mRNA from exon 4 of the <i>NijA</i> locus, even though exon 4 remains in the genome, indicating that the <i>D3</i> allele is a null. Error bars represent standard error of the mean.</p

Ninjurin A over-expression in the lymph gland causes nonapoptotic cell death.

<p>(<b>A</b>) Wild-type <i>w¹¹¹⁸</i> larva demonstrating background autofluorescence. (<b>B</b>) <i>hml>GFP</i> larva with GFP-positive differentiated hemocytes along posterior body wall and in lymph gland. (<b>C</b>) <i>hml>NijA,GFP</i> larva lacked GFP-positive cells. (<b>D</b>) <i>hml>hid,GFP</i> larva, a cell-death positive control, lacked GFP-positive cells. (<b>E</b>) Western blot of whole larval lysates probed with anti-GFP. <i>hml>NijA,GFP</i> and <i>hml>hid,GFP</i> larvae were devoid of GFP. (<b>F</b>) Quantification of three western blots probed for anti-GFP as in (E). GFP is virtually absent from <i>hml>NijA,GFP</i> and <i>hml>hid,GFP</i> larvae. Error bars represent standard error of the mean. (<b>G–I′</b>) Live partially dissected 3^rd instar larval lymph glands (arrows) were stained with acridine orange to detect cell death. Scale bars are 200 µM. tr: trachea; id: imaginal disc; vnc: ventral nerve cord; bl: brain lobe. (<b>G′</b>) <i>hml>GFP</i> larval glands did not stain with acridine orange. (<b>H′</b>) <i>hml>hid,GFP</i> glands, a cell-death positive control, stained with acridine orange. (<b>I′</b>) <i>hml>Nij</i>A,GFP glands stained with acridine orange, demonstrating that <i>NijA</i> induced cell death. (<b>J–L″</b>) Larval lymph glands were fixed, TUNEL labeled, and antibody stained. Scale bars are 50 µm. (<b>J–L</b>) Anti-Hemese staining labeled the lymph glands. (<b>J′–L′</b>) Anti-GFP staining shows no GFP-positive (hml+) hemocytes in <i>hml>hid,GFP</i> (K′) or <i>hml>NijA,GFP</i> (L′) larval glands. (<b>J″–L″</b>) TUNEL-labeled glands. (<b>J″</b>) Few TUNEL positive cells in <i>hml>GFP</i> negative control glands. (<b>K″</b>) Many TUNEL positive cells in <i>hml>hid,GFP</i> positive control glands. (<b>L″</b>) Few TUNEL positive-cells in the <i>hml>NijA,GFP</i> glands, indicating that <i>NijA</i> does not induce apoptosis. (<b>M</b>) Larvae expressing the apoptotic inhibitor p35 (<i>hml>p35,GFP)</i> displayed GFP-positive hemocytes similar to <i>hml>GFP</i> in (B). (<b>N–O</b>) p35 did not inhibit the NijA-induced loss of the GFP-positive cells in <i>hml>NijA,p35,GFP</i> larvae. (<b>P</b>) p35 inhibited the hid-induced loss of the GFP-positive cells in <i>hml>hid,p35,GFP</i> larvae, a positive control for p35 inhibition. In (A–D, M–P), anterior is up.</p

NijA appears to kill in a cell-autonomous manner.

<p>(<b>A–B</b>) Cells were transiently co-transfected with <i>pRmHa3-GFP</i> and various mutants of <i>pRmHa3-NijA</i> as indicated under each column; mock is empty <i>pRmHa3</i> vector. 48 h after induction, viability was assessed by trypan blue staining, and transfection status was assessed as GFP fluorescence. Wild-type <i>NijA</i> and most <i>NijA</i> mutants killed cells, whereas the mock control, the <i>D140A</i> mutant, and the N-terminal deletion (B) showed low levels of cell death. The sum of transfected live cells (GFP+) plus dead cells was relatively constant across samples despite the augmented or compromised capacity to kill cells, indicating that NijA kills the cell it transfects but not others. Data from 3 replicates are shown. Error bars indicate S.E.M. (<b>C</b>) Schematic showing topology of NijA (form A) protein and the extracellular region recognized by our polyclonal antibody. Amino acid residue numbers are indicated. (<b>D–G,I</b>) Immunofluorescence localization of wild-type NijA or NijA mutant forms expressed in S2 cells and stained with anti-NijA (D–G) or anti-c-Myc (I), both extracellular epitopes. For each construct, staining was performed on permeabilized cells to show NijA protein levels, and on unpermeabilized cells to show NijA cell-surface localization. Permeabilization status was verified by anti-tubulin staining. The merge image combines images for NijA (red), tubulin (cyan), DAPI (blue) and GFP fluorescence as a transfection control (green). Bar: 10 µm. (<b>H</b>) Diagram showing placement of the myc epitope for (I), necessary because the NijA antigenic region was deleted in this mutant.</p