fas3 mouse dshb  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a) Graphic representation of the Drosophila ovary and the early stages of oogenesis. TF: terminal filament, CC: cap cell, GSC: germline stem cell, GC: germ cell, EC: escort cell, a: anterior, c: central, p: posterior, FSC: follicle stem cell, pFC: prefollicle cell, MB: main body follicle cell, PC: polar cell, SC: stalk cell. Inset on the right highlights the apical to basal orientation of main body follicle cells. b) Average and percent expression of septate junction core components and tricellular septate junction (tSJ) components in escort cells and early follicle cell types identified in . Many septate junction components display high expression in polar cells and several components are enriched in escort cells. c) Germarium and early cyst stages stained for Nrx-IV::mCherry (magenta) and DAPI (blue). Nrx-IV::mCherry is highly expressed in escort cells (EC) and polar cells (arrowheads). d) Wildtype ovariole (109-30 ts > w 1118 ) stained for Cora. Cora is expressed in escort cells and follicle cells with high expression in mature stalk (arrow). e-i) Immunostaining (e-f) and associated quantification (i) of Cora staining in Stage 6 main body follicle. 109-30 ts was used to induce the respective RNAi. In wildtype Cora mostly localizes laterally. Knockdown of nrv2 , Nrx-IV or kune disturbs normal Cora localization. Label in e) illustrates how we divided the lateral membrane into the apical (“a”), lateral (“l”) and basal (“b”) portions. i) n = 6, 7, 7, 6 measurements for each condition. j-l) Germaria of the indicated genotype stained for DAPI (blue) and the follicle cell marker Fas3 (green or white in j’-l’). Germline cysts encapsulated by follicle cells in the germarium are marked by asterisks. m) Quantification of cysts in Region 2b in 109-30 ts crossed with the respective genotype. n = 37, 22, 21, 36, 42, 14, 15 germaria respectively. n-p) Stalk regions of the genotypes indicated in each respective row stained for Hts. Note that wildtype stalk forms a single row of cells (n) while knockdown of septate junction components results in double rowed stalks (o-p). q) Quantification of cells per stalk (Stage 4) in ovarioles from 109-30 ts crossed to the respective allele. n = 28, 11, 9, 12, 6, 28, 12 stalks respectively.

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Expressing, Staining, Immunostaining, Knockdown, Membrane, Marker

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a) Ovariole expressing RedStinger (magenta) under the 109-30-Gal4 driver and stained for Fas3 (green) and DAPI (blue). a’) RedStinger expression in white. b-c) Ovarioles of 109-30 ts crossed to w 1118 (b) or cora -RNAi (c) and stained for Cora. d-e) Ovarioles with pan-follicle cell depletion of Nrx-IV (d) or kune (e) stained for DAPI (blue) and Fas3 (green). f) Quantification of the frequency of ovarioles with more than two germline cysts in Region 2b of 109-30 ts crossed to the respective allele. For control genotypes the CyO positive sibling flies, which lack the 109-30-Gal4 allele, were dissected. P-values from Chi-squared test. n = 247, 236, 184, 219, 154, 232, 161, 252, 153, 150, 134, 158, 142 germaria respectively. g) Quantification of the stage of the second budded cyst in ovarioles with 109-30 ts driving the indicated allele. For significance testing we used stage numbers as values. n = 9, 10, 14, 12, 14, 11, 14 ovarioles respectively. h-i) Stalk regions of ovarioles of 109-30 ts crossed to Nrx-IV -RNAi (h) or kune -RNAi (i) stained for Hts. j) Quantification of the frequency of ovarioles with double row stalk (Stage 4 or later) of 109-30 ts crossed to the respective allele. Control flies were devoid of 109-30-Gal4. P-values from Chi-squared test. n = 197, 138, 149, 140, 135, 140, 136, 148, 144, 142, 133, 140, 147 ovarioles respectively.

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Expressing, Staining, Control

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a-e) Ovarioles of the respective genotypes stained for DAPI (blue) and EdU (magenta). Stalk cells are EdU negative and outlined in yellow. f-m) Immunostainings of ovarioles of 109-30 ts driving Nrx-IV -RNAi (f, h, j, l) or kune -RNAi (g, i, k, m) stained for DAPI (blue) and zfh1 (magenta) (f-g) with an inset showing zfh1 staining in white in the second stalk (f’-g’), or second stalks stained for DAPI (blue) and a cell type specific marker in green: aop (h-i), LamC (j-k) or eya (l-m). n) 109-30 ts > w 1118 ovariole stained for DAPI (blue), c-Dcp-1 (green) and Fas3 (white). n’) shows c-Dcp-1 (white). We detected c-Dcp-1 staining in Fas3 high polar cells but not in stalk cells. o) Quantification of Fas3 staining in ovarioles from 109-30 ts crossed to the respective allele. Fas3 intensity in Stage 4 stalk cells was normalized to Fas3 intensity in Region 2b follicle cells to circumvent influence of staining differences. n = 6, 7, 7, 7, 7, 7, 7 stalks respectively.

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a-b) Quantification of the percentage of proliferating cells in Region 2b (a) or main body follicle cells of Stage 5-6 cysts (b) in ovarioles of 109-30 ts crossed to the respective allele. a) n = 10, 11, 10, 12, 11, 11, 15 germaria. b) n ≥ 9, 11, 9, 8, 10, 11, 13 ovarioles. c-e) Germaria of 109-30 ts crossed to fz3::RFP and the respective allele and stained for DAPI (blue), RFP (magenta) and Fas3 (white). c’-e’) show the RFP channel in white. Yellow dotted line shows the Fas3 border between Region 2a and 2b. f) Associated quantification of fz3::RFP intensity in Region 2b for genotypes shown in (c-e). n = 8, 9, 6 germaria respectively. g-i) Ovarioles of the 109-30 ts driver crossed to the wildtype w 1118 (g), arm -RNAi (h) or dsh -RNAi (i) and stained for DAPI (blue) and Fas3 (green). g’-i’) show Fas3 in white. Asterisks mark Region 2b cysts. j) Quantification of cysts in Region 2b in the wildtype (109-30 ts > w 1118 ) or upon reduction of Wnt signaling by RNAi depletion of arm or dsh in follicle cells with 109-30 ts . n = 26, 21, 18 germaria respectively. k-m) Ovarioles of the respective genotypes stained for DAPI (blue) and zfh1 (magenta). Insets are magnified in k’-m’) and show zfh1 staining (white) in the second stalk. n-v) First stalks of ovarioles of the genotype indicated in the respective row stained for DAPI and for a cell type specific marker (green): aop (n-p), LamC (q-s), eya (t-v).

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a-f) Immunostainings of consecutive stages of stalk development of 109-30 ts crossed to w 1118 stained for E-Cad (a-f), Cora (a’-f’) and Phalloidin (a”-f”) in white. g-h) Maximum intensity projections and stalk cross sections of E-Cad staining in 109-30 ts driving Nrx-IV -RNAi (g) or kune -RNAi (h). i-l) Immunostainings of budding stalk regions of 109-30 ts driving Nrx-IV -RNAi (i, k) or kune -RNAi (j, l) stained for E-Cad and Baz (i-j) or Dlg and Phalloidin (k-l) as indicated. m) Ovariole with pan-follicle cell depletion of E-Cad (109-30 ts driving E-Cad -RNAi) stained for Fas3 (green) and DAPI (blue). Areas with cyst fusion and double row stalks are outlined in yellow.

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Staining

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a-c) Ovarioles of the wildtype (a) or with stalk cell specific knockdown of cora (b) or nrv2 (c) stained for DAPI (blue) and LamC (green). Insets are enlarged on the right and show LamC staining (white) in the second stalk. d) Quantification of cells per Stage 4 stalk in ovarioles of CG46339 ts crossed to the respective genotype. Stalk cell specific knockdown of septate junction components does not significantly increase stalk cell numbers or leads to only a mild increase. N = 9, 5, 5, 5, 5, 5, 5 stalks respectively. e-g) Ovarioles of the wildtype (e) or with polar cells specific knockdown of cora (f) or nrv2 (g) stained for DAPI (blue) and LamC (green). Insets on the right show the second stalk stained for LamC (white). h) Quantification of cells per stalk (Stage 4) in ovarioles with upd ts crossed to the respective genotype. n = 8, 8, 8, 8, 10, 6 stalks respectively. i-k) Ovarioles of 109-30 ts combined with the Jak-STAT reporter UAS-2xSTAT-eGFP and the respective allele as indicated. We stained for DAPI (blue), GFP (green) and Fas3 (white) (i-k). i’-k’) show the 2xSTAT-GFP channel (white). Insets on the right show the first or second stalk as indicated and stained for DAPI (blue), GFP (green) and Fas3 (white) on the left or 2xSTAT-GFP (white) on the right. l) Quantification of 2xSTAT-GFP in Region 2b, early stalk or mature stalk cells in 109-30 ts crossed to the respective genotype. n = 6, 5, 5, 14, 14, 14, 14, 14, 14 ovarioles respectively.

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Knockdown, Staining

Journal: bioRxiv

Article Title: Distinct Non-occluding Functions of Septate Junction Components in Signaling Pathway Regulation and Cell Polarity During Epithelial Development

doi: 10.1101/2024.09.06.611635

Figure Lengend Snippet: a) A germline cyst expressing Upd1::HA stained for HA (magenta), Fas3 (green) and DAPI (blue) and insets showing the indicated channels. Green line indicates the apical cap region in the Fas3 channel. Yellow outline in the DAPI channel marks the epithelium. b) Polar cells of ovarioles of 109-30 ts crossed to the indicated genotype and stained for E-Cad, Fas3, Phalloidin and DAPI as indicated (white). Apical side facing the germline cyst is oriented towards the bottom. The epithelium is outlined by a yellow dotted line in the DAPI channel. Note the presence of the apical cap in both examples of wildtype polar cells marked by a green line. c) Graphical overview of a polar cell cluster with apical cap and apical Upd1 enrichment. d) Quantification of Fas3 intensity of the apical polar cell membrane to apical main body follicle cell side in ovarioles of 109-30 ts crossed to the indicated genotype. n = 16, 20, 16, 18, 20, 18, 20 ovarioles respectively. e) Graph summarizing the functions of septate junction core components in early follicle cell development. Septate junctions allow normal FSC/pFC proliferation rates by promoting Wnt signaling activity which is vital for effective cyst encapsulation in Region 2b. Septate junctions further regulate stalk morphogenesis non-cell-autonomously by maintaining cell polarity in polar cells and thereby fine-tuning Jak-STAT signaling activity. Additionally, they cell-autonomously control stalk cell apical-basal polarity, cell adhesion and actin cytoskeleton to coordinate the intercalation process.

Article Snippet: The following antibodies were used: DSHB: mouse anti-Cora (1:1000, C566.9), mouse anti-Hts (1:100, 1B1), mouse anti-Fas3 (1:100, 7G10), mouse anti-LamC (1:100, LC28.26), mouse anti-Yan (1:100, 8B12H9), mouse anti-Eya (1:100, 10H6), rat anti-E-Cad (1:50, DCAD2), mouse anti-Dlg1 (1:50, 4F3).

Techniques: Expressing, Staining, Membrane, Activity Assay, Encapsulation, Control

Journal: PLOS ONE

Article Title: mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis

doi: 10.1371/journal.pone.0300337

Figure Lengend Snippet: List of fly stocks and reagents.

Article Snippet: Mouse monoclonal anti-Fasciclin 3 (Fas3) (7G10) , DSHB , .

Techniques: Transgenic Assay, shRNA, Imaging

Journal: PLOS ONE

Article Title: mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis

doi: 10.1371/journal.pone.0300337

Figure Lengend Snippet: (A) Cell identity markers in the testis stem cell niche. Germ cells are labeled with Vasa staining, hub cells (asterisk) with Fas3 and Traffic Jam (TJ) staining and early cyst cells with TJ staining, in a testis tip expressing nuclear GFP under the control of the early germ cell driver nanos TS , upon 5 days of induction at 29°C. GSCs (yellow dotted circles) are identified as Vasa + germ cells in contact with the hub and CySCs (arrowheads) are identified as TJ + cells in contact with the hub. (B) Phospho-dRpS6 (pS6) staining in the tip of wild-type ( y 1 , w 1 ) testis. The red arrow points to an example of spermatogonial cyst positive for pS6. E-Cadherin (DE-Cad) is used as a marker of hub cells. (C) pS6 staining in the tip of a mTor :: GFP testis. pS6 + GSCs (yellow) and spermatogonia (magenta) are circled in dotted lines in the right panel showing mTor::GFP expression alone. mTor::GFP is visualized by immunostaining with an anti-GFP antibody, as in the subsequent panels. (D) mTor::GFP expression pattern in a testis tip expressing RFP under the control of bam-GAL4 : VP16 . α-spectrin (α-spec) marks the fusome in germ cells. GSCs, in contact with hub cells, are circled in yellow and early spermatogonia that do not express RFP are circled in magenta. (E) mTor::GFP expression pattern in a testis tip co-stained with LaminC (LamC). LamC is expressed at high levels in hub cells (asterisk) and in spermatocytes (cyan dotted circles). In all panels, asterisks indicate the location of the hub at the apical tip of the testis. Scale bars: 15μm.

Article Snippet: Mouse monoclonal anti-Fasciclin 3 (Fas3) (7G10) , DSHB , .

Techniques: Labeling, Staining, Expressing, Control, Marker, Immunostaining

Journal: PLOS ONE

Article Title: mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis

doi: 10.1371/journal.pone.0300337

Figure Lengend Snippet: (A-B) Overall structure of single testes of the genotype nanos-GAL4/+;UAS-mTor TRiP-HMS00904 /tub-GAL80 ts , visualized by staining nuclei with DAPI (DNA) and contoured with yellow lines in non-induced control (flies maintained at 18°C, A) and upon early germ cell-specific depletion of mTor (flies reared at 18°C and shifted to 29°C at the adult stage for 7 days to induce mTor depletion in GSCs and early germ cells, B). Scale bar: 100μm. (C-D) Testis tips of non-induced (control, C) or mTor-depleted (D) nanos-GAL4/+;UAS-mTor TRiP-HMS00904 /tub-GAL80 ts flies with Vasa, α-spectrin (α-spec) and Fas3 stainings. Lower panels show maximum intensity projections of several images spanning the hub region (Z-projections). Scale bar: 15μm. (E) Number of germ cells with a spherical fusome per testis of the indicated genotypes: nanos TS >mTor RNAi1 — non induced (n = 37), nanos TS >mTor RNAi1 –7 days induction (n = 37), nanos TS >mTor RNAi2 –15 days induction (n = 25), nanos>white RNAi — expressed throughout development (n = 23) and nanos>raptor RNAi — expressed throughout development (n = 24). Means and standard deviations are shown. **** denotes statistical significance (p<0.0001) as determined with Mann-Whitney tests. (F-G) Testis tips of non-induced (control, F) or mTor-depleted (G) nanos-GAL4/+;UAS-mTor TRiP-HMS00904 /tub-GAL80 ts flies with phospho-Mad (pMad), α-spectrin and Fas3 stainings. Lower panels show pMad-associated fluorescence intensity represented as a color gradient with the lowest intensity in black and the brightest intensity in white. Scale bar: 15μm. (H) Number of pMad-positive germ cells per testis tip of the indicated conditions: nanos TS >mTor RNAi1 — non induced (n = 66), nanos TS >mTor RNAi1 –7 days induction (n = 71), nanos TS >mTor RNAi2 –15 days induction (n = 28), nanos>white RNAi — expressed throughout development (n = 20) and nanos>raptor RNAi — expressed throughout development (n = 20). Means and standard deviations are shown. **** denotes statistical significance (p<0.0001) as determined with Mann-Whitney tests. In all panels, asterisks indicate the hub.

Article Snippet: Mouse monoclonal anti-Fasciclin 3 (Fas3) (7G10) , DSHB , .

Techniques: Staining, Control, MANN-WHITNEY, Fluorescence

Journal: PLOS ONE

Article Title: mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis

doi: 10.1371/journal.pone.0300337

Figure Lengend Snippet: (A-B) Control (A) and mTor-depleted (B) testis tips with Vasa, α-spectrin and Fas3 stainings. Arrowheads signal large cytoplasmic areas devoid of Vasa staining in mTor-depleted germ cells. (C) Quantification of the number of testes with 0, 1–10, 11–20 or more than 20 germ cells with large cytoplasmic regions devoid of Vasa staining (later characterized as autolysosomes), in non-induced control flies (n = 46) and flies in which mTor depletion is induced in adult GSCs and early germ cells (n = 54). (D-E) Control (D) and mTor-depleted (E) testis tips with Lysotracker Red and Vasa stainings. Arrowheads signal Vasa-negative and Lysotracker-positive cytoplasmic areas in mTor-depleted germ cells. (F) Area of Atg8a+ vesicles (in μm 2 ) in non-induced control flies (n = 21 vesicles) and flies in which mTor depletion is induced in adult GSCs and early germ cells (n = 41 vesicles), carrying the pAtg8a-mCherry-Atg8a transgene. **** denotes statistical significance (p<0.0001) as determined with a two-tailed unpaired t-test. (G-H) Control (G) and mTor-depleted (H) testis tips ubiquitously expressing the EGFP-mCherry-Atg8a autophagic flux sensor. Magenta arrowheads signal yellow (EGFP and mCherry-positive) Atg8a puncta in germ cells and the yellow arrows point to red (mCherry-positive and EGFP-negative) Atg8a puncta in cyst cells. (I-J) Control (I) and mTor-depleted (J) testis tips stained with Ref(2)P, Vasa and Fas3. The inset in J is a magnification of the framed germ cell, showing Ref(2)P aggregates in the cytoplasm, outside of the Vasa-devoid cytoplasmic are denoted with an asterisk. Lower panels show Ref(2)P-associated fluorescence intensity represented as a color gradient with the lowest intensity in black and the brightest intensity in white. Testes shown in panels (A-E) and (I-J) are from flies of the genotype nanos-GAL4/+;UAS-mTor TRiP-HMS00904 /tub-GAL80 ts and testes in panels (G-H) are from flies of the genotype nanos-GAL4/pAtg8a-EGFP-mCherry-Atg8a;UAS-mTor TRiP-HMS00904 /tub-GAL80 ts . Control corresponds to non-induced conditions (flies maintained at 18°C) and nanos TS >mTor RNAi1 corresponds to induced conditions (flies raised at 18°C and shifted to 29°C for 7 days at the adult stage). The hub is indicated with an asterisk in (A-D) and is delimitated by white dotted lines in (E-H). For all images, scale bar: 15μm.

Article Snippet: Mouse monoclonal anti-Fasciclin 3 (Fas3) (7G10) , DSHB , .

Techniques: Control, Staining, Two Tailed Test, Expressing, Fluorescence

Journal: PLOS ONE

Article Title: mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis

doi: 10.1371/journal.pone.0300337

Figure Lengend Snippet: (A-C) Down-regulation of mTor in germ cells induces a concomitant increase in TOR activity in neighboring cyst cells. (A-B) Control (A) and mTor-depleted (B) testis tips with pS6, Vasa and TJ stainings. (C) Number of pS6- and TJ-double positive cells per testis tip in the indicated conditions: nanos TS >mTor RNAi1 — non induced (n = 42) and nanos TS >mTor RNAi1 - 7days induction (n = 46). Means and standard deviations are shown. **** denotes statistical significance (p<0.0001) as determined with Mann-Whitney tests. (D-F) Down-regulation of mTor in germ cells leads to a loss of early cyst cells. (D-E) Control (D) and mTor-depleted (E) testis tips with Eya, TJ and Fas3 stainings. The arrow in (E) points to an example of Eya-positive cyst cell in proximity to the hub. (F) Number of early cyst cells (TJ positive and Eya negative cells) per testis tip of the indicated conditions: nanos TS >mTor RNAi1 — non induced (n = 34) and nanos TS >mTor RNAi1 - 7days induction (n = 34). Means and standard deviations are shown. **** denotes statistical significance (p<0.0001) as determined with Mann-Whitney tests. Testes shown in panels (A-B) and (D-E) are from flies of the genotype nanos-GAL4/+;UAS-mTor TRiP-HMS00904 /tub-GAL80 ts . Control corresponds to non-induced conditions (flies maintained at 18°C) and nanos TS >mTor RNAi1 corresponds to induced conditions (flies raised at 18°C and shifted to 29°C for 7 days at the adult stage). The hub is indicated with an asterisk. For all images, scale bar: 15μm.

Article Snippet: Mouse monoclonal anti-Fasciclin 3 (Fas3) (7G10) , DSHB , .

Techniques: Activity Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Steroid signaling controls sex-specific development in an invertebrate

doi: 10.1101/2023.12.22.573099

Figure Lengend Snippet: (a) TFs are established in a control LL3 ovary ( tj>lacZ ). Engrailed (green) labels TF cells. (b) Blocking EcR activation in the somatic gonad ( tj>EcR DN ) prevents the establishment of terminal filaments in an L3 ovary. (c) In a control L3 testis, Fas3 (green) labels intact hub cells. (d) Activation of EcR by co-expressing EcR and Tai ( tj>EcR,tai ) in the male somatic gonad produces smaller hubs with visibly fewer cells. Fas3 (green) labels hub cells. (g) Quantification of hub cells per LL3 (blue dots) or L1 (yellow dots) testis in various genotypes. For statistical significance here and in subsequent figures, asterisks indicate statistical significance by Student’s t-test as follows: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Scale bars = 25 μm.

Article Snippet: The following antibodies were used: rabbit anti-Vasa (1:10,000; gift of Ruth Lehmann, Skirball Institute/NYU School of Medicine, NY, USA), guinea pig anti-Tj (1:1000), mouse anti-Fas3 (1:100; DSHB), rat anti-N-cadherin (1:20; DSHB), mouse anti-EcR (Ag10.2; 1:20; DSHB), mouse anti-Broad-core (25E9.D7; 1:40; DSHB), mouse anti-Broad-Z1 (1:50; DSHB), mouse anti-β-galactosidase (40-1a; 1:40; DSHB).

Techniques: Blocking Assay, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Steroid signaling controls sex-specific development in an invertebrate

doi: 10.1101/2023.12.22.573099

Figure Lengend Snippet: (a-b) Neither hub specification nor hub maintenance are compromised by blocking EcR activity ( tj>EcR- DN ). Fas3 (green) labels hub cells, Vasa (red) labels the germline, and Tj (blue) labels somatic cells. (c-e) Quantification of hub cell number (c), hub volume (d), and germline stem cell (GSC) number (e) upon blocking EcR activation in the somatic gonad. Scale bars = 10μm.

Article Snippet: The following antibodies were used: rabbit anti-Vasa (1:10,000; gift of Ruth Lehmann, Skirball Institute/NYU School of Medicine, NY, USA), guinea pig anti-Tj (1:1000), mouse anti-Fas3 (1:100; DSHB), rat anti-N-cadherin (1:20; DSHB), mouse anti-EcR (Ag10.2; 1:20; DSHB), mouse anti-Broad-core (25E9.D7; 1:40; DSHB), mouse anti-Broad-Z1 (1:50; DSHB), mouse anti-β-galactosidase (40-1a; 1:40; DSHB).

Techniques: Blocking Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Steroid signaling controls sex-specific development in an invertebrate

doi: 10.1101/2023.12.22.573099

Figure Lengend Snippet: (a-c) Representative images of early L1 (24-28h after egg lay, AEL) gonads, identified by the presence of a Fas3-positive hub (green). Ovaries lack a hub (a) while testes contain a Fas3-positive hub (green) by this stage (b). III staining. Co-expression of EcR and tai in the early somatic gonad (c) leads to a visibly smaller hub that contains significantly fewer hub cells than control L1 testes (see for quantification). Vasa (red) labels the germline, Tj (blue) labels somatic gonadal precursor cells, which give rise to the somatic gonad in both sexes. (d-e) Pupal testes upon EcR/Tai co-expression. In a control pupal testis (d), the germline and somatic stem cells are maintained by the presence of a hub (Fas3, green) and thus differentiating germ cells (Vasa, red) are observed in the testis. In some pupal testes, following EcR/Tai co-expression in the somatic gonad (e), no Fas3-positive hub cells are present and the absence of Vasa and DAPI staining indicates that both germline and somatic cell lineages have been lost. DAPI-positive muscle sheath cells can be found at the periphery of the testis. Scale bars = 10 μm.

Article Snippet: The following antibodies were used: rabbit anti-Vasa (1:10,000; gift of Ruth Lehmann, Skirball Institute/NYU School of Medicine, NY, USA), guinea pig anti-Tj (1:1000), mouse anti-Fas3 (1:100; DSHB), rat anti-N-cadherin (1:20; DSHB), mouse anti-EcR (Ag10.2; 1:20; DSHB), mouse anti-Broad-core (25E9.D7; 1:40; DSHB), mouse anti-Broad-Z1 (1:50; DSHB), mouse anti-β-galactosidase (40-1a; 1:40; DSHB).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Steroid signaling controls sex-specific development in an invertebrate

doi: 10.1101/2023.12.22.573099

Figure Lengend Snippet: (a-c) Chinmo expression in adult gonads and with EcR activation in the testis. In an adult ovary (a), Chinmo protein (green) is absent from the female somatic gonad (arrowheads). Chinmo is expressed in early female germ cells as previously observed . (b) In an adult testis, Chinmo is detectable in the hub and CySCs. (c) Chinmo expression is dramatically reduced upon EcR activation in the adult testis. (d-h) Expression of follicle cell markers Fas3 (red) and Castor (green) in adult gonads. Adult ovaries (d) show expression of Fas3 (red) and Castor (green) in early follicle cells, while in a control adult testis (e) Castor and Fas3 are absent from the cyst cell lineage. In adult testes, as seen in previous figures, Fas3 is expressed in hub cells. EcR activation in the testis (f) causes somatic feminization in the adult testis, characterized by the presence of Fas3- and Castor-positive somatic aggregates. Depletion of chinmo in the male somatic gonad (g) also causes somatic feminization as previously observed . Blocking EcR activation by over-expressing EcR DN (h) suppresses somatic feminization in the absence of Chinmo. (i) Quantification of feminization rescue in tj>chinmo RNAi testes by blocking EcR activation. An abbreviated description of phenotypic categories: 0=no feminization or germline defects; 1=germline defects but no feminization; 2=mild feminization; 3=moderate feminization; 4=severe feminization; 5=acellular testes. Feminization and acellularity are indicated on the graph in blue. More complete descriptions and visual examples of each phenotypic category can be found in Expression of Br-C (green) in the adult testis upon chinmo depletion. Br-C is expressed in follicle cells during mid-oogenesis in the adult ovary (j). Br-C is expressed at moderate levels in early cyst cells but is absent from differentiating cyst cells in an adult testis (k). In a tj>chinmo RNAi testis (l), Br-C is expressed in later cyst cells distal from the hub (hub and testis apex are out of frame in l and l’). Scale bars = 50 μm.

Article Snippet: The following antibodies were used: rabbit anti-Vasa (1:10,000; gift of Ruth Lehmann, Skirball Institute/NYU School of Medicine, NY, USA), guinea pig anti-Tj (1:1000), mouse anti-Fas3 (1:100; DSHB), rat anti-N-cadherin (1:20; DSHB), mouse anti-EcR (Ag10.2; 1:20; DSHB), mouse anti-Broad-core (25E9.D7; 1:40; DSHB), mouse anti-Broad-Z1 (1:50; DSHB), mouse anti-β-galactosidase (40-1a; 1:40; DSHB).

Techniques: Expressing, Activation Assay, Blocking Assay