Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: Slit and Robo expression patterns during the development of the visual system. (A) Diagram of the distribution of neurons in the L3 larval stage, horizontal view. R1–6 photoreceptors project their axon from the eye imaginal disc to the lamina (La), while R7–8 photoreceptors project them toward the medulla (Me). Perpendicular to the photoreceptor axons, the Eyeless (Ey+) neurons project their axons through the medulla. (B–M) Immunofluorescence of Slit and Robo (red) in an ey OK107 -GAL4 driving CD8-GFP (green) larva (L3 stage) showing the expression patterns in different developing neuropils. Ey + medulla neurons are delimited by the dotted line. (B–D) Expression pattern of Slit shows a homogeneous distribution and similar intensity in the medulla and lobula complex. (E–G) Robo1 expression pattern is similar to Slit expression. (H–J) With Robo2, we used Robo2-HA endogenously tagged for visualizing this receptor. Its expression pattern shows distribution in somas of T4/T5 neurons and high expression in the lobule complex, while weaker staining is observed in the medulla neuropil. (K–M) Robo3 is expressed in all optic lobe neuropils and shows a punctate distribution in somas of Ey + medulla neurons similar to Slit. La, lamina; Me, medulla; Lp, lobula plate; Lo, lobula. Schematic representation inspired by . N = 3 for all experiments. Single slice is presented. Scale bar: 30 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: Slit/Robo3 co-localization in Ey + neurons in the developing visual system. (A) Diagram of Ey + neurons in the L3 larval stage, frontal view. The growth cones of Ey + medulla neurons are located in the medulla (Me) next to the Lamina plexus (pLa), which delimits the medulla and lamina. We labeled with anti-Slit and anti-HA (endogenously tagged, Robo3-HA ). (a–D) Visualization of an area of the medulla shows that Slit and Robo3 have similar localization patterns and some punctate structures co-localize. The most enriched area for both proteins is next to the plexus region (a```) with Manders coefficient M1 = 0.53. There is also important co-localization in neuronal projections with M1 = 0.4 (a``) and in the Soma with M1 = 0.23 (a`) . N = 5. Single slice. (E) Slit-GFP reporter line labeled with anti-Robo3 (red, e` ) and GFP (green, e`` ). Visualization of medulla area development shows Robo3 presence in Slit + cells (arrow). N = 3. Single slice. AL: antennal lobe and MB: mushroom bodies. All images have a scale bar = 15 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: slit dui , robo3 3 , and Robo knockdown phenotypes in the optic lobe. (A) Diagram of the fly adult brain focused on the optic lobe, frontal view. Medulla (Me), R7–8 photoreceptor axons, and lobula complex (Lo and Lp) is indicated. (B) Immunofluorescence of the adult stage using anti-Chaoptin (photoreceptors) and N-Cadherin (neuropil) shows that robo3 3 null mutation in heterozygosity displays a control phenotype. (C) slit dui hypomorphic mutant has a disrupted medulla and ectopic photoreceptor fascicules (arrow). (D) robo3 3 null mutation in homozygosity displays a similar phenotype. (E) Slit knockdown (KD) in Ey + cells show similar phenotypes as the slit or robo mutations previously described. (F–J) Phenotypes of Robos KD in Ey + neurons. Experimental control (F) as well as Robo1 KD (G) and Robo2 KD (H) show a wild-type phenotype. (I) Robo3 KD phenotype is similar to robo3 and slit mutant animals. (J) Using another Robo3-RNAi line in a robo3 mutant background heterozygote also show a disrupted medulla phenotype. N = 10 for all experimental conditions. All images are from single slices. Scale bar: 30 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: Immunofluorescence, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: Rab GTPases co-localize with Slit and Robo3 in Ey + neurons in vivo . (A–C) Expression pattern of Rab5, Rab7, and Rab11 reporters tagged with GFP in Ey + neurons (larval stage) frontal view. For better visualization, Rab11-GFP and Rab7-GFP were labeled using anti-GFP. Rab5 and Rab11 are enriched in the axon growth cone (plexus region), while Rab7 is enriched in the Soma of Ey + neurons. (D–O) Slit and Robo3 immunofluorescence show different levels of co-localization with Rab proteins, indicated with arrow heads for somas and arrows for projections. Co-localization is presented using Manders coefficients. Rab5 has a higher level of co-localization with Slit in Soma (M = 0.71) (D) vs. projection (M1 = 0.46) (E) . Rab7 has similar levels of co-localization with Slit in Soma (M1 = 0.43) (H) and projection (M1 = 0.38) (I) . In the case of Rab11, high levels of co-localization with Slit are observed in both compartments (M1 = 0.85) (L–M) . On the other hand, Rab5 and Robo3 show similar levels of co-localization in Soma (M1 = 0.27) (F) and axon (M1 = 0.28) (G) . Rab7 shows low levels of co-localization with Robo3 in both compartments, soma (M1 = 0.15) (J), and projection (M1 = 0.14) (K) . Rab11 has the highest levels of co-localization with Robo3, M1 = 0.65 in the Soma (N) and M1 = 0.55 in the projection (O) . N = 5. All images are from single slices. Scale bar: 15 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: In Vivo, Expressing, Labeling, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: Robo3 co-localized with Rabs in Ey + neurons in vitro. Primary cell culture of Ey + neurons expressing Rab5-GFP ( N = 5, (A–B) ), Rab7-GFP ( N = 4, (C–D) ), and Rab11-GFP ( N = 4, (E–F) ) treated with mock or Slit-myc-Cherry conditioned medium (CM), respectively. Closed-up views: Robo3 (red), Rabs-GFP (green), and DNA (blue) are present in a`-f```. For staining the internalized protein, we performed an acidic wash and immunofluorescence against Robo3 (red). Arrows shows co-localization puncta. After 15 min treatment, Rab7-GFP shows higher levels of co-localization with Robo3 in the presence of Slit compared with the mock treatment (H) . Rab5 (G) and Rab11 (I) show no significant differences but there is a tendency toward increase co-localization with Slit-myc-Cherry treatment. (J) Schematic representation of Robo3 and Slit (information on ) shows an endocytic recycling route. Error bar: Mean ± SEM. The Mann–Whitney test, * p < 0.05. Images are Z projections from two slices, Scale bar: 15 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: In Vitro, Cell Culture, Expressing, Staining, Immunofluorescence, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: Genetic interaction between slit , robo3 , rab GTPases , and clathrin . (A) Schematic representation of an optic lobe in the adult stage. Close-up frontal views of the medulla neuropil. Immunofluorescence against Chaoptin (photoreceptors, green), N-Cadherin (neuropils, magenta), and DNA (blue) of flies expressing ey 3.5 -GAL80 and ey OK107 -GAL4 , which allows the expression of transgenes carrying the UAS promoter in Ey (+) medulla neurons, while repressing expression in the eye disc (Hoechst is included as counterstaining, blue). Dominant-negative (DN) forms of Rabs or RNAi for Clathrin were expressed. (B) slit 2 /+ experimental control shows some minor defects, consisting of occasional ectopic photoreceptor axons (arrow, N = 60). (C) robo3 3 /+ has a wild-type phenotype ( N = 60). (D) slit 2 /robo3 3 shows ectopic photoreceptor axons (arrow, N = 16). (E) ChcRNAi/+ flies display mild disorganization of photoreceptors, and it is the only GI that has a swelling axon (arrowhead, N = 30). (F) slit 2 /ChcRNAi flies show a medulla disruption phenotype ( N = 15), which is also seen in robo3 3 /ChcRNAi (G) , asterisk, N = 15). (H) rab5DN/+ ( N = 30) and (K) rab7DN/+ ( N = 30) show wild-type phenotypes. (I) slit 2 /rab5DN ( N = 15). (J) robo3 3 /rab5DN ( N = 15). (L) slit 2 /rab7DN ( N = 15) and (M) robo3 3 /rab7DN ( N = 15) display ectopic photoreceptor axons (arrow). (N) rab11DN/+ flies show mild photoreceptor disorganization ( N = 30), while (O) slit 2 /rab11DN ( N = 15) and robo3 3 /rab11DN (P) , N = 15) show strong photoreceptor disorganization and occasional disruption of the medulla. (Q) Graph shows the frequency of photoreceptor phenotypes evaluated: Wild-type, Type 1 (one ectopic photoreceptor axon), Type 2 (≥2 ectopic photoreceptor axon), Type 3 (mild disorganization of photoreceptors), Type 4 (strong disorganization of photoreceptors), and Type 5 (photoreceptor disorganized + ectopic axons). (R) Graph displays the frequency of medulla disrupted phenotypes. N = 15 for every GI experiment. Images are Z projections from five slices. Scale bar: 30 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: Immunofluorescence, Expressing, Dominant Negative Mutation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

doi: 10.3389/fcell.2022.874362

Figure Lengend Snippet: Genetic interactions between slit , robo3 , and Rho GTPases . (A) Schematic representation of an optic lobe in the adult stage. Close-up frontal views of the medulla neuropil. Immunofluorescence against Chaoptin (photoreceptor, green), N-Cadherin (neuropils, magenta), and DNA (blue) in the ey 3.5 -GAL80 and ey OK107 -GAL4 background. Dominant-negative constructs for small Rho GTPase proteins were expressed in Ey + medulla neurons as indicated. (B) slit 2 /+ shows few ectopic photoreceptor axons (arrow, n = 45). (C) robo3 3 /+ flies show a wild-type phenotype ( N = 45). (D) cdc42DN/+ flies display ectopic photoreceptor axons (arrow, N = 30). (E) slit 2 /cdc42DN flies display two or more ectopic photoreceptor axons ( N = 15). (F) robo3 3 /cdc42DN photoreceptor axons are disorganized ( N = 15). (G) racDN/+ presents an ectopic photoreceptor axon and disrupted medulla (asterisk, N = 30), but slit2/racDN (N = 15, H ) and robo3 3 /racDN ( N = 15, I ) flies show higher frequency of disrupted medulla phenotype. (J) rhoADN/+ flies show ectopic photoreceptor axons ( N = 30). (K) slit 2 /rhoADN ( N = 15) and (M) robo3 3 /rhoADN ( N = 15) display a similar phenotype to rhoADN/+ . (O) Graph showing the frequency of photoreceptors phenotypes that were evaluated: Wild-type, Type 1 (one ectopic photoreceptor axon), Type 2 (≥2 ectopic photoreceptor axons), Type 3 (mild disorganization of photoreceptors), Type 4 (strong disorganization of photoreceptors), and Type 5 (disorganized photoreceptors + ectopic axons). (P) Graph shows the frequency of the disrupted medulla phenotype. Images are Z projections from five slices. Scale bar: 30 μm.

Article Snippet: Primary monoclonal antibodies obtained from Developmental Studies Hybridoma Bank are mouse anti-Slit (C55.6D; 1:10), mouse anti-Robo1 (13c9; 1:50), mouse anti-Robo3 (14c9, 1:50), mouse anti-Chaoptin (24B10; 1:10), and rat anti N-Cadherin (DN-Ex #8; 1:10).

Techniques: Immunofluorescence, Dominant Negative Mutation, Construct

Journal: Journal of Bacteriology

Article Title: Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A , spo0B , and spo0F in Phage AP50c Infection of Bacillus anthracis

doi: 10.1128/JB.00739-13

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: It is temperature sensitive for replication and contains a chloramphenicol resistance gene for Gram-positive organisms, as well as a gene encoding the C9 variant of the Himar1 transposase and an erythromycin resistance gene, located between two inverted repeats that are transposed by the transposase. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid a Description Source or reference Strains Escherichia coli GM119 dam dcm mutant 36 C2925 dam dcm mutant New England Biolabs B. anthracis 34F 2 Sterne/pXO1 + /pXO2 − P. C. Hanna lab 7702 Sterne/pXO1 + /pXO2 − T. M. Koehler lab JB220 7702 ΔBAS2245 This study BAP350 7702 Δ csaB This study BA749 7702 ΔBAS0566 This study BA750 7702 Δ sap This study BA751 7702 Δ eag This study BA752 7702 ΔBAS1792 This study BA753 7702 ΔBAS3559 This study BA754 7702 Δ spo0A This study BA755 7702 Δ spo0F This study DP-B-5747 JB220 Δ spo0B This study B. cereus RS110 ATCC 4342 60 RS112 RTS100 ( B. anthracis -like) RS415 ATCC 25621 61 RS416 ATCC 43881 62 RS417 ATCC 14893 63 RS431 ATCC 27877 64 RS432 ATCC 7064 (NRS201) 60 RS436 B. cereus transition state strain CDC13100 47 RS437 B. cereus transition state strain CDC13140 47 RS438 B. cereus transition state strain CDC32805 47 RS710 B. cereus var.

Techniques: Plasmid Preparation, Mutagenesis, Expressing

Journal: Bioscience Reports

Article Title: Regulation of 5-oxo-ETE synthesis by nitric oxide in human polymorphonuclear leucocytes upon their interaction with zymosan and Salmonella typhimurium

doi: 10.1042/BSR20130136

Figure Lengend Snippet: ( A , B ) PMNLs (2×10 7 ) were incubated for 30 min at 37°C without additives or with 1 μM of different LPS forms or PMA (1–5 nM, as indicated), and then 2 mg/ml of zymosan was added for 15 min ( A ) or 5×10 8 S. typhimurium was added for 20 min ( B ). OZ or OS was added simultaneously with DEA NONOate (final concentration, 500 μM). The results represent the means±S.D. of four independent experiments, * P <0.05 and ** P <0.01, compared with the corresponding controls without PMA or LPS. ( C , D ) PMNLs (2×10 7 ) were incubated for 30 min at 37°C with 2 nM PMA, and then either 2 mg/ml OZ was added for 15 min ( C ) or 5×10 8 OS was added for 20 min ( D ). OZ or OS was added simultaneously with DEA NONOate at the indicated final concentration of NONOate (0.1–1 mM). The results represent the means±S.D. of four independent experiments, * P <0.05 and ** P <0.01, compared with the corresponding data without NO donor. ( E , F ) Time course of the 5-LOX reaction in PMNLs. PMNLs were incubated with 2 nM PMA for 30 min and then exposed to NO_OZ ( E ) or NO_OS ( F ) in a final concentration of 500 μM NONOate. The products generated by 5-LOX were extracted from the medium and separated using HPLC. The results are means±S.D. of three independent experiments performed in duplicate.

Article Snippet: HBSS (Hanks balanced salt solution) with calcium and magnesium but without phenol red and sodium hydrogen carbonate, Dulbecco's PBS with magnesium but without calcium, Dnp-Cl (1-chloro-2,4-dinitrobenzene), diamide and LPSs from Salmonella enterica serovar Typhimurium (smooth form derived from the strain ATCC 7823, Ra mutant TV119 and Re mutant SL1181) were from Sigma-Aldrich.

Techniques: Incubation, Concentration Assay, Generated