Journal: bioRxiv

Article Title: Dlg5 and Cadherins are key to peripheral glia integrity

doi: 10.1101/2022.11.01.514384

Figure Lengend Snippet: Identification of PDZ proteins required in glial cells of the peripheral nerve. A : Summary of PDZ protein RNAi screen. Details of RNAi lines used to target each candidate gene can be found in Table 1. Each box represents an individual RNAi line with green meaning wild type and magenta representing abnormal glial phenotypes. B-G : Longitudinal sections of peripheral nerves from control and selections of positive candidate identified in the RNAi screen. All glial membranes labeled with mCD8∷RFP (green) and axons immunolabeled with anti-Futsch (22C10; magenta). The pan-glial driver repo-GAL4 was used and crossed to: (B-B”) Control ( w[1118] ) peripheral nerve. (C-C”) Rho-GEF2-RNAi. Swollen regions are indicated (C’, yellow arrowheads) and these swellings were accompanied by defasciculation of the axons in that region (white arrowheads, C’’). (D-D”) Dlg1-RNAi. Glial membranes were disrupted (D’, yellow arrowhead). (E-E”) Scrib-RNAi. Glial membranes were abnormal with accumulations (E’, yellow arrowhead). (F-F”) Loco-RNAi. Peripheral nerves were swollen and vacuole-like structures were observed within the glial membranes (F’, yellow arrowheads). (G-G”) Dlg5-RNAi. Peripheral nerves had disrupted inner glial membranes (G’, yellow arrowheads). The morphology of axons in these nerves were unaffected (G’’). Scale bars: 15μm

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-Inx2 (1:500) ( ); mouse anti-Futsch/22C10 (1:1000, DSHB); rabbit anti-GFP (1:600, Life Technology); mouse anti-GFP (1:300, Novus Biologicals), rabbit anti-Cherry (1:300, Abcam), mouse anti-βPS (1:50, CF.6G11) , rabbit anti-HRP (1:500, Jackson ImmunoResearch, West Grove, PA); mouse anti-Futsch/22C10 (1:1000, DSHB); rabbit anti-Nrv2.1 (1:1000, Abcam).

Techniques: Labeling, Immunolabeling

Journal: bioRxiv

Article Title: Dlg5 and Cadherins are key to peripheral glia integrity

doi: 10.1101/2022.11.01.514384

Figure Lengend Snippet: Knockdown of Dlg5 in the peripheral glial leads to disruptions of the SPG and WG. A-B : Endogenously tagged Dlg5 (Dlg5∷GFP, green) reveals expression within glia and axons in the PNS. All glial membranes labeled with mCD8∷RFP (repo-GAL4, magenta)(A) or with axons immunolabeled with anti-Futsch (22C10, magenta)(B). C-D : Perineurial glia. The PG driver 46F-GAL4 crossed to w[1118] (C) or Dlg5-RNAi (D). The PG membranes (mCD8∷RFP, green) and axons (magenta) in both appeared normal in longitudinal and cross sections. E-F : Subperineurial glia. The SPG driver Gli-GAL4 crossed to w[1118](E) or Dlg5-RNAi (F) SPG membranes were labeled with mCD8∷RFP (green) and WG membranes with Nrv2∷GFP (magenta). The SPG membrane in control ( E’ ) was continuous along the length of the nerve. In Dlg5-RNAi nerves ( G’ ), the SPG was disrupted with breaks/gaps in the membrane (yellow arrowheads). The WG in both control (E’’) and Dlg5-RNAi (G’’) nerves extended normal processes along the length of the nerve. An SPG nuclei (asterisk) is indicated. G-H : Wrapping glia. The WG driver Nrv2-GAL4 crossed to w[1118] (G) and Dlg5-RNAi ( H ). The WG membranes were labeled with mCD8∷RFP (green) and axons labeled with anti-Futsch (22C10, magenta). WG in control nerves (G”) extended normal processes along the length of the nerve and in cross-sections surrounded the axons (magenta). WG in Dlg5-RNAi nerves had fewer and disrupted processes (H”, yellow arrowheads). In cross-sections the WG processes did not extend around the axons (yellow arrowhead). Scale bars: 15μm

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-Inx2 (1:500) ( ); mouse anti-Futsch/22C10 (1:1000, DSHB); rabbit anti-GFP (1:600, Life Technology); mouse anti-GFP (1:300, Novus Biologicals), rabbit anti-Cherry (1:300, Abcam), mouse anti-βPS (1:50, CF.6G11) , rabbit anti-HRP (1:500, Jackson ImmunoResearch, West Grove, PA); mouse anti-Futsch/22C10 (1:1000, DSHB); rabbit anti-Nrv2.1 (1:1000, Abcam).

Techniques: Expressing, Labeling, Immunolabeling

Journal: bioRxiv

Article Title: Dlg5 and Cadherins are key to peripheral glia integrity

doi: 10.1101/2022.11.01.514384

Figure Lengend Snippet: Knockdown of Ecad in the WG and SPG affects glial morphology. A-B : All glia. repo-GAL4 crossed to Dicer2 (control) (A) and Ecad-RNAi, Dicer2 (B) Peripheral nerves with all glial membranes labeled with mCD8∷GFP (green). SJ were immunolabeled with Dlg1 (magenta). Glial membranes (A’) and a linear SJ (A”) extends along the length of the nerve in control. With Ecad-RNAi, glial membranes are disrupted (yellow arrowheads, B,B’) and vacuole-like or hole-like structures observed (white arrowheads, B’B”) and the SJ disorganized. C-D : Wrapping glia. Nrv2-GAL4 crossed to Dicer2 (control)(C) and Ecad, Dicer2 (D). Longitudinal sections of peripheral nerves with WG membranes labeled with mCD8∷GFP (green) and axons immunolabeled with anti-Futsch (22C10, magenta). The WG membranes continuously extend along the length of control (green, C,C’). With Ecad-RNAi, WG membranes were discontinuous and disrupted (yellow arrowheads, D,D’). The axon morphology in Ecad-RNAi peripheral nerves looked similar to controls (magenta, D,D’’). E-F : Subperineurial glia. Gli-GAL4 crossed to Dicer 2 (control)(E) and Ecad-RNAi, Dicer2 (F) with the SPG membrane labeled with mCD8∷RFP (magenta) and the SJ immunolabeled with Dlg1 (green). The SPG membrane is continuous along the length of control (magenta, E”) and the SJ forms a continuous line (E’). In Ecad-RNAi nerves the membrane was discontinuous and disrupted (yellow arrowheads, F”) and whereas Dlg1 was lost in regions of the nerve where the SPG membrane was disrupted. Scale bars: 15μm

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-Inx2 (1:500) ( ); mouse anti-Futsch/22C10 (1:1000, DSHB); rabbit anti-GFP (1:600, Life Technology); mouse anti-GFP (1:300, Novus Biologicals), rabbit anti-Cherry (1:300, Abcam), mouse anti-βPS (1:50, CF.6G11) , rabbit anti-HRP (1:500, Jackson ImmunoResearch, West Grove, PA); mouse anti-Futsch/22C10 (1:1000, DSHB); rabbit anti-Nrv2.1 (1:1000, Abcam).

Techniques: Labeling, Immunolabeling

Journal: Disease Models & Mechanisms

Article Title: Nmnat mitigates sensory dysfunction in a Drosophila model of paclitaxel-induced peripheral neuropathy

doi: 10.1242/dmm.032938

Figure Lengend Snippet: Dendritic Futsch organization does not require Nmnat. (A-B′) Confocal projections showing immunostaining of microtubule-associated protein Futsch within sensory dendrites from larvae with C4da neuronal RNAi-mediated knockdown of Nmnat (B; ppk-Gal4>UAS-Dcr,UAS-NmnatRNAi ) and controls (A; ppk-Gal4>UAS-Dcr ). C4da neurons exhibit continuous Futsch staining throughout the major branches, even in those with substantial dendrite loss (B). Scale bar: 50 µm. (C) Futsch intensity measured within C4da neuronal compartments, i.e. cell body, major proximal dendrite branches and distal dendrite branches, in larvae with C4da RNAi-mediated knockdown of Nmnat and controls. Mean±s.e.m.; n =5 neurons from >4 larvae from each genotype, Student's t -test. (D-E′) Confocal projections of Futsch immunostaining and C4da expression of CD4:tdGFP in Nmnat heterozygous larvae (E; Δnmnat/+ ) and controls. (F) Futsch intensity measured within C4da neuronal compartments in larvae heterozygous for Nmnat and controls. Mean±s.e.m.; n =5 neurons from >4 larvae from each genotype, Student's t -test. * P <0.05.

Article Snippet: Secondary antibodies used at 1:250 dilutions were: Alexa-Fluor-555 anti-mouse for Futsch visualization, Alexa-Fluor-555 anti-guinea pig for Nmnat visualization, and Alexa-Fluor-647 anti-mouse IgG1 and Alexa-Fluor-568 anti-mouse IgG2a for Futsch and FasIII co-staining (Invitrogen).

Techniques: Immunostaining, Staining, Expressing

Journal: Disease Models & Mechanisms

Article Title: Nmnat mitigates sensory dysfunction in a Drosophila model of paclitaxel-induced peripheral neuropathy

doi: 10.1242/dmm.032938

Figure Lengend Snippet: Nmnat does not prevent paclitaxel-induced dendrite density or Futsch disruption. (A-D) Confocal projections showing ddaC sensory dendrites labeled with CD4:tdGFP from control genotype treated with vehicle (A) or paclitaxel (B), and larvae overexpressing Nmnat in C4da sensory neurons ( UAS-Nmnat ) treated with vehicle (C) or paclitaxel (D). Scale bar: 50 µm. (A′-D′) High magnification of dendritic regions boxed in A-D, showing immunostaining of microtubule-associated Futsch in C4da dendrites (arrowheads) and other classes of peripheral neurons. Scale bar: 50 µm. (E-G) Quantification of dendritic field (E), number of terminal branches (F) and terminal branch density (G) of the dorsal projections of ddaC neurons from ppk-Gal4 control larvae and larvae overexpressing Nmnat in C4da sensory neurons after 48 h treatment with vehicle or paclitaxel. Means±s.e.m.; n =14 neurons from >7 larvae form each genotype and treatment; one-way ANOVA with Tukey's multiple comparisons test. * P <0.05, **** P <0.0001; ns, nonsignificant.

Article Snippet: Secondary antibodies used at 1:250 dilutions were: Alexa-Fluor-555 anti-mouse for Futsch visualization, Alexa-Fluor-555 anti-guinea pig for Nmnat visualization, and Alexa-Fluor-647 anti-mouse IgG1 and Alexa-Fluor-568 anti-mouse IgG2a for Futsch and FasIII co-staining (Invitrogen).

Techniques: Labeling, Immunostaining