Journal: Neurotoxicology

Article Title: Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity

doi: 10.1016/j.neuro.2018.04.016

Figure Lengend Snippet: Examples of live imaging

Article Snippet: Target Method Brain region or cell type Reference Dendrites Transgenic lines controlled by isl2b or brn3c RGC specific promoters and transient expression driven by a Gal4/UAS system controlled by the same promoters RGCs (Mumm et al., 2006 ) Transgenic lines expressing membrane bound fluorescent proteins driven by either isl2b or brn3c RGC specific promoters RGCs (Choi et al., 2010 ) Membrane-targeted yellow fluorescent protein driven by the Purkinje cell specific aldoca (aldolase c, fructose-biphosphate a) promoter Purkinje cells (Tanabe et al., 2010 ) Dendrites and synaptic connections GAL4 driver controlled by the pan-neuronal goldfish alpha-1 tubulin promoter and UAS activator expresses dsRed (whole cell) and PSD95:GFP (presynaptic marker) Tectum (Niell et al., 2004 ) Synaptic connections Live staining using DiD (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine) and DiO (3,3’-dioctadecyloxacarbocyanine perchlorate) Spinal neurons (Jontes et al., 2000 ) Marked presynaptic terminals using GFP-linked N-cadherin, and verified by counterstaining for the synaptic vesicle protein SV2 Spinal neurons (RB) (Jontes et al., 2004 ) GFP-Synaptophysin and cytosolic DsRed, both driven by a Gal4/UAS system and targeted using an RGC-specific brn3C promoter Optic tectum and RGCs (Meyer and Smith, 2006 ) Transgenic line neurogenin1:GFP and fluorescently tagged synaptic proteins using the Gal4/UAS system driven by a RB neuron specific promoter Spinal neurons (RB) (Easley-Neal et al., 2013 ) Synaptic activity Calcium indicator dye directly injected into the tectal neuropil Tectum (Niell and Smith, 2005 ) The genetically encoded calcium indicator (GECI) transgenic lines under control of various neuronal promoters Various (Akerboom et al., 2012 ) Open in a separate window RGC = retinal ganglion cells RB = Rohon Beard cells Examples of live imaging 4.3.1.

Techniques: Transgenic Assay, Expressing, Marker, Staining, Activity Assay, Injection

Journal: Neurotoxicology

Article Title: Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity

doi: 10.1016/j.neuro.2018.04.016

Figure Lengend Snippet: Zebrafish resources for gene expression information

Article Snippet: Target Method Brain region or cell type Reference Dendrites Transgenic lines controlled by isl2b or brn3c RGC specific promoters and transient expression driven by a Gal4/UAS system controlled by the same promoters RGCs (Mumm et al., 2006 ) Transgenic lines expressing membrane bound fluorescent proteins driven by either isl2b or brn3c RGC specific promoters RGCs (Choi et al., 2010 ) Membrane-targeted yellow fluorescent protein driven by the Purkinje cell specific aldoca (aldolase c, fructose-biphosphate a) promoter Purkinje cells (Tanabe et al., 2010 ) Dendrites and synaptic connections GAL4 driver controlled by the pan-neuronal goldfish alpha-1 tubulin promoter and UAS activator expresses dsRed (whole cell) and PSD95:GFP (presynaptic marker) Tectum (Niell et al., 2004 ) Synaptic connections Live staining using DiD (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine) and DiO (3,3’-dioctadecyloxacarbocyanine perchlorate) Spinal neurons (Jontes et al., 2000 ) Marked presynaptic terminals using GFP-linked N-cadherin, and verified by counterstaining for the synaptic vesicle protein SV2 Spinal neurons (RB) (Jontes et al., 2004 ) GFP-Synaptophysin and cytosolic DsRed, both driven by a Gal4/UAS system and targeted using an RGC-specific brn3C promoter Optic tectum and RGCs (Meyer and Smith, 2006 ) Transgenic line neurogenin1:GFP and fluorescently tagged synaptic proteins using the Gal4/UAS system driven by a RB neuron specific promoter Spinal neurons (RB) (Easley-Neal et al., 2013 ) Synaptic activity Calcium indicator dye directly injected into the tectal neuropil Tectum (Niell and Smith, 2005 ) The genetically encoded calcium indicator (GECI) transgenic lines under control of various neuronal promoters Various (Akerboom et al., 2012 ) Open in a separate window RGC = retinal ganglion cells RB = Rohon Beard cells Examples of live imaging 4.3.1.

Techniques: Expressing, Generated

Journal: Development (Cambridge, England)

Article Title: Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis

doi: 10.1242/dev.056408

Figure Lengend Snippet: The SB tracheoblast clusters grow extensively during the L3 stage. (A) SB tracheoblasts of a stage 15 cut-Gal4; UAS-srcGFP (green) Drosophila embryo stained for Cut (red); three tracheal segments are shown overlaid on a phase-contrast image of the embryo. Membrane-tagged srcGFP highlights the close association of the SB with the embryonic epidermis. (B,B′) Early L2 (48 hours AEL) cut-Gal4; UAS-srcGFP (green) larva stained for DAPI (blue); all SB cells are Cut+. (C-C′) Early L2 (48 hours AEL) esg-Gal4 UAS-EGFP (green in C,C′) larva stained for Cut (red in C,C′) and DAPI (blue); all SB cells co-express Cut and Esg (outside the SB, Cut stains muscle nuclei). Arrowheads in B and C indicate the DT. (D-J) Development of the Tr5 SB tracheoblast cluster in btl-Gal4 UAS-GFP (green) animals stained for Cut (red). (D) Stage 15 embryo. (E) Early L2 larva (48 hours AEL). (F) Early L3 larva (72 hours AEL). (G) Mid-L3 larva (90 hours AEL). (H) Mid-L3 larva (100 hours AEL). (I) Wandering late L3 larva (120 hours AEL). (J) White prepupa (121 hours AEL). Brackets in D-F indicate the SB Cut+ cells. Scale bar: 20 μm.

Article Snippet: Numbers in parentheses correspond to Bloomington stock numbers: btl-Gal4 UAS-actGFP (#8807), UAS-srcGFP (#5432), fzr-lacZ G0326 (#12241), tub-Gal80 ts (#7016) ( McGuire et al., 2004 ), cut-Gal4 (PG142) ( Bourbon et al., 2002 ), UAS-cut (gift from B. Mathey-Prevot), UAS-cut RNAi (VDRC#5687) ( Dietzl et al., 2007 ), esg-Gal4 tub-Gal80 ts UAS-EGFP ( Micchelli and Perrimon, 2006 ), UAS-FLP ( Duffy et al., 1998 ), act5C-FRT-stop-FRT-lacZ ( Struhl and Basler, 1993 ) and hsFLP; act5C-FRT-stop-FRT-Gal4 UAS-GFP ( Ito et al., 1997 ).

Techniques: Staining

Journal: Development (Cambridge, England)

Article Title: Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis

doi: 10.1242/dev.056408

Figure Lengend Snippet: The transcription factors Cut, Hindsight and Escargot, and FGFR/Breathless, label the SB tracheoblasts in the late Drosophila larva. (A) Schematic of a Drosophila third instar larval tracheal system; blue box demarcates Tr4 and Tr5. (B) Schematic of the boxed region in A of an early larva (L1-L2), indicating the different tracheal branches (DB, dorsal branch; DT, dorsal trunk; TC, transverse connective; VB, visceral branch; SB, spiracular branch). The SB (red) corresponds to the original location of the SB tracheoblasts, whereas all other branches (green) correspond to gas-transporting Btl-positive tubes. (C) Schematic of the boxed region in A of a late L3 larva/early pupa; the SB cells (red) have proliferated and migrated on the TC, VB and DT. (D-D′) btl-Gal4 UAS-GFP (green in D,D′) L3 larva stained for Cut (red in D,D′) and DAPI (blue); Cut is also present in differentiated muscle cells with large nuclei. (E-E′) btl-Gal4 UAS-GFP (green in E,E′) L3 larva stained for Hnt (red in E,E′) and DAPI (blue); Hnt is expressed in all gas-transporting tracheal cells. (F,F′) esg-Gal4 UAS-GFP (green) late L3 larva stained for Cut (red) and DAPI (blue). White arrowheads show Esg+ Zone 4 cells and yellow arrows mark the Esg+ fusion cell on the DT. Cut is not expressed in the fusion cell. Brackets in D′,E′,F′ indicate Zone 2. (G,G′) Low-magnification view of the Tr4 and Tr5 of a btl-Gal4 UAS-GFP (green) L3 larva labeled with DAPI (blue). Scale bar: 20 μm in A-F′; 40 μm in G,G′. (H) Schematic of the four different Zones of the SB.

Article Snippet: Numbers in parentheses correspond to Bloomington stock numbers: btl-Gal4 UAS-actGFP (#8807), UAS-srcGFP (#5432), fzr-lacZ G0326 (#12241), tub-Gal80 ts (#7016) ( McGuire et al., 2004 ), cut-Gal4 (PG142) ( Bourbon et al., 2002 ), UAS-cut (gift from B. Mathey-Prevot), UAS-cut RNAi (VDRC#5687) ( Dietzl et al., 2007 ), esg-Gal4 tub-Gal80 ts UAS-EGFP ( Micchelli and Perrimon, 2006 ), UAS-FLP ( Duffy et al., 1998 ), act5C-FRT-stop-FRT-lacZ ( Struhl and Basler, 1993 ) and hsFLP; act5C-FRT-stop-FRT-Gal4 UAS-GFP ( Ito et al., 1997 ).

Techniques: Staining, Labeling

Journal: Development (Cambridge, England)

Article Title: Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis

doi: 10.1242/dev.056408

Figure Lengend Snippet: The SB consists of dividing Cut-positive cells and endocycling Hnt/Btl-positive cells. (A,A′) SB cells of btl-Gal4 UAS-GFP (green) L3 larva undergoing S-phase labeled with EdU (red in A,A′); EdU-positive cells are found throughout the SB. (B-B′) SB tracheoblasts of btl-Gal4 UAS-GFP (green) L3 larva stained for pH3 (red in B,B′) and Cyclin B (white in B′). DAPI is blue in A,B. (C,C′) DAPI quantification of Zone 1 and Zone 3 cells; at the late L3 stage, Zone 1 cells have approximately twice as much DNA than do Zone 3 cells (n=40 cells, P<0.001). (D) Number of pH3+ cells in different SB Zones. Zones 2 and 3 contain, on average, 2.75 and 0.66 pH3+ cells, respectively (n=24 SBs, P<0.001). (E-E′) Fzr-lacZ (red in E,E′) coincides with Hnt-positive Zone 1 tracheoblasts (green in E,E′). DAPI is blue in E. (F) Graph of heat-shock-induced mitotic clones in the different SB zones. Seventeen SB clusters were analyzed that each contained two or three clones, encompassing cells in Zone 2, Zone 3 and/or Zone 4. Zone 2 clones proliferate faster (larger size) compared with synchronous Zone 3 or 4 clones. Zone 4 clones are induced infrequently and contain one or two cells that do not proliferate during larval stages. (G) Tr5 tracheal metamere of a Fzr-lacZ 11 hour APF pupa stained for β-galactosidase (red), Cut (green) and DAPI (blue). (H) DAPI quantification of the different SB populations from images similar to the one in G. Cut+=Zone 3 cells; Fzr+SB=Zone 1 cells; Fzr+TR=mature tracheal cells. Scale bars: in A, 20 μm for A-E′; in G, 20 μm.

Article Snippet: Numbers in parentheses correspond to Bloomington stock numbers: btl-Gal4 UAS-actGFP (#8807), UAS-srcGFP (#5432), fzr-lacZ G0326 (#12241), tub-Gal80 ts (#7016) ( McGuire et al., 2004 ), cut-Gal4 (PG142) ( Bourbon et al., 2002 ), UAS-cut (gift from B. Mathey-Prevot), UAS-cut RNAi (VDRC#5687) ( Dietzl et al., 2007 ), esg-Gal4 tub-Gal80 ts UAS-EGFP ( Micchelli and Perrimon, 2006 ), UAS-FLP ( Duffy et al., 1998 ), act5C-FRT-stop-FRT-lacZ ( Struhl and Basler, 1993 ) and hsFLP; act5C-FRT-stop-FRT-Gal4 UAS-GFP ( Ito et al., 1997 ).

Techniques: Labeling, Staining, Clone Assay

Journal: Development (Cambridge, England)

Article Title: Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis

doi: 10.1242/dev.056408

Figure Lengend Snippet: SB larval tracheoblasts originate from multipotent embryonic Cut-positive cells. (A-A′) Early lineage clone of a UAS-FLP/cut-Gal4; UAS-srcGFP/act5C-FRT-stop-FRT-lacZ larva reared at 25°C; the Gal4 expression domain is GFP+ (green in A,A′) and the lineage-traced cells are LacZ+ (red in A,A′). All SB cells are LacZ+. (B-B′) Late lineage clone of a UAS-FLP/cut-Gal4 tub-Gal80ts; UAS-srcGFP/act5C-FRT-stop-FRT-lacZ larva kept at 18°C and shifted to 29°C 3 days before dissection; only cells in Zone 3 are LacZ+. (C,C′) X-gal staining of the adult abdomen of a UAS-FLP/cut-Gal4; UAS-srcGFP/act5C-FRT-stop-FRT-lacZ animal reared at 25°C, showing LacZ+ cells in the spiracles and tracheal tubes. C and C′ correspond to surface and internal focal planes, respectively. (D) X-gal staining of adult abdomen of a UAS-FLP/cut-Gal4 tub-Gal80ts; UAS-srcGFP/act5C-FRT-stop-FRT-lacZ lineage-traced animal showing restriction of signal to the spiracles. (E-E′) Early lineage clone of a UAS-FLP; btl-Gal4 UAS-srcGFP/act5C-FRT-stop-FRT-LacZ larva reared at 25°C; the Gal4 expression domain is GFP+ (green in E,E′) and the lineage-traced cells are LacZ+ (red in E,E′). The presence of LacZ+ cells in Zone 3 indicates that early btl-Gal4-expressing cells contribute to Zone 3. (F-F′) Late lineage clone of a UAS-FLP/tub-Gal80ts; btl-Gal4 UAS-srcGFP/act5C-FRT-stop-FRT-lacZ larva kept at 18°C and shifted to 29°C 3 days before dissection; only cells in Zone 1 are LacZ+, indicating that at this stage the SB cells are restricted. (G,G′) X-gal staining of adult abdomen of UAS-FLP/tub-Gal80ts; btl-Gal4 UAS-srcGFP/act5C-FRT-stop-FRT-lacZ lineage-traced animal showing restriction of the LacZ+ cells to the tracheal tubes. (G′) Twofold magnification of E. In C,D,G′, red arrowheads indicate the spiracles and yellow arrows indicate the tracheal tubes. In A,B,E,F, DAPI is blue. Scale bar: 20 μm in A-B′,E-F′.

Article Snippet: Numbers in parentheses correspond to Bloomington stock numbers: btl-Gal4 UAS-actGFP (#8807), UAS-srcGFP (#5432), fzr-lacZ G0326 (#12241), tub-Gal80 ts (#7016) ( McGuire et al., 2004 ), cut-Gal4 (PG142) ( Bourbon et al., 2002 ), UAS-cut (gift from B. Mathey-Prevot), UAS-cut RNAi (VDRC#5687) ( Dietzl et al., 2007 ), esg-Gal4 tub-Gal80 ts UAS-EGFP ( Micchelli and Perrimon, 2006 ), UAS-FLP ( Duffy et al., 1998 ), act5C-FRT-stop-FRT-lacZ ( Struhl and Basler, 1993 ) and hsFLP; act5C-FRT-stop-FRT-Gal4 UAS-GFP ( Ito et al., 1997 ).

Techniques: Expressing, Dissection, Staining

Journal: Development (Cambridge, England)

Article Title: Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis

doi: 10.1242/dev.056408

Figure Lengend Snippet: Cut is necessary for SB tracheoblast survival and morphogenesis of the adult airways. (A,A′) Wild-type late third instar larva stained for Cut (green), caspase 3 (red) and DAPI (blue). (B,B′) cutDB7 mutant clones (lack of GFP) stained with caspase 3 (red) and DAPI (blue). (C,C′) cutDB7 mutant clones (lack of GFP) stained with Cyclin B (red) and DAPI (blue). Cells in the upper clone found in Zone 2 have slightly larger nuclei. (D) Graph comparing cutDB7 mutant clones with their wild-type twin spots. Cells of 18 mosaic pairs were analyzed demonstrating that cutDB7 clones contain about half the number of cells compared with their wild-type twin spots. (E) Graph showing the average number of cells in cutDB7 clones and their wild-type twin spots (from D). (F,F′) tub-Gal80ts; btl-Gal4 UAS-srcGFP/UAS-cut larva reared at 25°C stained for Cut (red) and DAPI (blue). (G-G′) FLPout clones overexpressing cut (green) in Zone 1 suppress Fzr-lacZ expression (red in G,G′) and differentiation (small nuclei). DAPI is blue in G,G′. (H-H′) Higher magnification view of clone shown in G. Yellow lines delineate the clone in G,H. Scale bar in C′: 20 μm in A,A′,F,F′; 10 μm in B-C′,G-G′; 5 μm in H-H′.

Article Snippet: Numbers in parentheses correspond to Bloomington stock numbers: btl-Gal4 UAS-actGFP (#8807), UAS-srcGFP (#5432), fzr-lacZ G0326 (#12241), tub-Gal80 ts (#7016) ( McGuire et al., 2004 ), cut-Gal4 (PG142) ( Bourbon et al., 2002 ), UAS-cut (gift from B. Mathey-Prevot), UAS-cut RNAi (VDRC#5687) ( Dietzl et al., 2007 ), esg-Gal4 tub-Gal80 ts UAS-EGFP ( Micchelli and Perrimon, 2006 ), UAS-FLP ( Duffy et al., 1998 ), act5C-FRT-stop-FRT-lacZ ( Struhl and Basler, 1993 ) and hsFLP; act5C-FRT-stop-FRT-Gal4 UAS-GFP ( Ito et al., 1997 ).

Techniques: Staining, Mutagenesis, Clone Assay, Expressing