Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A ) Eve and Dbx expression in the NB7-1 lineage of wild-type and fd4 / fd5 mutant stage 17 embryos. Posterior view with dorsal oriented upward and ventral downward. The NB7-1 lineage is outlined with a yellow dashed line. Rightmost panels are 3D reconstruction of the NB7-1 lineage from the left three panels with Imaris Spots function. Scale bar: 2 μm. ( B–D ) Quantification of total ( B ), Eve+ ( C ), and Dbx+ ( D ) cells in each lineage. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. ( E ) UMNs in wild type and fd4 / fd5 mutant stage 17 embryos. The identity of UMNs is determined by the expression of marker Hb or Runt and the relative position of the cell within the hemisegment. The rightmost panels are summary cartoons from the left panels. Scale bar: 2 μm. ( F ) Quantification of each individual UMNs in each lineage. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. ( G ) Dbx and Cas expression in the NB7-1 lineage of wild-type and fd4 / fd5 mutant stage 17 embryos. Dorsal view with anterior oriented upward and posterior downward. Rightmost panels are 3D reconstruction of the NB7-1 lineage from the left three panels with Imaris Spots function. Scale bar: 2 μm. ( H–I ) Quantification. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotypes ( A–I ): wild type: fd4-gal4 , UAS-myr-sfGFP; fd4/fd5 mutant: fd4-gal4,fd5 1nt / Df(3R)BSC493 , UAS-myr-sfGFP . ( J ) Quantification of Eve+ cells in fd4 / fd5 mutant (the same data as C ) and vnd misexpression with en-gal4 in fd4 / fd5 mutant background. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotype: fd4 / fd5 mutant, fd4-gal4,fd5 1nt / Df(3R)BSC493 . ( K ) Summary of functions of spatial TFs, Fd4, and terminal selector genes in neuron identity.

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Expressing, Mutagenesis, Standard Deviation, Marker

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A–B ) Merged image of wild-type ( A ) and fd4/fd5 mutant ( B ) NB7-1 lineage axon (white) superimposed on the body wall muscles (blue) in the late stage 17 embryos. DO2 region (green box) is the axon target of early-born motor neurons, while LL4 region (magenta box) is the axon target of late-born neurons. Lateral view with dorsal side up. Genotype: wild type: fd4-gal4 , UAS-myr-sfGFP; fd4/fd5 mutant: fd4-gal4,fd5 1nt / Df(3R)BSC493 , UAS-myr-sfGFP . Scale bars: 10 μm. ( C–D ) Quantification of total length of the branched neurites in the boxed region from wild-type ( A ) and fd4 / fd5 mutant ( B ). Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test.

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Mutagenesis, Muscles, Standard Deviation

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A–B ) Eve and Dbx expression in wild type. En as a marker for segment boundary. Scale bars: 10 μm. ( C–D ) Eve and Dbx expression following Fd4 misexpression. En as a marker for segment boundary. Scale bars: 10 μm. ( E–F ) Quantification of Eve+ and Dbx+ cells in each hemisegment. Each dot represents an individual hemisegment. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotypes: wild type: y-w- ; Fd4 misexpression: sca-gal4 , UAS-fd4 .

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Expressing, Marker, Standard Deviation

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A–B ) Early-born ( A ) and late-born ( B ) Eve+ cells in wild type. Yellow arrowheads: Hb+ Eve+ ( A ) and Runt+ Eve+ ( B ) cells. Ventral view with anterior side up. White dashed lines indicate the segment midline. Genotype: y-w- . Scale bars: 5 μm. ( C–D ) Early-born ( C ) and late-born ( D ) Eve+ cells following Fd4 misexpression. Yellow arrowheads: Hb+ Eve+ ( C ) and Runt+ Eve+ ( D ) cells. We also found 17% ± 7% of Eve+ cells have a mixed fate (Hb+ Runt+). Ventral view with anterior side up. White dashed lines indicate the segment midline. Genotype: sca-gal4,UAS-fd4 . Scale bars: 5 μm. ( E–F ) Quantification. Each dot represents a hemisegment. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test.

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Standard Deviation

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A ) Schematic of spatial factor expression in one hemisegment of ventral nerve cord. ( B ) Fd4 misexpression in En+ cells in a stage 9 embryo. Overlayed green color shows Vnd expression domain, and overlayed red color shows Ind expression domain. Blue dashed lines outline the region where Fd4 is misexpressed by en-gal4 . White dashed lines, ventral midline. Genotype: en-gal4 , UAS-fd4,vnd-GFP-FPTB . ( C–D ) Fd4 misexpression in Vnd+ cells in a stage 9 embryo. Blue dashed lines outline the region where Fd4 is misexpressed by vnd-T2A-gal4 . White dashed lines, ventral midline. Genotype: vnd-T2A-gal4 , UAS-fd4 . ( E ) Schematic of temporal transcription factor expression from stage 9 to stage 12. ( F ) Fd4 misexpression in En+ cells in a stage 9 (left) and a stage 12 (right) embryo. Overlaid blue color shows the cells with Fd4 misexpressed with en-gal4 . White dashed lines, ventral midline. Genotype: en-gal4 , UAS-fd4 . Scale bar: 5 μm for all panels.

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Expressing

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A ) Expression of Ap and Eve in wild type (top row) or following Fd4 misexpression (bottom row) in NB5-6 using the NB5-6-specific lbe-gal4 driver. Scale bar: 2 μm. ( B–C ) Quantification of the number of RedStinger+ ( B ), Ap+ ( C ), and Eve+ ( C ) cells. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. ( D ) Expression of NB7-1 markers (Hb, Runt, Eve) in wild-type NB5-6 lineage and following Fd4 misexpression in the NB5-6 lineage. Scale bar: 2 μm. ( E ) Quantification of Hb+ Eve+ and Runt+ Eve+ cells. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. ( F ) Lateral view of lbe-gal4+ axon projection in wild type (top panel) and Fd4 misexpressed (bottom panel) embryos. The axons (white, arrowhead) were overlaying the body wall muscles (blue), and the muscles were labeled with antibody against Tropomyosin 1 (Tm1). Scale bars: 20 μm. ( G ) Quantification of percent of axons projected out of CNS to the body wall muscles in wild-type and Fd4 misexpressed embryos. ( H ) Expression of motor neuron marker pMad in ectopic Eve+ cells in Fd4 misexpressed NB5-6 lineage in newly hatched larvae. The number in the pMad panel shows the average number of pMad+ Eve+ cells per hemisegment. Scale bar: 2 μm. Genotypes: ( A–E, H ) wild type: lbe-gal4 , UAS-RedStinger ; Fd4 misexpression ( +UAS-fd4 ): lbe-gal4 , UAS-RedStinger , UAS-fd4 ; ( F–G ) wild type: 10xUAS-myr-smGdP.HA,lbe-gal4 . Fd4 misexpression ( +UAS-fd4 ): 10xUAS-myr-smGdP.HA, lbe-gal4 , UAS-fd4 .

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Expressing, Standard Deviation, Muscles, Labeling, Marker

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A–B ) Expression of serotonin (5-HT), Corazonin (Crz), and Eve in wild type ( A ) and following Fd4 misexpression in the NB7-3 lineage ( B ). Scale bar: 2 μm. ( C ) Quantification. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotypes: wild type: eg-gal4 , UAS-myr-sfGFP ; Fd4 misexpression ( +UAS-fd4 ): eg-gal4 , UAS-myr-sfGFP , UAS-fd4 .

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Expressing, Standard Deviation

Journal: eLife

Article Title: The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identity

doi: 10.7554/eLife.109188

Figure Lengend Snippet: ( A ) Expression of motor neuron markers Nkx6, Eve, and pMad in wild type (left column) and Fd4 misexpressed (right column) motor neuron in newly hatched larvae. Scale bars: 2 μm. ( B ) Quantification. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. ( C ) Dorsal view of three segments of wild type (top panel) and Fd4 misexpressed (bottom panel) neuronal projections. Yellow arrows indicate the fascicles projecting out from the neuropil. White arrowheads, ventral midline. Scale bars: 20 μm. ( D ) Quantification of fascicles exiting nervous system. Wild type (1±0; top panel). Fd4 misexpression (1.6±0.5; bottom panel). ( E, F ) Lateral view of eg-gal4+ motor neuron axon projection in wild type ( E ; left panels) and Fd4 misexpressed (F; right panels) embryos. Top two panels are the maximum projections of confocal image stacks of eg-gal4+ neurons. Middle and bottom panels are eg-gal4+ neuron axons reconstructed with Imaris (white), overlaying the body wall muscles (red). The muscles are labeled with antibody against Tropomyosin 1 (Tm1). The dashed lines indicate the boundary between dorsal and longitudinal (middle panels), and longitudinal and ventral muscles (middle and bottom panels). Scale bars: 50 μm. ( G ) Quantification of motor neuron axon lengths. Each black dot represents the length of an axon measured from the VNC. White diamonds indicate the average length. The error bars are standard deviation. ( H ) Summary. Genotypes: wild type: eg-gal4 , UAS-myr-sfGFP ; Fd4 misexpression ( +UAS-fd4 ): eg-gal4 , UAS-myr-sfGFP , UAS-fd4 .

Article Snippet: We used CRISPR to generate fd4-gal4 by replacing fd4 ORF with gal4 with the pHD-DsRed (Addgene plasmid #51434; http://n2t.net/addgene :51434; RRID: Addgene_51434 ) , which also contained 1 kb of homologous arms up- and downstream of ORF for homology-directed repair (HDR).

Techniques: Expressing, Standard Deviation, Muscles, Labeling

Journal: bioRxiv

Article Title: A GPI-anchored Ly6/uPAR superfamily gene belly roll is expressed in multiple peptidergic neurons in Drosophila melanogaster larvae

doi: 10.64898/2026.02.27.708413

Figure Lengend Snippet: (A) Overview of a method for visualizing bero expression patterns using bero-GAL4 T2A . (B) Confocal microscopy images of the expression pattern of bero-GAL4 T2A in CNS (a), lateral body wall (b) and anal pad (c) of Drosophila larvae. IPC: Insulin-like peptide producing cells; EH: Eclosion hormone producing neurons; BO: Bolwig organs; MG: Midline glia; AG: abdominal ganglion. (C) Confocal microscope stack images of bero-GAL4 T2A (magenta) or bero::YFP labeled cells (yellow) and ABLK neurons (cyan) in the CNS. Arrows indicate previously identified bero + cells, while triangles indicate unknown bero + cells. Scale bars: 50 μm.

Article Snippet: bero-GAL4 T2A strain was generated by inserting pBS-KS-attB2-SA(1)-T2A-Gal4-Hsp70 (Addgene: #62897) into MiMIC landing site of bero MI03849 strain (BDSC: 36397; WellGenetics Inc., Taipei, Taiwan) by φC31-mediated genomic integration, as previously described (Diao et al., 2015).

Techniques: Expressing, Confocal Microscopy, Microscopy, Labeling

Journal: bioRxiv

Article Title: A GPI-anchored Ly6/uPAR superfamily gene belly roll is expressed in multiple peptidergic neurons in Drosophila melanogaster larvae

doi: 10.64898/2026.02.27.708413

Figure Lengend Snippet: (A) A dot plot of neuropeptides and neurohormones in D-Is motor neurons, V-Is motorneurons, GPA2/GPB5 neurons, ABLK neurons and OK neurons. (B) Confocal microscope stacks showing bero::YFP labeled cells (yellow), AstA-GAL4 T2A labeled cells (magenta) and ABLK neurons (cyan) in the CNS. Scale bars: 50 μm or 10 µm. (C-E) Confocal microscope stacks showing ABLK neurons (yellow) and Dh44-GAL4 T2A labeled cells (C; magenta), Dh31 AC -GAL4 T2A labeled cells (D; magenta) or Dh31 D -GAL4 T2A labeled cells (E; magenta). Scale bars: 50 μm or 10 µm. (F) A heat map showing expression probability of Dh44, Dh31 AC and Dh31 D in ABLK neurons.

Article Snippet: bero-GAL4 T2A strain was generated by inserting pBS-KS-attB2-SA(1)-T2A-Gal4-Hsp70 (Addgene: #62897) into MiMIC landing site of bero MI03849 strain (BDSC: 36397; WellGenetics Inc., Taipei, Taiwan) by φC31-mediated genomic integration, as previously described (Diao et al., 2015).

Techniques: Microscopy, Labeling, Expressing