Fig 1.
Identification of RTKs involved in dendrite regeneration.
(A) Full dendrite arbor trace of a ddaC neuron before (left) and 24 h after (right) dendrite removal with a pulsed UV laser (red arrowheads). Regeneration was quantified 24 HPD using the widest diameter of the regenerating arbor (blue dotted line). The cell body and beginning of the axon are indicated in green. (B) Representative ddaC neurons immediately after dendrites were severed (left) and 24 HPD (right). Cells were labeled using the tester line ppk-Gal4, UAS-Dicer2;UAS-mCD8-GFP crossed to lines expressing the respective individual RNAi hairpins. The control targets the maternal γTub37C transcript not present in somatic tissues like neurons. (C) Quantification of dendrite regeneration. Bars show mean regeneration, error bars represent standard deviation. Sample size (within bar, bottom) represents number of cells analyzed, with one cell per animal. *P < 0.05, **P < 0.01, ***P < 0.001 with Mann–Whitney U test to compare mean rank. Quantitation is contained in S1 Data. Alk, anaplastic lymphoma kinase; da, dendritic arborization; ddaC, dorsal da neuron C; dnt, doughnut on 2; drl, derailed; GFP, green fluorescent protein; HPD, hours postdendrotomy; InR, insulin-like receptor; Nrk, neurospecific receptor kinase; ppk, pickpocket; Pvr, PDGF- and VEGF-receptor related; Ret, Ret oncogene; RNAi, RNA interference; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase; sev, sevenless; UAS, upstream activating sequence; γTub, γTubulin.
Fig 2.
Secondary screen of RTKs required for dendrite regeneration.
(A) Assay for ddaE dendrite regeneration. The dorsal comb dendrite was severed (red arrowhead) at time 0. The cell was imaged 24 h later, and new branches (green arrowheads) were counted. (B) Representative images of neurons expressing Dicer2, mCD8-GFP, and RNAi hairpins under control of the 221-Gal4 driver are shown. (C) Quantification of dendrite regeneration in neurons expressing different RNAi hairpins. Regeneration is represented by the number of new branch points 24 HPD; error bars are standard deviation; sample size (within bar, bottom) is number of animals from which one neuron was injured. *P < 0.05, **P < 0.01, ***P < 0.001 with Mann–Whitney U test to compare mean rank. Quantitation is contained in S1 Data. da, dendritic arborization; ddaE, dorsal da E; GFP, green fluorescent protein; HPD, hours postdendrotomy; InR, insulin-like receptor; Nrk, neurospecific receptor kinase; Ret, Ret oncogene; RNAi, RNA interference; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase.
Fig 3.
Ror is required for normal dendrite regeneration in ddaC and ddaE neurons.
(A) Diagram of the Ror gene and protein with 26-bp deletion resulting in null RorΔ26 mutant. The target regions of RNAi hairpins used in this work are mapped onto the gene. Domains in the protein include fz-like CRD, KD, TM, and TK. (B) Representative ddaC dendrite regeneration over the course of 72 HPD. The mutant line was RorΔ26/Δ26;ppk-Gal4, UAS-EB1-TagRFP-T crossed to RorΔ26/Δ26. Control flies used were yw;ppk-Gal4, UAS-EB1-TagRFP-T crossed to yw. Each neuron was imaged every 24 h to quantify regeneration over time in yw (control) or RorΔ26 null mutants. Quantitation is shown in (C). (D) Representative dendrite regeneration over time in ddaE neurons. Dorsal comb dendrites were severed (red arrowhead), and new branchpoints (green arrowheads) were counted at 24 h intervals. Mutant phenotype: RorΔ26/Δ26;221-Gal4, UAS-mCD8-GFP crossed to RorΔ26/Δ26. Control flies used were yw;221-Gal4, UAS-mCD8-GFP crossed to yw, and quantitation is shown in (E). Graphs show mean regeneration diameter, with error bars showing standard deviation (C) or added branch points (E). Sample size (within bar, bottom) represents individual neurons from each animal. *P < 0.05, **P < 0.01, ***P < 0.001 with Mann–Whitney U test to compare mean rank. Quantitation is contained in S1 Data. CRD, cysteine-rich domain; da, dendritic arborization; ddaC/E, dorsal da C/E; EB1, end binding protein 1; fz, frizzled; GFP, green fluorescent protein; HPD, hours postdendrotomy; KD, kringle domain; ppk, pickpocket; RFP, red fluorescent protein; RNAi, RNA interference; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase; TK, tyrosine kinase domain; TM, transmembrane domain; yw, yellow, white.
Fig 4.
Ror is not required for dendrite development.
(A) Images of ddaE neurons throughout larval development are shown from yw (control) or RorΔ26 mutant animals. Cells expressed mCD8-GFP under control of 221-Gal4. (B) Quantification of branch point number during ddaE development. Values are the mean of 10 animals per group. Error bars show standard deviation. (C) Representative images (top) and traces (bottom) of ddaC neurons from 3-day–old larvae. A Sholl analysis is shown in (D) and total dendrite length in (E). For the Sholl analysis, no difference was observed between nonlinear regression curves fitted as a fifth-order polynomial. Total dendrite length was unaffected in Ror mutants compared to controls using unpaired t test. Numbers on the bars indicate number of animals used. Quantitation is contained in S1 Data. da, dendritic arborization; ddaC/E, dorsal da C/E; GFP, green fluorescent protein; n.s., not significant; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase; yw, yellow, white.
Fig 5.
Axon regeneration is normal in Ror mutants.
(A) Diagram of ddaE axon regeneration, which occurs via conversion of a dendrite to a growing axon after proximal axon injury. Growth of the converted dendrite from the tip is measured 96 HPA. (B) Representative images of ddaE neurons at 0 h and 96 HPA in control and Ror knockdown neurons labeled with 221-Gal4,UAS-mCD8-GFP and expressing dicer2 and hairpin RNAs (for lower images). The green dotted line indicates converted dendrite. (C) Quantification of axonal regeneration normalized to arbor size. Only Wnd/DLK RNAi showed near-total abrogation of regeneration phenotype (P < 0.001). Mann–Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.001. Bars show mean, and error bars are standard deviation; sample size represents individual animals/neurons. Quantitation is contained in S1 Data. da, dendritic arborization; ddaE, dorsal da E; DLK, dual leucine zipper kinase; GFP, green fluorescent protein; HPA, hours postaxotomy; RNAi, RNA interference; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase; UAS, upstream activating sequence; wnd, wallenda; yw, yellow, white.
Fig 6.
Wnt signaling proteins contribute to dendrite regeneration.
(A) Quantification of ddaC dendrite regeneration diameter 24 HPD after RNAi knockdown of Wnt signaling proteins. (B) Example images of ddaC neurons 24 HPD are shown. (C) Representative images of ddaE regeneration following knockdown of Wnt signaling proteins. (D) Quantification of ddaE branchpoints added 24 HPD. Mann–Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars show standard deviation; sample size (within bar, bottom) represents individual animals/neurons. Quantitation is contained in S1 Data. arm, armadillo; arr, arrow; Axn, Axin; da, dendritic arborization; ddaC/E, dorsal da C/E; dsh, dishevelled; fz, frizzled; gish, gilgamesh; HPD, hours postdendrotomy; otk, off-track; RNAi, RNA interference; yw, yellow, white.
Fig 7.
dsh localization at baseline and during dendrite regeneration.
(A) UAS-dsh-GFP expressed under control of 221-Gal4 with UAS-mCD8-mRFPas a cell-shape marker at baseline (left) and 24 HPD (right). Red arrowheads denote cut sites; green arrowheads show new branch points positive for dsh-GFP, while white arrowheads show GFP-negative new branch points. (B) Quantification of dsh-GFP+ branchpoints at baseline and 24 HPD in control and Ror RNAi. For 24 HPD, the total number of branch points with dsh-GFP is shown in the 24 column, and newly added branch points are also shown separately. Mann–Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.001. Bars show mean; error bars are standard deviation; sample size represents individual animals/neurons. Quantitation is contained in S1 Data. dsh, dishevelled; GFP, green fluorescent protein; HPD, hours postdendrotomy; RNAi, RNA interference; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase; UAS, upstream activating sequence.
Fig 8.
Microtubule nucleation is required for normal dendrite regeneration.
(A) Representative images of ddaE dendrite regeneration in control, ɣTub, and plp RNAi neurons. (B) Number of branch points added 24 HPD is shown. (C) Representative ddaC arbors 24 HPD expressing RNAi hairpins targeting plp and γTub. (D) Quantitation of ddaC dendrite regeneration diameter 24 HPD is shown. (E) Example images of ddaE axon regeneration in Plp RNAi neurons are shown. (F) Mean normalized regeneration length (μm) is shown in the graph. Note: axon regeneration control data set is repeated (F) from Fig 5 for comparison. Error bars are standard deviation; sample size (within bar, bottom) represents individual animals/neurons. Mann–Whitney U test, *P < 0.05, **P < 0.01, ***P <0.001. Quantitation is contained in S1 Data. da, dendritic arborization; ddaC/E, dorsal da C/E; HPA, hours postaxotomy; HPD, hours postdendrotomy; plp, pericentrin-like protein; RNAi, RNA interference; γTub, γTubulin.
Fig 9.
Localization of γTub-GFP and a functional assay for nucleation in control and Ror RNAi neurons.
(A) Representative images of ddaE neurons expressing UAS-mCD8-RFP, UAS-Dicer2, and UAS-ɣTub-GFP under 221-Gal4 control. Neurons are shown before dendrite removal and 24 HPD. The cut sites are shown with red arrowheads. Areas of high-signal γTub-GFP at branch points are indicated with white arrowheads; low signal is indicated with gray arrowheads. (B) Quantitation of γTub-GFP fluorescence at branch points was calculated as (BP − nBP) = (fluorescence); insets show 2× magnification of indicated branch points. Sample size (within bar, bottom) represents individual neurons/animals. Linear regression, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Representative images of ddaC neurons expressing UAS-γTub-GFP and UAS-mCD8-RFP under ppk-Gal4 control are shown. White arrowheads indicate branch points with normal γTub-GFP signal, and gray arrowheads indicate low signal. (D) Quantification of γTub-GFP at branch points was calculated as in B. (E) Kymographs of EB1-GFP in dendrites of ddaE neurons are shown in E 8 h after axon injury. Kymographs show white EB1 comet tracking over 300 s. (F) Microtubule dynamics was quantified as EB1-GFP comet number in the comb dendrite of ddaE dendrite during 300 s live imaging (widefield, Zeiss; 63× Oil PlanApo 1× zoom). yw was used as control for RorΔ26 animals. *P <0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U test. Numbers on bars of all graphs are numbers of cells analyzed. Quantitation is contained in S1 Data. da, dendritic arborization; ddaC/E, dorsal da C/E; EB1, end binding protein 1; GFP, green fluorescent protein; HPD, hours postdendrotomy; ppk, pickpocket; RFP, red fluorescent protein; RNAi, RNA interference; Ror, RTK-like orphan receptor; RTK, receptor tyrosine kinase; UAS, upstream activating sequence; yw, yellow, white; γTub, γTubulin.