PMC

Representative tracings of class II ldaA (A–C) and class III v'pda (E–G) neurons from third instar larvae. Anterior is left and dorsal up and size bars represents 150 microns for ldaA and 100 microns for v'pda. (A) Wild-type (WT) ldaA neuron. Overexpression of the non-membrane bound Tutl AT02763 (B) and GH15753 (C) isoforms increased terminal dendritic branching in ldaA neurons. (D) Quantitative analyses of dendritic branching revealed a statistically significant increase with AT02763 and GH15753 overexpression relative to wild-type controls. (E) Wild-type (WT) v'pda neurons. Overexpression of the Tutl AT02763 isoform (F) increased terminal dendritic branching, whereas overexpression of the Tutl GH15753 isoform (G) decreased terminal dendritic branching in v'pda neurons. (H) Quantitative analyses of dendritic branching revealed a statistically significant increase with AT02763 and decrease with GH15753 overexpression relative to controls. The total n value for each genotype quantified is reported on the bar graph. Statistically significant p values are reported on the graph as follows (* = p<0.05; ** = p<0.01; *** = p<0.001; n.s. = not significant). Genotypes (A–H): WT: GAL4^C161,UASmCD8::GFP/+; AT02763: UAS-tutl^AT02763/+;+;GAL4^C161,UASmCD8::GFP/+; GH15753: UAS-tutl^GH15753/+;+; GAL4^C161,UASmCD8::GFP/+; HL01565: UAS-tutl^HL01565/GAL4^C161,UASmCD8::GFP; LD28224: UAS-tutl^LD28224/GAL4^C161,UASmCD8::GFP.

(A–D) Representative live confocal images of wild-type and Tutl isoform overexpressing class IV ddaC neurons collected at 20X magnification. Anterior is left and dorsal up and size bar represents 100 microns. (A) Wild-type (WT) class IV ddaC neuron. (B) Overexpression of the non-membrane bound Tutl AT02763 isoform reduces dendritic branching in ddaC neurons particularly at dendritic terminals. (C,D) Overexpression of the membrane bound Tutl HL01565 (C) and Tutl LD28224 (D) isoforms increases dendritic branching in ddaC neurons most evident at dendritic terminals. (E) Quantification of ddaC dendritic branch termini revealed a statistically significant decrease with AT02763 overexpression and increase with both HL01565 and LD28224 overexpression relative to wild-type controls. (F) Quantification of ddaC total dendritic length revealed no statistically significant (n.s, p>0.05) change with Tutl isoform overexpression relative to wild-type controls. The total n value for each genotype quantified is reported on the bar graphs. Statistically significant p values are reported on the graph as follows (** = p<0.01; *** = p<0.001; n.s. = not significant). Genotypes (A–D): WT: GAL4⁴⁷⁷,UASmCD8::GFP/+; AT02763: UAS-tutl^AT02763/+; GAL4⁴⁷⁷,UASmCD8::GFP/+; GH15753: UAS-tutl^GH15753/+; GAL4⁴⁷⁷,UASmCD8::GFP/+; HL01565: GAL4⁴⁷⁷,UASmCD8::GFP/+; UAS-tutl^HL01565/+; LD28224: GAL4⁴⁷⁷,UASmCD8::GFP/+; UAS-tutl^LD28224/+.

(A) Wild-type class II vdaA neuron. Note terminal branching complexity in inset. (B) tutl mutant class II vdaA neuron. As compared to wild-type, tutl mutant class II da neurons display an overall reduction in dendritic branching particularly with respect to terminal branching and to a lesser extent overall dendritic length. Note loss of terminal dendritic branches in inset. Images taken at 40X magnification and size bar represents 50 microns. (C) Quantitative analyses of overall dendritic length revealed a significant reduction in tutl mutant class II da neurons relative to wild-type controls (two way ANOVA, p<0.001). The n value for each class II da neuron subtype for both wild-type and tutl mutants is indicated on the bar graph. (D) Quantitative analyses of the total number of dendritic terminals revealed a significant reduction in tutl mutant class II da neurons relative to wild-type controls (two way ANOVA, p<0.001). The n value for each class II da neuron subtype for both wild-type and tutl mutants is indicated on the bar graph. (E) Quantitative analyses revealed an average 27% reduction in overall dendritic length relative to an average 46% reduction in the number of dendritic terminals in tutl mutant class II da neurons relative to wild-type controls. For these analyses each of the four class II da neuron subtypes (ddaB, ldaA, vdaA, vdaC) in both wild-type and tutl mutants was compared. The average percent reduction from wild-type in tutl mutants of each of the four subtypes is reported.

(A) Wild-type class IV v'ada neuron. (B) tutl mutant class IV v'ada neuron. As compared to wild-type, tutl mutant class IV da neurons display an overall reduction in dendritic length and dendritic field coverage. Images taken at 40X magnification and size bar represents 50 microns. (C) Quantitative analyses of overall dendritic length revealed a significant reduction in tutl mutant class IV da neurons relative to wild-type controls (p<0.001, two way ANOVA). The n value for each class IV da neuron subtype for both wild-type and tutl mutants is indicated on the bar graph. (D) Quantitative analyses of the total number of dendritic terminals revealed no significant (n.s.) reduction in tutl mutant class IV da neurons relative to wild-type controls (p>0.05, two way ANOVA). The n value for each class IV da neuron subtype for both wild-type and tutl mutants is indicated on the bar graph. (E) Quantitative analyses of total dendritic arbor demonstrate area is significantly reduced in tutl mutant class IV da neurons relative to wild-type controls (two way ANOVA, p<0.001). The n value for each class IV da neuron subtype is indicated on the bar graph. (F) Quantitative analyses revealed that in tutl mutants dendritic length is reduced by an average of 26% whereas terminals are reduced by approximately 10%. For these analyses each of the three class IV da neuron subtypes (ddaC, v'ada, and vdaB) in both wild-type and tutl mutants were compared. The average percent reduction from wild-type in tutl mutants of each of the three subtypes is reported.

(A) qPCR analysis reveals a significant 3.35 ± 1.26 fold increase in tutl genomic DNA in chromatin immunoprecipitated with a monoclonal anti-Cut antibody as compared to the normal mouse IgG control (n = 4). As a negative control, GAPDH2 levels are unchanged between anti-Cut and mouse IgG preparations (n = 4). (B–C) qPCR of RNA isolated from either wild-type (221 X Oregon-R) or Cut overexpressing (221 X UAS-cut) class I da neurons shows that in Cut overexpressing class I da neurons tutl mRNA is significantly 1.96 fold overexpressed compared with wild type class I neurons (B) (n = 4). cut mRNA was present at a significant 13.39 fold increase in Cut overexpressing class I da neurons compared with wild-type (C) (n = 4). (D) Quantitative mean pixel density analyses of Tutl protein expression revealed a 7–8 fold increase in Tutl immunoreactivity levels in class I neurons overexpressing UAS-cut (n = 8) relative to wild-type controls (n = 10). Statistically significant p values are reported on the graphs as follows (** = p<0.01; *** = p<0.001; n.s. = not significant). Genotypes: (B,C) 221 X Oregon-R corresponds to GAL4²²¹,UAS-mCD8::GFP/+; 221 X UAS-cut corresponds to UAS-cut/+;GAL4²²¹,UAS-mCD8::GFP/+. (D) WT corresponds to GAL4²²¹,UAS-mCD8::GFP/+; UAS-cut corresponds to UAS-cut/+; GAL4²²¹,UAS-mCD8::GFP/+.

(A,B) Live confocal images of representative class III da neuron MARCM clones at third larval instar. (A) Representative wild-type ddaA neuron. (B) Representative tutl mutant class III ddaA neuron. As compared to wild-type, tutl mutant class III da neurons display an overall reduction in dendritic branching, length, and field coverage. Arrows in denote correlate dendritic branches in wild-type (A) and tutl mutant (B) MARCM clones. Images were collected at 40X magnification in both panels and size bar represents 50 µm. (C) Quantitative analyses of overall dendritic length revealed a significant reduction in tutl mutant class III da neurons relative to wild-type controls (p<0.001, two way ANOVA). (D) Quantitative analyses of the total number of dendritic terminals revealed a significant reduction in tutl mutant class III da neurons relative to wild-type controls (p<0.01, two way ANOVA). The n value for each class III da neuron subtype for both wild-type and tutl mutants is indicated on the bar graph. (E) tutl is required for fully elaborating class III spine-like terminal branch protrusions. Quantitative analyses of the average length (in microns) of spine-like terminal branches reveal a significant reduction in tutl mutant (n = 24) class III da neurons relative to wild-type controls (n = 26) (p<0.01 Student's t-test). (F) Quantitative analyses revealed that in tutl mutants dendritic length is reduced by an average of 34% whereas terminals are reduced by 22%. For these analyses each of the five class III da neuron subtypes (ddaF, ddaA, ldaB, v'pda, vdaB) in both wild-type and tutl mutants were compared. The average percent reduction from wild-type in tutl mutants for each of the five subtypes is reported.

(A,B) Live confocal images of representative class I ddaE neuron MARCM clones at the third larval instar. (A) Representative wild-type (WT) ddaE neuron. (B) Representative tutl^c00018 mutant class I ddaE neuron. As compared to wild-type, tutl^c00018 mutant class I da neurons display no qualitative defects in overall dendritic branching or extension. Images were collected at 40X magnification in both panels and size bar represents 50 microns. (C) Quantitative analyses of overall number of dendritic terminals revealed no significant (n.s.) difference between wild-type and tutl mutants (Mann-Whitney rank-sum test, p>0.05). (D) Quantitative analyses of overall dendritic length revealed no significant difference between wild-type and tutl mutants (Mann-Whitney rank-sum test, p>0.05). The n value for both wild-type and tutl mutants is indicated on the bar graph. (E) Quantitative pairwise analyses of total number of dendritic terminals revealed no significant statistical difference between wild-type and trans-allelic tutl mutant class I ddaD and ddaE neurons (p>0.05, Student's t test) labeled by GAL4²²¹,UAS-mCD8::GFP. In vpda neurons, a significant reduction in the number of dendritic terminals was observed in only two trans-allelic combinations. (F) Quantitative pairwise analyses of total dendritic length generally revealed no significant statistical difference (p>0.05, Student's t test) between wild-type and trans-allelic tutl mutant class I da neurons with the exception of tutl^c00018/tutl^ex383 where there was a significant reduction in length in ddaE and vpda neurons. The total n value for each genotype quantified is reported on the bar graphs. Only statistically significant p values are reported on the graph as follows (* = p<0.05; ** = p<0.01; *** = p<0.001) and all other pairwise analyses were not significant.

(A) Neurons labeled with tutl-GAL4,UAS-mCD8::GFP. (B) Quantitative analyses of relative pixel intensity in tutl-GAL4 transgene driver expression reveal highest levels of expression in class III and IV da neurons, respectively, moderate levels in class II da neurons, and weak levels in class I da neurons. A total of 10 dorsal cluster da neurons images labeled with tutl-GAL4,UASmCD8::GFP were used to performed quantitative pixel intensity analyses (n = 10). (C) Confocal image of wild-type third instar larvae. Third instar larval filets were dissected, fixed and labeled with rabbit anti-Tutl antibodies to visualize endogenous Tutl protein expression. The image reveals differential expression levels of Tutl protein in da neuron cell bodies with highest expression in class III (ddaF/A) and class IV (ddaC) neurons, intermediate expression in class II (ddaB) and weaker expression in class I (ddaD/E) neurons consistent with the pattern observed with the tutl-GAL4 enhancer trap. Tutl protein expression is also observed on da neuron dendrites (arrows) and axons (arrowhead). (D–F) Third instar tutl^c00018 class II vdaA MARCM clone double labeled with rat anti-CD8 antibody (D) and rabbit anti-Tutl antibody (E). (E,F) Tutl staining is specifically absent from the vdaA MARCM clone, however is observed and differentially expressed in the adjacent ventral cluster da neuron cell bodies. Tutl IHC signal intensity reveals differential expression levels in da neuron subclass cell bodies with highest expression observed in the class III vdaD neuron followed by the class IV vdaB neuron and class II vdaC neuron. Tutl and CD8 signals optimized for cell body staining intensities in (E,F). Individual da neuron cell bodies are outlined by dashed lines. Size bars represent 50 µm and in all images anterior is left and dorsal up.

(A–F) Live confocal images of representative class I da neurons (ddaD, ddaE, vpda) at the third larval instar. Images were collected at 20X magnification and size bars represents 100 microns. (A,B) Representative images of wild-type dorsal ddaD/E neurons (A) and ventral vpda neuron (B) ectopically overexpressing Cut. (C,D) Representative images of dorsal ddaD/E neurons (C) and ventral vpda neuron (D) ectopically overexpressing Cut. Note, ectopic overexpression of Cut results in class I neurons displaying increased dendritic branching complexity characterized by a high incidence of short dendritic protrusions emanating from the primary branches as compared to wild-type. (E,F) Representative images of dorsal ddaD/E neurons (E) and ventral vpda neuron (F) in which ectopic overexpression of Cut is combined with the tutl^ex383 allele. As compared to Cut overexpression alone, introduction of one copy of the tutl^ex383 mutant allele results in strong suppression of the Cut phenotype particularly with respect to the incidence of short dendritic protrusions. (G) Quantitative analyses reveal that the total number of dendritic terminals is significantly reduced in animals carrying one copy of the tutl^ex383 mutant allele relative to Cut ectopic overexpression alone. (H) Quantitative analyses reveal a modest, yet statistically significant, decrease in overall dendritic length in animals carrying one copy of the tutl^ex383 mutant allele relative to Cut ectopic overexpression alone. The total n value for each neuron subtype is indicated on the bar graph. Statistically significant p values are reported on the graphs as follows (* = p<0.05; *** = p<0.001). Genotypes: (A,B) GAL4²²¹,UASmCD8::GFP/+; (C,D) UAS-cut/+; GAL4²²¹,UASmCD8::GFP/+; (E,F), UAS-cut/tutl^ex383; GAL4²²¹,UASmCD8::GFP/+.