Momento intramural

As putative downstream targets for cut and knot were identified using ectopic expression of the transcription factors in a da neuron subclass (Class I) where these proteins are not normally expressed, we wanted to understand the loss of function effect of the targets in a da neuron subclass where both the transcription factors are expressed in wild type. Using RNAi transgenic lines, we performed an in vivo genetic screen of the 58 genes in a wild type Class IV background, to identify the functionality of the targets. If a significant disruption in the dendritic architecture is observed by a specific RNAi line,

we can identify it as potential candidate for further suppression studies to validate its association with the transcription factor (cut/knot).

To weaken the position effect and/or RNAi efficacy, the genetic screen was performed using a minimum of two independent gene-specific UAS-RNAi lines (92% of genes screened). For the remaining 8% genes screened, only one gene specific UAS-RNAi line was available, and were screened. To avoid any screening bias, all transgenic RNAi lines were screened double-blind to the identity of the gene being analyzed and phenotypic analyses were performed at the third instar larval stage of development. The key morphological features that were analyzed included, total dendritic length, total dendritic branches, number of branches as a function of distance from the soma (Sholl), branch order distribution (reversed Strahler) and mean coverage area. Neuromorphometric quantitative analyses were used as criteria for positive RNAi hit selection based upon the dendritic phenotypes observed in our qualitative screen.

The genetic screening revealed approximately 72% (~42 out of 58) of the genes showing significant phenotypic effects on the Class IV dendritic architecture, while approximately 28% (16 out of 58), showed no phenotypic effect on the Class IV dendritic architecture. Qualitative evaluation of the 72% genes that exhibited phenotype, revealed that approximately 24% exhibited (~10 out of 42) severe reduction in dendritic arborization, with no to very few higher order branches in Class IV, whereas 48% (~20 out of 42) exhibited a moderate-to-mild phenotype, characterized as missing some higher order branches and/or abnormal distribution of branches along the proximal-distal axis of dendritic arbors. Quantitative neuromorphometric analysis of the images showed approximately 33% (~14 out of 42 genes), showing severe defects, while approximately 39% (~ 16 out of 42 genes) showed, medium to mild defects (Figures 2-4).

Figure 2-4 Quantitative evaluation of the phenotype of the RNAi lines

Heatmap representing the quantitative morphological analyses of the RNAi lines screened. Total dendritic length (TDL) and number of branches (NOB) parameters are represented in the heatmap where the value represents that percent change from controls. Values for RNAi lines, which are statistically significant from control (p<0.05) are represented in the heatmap, non- significant values are represented by 0. Pink indicates positive and Blue negative, with white indicating 0. Statistical test used t-test.

In our work flow, we focused on identifying putative targets for Cut and/or Knot, that were represented in functional categories pertaining to neuronal and cytoskeletal based biological processes. As a single gene is involved in different biological functions, the genes we screened are involved in a broad range of biological functions including cytoskeletal regulation (form3, RhoGAP18B, wdb, Ank2,

msps, cpa, cpb), ribosomal regulatory function (RpL7, RpL36A), neurogenesis (dmn, SkpA),

microtubule-based transport (dmn, ctp/Cdlc2, cpa, cpb), autophagy (wdb, ctp), dendritic pruning (SkpA) and chaperonin activity (T-cp1, CG7033). Representative images of the most severe phenotypic hits of the screen are shown in Figures 2-5 a, 2-6 a, and 2-7 a. These downstream putative targets for Cut/Knot, that produced significant phenotypes, were grouped into three major phenotypic categories based on quantitative neuromorphometric analyses— complexity suppresser, terminal tufted, and complexity enhancer.

Complexity suppressor: These group of genes are classified based on the severe reduction in total dendritic complexity, they produce when they are disrupted in Class IV da neurons. The genes that fall in this category are form3, RpL7, RpL36A, dmn, msps, T-cp1 and CG7033 reduces both total dendritic branches and total dendritic length, as well coverage area (Figure 2-5 a). Knockdown of

form3, RpL7, RpL36A and dmn, produced similar reductions in arbor morphology, characterized by a

loss of higher order branching, especially at the distal dendritic termini. These knockdowns likewise displayed the presence of short, fine dendrites emanating from primary branches in the region proximal to the soma. On the other hand, knockdown of msps, T-cp1 and CG7033 led to similar phenotypic defects, which likewise suppressed dendritic terminal branching and instead resulted in a shift of branching complexity to an intermediate location along the proximal-distal axis relative to the soma. Morphometric analyses of the number of branches (Figure 2-5 b) andtotal dendritic length (Figure 2-5 c) of the knocked down target genes, showed significant decrease in both the parameters, compared

to Class IV control.

Terminal tufted or Complexity shifters: The group of genes, when disrupted in Class IV da neurons, produce terminal tufting of short dendrites and reduced interstitial branching proximal to the cell body as compared to controls. The genes in this category are wdb, Ank2, RhoGAP18B, and

ctp/Cdlc2 (Figure 2-6 a). Within this gene group, the one exception is Ank2 disruption which displayed

an increase in aberrant short dendritic branches emanating first and second order branches relative to the cell body. Comparisons of dendritic terminals between these genes and controls reveals alterations in terminal branching, which in the knockdowns is characterized by clustered short dendritic branches giving a tufted appearance. Morphometric analyses revealed that all genes in this category have significant reductions in the number of branches (Figure 2-6 b) and total dendritic length (Figure 2-6 c).

Complexity enhancer: The final classificationof the screened genes, based on their phenotypic effect are the complexity enhancers, which when knocked down in Class IV neurons, causes an increase in dendritic complexity. The genes in this group are SkpA, cpa and cpb. (Figure2-7 a). Morphometric analysis of the knocked down genes compared to the control revealed, significant increase in Number of branches for SkpA and cpa, but an insignificant decrease in number of branches for cpb (Figure2-7

Figure 2.5 Complexity Reducers

Knockdown of kn targets reduces complexity of the Class IV da neurons:

Knockdown of form3 (form3-IR), Rpl-7 (Rpl-7-IR), Rpl-36A (Rpl-36A-IR), dmn (dmn-IR), msps

(msps-IR), T-cp1 (T-cp1-IR) and CG7033 (CG7033-IR), causes reduction in complexity in Class IV da-neurons (Control). (Figure 2-5 a). Quantitative analysis of knockdown phenotype revealed significant reduction: Statistical comparison of Number of branches (Figure 2-5 b) and Total dendritic length (Figure 2-5 c) of each of the knockdown kn targets compared to Class IV (Control), revealed significant reduction. Statistical analysis: ANOVA with Dunnet correction. ***= pvalue< 0.001. (Image 2-5 a adapted from Das et al. 2017, in revision.)

Figure 2.6 Terminal tufted or Complexity shifters

Knockdown of kn targets shifts complexity towards the terminals for the Class IV

da neurons: Knockdown of wdb (wdb-IR), Ank2 (Ank2-IR), RhoGap18B (RhoGap18B-IR), and

ctp/Cdlc2 (ctp/Cdlc2-IR), causes reduction in complexity near the cell body (highlighted by red box),

while increases the complexity at the terminals (highlighted by blue box) in Class IV da-neurons (Control).(Figure 2-6 a). Quantitative analysis of knockdown phenotype revealed significant decrease in quantitative parameters: Statistical comparison of Number of branches (Figure 2-6 b) and Total dendritic length (Figure 2-6 c) of each of the knockdown kn targets compared to Class IV (Control), revealed significant decrease in both the parameters. Statistical analysis: ANOVA with Dunnet correction. ***= pvalue< 0.0001, ***= pvalue< 0.001, *= pvalue< 0.05. (Image 2-6 a adapted from Das et al. 2017, in revision.)

Figure 2.7 Complexity enhancers

Knockdown of kn increases complexity in the Class IV da neurons: Knockdown of

SkpA (SkpA-IR), cpa (cpa-IR) and cpb (cpb-IR), causes increase in complexity in Class IV da-

neurons (Control).(Figure 2-7 a). Quantitative analysis of knockdown phenotype revealed significant increase in quantitative parameters: Statistical comparison of Number of branches (Figure 2-6 b) and Total dendritic length (Figure 2-6 c) of each of the knockdown kn targets

compared to Class IV (Control), revealed significant increase in both the parameters, except cpb-IR, which has a no-significant decrease in Number of branches parameter. Statistical analysis: ANOVA with Dunnet correction. ***= pvalue< 0.0001, ***= pvalue< 0.001, *= pvalue< 0.05. (Image 2-8 a adapted from Das et al. 2017, in revision.)