NCBP2 modulates neurodevelopmental defects of the 3q29 deletion in Drosophila and X. laevis models

The chromosome 3q29 deletion is associated with a range of neurodevelopmental disorders. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific knockdown of individual homologs of genes within the 3q29 region. We identified developmental, cellular and neuronal phenotypes for multiple homologs, potentially due to altered apoptosis and cell cycle mechanisms. We screened for 314 pairwise knockdowns of fly homologs of 3q29 genes, and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of the other homologs, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models. Our study suggests that NCBP2-mediated genetic interactions contribute to the neurodevelopmental features of the 3q29 deletion. IMPACT STATEMENT NCBP2 homologs in Drosophila and X. laevis enhance the neurodevelopmental phenotypes of other homologs of genes within the 3q29 deletion region, leading to disruptions in several cellular mechanisms.


INTRODUCTION 1
Rare copy number variants (CNVs), including deletions and duplications in the human 2 genome, significantly contribute to complex neurodevelopmental disorders such as 3 schizophrenia, intellectual disability/developmental delay, autism, and epilepsy (Girirajan et 4 al., 2011;Malhotra and Sebat, 2012). Despite extensive phenotypic heterogeneity associated 5 with recently described CNVs (Girirajan and Eichler, 2010), certain rare CNVs have been 6 linked to specific neuropsychiatric diagnoses. For example, the 22q11.2 deletion 7 (DiGeorge/velocardiofacial syndrome), the most frequently occurring pathogenic CNV, is 8 found in about 1-2% of individuals with schizophrenia (Karayiorgou et al., 2010(Karayiorgou et al., , 1995, and 9 animal models of several genes within the region show neuronal and behavioral phenotypes 10 on their own (Fenelon et al., 2011;Mukai et al., 2015). Similarly, the 1.6 Mbp recurrent 11 deletion on chromosome 3q29, encompassing 21 genes, was initially identified in individuals 12 with a range of neurodevelopmental features, including intellectual disability, microcephaly, 13 craniofacial features, and speech delay (Ballif et al., 2008;Mulle et al., 2010). Further studies 14 implicated this deletion as a major risk factor for multiple disorders (Glassford et al., 2016). 15 In fact, the deletion confers a >40-fold increase in risk for schizophrenia (Kirov et al., 2012;16 Mulle, 2015) as well as a >20-fold increase in risk for autism (Pollak et al., 2019). More  (Quintero-Rivera et al., 2010;Rutkowski et al., 2017). For example, DLG1 is a 27 scaffolding protein that organizes the synaptic structure at neuromuscular junctions (Budnik 28 et al., 1996), affecting both synaptic density and plasticity during development (Walch,29 disruptions in apoptosis and cell cycle pathways, leading to neuronal and developmental 1 defects in both model systems. These defects were further enhanced when each of the 2 homologs were concomitantly knocked down with homologs of NCBP2 in Drosophila 3 (Cbp20) and X. laevis (ncbp2), resulting in increased apoptosis and dysregulation of cell 4 cycle genes. Our results support an oligogenic model for the pathogenicity of the 3q29 5 deletion, and implicate specific cellular mechanisms for the observed developmental 6 phenotypes. 7 8 axonal targeting of sensory and motor neurons (Hing et al., 1999;Kim et al., 2003), in 1 addition to abnormal NMJ and mushroom body development (Ng and Luo, 2004;Parnas et 2 al., 2001). We sought to determine whether fly homologs for other genes in the 3q29 region 3 also contribute to defects in neuronal function, and therefore performed climbing assays for 4 motor defects and staining of larval brains for axonal targeting with pan-neuronal knockdown 5 of the fly homologs. Interestingly, Elav-GAL4 mediated pan-neuronal knockdown caused 6 partial larval or pupal lethality in dlg, Tsf2, and CG5543 (WDR53) flies (Figure 2A), and 7 about 30% of adult flies with knockdown of dlg1 did not survive beyond day 5 ( development. Furthermore, we found that flies with pan-neuronal knockdown of several 10 homologs of 3q29 genes, including dlg1 and Cbp20, exhibited a strong reduction in climbing 11 ability over ten days ( Figure 2B, Video 1), suggesting that these genes could contribute to 12 abnormalities in synaptic and motor functions (Sherwood et al., 2004). We next examined the 13 axonal projections of photoreceptor cells into the optic lobe by staining third instar larval 14 brains with anti-chaoptin. We found that GMR-GAL4 mediated eye-specific knockdown of  (Hing et al., 1999), and were similar to targeting defects 18 observed in models of other candidate neurodevelopmental genes, including the Drosophila stained the third instar larval eye discs for select knockdowns of individual homologs of 3q29 1 genes with anti-pH3 (phospho-Histone H3 (Ser10)) and Drosophila caspase-1 (dcp1),  Table 1). 19 20 Interactions between fly homologs of 3q29 genes enhance neuronal phenotypes 21 As knockdown fly models for homologs of multiple 3q29 genes showed a variety of 22 neuronal, developmental, and cellular defects, we hypothesized that interactions between 23 multiple genes in the 3q29 region could contribute to the neurodevelopmental phenotypes of 24 the entire deletion. We therefore generated GMR-GAL4 recombinant lines for nine fly involving Cbp20 as either a first or second-hit gene resulted in more severe eye phenotypes, 33 suggesting that reduced expression of Cbp20 drastically modifies the morphological 34 phenotypes of other homologs of 3q29 genes (Figure 3B-D). For further validation, we also 1 compared pairs of reciprocal crosses (i.e. Fsn/CG8888 versus CG8888/Fsn) and confirmed 2 concordant results for 19 out of 26 reciprocal interactions, including 14/16 reciprocal 3 interactions involving Cbp20 (Figure 3-Figure Supplement 1). We also found a non-4 significant increase in severity for dlg1/Pak knockdown flies using both RNAi and mutant 5 lines, concordant with enhanced neuromuscular junction and circadian rhythm defects 6 observed in mutant dlg1/Pak flies described by Grice and colleagues (Grice et al., 2015). 7 As Cbp20 knockdown enhanced the rough eye phenotypes of multiple homologs of 8 other 3q29 genes, we next tested for enhancement of other neuronal defects among flies with 9 knockdown of Cbp20 and other homologs of 3q29 genes. We found that the simultaneous 10 knockdown of Cbp20 with dlg1 or Fsn led to an increase in severity of axon targeting defects 11 ( Figure 3E). For instance, while knockdown of Cbp20 mostly led to mild-to-moderate axon 12 guidance defects, such as loss of R7-R8 axon projection into the medulla, we observed more 13 severe losses of projection across all of the axons with simultaneous knockdown of Cbp20 14 and dlg1 or Fsn (Figure 2-Figure Supplement 2). We also tested pan-neuronal Elav-GAL4 15 knockdown of select pairs of homologs, and found that both Cbp20/dlg1 and Cbp20/Fsn 16 significantly enhanced the severity of climbing defects observed with knockdown of Cbp20 17 ( Figure 3F, Video 2). Overall, these data suggest that Cbp20 interacts with other homologs 18 of genes in the 3q29 region to enhance the observed cellular and neuronal defects, suggesting 19 that NCBP2 is a key modifier of the developmental phenotypes associated with the deletion 20 ( Table 1). 21 To further characterize the functional effects of interactions between homologs of 22 3q29 genes, we analyzed changes in gene expression by performing RNA-sequencing of 23 heads from flies with select pan-neuronal knockdown of individual (Cbp20,dlg1,Fsn,and 24 Pak) and pairs (Cbp20/dlg1 and Cbp20/Fsn) of homologs of 3q29 genes. We identified

32
Furthermore, flies with knockdown of Cbp20 were enriched for dysregulated fly genes 33 related to metabolic processes, while knockdown of Fsn led to dysregulation of fly genes 34 involved in response to external stimuli and immune response. We also found that homologs 1 of the key signaling genes dysregulated in mouse models of the 3q29 deletion reported by 2 Baba and colleagues (Baba et al., 2019) were differentially expressed in our fly models for 3 homologs of 3q29 genes. In fact, knockdown of Fsn led to altered expression of all "early 4 immediate" signaling genes dysregulated in the deletion mouse model (Baba et al., 2019). 5 While dysregulated genes in Cbp20/dlg1 knockdown flies showed enrichments for protein 6 folding and sensory perception, Cbp20/Fsn knockdown flies were uniquely enriched for differentially-expressed homologs corresponding to human apoptosis genes in Cbp20/Fsn 10 knockdown flies, including homologs for the DNA fragmentation gene Sid (ENDOG) and the 11 apoptosis signaling genes tor (RET) and Hsp70Bb (HSPA1A). Furthermore, we found a 12 strong enrichment for fly genes whose human homologs are preferentially expressed in early 13 and mid-fetal brain tissues among the dysregulated genes in Cbp20/Fsn knockdown flies 14 (Figure 3-Figure Supplement 4D). These data suggest that Cbp20 interacts with other 15 homologs of genes in the 3q29 region to disrupt a variety of key biological functions, 16 including apoptosis and cell cycle pathways as well as synaptic transmission and metabolic 17 pathways, ultimately leading to enhanced neuronal phenotypes ( Table 1). 18 Finally, to complement the interactions among homologs of 3q29 genes that we 19 identified in Drosophila, we examined the connectivity patterns of 3q29 genes within human 20 gene interaction databases. Gene interaction networks derived from co-expression and  24 However, the average connectivity among 3q29 genes within a brain-specific interaction  where interactions between PRODH and COMT modulate neurotransmitter function 30 independently of other genes in the region (Paterlini et al., 2005). In fact, five genes in the 31 3q29 region, including NCBP2, PAK2, and DLG1, showed significantly higher connectivity 32 to other 3q29 genes compared with the average connectivity of random sets of genes ( Figure   33 3- Figure Supplement 5D). Interestingly, NCBP2 showed the highest connectivity of all 1 genes in the region, further highlighting its role as a key modulator of genes in the region. Interactions between Cbp20 and other homologs of 3q29 genes enhance apoptosis 4 defects 5 Cell death and proliferation are two antagonistic forces that maintain an appropriate number 6 of neurons during development (Yamaguchi and Miura, 2015). In fact, both processes have 7 been previously identified as candidate mechanisms for several neurodevelopmental 8 disorders (Ernst, 2016;Glantz et al., 2006;Pinto et al., 2010). While knockdown of Cbp20 9 with other homologs of 3q29 genes likely disrupts multiple cellular processes that contribute 10 towards the enhanced cellular defects, we next specifically investigated the role of apoptosis 11 towards these defects, as larval eye and wing discs with knockdown of Cbp20 showed strong 12 increases in apoptosis. We observed black necrotic patches on the ommatidia in adult eyes 13 with knockdown of Cbp20/dlg1 and Cbp20/Fsn, indicating an increase in cell death with Fsn. Cbp20/CG8888 knockdown flies also showed a decreased number of pH3-positive cells, 27 suggesting that both apoptosis and proliferation are affected by the interaction between these 28 two genes ( Figure 4F). 29 To validate apoptosis as a candidate mechanism for the cellular defects of flies with 30 knockdown of homologs of 3q29 genes, we crossed recombinant fly lines of Cbp20 and dlg1 31 with flies overexpressing Diap1 (death-associated inhibitor of apoptosis). Diap1 is an E3 32 ubiquitin ligase that targets Dronc, the fly homolog of caspase-9, and prevents the subsequent  suggesting that the neuronal defects observed in these flies could be attributed to increased 12 apoptosis. We further confirmed these mechanistic findings by observing increased severity 13 in cellular phenotypes upon overexpression of Dronc in Cbp20 and dlg1 knockdown flies.  cell cycle genes as well as 10 interactions with microcephaly genes. We found that 13 out of 34 15 homologs of neurodevelopmental genes, including all four microcephaly genes, enhanced 1 the phenotypes observed with knockdown of Cbp20 alone. Furthermore, knockdown of 2 Cbp20 or dlg1 enhanced the ommatidial necrotic patches observed with knockdown of arm 3 (CTNNB1) ( Figure 6B). Interestingly, we also found that knockdown of CG8888 and dlg1 4 suppressed the rough eye phenotypes observed with knockdown of Prosap (SHANK3), while interacts with DLG1 through the mediator protein DLGAP1 to influence post-synaptic 9 density in mice (Coba et al., 2018) and binds to proteins in the Rac1 complex, including 10 PAK2, to regulate synaptic structure (Duffney et al., 2015;Park et al., 2003). These results 11 suggest that homologs of 3q29 genes interact with key developmental genes in conserved 12 pathways to modify cellular phenotypes. 15 After identifying a wide range of neurodevelopmental defects due to knockdown of fly 16 homologs of 3q29 genes, we sought to gain further insight into the conserved functions of 17 these genes in vertebrate embryonic brain development using the Xenopus laevis model these homologs on X. laevis brain development at stage 47. To test this, we knocked down 27 each gene in half of the embryo at the two-cell stage, and left the other half uninjected to 28 create a side-by-side comparison of brain morphology ( Figure 7A). We performed whole-29 mount immunostaining with anti-alpha tubulin and found that reduction of ncbp2, fbxo45, 30 and pak2 each resulted in smaller forebrain and midbrain size compared with controls 31 (Figures 7A-C). We also found that simultaneous knockdown of ncbp2 with fbxo45 caused a 32 significant decrease in forebrain size and a trend towards decreased midbrain size compared 33 with ncbp2 knockdown (Figure 7A-C). Knockdown of pak2 with ncbp2 showed a similar 34 trend towards decreased forebrain size. Interestingly, the reduced brain volumes we observed 1 with knockdown of homologs of 3q29 genes in X. laevis recapitulate the reduced brain 2 volume observed in 3q29 deletion mice (Baba et al., 2019;Rutkowski et al., 2019), 3 suggesting multiple genes in the 3q29 region contribute to this deletion phenotype. We 4 further examined the effect of knocking down homologs of 3q29 genes on X. laevis eye 5 development at stage 42, and found that knockdown of these homologs caused irregular 6 shapes and decreased size compared with controls (Figure 7- Figure Supplement 2A-B). 7 The reductions in eye size were rescued to control levels when mRNA was co-injected along 8 with MO for each homolog (Figure 7-Figure Supplement 2C). Together, these data show 9 that individual and pairwise knockdown of homologs of 3q29 genes in X. laevis leads to 10 abnormal brain and eye morphology, confirming the conserved role of these genes during 11 vertebrate development.

12
To determine if the knockdown of homologs of 3q29 genes also disrupted apoptotic 13 processes in X. laevis, we tested whether overexpression of the X-linked inhibitor of 14 apoptosis gene (xiap) could rescue the observed developmental defects. We found that 15 overexpression of xiap rescued the midbrain and forebrain size deficits observed with ncbp2 16 knockdown to control levels ( Figure 7A-C). Similarly, we found that the decreased eye sizes  1C). We found that reduction of fbxo45 and ncbp2 expression each led to an increase in 22 cleaved caspase-3 levels compared with controls, which were restored to control levels with 23 concomitant overexpression of xiap ( Figure 7E). Caspase-3 levels were also enhanced when 24 fbxo45 and ncbp2 were knocked down together (Figure 7E), suggesting that these two 25 homologs contribute towards developmental phenotypes through increased apoptosis.

26
Overall, these results suggest involvement of apoptotic processes towards the developmental 27 phenotypes observed with knockdown of homologs of 3q29 genes in a vertebrate model 28 ( Using complementary Drosophila and X. laevis models, we interrogated individual genes, 2 genetic interactions, and cellular mechanisms potentially responsible for the 3 neurodevelopmental phenotypes associated with the 3q29 deletion. Our major findings were 4 recapitulated across both model systems (Table 1) and could also potentially account for the 5 developmental phenotypes reported in mouse models of the entire deletion. Several themes 6 emerge from our study that exemplify the genetic and mechanistic complexity of the 3q29 7 deletion.

8
First, our analysis of developmental phenotypes upon knockdown of homologs for 9 individual 3q29 genes showed that a single gene within the region may not be solely 10 responsible for the effects of the deletion. In fact, we found that knockdown of 12 out of 14 11 fly homologs showed developmental defects in Drosophila, while every fly homolog showed 12 an enhanced rough eye phenotype when knocked down along with at least one other homolog 13 (Figure 2). Although our study is limited to examining conserved cellular phenotypes of 14 homologs of 3q29 genes in Drosophila and X. laevis, evidence from other model organisms 15 also supports an oligogenic model for the deletion. In fact, knockout mouse models for 16 several 3q29 genes have been reported to exhibit severe developmental phenotypes, including 17 axonal and synaptic defects in Fbxo45 -/and embryonic lethality in Pak2 -/and Pcyt1a -/-18 knockout mice (Marlin et al., 2011;Saiga et al., 2009;Wang et al., 2005) (Figure 1-Figure   19 Supplement 3). Notably, Dlg1 +/or Pak2 +/mice did not recapitulate major developmental  Second, our screening of 161 crosses between pairs of fly homologs of 3q29 genes 1 identified 44 interactions that showed enhanced rough eye phenotypes, suggesting that 2 complex interactions among 3q29 genes could be responsible for the developmental defects 3 observed in carriers of the deletion ( Figure 8A). While we only tested a subset of all possible 4 interactions among the non-syntenic homologs of 3q29 genes in Drosophila, our results 5 highlight conserved mechanistic relationships between "parts", or the individual genes, 6 towards understanding the effects of the "whole" deletion. For example, knockdown of 7 Cbp20 enhanced the phenotypes of 11 out of 12 other fly homologs, suggesting that NCBP2 8 could be a key modulator of the deletion phenotype. NCBP2 encodes a subunit of the nuclear 9 cap-binding complex (CBC), which binds to the 5' end of mRNA and microRNA in the hallmark of an oligogenic model for the deletion. As these genetic interactions may vary 23 across different species, developmental timepoints, and tissues, the role of these interactions 24 should be more deeply explored using mouse and human cell culture models.

25
Third, we identified disruptions to several cellular processes due to both single and 26 pairwise knockdown of homologs in Drosophila and X. laevis models ( Table 1) (Table 1). We propose that NCBP2 could modify several cellular and molecular 1 processes that may not be directly related to apoptosis, but could instead lead to a cascade of 2 biological events that ultimately result in apoptosis ( Figure 8B). Apoptosis mechanisms are 3 well-conserved between Drosophila, X. laevis, and humans, with key genes such as XIAP 4 (Diap1), CASP2 (Dronc), CASP3 (DrICE), and CASP7 (Dcp-1) sharing the same roles in Benjamini-Hochberg correction). Although we focused on testing apoptosis phenotypes upon 10 knockdown of homologs of 3q29 genes, we note that apoptosis is potentially one of the many 11 cellular pathways disrupted by the 3q29 deletion ( Figure 8B). In fact, our data implicated  abnormal apoptosis in the early developing brain has been suggested as a possible mechanism 28 for the decreased number of neurons observed in individuals with autism and schizophrenia 29 (Courchesne et al., 2011;Glantz et al., 2006;Kreczmanski et al., 2007). For example,  Frappart et al., 2005). Overall, these findings highlight the importance of cell cycle-related 12 processes, particularly apoptosis and proliferation, towards modulating neuronal phenotypes 13 that could be responsible for developmental disorders.
14 In this study, the use of Drosophila and X. laevis models, both of which are amenable 15 to high-throughput screening of developmental phenotypes, allowed us to systematically disorders, which will hopefully allow for future discoveries of treatments for these disorders. RNAi lines of other homologs to achieve simultaneous knockdown of two genes (Figure 1). 1 We previously demonstrated that these two-hit crosses had adequate GAL4 to bind to two 2 independent UAS-RNAi constructs (Iyer et al., 2018).  Climbing assay 22 We set up fly crosses at 25°C with Elav-GAL4 to obtain pan-neuronal knockdown for select 23 homologs of 3q29 genes. For each RNAi line tested, groups of ten female flies were first 24 allowed to adjust at room temperature for 30 minutes and then transferred to a climbing 25 apparatus, made by joining two vials, and allowed to adjust for 5 minutes. The flies were 26 tapped down to the bottom, and the number of flies climbing past the 8 cm mark measured 27 from the bottom of the apparatus in 10 seconds was then counted (Videos 1-2). This assay 28 was repeated nine additional times for each group, with a one-minute rest between trials. The 29 sets of 10 trials for each group were repeated daily for ten days, capturing data from 100  Imaging of adult fly eyes and wings 1 We crossed RNAi lines with GMR-GAL4 and reared at 29°C for eye-specific knockdown and 2 beadex MS1096 -GAL4 at 25°C for wing-specific knockdown. For eye imaging, adult 2-3-day old 3 female progenies from the crosses were collected, immobilized by freezing at -80°C, 4 mounted on Blu-tac (Bostik Inc, Wauwatosa, WI, USA), and imaged using an Olympus 5 BX53 compound microscope with LMPLan N 20X air objective using a DP73 c-mount 6 camera at 0.5X magnification and a z-step size of 12.1μm. (Olympus Corporation, Tokyo, 7 Japan). We used CellSens Dimension software (Olympus Corporation, Tokyo, Japan) to Immunohistochemistry of eye and wing discs 30 Third instar larval and 44-hour-old pupal eye discs, reared at 29°C, and third instar larval 31 wing discs, reared at 25°C, were dissected in 1X phosphate-buffered saline (PBS) and fixed 32 in 4% paraformaldehyde for 20 minutes. The eye and wing discs were then washed thrice in 33 with 1% normal goat serum (NGS) for eye discs, or 1% bovine serum albumin (BSA) for 1 wing discs) for 30 minutes, and then incubated overnight with primary antibodies at 4°C.  AnalyzeParticles, and apoptotic cells in wing discs stained with dcp1 were analyzed using 32 manual counting.

33
Differential expression analysis of transcriptome data 1 We performed RNA sequencing (RNA-Seq) of samples isolated from three biological 2 replicates of 35 fly heads each for individual (Cbp20, dlg1, Fsn, Pak) and pairwise 3 (Cbp20/dlg1, Cbp20/Fsn) Elav-GAL4 mediated knockdowns of homologs of 3q29 genes. We 4 compared gene expression levels of each cross to VDRC control flies carrying the same 5 genetic background (GD or KK control lines crossed with Elav-GAL4). We prepared cDNA 6 libraries for the three biological replicates per genotype using TruSeq Stranded mRNA LT

32
Chemiluminescence detection was performed using Amersham ECL western blot reagent 33 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Band intensities were quantified by 34 densitometry in ImageJ and normalized to the control mean relative to beta-actin. Due to the 1 low number of replicates, we did not perform any statistical tests on data derived from these 2 experiments.
3 4 Human brain-specific network analysis of 3q29 gene interactions 5 We used a human brain-specific gene interaction network that was previously built using a 3q29 genes within the network, excluding genes without connectivity in the network from 10 final calculations. As a control, we also measured the connectivity of 500 randomly selected 11 genes with 100 replicates each of 20 other random genes. All network analysis was 12 performed using the NetworkX Python package (Hagberg et al., 2008).

14
Overlap between neurodevelopmental and apoptosis gene sets 15 We obtained a set of 1,794 genes annotated with the Gene Ontology term for apoptotic Whitney tests were used to analyze Drosophila functional data and human network data, as 31 several datasets were not normally distributed (p<0.05, Shapiro-Wilk tests for normality).

32
Climbing ability and survival data for each fly RNAi line across each experiment day were 33 analyzed using two-way and one-way repeated values ANOVA tests with post-hoc pairwise t-tests. We also used parametric t-tests to analyze Drosophila qPCR data and all X. laevis 1 data, as these data were either normally distributed (p>0.05, Shapiro-Wilk tests for normality) 2 or had a robust sample size (n>30) for non-normality. All p-values from statistical tests 3 derived from similar sets of experiments (i.e. Flynotyper scores for pairwise interactions, 4 dcp1 rescue experiments with Diap1) were corrected using Benjamini-Hochberg correction.     14 Finally, we tested a subset of three homologs of 3q29 genes in the X. laevis vertebrate model 15 system by injecting two-or four-cell stage embryos with GFP and morpholinos (MOs) for X. 16 laevis homologs of 3q29 genes to observe abnormal eye morphology, as well as injecting one 17 cell with GFP and MOs at the two-cell stage to observe abnormal brain morphology. We     Individual larval eye disc images were assigned mild, moderate or severe scores based on the severity of axon projection loss observed in each eye disc. We found that the mild to 1 moderate defects observed with knockdown of Cbp20 were enhanced with concomitant 2 knockdown of dlg1 or Fsn, while Diap1 overexpression partially rescued the defects 3 observed with knockdown of Cbp20 or dlg1. A list of full genotypes for fly crosses used in 4 these experiments is provided in Supplementary File 2.     along with two other large CNVs, 16p11.2 (red) and 22q11.2 deletion (green), within a brain-32 specific gene interaction network. Average connectivity is measured as the shortest weighted 33 distance between two genes, with lower values representing stronger connectivity. Genes within the 3q29 and 22q11.2 deletions were not significantly more connected to each other 1 (p>0.05, one-tailed Mann-Whitney test with Benjamini-Hochberg correction) than random 2 sets of 21 genes throughout the genome (grey). However, genes within the 16p11.2 region 3 were significantly more connected to each other than the random gene sets (p=0.003, one-   knockdown of Cbp20. While other homologs of 3q29 genes also interact with each other, our 32 data suggest that Cbp20 is a key modulator of cellular phenotypes within the deletion region.  boxes indicate phenotypes observed in the knockout models, while gray-shaded boxes 10 indicate a phenotype that was not tested in the knockout model. Neither Dlg1 +/nor Pak2 +/-11 knockout mice recapitulate the body and brain weight, spatial learning and memory, or 12 acoustic startle defects observed in the deletion mouse models. beadex-GAL4 (KK) beadex-GAL4 (GD) pH3 staining of larval wing discs for proliferation dcp1 staining of larval wing discs for apoptosis beadex-GAL4 (KK) beadex-GAL4 (GD)                  for L and S, 5'-TATCTGTGGTGGGAAGAAAAGGTCA-3' dlg1 for L, 5'-CAAATGAGGCAGCAACTTACTTTCT-3' pak2 for L and S, 5'-AGAGATAAATCCTACCTTTTTCTGT-3' standard control 5′-cctcttacctcagttacaatttata-3′ forward for L and S allele 5'-CCGACATACTGTGCAACCTG-3' reverse for L and S allele 5'-TGTCCAAGATCACCCGAATCC-3' dlg1 forward for L allele 5'-CTCTCCTATGAACCCGTCAC-3' reverse for L allele 5'-CCGGCCTCTATGAATTTGTG-3' pak2 forward for L and S allele 5'-AGGATAAACCACCAGCTCCTC-3' reverse for L and S allele 5'-GGGAGCCCATCTTTATCTGGTG-3' ODC1 control forward 5'-GCCATTGTGAAGACTCTCTCCATTC-3' reverse 5'-TTCGGGTGATTCCTTGCCAC-3'