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. 2009 Feb 1;18(3):454-62.
doi: 10.1093/hmg/ddn373. Epub 2008 Nov 7.

The Drosophila homologue of the Angelman syndrome ubiquitin ligase regulates the formation of terminal dendritic branches

Yubing Lu  1 , Fay Wang, Yan Li, Jacob Ferris, Jin-A Lee, Fen-Biao Gao
Affiliations

The Drosophila homologue of the Angelman syndrome ubiquitin ligase regulates the formation of terminal dendritic branches

Yubing Lu et al. Hum Mol Genet. .

Abstract

Angelman syndrome is a severe neurodevelopmental disorder mostly caused by loss-of-function mutations in the maternal allele of UBE3A, a gene that encodes an E3 ubiquitin ligase. Drosophila UBE3A (dUBE3A) is highly homologous to human UBE3A (hUBE3A) at the amino acid sequence level, suggesting their functional conservation. We generated dUBE3A-null mutant fly lines and found that dUBE3A is not essential for viability. However, loss of dUBE3A activity reduced dendritic branching of sensory neurons in the peripheral nervous system and slowed the growth of terminal dendritic fine processes. Several lines of evidence indicated that dUBE3A regulates dendritic morphogenesis in a cell autonomous manner. Moreover, overexpression of dUBE3A also decreased dendritic branching, suggesting that the proper level of dUBE3A is critically important for the normal dendritic patterning. These findings suggest that dendritic pathology may contribute to neurological deficits in patients with Angelman syndrome.

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Figures

Figure 1.
Figure 1.
Generation of dUBE3A mutant alleles. (A) Genomic organization of the dUBE3A locus. dUBE3A contains eight exons and is located on the chromosome 3L and flanked by genes CG6199 and CG7600. Arrows indicate the direction of transcription. (B) Schematic of dUBE3A mutant alleles generated with the P-element local hop-out approach. An EP element is inserted in the first exon of dUBE3A. Excision of this P-element resulted in the generation of deletion mutant alleles of dUBE3A. The size of each deletion was determined by the sequencing of PCR fragments covering the deletion sites. In line Δ15-8, a 1156 bp fragment from the P-element remained in the first exon of dUBE3A, creating an insertional mutant allele.
Figure 2.
Figure 2.
Absence of dUBE3A expression in the mutant alleles. (A) Quantitative PCR (qPCR) analysis of relative mRNA levels of dUBE3A and CG7600 in adult flies. Values are mean ± SEM. (B) Expression levels of dUBE3A protein in fly head extracts. Fly heads were used for western blot analysis because smaller dUBE3A fragments, presumably due to non-specific degradation, were observed when protein extracts from whole flies were used (not shown). This polyclonal antibody recognized several non-specific bands on western blot. However, the dUBE3A band of the predicted molecular weight was absent.
Figure 3.
Figure 3.
Dendritic phenotypes of ddaC neurons in dUBE3A mutant third instar larvae. GFP expression in ddaC neurons was under the control of Gal4477. (A) Gal4477, UAS-mCD8-GFP/+; +/+ larvae were used as controls. (B and C) Dendritic phenotypes were analyzed in Gal4477, UAS-mCD8-GFP/+; dUBE3AΔ15-8/dUBE3AΔ15-8 (B) and Gal4477, UAS-mCD8-GFP/+; dUBE3AΔ15-8/dUBE3AΔ3-8 (C) larvae at the third instar stage. (D) Quantification of the number of dendritic termini of ddaC neurons for genotypes in (A–C). Values are mean ± SEM. ***P < 0.001.
Figure 4.
Figure 4.
Genetic analysis of dUBE3A function in dendritic morphogenesis using the RNAi approach. (A) Western blot analysis of dUBE3A expression in extracts of heads from adult control flies, dUBE3AΔ15-8 homozygous mutants and flies expressing different UAS-RNAi constructs under the control of tubulin-Gal4. UAS-RNAi lines 2-3, 8-2, 9-1, 29-1 and 29-10 were generated in our laboratory; 45875 and 45876 were from the VDRC. The bracket indicates the non-specific bands recognized by the dUBE3A polyclonal antibody. Asterisks indicate dUBE3A; the top band is likely a post-translationally modified form. (B) A wild-type ddaC neuron in the A3 segment of a third instar larva. The number of dendritic ends of ddaC neurons in dUBE3AΔ15-8/+ is slightly reduced (See Fig. 7). (C) A ddaC neuron expressing UAS-dUBE3A RNAi (VDRC45875) in the dUBE3AΔ15-8/+ background. (D) A ddaC neuron expressing UAS-dUBE3A RNAi (VDRC45876) in the dUBE3AΔ15-8/+ background. (E) Quantification of dendritic ends from ddaC neurons in the A3 segment from larvae in panels (B–D). Values are mean ± SEM. ***P < 0.001.
Figure 5.
Figure 5.
MARCM analysis of the cell autonomous function of dUBE3A in the dendritic morphogenesis of ddaC neurons. (A) A control ddaC neuron in the A3 segment of a third instar larva. (B) A dUBE3AΔ3-8 homozygous mutant ddaC neuron in the A3 segment of a third instar larva. (C) A dUBE3AΔ15-8 mutant ddaC neuron. (D) Quantification of the number of terminal dendritic branches from ddaC neurons with the genotypes of (A–C). Values are mean ± SEM. *P < 0.05; ***P < 0.001.
Figure 6.
Figure 6.
Cell autonomous effects of dUBE3A on dendritic branching in ddaE and ddaF neurons. (A) A control ddaE neuron labeled by GFP. (B) A ddaE neuron homozygous for the dUBE3AΔ3-8 allele. (C) A ddaE neuron homozygous for the dUBE3AΔ15-8 allele. The GFP-labeled ddaE neurons in (A–C) were generated by the MARCM technique. (D) Quantification of the number of dendritic branches of ddaE and ddaF neurons of different genotypes. (E) A wild-type ddaE and a wild-type ddaF neuron labeled by GFP under the control of Gal4221. (F) A ddaE and a ddaF neuron expressing UAS-dUBE3A RNAi (VDRC45876) driven by Gal4221. (G) A ddaE and a ddaF neuron expressing UAS-dUBE3A RNAi (VDRC45875) driven by Gal4221. (H) Quantification of the number of dendritic branches of ddaE and ddaF neurons expressing GFP and RNAi constructs. Values are mean ± SEM. *P < 0.05; ***P < 0.001.
Figure 7.
Figure 7.
Genetic interaction between dUBE3A and eff in dendritic morphogenesis. (A) A control ddaC neuron in the A3 segment of a third instar larva. (B) A ddaC neuron in a heterozygous dUBE3AΔ15-8/+ third instar larva. (C) A ddaC neuron in a heterozygous eff/+ third instar larva. (D) A ddaC neuron in a transheterozygous dUBE3A/eff third instar larva. (E) Quantification of the number of dendritic termini of ddaC neurons in different genetic backgrounds. Values are mean ± SEM. *P < 0.05; ***P < 0.001.
Figure 8.
Figure 8.
dUBE3A overexpression phenotypes in the wing and the eye. (A) A wild-type wing from a 3-day-old vg-Gal4 fly. (B) A wing expressing dUBE3A under the control of the vg-Gal4. (C) A section of an eye from a 3-day-old GMR-Gal4 fly showing the regular arrangement of omatidia. (D) A section of an eye from a 1-day-old fly expressing dUBE3A under the control of GMR-Gal4. (E) A section of an eye from a 7-day-old fly expressing dUBE3A by the GMR-Gal4 showing extensive retinal degeneration.
Figure 9.
Figure 9.
The effects of ectopic expression of dUBE3A and hUBE3A on dendritic branching of a subset of Drosophila sensory neurons. (A) Wild-type ddae and ddaF neurons are labeled by GFP under the control of the Gal4221. (B) A representative image of ddaE and ddaF neurons that express dUBE3A. (C) A representative image of ddaE and ddaF neurons that express hUBE3A. (D) Quantifications of the number of dendirtic ends of ddaE and ddaF neurons with different genetic backgrounds as in (A–C). Values are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. The sample numbers for each genotype are listed in the middle of each column.
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