Kismet/CHD7 regulates axon morphology, memory and locomotion in a Drosophila model of CHARGE syndrome - PubMed (original) (raw)

. 2010 Nov 1;19(21):4253-64.

doi: 10.1093/hmg/ddq348. Epub 2010 Aug 17.

Affiliations

Kismet/CHD7 regulates axon morphology, memory and locomotion in a Drosophila model of CHARGE syndrome

David J Melicharek et al. Hum Mol Genet. 2010.

Abstract

CHARGE syndrome (CS, OMIM #214800) is a rare, autosomal dominant disorder, two-thirds of which are caused by haplo-insufficiency in the Chd7 gene. Here, we show that the Drosophila homolog of Chd7, kismet, is required for proper axonal pruning, guidance and extension in the developing fly's central nervous system. In addition to defects in neuroanatomy, flies with reduced kismet expression show defects in memory and motor function, phenotypes consistent with symptoms observed in CS patients. We suggest that the analysis of this disease model can complement and expand upon the existing studies for this disease, allowing a better understanding of the role of kismet in neural developmental, and Chd7 in CS pathogenesis.

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Figures

Figure 1.

Figure 1.

Adult phenotypes in Kismet knockdown flies. (A–E) Adult female flies. (A–C) Dorsal view. (D–E) Lateral view. (A) Wild-type (Canton S) fly. Note the position of the adult wings. (B and D) A fly expressing only the Daughterless-Gal4 reagent (Da-Gal4/ +) holds its wings normally. (C and E) A fly where Kis protein is ubiquitously knocked down (Da-Gal4/UAS:kis RNAi.a) holds its wings abnormally apart from its body. (F and G) Dorsal view of bristles on scutellum in (F) wild-type flies and (G) flies where Kis protein is knocked down in nervous tissue (Elav-Gal4/UAS:kis RNAi.a). Arrow notes duplication of bristles in Kis knockdown flies. (H and I) Adult wings from (H) wild-type and (I) Kis knockdown flies (Elav-Gal4/UAS:kis RNAi.a). Arrows denote presence of extra vein tissue near wing vein L2 (top arrow), the posterior crossvein (middle arrow) and wing vein L5 (bottom arrow). In cases not shown, _Da-Gal4/_+ and ELAV/+ flies show wild type phenotypes.

Figure 2.

Figure 2.

Kismet knockdown in muscles causes defective climbing behavior. (A–D) shows compiled climbing ability of wild-type, control and Kis knockdown flies at days 2–10. (A) Ubiquitous expression of UAS:Kis RNAi.a (Da-Gal4/UAS:Kis RNAi.a) shows severely reduced climbing ability compared with wild-type, Da-Gal4/+ and UAS:Kis RNAi.a/+ outcrossed control flies. (B) Kis knockdown restricted to muscles (DJ757-Gal4/UAS:Kis RNAi.b) also shows a strong reduction in climbing ability compared with wild-type, DJ757-Gal4/+ and UAS:Kis RNAi.b/+ outcrossed controls. (C) Kis knockdown in mushroom body neurons and the ventral nerve cord (c309-Gal4/UAS:Kis RNAi.b) shows a strong reduction in climbing ability compared to wild-type, c309-Gal4/+ and UAS:Kis RNAi.b/+ outcrossed controls. (D) Kis knockdown in the giant fiber inter-neurons (OK307-Gal4/UAS:Kis RNAi.b) shows a small but significant effect compared with wild-type, OK307-Gal4/+ and UAS:Kis RNAi.b/+ outcrossed controls. Error bars represent ± SEM. In all cases, double asterisk indicates P < 0.001 and asterisk indicates P < 0.05 compared with controls.

Figure 3.

Figure 3.

Kismet is widely expressed in the developing larval central nervous system. (A–C and G–I) Developing brain lobe of wild-type late third instar central nervous system. (D–F) Developing ventral nerve cord of wild-type late third instar larval central nervous system. (A–F) are whole brain reconstructions from individual confocal image slices. (A) Kis expression (red), Atonal expression (blue) and Daughterless expression (green) in wild-type brain lobe. Upper arrow denotes the Atonal-expressing DCNs. Lower arrow denotes the developing optic lobe. (B) Daughterless expression (white) from (A). Arrow denotes Daughterless expression in DCNs. (C) Kis expression (white) from (A). Arrow denotes Kis expression in DCNs. (D) Kis expression (blue) and Daughterless expression (green) in developing ventral nerve cord. (E) Daughterless expression (white) from (D). (F) Kis expression (white) from (D). (G) GFP expression (green) in the membrane as driven from the 201Y-Gal4 driver (201Y-Gal4/UAS:CD8-GFP) to denote the location of the γ-Kenyon neurons. Kis expression (red). Note strong co-localization. (H) Kis expression (white) from (G). (I) GFP expression (white) from (G).

Figure 4.

Figure 4.

Kismet knockdown flies can learn, but are deficient in their immediate recall memory. (A–C) denote learning during the first 10 (gray columns) and last 10 minutes (white columns) of the training phase during the courtship suppression assay. Genotypes are indicated. Note normal response of Kis knockdown mutants. (D–F) Panels denote immediate recall memory (0–2 min post-training) of trained flies (gray columns) when compared with sham-trained matching genotypes (white columns). Kis knockdown showed no significant difference between trained and sham-trained flies, indicative of no immediate recall memory of training. Error bars represent ± SEM. In all cases, double asterisk indicates P < 0.001 and asterisk indicates P < 0.05 compared with controls.

Figure 5.

Figure 5.

Kismet is required for proper axon pruning and axon migration in developing mushroom bodies. All panels show GFP in Kenyon neurons by MARCM analysis. (A and B) Pupal brains 18–20 h after puparium formation (apf). (C and D) Adult brains 48 h after eclosion. (A) Wild-type MARCM clones (FRT 40A) in pupal brains driven by 201Y-Gal4 show remnants of proper axonal pruning of γ neurons that were previously populating the α lobes (arrow). (B) kis LM27 homozygous mutant MARCM clones display unpruned γ axons that continue to populate the α lobes (arrow). (C) Wild-type MARCM clones (FRT 40A) in adult brains driven by OK107-Gal4 show normal pattern of α, α′, β, β′ and γ axons innervating the mushroom body lobes, as labeled. (D) kis LM27 homozygous mutant MARCM clones in these adult brains often display abnormal axon migration beyond the midline (demarcated by the arrowhead).

Figure 6.

Figure 6.

Kismet is required for proper DC position and axon migration in developing DCNs. All panels show GFP in DCNs by MARCM analysis. (A and B) Late third instar larval brains. Both images show the left hemisphere. Anterior is up. Medial is right. Lateral is left. (C–F) Adult brains 48 h after eclosion. (A) Wild-type MARCM clones (FRT 40A) in larval DCNs. Note position of the soma cluster axon bundles (parallel to vertical arrow) compared with commissural axon bundles (below horizontal arrow). (B) kis LM27 homozygous mutant MARCM clones display abnormal positioning of soma cluster compared with commissural axon bundles (left arrow), soma that have developed outside of the normal cluster (arrowhead) and disrupted commissural axon migration (right arrow). (C) Wild-type MARCM clones (FRT 40A) in adult DCNs displaying normal dendritic morphology on ipsilateral brain hemisphere. (D) kis LM27 homozygous mutant MARCM clones in adult brains. Overall morphology of dendritic field appears normal. (E) Wild-type MARCM clones (FRT 40A) in adult DCNs displaying normal axonal morphology on contralateral brain hemisphere. (F) kis LM27 homozygous mutant MARCM clones in adult brains. Arrow displays abnormal and reduced number of axonal extensions from lobulla into the medulla.

Figure 7.

Figure 7.

Kismet is required for proper photoreceptor axon migration and eye development. (A–D) Late third instar larval retinas with attached brains. (E–H) Adult eyes, anterior right. (A) Wild-type retina with attached larval brain stained for the Futsch protein (white) display normal axonal bundles in the optic stalk that innervate the developing optic lobes. (B) kis LM27 homozygous mutant clones in the developing retina stained for Futsch (white) display abnormal axonal migration into the developing brain. Arrow indicates defasciculation of axon bundles in the optic stalk. (C) Cross-section of kis LM27 homozygous mutant clones in the developing retina posterior to the morphogenetic furrow. Heterozygous (control) tissue is denoted by the presence of GFP (green). Homozygous clones are denoted by lack of GFP. Retinas are stained with Futsch (magenta). Note normal positioning of ommatidia clusters near the apical (top) portion of the retina. (D) Futsch stain (white) from (C). (E) Normal eye expressing only eyeless:Flip. (F) Adult eye showing kis LM27 homozygous mutant clones (in white). (G) GMR-Gal4 adult eye raised at 25°C displays normal morphology. (H) GMR-Gal4 / UAS:kis RNAi.b adult eye raised at 25°C displays abnormal morphology and glassy appearance.

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