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First published online 6 February 2008
doi: 10.1242/dev.009530

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Auxilin is essential for Delta signaling

Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.

View larger version (114K): [in this window] [in a new window]   Fig. 1. Auxilin is required only in the Delta signaling cells. (A) Structure of Drosophila Auxilin. The vertical black bars within the Clathrin-binding domain are DPF motifs that in GAK bind AP2 (Umeda et al., 2000; Korolchuk and Banting, 2002). Approximate sites of the mutations in three aux nonsense mutant alleles and one missense allele (K47) are indicated (Eun et al., 2007). (B,B') Confocal image of an aux727/auxD128 clone (outlined) marked by the absence of GFP. Clones were generated in larvae of the genotype w, eyFLP; FRT42D gaux+, Ubi-GFP/FRT42D; aux727/auxD128. (C) Diagram of three Notch/Delta signaling events near the morphogenetic furrow. I, proneural enhancement; II, lateral inhibition; III, R-cell restriction. Numbers at top indicate rows of developing ommatidia. Numbers inside circles indicate R-cells. Blue cells are ectopic R-cells when R-cell restriction fails. mf, morphogenetic furrow; a, anterior. (D-G') Confocal images of immunolabeled third instar larval eye discs. (D,D') aux727/auxD136 clone (outlined), marked by absence of GFP, generated in larvae of genotype w, eyFLP; FRT42D gaux+, Ubi-GFP/FRT42D; aux727/auxD136. R-cell nuclei are Elav+. Why Elav+ cells are observed that are not directly adjacent to the clone edge is not known. This was observed also in lqf- and Dl- clones (Baker and Yu, 1996; Overstreet et al., 2004). (E,E') aux727/auxD136 clone expressing β-galactosidase from a Delta enhancer trap (Dl-lacZ) generated in larvae of genotype w, eyFLP; Tub-aux+, Ubi-GFP, FRT40A/FRT40A; auxD136 Dl-lacZ/aux727. The arrow indicates furrow. (F,F') aux- clone generated as in B, outlined. The arrow indicates furrow. (G,G') aux- clone generated as in D, outlined. Arrows indicate three aux- E(spl)+ nuclei. There are few aux- E(spl)+ nuclei observed because most of the cells at the clone border are R-cells (Elav+, see D), which do not express E(spl). Also, additional E(spl)+ nuclei are observed in other planes. Scale bar: 20 µm in B,B',D-F'; 10 µm in G,G'.

View larger version (140K): [in this window] [in a new window]   Fig. 2. Auxilin is required for some Delta internalization. Confocal images of immunolabeled third instar larval eye discs. Black-and-white images (A-D,E,F) show Delta protein. Arrowheads indicate furrow. (A) Wild type (wt). (B) Nts1 disc at 29°C for 6 hours. (C) lqf- clone (outlined). (D) lqf hypomorph heterozygous for strong neur mutation. (E-E'') auxK47/auxD136 showing Delta and F-actin (plasma membrane). (F-F'') aux727/auxD128 clone (outlined) marked by absence of GFP, generated in larvae of genotype w, eyFLP; FRT42D gaux+, Ubi-GFP/FRT42D; aux727/auxD128. (G) Same clone as in F, but more basal. Arrowheads indicate some of the numerous puncta. Scale bar: 10 µm in G for A-G.

View larger version (50K): [in this window] [in a new window]   Fig. 3. The Clathrin-binding and J domains of Auxilin are sufficient and the J domain is necessary for Delta signaling. (A) Drosophila Auxilin (1165 amino acids) and four transgene constructs. To the right is a summary of the complementation test results. UAS, UAS-cDNA; gDNA, genomic DNA-based constructs; +, indicates complementation of all aux- mutant phenotypes including lethality (rescue); -, indicates no activity (no rescue). The aux- genotypes tested for complementation are aux727/auxD128 (strong) and auxK47/auxD128 (hypomorphic). Three independent UAS-auxilinJ lines were tested, and in an aux727/+ background, two lines showed aux mutant-like wing and eye phenotypes (data not shown) indicative of dominant-negative activity. No auxilinK+PTEN lines are dominant negative. The auxilinJ line tested for complementation expresses protein; it has dominant-negative activity and protein is detected in immunolabeled eye discs (data not shown). (B) Confocal images of a facet (diagram of cell outlines at far right) in wild-type third instar larval eye disc immunolabeled with anti-Auxilin antibodies and phalloidin (F-actin).

View larger version (154K): [in this window] [in a new window]   Fig. 4. A Chc+ transgene suppresses aux eye and wing mutant phenotypes. (A) auxK47/auxD128 Drosophila wing. (B) auxK47/auxD128 wing with one copy of PChc+. The wing is indistinguishable from that of wild type. (C) Bar chart showing the degree of suppression of auxK47/auxD128 eyes by a single copy of PChc+. For each genotype, 51-124 facets in each of ten eyes were examined. Error bars represent one standard deviation from the mean calculated for each eye. The effect of PChc+ is significant (Student's t-test, P<0.001). (D-F) Tangential sections of adult eyes are shown as examples of data tabulated in C. PChc+ complements Chc1 completely (Bazinet et al., 1993). The mutant phenotype of aux hypomorphs reflects some defects in lateral inhibition (see Fig. 2E), and mainly defects in R-cell restriction. The same phenomenon was observed for lqf hypomorphs (Overstreet et al., 2004). This observation might reflect a particular requirement for efficient Delta signaling in R-cell restriction, or mechanistic differences in signaling at different times during eye development.

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