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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01417

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Delta proteins and MAGI proteins: an interaction of Notch ligands with intracellular scaffolding molecules and its significance for zebrafish development

Gavin J. Wright^1,2,*, Jonathan D. Leslie^1,*, Linda Ariza-McNaughton¹ and Julian Lewis^1,

¹ Vertebrate Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
² Vertebrate Functional Proteomics Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK

View larger version (52K): [in a new window]   Fig. 1. Biochemical purification and identification of proteins that interact with a peptide corresponding to the C terminus of an –ATEV Delta. (A) Proteins purified using a C-terminal peptide of human Delta1 from lysates prepared from either an adult mouse whole brain (left panel) or the human neuroblastoma NB100 cell line (right panel) were resolved by SDS-PAGE under reducing conditions and identified by tryptic peptide mass spectrometry. Proteins that could be confidently identified have been labelled. (B) Domain structure of the MAGI family of proteins.

View larger version (88K): [in a new window]   Fig. 2. Expression pattern of magi1 in the zebrafish embryo. (A) Early stages analysed by RT-PCR (data replicated in three separate experiments). Maternal mRNA present in the 0 hpf egg has largely disappeared by 3 hpf, with fresh (zygotically synthesized) transcripts appearing by 6 hpf. EF1 was used as a positive control. (B-E) In situ hybridization patterns. (B) Lateral view at bud stage (10 hpf), showing diffuse expression. (C) Dorsal view at 24 hpf, showing expression still diffuse but strongest in the neural tube. (D) Dorsal view of the head of a whole mount at 72 hpf, showing expression in the retinae and, most strongly, in the diencephalon and telencephalon. (E) Transverse section of embryo stained as a whole mount at 72 hpf, showing expression in the hindbrain and in sensory hair cells in the ear (red arrow).

View larger version (53K): [in a new window]   Fig. 3. Zebrafish MAGI1 binds directly and specifically to zebrafish DeltaC and DeltaD. (A) Beads were coated with peptides corresponding to the C-terminal 27 or 26 amino acids of DeltaC (top panel) or DeltaD (bottom panel) either with the terminal valine (V) or without (E). The beads were then incubated with purified proteins corresponding to each of the six individual PDZ domains of zebrafish MAGI1 (0 to 5). Beads were then washed and bound proteins eluted and resolved by SDS-PAGE under reducing conditions. Both DeltaC and DeltaD peptides that terminated in the ATEV motif interacted directly and selectively with the PDZ4 domain alone, while control peptides that lacked the terminal valine (ending –ATE) showed no binding to any PDZ domain. (B,C) HEK293T cells cotransfected with plasmids coding for MAGI1-EGFP (green) and either full-length DeltaD (red in B) or DeltaD lacking its terminal valine (DeltaD-TE*; red in C). Both forms of DeltaD were detected with zdd2 anti-DeltaD monoclonal antibody. DeltaD, but not DeltaD-TE*, recruits MAGI1 to the plasma membrane [compare regions indicated by arrows in (B) and (C)]. 56 out of 59 cells transfected with DeltaD showed MAGI1 membrane recruitment, but only 1 out of 37 transfected with DeltaD-TE* did so. (D,E) Confocal sections of somite cells in 10-14 somite stage zebrafish embryos injected with 20 pg mRNA encoding for MAGI1-EGFP at the two- to four-cell stage, either (D) alone or (E) with coinjection of 5 ng MO[dlD-V]. MAGI1-EGFP was detected with an anti-GFP antibody and endogenous DeltaD was detected with zdd2. Note co-localization of DeltaD and MAGI1 in (D) but not (E). Scale bars: 10 µm.

View larger version (53K): [in a new window]   Fig. 4. A morpholino, MO[dlD-V], targeted to the intron-exon boundary responsible for the addition of the DeltaD terminal valine residue disrupts splicing and selectively removes the PDZ domain binding site for up to 72 hours of development. (A) The genomic structure of the zebrafish DeltaD gene surrounding the PDZ domain binding site, indicating the amino acids at the boundaries of each exon, the MO[dlDV] morpholino annealing site, and primer binding sites used to monitor the effects on splicing by RT-PCR in (B). (B) Effects on deltaD mRNA splicing, monitored by RTPCR, in embryos injected with MO[dlD-V] at the one-cell stage and left to develop until the shield stage (6 hpf). Total RNA was extracted from 20 embryos for each dose, reverse transcribed and amplified by PCR. PCR products were cloned and fully sequenced. Uninjected embryos produced a 370 bp band that corresponds to the correctly spliced transcript, coding for a protein that ends –ATEV. The amount of correctly spliced product decreases as the amount of injected morpholino increases; at a dose of 5 ng or more per embryo, no correctly spliced product is observable. Products at 469 and 269 bp were mis-spliced as shown and correspond to proteins lacking the terminal ATEV. The band at 330 bp was not characterized but probably represents the use of a cryptic splice donor site. The 546 bp band may result from small amounts of contaminating genomic DNA in the extracted RNA. The separate small panel below shows RT-PCR analysis of the same cDNA using oligonucleotides targeted to an upstream region of the deltaD transcript (nucleotides 389-923) that is unaffected by MO[dlD-V]. The total quantity of deltaD mRNA is not significantly altered by the MO injections. (C) Time course of the effect. Embryos were injected and allowed to develop until the indicated stages, and RT-PCR was performed as in B.

View larger version (101K): [in a new window]   Fig. 5. Effects of morpholino injections that block the interaction of DeltaD with MAGI1. (A-E) Dorsal views of the midbrain-hindbrain region in live embryos at 24 hpf. (A) Uninjected wild-type control. (B) Wild-type embryo injected with 5 ng of MO[dlD-V]; note narrowed third (III) and fourth (IV) ventricles and irregular texture due to dying cells in the walls of the hindbrain. (C) Similar phenotype produced by injection of 10 ng of MO[MAGI1]. (D) after eight (aei) uninjected embryo; note normal morphology. (E) aei embryo injected with 5 ng of MO[dlD-V]; note phenotype similar to that seen in (B) and (C). (F-H) Lateral views of live embryos at 24 hpf showing somite segmentation. Somite boundaries are disorganized below the eighth somite in aei (H), but are unaffected by MO[dlD-V] treatment (G). (I-K) Lateral views, anterior towards the left, of embryos at 26 hpf analysed by in situ hybridization for col2a1 expression to mark floor-plate (fp) and hypochord (hc). aei embryos exhibit a reduction in hypochord cell number. This effect is not observed upon MO[dlD-V] treatment (J). Scale bar: 50 µm for I,J,K.

View larger version (99K): [in a new window]   Fig. 6. Disruption of the DeltaD-MAGI interaction causes mislocalization of Rohon-Beard neurons. (A-D) Dorsal views of embryos at 16 hpf stained by in situ hybridization for islet1 (black) to mark Rohon-Beard neurons (as well as other primary neurons below the plane of focus). myoD (brown) expression serves as a reference for somite number and position. aei embryos (C,D) show a 1.6-fold increase in the number of Rohon-Beard cells compared to wild-type embryos (A), whereas the number of these cells is only slightly increased in wild-type embryos injected with MO[dlD-V] (B). The MO[dlD-V] embryos are abnormal, however, in that many of the Rohon-Beard cells stray into the midline region. The proportion of such mislocalized cells is not affected by the morpholino in aei embryos, where DeltaD is missing (C,D). (E,F) Cell counts. The distribution of the neurons was quantified for a region corresponding approximately to somites 5 to 10; 10-13 embryos were analysed for each condition. Error bars represent s.e.m. Scale bar: 50 mm for A-D.