Boundary formation in Drosophila wing: Notch activity attenuated by the POU protein Nubbin

Science

Volumn 281, Issue 5375, 1998, Pages 409-413

  Neumann, Carl J ^a,b   Cohen, Stephen M ^a  

^a EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

^b MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

CELL PROTEIN; TRANSCRIPTION FACTOR POU; UNCLASSIFIED DRUG;

ARTICLE; CELL ACTIVATION; CELL INTERACTION; DEVELOPMENT; DROSOPHILA; GENE OVEREXPRESSION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN;

ANIMALS; CALCIUM-BINDING PROTEINS; DROSOPHILA; DROSOPHILA PROTEINS; ENHANCER ELEMENTS (GENETICS); GENE EXPRESSION REGULATION; GENES, INSECT; HOMEODOMAIN PROTEINS; INSECT PROTEINS; INTERCELLULAR SIGNALING PEPTIDES AND PROTEINS; LIGANDS; MEMBRANE PROTEINS; MUTATION; N-ACETYLGLUCOSAMINYLTRANSFERASES; NUCLEAR PROTEINS; POU DOMAIN FACTORS; PROTO-ONCOGENE PROTEINS; RECEPTORS, NOTCH; SIGNAL TRANSDUCTION; TRANSCRIPTION FACTORS; WING;

EID: 0032540875     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5375.409     Document Type: Article

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33. dpp-GAL4 was used to direct expression of UAS-Notch[intra] or UAS-Notch[intra] and UAS-Nubbin (parental genotypes: dppGAL4/TM6B X UAS-Nub/+; UAS-Notch[intra]/+). Larvae were triply labeled with rat antibody to Notch (anti-Notch) [R. G. Fehon et al., Cell 61, 523 (1990)], mouse anti-Nubbin (27), and rabbit anti-Wingless [P. Bhanot et al., Nature 382, 225 (1996)] to verify the genotype and assess the effects of Nubbin on Notch[intra] induction of Wg expression. Preliminary results suggest that genetic interactions occur between UAS-Nubbin and UAS-Notch[intra] in other developmental contexts (14). Further work will be required to determine whether this reflects a conserved regulatory relationship outside the developing wing.
34. We considered the possibility that the effects of nubbin could be mediated indirectly by effects on expression of Notch ligands. Delta is up-regulated in nubbin clones (14). Delta and Serrate can induce wingless expression but do so nonautonomously (1, 4, 5, 21, 25). If the effects of nubbin clones were mediated by increased expression of Delta or Serrate, we would expect wingless to be expressed in cells adjacent to the clone as well as in nubbin mutant cells. However, nubbin clones induce Wingless and vestigial strictly cell autonomously. Nubbin overexpression also argues against an indirect effect mediated by Delta and Serrate. If the effects were due to reduced expression of Notch ligands, Wingless expression should be rescued at the edge of the clone of Nubbin-expressing cells (as observed in Serrate or Delta mutant clones) (26), but it is not (Fig. 4).
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37. Clones of cells overexpressing Nubbin were produced by using a combination of the flip-out and Gal4 systems (F. Pignoni and S. L Zipursky, ibid., p. 271) in larvae of genotype Actin>CD2>Gal4; UAS-Nubbin; UAS-lacZ. Clones in which the flip-out cassette is excised will express GAL4 and direct expression of both Nubbin and β-Gal, which are under UAS control. Mouse anti-Wg has been described [W. J. Brook and S. M. Cohen, Science 273, 1373 (1996)].
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39. Bacterially expressed Nubbin protein was recovered as a pellet of insoluble inclusion bodies and solubilized in 2 M guanidine isothiocyanate in phosphate-buffered saline (PBS). Footprinting reactions were carried out as described [P. Rorth and D. Montell, Genes Dev. 6, 2299 (1992)]. Nubbin protein was diluted with a solution of 50% glycerol and 1% bovine serum albumin in PBS to 1:10, 1:30, 1:90, and 1:270. We incubated 1 μ1 of each dilution with 10 fmol of end-labeled DNA per 20 μl of footprinting reaction. To produce vestigial enhancer mutants, we modified the wild-type intron 2 enhancer (2) by polymerase chain reaction to introduce an Xho I site at the 5′ end and cloned this into Casper-HS43-AUG-β-Gal cut with Xho I-Eco RI. Expression of the modified enhancer is indistinguishable from the original version (2). Nubbin-binding sites were deleted by cutting the modified enhancer plasmid at unique Sac II and Sph I sites and re-ligating after end repair.
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44. We thank M. Mílán for help with nubbin clones, M. Ng for constructing the UAS-Nub, R. Nusse and S. Artavanis-Tsakonas for antibodies, F. Pignoni and S. Bray for fly strains, K. Huebner and H. Schoeler for advice on POU proteins, A.-M. Voie for technical assistance, P. Rørth for advice on footprinting, and S. Carroll for communicating unpublished results on mutations of the vestigial intron 2 enhancer.