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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00869

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Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning

Luis M. Escudero^1,*, Shu-Yi Wei^2,*, Wei-Hsin Chiu², Juan Modolell^1, and Jui-Chou Hsu^2,

¹ Centro de Biología Molecular Severo Ochoa, C.S.I.C. and U.A.M., Cantoblanco, 28049 Madrid, Spain
² Institute of Molecular Medicine, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30034, Republic of China

View larger version (94K): [in a new window]   Fig. 1. ed mutations promote generation of extra bristles in the notum of Drosophila. (A-C) Nota from wild-type, ed1X5/edslH8 and sca-Gal4; UAS-edIII flies, respectively. On B, note the extra macrochaetae arising near the extant ones and the increased density of microchaetae with respect to A. (C) Expression of UAS-edIII partially suppressed macrochaetae (such as the dorsocentrals and scutellar, and some microchaetae) and also caused occasional duplication of some macrochaetae (arrowhead). (D,E,E') Prospective notum of a third instar wing disc harbouring ed1x5 homozygous clones (absence of the GFP green fluorescence, arrowheads). Boxed area in A is shown at greater magnification in E and E' (E', green channel only). Note the very small size of the clones, which in some cases consist of a single cell (arrowheads in D,E,E') that has been singled out as an SOP (red, Sens marker). One of the two ed1x5 SOPs corresponds to an ectopic SO, because only two SOPs arise from a wild-type DC proneural cluster. The twin wild-type clones (bright green) consist of many cells, indicating the poor viabilty of the ed1x5 homozygous cells. (F) An extra DC f36a ed1x5 macrochaeta (red arrowhead) that may have arisen from a clone similar to those in E, but which probably consisted of more than a single cell, as two mutant microchaetae have developed adjacent to the extra macrochaeta. Black arrowheads indicate the ADC and PDC macrochaetae. (G) ed1x5 M+ clones, induced at 72-96 hours after egg laying (absence of green marker) survive well in a M+/– background and promote development of extra SOPs (red, Sens marker) when they include cells of a proneural cluster, in this case the DC one. No extra SOPs were observed outside the ed1x5 M+ clones. (H,I) Groups of extra bristles (labeled with f36a) develop within ed1x5 M+ clones. Five f36a DC macrochaetae are shown in H (red arrow) and a patch of f36a microchaetae in I (red arrow; clones were induced at 48-72 hours after egg laying). The increased density of microchaetae can be seen by comparing with the same region of a wild-type notum (J). Asterisks indicate the ADC macrochaeta. Black arrowhead in H indicates a f+ DC macrochaeta displaced from its position by the ed1x5 extra macrochaetae. (K,L) Prospective wing of third instar discs harbouring ed1x5 homozygous clones (absence of the GFP green fluorescence) in a wild-type or UAS-edIII/C765-Gal4 background, respectively. The expression of UAS-edIII largely increased the number of surviving clones and that changed their contours from smooth to uneven.

View larger version (43K): [in a new window]   Fig. 2. Accumulation of Sc, Sens and E(spl)-m8 mRNA in wild-type (A,C) and C253-Gal4; UAS-edECD/UAS-edECD (B,D) wing discs. (A,B) Notum regions showing that the levels of Sc protein in proneural clusters (green) are not significantly modified, except for the presence of extra SOPs in the disc expressing UAS-edECD (B). These also accumulate Sens protein (red channel), as shown in magnified images (insets) of the anterior and posterior notopleural (ANP, PNP) and dorsocentral (DC) proneural clusters. As many extra SOPs develop late, their accumulation of Sens is lower than that of earlier emerging SOPs. (C,D) Expression of E(spl)-m8 is decreased in the proneural clusters of discs expressing the Ed dominant-negative protein. Images show representative discs from a sample of 41 wild-type and 22 UAS-edECD-expressing discs. (E) Notum from a fly with the same genotype (C253-Gal4; UAS-edECD/UAS-edECD) of the discs shown in B and D. Note the increased number of macrochaetae arising from proneural clusters (compare with Fig. 1A). (F) Physical structure of Ed derivatives overexpressed in UAS constructs. Ed contains the extracellular and transmembrane (TM, red) domains, followed by the 315 amino acid intracellular domain. Edintra only contains the intracellular domain. EdECD lacks the extracellular domain but contains the TM and intracellular domain. EdECD-48 and EdECD-124 are similar to EdECD but lack the C-terminal 48 and 124 amino acids, respectively. As overexpressed with either ap-Gal4, sca-Gal4 or C253-Gal4, only UAS-edECD exhibited ectopic macrochaetae and increased density of microchaetae, and is referred as a dominant negative (DN). Overexpression of the rest of UAS constructs had no effect on bristle pattern (–).

View larger version (132K): [in a new window]   Fig. 3. Synergistic interaction between ed and the N pathway. (A-C) Nota of flies expressing in proneural clusters (C253-Gal4 driver) either UAS-edECD (abbreviated edDN) (A), UAS-NDN (B), or UAS-NDN plus UAS-edECD (C). Note the strongly enhanced neurogenic phenotype in C: the replacement of extant macrochaetae by tufts of bristles (arrowheads) and the loss of many macro and most microchaetae. (D) Notal dorsocentral region of a C253; UAS-NDN; UAS-edECD pupa stained with 22C10 antibody. Clusters of neurons appear at the sites of the developing sensory organs, as a result of the loss of N signaling, which leads to the differentiation of the descendants of the pIIa precursor cells as extra neurons. In the wild type, only a single neuron innervates each notum bristle. Inset depicts part of the third instar notum region of a C253; UAS-NDN; UAS-edECD larva stained with anti Sens antibody. Note the large clusters of SOPs at the DC and NP positions. (E,F) Nota of N55e11 (E) and N55e11; ed1x5/edslH8 flies (F). Similar to C, the simultaneous decrease of N and ed functions increases the neurogenic phenotype manifested by the almost complete absence of microchaetae (compare with ed1x5/edslH8 notum, Fig. 1B). (G-I) Nota of flies expressing in proneural clusters (sca-Gal4 driver) either UAS-edECD (G), UAS-DlDN (H) or UAS-DlDN plus UAS-edECD (I). The neurogenic phenotype caused by a decrease of Dl function is potentiated by the simultaneous decrease of ed function, resulting in large tufts of macrochaetae and increased density of microchaetae.

View larger version (76K): [in a new window]   Fig. 4. ed acts in the canonic N pathway, before the cytoplasmic release of NICD. (A,B) Removal of most microchaetae by expression of hs-Nicd in 0-8 hours-old pupae (A) is not rescued by UAS-edECD driven by sca-Gal4 (B). The control shown in Fig. 3G indicates that UAS-edECD driven by sca-Gal4 was active during microchaetae determination, as it increased microchaetae density. The transformation of macrochaetae to double sockets, owing to the excess of N signaling during differentiation, is clearly visible in some cases (arrowheads). (C,D) In a similar experiment, the weak removal of microchaetae by hs-NECN (C) is not rescued by UAS-edECD (D). (E,F) The low density of microchaetae typical of Nmcd1/+ (E) is not rescued by decreasing ed function (F, ed1X5/edslH8 combination). (G,H) The absence of macrochaetae characteristic of AxM1/+ (G) is not rescued by ed1X5/edslH8 (H).

View larger version (91K): [in a new window]   Fig. 5. Ed colocalizes with N at the zonula adherens. (A-C) Third instar larval wing discs were double-labeled with anti-Ed antibodies (green) and anti-Arm antibodies (red). Both Ed and Arm are colocalized at the zonula adherens. (D-F) Wing discs were double labeled with anti-Ed antibodies (green) and anti-NICD antibodies (red). N colocalizes with Ed to the zonula adherens. Insets in A-F show the corresponding z sections along the apicobasal axis of the epithelium; apical is towards the top.

View larger version (63K): [in a new window]   Fig. 6. ed produces a neurogenic phenotype in the Drosophila embryo. (A) Cuticle of a wild-type embryo showing the characteristic ventral denticle belts. (B) In ed germline clone embryos, lacking both maternal and zygotic ed expression, there is a ventral hole in the cuticle. (C) A wild-type embryo stained for the neuronal marker ELAV exhibits the condensed central nervous system. (D) An ed germline clone embryo displays a disorganized central nervous system with increased number of ELAV-positive cells, a phenotype typical of reduced N signaling.

View larger version (139K): [in a new window]   Fig. 7. Egfr acts antagonistically to Ed in bristle patterning, but it does not affect the levels of N signaling in proneural clusters. (A-D) Nota of flies expressing in proneural clusters (sca-Gal4 driver) either UAS-Egfr (A), UAS-Egfr plus UAS-edECD (B), UAS-rafgof (C), or UAS-rafgof plus UAS-edECD (D). There is a strongly enhanced tufting effect caused by co-expression in B and D. (E,F) Nota of ap-Gal4;UAS-EgfrDN (E) and ed1x5/edslH8 ap-Gal4;UAS-EgfrDN (F) flies. The ectopic bristle phenotype of ed1x5/edslH8 mutant flies is partially suppressed by the simultaneous decrease of Egfr function (compare with Fig. 1B). Arrow and arrowhead in F indicates the positions where ADC and PPA bristles are respectively eliminated. (G-I) Accumulation of E(spl)-m8 mRNA in the notum region of a wild-type disc (G) and in discs overexpressing either UAS-ras1V12 (H) or UAS-aos (I) in proneural clusters (C253-Gal4 driver). No significant differences were observed by comparing discs of similar stages from a total of 22 wild-type, 19 UAS-ras1V12 and 16 UAS-aos discs examined.