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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00796

Development 130, 6295-6304 (2003)
Published by The Company of Biologists 2003
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echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

Amina Ahmed1,*, Shweta Chandra1,*, Marta Magarinos2 and Harald Vaessin1,2,3,{dagger}

1 Molecular, Cellular and Developmental Biology Program, Center for Molecular Neurobiology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
2 Ohio State Biochemistry Program, Center for Molecular Neurobiology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
3 Department of Molecular Genetics, Center for Molecular Neurobiology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA



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  		Fig. 1. ed expression pattern during embryogenesis. ed RNA (A,B) and protein (C-H) expression in wild-type embryos. In stage 9 embryos (A-D), differential expression of ed is evident. Whereas ed is expressed in the neuroectodermal (NE) and mesodermal (ME) cell layer, no expression is detected in the delaminated neuroblasts. (B,D) Enlarged lateral views of the ventral regions of embryos in A and C, respectively. The NE and ME cell layer are indicated and the bracket spans the layer where delaminated neuroblasts are located. (E) By stage 11, Ed expression ceases in the ventral neurogenic region and becomes restricted to the tracheal pits, marked by the asterisk, and the epidermis. At late stage 13, Ed can be detected in a subset of cells in the developing CNS and also in the epidermis (F). During stage 14, Ed expression is limited to the anterior and posterior epidermal stripes (H). At stage 16, Ed is highly localized to the axons with a higher concentration in the posterior commissures (G). A and P refer to anterior and posterior commissure, respectively.

 


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  		Fig. 2. Embryonic phenotypes associated with ed mutations. Ventral views of wild-type (WT) (A) and ed2B8/ed2B8 (B) embryos stained with anti-HRP antibody. ed2B8 homozygous embryos exhibit a hyperplasia of the CNS. This hyperplasia is accompanied by loss of epidermal structures. (C-F) Lateral views of the embryos. Cuticle preparations of WT (C) and ed2B8/ed2B8 (D) embryos. Asterisks in C mark denticle belts, characteristic structures of the ventral cuticle. ed2B8 homozygous embryos show an extensive loss of ventral and procephalic cuticle. (E,F) Lateral views of WT (E) and ed2B8 homozygous (F) embryos labeled with anti-Deadpan antibody. Arrowheads in F mark the ectopic neuroblasts present in ed mutant embryos.

 


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  		Fig. 3. Adult phenotypes associated with ed mutations. (A) Wing of wild-type (WT) and (B) ed2B8/edts flies. ed2B8/edts flies shifted to 29°C for three days at end of larval stage 2 show wings with irregular thickening of wing vein II and/or slight notching (indicated by the bracket in B) of the distal margin. In addition, extra macrochaetae and an increased density of microchaetae are evident on the thorax of ed2B8/edts flies (F), compared with the WT (E). Arrowheads point to ectopic bristles. Foreleg of WT (C) and edk01102/edm1 (D) males. The sex combs are malformed.

 


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  		Fig. 4. EdExt has dominant-negative activity. (A) Control wing of UAS-EdExt/+ fly. (B) UAS EdExt/69B GAL4 fly wing have irregular wing vein II. (C) T80 GAL4/+; UAS-EdExt/+ fly wing exhibits irregular wing vein II and rare notches (indicated by the bracket) in the distal wing margin. (D) Partial dorsal thorax of control (UAS-EdExt/+) fly showing the normal number of sensory bristles on the scutellum. (E) Scutellums of T80 GAL4/+; UAS-EdExt/+ flies show extra sensory bristles (indicated by arrowheads). The extra sensory bristle phenotype is more evident when pnr GAL4 is used to mediate EdExt expression. A loss of epithelium is also noticeable (F). (G) Magnification of a part of the mesothoracic region of the thorax in F. The microchaetae are missing (asterisk) or quadrupled (arrows).

 


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  		Fig. 5. Genetic interactions between ed and genes of the Notch signaling pathway. Anti-HRP labeling of neurons in the embryos (A-H). (AD) Lateral views; (E-H) ventral views. Embryos in A-H were grown at 29°C. (A,E) Wild-type (WT) embryos. (B,F) Examples of the extent of hyperplasia of the CNS evident in ed2B8/edts embryos. Ectopic expression of Nact in the parasegments 4-6 mediated by the Kr-GAL4 driver, results in the suppression of neuronal cell fate in this region (C,G). edts:UAS-Nact/ed2B8; Kr-GAL4/+ embryos show strong suppression of the Nact overexpression phenotype, resulting in a near WT morphology (D,H). (I) Control wing of Dlvia1/+ fly. (J) edts/edm1 flies grown at 25°C show a mild thickening of wing vein II. (K) edts/edm1; Dlvia1/+ flies grown at 25°C show an enhancement of the thick wing vein II phenotype and show additional wing vein material (indicated by the arrowhead) in the posterior cell. (L) edts/+heterozygous flies raised at 29°C show a WT wing morphology. (M) E(spl)8D06/+ flies raised at 29°C show extra wing vein material (arrowhead) in the posterior cell with low penetrance. (N) edts/+; E(spl)8D06/+ flies at 29°C display more ectopic vein material (arrowhead) with full penetrance.

 


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  		Fig. 6. The dominant-negative activity of EdExt is modulated by the Notch signaling pathway. (A-D) SEM of anterior midline region of adult thorax. (A) Normal microchaetae distribution in wild type (WT) flies. Ectopic expression of EdExt mediated by Eq-GAL4 driver results in a mild increase in the microchaetae density (B). Ectopic expression of Dl also results in an increased microchaetae density (C). (D) UASEdExt/+; UAS-Dl/Eq-GAL4 flies show significantly increased microchaetae density. (E) Arrangement of sensory bristles on the notum of an UAS-EdExt/+ fly. An extra bristle can be seen rarely. pnr-GAL4-mediated ectopic expression of EdExt results in formation of extra bristles, which is accompanied by loss of epidermis (F). Overexpression of E(spl)m7 results in suppression of sensory bristle formation, resulting in a bald mesothorax region (G). Overexpression of E(spl)m7 completely suppresses the phenotype of EdExt overexpression (H). Thus UAS-EdExt/+; UAS-E(spl)m7/pnr-GAL4 flies (H) are indistinguishable from UAS-E(spl)m7/pnr-GAL4 flies (G).

 

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© The Company of Biologists Ltd 2003