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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01120

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Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein

¹ The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston, Brisbane 4029, Australia
² Department of Zoology and Entomology, University of Queensland, Brisbane 4072, Australia
³ Central Clinical School, University of Queensland, Brisbane 4029, Australia

View larger version (26K): [in a new window]   Fig. 1. Distribution of microarray clones into expression profile categories during normal and anti-Hemps antibody-treated metamorphosis. Proportion of genes affected by anti-Hemps treatment at 30 minutes (A) and 4 hours (B) post-induction (PI) in each of the seven normal expression profiles. Red, greater than 2-fold increase in mRNA abundance in treated post-larvae; yellow, less than 2-fold change in mRNA abundance in treated post-larvae; blue, greater than 2-fold decrease in mRNA abundance in treated post-larvae. The figures to the right of each profile represent the percentage of genes in each of the categories affected by anti-Hemps antibody.

View larger version (31K): [in a new window]   Fig. 2. Expression of Hemps normalised to Hec-coA compared with Hemps northern analysis. (A) The expression of Hec-coA was compared with the expression of Hec-Ubiq through developmental stages egg, gastrula, mid-tailbud, hatched, competent, 30 minutes post-induction (PI), 4 hours PI, 16 hours PI, 24 hours PI and juvenile. (B) The expression of Hemps through developmental stages egg to juvenile normalised to Hec-coA using quantitative real-time RT-PCR (QPCR). (C) Densitometry analysis of Hemps expression using northern analysis shows a similar expression profile to that derived using QPCR (Eri et al., 1999).

View larger version (20K): [in a new window]   Fig. 3. Validation of the expression levels between normal metamorphosing larvae and anti-Hemps antibody-treated larvae using quantitative real-time RT-PCR (QPCR) analysis at 30 minutes post-induction (PI; A) and 4 hours PI (B). Clones selected are Hec-bcsX1, Hec-meta1, Hec-meta2a, Hec-meta2, Hec-sap, Hec-mbl, Hec-shm, Hec-rab, Hec-nopp34, Hec-hsup and one unknown gene, Ac925 (left to right). Shown are the ratios (treated:normal) derived by the microarrays and QPCR. A ratio >1 represents a gene with expression that is upregulated when treated with anti-Hemps antibody. A ratio <1 represents a gene with expression that is downregulated when treated with anti-Hemps antibody. An asterisk indicates a significant difference between treated and untreated larvae.

View larger version (47K): [in a new window]   Fig. 4. Relative transcript abundances of 15 genes during Herdmania embryogenesis and metamorphosis normalised to Enoyl-CoA-hydrotase gene expression. Normalized relative transcript abundances are shown relative to time in hours since fertilization. Embryogenesis = 0-10 hours. Larvae hatch at 10 hours and are competent by about 14 hours; dashed lines represents onset of competence and induction of metamorphosis. Early metamorphosis = 14-40 hours. Relative transcript abundance data at 8 days, when the juvenile body plan is clearly evident, are shown for each gene. Error bars indicate standard deviation. Genes that have an altered expression when treated with the anti-Hemps antibody are indicated by an asterisk.

View larger version (110K): [in a new window]   Fig. 5. Localised expression of five genes that are transiently expressed during larval development and early metamorphosis. Whole-mount in-situ hybridisation of competent larvae (left plates in each row), 30-minute post-induction (PI) post-larvae (middle plates in each row) and 4-hour PI post-larvae (right plates in each row); anterior to the right. (A-C) Hec-pnx1, (D-F) Hec-smdp1, (G-I) Hec-mbl, (J-L) Hec-meta2a, (M-O) Hec-meta2. Scale bar: 50 µm.

View larger version (102K): [in a new window]   Fig. 6. Localised expression of five genes that are differentially expressed during larval development and early metamorphosis. Whole-mount in-situ hybridisation of competent larvae (left plates in each row), 30-minute post-induction (PI) post-larvae (middle plates in each row) and 4-hour PI post-larvae (right plates in each row); anterior to the right. (A-C) Hec-sap, (D-F) Hec-cip1, (G-I) Hec-cip2a, (J-L) Hec-cip2B, (M-O) Hec-lp1. Scale bar: 50 µm.