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Isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening

Calestani, Cristina and Rast, Jonathan P. and Davidson, Eric H. (2003) Isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening. Development, 130 (19). pp. 4587-4596. ISSN 0950-1991. doi:10.1242/10.1242/dev.00647.

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New secondary mesenchyme specific genes, expressed exclusively in pigment cells, were isolated from sea urchin embryos using a differential screening of a macroarray cDNA library. The comparison was performed between mRNA populations of embryos having an expansion of the endo-mesodermal territory and embryos blocked in secondary mesenchyme specification. To be able to isolate transcripts with a prevalence down to five copies per cell, a subtractive hybridization procedure was employed. About 400 putative positive clones were identified and sequenced from the 5' end. Gene expression analysis was carried out on a subset of 66 clones with real time quantitative PCR and 40 clones were positive. This group of clones contained sequences highly similar to: the transcription factor glial cells missing (gcm); the polyketide synthase gene cluster (pks-gc); three different members of the flavin-containing monooxygenase gene family (fmo); and a sulfotransferase gene (sult). Using whole mount in situ hybridization, it was shown that these genes are specifically expressed in pigment cells. A functional analysis of the S. purpuratus pks and of one S. purpuratus fmo was carried out using antisense technology and it was shown that their expression is necessary for the biosynthesis of the sea urchin pigment echinochrome. The results suggest that S. purpuratus pks, fmo and sult could belong to a differentiation gene battery of pigment cells.

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Additional Information:© 2003 The Company of Biologists Limited. Accepted 4 June 2003. First published online August 18, 2003. We thank the robotics macroarraying facility of the Caltech Beckman Institute. Miki Yun and Sangeeta Bardhan helped with the sequencing and Katherine Carlsmith helped with embryo injections. We also thank Titus Brown for assistance with the bioinformatic analysis. We are grateful to Prof. Dave McClay for kindly providing the dnN construct. We thank Dr Andrew Ransick and Dr Veronica Hinman for critical review of the manuscript, and Prof. Jack Richards and Dr Ivan Dmochowski for useful discussions. This work was supported by NIH grants HD-37105, RR-06591 and RR-15044, and by the Lucille P. Markey Charitable Trust.
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Lucille P. Markey Charitable TrustUNSPECIFIED
Subject Keywords:Sea urchin, Pigment cells, Mesoderm specification, SMC, Notch signaling, Macroarray
Issue or Number:19
Record Number:CaltechAUTHORS:CALdev03
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12893
Deposited By: Archive Administrator
Deposited On:09 Jan 2009 07:03
Last Modified:08 Nov 2021 22:33

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