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New Early Zygotic Regulators Expressed in Endomesoderm of Sea Urchin Embryos Discovered by Differential Array Hybridization

Ransick, Andrew and Rast, Jonathan P. and Minokawa, Takuya and Calestani, Cristina and Davidson, Eric H. (2002) New Early Zygotic Regulators Expressed in Endomesoderm of Sea Urchin Embryos Discovered by Differential Array Hybridization. Developmental Biology, 246 (1). pp. 132-147. ISSN 0012-1606. doi:10.1006/dbio.2002.0607.

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Genes that are upregulated by LiCl treatment of sea urchin embryos and/or downregulated by injection into the egg of mRNA encoding an internal fragment of cadherin (Cad) were detected in a differential macroarray screen. The method was that recently described by J. P. Rast et al. (2000, Dev. Biol. 228, 270–296). Almost 105 clones from a 12-h cDNA library were screened. Measurements on internal standards showed that the screening procedure was sufficiently sensitive to afford detection of differentially expressed mRNAs of the most rare class, those present in only a few copies per average cell. The injection of Cad mRNA, which specifically blocks nuclearization of β-catenin, resulted in many-fold decreases in the levels of transcripts of a suite of marker genes expressed zygotically during endomesoderm specification. These measurements substantiated the use of Cad mRNA as the basis for a differential screen for discovery of new endomesodermal genes. By use of the newly developed BioArray software for analysis of macroarray screens, 1106 clones representing differentially expressed genes and yielding useful sequence were recovered. The 367 clones that gave significant BLASTX matches to known cellular proteins fell into 264 nonredundant sequence classes. Those of particular interest for this work were clones encoding DNA-binding transcription factors, signal transduction pathway components, proteases, kinases, and phosphatases. Quantitative PCR analysis of 66 such selected clones revealed that the large majority of these clones had been selected because they are upregulated by LiCl treatment, which affects the expression of a much greater diversity and number of genes than are involved in endomesoderm specification. Seven transcript species were identified that responded sharply to injection of Cad mRNA, and that are not represented in maternal mRNA. Six of those encode transcription factors. We focused on three transcription factor genes of this set that were previously unknown in sea urchin embryos. By whole-mount in situ hybridization, these genes are expressed in specific domains of the endomesodermal territory. They are: (1) Speve, an evenskipped orthologue expressed very early in all vegetal blastomeres and then gradually shifting to veg1 derivatives by the mesenchyme blastula stage; (2) Spgcm, an orthologue of the fruit fly gene glial cells missing, which is first expressed specifically and exclusively in part of the prospective secondary mesenchyme (mesodermal) domain at late-cleavage blastula stage; and (3) Spfoxc, which is first expressed in the early blastula only in the four small micromeres, and later only expressed in that coelomic pouch which gives rise to the mesoderm of the ventral surface of the adult rudiment.

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Additional Information:© 2002 Elsevier Science (USA). Received for publication December 18, 2001. Revised January 28, 2002. Accepted January 28, 2002. We thank all of those associated with the Davidson Laboratory who made invaluable contributions to this project, including Mi Ryong Yun, Chi H. Yun, Niñon Le, Katherine Carlsmith, and Albert Nguyen. We also thank the robotics macroarraying facility of the Caltech Beckman Institute. This work was supported by NIH Grants HD37105, RR-06591, and RR15044 (to E.H.D.).
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Subject Keywords: endomesoderm; macroarray; subtractive hybridization; sea urchin; embryo; specification; zygotic
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Record Number:CaltechAUTHORS:20160114-071950323
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Official Citation:Andrew Ransick, Jonathan P. Rast, Takuya Minokawa, Cristina Calestani, Eric H. Davidson, New Early Zygotic Regulators Expressed in Endomesoderm of Sea Urchin Embryos Discovered by Differential Array Hybridization, Developmental Biology, Volume 246, Issue 1, 1 June 2002, Pages 132-147, ISSN 0012-1606, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63666
Deposited By: Ruth Sustaita
Deposited On:14 Jan 2016 19:48
Last Modified:10 Nov 2021 23:19

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