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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00647

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

Cristina Calestani, Jonathan P. Rast and Eric H. Davidson*

Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA



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  		Fig. 1. Differential macroarray screen. (A) Digital image of macroarray colony filters hybridized with a radiolabeled RNA probe synthesized from LiCl-treated embryos (Before subtraction) and with a radiolabeled RNA probe synthesized after subtractive hybridization (After subtraction). Before the subtractive hybridization, two different 500 nucleotide bacteriophage {lambda} sequences ({lambda}1 and {lambda}2) were added to the selectate at a concentration equivalent to five copies/average cell and were also spotted onto the library filters. A magnification of the spots corresponding to the {lambda} and ubiquitin clones are shown. The {lambda} clones became detectable only after subtraction while the ubiquitin sequence, common to both selectate and driver, was not enriched after subtraction. (B) Table showing the level of enrichment obtained by the subtractive hybridization, measured by QPCR as enrichment of the {lambda} sequences relative to the ubiquitin sequence. QPCR amplification of the {lambda} and ubiquitin sequences was carried out on an aliquot of the subtractive hybridization reaction before HAP chromatography (Before HAP) and on the single-strand fraction after HAP chromatography (After HAP). The differences in cycle threshold (Ct) between the {lambda} sequences and ubiquitin were calculated ({Delta}Ct Ubiq); Ct values were means of triplicates. The enrichment values are the cycle amplification efficiency (1.9) raised to the power of the difference [(before HAP {Delta}CtUbiq)-(after HAP {Delta}CtUbiq)].

 


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  		Fig. 2. Spot intensity enhancement of macroarray clones after subtractive hybridization. Data for one filter are shown as a representative example. On the x-axis are the clone numbers and on the y-axis are the ranked spot intensity ratios of the after (Isub) and before subtraction (Iunsub) screen. To select the putative pigment cell specific clones, an enrichment threshold value (broken line) was determined based on the range of enrichment values of the 24 {lambda} phage clones spotted onto each filter. Most clones with enrichment values below threshold are clones the spot intensity of which is very close to the background both before and after subtraction. The spot intensity values of the selected group of enriched clones were checked on the dnN (driver) screen. The clones for which spot intensity was above background were discarded as false positive.

 


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  		Fig. 3. Whole mount in situ hybridization of pigment cell specific genes. In situ hybridization with antisense RNA DIG-labeled probe for the SpPks gene (A-D), SpFmo1 gene (E-H) and SpSult gene (I-L) on embryos at different developmental stages: hatched blastula side view (A,E,I) and vegetal view (B,F,J); gastrula side view (C,G) and apical view (K); pluteus oral view (D,H,L). In situ hybridization for SpFmo2 and SpFmo3 were also carried out (data not shown). All the genes examined are expressed in pigment cell precursors at the blastula stage and their expression is maintained in pigment cells throughout the gastrula and pluteus stages.

 


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  		Fig. 4. SpPks and SpFmo1 antisense morpholino oligonucleotide injected embryos. (A-C) Aboral view of prism stage embryos injected with 200 µM antisense morpholino oligonucleotide against the SpPks (B) and the SpFmo1 (C) genes and with a 200 µM random sequence morpholino oligonucleotide (A). The embryos injected with the SpPks and SpFmo1 morpholinos did not show accumulation of the echinochrome pigment. (D-F) Whole mount in situ hybridization with antisense RNA DIG-labeled probe for SpSult on prism stage embryos injected with a random sequence morpholino oligonucleotide (D), or with the SpPks (E) or the SpFmo (F) morpholino oligonucleotides. (E,F) Pigment cells are present in embryos lacking SpPKS or SpFMO.

 

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© The Company of Biologists Ltd 2003