Cristina Calestani

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

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.

Cis-regulatory analysis of the sea urchin pigment cell gene polyketide synthase

The Strongylocentrotus purpuratus polyketide synthase gene (SpPks) encodes an enzyme required for the biosynthesis of the larval pigment echinochrome. SpPks is expressed exclusively in pigment cells and their precursors starting at blastula stage. The 7th-9th cleavage Delta-Notch signaling, required for pigment cell development, positively regulates SpPks. In previous studies, the transcription factors glial cell missing (SpGcm), SpGatae and kruppel-like (SpKrl/z13) have been shown to positively regulate SpPks. To uncover the structure of the Gene Regulatory Network (GRN) regulating the specification and differentiation processes of pigment cells, we experimentally analyzed the putative SpPks cis-regulatory region. We established that the -1.5kb region is sufficient to recapitulate the correct spatial and temporal expression of SpPks. Predicted DNA-binding sites for SpGcm, SpGataE and SpKrl are located within this region. The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. These results demonstrate that the sea urchin GRN for pigment cell development is quite shallow, which is typical of type I embryo development.

Expression pattern of polyketide synthase-2 during sea urchin development.

Polyketide synthases (PKSs) are a large group of proteins responsible for the biosynthesis of polyketide compounds, which are mainly found in bacteria, fungi, and plants. Polyketides have a wide array of biological functions, including antibiotic, antifungal, predator defense, and light responses. In this study, we describe the developmental expression pattern of pks2, one of two pks found in the sea urchin genome. Throughout development, pks2 expression was restricted to skeletogenic cells and their precursors. Pks2 was first detected during the blastula stage. The transcript level peaked at hatched blastula, when all skeletogenic cell precursors expressed pks2. This was followed by a steady decline in expression in the skeletogenic cells on the aboral side of the embryo. By the prism stage, pks2 expression was limited to only 3-4 skeletogenic cells localized on the oral side.

Genome-wide assessment of differential effector gene use in embryogenesis.

Six different populations of cells were isolated by fluorescence-activated cell sorting from disaggregated late blastula- and gastrula-stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered bacterial artificial chromosomes producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicated the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1-2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus, at the effector gene level, a dramatic, cell type-specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred. Highlighted article: Spatially distinct populations of the early sea urchin embryo display profound differences in effector gene expression before morphological differences can be distinguished.

These Colors Don’t Run: Regulation of Pigment—Biosynthesis in Echinoderms

Pigment production is an important biological process throughout the tree of life. Some pigments function for collecting light energy, or for visual identification, while others have dramatic antimicrobial functions, or camouflage capabilities. The functions of these pigments and their biosynthesis are of great interest if only because of their diversity. The biochemistry of echinoderm pigmentation has been intensively studied for many years, and with more recent technologies, the origin and functions of these pigments are being exposed. Here we summarize the major pigment types in biology and emphasize the status of the field in echinoderms, taking full advantage of the new genomic and technologic resources for studying these important animals and their beautiful pigmentation.