Localization of a family of MRNAS in a single cell type and its precursors in sea urchin embryos.

Abstract

Spec 1 mRNAs increase 100-fold in abundance per embryo during early sea urchin development. Previous studies indicated an enrichment of this mRNA in ectoderm fractions of gastrulae and plutei. We have determined the precise localization of this mRNA by in situ hybridization techniques. In pluteus larvae, the mRNA is highly restricted to a set of morphologically uniform ectoderm cells in the dorsal part of the embryo.The mRNA is not detectable in other regions of ectoderm or in endoderm and mesoderm. The pattern of localization is already established at the gastrula stage, before these cells are distinguishable by morphological criteria. This pattern of distribution of Spec 1 mRNA is distinct from that of bulk poly(A)+ mRNA. Measurements of the amount of Spec 1 mRNA per embryo and the number of cells containing this RNA indicate that there are about 500 Spec 1 mRNA molecules per cell at the pluteus stage and probably twice as many at the gastrula stage. These results indicate that the sensitivity of the in situ hybridization method allows detection of sequences that comprise '-0.05% of the embryo mRNA nucleotides.The sea urchin embryo is perhaps the best-characterized system with respect to developmental modulation in gene expression (1-4). Recent analysis of a large number of different mRNAs indicates that few undergo greater than 10-fold modulation in abundance per embryo during early development (5). It is likely that such measurements of whole embryo concentration underestimate larger cell lineage-specific differences in gene activity that either reflect or effect determination and differentiation. To date, no set of gene products has been shown to be cell type specific during sea urchin embryogenesis. More accurate analyses of determination and the onset of differentiation require studies of expression of individual genes in different cell lineages of developing embryos.Recently, cell fractionation techniques have been used to identify a set of mRNAs and their associated proteins that are enriched in ectoderm of plutei (1,6). These studies showed that mRNAs complementary to the cDNA clone pSpecl increase markedly in concentration during embryogenesis beginning at early blastula stage. In vitro translation of mRNAs hybridizing to pSpecl revealed a family of similar small acidic proteins whose quantitative regulation during development parallels that of their mRNAs.In the experiments reported here, we used an improved in situ hybridization technique to map the distribution of Spec 1 mRNA precisely and to quantitate its concentrations in individual cells of developing embryos.MATERIALS AND METHODS Embryo Culture and Tissue Preparation. Strongylocentrotus purpuratus were obtained from Pacific Biomarine (Venice, CA) or from Patrick Leahy (Kerckhoff Marine Laboratory, California Institute of Technology). Embryos were cultured by standard techniques, fixed in 1% glutaraldehyde, embedded in paraffin, and sectioned and the sections were treated with proteinase K and acetic anhydride as described (7,8).Prepar...

A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

“…The latter is present at the prism stage in 300,000 copies or 750 copies per aboral ectoderm cell (32,33). This comparison suggested that SUM-1 was a rare mRNA on a per embryo basis.…”

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

“…1). These cells normally express Spec1 (Lynn et al, 1983;Wikramanayake et al, 1995) (Fig. 5N,P, white arrows).…”

Spec3: embryonic expression of a sea urchin gene whose product is involved in ectodermal ciliogenesis.

“…Analysis of serial sections leads us to believe that the section shown in Figure 2g passes through the border of presumptive aboral and oral ectoderm. These are regions of contiguous cells, each comprising approximately one hemisphere of differentiating ectoderm (for example, see Lynn et al 1983). The border between these regions contains cells of the presumptive ciliary band, and the unformly high labeling intensities observed here over ectoderm are consistent with this interpretation.…”

“…An impressive variety of molecular probes has been used in a number of investigations, including ones that have identified the origins and fates of several embryonic cell lineages (Lynn et al 1983;Gibson and Burke 1985;Cox et al 1986;Pehrson and Cohen 1986), studied the effects of embryonic perturbations on determination and differentation (Nemer 1986;Wessel and McClay 1987), and identified novel proteins associated with either the differentiation of embryonic cell types Wessel and McClay 1985;Leaf et al 1987) or the generation of embryonic structures (Wessel et al 1984;Benson et al 1987). …”