We previously established a system for examining mRNA expression by performing in situ hybridization of juveniles of E. fluviatilis that are hatched from gemmules (Funayama et al.,2005a). We previously defined six developmental stages of this hatching process (Funayama et al.,2005a), and in the present study, we used sponges at developmental stage 2 (Fig. 1C), because both the differentiation of megasclerocytes from stem cells (called archeocytes) and the production of megascleres in the cytoplasm of megasclerocytes are actively occurring at this stage (Funayama et al.,2005b). Because the cDNA sequences encoding mature protein regions of the various E. fluviatilis silicatein isoforms are quite similar, we used cDNA fragments encoding the N-terminal pro-region (the region upstream of the arrowhead in Fig. 2A) as templates for synthesizing the probes to avoid cross-hybridization (see the Experimental Procedures section for details). We confirmed that our whole-mount in situ hybridization was sensitive enough to detect silicatein mRNA using approximately 300-bp probes, because we could detect the expression of Ef silicatein-M1 (former Ef silicatein) mRNA in round (Fig. 3A, black arrowhead) or spreading (Fig. 3A, black arrow) nonspiculous cells (which were presumably archeocytes committed to the megasclerocyte lineage) and in cells containing long monaxonal spicules that were in various stages of formation, including short, immature (Fig. 3A, white arrowheads) and long, mature spicules (Fig. 3A, white arrows), as described in our previous report (Funayama et al.,2005b). From these results, we concluded that our in situ hybridization method is sufficiently sensitive to detect silicatein mRNA expression using probes of around 300 bp. Using these probes, we examined the expression of each isoform in sponges at developmental stage 2 (Fig. 3), and quantitatively analyzed the length of cells positive for the expression of each isoform (Fig. 4). We measured the length of cells positive for each silicatein isoform (Fig. 4, filled columns) and the length of cells containing megascleres but negative for expression of each silicatein isoform (Fig. 4, open columns). According to this analysis, the length of cells containing mature megascleres was up to 250 μm. It was clearly confirmed that the expression of Ef silicatein-M1 mRNA was active in nonspiculous cells (under 30 μm in cell length) and in megasclerocytes that were producing megascleres but it was down-regulated after megascleres had grown to 150–200 μm, when the megascleres had grown close to their mature length (Fig. 4A). The expression pattern of Ef silicatein-M2 was similar to that of Ef silicatein-M1. It was expressed in nonspiculous cells (Fig. 3B, black arrowhead) and megasclerocytes containing megascleres of various lengths, including short, immature (Fig. 3B, white arrowheads), and long, mature (Fig. 3B, white arrow) spicules. Like the expression of Ef silicatein-M1, the expression of Ef silicatein-M2 mRNA was down-regulated after megascleres had grown to 150–200 μm (Fig. 4B). On the other hand, Ef silicatein-M3 and Ef silicatein-M4 were expressed in nonspiculous cells (Fig. 3C,D black arrowheads) and megasclerocytes containing short, immature megascleres (Fig. 3C,D white arrowheads), but not in cells containing long, mature megascleres (Fig. 3C,D white arrows). Especially, in the case of Ef silicatein-M4, strong positive signals were mainly detected in cells that did not contain any spicules or in cells containing very short, immature spicules (Fig. 3D, black and white arrowheads). The analysis of cell size indicated that the expression of Ef silicatein-M3 was down-regulated after megascleres had grown to 100–150 μm and was negative in cells containing megascleres larger than 150 μm, and that Ef silicatein-M4 was expressed only in cells containing megascleres shorter than approximately 100 μm (Fig. 4C,D). Ef silicatein-G1 and -G2 were expressed in nonspiculous cells and megasclerocytes containing short, immature or long, mature spicules, but their weak signals even with long coloring reaction times indicated that their expression was weaker than that of the other isoforms (Fig. 3E,F, cells indicated by black arrows). For the cell size measurement analysis, longer coloring reaction time was required to detect signals of these isoforms. This analysis indicated that the number of Ef silicatein-G1- or -G2-positive cells containing longer spicules (100–300 μm) was similar to the number of Ef silicatein-M1 or -M2-positive cells, whereas the number of cells positive for Ef silicatein-G1 or -G2 under and 100 μm or shorter in length was smaller than that of Ef silicatein-M1- or -M2-positive cells of that size (Fig. 4E,F). As low-level expression and the high background staining caused by the longer coloring reaction time make it difficult to distinguish positive cells, the number of cells expressing Ef silicatein -G1 or -G2 might have been undercounted. Even considering this undercounting, the expression level of Ef silicatein-G2 in megasclerocytes having longer spicules seemed to be relatively higher than that in nonspiculous cells or megasclerocytes containing short megascleres. In summary, mRNAs of four silicateins, Ef silicatein-M1, Ef silicatein-M2, Ef silicatein-M3, and Ef silicatein-M4, were expressed in megasclerocytes, while those of Ef silicatein-G1 and Ef silicatein-G2 were not robustly expressed in these cells.
Figure 3. A–F: Whole-mount in situ hybridization using probes of Ef silicatein-M1 (A), Ef silicatein-M2 (B), Ef silicatein-M3 (C), Ef silicatein-M4 (D), Ef silicatein-G1 (E), and Ef silicatein-G2 (F) at developmental stage 2. In each panel, low-magnification images of sponges hybridized with antisense and sense probes are shown on the left and three (A–D) or one (E,F) high-magnification images of cells hybridized with antisense probes are shown on the right. A–D: High-magnification images, round or spreading nonspiculous cells with positive signals are shown on the top, cells containing short immature spicules on the middle and images of cells containing long, mature megascleres on the bottom. Black arrowheads in high-magnification images indicate round spicule-lacking cells with positive signals. The black arrow in A indicates spread cells which lack spicules. White arrowheads and white arrows in the right images indicate cells containing short, immature and long, mature megascleres, respectively. E,F: Black arrows in the high-magnification images indicate cells with weak positive signals. In every low-magnification image, circles drawn with white dashed lines indicate the position of gemmule shells.
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