Figure 5.

Immunohistochemistry of Epith-2 internalization during mesenchyme ingression in H. pulcherrimus [(A–D); 6-μm thick Polywax sections], purified primary mesenchyme cells [PMC; (E,E’)] and the fate of Epith-2 in ingressed PMCs by immunoblotting in T. hardwicki (F,G). (A) An early mesenchyme blastula. Scale bar, 40 μm. (A’) Phase-contrast micrograph of the same section as (A). (B) Higher magnification image of the box in (A). Arrows, cytoplasmic anti-Epith-2 mAb-positive dots in primary mesenchyme cells (PMC). Scale bar, 10 μm. (C) Late gastrula. Scale bar, 40 μm. (C’) Phase-contrast micrograph of the same section as (C). Arrow, PMC aggregate near the blastopore. (D) Higher magnification image of the box in (C). Arrows, anti-Epith-2 mAb-positive dots in secondary mesenchyme cells (SMC). Scale bar, 10 μm. (E) Isolated PMCs stained with anti-P4 mAb. Scale bar, 30 μm. (E’) Phase-contrast micrograph of the same cells as (E). (F) A chart showing the sample preparation of the immunoblotting shown in (G). Samples in the broken-line box contain anti-P4 mAb and anti-mouse IgG Ab-tagged-magnetized Microbeads. Samples in the shaded box were examined with anti-Epith-2 mAb and anti-mouse IgG Ab. Samples in dark box were examined only with anti-mouse IgG Ab. (G) Epith-2 in the cytoplasm of PMCs analyzed with anti-Epith-2 mAb (lanes 1–6) and secondary antibody (Ab) alone (lanes 7, 8). Lane 1; anti-P4 mAb and anti-mouse IgG Ab-tagged-magnetized Microbeads-treated PMC fraction (PMCs), lane 2; anti-P4 mAb-treated epithelial cell fraction (Epithelial cells), lane 3; anti-P4 mAb-treated dissociated mesenchyme blastulae (Diss. Bl.), lane 4; whole mesenchyme blastulae (Whole mBl), lanes 5, 7; anti-P4 mAb alone (P4 mAb), lanes 6, 8; anti-mouse IgG Ab-tagged magnetic Microbeads alone (Microbeads). Arrows, Epith-2 at 160 kDa region. Arrowheads, IgG of primary Ab.