Fig. 6

Bisection and diI labeling of Eunapius fragilis larvae: fate restrictions of anterior and posterior cells. a Body plan of a freshwater sponge larva. The anterior half of the larva is characterized by a larval cavity, and the posterior half contains a mixture of differentiated and undifferentiated cells. Boxed areas, insets: scanning electron micrographs (SEM, scales: 10 μm) of fractures from the anterior half (left) and posterior (right), showing archaeocytes (ar, pseudo-coloured green or pink), collagen (col) and other cells present throughout the larva. The surface of the larva is covered in small ciliated cells (~2 × 6 μm; cells with cilia, cil). The larval cavity is lined by pinacocytes (pn). b A settled anterior hemisphere lacking an osculum, choanocytes and canals. b′) These settled anterior halves contained only archaeocytes (ar). c) A fractured settled posterior hemisphere showing a normal pinacoderm (pn) and ostia (arrow) with multiple oscula (arrowheads). c′) Normal chaonocyte chambers (ch) in a settled posterior hemisphere. d) Posterior halves also failed to settle in 50% of cases and remained floating; these had a normal pinacoderm (pn), multiple oscula (arrowheads), and a spicule skeleton (arrows). d′) Fractured specimen showing canals (c) and choanocyte chambers (ch). b′) and c′) nuclei = blue (Hoechst), actin = green (Bodipy fluorescein-phallacidin), tubulin = red (anti-tubulin, Alexa 594 2° antibody). e Overview of labelled cells from anterior or posterior diI tattoos. Cell lineage tracer dye (DiI) is shown in red, nuclei are blue (Hoechst). f) Cells from posterior and anterior poles of larvae are fated to become various cell types including basopinacocytes (bpn), choanocytes (ch), sclerocytes (scl) and archaeocytes (ar). g) Cells at the posterior pole give rise to the osculum (bottom panels show boxed areas; DF = dark field). Scales: b), c), e) and g) 100 μm; b′) and c′) and d) 25 μm; d′) 10 μm; and f) 50 μm. Diagrams are modified from versions appearing in [61]