Cell labelling and lineage-tracing through metamorphosis in Amphimedon queenslandica. A, D: Distributions of labelled cells in free-swimming larvae. Anterior (swimming axis) is to the bottom. B, C, E, F: Descendants of the labelled cells in juveniles. Nuclei are stained with DAPI. In E and F, the juveniles are labelled with an antibody against tyrosinated tubulin (tyrTub). A: Longitudinal confocal sections through the centre of a free-swimming larva incubated with CM-DiI, showing strong labelling in ciliated epidermal cell types, the columnar epithelial cell (co) and the flask cell (fc) (inset), with little labelling in inner cell mass (icm). B: Choanocytes in chambers (ch). In some cases, a subset of choanocytes is CM-DiI-labelled in a single choanocyte chamber (arrowhead in inset), suggesting that multiple precursor cells can be involved in development of a single chamber. C: Labelled exopinacocytes (arrowheads). D: A confocal longitudinal section through the centre of a free-swimming larva pulse-labelled with EdU. Note that the labelled cells localise in the inner cell mass (icm) and are likely to be proliferating archeocytes with characteristic large nucleoli (nu) [see Additional file 3: Figure S3]. E: An EdU-positive choanocyte in the chamber (arrowhead). F: An EdU-positive exopinacocyte (arrowhead). Other abbreviations: ep, outer layer epithelium; ci, cilium; ex external environment; mh, mesohyl. Scale bars: 100 μm (A, D), 10 μm (B, C, E, F, inset in D), 5 μm (inset in A). DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2′-deoxyuridine.