Fig. 1. Multiplexed clonal analysis demonstrates multipotency of premigratory cardiac neural crest cells.

a Sequences encoding H2B-RFP, H2B-YFP, H2B-CFP, Mito-CFP, Mem-YFP, and Utrophin-Scarlet were cloned downstream of the RSV promoter of the RIA vector for combinatorial infection. b Schematic diagram of in vivo slice culture. At HH21, a ~500 μm thick slice was obtained posterior to the otic placode. The slice was then embedded in 1% agarose and imaged using inverted confocal microscopy. A, anterior; P, posterior; D, dorsal; V, ventral. c Schematic diagram of cardiac neural crest-derived tissues (gray). Dorsal neural tube (dNT), cranial nerve nine (CN-IX), pharyngeal arch arteries (PAA) and outflow tract (OFT) were imaged and scored for cells with identical clonal signatures. d A tiled multiplex image of the cardiac slice, representative of 23/23 embryos. CN-IX, PAA, OFT and Schwann cells (arrowhead) were infected by a mixture of RIAs. Red signal along the pharyngeal endoderm is autofluorescence from tissue debris common in chick explant cultures. e–h Clonally related cells (ID: Supplementary Data 1a-84) infected by three viruses (H2B-RFP, H2B-YFP, Mito-CFP, arrowhead) were identified in all four cardiac neural crest derivatives: dNT (e), CN-IX (f), PAA (g), OFT (h). In (h), RFP and YFP signals did not align in the OFT as a result of heart beating during image acquisition. i Bar graph showing distribution of double (blue) and triple (orange) infected clones. j, k In clones that appeared to be restricted in one location, cells could acquire distinct fates. At E7, one cell in a rare clone near CN-IX expressing H2B-RFP, Mem-YFP and Mito-CFP differentiated into HuC/D expressing neuron (j), while another cell of the same clone remained HuC/D negative (k). Scale bars: d 500 μm; e–h 100 μm; j, k 60 μm.