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Fig. S10
+2.0drl drives specific reporter expression in lamprey, Ciona, and amphioxus
(a-d) Whole-mount ISH for pmHandA marks the LPM in lamprey embryos from neurula to hatching stages (st 18-25). (a) Expression is first detectable as bilateral stripes in the PLPM at st 18. (b,c) Later, the PLPM expression expands covering the yolk, and pmHandA is upregulated in the heart tube, branchial region, and caudal to the heart. Lateral and dorsal views are shown with the anterior (a) of the embryo to the left. (i-k) Transverse sections of fixed st 21-22 embryos. The schematics indicate the plane of sectioning in blue and sections are shown with dorsal to the top. The schematic transverse section represents k-III. (i) ISH for pmHandA at st 21 marking the LPM (black arrowheads). (j,k) ISH for EGFP in transverse sections of +2.0drl:EGFP transient transgenic lamprey embryos fixed at st 21-22, showing enhancer activity in the anterior mesendoderm subjacent to the ectoderm (black arrowheads), in the pharyngeal endoderm (k-III, white arrowhead), in the pharyngeal mesoderm (k, black arrowheads), and in some embryos in the ectoderm as commonly observed unspecific expression of reporter plasmids (k-II, black arrowheads). (l,m) Immunostaining for EGFP in Ciona larvae embryo (st 26) expressing drl reporters (green) and Mesp:H2B-mCherry to track the B7.5 nuclei cell lineage (red). (L) Expression of +2.0drl-driven EGFP reporter in the larvae is stained in the mesenchymal lineage (white asterisks) and in the B7.5 cell progeny including ASM precursors (ASMP) (white arrow) and both cardiac first and second heart precursors (FHPs and SHPs) (white arrowheads), zoomed in box. Anterior to the left. (m) Proportion of larvae embryos expressing both GFP and mCherry in the B7.5 lineage when co-electroporated drl reporter and Mesp:H2B-mCherry in comparison to the control. N = number of electroporated larval halves. (n-p) Confocal Z-stack of amphioxus embryo at mid-neurula stage (6/7 ss), injected with +2.0drl:EGFP, showing specific reporter activity in the lateral endoderm (arrowhead), and ventral half of the somites (n=61/65, arrows). Embryos counterstained with Phalloidin (red), embryos/larvae with DAPI (blue), and anti-acetylated tubulin antibody (magenta). Lateral view shown as 3D-rendering (n) or Z-stack sagittal sections (o), anterior to the left and dorsal to the top. At early larvae stage (p), the activity of +2.0drl reporter was observed in the developing pharynx (n=15/30, arrowhead). Branchial region (br), heart (h), neural folds (nf), neural tube (nt), notochord (n), pharynx (ph), and yolk (y) labeled (i-k). Scale bars (i-k) 200 μm, (l) 25 μm, (n,p) 50 μm.