Figure 3. Specification of heart vs atrial siphon muscle precursors in M. occidentalis.

(A) First and second asymmetric division of TVCs in M. occidentalis embryos. B7.5 lineage was visualized by electroporation of Moocci.Mesp>H2B::GFP (green). The first division occurs at ∼6 hpf at 24°C, and the second division occurs at ∼7.5 hpf at 24°C. (B) Result of two asymmetric divisions of TVCs on left side of embryo electroporated with Moocci.Mesp>H2B::GFP (green). At 8 hpf at 24°C, a cluster of 6 cells derived from the TVCs is located in the ventro-lateral region of the trunk. From top to bottom: 2 atrial siphon muscle founder cells (ASMFs), 2 second heart precursors (SHPs), and 2 first heart precursors (FHPs). (C) In situ hybridization (ISH, red) for Moocci.Tbx1/10 + β-galactosidase immunodetection (IHC, green) in embryos electoporated with Moocci.Mesp>nls::lacZ. Moocci.Tbx1/10 nascent transcripts are detected as two dots in the nuclei of Secondary TVCs (STVCs), between the first and second asymmetric divisions. (D) ISH + IHC for Moocci.Ebf (red) in embryos electoporated with Moocci.Mesp>nls::lacZ (green), revealing Moocci.EBF expression in ASMFs after the second asymmetric division. (E) ISH for Moocci.Ebf in a swimming larva, viewed dorsally, revealing Moocci.Ebf+ migrating atrial siphon muscle precursors (ASMPs, arrowheads). (F) Lateral view of a M. occidentalis juvenile (>100 hpf) electroporated with Moocci.Mesp>H2B::GFP. GFP + nuclei reveal contributions of B7.5 lineage to atrial siphon muscle-derived pharyngeal muscles (PhMs) and heart. (G) Diagram of the TVC divisions giving rise to the pharyngeal muscles and heart of the adult. (H) Ventral view of a M. occidentalis embryo electroporated with Moocci.Mesp>H2B::GFP, just after the first asymmetric division of the TVCs. (H′) Inset from (H) is focused on the distance between FHPs on either side of the embryo (mean = 57.6 μm, n = 9). (I) Ventrolateral view of a C. intestinalis embryo electroporated with Ciinte.Mesp>H2B::GFP, right after the first asymmetric division. (I′) The distance between FHPs from either side is very small (mean = 7.5 μm, n = 14) since they contact each other at the midline to form a single cluster of cells. (J) Double ISH for Moocci.Dlx.a (green) and Moocci.Ebf (red) in a M. occidentalis larva, viewed dorsally. Moocci.Dlx.a marks the siphon primordia, while Moocci.Ebf marks siphon muscle precursors. Arrowheads point to atrial siphon muscle precursors (ASMPs), which have migrated dorsally but do not encounter an atrial siphon primordium around which to encircle. In contrast, oral siphon muscle precursors form a ring around the oral siphon primordium (OSP). Other Moocci.Ebf+ cells seen are neurons or neuronal precursors in the central nervous system (cns). (K) Double ISH for Ciinte.Dlx.a (green) and Ciinte.Ebf (red) in a C. intestinalis larva viewed dorsolaterally. Arrowhead indicates Ciinte.Ebf+ ASMPs on one side of the embryo encircling one of two bilaterally paired atrial siphon primordia (ASP). The same configuration of Ciinte.Ebf+ muscle precursors is seen encircling the OSP.

DOI: http://dx.doi.org/10.7554/eLife.03728.013

Figure 3—figure supplement 1. Mosaic transgene labeling of M. occidentalis suggests bilateral origin of juvenile heart.

(A) Dorso-lateral view of a metamorphosing juvenile (>100 hpf) developing from embryo electroporated with Moocci.Mesp>H2B::GFP. H2B::GFP labels nuclei of right side of and subset of atrial siphon/body-wall muscles. (B) Magnified view of boxed area in A. Mosaicism of H2B::GFP-labeling of juvenile tissues is interpreted as reflecting left/right mosaic uptake and/or retention of Moocci.Mesp>H2B::GFP plasmid during embryonic stages.

DOI: http://dx.doi.org/10.7554/eLife.03728.014

Figure 3—figure supplement 2. Delayed atrial siphon primordium specification in M. occidentalis.

(A) Dorsal view of hatched larva (13 hpf) stained with Alexa Fluor 546 phalloidin, showing oral siphon primordium (OSP) as a distinct structure, while the atrial siphon primordium (ASP) has not formed yet. (B) Dorsal view of metamorphosing juvenile (>72 hpf) stained with Alexa Fluor 546 phalloidin showing fully formed ASP as a rosette of apically constricted cells. OSP is out of the plane of view. (C) Dorsal view of M. oculata hatched larva stained with Alexa Fluor 633 phalloidin, showing formation of both OSP and ASP at this stage.

DOI: http://dx.doi.org/10.7554/eLife.03728.015

Figure 3—figure supplement 3. Cardiopharyngeal development in C. intestinalis vs. M. occidentalis.

Schematic diagram comparing cardiopharyngeal development in C. intestinalis (top) and M. occidentalis (bottom) embryos. The B7.5 cell lineage, which is identical in the two species, is shown in the middle, and each progenitor type is color-coded to match the embryo diagrams. Dashed line in left-most drawings indicates embryonic midline. Although both FHPs and SHPs contribute to the juvenile heart, the relative contributions of their descendants have yet to be elucidated, in either Molgula or Ciona. Refer to text for other details.

DOI: http://dx.doi.org/10.7554/eLife.03728.016