Early Chordate Origins of the Vertebrate Second Heart Field

Abstract

The vertebrate heart is formed from diverse embryonic territories, including the first and second heart fields. The second heart field (SHF) gives rise to the right ventricle and outflow tract, yet its evolutionary origins are unclear. We found that heart progenitor cells of the simple chordate Ciona intestinalis also generate precursors of the atrial siphon muscles (ASMs). These precursors express Islet and Tbx1/10, evocative of the splanchnic mesoderm that produces the lower jaw muscles and SHF of vertebrates. Evidence is presented that the transcription factor COE is a critical determinant of ASM fate. We propose that the last common ancestor of tunicates and vertebrates possessed multipotent cardiopharyngeal muscle precursors, and that their reallocation might have contributed to the emergence of the SHF.

The vertebrate heart initially forms as a tube from a population of precursor cells termed the first heart field (FHF). Cells from the adjacent second heart field (SHF) are then progressively added to the developing heart (1, 2). In avian and mammalian hearts, the FHF contributes mainly to the left ventricle, whereas the SHF gives rise to the outflow tract and large portions of the right ventricle and atria. Both fields arise from common mesodermal progenitors, although the detailed lineage relationships between FHF and SHF remain uncertain (1, 3). SHF-like territories have been identified in frog (4, 5), zebrafish (6), and lamprey (7), yet evidence for a deeper evolutionary origin remains obscured by the absence of a clear SHF in invertebrates (8).

Tunicates are the sister group to the vertebrates (9). Studies on a model tunicate, the ascidian Ciona intestinalis, have revealed conserved regulatory mechanisms underlying chordate heart development (10). The Ciona heart arises from a pair of blastomeres (named the B7.5 cells, by Conklin’s nomenclature) in gastrulating embryos (11). Localized expression of MesP in B7.5 cells determines their competence to form the heart (Fig. 1A) (12, 13). Subsequently, fibroblast growth factor signaling induces expression of FoxF and the heart determinants NK4 (tinman/Nkx2.5), GATAa, and Hand-like/NoTrlc in the anterior B7.5 granddaughter cells [the trunk ventral cells (TVCs)] (Fig. 1B) (13, 14). FoxF activates downstream target genes that control the migration of the TVCs to the ventral trunk region (Fig. 1C) (14, 15).

Fig. 1.

Contribution of trunk ventral cells (TVCs) to heart and atrial siphon muscles (ASMs). (A) Immunodetection of β-gal expression (green) in B7.5 cells in a gastrulating embryo transfected with MesP>lacZ transgene. (B) Visualization of MesP>lacZ (green) and FoxF[TVC enhancer]>mCherry (red) expression in B7.5 descendants in a tailbud-stage embryo. (C) Expression of a MesP>Histone2B(H2B)∷GFP fusion protein (green) in a tadpole. (D) Visualization of MesP>H2B∷CFP (green) in a stage 38 juvenile [~100 hours post-fertilization (hpf)]. Expression is visible in the heart (arrowhead), ASMs, and longitudinal muscles (LoMs) (arrows). MesP is activated only in the B7.5 pair of cells at the gastrula stage. a.s., atrial siphon; o.s., oral siphon. (E) Frames from movie S1 (time points 0 to 2) and S2 (time points 3 to 5). Dashed line indicates ventral midline. Right-side cells are partially visible at time points 1 and 2. Stereotyped cell divisions (see text) result in four lateral TVCs on either side of the embryo flanking ~16 medial TVCs. The four lateral TVCs on either side detach and migrate to form ASMs. Medial cells form the heart. Cells were visualized as two independent time-lapse sequences of embryos transfected with MesP>H2B∷GFP/CFP. (F) Cartoon representing the events in (E). (G and H) Frames from movie S3 showing left-side ASM precursors expressing MesP>H2B∷mCherry (red) and MesP>PH∷GFP (green). ASM precursors encircle the siphon primordium, between 21 hpf (G) and 23 hpf (H). Scale bars, 50 μm [(A) to (D)], 20 μm (E). Asterisks identify anterior tail muscles (ATMs).

After metamorphosis, descendants of the B7.5 lineage give rise to the heart (Fig. 1D). B7.5 descendants also generate the atrial siphon muscles (ASMs) that surround the excurrent openings of the peribranchial atrium (Fig. 1D), as well as longitudinal muscles (LoMs) arising from the ASMs during metamorphosis (fig. S7). The contribution of the B7.5 lineage to ASMs is consistent with conventional lineage tracing performed in the distantly related ascidian Halocynthia roretzi (16). Cardiomyocytes and ASMs are distinguished by expression of the myosin heavy chain (MHC) genes MHC2 and MHC3 (17), respectively (fig. S3). Thus, they constitute distinct muscle types arising from common progenitor cells.

Live imaging of the B7.5 lineage cells allowed the characterization of events leading to the separation of heart and ASMs (Fig. 1E). After their migration to the ventral trunk region, each TVC undergoes two successive asymmetric divisions along the mediolateral axis to produce six cells on either side of the ventral midline (Fig. 1, E and F, time points 0 to 2; movie S1). The larger daughter cells (lateral TVCs) are positioned lateral to the smaller medial TVCs. Subsequent symmetric cell divisions result in an array of ~24 cells: eight lateral TVCs (four on either side) bracketing 16 medial TVCs (Fig. 1, E and F, time points 3 to 5; movie S2) (18).

A second migration occurs several hours after hatching. Each group of lateral TVCs detaches from the medial TVCs and migrates dorsally as a polarized cluster of cells on either side of the trunk. They eventually form a ring of cells underneath the atrial siphon primordia (Fig. 1, G and H, and movie S3). Targeted inhibition of TVC specification blocked the formation of ASMs (fig. S4). These observations demonstrate that the lateral TVCs correspond to the precursors of the ASMs.

The ASMs are evocative of vertebrate jaw muscles arising from lateral/splanchnic mesoderm (SpM): Ciona TVCs and vertebrate anterior SpM both express orthologs of Nkx2.5 and FoxF and derive from progenitors that expressed MesP during gastrulation (5, 19, 20). In chick and mouse embryos, much of the anterior SpM gives rise to the SHF, but some precursors migrate into the first branchial arch and form intermandibular muscles (21, 22). A key marker of the anterior SpM and SHF is the LIM-homeodomain transcription factor Islet1 (Isl1) (4–6, 21). The single Ciona Islet (23) gene is expressed in several tissues including the ASM precursors, which maintain Islet expression during their migration away from the medial TVCs (fig. S1). The latter possibly show weak and transient Islet expression, which is reminiscent of that reported in the FHF of vertebrates (19).

Islet expression was further characterized using defined enhancers (fig. S2). Reporter transgenes containing ~3.2 kb of the Islet 5′ flanking region exhibited localized expression in the lateral TVCs, and in ASMs in juveniles (Fig. 2, A to C). In contrast, MesP reporter transgenes labeled the entire B7.5 lineage, including both ASMs and heart (e.g., Fig. 1, C and D, and fig. S5). The heart primordium is situated ventrally and medially to the Islet⁺ ASM progenitors (Fig. 2A). This is reminiscent of the positioning of the FHF relative to Isl1⁺ SHF/pharyngeal mesoderm in basal vertebrates (4, 5, 7). Furthermore, LoM precursors segregating from the ASMs express the Ciona ortholog of Tbx1 (24), an important regulator of SHF and pharyngeal mesoderm development in vertebrates (25) (Fig. 2, D to F, and fig. S7). Taken together, these results suggest homology between the ASM/LoM precursors of tunicates and the progenitors of lower jaw muscles and SHF of vertebrates.

Fig. 2.

ASM-specific gene expression. (A) Dorsal view of electroporated larva exhibiting mosaic incorporation (left side) of Islet>GFP (green) and MesP>H2B∷mCherry (red) transgenes at 20 hpf, before the migration of the lateral TVCs. Islet>GFP expression is restricted to lateral TVCs. Dotted line indicates midline; M and L indicate medial and lateral, respectively. (B) Lateral view of larva expressing same transgenes as in (A) at 24 hours, after migration of Islet>GFP-positive lateral TVC descendants around the atrial siphon primordium. D, dorsal; V, ventral. (C) Juvenile (~100 hpf) raised from embryo transfected with Islet>H2B∷mCherry (red), with transgene expression visible around atrial siphons (arrow) and longitudinal muscles (arrowheads), but not heart (Ht). F-actin is stained by phallacidin (blue-green). Scale bar, 100 μm. (D) Magnified view (see fig. S7) of LoMs (arrowhead) segregating from ASMs (arrows) during metamorphosis, visualized by MesP>lacZ expression (red). Panel width is ~100 μm. (E) In situ hybridization of Tbx1/10 (green). (F) Merged view of (D) and (E), showing activation of Tbx1/10 in LoMs. (G) COE expression in lateral TVCs at 20 hpf revealed by in situ hybridization. Dotted line indicates ventral midline.

Preliminary functional assays suggest that Islet is not instructive for the specification of ASMs (fig. S10). In the course of these studies, we found that the transcription factor Collier/Olf1/EBF (COE) is expressed early in the ASM precursors (Fig. 2G and fig. S11). To determine whether this localized expression is instructive for ASM specification, we used the FoxF TVC enhancer to misexpress COE in all TVCs (13). In 96% of transfected embryos, all TVC descendants migrated toward the atrial siphon placodes and expressed Islet>GFP (Fig. 3, A and B, fig. S6, and movie S4). More than half (56%) of the transfected embryos grew into juveniles that lacked a heart but still had ASMs (Fig. 3, D, E, and G to J, and fig. S6), which suggests that COE is sufficient to specify a lateral TVC identity and subsequent ASM fate.

Fig. 3.

COE controls ASM specification. (A to C) Larvae cotransfected with MesP>H2B∷mCherry (red), Islet>GFP (green), and (A) FoxF>lacZ, (B) FoxF>COE, or (C) FoxF>COE∷WRPW. (B) All TVCs are transformed into ASM precursors (arrows). No heart primordium is formed (dashed triangle). (C) All TVC descendants form a heart (arrowhead) with no Islet>GFP expression. (D) Juveniles raised from embryos transfected with MesP>H2B∷mCherry (red), counterstained with phallacidin (blue-green). H2B∷mCherry⁺ B7.5 descendants populate the heart (Ht) and ASMs (arrows) (residual larval muscle staining indicated by asterisk). (E) Cotransfection with the FoxF>COE transgene, resulting in no heart (usual location indicated by dashed circle) but normal ASMs (arrow). (F) Coexpression of the FoxF>COE:WRPW transgene. TVCs form an expanded heart and there are no ASMs (arrow). (G and H) In situ hybridization on wild-type juveniles, showing MHC3 and MHC2 expression in ASMs (arrow) and heart, respectively. (I and J) FoxF>COE results in loss of MHC2 expression (dashed circle), but not MHC3 expression (arrow). (K and L) FoxF>COE∷WRPW abolishes MHC3 expression (arrow) and leads to expanded MHC2 expression. Scale bars, 50 μm; o.s., oral siphons.

Similar misexpression of a repressor form of COE (COE∷WRPW) resulted in the reciprocal phenotype: All TVC descendants remained in the ventral trunk, and Islet>GFP expression was abolished (Fig. 3, A and C, and fig. S6). Upon metamorphosis, TVC descendants differentiated into enlarged hearts (Fig. 3, D, F to H, K, and L, and fig. S8). Inhibition of COE function thus transforms the entire TVC lineage into heart tissue, indicating that COE activity is required for ASM specification.

The COE homolog Collier/Knot is involved in muscle type specification in Drosophila (26), but a role for COE in vertebrate SHF or jaw muscle development has not been reported. As a first step toward determining whether COE factors might play a conserved role in vertebrates, we performed in situ hybridization of the COE orthologs Xebf2 and Xebf3 in Xenopus tropicalis embryos (Fig. 4, A to C, and fig. S9). Xebf2 expression was seen in Nkx2.5⁺/Isl1⁺ anterior lateral mesoderm, where Tbx1 is also expressed (5).

Fig. 4.

Comparison to vertebrate pharyngeal mesoderm. (A to C) Expression of the COE ortholog Xebf2 and anterior lateral mesoderm/SHF markers Nkx2.5 and Islet1 in X. tropicalis embryos at NF stage 20. (A) Expression of Xebf2 (white arrow) partially overlaps that of Islet1 (B) and Nkx2.5 (C) in pharyngeal mesoderm lateral to the heart primordium. Scale bar, 500 μm. (D and E) The cardiopharyngeal lineages of Ciona (D) and vertebrates (E). (D) Summary of differential expression of selected regulatory genes in the B7.5 lineage. (E) Expression of orthologous genes in mouse development. Evolutionary reallocation of Islet1⁺ cardiopharyngeal precursors toward the heart might have given rise to the SHF.

The preceding results suggest that the last common ancestor of tunicates and vertebrates had a population of cardiopharyngeal mesoderm, which (i) arose from MesP-expressing early mesoderm, (ii) expressed orthologs of FoxF and Nkx2.5, and (iii) had the potential to give rise to both heart tissue and pharyngeal muscles, which (iv) correlated with differential maintenance of Islet expression. Moreover, COE might be a conserved determinant of chordate pharyngeal mesoderm development.

We propose that the reallocation of Islet⁺ cardiopharyngeal progenitors among the heart and cranial myogenic fields supported the emergence of the SHF (Fig. 4, D and E). In Ciona, Islet⁺ cells do not contribute to the heart, but targeted expression of COE∷WRPW was sufficient to convert them into cardiomyocytes. It is conceivable that the reallocation of Islet⁺ pharyngeal muscle progenitors toward SHF depended on the intercalation of cardiac regulatory network components (e.g., GATA4, Mef2c) downstream of Islet (27).

An ancient connection between heart and craniofacial muscles has been proposed on the basis of the role of an Nkx2.5 ortholog in the pharynx of the nematode Caenorhabditis elegans, which lacks a heart (28). Haikouella lanceolata, a fossil chordate from the Lower Cambrian, shows a putative heart adjacent to a muscularized pharyngeal atrium (29). The shared lineage of cardiomyocytes and pharyngeal muscles in tunicates hints that a pool of common precursors could have formed both heart and pharyngeal atrium muscles in a Haikouella-like ancestor. Northcutt and Gans (30) proposed that muscular ventilation of the pharyngeal arches was a key transition in the evolution of the vertebrates. Therefore, the cardiopharyngeal mesoderm could have been instrumental in the coevolution of circulatory, respiratory, and feeding functions in tunicates and vertebrates.