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Abstract
BACKGROUND: Mesoderm is generally considered to be a germ layer that is unique to Bilateria, and it develops into diverse tissues, including muscle, and in the case of vertebrates, the skeleton and notochord. Studies on various deuterostome animals have demonstrated that fibroblast growth factor (FGF) signaling is required for the formation of many mesodermal structures, such as vertebrate somites, from which muscles are differentiated, and muscles in sea urchin embryos, suggesting an ancient role of FGF signaling in muscle development. However, the formation of trunk muscles in invertebrate chordates is FGF-independent, leading to ambiguity about this ancient role in deuterostomes. To further understand the role of FGF signaling during deuterostome evolution, we investigated the development of mesodermal structures during embryogenesis and metamorphosis in Ptychodera flava, an indirect-developing hemichordate that has larval morphology similar to echinoderms and adult body features that are similar to chordates.
RESULTS: Here we show that genes encoding FGF ligands, FGF receptors and transcription factors that are known to be involved in mesoderm formation and myogenesis are expressed dynamically during embryogenesis and metamorphosis. FGF signaling at the early gastrula stage is required for the specification of the mesodermal cell fate in P. flava. The mesoderm cells are then differentiated stepwise into the hydroporic canal, the pharyngeal muscle and the muscle string; formation of the last two muscular structures are controlled by FGF signaling. Moreover, augmentation of FGF signaling during metamorphosis accelerated the process, facilitating the transformation from cilia-driven swimming larvae into muscle-driven worm-like juveniles.
CONCLUSIONS: Our data show that FGF signaling is required for mesoderm induction and myogenesis in the P. flava embryo, and it is reiteratively used for the morphological transition during metamorphosis. The dependence of muscle development on FGF signaling in both planktonic larvae and sand-burrowing worms supports its ancestral role in deuterostomes.
Fig. 1. FGF ligand genes are dynamically expressed during P. flava embryogenesis. In situ hybridization of fgf8/17/18 (A1-A5), fgfa (B1-B5), fgfb (C1-C5), fgfc (D1-D5) and fgfd (E1-E5) at different embryonic stages. All embryos are shown from a lateral view and oriented with the mouth to the left, except for panel D5. The embryo in D5 is shown from the ventral side. Embryos shown in C3 and D4 were photographed with the focal plane on the ectoderm. Inserts in A4, C3, D4 and D5 are shown from the ventral side, the apical surface, the ventral surface, and the lateral side, respectively. The expression patterns for all ligands are schematically summarized in F1-F7. Each stage is shown and different genes are indicated with different colors. The blue arrowheads in panels A2-A4 and B4-B5 indicate ectodermal expression. Black arrows in B4-B5 indicate the animal part of the protocoel, and the hydroporic canal is indicated by a black arrowhead. The four vertical stripes of fgfb expression are labeled with lowercase letters (a, b, c, and d) in the C3 insert. Two of the four stripes are circled by white dashed lines in C3 and labeled with lowercase letters (a and b), corresponding to the labels in the insert. Red arrows and arrowheads in panels D4-D5 and F6-F7 indicate the expression of fgfc in preoral and postoral ciliary bands. Green arrowheads in E4-E5 indicate the expression of fgfd in the ventral tip of the mesoderm. All panels are shown in the same scale, according to the scale bar in A1
Fig. 2. FGF receptor genes are expressed in mesodermal cells. In situ hybridization of fgfra1 (A1-A5), fgfra2 (B1-B5) and fgfrb (C1-C5) at different embryonic stages. The embryos are shown from a lateral view and oriented with the mouth to the left. Red arrows in A5 and B5 indicate the expression in the sphincter. Blue arrows in C4-C5 indicate ectodermal fgfrb expression domains. Black asterisks mark the hydropore in A5-C5. The expression patterns of the FGF receptor genes in the developing mesodermal cells and other embryonic territories are summarized schematically at the corresponding stages in D1-D4. All panels are shown at the same scale, according to the scale bar in A1
Fig. 3. FGF signaling is required for mesoderm induction. Phenotypes of embryos at 43 hpf (A1-F1) and 73 hpf (A2-F2) after treatment with FGF signaling inhibitors (B1-D2) or bFGF protein (F1-F2) upon fertilization. Control embryos were treated with DMSO or 0.1% BSA. The concentrations of each drug or protein are indicated in each panel. All embryos are shown from a lateral view with mouth on the left. All panels have the same scale, with the scale bar marked in A1. Abbreviations: me: mesoderm; en: endoderm
Fig. 4. FGF signaling stepwise regulates the formation of the mesoderm-derived structures. (A1-A5) Panels show the morphology of wild type embryos at the time points indicated by the solid yellow circles. Treatments were performed at the same time points, and embryos were observed at the time points indicated by the blue circles. (B1-B6) Phenotypes of the tornaria larvae treated with DMSO (B1) or PD173074. PD173074 was applied at different stages of development, indicated by the yellow circles on the left (B2-B6). (C1-D4) The tornaria larvae (73 hpf) treated with PD173074 (C2-C6) or bFGF (D2-D4) at different developmental stages were stained with Phalloidin (green). The penetrance of the drug effects was high (> 99%) when treated at either 18 or 40 hpf. When treated at 23, 26 or 31 hpf, ~ 70% of the larvae did not exhibit a muscle string. The efficiency of bFGF protein was consistently high (> 99%) for all the treatments. The larvae were counterstained with Hoechst 33,342 for nuclei (blue). e Illustration of a tornaria larva with mesodermal structures in red. The pharyngeal muscle, the muscle string and the hydroporic canal are indicated. The scale bar in A1 is shown for panels A1-B6, and the scale bar in C1 is shown for panels C1-D4. Abbreviations: ms, muscle string; pm, pharyngeal muscle; hc, hydroporic canal
Fig. 5. FGF signaling regulates the expression of the mesodermal transcription factor genes in the presumptive mesoderm. In situ hybridization for snail (A1-A3), foxc (B1-B3), foxf (C1-C3), twist (D1-D3) and foxa (E1-E3) in 26 hpf embryos treated with DMSO (A1-E1) or 2.5 μM PD173074 at 18 hpf (A2-E2) or 23 hpf (A3-E3). All panels are shown in the same scale, according to the scale bar (100 μm) in A1
Fig. 6. FGF signaling regulates the expression of the mesodermal and myogenic genes. Expression patterns of foxc (A1-B4), foxf (C1-D4), myocardin (E1-F4), stMHC (G1-H4) and fgfa (I1-J4) were analyzed in 43 hpf or 73 hpf embryos treated with DMSO or 2.5 μM PD173074 at various developmental stages (indicated by the yellow circles on the left). Embryos were observed from the lateral side with the mouth to the left. All panels are shown in the same scale, according to the scale bar (100 μm) in A1. Green arrows in A3, B3, F3 and H3 mark expression of the indicated gene at the ventral tip of the mesoderm/pharyngeal muscle. Blue arrow in J4 indicates the expression of fgfa in the hydroporic canal
Fig. 7. Muscle fibers are extensively generated during metamorphosis. Morphological changes during the P. flava transition from the Spengel (a-c) to the Agassiz (d-e) and then to the juvenile stage (f-g). Fully developed protocoel, mesocoel and metacoel are outlined by white dashed lines in panel a. Phalloidin staining (green) revealed the distribution of muscle fibers in the protocoel of the Spengel larva (b and c, viewed from the lateral and the apical side, respectively), and in the proboscis and trunk regions at the Agassiz (e) and the juvenile (g) stages. The asterisks in (b) and (c) indicate the position of the mouth. Nuclei were counterstained with Hoechst 33,342 (blue). Scale bar: 1Â mm. Abbreviations: pc, protocoel; mesoc, mesocoel; metac, metacoel; prob., proboscis; col., collar
Fig. 8. The effect of FGF signaling on sand-induced metamorphosis. a-c The transforming rate of the Spengel larvae after 2 days of incubation with the sand and PD173074, U0126 (a), SU5402 (b) or bFGF protein (c) at indicated concentrations. The transformation rate was calculated by dividing the total number of the Spengel larvae used in the experiment with the sum up number of the Spengel larvae transformed into the Agassiz, transforming Agassiz and juvenile stages. d The percentages of the Spengel larvae that transformed into the Agassiz, transforming Agassiz, or juvenile stages after 2 days of incubation with sand, sterilized sand, or without sand in the presence of bFGF (+) or BSA (−) protein are shown. Every experiment was repeated at least three times except the U0126 treatment, which was conducted only once. N.S: not statistically significant
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