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Figure 10. Time course of pax7 mRNA expression in Xenopus laevis. Lateral views, anterior to the left. Embryonic stages are indicated at the top. Inset in (D): pharyngeal arches and head mesenchyme were dissected away from the left side to reveal the brain. Up to stage 36, Pax7 expression is confined to the central nervous system including the ventral diencephalon (arrowhead), the hypophysis (hp), and the frontonasal neural crest cells. Weak expression is also seen in the somites. From stage 39 onwards, weak expression can be detected in craniofacial muscle anlagen. Abbreviations as before and: cg, cement gland; hp, hypophysis; ht, heart; first arch derived muscle: im, m. intermandibularis anlage; lm, m. levatores mandibulae anlage; second arch derived muscle: ih, m. interhyoideus anlage; oh, m. orbitohyoideus anlage; qh, m. quadrato-hyoangularis anlage; q/oh, common oh and qh precursor.
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Figure 11. Time course of head mesoderm and muscle gene expression in Xenopus laevis. Same stages, views, and abbreviations as in Figure 11; markers are indicated on the left. Note that msc, myf5, myod, myog, and desmin are expressed before, mrf4 concomitant with the onset of pax7 expression.
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Figure 12. Sarcomeric myosin (A,C,E,G,I) and pax7 protein expression (B,D,F,H,J) in st26 Xenopus laevis control embryos and in craniofacial muscle anlagen embryos at st40. (K–M) GFP expression and pax7 protein expression in a st40 cardiac actin; GFP embryo. (A,B,C,D,G,H,K,L) lateral views, rostral to the left, dorsal; to the top; (E,F,I,J) ventral views, rostral to the left. In (A–F) a HRP-coupled secondary antibody was used, in (G–J) a secondary antibody coupled to Alexa fluor 594. In (K–M), Alexa fluor 488 and 594 coupled secondary antibodies were used to detect anti GFP and pax7 primaries, respectively. The staining at st26 recapitulates the known myosin and pax7 expression patterns. At st40, the strong expression of sarcomeric myosin and the activity of the cardiac actin promoter indicate that in particular in muscle anlagen associated with the first (mandibular) and second (hyoid) pharyngeal arch, muscle differentiation is well under way. In these muscle anlagen, a faint expression is visible for pax7. The pattern is punctuate, as expected for a nuclear localisation of the pax7 protein. Abbreviations as in Figures 1, 10, 11 and: somite derived muscle: gh, m. geniohyoideus anlage.
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Figure 1. Time course of Pax7 expression in the chicken embryo; stages of development are indicated at the top of the panel. (A–L) Expression of Pax7 mRNA with (A-Ci) dorsal views, anterior to the top, (D–M) lateral views of the right side of embryos, anterior to the top, dorsal to the left. In (G), cranial ganglia are revealed by Isl1 expression (red staining). In (L), the eye was removed after staining. (M) Expression of Pax7 protein; lateral view of the left side of an embryo, anterior to the top, dorsal to the right, the eye has been removed before staining. The onset of Pax7 expression in craniofacial muscle anlagen is demarcated by green frames to the respective image. Throughout the first 5 days of development, Pax7 is well detectable in the central nervous system, cranial neural crest cells and the somites and somite-derived muscle precursors/embryonic muscle stem cells. In muscle anlagen derived from the paraxial head mesoderm, expression is first seen at stage HH18 in the lateral rectus eye muscle anlage, the only head muscle to express some trunk markers. In the other head mesoderm derived muscles, Pax7 mRNA can be detected in strongly stained specimen at stage 22; expression becomes more robust at stages 23–24 and is followed by protein expression at HH25. Abbreviations: cns, central nervous system; do, dorsal oblique eye muscle anlage; dr, dorsal rectus eye muscle anlage; hg/hb, hypoglossal/hypobranchial muscle anlage; lr, lateral rectus eye muscle anlage; ma, mandibular arch muscle anlage (jaw closure muscles); mr, medial rectus eye muscle anlage; ncc, neural crest cells; npb, neural plate border; nt, neural tube; ps, primitive streak; s/s1, somite/ somite 1; vo, ventral oblique eye muscle anlage; vr, ventral rectus eye muscle anlage; V, 5th cranial ganglion (trigeminal ganglion).
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Figure 2. Series of frontal (B–J) and cross (K–M) sections of a HH22 chicken head, stained for the expression of Pax7 mRNA, the plane and order of sections in indicated in (A). (B–J) anterior to the top, lateral to the right; (K–M) dorsal to the top. Abbreviations as in Figure 1. The position of the Pax7 signals beneath the trigeminal ganglion, beneath the eye and in the core of the mandibular arch confirms that these are expression domains associated with developing head muscles.
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Figure 3. Time course for the mRNA expression of head mesoderm markers in chicken embryos at HH10 (dorsal views) and HH13/14-E4 (lateral views); in (D), cranial nerves are revealed with the RMO270 antibody (brown staining). Gene names are displayed on the left of the panel; developmental stages are indicated at the top. The onset of marker gene expression is demarcated by a green frame, for genes being expressed earlier than HH10, frames are displayed in magenta. Abbreviations as in Figure 1 and: a/hm, anterior head mesoderm; ect, surface ectoderm; eom, extraocular muscle anlagen; end, endoderm; lam, lateral mesoderm; ht, heart; ov, otic vesicle; pam, pharyngeal arch muscle anlagen; p/hm, posterior head mesoderm. The open arrowhead in (H,J) points at Alx4 expression in the mandibular arch ectoderm and in (V–Y) at Tbx1 expression in the posterior ectoderm of the hyoid (2nd pharyngeal) arch. Note that all head mesoderm markers begin their expression well before Pax7. With the exception of Alx4 which from HH13/14 onwards mainly labels cranial neural crest cells and Tcf21/Capsulin which throughout has lower expression levels than its paralog MSc/MyoR, all head mesoderm markers continue to strongly label the myogenic head mesoderm. Their expression domains are wider than that of Pax7, whose expression domain is nested in the expression domain of the head mesoderm genes (compare Figures 1, 4).
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Figure 4. Time course for the mRNA expression of Mrf transcriptions factors in chicken embryos from HH10-E4; orientation of specimen and frames indicating the onset of expression as in Figure 3. Gene names and developmental stages are displayed as in Figure 3, Abbreviations as in Figures 1, 3 and: hy, hyoid arch. Myf5 and MyoD indicate the myogenic commitment of precursor cells and are expressed in developing craniofacial muscle anlagen from HH13/14 (Myf5) and HH16 (MyoD) onwards, i.e., significantly before the onset of Pax7. Expression of Myogenin (MyoG/Mgn) indicates the entry of cells into muscle differentiation and commences at E3.5-4, i.e., about the same time as Pax7 (compare with Figure 1).
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Figure 5. Time course in chicken embryos from HH10-E4 for markers indicating the cohesion of muscle anlagen (Cadherin 4–mRNA expression) and terminal differentiation [Troponin I 1 (Tnni1)–mRNA expression; sarcomeric Myosin–MF20 antibody staining]. Orientation of specimen and frames indicating the onset of expression as in Figures 3, 4; abbreviations as in Figures 1, 3, 4. The time course of Cadherin 4 expression resembles that of MyoD, and Tnni1 expression commences in many craniofacial muscle anlagen E3, i.e., both are expressed before or at the onset of Pax7 expression. Sarcomeric Myosins can be detected at E3.5-4, simultaneous to the onset of Pax7.
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Figure 6. Time course for the mRNA expression of trunk pre-myogenic genes; embryos are displayed and annotated as in Figures 3–5. Abbreviations as before and: na, nasal pit. (A–E)
Pax3 labels the central nervous system, the frontonasal neural crest, the trigeminal ganglion, the somites and the somite-derived hypobranchial and limb muscle precursors, but remains absent from genuine craniofacial muscle anlagen. (F–J)
Paraxis expression overlaps with that of Pax3 and 7 in the somite-derived muscle precursors and in the frontonasal crest. Similar to Pax7, Paraxis is also expressed in the lateral rectus eye muscle, but is absent from all other craniofacial muscles. Six1
(K–O) and Eya1
(P–T) are expressed in the head mesoderm before and at HH10. From that stage onwards mesoderm expression becomes somewhat obscured by the overlying expression in neural crest cells. However, Six1 (but not Eya1) remains detectable in craniofacial muscle anlagen.
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Figure 7. Time course of Pax7 expression in the mouse. (A–D)
Pax7 mRNA expression from E9.5-E12.5 of development; lateral views of the right side of embryos, anterior to the top. Expression is readily detectable in the developing central nervous system, emigrating neural crest cells (prolonged expression in the frontonasal neural crest) and the somites. Head muscle anlagen show expression first at E10.5. (E) Serial cross sections of the mandibular arch at E12.5, dorsal to the top; (F,G) higher magnifications of the areas indicated by the boxes in (E) and stained for Dapi and MyoD protein (Fi,ii) or Dapi and Pax7 protein (Gi,ii). Note that MyoD and Pax7 domains overlap. (H,I) Serial frontal sections of the mandible at birth (P0), dorsal to the top, lateral to the left. (H) Sirius Red staining showing muscle fibers in yellow and bone and connective tissue in red. (I) Dapi staining of the same region, with (Ii) showing a magnification of the cheek and the floor of the mouth as indicated in (I). Skeletal muscle fibers are shown in red. (J) Subsequent section stained for Pax7 protein in red. Note the punctate, nuclear staining for Pax7, associated with the Myosin-positive muscle fibers. (K–Q) Lineage tracing of Pax7 expressing cells, revealed by beta galactosidase staining; lateral views of the right side of embryos, dorsal to the top. With a delay of 1 day, cells with a history of Pax7 expression can be detected in the central nervous system, the trigeminal ganglion, the frontonasal neural crest and the somites. In craniofacial muscle anlagen, cells with a history of Pax7 expression can be detected between E11.5 and E12.5, with a more robust staining appearing at E13.5. Eventually, all craniofacial muscles are stained and the staining is found in muscle fibers, indicating that, similar to the trunk, Pax7-positive cells contribute to fetal and perinatal muscle growth. Abbreviations as in Figures 1, 3, 4 and: fl, forelimb; ISH, in situ hybridisation; ms, masseter; te, temporalis muscle; wt, wildtype.
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Figure 8. mRNA Expression of mouse head mesoderm markers and markers for myogenic commitment before and at the onset of Pax7 expression. Lateral views, dorsal to the top. Stages of development are indicated at the top of the panel, gene names on the left. (A,B)
Musculin expression commences before E9.5 (not shown); at E9.5-10.5, the gene is widely expressed in the myogenic head mesoderm. Myf5
(C,D) and MyoD
(E,F) expression commences at E9.5, i.e., before the onset of Pax7. (G,H)
Myogenin expression is not yet detectable at these stages and commences slightly later at E11.5 (not shown).
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Figure 9. Lineage tracing of MyoD expressing cells in MyoDiCre/+ R26NG embryos, revealed by anti-GFP antibody (green) staining. (A–D) Lateral views of the right side of E10.5-E13.5 embryos; the dotted line indicates the sectional plane in (F–I). (Ei-iii) Lateral views of the left side of an E11.5 embryo, stained for Pax7 mRNA (blue) and GFP protein (green); dorsal to the top. (F) Frontal section of an E13.5 embryo, stained for Pax7 protein (red), GFP (green), and Dapi (blue). (G) Detail of the ventral rectus eye muscle, (H) detail of the tongue, (I) detail of the masseter as indicated in (D,F). The widely distributed bright green (F) or yellow cells (G–I) are autofluorescing blood cells. Cells with a history of MyoD expression can readily be detected at E10.5 and 11.5, first in the mandibular and hyoid arch, then in the developing extraocular muscles. In head-mesoderm-derived muscles, Pax7 mRNA and subsequent protein expression colocalises with that of MyoD-Cre driven GFP, and Pax7 containing nuclei reside in GFP expressing cells. In contrast, in the somite-derived tongue muscle, most Pax7-positive nuclei are not located in GFP expressing cells. Abbreviations as in Figures 1, 4, 5 and: eom, developing extraocular muscles.
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Figure 13. pax7a mRNA, myod mRNA, and sarcomeric myosin expression (MF20 antibody staining) in 72 hpf zebrafish larvae, lateral views, anterior to the left; markers are indicated on the left of the panel. While myogenic markers show robust expression in craniofacial muscle anlagen, pax7 mRNA is barely detectable (arrowheads). Abbreviations as before and: am, adductor mandibulae; hh, hyohyoideus; hpf, hours post fertilization; ih, interhyoideus; im/a, intermandibularis anterior muscle; im/p, intermandibularis posterior muscle; m, mouth; pf, pectoral fin; sh, sternohyoideus.
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Figure 14. Proposed model for the emergence of craniofacial muscle stem cells: the bi-potential head mesodermal cells commit to myogenesis before adapting a muscle stem cell state, and then a lateral inhibition mechanism initiated by the differentiating cells controls the simultaneous production of functional muscle and the maintenance of the stem cell pool.
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msc (musculin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
Key: hy= hyoid arch; ov= otic vesicle.
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msc (musculin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.
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Figure 11 A- D. Time course of msc (musculin) gene expression in Xenopus laevis, lateral views, anterior left, dorsal up.
Key: pam: pharygeagel arch musculature; hp= hypophysis; ht= heart; hy = hyoid arch; im= m. intermandibularis primordium; lm= m. levatores mandibulae primordium; ih=m. interhyoideus primordium; oh= m. orbitohyoideus primordium; qh= m. quadrato-hyoangularis primordium; q/oh=common oh and qh precursor; ov= otic vesicle
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Figure 11 E- H'. Time course of myl5 () gene expression in Xenopus laevis, lateral views, anterior left, dorsal up.
Key: pam: pharygeagel arch musculature; hp= hypophysis; ht= heart; hy = hyoid arch; im= m. intermandibularis primordium; lm= m. levatores mandibulae primordium; ih=m. interhyoideus primordium; oh= m. orbitohyoideus primordium; qh= m. quadrato-hyoangularis primordium; q/oh=common oh and qh precursor; ov= otic vesicle
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Figure 11 A-Li. Time course of myod1 (myogenic differentiation 1) gene expression in Xenopus laevis, NF stage 33 & 34 to NF stage 40,lateral views, anterior left, dorsal up.
Key: pam: pharygeagel arch musculature; hp= hypophysis; ht= heart; hy = hyoid arch; im= m. intermandibularis primordium; lm= m. levatores mandibulae primordium; ih=m. interhyoideus primordium; oh= m. orbitohyoideus primordium; qh= m. quadrato-hyoangularis primordium; q/oh=common oh and qh precursor; ov= otic vesicle
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Figure 11 M-Pi. Time course of myog (myogenic factor 4) gene expression in Xenopus laevis, NF stage 33 & 34 to NF stage 40, lateral views, anterior left, dorsal up.
Key: pam: pharygeagel arch musculature; hp= hypophysis; ht= heart; hy = hyoid arch; im= m. intermandibularis primordium; lm= m. levatores mandibulae primordium; ih=m. interhyoideus primordium; oh= m. orbitohyoideus primordium; qh= m. quadrato-hyoangularis primordium; q/oh=common oh and qh precursor; ov= otic vesicle
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Figure 11 Q-Ti. Time course of myf6 (myogenic factor 6) gene expression in Xenopus laevis, NF stage 33 & 34 to NF stage 40, lateral views, anterior left, dorsal up.
Key: pam: pharygeagel arch musculature; hp= hypophysis; ht= heart; hy = hyoid arch; im= m. intermandibularis primordium; lm= m. levatores mandibulae primordium; ih=m. interhyoideus primordium; oh= m. orbitohyoideus primordium; qh= m. quadrato-hyoangularis primordium; q/oh=common oh and qh precursor; ov= otic vesicle
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Figure 11 U-Xi. Time course of des.1 (desmin, gene 1) gene expression in Xenopus laevis, NF stage 33 & 34 to NF stage 40, lateral views, anterior left, dorsal up.
Key: pam: pharygeagel arch musculature; hp= hypophysis; ht= heart; hy = hyoid arch; im= m. intermandibularis primordium; lm= m. levatores mandibulae primordium; ih=m. interhyoideus primordium; oh= m. orbitohyoideus primordium; qh= m. quadrato-hyoangularis primordium; q/oh=common oh and qh precursor; ov= otic vesicle
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