Relationship between neural crest cells and cranial mesoderm during head muscle development

Abstract

Background: In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/principal findings: Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1(-/-) mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/significance: This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.

Figure 1. Comparison of MyoR and MyoD expression domains during chick first branchial arch development.

(A) Lateral view of a HH20 chick embryo hybridized with a MyoR probe. HH20 (B,C), HH22 (D,E) HH24 (F,G) and HH26 (H,I) embryos were frontally sectioned at the head level. The plane of section is indicated in the panel A. Adjacent sections from each stage were hybridized with DIG-labelled antisense probes for either MyoR (B,D,F,H) or MyoD (C,E,G,I). MyoR delineates the myogenic core of the first branchial arch (B,D,F) and is subsequently expressed in all branchiomeric muscles (H). MyoD transcripts are first detected in a lateral sub-region of the MyoR domain at HH20 (B,C, arrows) and HH22 (D,E, arrows), then spread progressively from lateral to medial regions to overlap with the MyoR domain in branchiomeric muscles (E,G,I). (D–G) Arrows point to the lateral domains of the core, while arrowheads show the medial domain of the core. (H,I) Arrows indicate the branchiomeric muscles expressing both MyoR (H) and MyoD (I). (A) Arrowheads indicate the hypaxial lips at the interlimb level, expressing MyoR. BA1, first branchial arch, di, diencephalon; e, eye; fl, forelimb; hl, hindlimb; mb, mandibular arch; mes, mesenchephalon; MR, medial rectus; mx, maxillary arch; ph, pharynx; tel, telencephalon; VO, ventral oblique; VR, ventral rectus.

Figure 2. Comparison of MyoR and MyoD expression in extra-ocular muscles.

HH22 (A,B), HH24 (C,D), HH26 (E,F), HH30 (G,H) chick embryos were frontally sectioned at the head level. Adjacent sections from each stage were hybridized with DIG-labelled antisense probes for either MyoR (A,C,E,G) or MyoD (B,D,F,H). E12.5 mouse embryos were sagitally sectioned at the head level and hybridized with DIG-labelled antisense probes for either mMyoR (I) or mMyoD (J). In the chick embryo, At HH22, the ventral oblique is the first ocular muscle to express MyoR and MyoD (A,B). At HH 24, MyoR transcripts are observed faintly in the dorsal rectus, strongly in lateral rectus (C), while MyoD is expressed in both dorsal rectus and lateral rectus muscles and in branchiomeric muscles (D). At HH26 stage, the ventral and dorsal obliques harbour strong MyoR (E) and MyoD (F) expression. At HH30, when the medial and ventral rectus muscles are individualized, MyoD is expressed in both muscles, while MyoR is expressed in ventral rectus but not in medial rectus (G,H). (H) The innervation is labelled with HNK1 antibody, two arrowheads point to nerves. The optic nerve is also labelled in light brown with the HNK1 antibody. (G) The absence of MyoR expression in the medial rectus is indicated by arrows. (I,J) In E12.5 mouse embryos, extra ocular muscles, labelled by white asterisks (J) expressed both mMyoR (I) and mMyoD (J). di, diencephalon; DO, dorsal oblique; DR, dorsal rectus; LR, lateral rectus; mBA1, first branchial arch muscles; MR, medial rectus; mx, maxillary arch; ON, optic nerve; ph, pharynx; VO, ventral oblique; VR, ventral rectus.

Figure 3. Neural crest cells visualized with AP2α expression are observed inside the mesodermal core of the first branchial arch.

Adjacent transverse sections of chick embryos at the level of the first branchial arch at 22 (A,B), 29 (C,D) and 32 (E,F) somite-stages were hybridized with MyoR (A,C,E) and AP2α (B,D,F) probes. The mesodermal core is visualized with MyoR expression (A,C,E). AP2α is expressed in all neural crest cells within the arch and in the surface ectoderm (B,D,F). The AP2α-positive surface ectoderm is arrowed in (B,D,F). AP2 α positive cells are also observed inside the MyoR-positive domain (B,D,F). The inset in F shows an enlargement of the mesodermal core, where the AP2 α-positive cells are indicated by arrowheads. Frontal sections of E9.5 mouse embryos from Myf5-nlacZ mice were immunostained using an anti-β-galactosidase antibody to visualise mMyf5 expression (G, green) and an anti-AP2 antibody to detect mAP2α location (H, red). I is a merged picture of G and H. AP2α-positive cells (red) are observed in the mesodermal core delineated by Myf5-postive cells (green). Hoechst staining in blue indicate nuclei. a, aortic arch; BA1, first branchial arch; BA2, second branchial arch; ecto, surface ectoderm; endo, pharyngeal endoderm; No, notochord; ph, pharynx.

Figure 4. Neural crest cells invade the cephalic mesoderm.

(A) Schematic representation of the chick neural fold replacement by its quail counterpart (red) performed on 5/6-somite stage quail and chick embryos. (B–F) Transverse sections of quail–chick chimeras at 16 (B,C,D), 18 (E) and 21 (F) somite-stages, at the level of the future first branchial arch were hybridized with the MyoR probe followed by immunohistochemistry using the QCPN mAb. (C) is a higher magnification of (B). (B,C,D) corresponds to sections from the same embryo, (B,C) being slightly (80 µm) more rostral than (D). The asterisk in B marks the position of the graft. (B–D) At the future first branchial arch level, the QCPN-positive cells progressively invaded the cephalic mesoderm expressing MyoR, in a rostral to caudal manner. At 18 and 21 somite-stages (E,F), QCPN positive cells are observed inside the MyoR-positive domains. a, aortic arch; ecto, ectoderm; endo, pharyngeal endoderm; mes, mesencephalon; ph, pharynx.

Figure 5. Relationship between neural crest cells and muscle precursor cells during branchial arch development.

(A–C) Adjacent frontal sections from HH20 quail-chimeras at the first branchial level were hybridized with MyoR (A) and MyoD (B) probes and then incubated with QCPN antibody or uniquely incubated with QCPN antibody (C). (D–G) Adjacent frontal sections from HH23 (D,E) and HH27 (F,G) quail-chimeras at the first branchial level were hybridized with MyoR (D), MyoD (F) or Scleraxis (G) probes and then incubated with the QCPN antibody or directly incubated with the QCPN antibody (E). QCPN-positive cells are observed in the MyoR- and MyoD-positive domains of the core at HH21 (A–C) and HH23 (D,E). From HH27 there is an increase of QCPN-positive cells inside the muscles (F,G).

Figure 6. Head tendons express Scleraxis and are of quail origin.

(A) In situ hybridization to HH24 embryos with the Scleraxis probe. Adjacent frontal sections of HH24 (B,C) and HH26 (D–H) embryos were hybridized with the Scleraxis (B,D,F,G) and MyoD (C,E,H) probes were followed by an immunohistochemistry using the MF20 antibody to reveal differentiated muscle fibres. MF20 is visible in (F,G). (A,B) Arrows point to the Scleraxis expression domain in tendon primordia at HH24. At HH26, Scleraxis labels branchiomeric tendons (D,E) and eye tendons (F–H). (I–M) Adjacent saggital sections of HH28 quail-chick chimeras, were hybridized with the MyoD (I,K) and Scleraxis (J,L,M) probes followed by immunohistochemistry using the QCPN antibody. QCPN is visible in (I,JM). The Scleraxis-positive tendons associated with the extra ocular muscle (I,J) or with a jaw operating–muscle (K–M) are of quail origin. (M) is a higher magnification of (L).

Figure 7. Scleraxis expression is normally established in the absence of differentiated muscles in the first branchial arch of E12.5 Tbx1 ^−/− mice.

Adjacent saggital sections of either wild-type (A,C,E) or Tbx1 ^−/− mutant mice (B,D,F) were hybridized with mMyoD (A,B) or mScleraxis (C–F) probes. (C–F) After in situ hybridization with a mouse Scleraxis probe, the differentiated myofibres were detected using MF20 antibody. Tbx1 ^−/− mutant mice display a loss of branchiomeric-derived muscles, highlighted by the absence of mMyoD expression (B, black arrow) and MF20 labelling (D, F black arrows) compared to the wild type situation (A,C,E, black arrows). Despite the absence of branchiomeric muscles, mScleraxis expression pattern remains unchanged in mutant mice (D,F black arrowheads) compared to wild-type (C,E black arrowheads). Green arrows (A,B) and arrowheads (C,D) point to the non-affected extraocular muscles and tendons, respectively in control (A,C) and Tbx1 ^−/− mutant mice (B,D). (E,F) are high magnifications of (C,D) respectively. The open arrow in F indicates Scleraxis domain that has spread to the space left by the absent muscle.

Figure 8. Scleraxis expression is lost in the absence of differentiated muscles in the first branchial arch of E15.5 Tbx1 ^−/− mice.

Adjacent saggital sections of either E15.5 wild-type (A,C,E) or E15.5 Tbx1 ^−/− mutant mice (B,D,F) were hybridized with mMyoD (A–D) or mScleraxis (E,F) probes. (E,F) After in situ hybridization with a mouse Scleraxis probe (blue), differentiated myofibres were detected using MF20 antibody (light brown). (C,D) are higher magnification of A,B), respectively, focusing on the mandible. Tbx1 ^−/− mutant mice display loss of branchiomeric muscles, highlighted by the absence of both mMyoD expression (B,D) and MF20 labelling (F) in the mandibula, compared to the wild type situation (A,C,E). The anterior digastric muscle is arrowed in the control mandible (C,E), while residual muscle masses are indicated by an arrow (D,F) in similar mandibular regions of Tbx1^−/− mutant mice. Non-branchiomeric muscles are not affected in E15.5 Tbx1 ^−/− embryos. (E) Scleraxis expression is observed in tendons associated with the anterior digastric muscle in the wild type situation (arrowheads). (F) Scleraxis expression is lost in the absence of muscles in E15.5 Tbx1^−/− mutant mice, while Scleraxis expression is normally associated with non-branchiomeric muscles (green arrowheads). Mb, mandibular, mx, maxillary; t, tongue.