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First published online 9 January 2008
doi: 10.1242/dev.007989

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The contribution of Islet1-expressing splanchnic mesoderm cells to distinct branchiomeric muscles reveals significant heterogeneity in head muscle development

¹ Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel
² University of California-San Diego, Skaggs School of Pharmacy, La Jolla, CA 92093, USA.

View larger version (72K): [in this window] [in a new window]   Fig. 1. Histochemical and fluorescence in situ hybridizations identify the molecular boundaries of the AHF/SpM in the craniofacial mesoderm at cardiac crescent stages. The molecular identities of distinct cell populations in the head were determined, using histochemical in situ hybridization (ISH) and a double-fluorescence ISH method in St. 8+ chick embryos. AHF/SpM cells are delineated by dashed ellipses. (A-D) Whole-mount ISH for the indicated genes. Dashed lines indicate the plane of the stained transverse sections (A'-D'). Nkx2.5 and Isl1 are expressed in a broader portion of the SpM (defined as the AHF/SpM), whereas C-actin and Gata4 are expressed in the more-differentiated SpM. (E-E'') Fluorescent ISH for Isl1 (E) and Nkx2.5 (E'), and their overlay (E''), showing that both genes are expressed in both the differentiated SpM and AHF/SpM. (F-F'') Fgf10 (F), Nkx2.5 (F') and overlay (F''). (G-G'') The expression of CPM marker Cyp26c1 (G) and the AHF/SpM marker Nkx2.5 (G') defining the boundary between the CPM and the AHF/SpM (G''). (H-H'') Expression of the differentiated SpM marker Gata4 (H) and Nkx2.5 (H'). The merged image (H'') demonstrates that the AHF can be molecularly identified as comprising Nkx2.5+ and Gata4- cells. (I-I'') Tbx20 expression (I) marking both differentiated SpM and AHF/SpM. Tbx5 expression (I') labeled only the differentiated SpM. The merged image (I'') shows that the AHF can be molecularly identified as comprising Tbx20+/Tbx5- cells. nt, neural tube; no, notochord; CPM, cranial paraxial mesoderm; AHF/SpM, anterior heart field/splanchnic mesoderm.

View larger version (112K): [in this window] [in a new window]   Fig. 2. Expression of Isl1 protein in the AHF and in myogenic progenitors of the branchial arches. Immunofluorescence staining for Isl1 at different stages of chick embryonic development [as illustrated in the top row, modified from The Atlas of Chick Development (Bellairs and Osmond, 1998)]. (A-C) Isl1 is expressed in the SpM and the pharyngeal endoderm at St. 8. (D-F) At St. 12, Isl1 is restricted to the ventral pharynx and the AHF/SpM at different anterior-posterior levels. Myocardial cells in the linear heart tube show no Isl1 expression. (G-I) Isl1 expression (St. 18) is detected in the mesodermal core of BA1-2 but not in the neural crest-derived mesenchyme (nc). Isl1 is also expressed in the aortic sac (as, H). (A'-I') Higher magnifications of the regions indicated by the dashed squares in A-I. Arrows in E' show Isl1 expression in the border of the SpM. Arrows in G' and I' the expression of Isl1 in the mesoderm of BA1 and BA2, respectively. nt, neural tube; ph, pharynx; ecto, ectoderm; endo, endoderm; ba, branchial arch; SpM, splanchnic mesoderm; hrt, heart; oft, outflow tract; ov, otic vesicle; CPM, cranial paraxial mesoderm; mes, mesoderm.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Regionalization of the myogenic core of BA1 by CPM and SpM cells. (A,A') Injection of DiI to the CPM (A) of a St. 8 chick embryo. After 36 hours incubation, the labeled cells streamed toward BA1. Notably, some of the CPM cells were seen in the distal outflow tract (A'). (A'',A''') Injection of DiI into the AHF/SpM (A'') of a St. 8 embryo. After 36 hours incubation, labeled cells populated the outflow tract; some of the cells were seen in the distal portion of BA1 (A'''). (B,B') Double injection of DiI (CPM) and DiO (AHF/SpM) into a St. 8 embryo (B) as shown in the transverse section (B') (n=22). (C-C'') After 18 hours incubation (C), at St. 11, the DiI was found in the CPM (C') anterior to the location of the DiO in the outflow tract (C''). (D-D'') After 24 hours (St. 12, D) the DiI was found at the most-proximal area of the developing BA (D'), as well as in the outflow tract (yellow staining in D''). The DiO was located in the distal part of the forming BA (D') and in the cardiac outflow tract (D''). (E-E'') After 48 hours (St. 16, E) the DiI was found in the most-proximal area of the mesoderm core, whereas the DiO was located in the distal part of the BA1 core (E'). BA1 viewed at higher resolution (E'') demonstrates that the dye is restricted to the mesodermal core, and doesn't appear in the neural crest cells (nc) surrounding the core. (F,G) DiO was injected into the AHF/SpM of St. 8 embryos that were subsequently immunostained (red) for the AHF markers Isl1 (F) and Nkx2.5 (G). Co-localization of the DiO and the two AHF markers is shown in the distal part of the arch. Note that the DiO only labeled mesoderm cells (Isl1+ and Nkx2.5+), and not neural crest cells. oft, outflow tract; nt, neural tube; BA1, branchial arch 1; mes, mesoderm; hrt, heart; ph, pharynx; ecto, ectoderm; CPM, cranial paraxial mesoderm; AHF/SpM, anterior heart field/splanchnic mesoderm; ov, otic vesicle.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Distinct programs underlie the development of CPM- and SpM-derived jaw muscles in the chick embryo. (A-A''') Scheme (A) of the dye labeling experiment of proximal and distal BA1 myoblasts (A'). The mandibular adductor complex is illustrated in A'' and the intermandibular muscle is shown in A'''. Co-staining of MyoD and DiO is shown in these muscles (A'',A'''). (B-B'') Transverse section of BA1 in St. 20 embryos stained for Myf5 and Isl1 (B') and Myf5 with Pax7 (B''). (C) Schematic depicting the distinct cellular origins and molecular identities of the branchiomeric muscles. (D-E') A transverse section through the adductor (add) muscle complex at St. 26 stained for the markers indicated. Note that Isl1 shows no apparent expression in this region (D''). (F-F''') A transverse section through the intermandibular (im) muscle (F) stained for Isl1 and Myf5 (F'). Note the co-localization of Isl1 and Myf5 in the enlargement (F''). Staining for MHC and Isl1 shows the lack of MHC in the Isl1+ cells in the intermandibular anlagen (F'''). (G-G''') Similar sections stained for Isl1 and MyoD (G'). Note the mutually exclusive expression of Isl1 and MyoD, as shown in the enlargement (G''). Isl1 was restricted to the intermandibular muscle, and was not expressed in the genioglossal (g) muscle (arrow). A sequential section (G''') was stained for Pax7 (red) and MHC (blue). Pax7 and Isl1 were not expressed in the same muscles (arrow). (H-H''') Staining for Isl1, MyoD, MHC and Pax7 at the intermandibular region of St. 31, indicating the loss of Isl1 along with the upregulation of MHC and Pax7. Scale bars: 50 µm.

View larger version (129K): [in this window] [in a new window]   Fig. 5. Contribution of Isl1+ progenitors and their progeny to a subset of branchiomeric muscles in the mouse. (A,A') Expression of the lacZ reporter in the mesodermal core (white arrowhead) of BA1 in E10.5 Isl1-Cre/R26R whole-mount (A) or sectioned (A') embryo. Dashed line in A indicates plane of section. (B) Illustration of the various sections of the E16.5 Isl1-Cre/R26R embryos, modified from the Atlas of Mouse Development (Kaufman, 1992). (C-F) X-Gal staining for the lacZ reporter in transverse sections of an E16.5 Isl1-Cre/R26R embryo. The Isl1 lineage gives rise to multiple tissues including branchiomeric muscles (C,D,E), the salivary glands (arrowhead), and the oropharynx (arrow). (G-H') Immunostaining for β-gal (G,H) and MF20 (MHC; G',H') on serial frontal paraffin sections of an E16.5 Isl1-Cre/R26R embryo (shown in B). For clarity, a half-section of β-gal was combined with the adjacent half of the MF20 staining. Note the co-expression of β-gal and MF20 in a subset of branchiomeric muscles, but not in tongue or extraocular muscles (eom, marked by squares in G,G'). The masseter (ma) has both superficial and deep masses and the pterygoid (pt) has medial and lateral masses (H'). nt, neural tube; ov, otic vesicle; nc, neural crest; mes, mesoderm; t, tongue; my, mylohyoid; ad, anterior digastric; g, genioglossal; co, constrictor; st, stylohyoid; pd, posterior digastric; tm, temporalis.

View larger version (96K): [in this window] [in a new window]   Fig. 6. The Wnt/ β-catenin signaling pathway negatively regulates specification and differentiation of cells from the SpM and CPM in chick embryos. (A) Schematic representation of the experimental procedure showing the electroporation of the construct into a St. 3c embryo, which was analyzed 16 hours later, at St. 8. (B-D) Fluorescent ISH of the St. 8 electroporated embryos for the markers indicated. As a result of Wnt3a activity, the expression levels of Tbx5 (B), Nkx2.5(C) and Gata4 (D) transcripts were reduced on the electroporated side (arrows; n=14). (E) Representation of the experimental procedure showing the electroporation of the pCIG-Wnt3a-IRES-GFP construct into a St. 8 embryo, and its analysis at St. 12 (note a reverse cardiac looping). (F,G) St. 12 embryos stained for Isl1 (F) and Nkx2.5 (G) following electroporation with pCAGG-GFP (arrows). The normal expression of both proteins was not affected (arrowheads). (F',G') St. 12 embryos, electroporated with pCIG-Wnt3a-IRES-GFP marked by the GFP expression in the surface ectoderm. Both Isl1 (F') and Nkx2.5 (G') levels in the SpM of the AHF (arrowheads) were reduced on the electroporated side (n=17). (H-I') Whole-mount ISH for Tbx1 and capsulin in St. 13 embryos electroporated with either pCAGG-GFP or pCIG-Wnt3a-IRES-GFP. Both Tbx1 (H') and capsulin (I') levels in the BA were reduced (arrows) on the Wnt3a-electroporated side (n=9). nt, neural tube; hrt, heart; ph, pharynx.

View larger version (92K): [in this window] [in a new window]   Fig. 7. Antagonists of the Wnt/ β-catenin pathway promote Isl1 and Nkx2.5 expression in AHF/SpM and branchial arches. (A,B) Representation of the electroporation of pCIG-sFrp2-IRES-GFP together with pCIG-sFrp3-IRES-GFP constructs between St. 8 and St. 12. Dashed line indicates the plane of the transverse sections. (C) Immunostaining for Nkx2.5 at St. 12 indicating that Nkx2.5 protein level increased (arrowhead) on the electroporated side (arrow). (D) Immunostaining for Isl1 at St. 12. GFP expression in the surface ectoderm marks cells expressing the sFrp2 and sFrp3 proteins. Isl1 level in the SpM (arrowhead) was increased on the electroporated side (n=7). (E,F) Fz8-IgG-soaked bead insertion (E, St. 8) and the same embryo 23 hours later (F, St. 14). (G-I) Transverse sections showing the location of the implanted bead (blue circle in G,I and dashed circle in H). (G'-I') Immunostaining of sections G-I. As a result of the Fz8-IgG activity, both Nkx2.5 (G',H') and Isl1 (I') levels in BA1 increased around the bead (n=4). EP, electroporation; nt, neural tube; BA1, branchial arch 1; hrt, heart; ph, pharynx.

View larger version (67K): [in this window] [in a new window]   Fig. 8. Model for the molecular regulation of the boundaries within the cardio-craniofacial mesoderm in chick embryos. (A) At St. 8, the cardio-craniofacial mesoderm can be divided into CPM (blue), AHF/SpM (green) and differentiated SpM (red). (B) At St. 10, the division of the cardio-craniofacial mesoderm becomes more obvious. The AHF/SpM (green) is located underneath the pharynx, just dorsal to the heart. (C) At St. 18, both CPM and SpM contribute to BA1 and to the cardiac outflow tract. The distal part of BA1 mesoderm is SpM-derived (green) and is Isl1+, whereas the proximal region is CPM-derived (blue) and Myf5+. In the cardiac outflow tract, the contribution of the AHF/SpM is greater than that of the CPM, the contribution of which is seen in the more-distal part of the heart. (D) Heart formation is regulated by a combination of positive and negative signals from surrounding tissues. Whereas a signal(s) from the anterior endoderm works to promote heart formation in concert with BMP signals from the anterior lateral mesoderm, Wnt signals from the axial tissues (orange) repress heart formation in the CPM. (E) This study demonstrates that Wnt signaling can repress Isl1 and Nkx2.5 in the AHF/SpM, whereas treatment with Wnt antagonists (sFRP, purple) promotes Isl1 and Nkx2.5 expression. EP, electroporation; fg, foregut; nt, neural tube; da, dorsal aorta; n, notochord; h, heart; lm, lateral mesoderm; CPM, cranial paraxial mesoderm; AHF/SpM, anterior heart field/splanchnic mesoderm. Electron micrographs shown in A and B were reproduced with permission from Prof. Schoenwolf (University of Utah School of Medicine, Salt Lake City, UT).