Skeletal muscles of the head and trunk originate in distinct lineages with divergent regulatory programmes converging on activation of myogenic determination factors. Branchiomeric head and neck muscles share a common origin with cardiac progenitor cells in cardiopharyngeal mesoderm (CPM). The retinoic acid (RA) signalling pathway is required during a defined early time window for normal deployment of cells from posterior CPM to the heart. Here, we show that blocking RA signalling in the early mouse embryo also results in selective loss of the trapezius neck muscle, without affecting other skeletal muscles. RA signalling is required for robust expression of myogenic determination factors in posterior CPM and subsequent expansion of the trapezius primordium. Lineage-specific activation of a dominant-negative RA receptor reveals that trapezius development is not regulated by direct RA signalling to myogenic progenitor cells in CPM, or through neural crest cells, but indirectly through the somitic lineage, closely apposed with posterior CPM in the early embryo. These findings suggest that trapezius development is dependent on precise spatiotemporal interactions between cranial and somitic mesoderm at the head/trunk interface.

The vertebrate neck and its associated muscles arise from a transition zone at the interface between cranial and somitic mesoderm (Sambasivan et al., 2011). Cranial mesoderm gives rise to branchiomeric head and neck muscles, which originate in the core of bilateral pharyngeal (or branchial) arches, as well as extraocular muscles, whereas somitic mesoderm gives rise to trunk and limb muscles, as well as infrahyoid neck and tongue muscles. Although myogenic regulatory factors (MRFs) of the MyoD family control determination and differentiation in all skeletal muscle primordia, the upstream control of head and trunk myogenesis differs (Grifone and Kelly, 2007; Hacker and Guthrie, 1998; Lescroart et al., 2022; Mootoosamy and Dietrich, 2002; Schubert et al., 2019; Tajbakhsh et al., 1997). The activation of MRF genes in branchiomeric muscle progenitor cells is regulated by a combination of transcription factors, including TBX1, TCF21, PITX2, LHX2, SIX1, SIX2, SIX5 and ISL1, in contrast to regulation by PAX3 and SIX transcription factors in somitic myogenesis (Dong et al., 2006; Harel et al., 2012, 2009; Kelly et al., 2004; Lu et al., 2002; Moncaut et al., 2012; Wurmser et al., 2023). The upstream signalling pathways regulating myogenesis also differ in the head and trunk, for example Wnt signalling promotes trunk but negatively regulates head myogenesis (Tirosh-Finkel et al., 2006; Tzahor et al., 2003; von Scheven et al., 2006).

Clonal analysis and genetic lineage-tracing experiments have shown that branchiomeric, but not somitic muscle, is related to myocardium (Diogo et al., 2015; Lescroart et al., 2015, 2010; Tzahor and Evans, 2011). This reflects the sequential formation of pharyngeal arches along the anterior-posterior axis as cardiac progenitor cells of the second heart field (SHF) are deployed to the growing heart tube. Cranial mesoderm giving rise to branchiomeric and SHF-derived parts of the heart is known as cardiopharyngeal mesoderm (CPM), an evolutionarily conserved multipotent progenitor cell population (Adachi et al., 2020; Diogo et al., 2015; Gopalakrishnan et al., 2015; Lescroart et al., 2022). Defects in CPM development have been implicated in human genetic syndromes and congenital disease associated with craniofacial and cardiac defects, including muscle hypotonia and postural defects (Digilio et al., 2003; Scambler, 2010).

Neck muscles comprise branchiomeric as well as somite-derived infrahyoid and dorsal neck muscles (Adachi et al., 2018; Heude et al., 2018). The trapezius neck muscle originates in posterior CPM and progressively extends into the thoracic region, spanning the head trunk transition zone, ultimately controlling movement of the scapula and spine, and integrating movement of the head and trunk. The trapezius muscle evolved with the jaw during vertebrate radiation, and recent evidence suggests it originated from a posterior branchial arch muscle as the pectoral girdle emerged in the common ancestor of jawed vertebrates (Brazeau et al., 2023; Naumann et al., 2019, 2017; Sefton et al., 2016). Innervation of the trapezius muscle by cranial nerve XI is consistent with a branchiomeric identity (Heude et al., 2018). However, because of its posterior position, the branchiomeric versus somitic origin of the trapezius muscle has been debated (Noden, 1983). Theis and colleagues showed that the avian homologue of the trapezius muscle, the cucullaris, is derived from progenitor cells in lateral plate mesoderm adjacent to the first three somites (Theis et al., 2010). More recently, retrospective clonal analysis has shown that the trapezius muscle arises from common progenitor cells with myocardium of the atria and outflow tract of the heart, consistent with an origin in posterior CPM (Lescroart et al., 2015). Genetic lineage-tracing experiments further support a branchiomeric origin of the trapezius muscle (Heude et al., 2018; Huynh et al., 2007; Lescroart et al., 2015; Theis et al., 2010) and neural crest and lateral plate mesoderm contribute to connective tissue associated with the trapezius, reflecting its position bridging the head trunk interface (Durland et al., 2008; Heude et al., 2018; Matsuoka et al., 2005; Noden, 1983). Consistent with a branchiomeric origin, the trapezius is absent in Tbx1−/− embryos and present in Myf5−/−;Pax3−/− embryos (Kelly et al., 2004; Tajbakhsh et al., 1997; Theis et al., 2010). However, the signalling pathways and cellular mechanisms underlying the development of this muscle remain elusive.

The retinoic acid (RA) signalling pathway, whereby the vitamin A derivative RA modulates transcription by nuclear receptors acting through RA-responsive target sites, plays multiple essential functions in patterning, segmentation and differentiation during embryonic development (Cunningham and Duester, 2015). High levels of RA signalling in the anterior somites play crucial roles in patterning adjacent tissues, and perturbation of RA signalling results in defects in pharyngeal and cardiac development (Begemann et al., 2001; Matt et al., 2003; Niederreither et al., 2003; Quinlan et al., 2002; Stefanovic and Zaffran, 2017; Wendling et al., 2000; Xavier-Neto et al., 2015). Exposure to BMS493, a pan-RA receptor inverse agonist, on embryonic day (E) 8, leads to hypoplasia of the caudal pharyngeal region and defective endodermal, mesodermal and neural crest development (Wendling et al., 2000). RA synthesis at this time point is regulated by the retinaldehyde dehydrogenase RALDH2 (ALDH1A2), which defines the posterior limit of the SHF (Ryckebusch et al., 2008; Sirbu et al., 2008). Perturbation of the RA pathway during the E8 time window impairs SHF addition at the venous pole of the heart leading to atrial septal defects (De Bono et al., 2018; Hochgreb et al., 2003). RA signalling has also been implicated in regulating the alignment of neural and mesodermal tissues at the head–trunk boundary (Begemann et al., 2001; Kuratani et al., 1998; Lee and Skromne, 2014). In addition, multiple studies have shown that RA signalling promotes skeletal muscle development. RA induces myogenesis in stem cells, acting upstream of and in direct interaction with MRFs (Albagli-Curiel et al., 1993; Arnold et al., 1992; Edwards and McBurney, 1983; Froeschlé et al., 1998). During limb muscle development, RA signalling plays iterative roles in indirectly controlling migration of myogenic progenitor cells into the limb field, maintaining premyogenic cell populations, and regulating their differentiation and patterning (Hamade et al., 2006; Mic and Duester, 2003; Reijntjes et al., 2010; Vermot et al., 2005b). RA signalling also plays important roles in extraocular muscle patterning and negatively regulates Tbx1 expression during early patterning of avian head mesoderm (Bothe et al., 2011; Comai et al., 2020). Moreover, a recent study has shown that excess RA signalling leads to loss and hypoplasia of developing branchiomeric muscles in anterior pharyngeal arches by reducing progenitor cell survival (Wang et al., 2021).

As cardiac derivatives of posterior CPM require RA signalling during an early developmental time window, we investigated whether RA signalling is also required for trapezius muscle development in the mouse embryo using pharmacological and genetic approaches. We demonstrate that trapezius muscle development selectively requires RA signalling during early embryonic stages. Lineage-specific activation of a dominant-negative RA receptor reveals that, unexpectedly, this effect is not mediated by direct RA signalling to trapezius progenitor cells, but indirectly through activation of secondary events that license trapezius progenitor cells to expand into the trunk territory.

Development of the trapezius muscle requires retinoic acid signalling

Genetic lineage tracing using Mef2c-AHF-Cre;ROSA26-lacZ (R26-lacZ) allows visualization of branchiomeric skeletal muscles of the head and neck, including cervical (acromiotrapezius) and thoracic (spinotrapezius) components of the trapezius muscle (Heude et al., 2018; Lescroart et al., 2015). Given that the trapezius muscle is clonally related to myocardium at the venous pole of the heart, normal development of which depends on the RA signalling pathway (De Bono et al., 2018; Lescroart et al., 2015), we investigated the impact of blocking RA signalling on neck muscle development. Mef2c-AHF-Cre;R26-lacZ embryos were exposed to a pan-RA receptor (RAR) inverse agonist, BMS493, during an early developmental time window in which RA signalling has been shown to be required for posterior pharyngeal and venous pole development (Fig. 1A) (De Bono et al., 2018; Wendling et al., 2000). BMS493 was administered by oral gavage to pregnant females (10 mg/kg) at E7.75 and E8.25, and embryos were collected and analysed at E14.5 and E15.5 (Fig. 1A-D). In Mef2c-AHF-Cre;R26-lacZ embryos exposed to vehicle alone, whole-mount X-gal staining labelled branchiomeric muscles, including cervical and thoracic components of the trapezius muscle (Fig. 1B,C). In contrast, BMS493-treated embryos presented a range of trapezius muscle defects that included unilateral or bilateral absence, shortening, or hypoplasia (Fig. 1B,C, Table S1). These defects affected either the spinotrapezius alone, or both acromiotrapezius and spinotrapezius muscles. The observed pattern of stochastic muscle defects suggests that RA signalling is required for robust bilateral development of the trapezius muscle. Branchiomeric head muscles derived from anterior CPM, including jaw closing muscles (the temporalis and masseter) and facial expression muscles, formed normally in embryos with severe trapezius muscle defects (Fig. 1B,C). Immunofluorescence with myosin heavy chain antibody (MF20) at E14.5 further demonstrated defects in trapezius muscle development in embryos exposed to BMS493 at E7.75 and E8.25, whereas other muscles, including the sternocleidomastoid muscle derived from posterior arch mesoderm, formed normally, revealing the selective sensitivity of the developing trapezius to reduced RA signalling (Fig. 1D, Fig. S1). Cardiac defects, including common arterial trunk and atrioventricular septal defects, were observed in BMS493-treated embryos, consistent with previous results (Fig. S2) (De Bono et al., 2018).

Fig. 1.

Blocking retinoic acid signalling during an early developmental time window impairs trapezius muscle development. (A) Experimental strategy used to label the trapezius muscle with Mef2c-AHF-Cre and R26-lacZ after BMS493 exposure during early development, followed by analysis at E14.5-15.5. (B,C) Lateral (B) and dorsal (C) views of embryos at E15.5 showing β-galactosidase labelling in skeletal muscles of the head and neck, including the acromiotrapezius (atrap) and spinotrapezius (strap) muscles, in embryos exposed to vehicle (left) (n=12) and BMS493 at E7.75 and E8.25 with absence (asterisks) or hypoplasia (arrows) of the trapezius muscles (n=14). (D) Immunostaining of MF20 on vehicle (n=5) and BMS493 (E7.75+8.25; n=4) treated embryos at E14.5. Neck and shoulder level sections showing shortening and thinning (arrows) of the acromiotrapezius muscle, and hypoplastic spinotrapezius muscles in BMS493-treated embryos. Other skeletal muscles are unaffected. (E) Lateral views of Mef2c-AHF-Cre;R26-lacZ embryos at E14.5 exposed to vehicle (left) (n=8) and BMS493 at single time points of E7.75 (n=8), E8.25 (n=16) and E9.5 (n=9). Note the absence (asterisk) of the trapezius muscle after BMS493 exposure at E8.25. acr, acromion; atrap, acromiotrapezius; epm, epaxial muscle; ifs, infraspinatus muscle; ma, masseter muscle; rm, rhomboid major muscle; sa, serratus anterior muscle; scp, scapula; ss, supraspinatus muscle; strap, spinotrapezius; te, temporalis muscle. Scale bars: 200 µm.

Fig. 1.

Blocking retinoic acid signalling during an early developmental time window impairs trapezius muscle development. (A) Experimental strategy used to label the trapezius muscle with Mef2c-AHF-Cre and R26-lacZ after BMS493 exposure during early development, followed by analysis at E14.5-15.5. (B,C) Lateral (B) and dorsal (C) views of embryos at E15.5 showing β-galactosidase labelling in skeletal muscles of the head and neck, including the acromiotrapezius (atrap) and spinotrapezius (strap) muscles, in embryos exposed to vehicle (left) (n=12) and BMS493 at E7.75 and E8.25 with absence (asterisks) or hypoplasia (arrows) of the trapezius muscles (n=14). (D) Immunostaining of MF20 on vehicle (n=5) and BMS493 (E7.75+8.25; n=4) treated embryos at E14.5. Neck and shoulder level sections showing shortening and thinning (arrows) of the acromiotrapezius muscle, and hypoplastic spinotrapezius muscles in BMS493-treated embryos. Other skeletal muscles are unaffected. (E) Lateral views of Mef2c-AHF-Cre;R26-lacZ embryos at E14.5 exposed to vehicle (left) (n=8) and BMS493 at single time points of E7.75 (n=8), E8.25 (n=16) and E9.5 (n=9). Note the absence (asterisk) of the trapezius muscle after BMS493 exposure at E8.25. acr, acromion; atrap, acromiotrapezius; epm, epaxial muscle; ifs, infraspinatus muscle; ma, masseter muscle; rm, rhomboid major muscle; sa, serratus anterior muscle; scp, scapula; ss, supraspinatus muscle; strap, spinotrapezius; te, temporalis muscle. Scale bars: 200 µm.

To define the embryonic time window during which trapezius muscle development is dependent on RA signalling, Mef2c-AHF-Cre;R26-lacZ embryos were analysed at E14.5 after exposure to a single dose of BMS493 at either E7.75, E8.25 or E9.5. Major trapezius muscle defects, including both unilateral and bilateral hypoplasia, were only observed in embryos after RA signalling was inhibited at E8.25 (Fig. 1E, Table S2). In contrast, BMS493 treatment at E7.75 or E9.5 resulted in a low incidence of minor, or no, defects in trapezius development, respectively (Fig. 1E, Table S2). Cardiac defects affecting the conotruncal region of the heart were observed after BMS493 exposure at all time points (Fig. S2) (De Bono et al., 2018). In a complementary approach to oral gavage, BMS493 was administered by intraperitoneal injection at E7.75 and E8.25. Analysis of E14.5 embryos displayed a similar range of trapezius muscle defects to those after oral gavage, including unilateral or bilateral absence, shortening, or hypoplasia (Fig. S3, Table S2). Increased overall severity of the trapezius phenotypes following intraperitoneal injection of BMS493 is likely to reflect improved delivery compared with gavage. Consistent with this, intraperitoneal injection of a lower dose of BMS493 (5 mg/kg) led to milder trapezius muscle defects (Fig. S3). Together, these results demonstrate that RA signalling is required for trapezius muscle development on early E8, overlapping with the time window of RA susceptibility in venous pole progenitor cells within the SHF (De Bono et al., 2018).

Retinoic acid signalling is required for robust activation of myogenic determination factors in posterior pharyngeal mesoderm

We next investigated when exposure to BMS493 during the early developmental time window impacts on trapezius muscle development. The onset of myogenesis in the trapezius primordium was investigated at E10.5 by analysis of the expression pattern of the MRF genes MyoD (Myod1) and Myf5. In control embryos, MyoD was expressed at all sites of skeletal myogenesis, including the mesodermal core of pharyngeal arches 1, 2 and 3-6, as well as in the myotomal compartment of the somites, somite-derived hypoglossal cord, and forelimb muscle masses (Fig. 2A). Embryos exposed to BMS493 at E7.75 and E8.25 showed normal MyoD expression in anterior pharyngeal arch mesoderm, as well as somites and their derivatives; in contrast, the accumulation of MyoD transcripts in posterior pharyngeal mesoderm was severely reduced compared with control (Fig. 2B). MyoD labelling of the hypoglossal cord, derived from the most anterior somites, was also reduced, consistent with hypoplasia of the posterior pharyngeal region following BMS493 treatment (Wendling et al., 2000). Similarly to the range of phenotypes observed at foetal stages, we observed variability in the extent of MyoD activation in the posterior pharyngeal region, differing between the left and right sides of individual embryos (Fig. 2C). Expression of Myf5 was almost undetectable at this stage in posterior pharyngeal arches in both control and BMS493-treated embryos, indicating earlier activation of MyoD than Myf5 in this region and reflecting divergence of the myogenic programmes operating in anterior and posterior pharyngeal arches (Fig. S4A-C). We further investigated Myf5 expression at E11.5 by visualizing β-galactosidase activity in embryos carrying a Myf5-nlacZ transgene (y96-Myf5-nlacZ-16), which revealed a highly selective reduction of transgene expression in the trapezius primordium in BMS493-treated embryos (Fig. 2D,E). Asymmetric downregulation of Myf5 transgene expression was observed in the trapezius anlagen, consistent with the later asymmetric defects in trapezius muscles (Fig. 2F). A reduction of Mef2c-AHF-Cre lineage-labelled cells was also observed in the emerging trapezius primordium at E11.5 after BMS493 treatment (Fig. S4D).

Fig. 2.

Myogenic determination factor gene expression in embryos exposed to BMS493 during early development. (A-F) Lateral views of in situ hybridization for MyoD at E10.5 (A-C) and X-gal-stained embryos carrying a Myf5-nlacZ transgene at E11.5 (D-F). Embryos exposed to vehicle (A,D; n=8 and 6) or BMS493 at E7.75 and E8.25 (B,C and E,F; n=7 and 8) are shown. MyoD transcripts and X-gal-stained cells accumulate in the myotomal compartment of somites (my), hypoglossal cord (hc), forelimb (fl) and mesodermal core of pharyngeal arches (pa) 1, 2 and 3-6. Note the selective failure of myogenesis in the region of arches 3-6 (black arrowheads) that contains the trapezius anlagen (trap) following BMS493 exposure. C and F show left-right asymmetric distribution of hypoplastic muscle progenitor cells in the posterior pharyngeal region (arrowheads).

Fig. 2.

Myogenic determination factor gene expression in embryos exposed to BMS493 during early development. (A-F) Lateral views of in situ hybridization for MyoD at E10.5 (A-C) and X-gal-stained embryos carrying a Myf5-nlacZ transgene at E11.5 (D-F). Embryos exposed to vehicle (A,D; n=8 and 6) or BMS493 at E7.75 and E8.25 (B,C and E,F; n=7 and 8) are shown. MyoD transcripts and X-gal-stained cells accumulate in the myotomal compartment of somites (my), hypoglossal cord (hc), forelimb (fl) and mesodermal core of pharyngeal arches (pa) 1, 2 and 3-6. Note the selective failure of myogenesis in the region of arches 3-6 (black arrowheads) that contains the trapezius anlagen (trap) following BMS493 exposure. C and F show left-right asymmetric distribution of hypoplastic muscle progenitor cells in the posterior pharyngeal region (arrowheads).

Given that MyoD is activated in the trapezius muscle primordium 48 h after the time of RA sensitivity, we investigated the impact of RA inhibition on upstream regulators of the branchiomeric myogenic programme. In situ hybridization revealed that Tbx1 and Tcf21, encoding upstream transcriptional regulators of branchiomeric myogenesis, were expressed in the posterior pharyngeal region at E9.5 following BMS493 treatment at E7.75 and E8.25 (Fig. 3A,B). Immunofluorescence confirmed that TBX1 was expressed in posterior pharyngeal mesoderm after RA inhibition (Fig. 3C). In contrast, the TBX1-positive fourth pharyngeal pouch was not observed, consistent with abnormal specification of caudal pharyngeal endoderm (Fig. 3C) (Wendling et al., 2000). ISL1, another upstream regulator of CPM, was also expressed in posterior pharyngeal mesoderm after BMS493 treatment (Fig. 3D). Together, these observations indicate that RA signalling impacts on posterior pharyngeal myogenesis after the activation of Tbx1, Isl1 and Tcf21 and before robust MyoD and Myf5 gene expression.

Fig. 3.

Expression of pharyngeal mesodermal regulatory genes in embryos exposed to BMS493 during early development. (A,B) Lateral views of embryos after in situ hybridization for Tbx1 (A) and Tcf21 (B) at E9.5. Tbx1 is expressed in mesoderm at the level of pharyngeal arches (pa) 1, 2 and 3-6 in embryos exposed to vehicle (n=9) and BMS493 at E7.75 and E8.25 (n=10). Tcf21 transcripts accumulate in pharyngeal mesoderm including posterior arches at E10.5 after vehicle (n=5) and BMS493 exposure at E7.75 and E8.25 (n=3). (C,D) Immunofluorescence for TBX1 (C) and ISL1 (D) on transverse sections at E9.5 showing accumulation in pharyngeal mesoderm in the region of the trapezius anlagen (arrowheads) in control (n=6) and BMS493-treated embryos (n=7); note the absence of the TBX1-positive fourth pharyngeal pouch (pp4) in embryos exposed to BMS493. acv, anterior cardinal vein; da, dorsal aorta; mn, motoneurons; pa, pharyngeal arch; scl, sclerotome; vp, venous pole. Scale bar: 100 µm.

Fig. 3.

Expression of pharyngeal mesodermal regulatory genes in embryos exposed to BMS493 during early development. (A,B) Lateral views of embryos after in situ hybridization for Tbx1 (A) and Tcf21 (B) at E9.5. Tbx1 is expressed in mesoderm at the level of pharyngeal arches (pa) 1, 2 and 3-6 in embryos exposed to vehicle (n=9) and BMS493 at E7.75 and E8.25 (n=10). Tcf21 transcripts accumulate in pharyngeal mesoderm including posterior arches at E10.5 after vehicle (n=5) and BMS493 exposure at E7.75 and E8.25 (n=3). (C,D) Immunofluorescence for TBX1 (C) and ISL1 (D) on transverse sections at E9.5 showing accumulation in pharyngeal mesoderm in the region of the trapezius anlagen (arrowheads) in control (n=6) and BMS493-treated embryos (n=7); note the absence of the TBX1-positive fourth pharyngeal pouch (pp4) in embryos exposed to BMS493. acv, anterior cardinal vein; da, dorsal aorta; mn, motoneurons; pa, pharyngeal arch; scl, sclerotome; vp, venous pole. Scale bar: 100 µm.

Retinoic acid signalling is not required in the Mef2c-AHF-Cre lineage for trapezius muscle development

To complement our pharmacological approach, we used mouse genetics to downregulate RA signal reception selectively in CPM. RARa403 is a dominant-negative version of the retinoic acid receptor alpha, with a C-terminal truncation impairing ligand dependent activation (Damm et al., 1993). A RARa403 cDNA placed after a floxed transcriptional stop sequence at the Rosa26 locus allows Cre-dependent reduction of RA signal reception in specific genetic lineages (Rosselot et al., 2010). The Mef2c-AHF-Cre transgene, expressed from E7.5, was used to conditionally activate RARa403 in CPM, including the trapezius primordium (Fig. 4A) (Lescroart et al., 2015; Verzi et al., 2005). MF20 immunofluorescence on E14.5 sections revealed that the trapezius muscle was present in Mef2c-AHF-Cre;RARa403 embryos and indistinguishable from that of control littermates (Fig. 4B,C). Although the trapezius muscle was unaffected, Mef2c-AHF-Cre;RARa403 embryos displayed defects in septation of the cardiac outflow tract, another CPM derivative (Fig. 4D). Similarly, MyoD in situ hybridization at E12.5 revealed normal development of the trapezius anlagen in Mef2c-AHF-Cre;RARa403 embryos (Fig. S5). Downregulation of RA signal reception in the Mef2c-AHF-Cre lineage thus leads to failure of cardiac outflow tract septation, but does not result in defects in trapezius muscle development.

Fig. 4.

The trapezius muscle forms normally after conditional activation of a dominant-negative RARa transgene in pharyngeal mesoderm. (A) Genetic cross showing Mef2c-AHF-Cre activation of the conditional RARa403 dominant-negative transgene at the ROSA26 locus. (B-D) Immunostaining of MF20 on transverse sections at the level of the acromiotrapezius (B), spinotrapezius (C) and heart (D) from control (left) and Mef2c-AHF-Cre;RARa403 embryos (right). Note the normal development of the trapezius muscle in the Mef2c-AHF-Cre;RARa403 embryo (arrowheads) despite failure of outflow tract division into the ascending aorta and pulmonary trunk, leading to a common arterial trunk. acr, acromion; ao, ascending aorta; cat, common arterial trunk; epm, epaxial muscle; pt, pulmonary trunk; rm, rhomboid major muscle; rv, right ventricle; sa, serratus anterior muscle; scp, scapula; ss, supraspinatus muscle; tm, teres major muscle. Scale bars: 500 µm.

Fig. 4.

The trapezius muscle forms normally after conditional activation of a dominant-negative RARa transgene in pharyngeal mesoderm. (A) Genetic cross showing Mef2c-AHF-Cre activation of the conditional RARa403 dominant-negative transgene at the ROSA26 locus. (B-D) Immunostaining of MF20 on transverse sections at the level of the acromiotrapezius (B), spinotrapezius (C) and heart (D) from control (left) and Mef2c-AHF-Cre;RARa403 embryos (right). Note the normal development of the trapezius muscle in the Mef2c-AHF-Cre;RARa403 embryo (arrowheads) despite failure of outflow tract division into the ascending aorta and pulmonary trunk, leading to a common arterial trunk. acr, acromion; ao, ascending aorta; cat, common arterial trunk; epm, epaxial muscle; pt, pulmonary trunk; rm, rhomboid major muscle; rv, right ventricle; sa, serratus anterior muscle; scp, scapula; ss, supraspinatus muscle; tm, teres major muscle. Scale bars: 500 µm.

Cells responding to retinoic acid signalling during the early developmental time window contribute to cells intercalated between trapezius muscle fibres

The above results indicate that, although RA signalling is required for trapezius muscle formation in an early developmental time window, RA signal reception is not required in the Mef2c-AHF-Cre lineage. To identify the later fate of cells responding to RA during the time window of RA sensitivity, we carried out genetic lineage tracing using a tamoxifen-inducible Cre transgene activated by a retinoic acid response element (RARE-CreERT2), with Rosa26-mTmG and Rosa26-tdTomato conditional reporter lines (Comai et al., 2020; Da Silva et al., 2021). Pregnant females were injected with tamoxifen at E7.5 to induce RARE-driven Cre activity during the early time window and embryos analysed at E14.5 (Fig. 5A). Tamoxifen injection at E7.5 resulted in labelling of posterior regions of the embryo, including mosaic labelling of surface ectoderm, but not craniofacial regions (Fig. 5B). Both cervical and thoracic elements of the trapezius muscle appeared to be labelled by GFP (Fig. 5B). However, immunofluorescence using GFP and MF20 antibodies revealed minimal labelling of muscle fibres in the developing trapezius; in contrast, GFP was observed in MF20-negative cells intercalated between muscle fibres (Fig. 5C,D). Immunofluorescence with markers of muscle fibres (MF20), connective tissue (TCF4), endothelial cells (PECAM-1) and neuronal cells (TUJ-1; TUBB3) was performed to determine the cell-type identity of these intercalated cells. We observed expression of a conditional tdTomato reporter gene in a subset of TCF4-positive and a smaller fraction of PECAM-1-positive cells within the trapezius muscle following induction of RARE-driven CreERT2 activity at E7.5 (Fig. 5E, Fig. S6A). Cells responding to RA signalling during the early time window therefore give rise to cells of different identities, predominantly connective tissue and endothelial cells, with minimal labelling of muscle fibres.

Fig. 5.

Genetic lineage tracing of cells that respond to retinoic acid signalling during the early developmental time window reveals non-myogenic contributions to the trapezius muscle. (A) Experimental strategy used to genetically label RA-responding cells. Nuclear localization of CRE encoded by RARE-CreERT2 targets recombination of mTmG to activate a GFP reporter following tamoxifen injection at E7.5; embryos were analysed at E14.5. pCA, CMV enhancer and chicken β-actin core promoter. (B) Lateral view of a RARE-CreERT2;mTmG embryo at E14.5 showing GFP labelling in surface ectoderm and the region of the developing acromiotrapezius and spinotrapezius muscles (n=10). Dotted lines indicate section levels for C and D. (C,D) MF20 and GFP immunostaining on sections of E14.5 RARE-CreERT2;mTmG embryos at the level of the acromiotrapezius (C) and spinotrapezius (D) muscles. The boxes highlight the areas magnified in panels to the right. Note that GFP-labelled cells (arrowheads) colocalize predominantly with cells intercalated between MF20-positive muscle fibres. (E) Immunostaining for tdTomato (green), MF20 (red) and TCF4 or PECAM-1 (blue) in sections from the spinotrapezius muscle of E14.5 RARE-CreERT2;tdTomato embryos exposed to tamoxifen at E7.5 (n=3). Examples of tdTomato-positive cells co-labelled with TCF4 or PECAM-1 are indicated by filled or empty white arrows, respectively, and lineage-only labelled cells by white arrowheads. (F) Immunostaining for YFP (green), MF20 (red) and TCF4 or PECAM-1 (blue) in the spinotrapezius muscle of E14.5 Pax3-Cre;RYFP embryos (n=4). The Pax3-Cre genetic lineage does not label trapezius MF20-positive myofibres but contributes to cells intercalated between muscle fibres. Examples of YFP-positive cells co-labelled with TCF4 or PECAM-1 are indicated by filled or empty white arrows, respectively, and lineage-only labelled cells by white arrowheads. acr, acromion; atrap, acromiotrapezius; scp, scapula; strap, spinotrapezius. Scale bars: 100 µm (C,D); 50 µm (E,F).

Fig. 5.

Genetic lineage tracing of cells that respond to retinoic acid signalling during the early developmental time window reveals non-myogenic contributions to the trapezius muscle. (A) Experimental strategy used to genetically label RA-responding cells. Nuclear localization of CRE encoded by RARE-CreERT2 targets recombination of mTmG to activate a GFP reporter following tamoxifen injection at E7.5; embryos were analysed at E14.5. pCA, CMV enhancer and chicken β-actin core promoter. (B) Lateral view of a RARE-CreERT2;mTmG embryo at E14.5 showing GFP labelling in surface ectoderm and the region of the developing acromiotrapezius and spinotrapezius muscles (n=10). Dotted lines indicate section levels for C and D. (C,D) MF20 and GFP immunostaining on sections of E14.5 RARE-CreERT2;mTmG embryos at the level of the acromiotrapezius (C) and spinotrapezius (D) muscles. The boxes highlight the areas magnified in panels to the right. Note that GFP-labelled cells (arrowheads) colocalize predominantly with cells intercalated between MF20-positive muscle fibres. (E) Immunostaining for tdTomato (green), MF20 (red) and TCF4 or PECAM-1 (blue) in sections from the spinotrapezius muscle of E14.5 RARE-CreERT2;tdTomato embryos exposed to tamoxifen at E7.5 (n=3). Examples of tdTomato-positive cells co-labelled with TCF4 or PECAM-1 are indicated by filled or empty white arrows, respectively, and lineage-only labelled cells by white arrowheads. (F) Immunostaining for YFP (green), MF20 (red) and TCF4 or PECAM-1 (blue) in the spinotrapezius muscle of E14.5 Pax3-Cre;RYFP embryos (n=4). The Pax3-Cre genetic lineage does not label trapezius MF20-positive myofibres but contributes to cells intercalated between muscle fibres. Examples of YFP-positive cells co-labelled with TCF4 or PECAM-1 are indicated by filled or empty white arrows, respectively, and lineage-only labelled cells by white arrowheads. acr, acromion; atrap, acromiotrapezius; scp, scapula; strap, spinotrapezius. Scale bars: 100 µm (C,D); 50 µm (E,F).

Similarities between RA-responding and Pax3-Cre genetic lineage contributions to non-myogenic cells of the developing trapezius muscle

Genetic lineage analysis has shown that Pax3 is expressed in progenitor cells of somite-derived muscles but not branchiomeric muscles (Heude et al., 2018; Lescroart et al., 2015). Although trapezius muscle fibres are unlabelled by the Pax3-Cre genetic lineage, non-myogenic lineage labelled cells have been observed between unlabelled muscle fibres (Theis et al., 2010). We used Pax3-Cre to show, consistent with previous results, that whereas the Pax3-Cre lineage contributes to myofibers in surrounding somite-derived skeletal muscles, trapezius muscle fibres are unlabelled (Fig. 5F, Figs S6B and S7A). Moreover, within the trapezius muscle the Pax3-Cre lineage contributes to intercalated cells, showing a similar contribution to that of early RA-responding cells after tamoxifen injection at E7.5 (Fig. 5C-F). Pax3 is expressed in somitic and neural crest cell lineages (Buckingham and Relaix, 2007; Epstein, 2000); the somitic contribution was deduced by comparison with the Wnt1-Cre genetic lineage, which labels neural crest cells and their descendants (Danielian et al., 1998). Similarly to cells responding to RA in the early embryo, the contribution of the Pax3-Cre lineage to the developing trapezius included subsets of TCF4- and PECAM-1-positive cells; in contrast, the Wnt1-Cre lineage contribution is minor and only labelled a small number of TCF4 positive cells in the spinotrapezius muscle (Fig. 5F, Fig. S7). We also investigated Pax3-Cre lineage contributions in BMS493-treated embryos and observed lineage-labelled, TCF4-positive interstitial cells associated with hypoplastic trapezius muscles (Fig. S8). These results suggest that cells in the somitic Pax3-Cre lineage respond to RA during the early time window and contribute to connective tissue and endothelial cells associated with the trapezius muscle.

Retinoic acid signalling is required in the somitic but not the neural crest lineage for trapezius muscle development

We next investigated whether activation of RARa403 using Pax3-Cre impacts trapezius muscle development. We also analysed Wnt1-Cre;RARa403 embryos, in which RA signal reception is downregulated in the neural crest but not somitic lineage. Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos were collected at E12.5 and myogenesis was evaluated by MyoD in situ hybridization. In Pax3-Cre;RARa403 embryos, normal expression of MyoD transcripts was observed in forming somite-derived trunk and limb muscles, including epaxial back muscles, as well as in branchiomeric muscles derived from the first and second pharyngeal arches (Fig. 6A,B). The trapezius primordium, however, was absent or extremely hypoplastic (Fig. 6A,B). An ectopic or residual site of MyoD expression was observed in the neck region corresponding approximately to the location of the acromiotrapezius primordium in control embryos (Fig. 6B). In Wnt1-Cre;RARa403 embryos, MyoD in situ hybridization at E12.5 showed a similar expression pattern to that of control embryos, indicating normal trapezius development (Fig. 6C). Histological analysis confirmed the absence of the trapezius anlagen in Pax3-Cre;RARa403 but not Wnt1-Cre;RARa403 embryos (Fig. 7A). Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos die by E13.5, precluding analysis of the trapezius muscle at foetal stages. Cardiac outflow tract septation, a process driven by neural crest cell influx, failed in both Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos (Fig. 7B). A severe midfacial cleft was also observed in both genotypes (Fig. 7C). In addition, MyoD in situ hybridization revealed defects in extraocular muscle development, including the failure of inferior oblique muscle formation, in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos (Fig. 7D). This phenotype resembles that following inactivation of RARβ and RARγ in neural crest cells, further supporting a role of RA signalling in neural crest-derived mesenchyme adjacent to myogenic progenitor cells for extraocular muscle anlagen patterning and splitting (Comai et al., 2020). Together, these findings demonstrate common phenotypes caused by downregulation of RA signalling in neural crest cells in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos, whereas trapezius development is perturbed only in Pax3-Cre;RARa403 embryos. This suggests that RA signalling through Pax3 lineage-positive somitic mesoderm regulates development of the CPM-derived trapezius muscle, likely during the early time window of BMS493 sensitivity.

Fig. 6.

The trapezius muscle fails to develop after conditional activation of a dominant-negative RARa transgene in the Pax3-Cre, but not Wnt1-Cre, genetic lineage. (A-C) Control (A; n=16), Pax3-Cre;RARa403 (B; n=8) and Wnt1-Cre;RARa403 (C; n=7) E12.5 embryos after MyoD in situ hybridization, showing embryonic muscle anlagen. High magnification of left and right lateral views (central panels) and dorsal views (right panels) are shown. The trapezius primordium (black arrowheads) is evident in control and Wnt1-Cre;RARa403 embryos but is absent in Pax3-Cre;RARa403 embryos. In the dorsal view, the loss of trapezius muscle and persistence of an epaxial somite-derived back muscle (arrows) is apparent. Note an ectopic spot of MyoD expression (white arrowheads) in the neck region anterior to the forelimb (fl). MyoD is expressed normally in muscle masses derived from the first (1) and second (2) arches, respectively, in embryos of all three genotypes; similarly, the primordia of other muscles are unaffected, including the deltoid (d), cutaneous maximus (cm) and epaxial back muscles (e).

Fig. 6.

The trapezius muscle fails to develop after conditional activation of a dominant-negative RARa transgene in the Pax3-Cre, but not Wnt1-Cre, genetic lineage. (A-C) Control (A; n=16), Pax3-Cre;RARa403 (B; n=8) and Wnt1-Cre;RARa403 (C; n=7) E12.5 embryos after MyoD in situ hybridization, showing embryonic muscle anlagen. High magnification of left and right lateral views (central panels) and dorsal views (right panels) are shown. The trapezius primordium (black arrowheads) is evident in control and Wnt1-Cre;RARa403 embryos but is absent in Pax3-Cre;RARa403 embryos. In the dorsal view, the loss of trapezius muscle and persistence of an epaxial somite-derived back muscle (arrows) is apparent. Note an ectopic spot of MyoD expression (white arrowheads) in the neck region anterior to the forelimb (fl). MyoD is expressed normally in muscle masses derived from the first (1) and second (2) arches, respectively, in embryos of all three genotypes; similarly, the primordia of other muscles are unaffected, including the deltoid (d), cutaneous maximus (cm) and epaxial back muscles (e).

Fig. 7.

Myogenic, cardiac and craniofacial defects following conditional activation of a dominant-negative RARa transgene in the Pax3-Cre and Wnt1-Cre genetic lineages. (A,B) Haematoxylin and Eosin-stained transverse sections at E12.5 of the shoulder (A) and cardiac outlet region (B) showing loss of trapezius anlagen (black arrowheads) in Pax3-Cre;RARa403 embryos and failure to divide the outflow tract into the ascending aorta and pulmonary trunk leading to a common arterial trunk in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos. (C) Whole-mount frontal views of the head region showing a midfacial cleft (black arrowheads) in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos and exencephaly (white arrowhead) in Pax3-Cre;RARa403 embryos. (D) Lateral views of the head region following MyoD in situ hybridization at E11.5 reveals defects in extraocular muscle development (white arrows) in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos; the inferior rectus muscle primordium (black arrow) is present only in control embryos. ao, aorta; cat, common arterial trunk; e, eye; m, masseter muscle anlage; pt, pulmonary trunk; scp, scapula. Scale bars: 200 µm (A,B).

Fig. 7.

Myogenic, cardiac and craniofacial defects following conditional activation of a dominant-negative RARa transgene in the Pax3-Cre and Wnt1-Cre genetic lineages. (A,B) Haematoxylin and Eosin-stained transverse sections at E12.5 of the shoulder (A) and cardiac outlet region (B) showing loss of trapezius anlagen (black arrowheads) in Pax3-Cre;RARa403 embryos and failure to divide the outflow tract into the ascending aorta and pulmonary trunk leading to a common arterial trunk in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos. (C) Whole-mount frontal views of the head region showing a midfacial cleft (black arrowheads) in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos and exencephaly (white arrowhead) in Pax3-Cre;RARa403 embryos. (D) Lateral views of the head region following MyoD in situ hybridization at E11.5 reveals defects in extraocular muscle development (white arrows) in Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos; the inferior rectus muscle primordium (black arrow) is present only in control embryos. ao, aorta; cat, common arterial trunk; e, eye; m, masseter muscle anlage; pt, pulmonary trunk; scp, scapula. Scale bars: 200 µm (A,B).

Retinoic acid signalling is required in the Pax3 lineage for occipital somite development

The onset of branchiomeric myogenesis was assessed by MyoD in situ hybridization at E10.5. In contrast to sites of myogenesis in the anterior pharyngeal arches, myogenesis in the posterior pharyngeal arches was defective in Pax3-Cre;RARa403 embryos. MyoD-expressing cells were restricted to an intense spot in the posterior pharyngeal region that was more compact than the equivalent MyoD labelling in control embryos (Fig. 8A,B). At E11.5, this pool of myogenic progenitor cells failed to expand caudally compared with control embryos (Fig. S9). MyoD transcripts accumulated in the myotomal compartment of somites in Pax3-Cre;RARa403 embryos; however, expression was absent or severely reduced in the anterior-most occipital somites (Fig. 8B). Moreover, the hypoglossal cord was not detected in Pax3-Cre;RARa403 embryos (Fig. 8B). This structure comprises myogenic progenitor cells derived from the hypaxial region of occipital somites and its absence is consistent with defective occipital somite development. In contrast, in Wnt1-Cre;RARα403 embryos both branchiomeric and somitic myogenesis were indistinguishable from the situation in control embryos (Fig. 8C). In particular, development of the trapezius anlagen, hypoglossal cord and occipital somites was unaffected (Fig. 8C), suggesting that the Pax3-Cre;RARa403 muscle phenotype results from reduced RA signal reception in the somitic, rather than the neural crest cell lineage.

Fig. 8.

Defects in anterior somite development co-occur with abnormal development of the trapezius primordium on dominant-negative RARa activation in the Pax3 genetic lineage. (A-C) Control (C; n=32), Pax3-Cre;RARa403 (B; n=12) and Wnt1-Cre;RARa403 (C; n=6) E10.5 embryos after MyoD whole-mount in situ hybridization. Magnified views of the left (middle) and right (right) sides at the head trunk interface are shown. MyoD is expressed in the myotomal compartment of somites (numbered), hypoglossal cord, forelimb muscle mass, and mesodermal core of pharyngeal arches 1, 2 and 3-6. In Pax3-Cre;RARa403 embryos, MyoD activation in arches 3-6 remains as an intense spot of labelling (arrowheads) that fails to extend posteriorly; note the reduction in the number of somites anterior to the forelimb. Absence of the hypoglossal cord is consistent with defective occipital somite development. In contrast, myogenesis in Wnt1-Cre;RARa403 embryos proceeds normally and the distribution of MyoD transcripts is equivalent to that in control embryos. fl, forelimb muscle mass; h, heart; hc, hypoglossal cord; pa, pharyngeal arch.

Fig. 8.

Defects in anterior somite development co-occur with abnormal development of the trapezius primordium on dominant-negative RARa activation in the Pax3 genetic lineage. (A-C) Control (C; n=32), Pax3-Cre;RARa403 (B; n=12) and Wnt1-Cre;RARa403 (C; n=6) E10.5 embryos after MyoD whole-mount in situ hybridization. Magnified views of the left (middle) and right (right) sides at the head trunk interface are shown. MyoD is expressed in the myotomal compartment of somites (numbered), hypoglossal cord, forelimb muscle mass, and mesodermal core of pharyngeal arches 1, 2 and 3-6. In Pax3-Cre;RARa403 embryos, MyoD activation in arches 3-6 remains as an intense spot of labelling (arrowheads) that fails to extend posteriorly; note the reduction in the number of somites anterior to the forelimb. Absence of the hypoglossal cord is consistent with defective occipital somite development. In contrast, myogenesis in Wnt1-Cre;RARa403 embryos proceeds normally and the distribution of MyoD transcripts is equivalent to that in control embryos. fl, forelimb muscle mass; h, heart; hc, hypoglossal cord; pa, pharyngeal arch.

Juxtaposition of posterior cardiopharyngeal mesoderm with anterior somites

The distribution of Pax3 transcripts at the time of RA sensitivity for trapezius muscle development was investigated by RNAscope fluorescent in situ hybridization between E7.75 and E8.75. The expression profile of Pax3 was compared with that of Tbx1, which is expressed in CPM including trapezius progenitor cells (Fig. S10) and is required for trapezius muscle development (Huynh et al., 2007; Kelly et al., 2004; Theis et al., 2010). Consistent with published results, Pax3 transcripts were observed in the neural plate, dorsal neural tube, migrating neural crest-derived mesenchyme, and somitic mesoderm (Fig. 9). Our results revealed a boundary along the anterior-posterior axis between Pax3-positive somitic paraxial mesoderm and Tbx1-positive cranial mesoderm at E7.75 (Fig. 9A). Mesoderm immediately lateral to the most anterior, or occipital, somites was positive for Tbx1 (Fig. 9A-C). This region of the mesoderm has been shown to contain trapezius progenitor cells (Theis et al., 2010). Pax3-expressing cells, in which RA signalling is required for trapezius muscle development, are thus juxtaposed with Tbx1-expressing mesodermal cells during the time window of BMS493 sensitivity. Investigation of the distribution of Pax3 and Tbx1 transcripts in E8.5 Pax3-Cre;RARa403 and Wnt1-Cre;RARa403 embryos revealed that Tbx1 transcript accumulation was indistinguishable from that in wild type. However, reduced Pax3 expression revealed failure of normal development of the occipital somites in Pax3-Cre;RARa403, but not Wnt1-Cre;RARa403 embryos. Whole-mount in situ hybridization of Tbx1 at E8.5 and Tcf21 at E9.5 confirmed normal expression of these CPM genes in Pax3-Cre;RARa403 embryos, and analysis of the somitic marker Tcf15 (also known as paraxis) revealed failure of anterior somite development (Fig. S11). The juxtaposition between occipital somites and trapezius progenitor cells in posterior CPM is thus disrupted in Pax3-Cre;RARa403 embryos (Fig. 9D). Together, these results suggest that RA signal reception in occipital somites is required for normal development of the trapezius muscle primordium in adjacent CPM.

Fig. 9.

Pax3­- and Tbx1-expressing cells are juxtaposed in the early mouse embryo. (A-C) Fluorescent RNAscope in situ hybridization showing Tbx1 (green) and Pax3 (red) transcript distribution at E7.75 (A; n=4), E8.25 (B; n=10) and E8.75 (C; n=7). At E7.75, Tbx1 transcripts are initially observed in the cranial mesoderm rostral to Pax3-positive somitic mesoderm. Pax3 is also expressed in the neural plate. Optical section showing low level Tbx1 expression in mesoderm lateral to Pax3-positive cells in the first somites. At E8.25 and E8.75, Tbx1-positive pharyngeal mesoderm is observed in pharyngeal arches 1 and 2 and the posterior pharyngeal region (arrowheads), including mesoderm lateral to the most anterior (occipital) somites; Pax3 is expressed in the neural tube and somitic mesoderm. (D) Comparison of Pax3 and Tbx1 expression at E8.75 in control (top; n=3), Pax3-Cre;RARa403 (middle; n=17) and Wnt1-Cre;RARa403 (bottom; n=5) embryos. In Pax3-Cre;RARa403 embryos, the posterior expansion of Tbx1-positive pharyngeal mesoderm appears normal (arrowheads), whereas the adjacent Pax3 labelling is reduced and the most anterior somites (1 and 2) are not apparent. Pax3 expression appears normal in Wnt1-Cre;RARa403 embryos at E8.75. cm, cranial mesoderm; hf, head fold. lm, mesoderm lateral to the first somites; np, neural plate; nt, neural tube; pa, pharyngeal arch; s, somites; sm, somitic mesoderm. Scale bars: 50 μm.

Fig. 9.

Pax3­- and Tbx1-expressing cells are juxtaposed in the early mouse embryo. (A-C) Fluorescent RNAscope in situ hybridization showing Tbx1 (green) and Pax3 (red) transcript distribution at E7.75 (A; n=4), E8.25 (B; n=10) and E8.75 (C; n=7). At E7.75, Tbx1 transcripts are initially observed in the cranial mesoderm rostral to Pax3-positive somitic mesoderm. Pax3 is also expressed in the neural plate. Optical section showing low level Tbx1 expression in mesoderm lateral to Pax3-positive cells in the first somites. At E8.25 and E8.75, Tbx1-positive pharyngeal mesoderm is observed in pharyngeal arches 1 and 2 and the posterior pharyngeal region (arrowheads), including mesoderm lateral to the most anterior (occipital) somites; Pax3 is expressed in the neural tube and somitic mesoderm. (D) Comparison of Pax3 and Tbx1 expression at E8.75 in control (top; n=3), Pax3-Cre;RARa403 (middle; n=17) and Wnt1-Cre;RARa403 (bottom; n=5) embryos. In Pax3-Cre;RARa403 embryos, the posterior expansion of Tbx1-positive pharyngeal mesoderm appears normal (arrowheads), whereas the adjacent Pax3 labelling is reduced and the most anterior somites (1 and 2) are not apparent. Pax3 expression appears normal in Wnt1-Cre;RARa403 embryos at E8.75. cm, cranial mesoderm; hf, head fold. lm, mesoderm lateral to the first somites; np, neural plate; nt, neural tube; pa, pharyngeal arch; s, somites; sm, somitic mesoderm. Scale bars: 50 μm.

RA signalling is required during E8 to pattern the posterior pharyngeal region (Mark et al., 2004; Wendling et al., 2000). Reduced RA signalling during this time window leads to failure to upregulate Tbx5 in posterior CPM, resulting in abnormal SHF addition to the venous pole of the heart and atrial septal defects (De Bono et al., 2018). Here, we show that formation of a posterior CPM-derived skeletal muscle is also RA dependent. Indeed, perturbing RA signalling has a strikingly selective effect on the trapezius, whereas the primordia of muscles from anterior arches, as well as somite-derived muscles, develop normally. Recent evidence that elevated RA exposure leads to reduced survival of branchiomeric muscle progenitor cells in anterior arches is consistent with a requirement for RA signalling in posterior, but not anterior, CPM (Wang et al., 2021). Our results reveal that RA signalling impacts MRF gene expression after activation of the early CPM regulators TBX1, ISL1 and TCF21, and that activation of Myf5 is delayed compared with MyoD in posterior CPM, reinforcing findings based on genetic lineage tracing that specific regulatory pathways control muscle development in the neck region (Heude et al., 2018). The presence of asymmetric hypoplastic trapezius muscles after BMS493 exposure is reminiscent of the stochastic branchiomeric muscle formation seen in Tbx1 null embryos (Kelly et al., 2004; Sambasivan et al., 2009), suggesting that RA signalling is required for robust bilateral activation of MRF expression in posterior CPM. Moreover, the posterior CPM specific programme appears to be evolutionarily conserved, as zebrafish raldh2 mutant (neckless) and tbx1 mutant (van gogh) embryos exhibit defective gill muscle development (Begemann et al., 2001; Piotrowski and Nüsslein-Volhard, 2000). The trapezius muscle and atrial septum are synapomorphies of jawed vertebrates and lobe-finned fishes, respectively (Ericsson et al., 2013; Jensen et al., 2019; Kuratani, 2008; Trinajstic et al., 2013), and the integration of RA dependence in the CPM genetic programme may have contributed to the evolution of these structures.

Although RA signalling has been shown to play multiple roles during myogenesis, our data suggest that the effect of RA signalling on trapezius muscle development is indirect. Genetic lineage tracing revealed that cells responding to RA during the E8 time window do not contribute significantly to trapezius muscle fibres, suggesting that RA signalling affects trapezius development indirectly through other cell types. Earlier work has pointed to the importance of RA-dependent patterning of pharyngeal endoderm in caudal pharyngeal development (Wendling et al., 2000). However, our results suggest that cells responding to RA at this early time point are progenitors of cells that later intercalate between CPM-derived trapezius muscle fibres. Non-myogenic cells, including muscle connective tissue and vasculature, play essential roles in muscle migration, patterning and attachment (Mayeuf-Louchart et al., 2016; Nassari et al., 2017; Rinon et al., 2007; Tozer et al., 2007). Recent work has shown that RA signalling to neural crest-derived mesenchyme is required for extraocular muscle development (Comai et al., 2020). During branchiomeric myogenesis, neural crest-derived cells are required after the onset of MRF activation for muscle patterning, migration and differentiation (Rinon et al., 2007). CPM also contributes to muscle connective tissue in branchiomeric and somite-derived neck muscles and plays a role in muscle patterning (Adachi et al., 2020). In the case of the trapezius muscle, genetic and experimental lineage studies have shown that both neural crest cells and lateral plate mesoderm contribute to intercalated non-myogenic cells (Adachi et al., 2020; Durland et al., 2008; Heude et al., 2018; Matsuoka et al., 2005; Noden, 1983; Theis et al., 2010). Our analysis shows that the Pax3-Cre genetic lineage makes predominant contributions to non-myogenic intercalated cells in the developing trapezius, in particular to the spinotrapezius muscle, which extends deep into the thoracic region. As this contribution appears more extensive than that made by the Wnt1-Cre lineage, we conclude that Pax3-expressing cells in somitic mesoderm are a major source of intercalated cells in the spinotrapezius muscle. This reveals a previously unrecognized combination of myofibre and connective tissue sources likely to be important for trapezius muscle development and patterning.

Further support for an indirect effect of RA signalling was provided by the observation that trapezius development is normal on conditional activation of a dominant-negative RA receptor in CPM. The Mef2c-AHF-Cre transgene is active in CPM from E7.5, prior to the window of BMS493 sensitivity, and labels the trapezius primordium as it emerges in posterior CPM at E10.5 (Lescroart et al., 2015; Verzi et al., 2005; this study). Cardiac defects, including a common arterial trunk, were observed in Mef2c-AHF-Cre;RARa403 embryos, in agreement with prior evidence for cell-autonomous requirements for RA signalling in the SHF (Li et al., 2010). Moreover, consistent with the similar contributions of the RARE-CreERT2 and Pax3-Cre genetic lineages to intercalated cells in the developing trapezius, activation of RARa403 in the Pax3-Cre lineage led to a striking defect in trapezius muscle development. As in embryos exposed to BMS493 at E8, expression of Tbx1 and Tcf21 was observed in posterior CPM, but MyoD activation was abnormal and restricted to a small region from which cells failed to extend normally in a caudal direction. Although both pharmacological and genetic reduction of RA signalling have a selective effect on trapezius development, differences between the phenotypes may reflect the permanent reduction of RA signal reception in the Pax3-Cre lineage compared with the transient global reduction on BMS493 exposure, where endodermal patterning defects may also contribute to the phenotype (Wendling et al., 2000). We observed that Pax3-Cre and Wnt1-Cre activated RARa403 resulted in a midfacial cleft and common arterial trunk, presumably as a result of downregulation of RA signalling in neural crest cells. Of note, an earlier study found no cardiovascular defects when Wnt1-Cre was used to activate an independently generated RARa403 transgene (Li et al., 2012); the increased severity in our study may be due to structural differences in the two RARa403 alleles. We also observed extraocular muscle defects on activation of RARa403 in the Pax3-Cre and Wnt1-Cre lineages, reinforcing the conclusion that RA signalling to neural crest-derived periocular mesenchyme is essential for extraocular muscle patterning (Comai et al., 2020). However, normal development of the trapezius anlagen on RARa403 activation using Wnt1-Cre strongly suggests that the target of RA signalling required for trapezius development is somitic mesoderm, where Pax3, but not Wnt1, is expressed.

Comparison of Tbx1 and Pax3 transcript distribution at E8 revealed that these genes are expressed in closely apposed cell populations: Pax3 in somitic mesoderm and Tbx1 in mesoderm immediately lateral to the most anterior occipital somites. This domain of Tbx1 expression appears to be a posterior expansion of CPM that prefigures the later expansion of the trapezius primordium into the trunk region (Heude et al., 2018; Theis et al., 2010; this study). Thus, branchiomeric and somitic mesoderm are juxtaposed at the time that development of the trapezius primordium depends on RA signalling. Occipital somites are distinct from more posterior somites and give rise to the cranial occipital bone as well as migratory myogenic progenitor cells that leave the occipital somites to contribute to the hypoglossal cord and ultimately tongue and infrahyoid muscles. RA signalling has been previously implicated in anterior-posterior patterning of paraxial mesoderm and in segmentation of somitic mesoderm, notably playing roles in coordinating somite formation on the left and right sides of the embryo, as well as in sclerotome development (Maden et al., 2000; Vermot et al., 2005a; Wilson et al., 2009). In embryos in which RARa403 is activated in the Pax3-Cre genetic lineage, we observed a distinct somitic phenotype: a severe defect in formation or maintenance of the most anterior occipital somites and absence of the hypoglossal cord consequent to somite loss. Somitic mesoderm in Pax3-Cre;RARa403 embryos is thus not available to provide cells to the developing trapezius analgen. This cellular contribution is likely to be essential for posterior expansion of the trapezius anlagen that prefigures muscle attachment to cervical and thoracic vertebrae. Indeed, we show that the Pax3-Cre lineage contributes interstitial cells to hypoplastic trapezius muscles observed after BMS493 exposure. Theis et al. have proposed that Wnt signalling, which promotes somitic but inhibits branchiomeric myogenesis (Tzahor et al., 2003), may delay differentiation of the branchiomeric trapezius primordium after crossing the head/trunk interface, allowing its subsequent posterior extension (Theis et al., 2010). We suggest that, potentially in addition to this model, occipital somites also make a cellular contribution to the developing trapezius analgen that would promote this expansion, licensing and escorting trapezius growth into the trunk territory. Given that the interface between somitic and lateral plate mesoderm, known as the lateral somitic frontier, is a region of signal exchange (Burke and Nowicki, 2003), somite-derived signals, potentially RA dependent, may also be required for trapezius muscle expansion. Future experiments will define the RA-dependent downstream cellular and signalling contributions from somitic mesoderm that are necessary for normal trapezius development.

Emergence of the trapezius muscle in the tetrapod lineage has enabled coordinated movement of the head and trunk as well as shoulder mobility. Our results suggest that CPM derivatives as well as RA signalling have played major roles in coordinating tissue development at the head/trunk interface over vertebrate evolution and more generally illustrate how evolutionary novelty can arise at the boundary between different cell populations and embryonic environments (Adachi et al., 2018; Burke and Nowicki, 2003). Moreover, perturbation of such processes may be involved in the etiology of myopathies affecting individual muscles or muscle groups, suggesting that further understanding of cellular origins and tissue crosstalk will provide evolutionary and clinical insights into the vertebrate musculature.

Mice

The following mouse lines were used: Ai14 (tdTomato) (Madisen et al., 2010), Mef2c-AHF-Cre (Verzi et al., 2005), Pax3-Cre (Engleka et al., 2005), RARE-CreERT2 (Da Silva et al., 2021), Tbx1-Cre (Huynh et al., 2007), ROSA26-mTmG (Muzumdar et al., 2007), ROSA26-H2B-EGFP (Abe et al., 2011), ROSA26-RARa403 (Rosselot et al., 2010), ROSA26-lacZ (Soriano, 1999), ROSA26-YFP (Srinivas et al., 2001), Wnt1-Cre (Danielian et al., 1998) and y96-Myf5-nlacZ (Hadchouel et al., 2000). Mice were maintained on a mixed C57Bl/6 and CD1 background and genotyped by PCR. Noon of the day of the vaginal plug observation was defined as E0.5. Animal experiments were carried out in agreement with national and European law and approved by the Ethics Committee for Animal Experimentation of Marseilles and the French Ministry for National Education, Higher Education and Research and the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (G2023-038C5 and A2023-046C7). Authorisation for projects using animals for scientific purposes (APAFIS): #10266-2017061618121519 v3 and #44241-2023072110393026 v4.

Oral gavage and intraperitoneal injection experiments

For oral gavage, BMS493 (Bristol Myers Squibb; Sigma-Aldrich, B6688), a pan-retinoic acid receptor synthetic retinoid inverse agonist, was diluted in ethanol at 10−2 M and this solution was further diluted with sunflower oil (1:2.5 v/v) and administrated at 10 mg/kg weight to pregnant mice at times corresponding to E7.75 and E8.25. Ethanol was diluted with sunflower oil at the same concentration and administrated by oral gavage for control pregnant mice. For intraperitoneal injections, BMS493 was diluted in dimethyl sulfoxide (DMSO) at 10 mg/ml and then diluted four times in PBS and injected intraperitoneally at 5 or 10 mg/kg to pregnant mice at times corresponding to E7.75, E8.25 and E9.5 (De Bono et al., 2018). Control mice were injected with DMSO in PBS at the same dilution.

Tamoxifen injection

Tamoxifen (Sigma-Aldrich, T5648) was dissolved in 100% ethanol at 100 mg/ml, diluted in sunflower oil (Sigma-Aldrich) to 20 mg/ml and 100 μl was injected intraperitoneally to pregnant females at E7.5.

Histological sections

Embryos were dissected in 1× PBS at 4°C, fixed in 4% paraformaldehyde (PFA) for 1 h, and dehydrated prior to embedding in paraffin (Sigma-Aldrich, Paraplast X-tra, P3808). Sagittal or transverse sections (10 μm thick) were mounted on Superfrost Plus slides (Thermo Fisher Scientific) and stained in Haematoxylin and Eosin solutions (Merk Sigma-Aldrich, Mayer's Hematoxylin solution, MHS32 and Alcoholic Eosin Y solution, HT110132). Images were taken with a Zeiss AxioZoom V16 microscope equipped with an Axiocam 512 colour camera.

Whole-mount X-gal staining

Embryos were dissected in PBS, fixed in 4% PFA for 15 min at 4°C and washed in PBS. Embryos were incubated in X-gal staining solution [0.1% X-gal (Millipore, 4063-102), 2 mM MgCl2, 0.01% deoxycholate, 0.01% Nonidet P40, 5 mM potassium hexacyanoferrate (II) trihydrate (Sigma-Aldrich, P3289) and 5 mM potassium hexacyanoferrate (III) (Sigma-Aldrich, 244023)] at 37°C. After incubation, embryos were washed in PBS, refixed in 4% PFA for 1 h at 4°C, and images taken with a Zeiss microscope AxioZoom V16 and Axiocam 512 colour camera.

In situ hybridization

Embryos were dissected in 4°C DEPC (Sigma-Aldrich, D5758)-treated PBS and fixed in 4% PFA for 24-72 h at 4°C, then washed in PBS/DEPC and dehydrated in sequential ethanol baths (25%, 50%, 80% and 100%). Embryos were stored at −20°C in 100% ethanol for at least 24 h. After bleaching in 20% H2O2/ethanol solution for 1 h at room temperature, embryos were rehydrated in ethanol/PBS/Tween 20 (80%, 50% and 25% ethanol/PBS/DEPC water and 0.1% Tween 20) and washed in PBST (PBS with 0.1% Tween 20). Embryos were permeabilized with Proteinase K (Gibco fungal, 25530-03) treatment (duration adapted to the stage of the embryo) at room temperature and the treatment was stopped with glycine (Millipore, 1.04201.0100). Embryos were then postfixed in 4% PFA/0.2% glutaraldehyde (Appli Chem, A0589.0100) for 20 min, washed, and incubated in prehybridization solution [50% deionized formamide (Millipore, Omnipur, 4650-500ML), 5× SSC (Euromedex, EU0300-C), 50 µg/ml yeast total RNA (Sigma-Aldrich, R6625), 1% SDS (Euromedex, EU0660), 50 µg/ml heparin (Sigma-Aldrich, H3393-500KU)] for 2-3 h before hybridization with digoxygenin-labelled probe at 0.5 µg/ml overnight at 70°C. Embryos were washed in solution 1 at 70°C (50% formamide, 5× SSC and 1% SDS), solution 2 at 65°C (50% formamide and 2× SSC), mixed (1/1) solution 2 and solution 3 (0.5 M NaCl, 10 mM Tris-HCl pH 7.5 and 0.05% Tween 20) at 65°C, and solution 3 at room temperature. Embryos were incubated in 100 µg/ml RNase A (Sigma-Aldrich, R5500-100) in solution 3 for 30 min twice at 37°C, then washed in solution 3. After washing with TBST (140 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl pH 7.5 and 0.1% Tween 20), blocking was carried out in 10% sheep serum (Sigma-Aldrich, S2263) in TBST for 2 h at room temperature and then embryos were incubated in anti-digoxigenin antibody (Roche, anti-digoxigenin-AP fab fragment, 11093274910) at 4°C overnight. Embryos were repeatedly washed with TBST, and subsequently in NTMT (100 mM NaCl, 100 mM Tris-HCl pH 9.5, 50 mM MgCl2 and 1% Tween 20) for 20 min three times at room temperature, before being placed in BM Purple (Roche, 11442074001). Embryos were washed in TBST and then post-fixed in 4% PFA. Images were taken with a Zeiss AxioZoom V16 microscope and Axiocam 512 colour camera. The following riboprobes were used: MyoD, Myf5, Tcf21, Tbx1 (Kelly et al., 2004). A Tcf15 riboprobe was amplified by PCR from genomic DNA using the following primers: 5′ primer, GGGCAGCTGCTTGAAAGTGA; 3′ primer (incorporating a T7 RNA polymerase promoter), TAATACGACTCACTATAGGCCAGCTAGGCCTGGATGGC.

RNAscope assay

All the experiments were performed with the RNAscope multiplex fluorescent detection kit v2 (ACDBio, 322110). Embryos were dissected in 4°C DEPC (Sigma-Aldrich, D5758)-treated PBS and fixed in 4% PFA for 24-72 h at 4°C. Embryos were progressively dehydrated in sequential methanol baths (25%, 50%, 75% and 100%) and stored at −20°C in 100% methanol for at least 24 h. Embryos were rehydrated in methanol baths containing 0.1% Tween 20, permeabilized with the Protease Plus reagent (ACDBio, 322381), then washed in PBST (0.01% Tween 20) three times. Probes C1+C2 were prepared at a ratio of 50:1 and incubated at 40°C for 10 min. Embryos were then incubated with the probes mix overnight at 40°C. Embryos were washed in PBST/0.2% SSC at room temperature for 8 min, three times, and then fixed for 10 min in 4% PFA at room temperature. Embryos were further washed in PBST/0.2% SSC at room temperature for 8 min, three times. Amplification steps were then performed according to ACDBio instructions, using TSA-Cy3 and TSA-Cy5 (PerkinElmer, NE744B001KT). ACDBio probe references: Pax3 455801; Tbx1 481911. Embryos were imaged using a confocal microscope Zeiss LSM880 with Airyscan.

Immunofluorescence on paraffin sections

After dissection in PBS, embryos were fixed in 4% PFA for 1 h at 4°C and dehydrated prior to embedding in paraffin (Sigma-Aldrich, Paraplast X-tra P3808). Sagittal or transverse sections (10 μm thick) cut on a microtome (Leica, RM2255) were mounted on Superfrost Plus slides (Thermo Fisher Scientific). Sections were deparaffinized in xylene (Carlo Erba reagents, 492301), rehydrated and then boiled in Antigen Unmasking Solution (Vector Laboratories, H-3300) for 15 min. Slides were washed three times in PBST (0.05% Tween 20) and incubated in TNB blocking buffer (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl and 0.5% blocking reagent) for 1 h at room temperature prior to incubation with primary antibodies in TNB overnight at 4°C. Sections were then incubated with secondary antibodies and Hoechst 33258 (1/1000, Merck Sigma-Aldrich, 861405) for 1 h at room temperature. After washing three times in PBST, slides were mounted in Fluoromount-G (SouthernBiotech, 0100-01). For antibodies requiring amplification, endogenous peroxidase activity was quenched by 3% H2O2 treatment for 30 min prior to the blocking step, and the secondary antibody was biotinylated. Tyramide signal amplification (TSA) was performed using streptavidin-HRP (horseradish peroxidase) for 1 h at room temperature followed by incubation for 3-4 min in TSA-Fluorescein or TSA-Cy3 diluted in amplification reagent (PerkinElmer, TSA-plus Fluorescein system kit NEL701A001KT or Cyanine 3 systems kit NEL704A001KT). The following antibodies were used in this study: mouse anti-ISL1 [1/100, Developmental Studies Hybridoma Bank (DSHB), 39.4D5 and 40.2D6], chicken anti-GFP (1/500, Aves Labs, GFP-1020), mouse anti-MHC1 (1/100, MF20, DSHB), rabbit anti-TCF4 (1/200, Cell Signaling Technology, 2569), rabbit anti-TBX1 (1/100, LSBio, Ls-C31179), rat anti-PECAM-1 (1/500, BD Biosciences, BDB553370), mouse anti-β3-tubulin (1/500, Tuj1, BioLegend, 801202), Alexa Fluor 488 donkey anti-chicken (1/500, Jackson ImmunoResearch, 703545155), AlexaFluor 568 donkey anti-mouse (1/500, Life Technologies, A10037) and Alexa Fluor 647 donkey anti-rat (1/500, Jackson ImmunoResearch, 712605153). The TSA Plus system was used to detect TBX1. Sections were photographed using a Zeiss AxioZoom V16 microscope and Axiocam 512 colour camera, a Zeiss AxioImager Z1 Apotome1.2, or a Keyence BZ-X700 fluorescent microscope.

Immunofluorescence on cryosections

After dissection in PBS, embryos were fixed in 4% PFA for 1 h at 4°C and then rinsed three times in PBS. After 2 h progressive saccharose (Carl Roth, 4621.1) baths (10% and 15%), embryos were kept overnight at 4°C in 30% saccharose and then transferred to OCT (VWR, 361603E) for 3 h. Embryos were then embedded in fresh OCT in cryomolds on dry ice. Sections were made using a cryostat (Leica, CM3050 S), placed on Superfrost Plus slides (Thermo Fisher Scientific, 631-9483), and stored at −70°C. Slides were washed in PBST (0.05% Tween 20) three times for 5 min, then permeabilized in PBS/0.2% Triton X-100 (Euromedex, 2000-B) for 15 min and bleached in 3% H2O2/PBS for 10 min. The slides were then washed in PBST three times for 5 min each wash and then placed in a blocking solution [PBS with 2% bovine serum albumin (Sigma-Aldrich, A7030) and 0.05% Saponin (Millipore, 558255)] for 1 h. Slides were incubated with the primary antibodies diluted in the blocking solution overnight at 4°C. Slides were thoroughly rinsed in PBST and incubated with the secondary antibodies and Hoechst 33258 (1/1000, Merck Sigma-Aldrich, 861405) diluted in the blocking solution for 1 h at room temperature. The slides were mounted in Fluoromount-G (SouthernBiotech) and imaged on a Zeiss AxioImager Z1 Apotome1.2, or a Zeiss AxioZoom V16 microscope equipped with an Axiocam 512 colour camera.

Sample sizes

The number of embryos analysed per experiment was at least three and is given in the relevant figure legends. Samples were not excluded and experimenters were aware of genotype.

We are grateful to our colleagues in the Kelly lab for helpful discussions, and Serge Van de Pavert, Françoise Helmbacher and Antonio Baldini for providing Rosa26-RARa403 mice, Pax3-Cre mice and Tbx1-Cre;mTmG embryos, respectively. We thank the IBDM imaging and animal facilities, the France-BioImaging/PICsL infrastructure (ANR-10-INSB-04-01) and the TMDU experimental animals and Research core centres.

Author contributions

Conceptualization: N.A., R.G.K.; Methodology: C.E.D., C.D.B., C.C., N.A., R.G.K.; Validation: C.E.D., N.A.; Formal analysis: C.E.D., C.R., C.D.B., C.C., E.J., N.A., R.G.K.; Investigation: C.E.D., C.R., C.D.B., C.C., E.J., F.L., S.Z., N.A., R.G.K.; Resources: F.L., S.Z.; Data curation: C.E.D., C.R., C.C., N.A.; Writing - original draft: C.D.B., N.A., R.G.K.; Writing - review & editing: C.E.D., C.R., C.D.B., C.C., E.J., F.L., S.Z., N.A., R.G.K.; Visualization: C.E.D., C.R., C.C., N.A.; Supervision: N.A., R.G.K.; Project administration: R.G.K.; Funding acquisition: R.G.K.

Funding

This study received support from the AFM-Téléthon (24396), Agence Nationale de la Recherche (ANR-19-CE13-0008 and ANR-22-CE13-0047), Fondation Leducq (TNE 15CVD01), Fondation pour la Recherche Médicale (FDT2022040147500), and a Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (23K06316). R.G.K. is an INSERM (Institut National de la Santé et de la Recherche Médicale) research fellow.

Data availability

All relevant data can be found within the article and its supplementary information.

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Competing interests

The authors declare no competing or financial interests.

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