Hedgehog Signaling in Tongue Muscle Development: A Comparative Perspective With Limb Myogenesis

Abstract

The mammalian tongue is an intricate skeletal muscle organ. From its initial formation to maturation, tongue muscle development involves precisely coordinated processes during embryonic and fetal phases of myogenesis. Extensive research on the regulatory pathways involved in tongue epithelial taste organ development has shown that the Hedgehog (HH) signaling pathway is vital to the formation and epithelial patterning of the tongue and taste organs. Emerging evidence also points to its involvement in the initial formation and spatial patterning of the tongue muscle. HH signaling is a well-established regulator of skeletal muscle development, particularly in limb myogenesis. However, structural and functional differences between limb and tongue muscles, as well as variations in their HH signaling regions, prevent the direct application of findings from limb muscles to the tongue. Consequently, a comprehensive comparative analysis is essential to establish the conserved and divergent mechanisms by which HH signaling operates in these distinct muscle systems. A detailed mechanistic understanding of HH signaling during lingual muscle formation and maturation is vital for fully elucidating its role in tongue function. Further, lingual myogenesis studies pave the way for potential regenerative therapeutic strategies for congenital anomalies and acquired conditions affecting the tongue. Thus, understanding the regulatory mechanisms of tongue muscle development has both biological and clinical importance. This review explores the role of HH signaling throughout the key stages of embryonic tongue muscle development (including myoblast determination, proliferation, differentiation, patterning, and maturation) and compares its role in limb myogenesis.

Graphical abstract

Keywords

skeletal muscle / tongue / muscle development / signal transduction / taste

1. Introduction

The mammalian tongue is a highly specialized skeletal muscle [1]. However, unlike other skeletal muscles, intrinsic tongue muscles are covered by dorsal epithelium and lamina propria comprising mesenchymal cells. Skeletal muscle development in the embryo begins with somatic epithelial cells transitioning into mesenchymal cells, which then differentiate into muscle cells [2, 3]. This process, known as myogenesis, involves distinct stages regulated by specific myogenic regulatory factors (MRFs). The MRFs are further modulated by signaling pathways, including Hedgehog (HH), Wnt, Notch, bone morphogenetic protein (BMP) and transforming growth factor beta (TGF-\beta) [4, 5, 6, 7, 8]. Despite having established roles in tongue epithelium and/or mesenchyme development, homeostasis and regeneration, HH signaling role in lingual muscles remains understudied [9, 10]. In this review, we evaluate HH signaling regulation of lingual muscle development in comparison to other skeletal muscles, using limb muscle as the best-characterized reference, since HH regulation of myogenesis has been extensively studied in the limb. We observed that growth and development of tongue muscles diverge significantly from those of limb skeletal muscles. Thus, findings from limb skeletal muscle research cannot be directly extrapolated to the tongue. We propose that further studies are needed to understand the role of HH signaling in lingual muscle maturation.

2. Myogenesis

The tightly regulated process of myogenesis involves several distinct stages: myoblast determination, migration, proliferation, differentiation, patterning, and maturation (Fig. 1). It occurs in two distinct phases: embryonic and fetal myogenesis [11]. Embryonic myogenesis initiates the process of muscle development with somites (blocks of mesoderm) differentiating into the dermomyotome (transient epithelium), which delaminates and gives rise to the myotome, the source of skeletal muscle precursors [2, 3]. During the determination stage, mesodermal muscle precursors migrate to their target locations, commit to the myogenic lineage, and develop into myoblasts. This commitment and migration are regulated by transcription factors, primarily Paired Box 3 (Pax3) and its orthologue, Pax7.

As muscle progenitor cells migrate to their destination, they undergo continuous proliferation [12]. Pax3 and Pax7 activate MRFs Myogenic Factor 5 (Myf5) and Myogenic differentiation 1 (MyoD), driving myoblast proliferation [13]. The differentiation stage in the target locations commences with myoblasts exiting the cell cycle and differentiating into myocytes under the antiproliferative effect of myogenin. As myoblasts differentiate, they begin to align, express muscle-specific proteins, and prepare to fuse with other myocytes to form multinucleated myotubes. This aggregation into myotubes marks the transition from progenitor cell to functional muscle cell [14]. Thus, myogenic differentiation is coordinated with an intricate fusion pattern to create their spatial organization [15]. In the final maturation stage, myotubes develop into mature muscle fibers, acquire striations, establish innervation, and develop contractile function. The contractile protein myosin heavy chain (MyHC), predominantly MyHC-embryonic, regulates muscle fiber size, number, and type, and myogenic differentiation genes [16]. Several other MRFs are also required to precisely control myogenesis stages [17].

Embryonic myogenesis, characterized by the formation of primary myofibers, occurs between embryonic day (E) 10 and E12.5 in mouse limb muscle. Fetal myogenesis, which gives rise to secondary myofibers, happens between E14.5 and E17.5 [18]. While embryonic myogenesis is crucial for the initial formation of the organ, fetal myogenesis establishes the muscular foundation necessary for critical postnatal functions [19]. Marked by a shift to fetal myoblasts, which are distinct from their embryonic counterparts, fetal myogenesis promotes muscle proliferation, differentiation, patterning, and functional maturation [11]. This process also establishes the satellite cell population necessary for postnatal muscle growth and regeneration [20]. Thus, the regulation of both embryonic and fetal myogenesis is necessary for proper muscle formation and growth in developing limbs.

3. Tongue Muscle Formation

Tongue muscle development begins at E10.5 (Fig. 2) with the formation of a central median lingual swelling on the first mandibular arch, followed by a lateral lingual swelling on each side comprising an epithelium and cranial neural crest cell (CNCC)-derived mesenchyme [21]. Muscle progenitors migrate from the somites into the tongue primordium within the lingual swellings starting at E11.5. These lingual swellings subsequently fuse to form the anterior two-thirds of the tongue; the posterior third arises from the third and fourth branchial arches [21]. Following fusion of the lingual swellings, myogenic progenitors develop into myofibers that occupy most of the tongue. HH signaling is known to regulate myoblast determination, tongue formation [22], and initial myofiber patterning [23], but its role in muscle proliferation, differentiation, and maturation in tongue remains poorly understood.

4. Tongue Muscles are Different From Other Skeletal Muscles

4.1 Origin

Skeletal muscles share fundamental characteristics but also exhibit distinct structural and functional differences. All skeletal muscles are composed of striated muscle fibers and require neural input for contraction [24]. However, while both limb and tongue belong to a migratory lineage of somite [21, 25], their origin, function, organization, and innervation vary significantly. All limb muscles originate from the segmented paraxial mesoderm (somites) [26]. In contrast, tongue muscles have a mixed origin, deriving primarily from the occipital somites [27] with contributions from the cranial mesoderm [28] (Table 1).

4.2 Function and Composition

Limb muscles support voluntary movements, postural stability, and locomotion, while tongue muscles assist with mastication, speech articulation, and swallowing (Table 1). Their differing functional requirements for contraction speed, energy metabolism, and fatigue resistance determine their muscle fiber composition [29, 30]. Broadly, skeletal muscles contain one slow-twitch and three major fast-twitch fiber types, with variations in their distribution based on functional demands [30]. For example, the soleus is composed predominantly of a slow-twitch fiber (~70%), while other leg muscles contain significantly less (\le50%) slow-twitch fibers [31]. By comparison, the anterior tongue tip consists mainly of fast-twitch fibers (~75%), while the posterior half of the tongue is mostly slow-twitch fibers (40–54%) [32]. Furthermore, the fiber types are comparable in size in the tongue, whereas slow muscle fibers are smaller in the limbs [33].

4.3 Organization and Innervation

Muscle organization also differs dramatically between limb and tongue. Limb muscle masses give rise to four major muscle types (flexors, extensors, adductors, abductors) and over 50 muscle fiber types that remain anatomically and functionally separate [34]. Limb muscles have distinct fascicles and defined tendons that attach to bone (Table 1), allowing for efficient force transmission. The tongue, however, incorporates a unique combination of intrinsic and extrinsic muscle fiber types [35] (Fig. 2). Its four intrinsic muscles (superior longitudinal, inferior longitudinal, vertical, and transverse muscle fibers) are entirely contained within the tongue and lack bony attachments. They interweave in complex, multidirectional layers to enable fine motor control for shaping, elongating, and thickening the tongue [33]. The extrinsic muscles (genioglossus, hyoglossus, styloglossus, and palatoglossus) originate from external bony structures and insert into the tongue to facilitate larger positional movements such as protrusion, retraction, and elevation [36]. Together, these two types of muscle fibers enable both the fine motor control and gross movement necessary for the tongue’s diverse functions [36].

Motor neurons originating from the spinal cord innervate limb muscles [37]. In contrast, tongue muscles are predominantly innervated by cranial motor neurons of the hypoglossal nerve [38] with additional vagus nerve innervation to the extrinsic palatoglossus muscle [36]. Thus, the neural control of limb is distinct from tongue.

4.4 MRFs Regulation

The MRFs also regulate myogenesis differently in the tongue and limb. Myf5 and MyoD are activated simultaneously in the limb, whereas the tongue exhibits a sequential activation, with Myf5 preceding MyoD [39]. Further, myoblasts contributing to tongue formation predominantly express Myf5 rather than MyoD [40]. Myf5 loss can be compensated in both limb [41] and tongue [39]. However, while MyoD loss can be compensated in the limb [42], in the tongue it causes reduced muscle formation resulting in microglossia [39]. Additionally, myogenin-null embryos exhibit more severe defects in tongue muscle development compared to the limb [43]. These differences may reflect distinct myogenic timelines, as embryonic and fetal myogenesis are temporally separated in the limb but overlap in the tongue [44].

4.5 Signaling Regulation

The distinct myogenic phases, embryonic and fetal, are differentially regulated in limb and tongue. For example, Wnt signaling in limb regulates fetal myogenesis but not embryonic myogenesis [45]. Further, Wnt signaling is required for limb muscle cell precursor number and myofiber quantity. In contrast, Wnt5a ligand is expressed in developing tongue muscle cells between E12.5–E14.5 and not apparent in mature myofibers after E15.5 [46]. Muscle-specific Wnt signaling regulates lingual myoblast fusion and differentiation at E14.5 [47], while epithelial specific Wnt signaling controls the number of muscle progenitor cells and their proliferation in tongue [48]. Further, it has been proposed that the tongue muscle progenitor proliferation regulated by Wnt signaling is through its upstream control of Notch signaling [48]. In limb, Notch signaling has established roles in maintaining muscle stem cells by inhibiting differentiation [6, 49]. Recently, Notch1 signaling has been shown to have roles in limb muscle fiber type composition and myofiber maturation [50].

In limb, BMP signaling plays a dual role: promoting embryonic myogenesis while inhibiting muscle differentiation during fetal myogenesis [51]. While BMP is also critical for craniofacial development [52], its specific role in regulating tongue muscle formation has not yet been investigated. TGF-\beta signaling inhibits limb myoblast fusion and myoblast differentiation during embryogenic and fetal myogenesis, respectively [53, 54]. In contrast, TGF-\beta signaling regulates lingual myoblast proliferation and differentiation at or before E13.5 [55], with no data available beyond this stage. Collectively, Wnt, Notch, BMP and TGF-\beta pathways play important roles in muscle development, although their temporal activity and mechanisms of action diverge between limb and tongue. Growing evidence indicates that HH signaling interacts with many of these pathways in cancer context [56, 57]. For example, Sonic HH (SHH) signaling is downstream of Notch signaling [58], but acts upstream of Wnt signaling [59]. BMP signaling inhibits effects of SHH [60] and thus can have opposing roles as to HH signaling [61]. On the other hand, TGF-\beta can promote GLI2 transcription factor and thus activate non-canonical HH signaling [62]. However, direct evidence of crosstalk between HH signaling and these pathways in myogenesis is currently limited. Importantly, HH signaling has vital roles in craniofacial development, including tongue formation and papillae patterning [59]. Whether it acts as a central regulator of tongue myogenesis is our key focus here.

5. Hedgehog Signaling

5.1 HH Pathway Components

The HH signaling pathway is a well-established regulator of embryonic tissue development [63]. It requires a membrane receptor Patched1 (PTCH1) that inhibits another membrane receptor, Smoothened (SMO), in the absence of HH ligand. There are three studied HH ligands: SHH, Indian HH (IHH), and Desert HH (DHH) [64]. Among these, SHH expression is extensively reported in the tongue, while both SHH and IHH have been implicated in limb muscle. Importantly, SHH is dual lipidated which limits its free diffusion [65] and thus require additional co-receptors, Growth Arrest Specific 1 (GAS1), Cell-adhesion molecule-related/downregulated by oncogenes (CDON) and brother of CDON (BOC), for ligand reception [66]. Specifically, the dual-lipidated SHH is recruited at the cell surface by the cell adhesion molecules CDON and BOC, which are members of the immunoglobulin superfamily. GAS1, a glycosylphosphatidylinositol (GPI)-anchored membrane protein, subsequently recognizes the ligand and removes its lipid modifications, facilitating SHH binding to PTCH1. When the ligand binds to PTCH1, the SMO inhibition is relieved to modulate downstream signaling. The final effectors of the HH pathway are the GLI transcription factors (GLI1, GLI2, and GLI3), which activate or inhibit target gene expression [67].

5.2 HH Signaling Regulation of MRFs

SHH decreases Pax3 and Pax7 expression and promotes the expression of other MRFs (Fig. 1) [68, 69]. Although SHH does not directly bind to the MRF, Myf5, its expression is dependent on HH signaling via GLI transcription factors, for both initial and continuous expression [70, 71, 72]. GLI1 and GLI2 can also interact with various regulatory elements of the MRF, MyoD [70, 73, 74]. Further, MyoD regulation can be independent of SHH [75], possibly due to cross-talk of GLI2 with other pathways [73]. The GLI regulation is also observed for myogenin [73, 76]. HH signaling also reportedly mediates expression of MyHC in mussel larval stages [77], in vitro muscle cells [78], and mouse embryonic cardiac tissue [79]. This highlights the versatility and importance of HH signaling in different myogenesis stages.

5.3 HH Signaling Activity in Limb and Tongue

The expression patterns of HH signaling and its components differ significantly between the tongue and limbs (Fig. 3). In the limb, SHH ligand is expressed in the posterior limb bud mesenchyme, specifically within the zone of polarizing activity, (Fig. 3A, yellow) creating a gradient of GLI3 repressor [80, 81]. This asymmetric distribution of GLI isoforms ensures HH pathway activation in the posterior limb while maintaining pathway repression in the anterior limb. Expression of HH receptor SMO and target gene GLI1 indicates active HH signaling in muscle lineage cells (Pax3+, Myf5+) as well as in SHH surrounding lateral plate-derived mesenchymal cells [42].

Conversely, in the embryonic tongue, SHH is present in the entire epithelium of the mandibular arches and the developing tongue [82, 83, 84] (Fig. 3B). Its downstream signaling components, including the SMO receptor and GLI transcriptional activators, are expressed in both the epithelium and the CNCC-derived mesenchyme [83]. However, by E18.5, as the epithelium differentiates into taste and non-taste epithelium, SHH expression becomes restricted to taste cells, and HH signaling occurs only in cells of the taste epithelium [83]. Our recent studies confirm the absence of HH signaling, as indicated by Gli1 expression, in embryonic tongue muscles [85]. Notably, epithelial SHH in the tongue primarily signals to the CNCC-derived mesenchyme, which in turn supports tongue muscle development [23] — a notably distinctive mechanism from the mesoderm-directed HH signaling observed in limb muscle formation. Overall, the HH pathway in tongue development exhibits greater complexity and temporal regulation than in limb development.

5.4 HH Signaling Regulation of Limb and Tongue Muscles

Mouse, chick and zebrafish have conserved steps of skeletal muscle development parallel to humans [86, 87]. Studies using vertebrate models or in vitro mouse or chick cells have determined that HH signaling regulates distinct stages of both limb and tongue myogenesis (Table 2, Ref. [22, 23, 42, 73, 83, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102]). Specifically, in limb, Shh ligand is responsible for muscle migration, proliferation, fusion, differentiation of slow fiber types, muscle size and muscle mass. In the tongue, limited studies indicate its critical role in tongue formation and initial myofiber arrangement. Similarly, the HH receptor Smo affects all stages of limb myogenesis; in the tongue, its primary function lies in myoblast migration-mediated tongue formation. Additionally, Smo overexpression can disorganize lingual muscle structure.

Another HH receptor, Gas1, also plays a vital role in skeletal myogenesis. Although in limb in vitro, it enhances myotube formation and myoblast differentiation, our recent studies in tongue with in vivo Gas1 deletion suggest it also has roles in muscle cell proliferation, differentiation and maturation during fetal myogenesis [85]. Intriguingly, these effects are not due to muscle-cell specific deletion. Global deletion, including additional epithelium and stromal Gas1 deletion, results in altered lingual intrinsic muscles that consequently affect tongue shape and size [85]. Further, Gas1 functions via alternate pathways to HH signaling. On the other hand, Gli transcription factors initiate MRF activation in limb (Fig. 1), while in tongue its deletion from mesenchyme results in aglossia [87]. Overall, numerous studies indicate essential roles of HH signaling in limb myogenesis. However, no similarly comprehensive body of research exists for tongue myogenesis (Table 2). Available studies indicate vital roles of HH signaling in tongue formation and initial myofiber patterning, but the regulation once the muscles are arranged remains understudied.

6. Myogenesis Stage-Specific Comparison Between Tongue and Limb Muscle for HH Signaling Regulation

6.1 Determination and Migration

In limbs, SHH organizes myoblast distribution along multiple axes and is crucial for distal migration [88]. It directs the anteroposterior axis non-cell-autonomously through lateral plate-derived limb mesenchyme [42], while also acting cell-autonomously on a subset of limb myoblasts to regulate autopod muscle formation [88]. Loss of SHH delays MRF activation, disrupting muscle formation, but does not prevent the proximal migration of myoblasts into the limb bud [88].

Tongue myoblast precursors migrate as a cohesive strand along a distinct and complex pathway [103]. Myoblast determination in the tongue occurs in a posterior-to-anterior direction [23]. Interactions between CNCC- and mesoderm-derived cells contribute to this process through HH signaling [104]. However, reduced SHH expression or inhibition of SHH signaling in CNCC-derivatives does not appear to directly impact myoblast determination (Table 2). In contrast, epithelial SHH acts non-cell-autonomously through mandibular CNCC-derived mesenchyme to guide the unidirectional myoblast migration to the tongue primordium [21]. Deletion of Shh, membrane receptor Smo, or transcription factors Gli2 and Gli3 in CNCC-derivatives leads to aglossia due to impaired myoblast migration [23, 82, 84, 87, 96, 101, 102] (Fig. 4A). This demonstrates the necessity of non-cell-autonomous HH signaling for the migration of tongue myoblast precursors and subsequent muscle formation.

Together, in the limb, SHH functions both cell-autonomously within myoblasts and non-cell-autonomously through limb mesenchyme to control myoblast distribution and distal migration. In contrast, in the tongue, SHH signaling operates solely in a non-cell-autonomous manner from the epithelium to CNCC-derived mesenchyme, orchestrating myoblast migration but not determination.

6.2 Proliferation

Determined myoblasts undergo extensive proliferation while maintaining an undifferentiated state, a process essential for increasing muscle mass [105]. HH-responding Gli1+ cells are present in limb muscle during and after myoblast migration, suggesting ongoing HH pathway activity in muscle progenitors [80, 94]. In the limb, SHH deletion or loss of cell-autonomous HH signaling does not impair the proliferation of embryonic myoblasts in mice [88]. Instead, non-myogenic tissues appear to influence myogenic proliferation and differentiation [106]. In the posterior hindlimb of late chick embryos, SHH indirectly supports myoblast proliferation by maintaining cells in an undifferentiated state, a stage equivalent to E14 in mice [95]. Additionally, ectopic SHH expression in chick muscle cells has been shown to induce myoblast proliferation in vitro [94], reinforcing a potential cell-intrinsic role for HH signaling in promoting limb myoblast proliferation under specific conditions.

Tongue mandibular explant cultures treated with SMO inhibitors exhibit microglossia [83, 99] (Fig. 4B), primarily due to decreased proliferation of CNCC-derived mesenchymal cells [99]. Interestingly, despite the reduction in CNCC-derivatives proliferation upon HH pathway inhibition, the proliferation of myoblasts and expression of key MRFs such as MyoD and Myf5 remain unaffected [99]. This might be due to that fact that CNCCs support the proliferation of myogenic precursors through paracrine signaling, particularly via TGF-\beta pathways [107, 108].

Some in vivo studies of the tongue show that reduction of Shh expression in the epithelium or decreased HH pathway activity in CNCCs, as indicated by reduced expression of target genes Gli1 and Ptch1, does not disrupt either CNCC-derived mesenchyme or myogenic cells proliferation [23, 101, 104]. These results contrast with studies in which CNCC-specific Smo haploinsufficiency resulted in diminished mesenchymal proliferation [84] and that reduction of SHH in distal ectoderm reduces myoblast proliferation at E11.5 [82]. Such discrepancies may reflect potential context-dependent effects or differences in experimental design, timing, or degree of pathway suppression. Our recent research indicates HH-independent roles of receptor Gas1 in regulating Pax7+ myoblast numbers and muscle cell proliferation at E18.5, potentially as a compensatory response to reduced numbers of mature fibers and an increased demand for new fiber formation [85].

Overall, studies on limb and tongue myogenesis suggest that HH signaling may not be not essential for embryonic myoblast proliferation. However, the findings in limb muscle showing that ectopic SHH can induce proliferation underscore its context-dependent role. In contrast, during tongue development, alternative pathways or compensatory mechanisms may regulate myogenic proliferation independently of HH input.

6.3 Differentiation

Limb muscles contain variable distributions of both slow and fast fibers (Table 1). HH signaling promotes slow muscle fiber differentiation by inducing slow MyHC expression in a larger precursor pool of myoblast to suppress fast myofiber formation [90, 109, 110]. Overexpression of SHH leads to an increased proportion of slow fibers, whereas continuous deletion disrupts overall muscle formation by E16.5 [89]. Fast fiber formation in the absence of HH signaling in zebrafish studies further support this finding [91, 92]. IHH deletion also results in defective muscle differentiation [93]. Notably, SHH expression ceases in the limb at E16.5 in mice but Ptch1 remains in fetal myoblasts, indicating a possible transition to the IHH signaling seen in the developing bone anlagen [93]. This shift from SHH to IHH highlights the dynamic regulation of HH signaling in limb muscle development and the necessity of maintaining proper signaling levels for muscle fiber differentiation.

Fast-twitch fibers predominate in tongue muscles, with a smaller population of slow-twitch fibers also present (Table 1). Notably, slow fibers are especially enriched in the posterior region of the extrinsic genioglossus muscle [32, 111]. If Shh is deleted after myoblast migration, muscle fiber differentiation into intrinsic or extrinsic types are unimpaired [41] (Fig. 4C). This indicates less dependence on HH signaling in tongue muscle fiber differentiation than for limb muscle differentiation. On the other hand, alternative pathways, including the TGF\beta-Smad4-Fgf6 cascade, as well as fibroblast growth factor (FGF), and Notch, signaling, play more prominent roles in myoblast fusion and terminal differentiation in the tongue [48, 55, 107, 112]. Although components of these pathways such as suppression of FGF through Foxf2 are regulated by HH signaling [112, 113], direct evidence addressing the role of HH signaling in fiber-type specification is limited. Our recent research further suggests that Gas1 deletion reduces overall differentiated muscle cells and MyHC-embryonic expression through mechanisms independent of canonical HH signaling [85].

The slow and fast fiber specification is responsive to innervation [114, 115], which itself can be shaped by CNCC-derived mesenchyme [116, 117]. We hypothesize that HH signaling might regulate fiber specification indirectly through its role in CNCC rather than directly on myogenic cells [118]. In summary, no studies to date have definitively shown that HH signaling directly determines slow vs. fast fiber fate in lingual myogenesis.

Given the necessity of HH signaling in slow fiber-type specification in limb muscles and the relative scarcity of slow fibers in the tongue, the direct impact of HH signaling on fast fiber differentiation in tongue remains unclear and warrants further investigation.

6.4 Patterning

HH signaling plays a multifaceted role in dictating the spatiotemporal organization of limb musculature. It has been shown that non-myogenic tissues, particularly muscle mesenchyme, contribute significantly to muscle patterning [119, 120]. Conditional Smo deletion in such non-muscle limb mesenchyme resulting in a significant loss of antero-posterior muscle patterning while the failure of cell-autonomous removal of Smo activity in the somites themselves to affect this patterning reinforces its non-cell-autonomous role [42]. Myoblasts have receptors for mesenchymal extracellular matrix (ECM) components comprising heparan sulphate proteoglycans [121, 122], which can bind with SHH and mediate distinct patterning [123]. Separately, ectopic expression of Shh in the anterior compartment of the chick wing leads to dramatic muscle re-patterning, with anterior muscles adopting posterior-like characteristics in a mirror-image arrangement [124].

In the mouse tongue, SHH is not present in muscle cells or ECM of embryonic tongue [83, 85]. However, it can still control intrinsic muscle patterning via its interaction with ptch1+ or Smo+ CNCC-derivatives [23]. SHH mediation in the development of lingual tendons also aids indirectly by providing a scaffold for the normal patterning of intrinsic muscle fibers. In mice with Shh deletion from E10.5, the normal striated architecture of the intrinsic muscles was lost and tongue size reduced at E14.5 (Fig. 4B). Once lingual muscles are migrated, Ptch1/HH signaling inhibition in CNCCs specifically leads to hypoglossia, tongue clefting, and disorganized myotube arrangement at E14.5 (Fig. 4C). However, overexpression of Shh in K14+ epithelium at this stage distorts tongue architecture but retains normal arrangement of the intrinsic muscles (Fig. 4D). Further, deletion of Shh after the initial arrangement of muscle fibers results in subtle organizational disruptions (Fig. 4E), suggesting temporal SHH loss at later stages produces milder effects. Notably, a reduction, but not complete elimination, of SHH does not alter lingual muscle patterning [23, 125] (Fig. 4F). The presence of multiple SHH sources in the tongue [9, 126, 127] may account for the graded severity of tongue defects. In contrast, Smo overexpression in CNCC-derivatives resulted in a disorganized muscular structure [22] (Fig. 4F) while pharmacological SMO overactivation caused the formation of bifid tongue [97] (Fig. 4G).

Additionally, deletion of HH co-receptors Gas1 and Boc affects pharyngeal tongue formation at E15.5 [128]. Anterior tongue formation occurred but muscle patterning was not investigated. Expression of HH signaling target gene and activator Gli1 and HH co-receptors Gas1, Cdon, and Boc has previously been observed in embryonic tongue at E11.5 [129]. We recently identified Gas1, both gene and protein, as well as CDON and BOC in the embryonic lingual muscles at E18.5 [85]. Intriguingly, constitutive deletion of Gas1 alters tongue morphology and size as well as the number and size of myofibers at E18.5 (Fig. 4H). HH-responding Gli1+ cells are notably absent in lingual muscles at this stage [83, 85], and their expression in the epithelium and mesenchyme remains unchanged following Gas1 deletion. These findings suggest that the role of HH signaling in muscle organization during tongue development may involve non-canonical or indirect mechanisms.

Overall, an intricate interplay between SHH and the ECM is fundamental to limb muscle patterning. In contrast, in the tongue, SHH acts through CNCC-derived mesenchyme and tendon scaffolding, rather than the ECM, in a temporally regulated manner.

6.5 Maturation

A hallmark of muscle cell maturational progression is the sequential expression of distinct MyHC isoforms. The MyHC isoform expression, including embryonic and neonatal, is tightly regulated during muscle embryonic maturation [130, 131]. Studies on myogenesis regulation have primarily focused on the differentiation of MyHC into slow and fast fibers. However, no literature is currently available for studies specifically focusing on the regulation of neonatal and embryonic MyHC isoforms by HH signaling in either tongue or limb muscles. Our recent studies show that Gas1 deletion significantly reduced MyHC-embryonic expression, but not MyHC-neonatal, without disrupting HH signaling [85].

The development of the NMJ is also a critical step in muscle maturation, enabling effective communication between motor neurons and muscle fibers [132]. SHH is identified as a key regulator in motor neuron development [133, 134]. HH signaling is also essential for the development and patterning of the central nervous system and neural tube, influencing the fate and proliferation of neural progenitors [135, 136]. However, its direct involvement in NMJ formation or maturation in limb or tongue muscles during embryonic development has not been established. We observed a significant reduction in NMJ area following embryonic deletion of Gas1, as well as a percentage overlap between nerve terminals and muscle endplates [85]. Whether these NMJ structural changes in Gas1 mutants translate into muscle functional deficits remains unknown.

Taken together, the role of HH signaling in regulating muscle maturation in both the tongue and limb warrants further investigation. Our recent findings suggest that HH co-receptor Gas1 supports muscle maturation through non-canonical HH signaling.

7. Conclusion

Overall, HH signaling is essential for embryonic myogenesis in both limb and lingual skeletal muscles. Numerous studies have demonstrated that the loss of key HH pathway components, such as Shh or Smo, leads to significant muscle dysmorphogenesis, though the nature and severity of these defects differ between the limb and tongue (Table 2, Fig. 4). These findings underscore the distinct and context-dependent roles of HH signaling in limb versus tongue muscle development. However, important gaps remain in our understanding of HH signaling in the lingual muscle, particularly during the stages of myoblast proliferation, differentiation, and fiber maturation. Further studies are needed to elucidate HH-mediated fetal myogenesis during tongue embryonic development. A detailed understanding of the pathway regulation would advance our fundamental knowledge of tongue muscle growth and maturation as well as potentially offer new therapeutic avenues for muscle regeneration and repair in congenital or acquired disorders.

References

[1] Doyle ME, Premathilake HU, Yao Q, Mazucanti CH, Egan JM. Physiology of the tongue with emphasis on taste transduction. Physiological Reviews. 2023; 103: 1193–1246. https://doi.org/10.1152/physrev.00012.2022.

[2] Kim DH, Xing T, Yang Z, Dudek R, Lu Q, Chen YH. Epithelial Mesenchymal Transition in Embryonic Development, Tissue Repair and Cancer: A Comprehensive Overview. Journal of Clinical Medicine. 2017; 7: 1. https://doi.org/10.3390/jcm7010001.

[3] Gros J, Manceau M, Thomé V, Marcelle C. A common somitic origin for embryonic muscle progenitors and satellite cells. Nature. 2005; 435: 954–958. https://doi.org/10.1038/nature03572.

[4] Hernández-Hernández JM, García-González EG, Brun CE, Rudnicki MA. The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration. Seminars in Cell & Developmental Biology. 2017; 72: 10–18. https://doi.org/10.1016/j.semcdb.2017.11.010.

[5] Shirakawa T, Toyono T, Inoue A, Matsubara T, Kawamoto T, Kokabu S. Factors Regulating or Regulated by Myogenic Regulatory Factors in Skeletal Muscle Stem Cells. Cells. 2022; 11: 1493. https://doi.org/10.3390/cells11091493.

[6] Gioftsidi S, Relaix F, Mourikis P. The Notch signaling network in muscle stem cells during development, homeostasis, and disease. Skeletal Muscle. 2022; 12: 9. https://doi.org/10.1186/s13395-022-00293-w.

[7] Mierzejewski B, Ciemerych MA, Streminska W, Janczyk-Ilach K, Brzoska E. miRNA-126a plays important role in myoblast and endothelial cell interaction. Scientific Reports. 2023; 13: 15046. https://doi.org/10.1038/s41598-023-41626-z.

[8] Xie Y, Su N, Yang J, Tan Q, Huang S, Jin M, et al. FGF/FGFR signaling in health and disease. Signal Transduction and Targeted Therapy. 2020; 5: 181. https://doi.org/10.1038/s41392-020-00222-7.

[9] Kumari A, Ermilov AN, Grachtchouk M, Dlugosz AA, Allen BL, Bradley RM, et al. Recovery of taste organs and sensory function after severe loss from Hedgehog/Smoothened inhibition with cancer drug sonidegib. Proceedings of the National Academy of Sciences of the United States of America. 2017; 114: E10369–E10378. https://doi.org/10.1073/pnas.1712881114.

[10] Mistretta CM, Kumari A. Hedgehog Signaling Regulates Taste Organs and Oral Sensation: Distinctive Roles in the Epithelium, Stroma, and Innervation. International Journal of Molecular Sciences. 2019; 20: 1341. https://doi.org/10.3390/ijms20061341.

[11] Messina G, Biressi S, Monteverde S, Magli A, Cassano M, Perani L, et al. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell. 2010; 140: 554–566. https://doi.org/10.1016/j.cell.2010.01.027.

[12] Pownall ME, Gustafsson MK, Emerson CP, Jr. Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos. Annual Review of Cell and Developmental Biology. 2002; 18: 747–783. https://doi.org/10.1146/annurev.cellbio.18.012502.105758.

[13] Kassar-Duchossoy L, Gayraud-Morel B, Gomès D, Rocancourt D, Buckingham M, Shinin V, et al. Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature. 2004; 431: 466–471. https://doi.org/10.1038/nature02876.

[14] Abmayr SM, Pavlath GK. Myoblast fusion: lessons from flies and mice. Development (Cambridge, England). 2012; 139: 641–656. https://doi.org/10.1242/dev.068353.

[15] Toulouse G, Jarassier W, Jagot S, Morin V, Le Grand F, Marcelle C. Early lineage segregation of primary myotubes from secondary myotubes and adult muscle stem cells. Nature Communications. 2025; 16: 7858. https://doi.org/10.1038/s41467-025-61767-1.

[16] Agarwal M, Sharma A, Kumar P, Kumar A, Bharadwaj A, Saini M, et al. Myosin heavy chain-embryonic regulates skeletal muscle differentiation during mammalian development. Development (Cambridge, England). 2020; 147: dev184507. https://doi.org/10.1242/dev.184507.

[17] Wu J, Yue B. Regulation of myogenic cell proliferation and differentiation during mammalian skeletal myogenesis. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie. 2024; 174: 116563. https://doi.org/10.1016/j.biopha.2024.116563.

[18] Biressi S, Molinaro M, Cossu G. Cellular heterogeneity during vertebrate skeletal muscle development. Developmental Biology. 2007; 308: 281–293. https://doi.org/10.1016/j.ydbio.2007.06.006.

[19] Feng LT, Chen ZN, Bian H. Skeletal muscle: molecular structure, myogenesis, biological functions, and diseases. MedComm. 2024; 5: e649. https://doi.org/10.1002/mco2.649.

[20] Lepper C, Conway SJ, Fan CM. Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements. Nature. 2009; 460: 627–631. https://doi.org/10.1038/nature08209.

[21] Parada C, Han D, Chai Y. Molecular and cellular regulatory mechanisms of tongue myogenesis. Journal of Dental Research. 2012; 91: 528–535. https://doi.org/10.1177/0022034511434055.

[22] Xu J, Liu H, Lan Y, Jiang R. The transcription factors Foxf1 and Foxf2 integrate the SHH, HGF and TGFβ signaling pathways to drive tongue organogenesis. Development (Cambridge, England). 2022; 149: dev200667. https://doi.org/10.1242/dev.200667.

[23] Okuhara S, Birjandi AA, Adel Al-Lami H, Sagai T, Amano T, Shiroishi T, et al. Temporospatial sonic hedgehog signalling is essential for neural crest-dependent patterning of the intrinsic tongue musculature. Development (Cambridge, England). 2019; 146: dev180075. https://doi.org/10.1242/dev.180075.

[24] McCuller C, Jessu R, Callahan AL. Physiology, skeletal muscle. StatPearls Publishing: Treasure Island (FL). 2023.

[25] Buckingham M, Bajard L, Chang T, Daubas P, Hadchouel J, Meilhac S, et al. The formation of skeletal muscle: from somite to limb. Journal of Anatomy. 2003; 202: 59–68. https://doi.org/10.1046/j.1469-7580.2003.00139.x.

[26] Tani S, Chung UI, Ohba S, Hojo H. Understanding paraxial mesoderm development and sclerotome specification for skeletal repair. Experimental & Molecular Medicine. 2020; 52: 1166–1177. https://doi.org/10.1038/s12276-020-0482-1.

[27] Yamane A. Embryonic and postnatal development of masticatory and tongue muscles. Cell and Tissue Research. 2005; 322: 183–189. https://doi.org/10.1007/s00441-005-0019-x.

[28] Czajkowski MT, Rassek C, Lenhard DC, Bröhl D, Birchmeier C. Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle. Developmental Biology. 2014; 395: 307–316. https://doi.org/10.1016/j.ydbio.2014.09.005.

[29] Zaidi FN, Meadows P, Jacobowitz O, Davidson TM. Tongue anatomy and physiology, the scientific basis for a novel targeted neurostimulation system designed for the treatment of obstructive sleep apnea. Neuromodulation: Journal of the International Neuromodulation Society. 2013; 16: 376–86; discussion 386. https://doi.org/10.1111/j.1525-1403.2012.00514.x.

[30] Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiological Reviews. 2011; 91: 1447–1531. https://doi.org/10.1152/physrev.00031.2010.

[31] Edgerton VR, Smith JL, Simpson DR. Muscle fibre type populations of human leg muscles. The Histochemical Journal. 1975; 7: 259–266. https://doi.org/10.1007/BF01003594.

[32] Stål P, Marklund S, Thornell LE, De Paul R, Eriksson PO. Fibre composition of human intrinsic tongue muscles. Cells, Tissues, Organs. 2003; 173: 147–161. https://doi.org/10.1159/000069470.

[33] Sanders I, Mu L. A three-dimensional atlas of human tongue muscles. Anatomical Record (Hoboken, N.J.: 2007). 2013; 296: 1102–1114. https://doi.org/10.1002/ar.22711.

[34] Lieber RL, Ward SR. Skeletal muscle design to meet functional demands. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2011; 366: 1466–1476. https://doi.org/10.1098/rstb.2010.0316.

[35] Sakamoto Y. Structural arrangement of the intrinsic muscles of the tongue and their relationships with the extrinsic muscles. Surgical and Radiologic Anatomy: SRA. 2018; 40: 681–688. https://doi.org/10.1007/s00276-018-1993-5.

[36] Dotiwala AK, Samra NS. Anatomy, head and neck, tongue. StatPearls Publishing: Treasure Island (FL). 2023.

[37] Zayia LC, Tadi P. Neuroanatomy, motor neuron. StatPearls Publishing: Treasure Island (FL). 2025.

[38] Kim SY, Naqvi IA. Neuroanatomy, cranial nerve 12 (hypoglossal). StatPearls Publishing: Treasure Island (FL). 2022.

[39] Zhong Z, Zhao H, Mayo J, Chai Y. Different requirements for Wnt signaling in tongue myogenic subpopulations. Journal of Dental Research. 2015; 94: 421–429. https://doi.org/10.1177/0022034514566030.

[40] Dalrymple KR, Prigozy TI, Mayo M, Kedes L, Shuler CF. Murine tongue muscle displays a distinct developmental profile of MRF and contractile gene expression. The International Journal of Developmental Biology. 1999; 43: 27–37.

[41] Haldar M, Karan G, Tvrdik P, Capecchi MR. Two cell lineages, myf5 and myf5-independent, participate in mouse skeletal myogenesis. Developmental Cell. 2008; 14: 437–445. https://doi.org/10.1016/j.devcel.2008.01.002.

[42] Hu JKH, McGlinn E, Harfe BD, Kardon G, Tabin CJ. Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation. Genes & Development. 2012; 26: 2088–2102. https://doi.org/10.1101/gad.187385.112.

[43] Valdez MR, Richardson JA, Klein WH, Olson EN. Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4. Developmental Biology. 2000; 219: 287–298. https://doi.org/10.1006/dbio.2000.9621.

[44] Kawamoto S, Hani T, Fujita K, Taya Y, Sasaki Y, Kudo T, et al. Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue. Journal of Oral Biosciences. 2023; 65: 88–96. https://doi.org/10.1016/j.job.2023.01.003.

[45] Hutcheson DA, Zhao J, Merrell A, Haldar M, Kardon G. Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for beta-catenin. Genes & Development. 2009; 23: 997–1013. https://doi.org/10.1101/gad.1769009.

[46] Asada N, Suzuki K, Sunohara M. Spatiotemporal distribution analyses of Wnt5a ligand and its receptors Ror2, Frizzled2, and Frizzled5 during tongue muscle development in prenatal mice. Annals of Anatomy = Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft. 2023; 245: 152017. https://doi.org/10.1016/j.aanat.2022.152017.

[47] Suzuki A, Minamide R, Iwata J. WNT/β-catenin signaling plays a crucial role in myoblast fusion through regulation of nephrin expression during development. Development (Cambridge, England). 2018; 145: dev168351. https://doi.org/10.1242/dev.168351.

[48] Zhu XJ, Yuan X, Wang M, Fang Y, Liu Y, Zhang X, et al. A Wnt/Notch/Pax7 signaling network supports tissue integrity in tongue development. The Journal of Biological Chemistry. 2017; 292: 9409–9419. https://doi.org/10.1074/jbc.M117.789438.

[49] Vargas-Franco D, Kalra R, Draper I, Pacak CA, Asakura A, Kang PB. The Notch signaling pathway in skeletal muscle health and disease. Muscle & Nerve. 2022; 66: 530–544. https://doi.org/10.1002/mus.27684.

[50] Yamashita AMS, Garay BI, Kim H, Bosnakovski D, Abrahante JE, Azzag K, et al. Effect of Notch1 signaling on muscle engraftment and maturation from pluripotent stem cells. Stem Cell Reports. 2025; 20: 102396. https://doi.org/10.1016/j.stemcr.2024.102396.

[51] Borok MJ, Mademtzoglou D, Relaix F. Bu-M-P-ing Iron: How BMP Signaling Regulates Muscle Growth and Regeneration. Journal of Developmental Biology. 2020; 8: 4. https://doi.org/10.3390/jdb8010004.

[52] Ueharu H, Mishina Y. BMP signaling during craniofacial development: new insights into pathological mechanisms leading to craniofacial anomalies. Frontiers in Physiology. 2023; 14: 1170511. https://doi.org/10.3389/fphys.2023.1170511.

[53] Cusella-De Angelis MG, Molinari S, Le Donne A, Coletta M, Vivarelli E, Bouche M, et al. Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis. Development (Cambridge, England). 1994; 120: 925–933. https://doi.org/10.1242/dev.120.4.925.

[54] Melendez J, Sieiro D, Salgado D, Morin V, Dejardin MJ, Zhou C, et al. TGFβ signalling acts as a molecular brake of myoblast fusion. Nature Communications. 2021; 12: 749. https://doi.org/10.1038/s41467-020-20290-1.

[55] Han A, Zhao H, Li J, Pelikan R, Chai Y. ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions. Molecular and Cellular Biology. 2014; 34: 3120–3131. https://doi.org/10.1128/MCB.00623-14.

[56] Iluta S, Nistor M, Buruiana S, Dima D. Notch and Hedgehog Signaling Unveiled: Crosstalk, Roles, and Breakthroughs in Cancer Stem Cell Research. Life (Basel, Switzerland). 2025; 15: 228. https://doi.org/10.3390/life15020228.

[57] Pelullo M, Zema S, Nardozza F, Checquolo S, Screpanti I, Bellavia D. Wnt, Notch, and TGF-β Pathways Impinge on Hedgehog Signaling Complexity: An Open Window on Cancer. Frontiers in Genetics. 2019; 10: 711. https://doi.org/10.3389/fgene.2019.00711.

[58] Ma L, Li C, Lian S, Xu B, Yuan J, Lu J, et al. ActivinA activates Notch1-Shh signaling to regulate proliferation in C2C12 skeletal muscle cells. Molecular and Cellular Endocrinology. 2021; 519: 111055. https://doi.org/10.1016/j.mce.2020.111055.

[59] Xu J, Iyyanar PPR, Lan Y, Jiang R. Sonic hedgehog signaling in craniofacial development. Differentiation; Research in Biological Diversity. 2023; 133: 60–76. https://doi.org/10.1016/j.diff.2023.07.002.

[60] Manzari-Tavakoli A, Babajani A, Farjoo MH, Hajinasrollah M, Bahrami S, Niknejad H. The Cross-Talks Among Bone Morphogenetic Protein (BMP) Signaling and Other Prominent Pathways Involved in Neural Differentiation. Frontiers in Molecular Neuroscience. 2022; 15: 827275. https://doi.org/10.3389/fnmol.2022.827275.

[61] Robertson RA, Knight HG, Lipovsky C, Ren J, Chi NC, Yelon D. Hedgehog and Bmp signaling pathways play opposing roles during establishment of the cardiac inflow tract in zebrafish. bioRxiv. 2025. https://doi.org/10.1101/2025.07.19.665705. (preprint)

[62] Liu R, Yu Y, Wang Q, Zhao Q, Yao Y, Sun M, et al. Interactions between hedgehog signaling pathway and the complex tumor microenvironment in breast cancer: current knowledge and therapeutic promises. Cell Communication and Signaling: CCS. 2024; 22: 432. https://doi.org/10.1186/s12964-024-01812-6.

[63] Briscoe J, Thérond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nature Reviews. Molecular Cell Biology. 2013; 14: 416–429. https://doi.org/10.1038/nrm3598.

[64] Jing J, Wu Z, Wang J, Luo G, Lin H, Fan Y, et al. Hedgehog signaling in tissue homeostasis, cancers, and targeted therapies. Signal Transduction and Targeted Therapy. 2023; 8: 315. https://doi.org/10.1038/s41392-023-01559-5.

[65] Grover VK, Valadez JG, Bowman AB, Cooper MK. Lipid modifications of Sonic hedgehog ligand dictate cellular reception and signal response. PloS One. 2011; 6: e21353. https://doi.org/10.1371/journal.pone.0021353.

[66] Wierbowski BM, Petrov K, Aravena L, Gu G, Xu Y, Salic A. Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand. Developmental Cell. 2020; 55: 450–467.e458. https://doi.org/10.1016/j.devcel.2020.09.017.

[67] Hui CC, Angers S. Gli proteins in development and disease. Annual Review of Cell and Developmental Biology. 2011; 27: 513–537. https://doi.org/10.1146/annurev-cellbio-092910-154048.

[68] Otto A, Schmidt C, Patel K. Pax3 and Pax7 expression and regulation in the avian embryo. Anatomy and Embryology. 2006; 211: 293–310. https://doi.org/10.1007/s00429-006-0083-3.

[69] Cairns DM, Sato ME, Lee PG, Lassar AB, Zeng L. A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3. Developmental Biology. 2008; 323: 152–165. https://doi.org/10.1016/j.ydbio.2008.08.024.

[70] Gustafsson MK, Pan H, Pinney DF, Liu Y, Lewandowski A, Epstein DJ, et al. Myf5 is a direct target of long-range Shh signaling and Gli regulation for muscle specification. Genes & Development. 2002; 16: 114–126. https://doi.org/10.1101/gad.940702.

[71] Francetic T, Li Q. Skeletal myogenesis and Myf5 activation. Transcription. 2011; 2: 109–114. https://doi.org/10.4161/trns.2.3.15829.

[72] Teboul L, Summerbell D, Rigby PWJ. The initial somitic phase of Myf5 expression requires neither Shh signaling nor Gli regulation. Genes & Development. 2003; 17: 2870–2874. https://doi.org/10.1101/gad.1117603.

[73] Voronova A, Coyne E, Al Madhoun A, Fair JV, Bosiljcic N, St-Louis C, et al. Hedgehog signaling regulates MyoD expression and activity. The Journal of Biological Chemistry. 2013; 288: 4389–4404. https://doi.org/10.1074/jbc.M112.400184.

[74] Gerber AN, Wilson CW, Li YJ, Chuang PT. The hedgehog regulated oncogenes Gli1 and Gli2 block myoblast differentiation by inhibiting MyoD-mediated transcriptional activation. Oncogene. 2007; 26: 1122–1136. https://doi.org/10.1038/sj.onc.1209891.

[75] Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature. 1996; 383: 407–413. https://doi.org/10.1038/383407a0.

[76] McDermott A, Gustafsson M, Elsam T, Hui CC, Emerson CP, Jr, Borycki AG. Gli2 and Gli3 have redundant and context-dependent function in skeletal muscle formation. Development (Cambridge, England). 2005; 132: 345–357. https://doi.org/10.1242/dev.01537.

[77] Tang Y, Wang YQ, Ni JY, Lin YT, Li YF. Hedgehog signaling is required for larval muscle development and larval metamorphosis of the mussel Mytilus coruscus. Developmental Biology. 2024; 512: 57–69. https://doi.org/10.1016/j.ydbio.2024.05.007.

[78] Kim JT, Zhou Y, Jeon DH, Kwon JW, Lee GY, Son HM, et al. Abstract 2322 Caudatin ameliorated muscle atrophy via Hedgehog signaling in C2C12 cells. Journal of Biological Chemistry. 2024; 300: 106046. https://doi.org/10.1016/j.jbc.2024.106046.

[79] Rowton M, Perez-Cervantes C, Hur S, Jacobs-Li J, Lu E, Deng N, et al. Hedgehog signaling activates a mammalian heterochronic gene regulatory network controlling differentiation timing across lineages. Developmental Cell. 2022; 57: 2181–2203.e2189. https://doi.org/10.1016/j.devcel.2022.08.009.

[80] Ahn S, Joyner AL. Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell. 2004; 118: 505–516. https://doi.org/10.1016/j.cell.2004.07.023.

[81] Lopez-Rios J. The many lives of SHH in limb development and evolution. Seminars in Cell & Developmental Biology. 2016; 49: 116–124. https://doi.org/10.1016/j.semcdb.2015.12.018.

[82] Zhang W, Yu J, Fu G, Li J, Huang H, Liu J, et al. ISL1/SHH/CXCL12 signaling regulates myogenic cell migration during mouse tongue development. Development (Cambridge, England). 2022; 149: dev200788. https://doi.org/10.1242/dev.200788.

[83] Liu HX, Maccallum DK, Edwards C, Gaffield W, Mistretta CM. Sonic hedgehog exerts distinct, stage-specific effects on tongue and taste papilla development. Developmental Biology. 2004; 276: 280–300. https://doi.org/10.1016/j.ydbio.2004.07.042.

[84] Jeong J, Mao J, Tenzen T, Kottmann AH, McMahon AP. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes & Development. 2004; 18: 937–951. https://doi.org/10.1101/gad.1190304.

[85] Audu GC, Rohan SY, Kumari A. Gas1 regulates embryonic tongue muscle proliferation, differentiation and maturation via alternative pathways to Hedgehog signaling. Development (Cambridge, England). 2025; 152: dev204868. https://doi.org/10.1242/dev.204868.

[86] Costa ML, Jurberg AD, Mermelstein C. The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis. Frontiers in Physiology. 2021; 12: 668600. https://doi.org/10.3389/fphys.2021.668600.

[87] Elliott KH, Chen X, Salomone J, Chaturvedi P, Schultz PA, Balchand SK, et al. Gli3 utilizes Hand2 to synergistically regulate tissue-specific transcriptional networks. eLife. 2020; 9: e56450. https://doi.org/10.7554/eLife.56450.

[88] Anderson C, Williams VC, Moyon B, Daubas P, Tajbakhsh S, Buckingham ME, et al. Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity. Genes & Development. 2012; 26: 2103–2117. https://doi.org/10.1101/gad.187807.112.

[89] Krüger M, Mennerich D, Fees S, Schäfer R, Mundlos S, Braun T. Sonic hedgehog is a survival factor for hypaxial muscles during mouse development. Development (Cambridge, England). 2001; 128: 743–752. https://doi.org/10.1242/dev.128.5.743.

[90] Li X, Blagden CS, Bildsoe H, Bonnin MA, Duprez D, Hughes SM. Hedgehog can drive terminal differentiation of amniote slow skeletal muscle. BMC Developmental Biology. 2004; 4: 9. https://doi.org/10.1186/1471-213X-4-9.

[91] Wu P, Yong P, Zhang Z, Xu R, Shang R, Shi J, et al. Loss of Myomixer Results in Defective Myoblast Fusion, Impaired Muscle Growth, and Severe Myopathy in Zebrafish. Marine Biotechnology (New York, N.Y.). 2022; 24: 1023–1038. https://doi.org/10.1007/s10126-022-10159-3.

[92] Shi J, Cai M, Si Y, Zhang J, Du S. Knockout of myomaker results in defective myoblast fusion, reduced muscle growth and increased adipocyte infiltration in zebrafish skeletal muscle. Human Molecular Genetics. 2018; 27: 3542–3554. https://doi.org/10.1093/hmg/ddy268.

[93] Bren-Mattison Y, Hausburg M, Olwin BB. Growth of limb muscle is dependent on skeletal-derived Indian hedgehog. Developmental Biology. 2011; 356: 486–495. https://doi.org/10.1016/j.ydbio.2011.06.002.

[94] Teixeira JD, de Andrade Rosa I, Brito J, Maia de Souza YR, Paulo de Abreu Manso P, Machado MP, et al. Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis. Biochemical and Biophysical Research Communications. 2018; 507: 496–502. https://doi.org/10.1016/j.bbrc.2018.11.071.

[95] Bren-Mattison Y, Olwin BB. Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage. Developmental Biology. 2002; 242: 130–148. https://doi.org/10.1006/dbio.2001.0528.

[96] Xu J, Liu H, Lan Y, Adam M, Clouthier DE, Potter S, et al. Hedgehog signaling patterns the oral-aboral axis of the mandibular arch. eLife. 2019; 8: e40315. https://doi.org/10.7554/eLife.40315.

[97] Mao C, Jiang Y, Li Z, Zhou W, Lai Y, Wang C, et al. Embryonic exposure to Smoothened Agonist disrupts tongue development in mice. Developmental Biology. 2025; 524: 36–46. https://doi.org/10.1016/j.ydbio.2025.04.018.

[98] Hirsinger E, Stellabotte F, Devoto SH, Westerfield M. Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors. Developmental Biology. 2004; 275: 143–157. https://doi.org/10.1016/j.ydbio.2004.07.030.

[99] Torii D, Soeno Y, Fujita K, Sato K, Aoba T, Taya Y. Embryonic tongue morphogenesis in an organ culture model of mouse mandibular arches: blocking Sonic hedgehog signaling leads to microglossia. In Vitro Cellular & Developmental Biology. Animal. 2016; 52: 89–99. https://doi.org/10.1007/s11626-015-9951-6.

[100] Leem YE, Han JW, Lee HJ, Ha HL, Kwon YL, Ho SM, et al. Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK. Cellular Signalling. 2011; 23: 2021–2029. https://doi.org/10.1016/j.cellsig.2011.07.016.

[101] Millington G, Elliott KH, Chang YT, Chang CF, Dlugosz A, Brugmann SA. Cilia-dependent GLI processing in neural crest cells is required for tongue development. Developmental Biology. 2017; 424: 124–137. https://doi.org/10.1016/j.ydbio.2017.02.021.

[102] Billmyre KK, Klingensmith J. Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation. Developmental Dynamics. 2015; 244: 564–576. https://doi.org/10.1002/dvdy.24256.

[103] Mackenzie S, Walsh FS, Graham A. Migration of hypoglossal myoblast precursors. Developmental Dynamics. 1998; 213: 349–358. https://doi.org/10.1002/(SICI)1097-0177(199812)213:4<349::AID-AJA1>3.0.CO;2-6.

[104] Kawasaki M, Kawasaki K, Sari FT, Kudo T, Nihara J, Kitamura M, et al. Cell-cell interaction determines cell fate of mesoderm-derived cell in tongue development through Hh signaling. eLife. 2024; 13: e85042. https://doi.org/10.7554/eLife.85042.

[105] Esteves de Lima J, Blavet C, Bonnin MA, Hirsinger E, Comai G, Yvernogeau L, et al. Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning. Nature Communications. 2021; 12: 3851. https://doi.org/10.1038/s41467-021-24157-x.

[106] Nassari S, Duprez D, Fournier-Thibault C. Non-myogenic Contribution to Muscle Development and Homeostasis: The Role of Connective Tissues. Frontiers in Cell and Developmental Biology. 2017; 5: 22. https://doi.org/10.3389/fcell.2017.00022.

[107] Han D, Zhao H, Parada C, Hacia JG, Bringas P, Jr, Chai Y. A TGFβ-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development. Development (Cambridge, England). 2012; 139: 1640–1650. https://doi.org/10.1242/dev.076653.

[108] Hosokawa R, Oka K, Yamaza T, Iwata J, Urata M, Xu X, et al. TGF-beta mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis. Developmental Biology. 2010; 341: 186–195. https://doi.org/10.1016/j.ydbio.2010.02.030.

[109] Elworthy S, Hargrave M, Knight R, Mebus K, Ingham PW. Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity. Development (Cambridge, England). 2008; 135: 2115–2126. https://doi.org/10.1242/dev.015719.

[110] Jackson HE, Ingham PW. Control of muscle fibre-type diversity during embryonic development: the zebrafish paradigm. Mechanisms of Development. 2013; 130: 447–457. https://doi.org/10.1016/j.mod.2013.06.001.

[111] Fogarty MJ, Sieck GC. Tongue muscle contractile, fatigue, and fiber type properties in rats. Journal of Applied Physiology (Bethesda, Md.: 1985). 2021; 131: 1043–1055. https://doi.org/10.1152/japplphysiol.00329.2021.

[112] Kitamura T, Kitamura YI, Funahashi Y, Shawber CJ, Castrillon DH, Kollipara R, et al. A Foxo/Notch pathway controls myogenic differentiation and fiber type specification. The Journal of Clinical Investigation. 2007; 117: 2477–2485. https://doi.org/10.1172/JCI32054.

[113] Everson JL, Fink DM, Yoon JW, Leslie EJ, Kietzman HW, Ansen-Wilson LJ, et al. Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis. Development (Cambridge, England). 2017; 144: 2082–2091. https://doi.org/10.1242/dev.149930.

[114] Maggs AM, Huxley C, Hughes SM. Nerve-dependent changes in skeletal muscle myosin heavy chain after experimental denervation and cross-reinnervation and in a demyelinating mouse model of Charcot-Marie-Tooth disease type 1A. Muscle & Nerve. 2008; 38: 1572–1584. https://doi.org/10.1002/mus.21106.

[115] Khodabukus A. Tissue-Engineered Skeletal Muscle Models to Study Muscle Function, Plasticity, and Disease. Frontiers in Physiology. 2021; 12: 619710. https://doi.org/10.3389/fphys.2021.619710.

[116] Méndez-Maldonado K, Vega-López GA, Aybar MJ, Velasco I. Neurogenesis From Neural Crest Cells: Molecular Mechanisms in the Formation of Cranial Nerves and Ganglia. Frontiers in Cell and Developmental Biology. 2020; 8: 635. https://doi.org/10.3389/fcell.2020.00635.

[117] Fabian P, Crump JG. Reassessing the embryonic origin and potential of craniofacial ectomesenchyme. Seminars in Cell & Developmental Biology. 2023; 138: 45–53. https://doi.org/10.1016/j.semcdb.2022.03.018.

[118] Calloni GW, Glavieux-Pardanaud C, Le Douarin NM, Dupin E. Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104: 19879–19884. https://doi.org/10.1073/pnas.0708806104.

[119] Hasson P, DeLaurier A, Bennett M, Grigorieva E, Naiche LA, Papaioannou VE, et al. Tbx4 and tbx5 acting in connective tissue are required for limb muscle and tendon patterning. Developmental Cell. 2010; 18: 148–156. https://doi.org/10.1016/j.devcel.2009.11.013.

[120] Besse L, Sheeba CJ, Holt M, Labuhn M, Wilde S, Feneck E, et al. Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis. Cell Reports. 2020; 30: 3552–3565.e3556. https://doi.org/10.1016/j.celrep.2020.02.037.

[121] Brandan E, Gutierrez J. Role of skeletal muscle proteoglycans during myogenesis. Matrix Biology: Journal of the International Society for Matrix Biology. 2013; 32: 289–297. https://doi.org/10.1016/j.matbio.2013.03.007.

[122] Mukund K, Subramaniam S. Skeletal muscle: A review of molecular structure and function, in health and disease. Wiley Interdisciplinary Reviews. Systems Biology and Medicine. 2020; 12: e1462. https://doi.org/10.1002/wsbm.1462.

[123] Rodgers KD, San Antonio JD, Jacenko O. Heparan sulfate proteoglycans: a GAGgle of skeletal-hematopoietic regulators. Developmental Dynamics. 2008; 237: 2622–2642. https://doi.org/10.1002/dvdy.21593.

[124] Duprez D, Lapointe F, Edom-Vovard F, Kostakopoulou K, Robson L. Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud. Mechanisms of Development. 1999; 82: 151–163. https://doi.org/10.1016/s0925-4773(99)00040-4.

[125] Sagai T, Amano T, Tamura M, Mizushina Y, Sumiyama K, Shiroishi T. A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings. Development (Cambridge, England). 2009; 136: 1665–1674. https://doi.org/10.1242/dev.032714.

[126] Kumari A, Franks NE, Li L, Audu G, Liskowicz S, Johnson JD, et al. Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis. PloS One. 2024; 19: e0294835. https://doi.org/10.1371/journal.pone.0294835.

[127] Donnelly CR, Kumari A, Li L, Vesela I, Bradley RM, Mistretta CM, et al. Probing the multimodal fungiform papilla: complex peripheral nerve endings of chorda tympani taste and mechanosensitive fibers before and after Hedgehog pathway inhibition. Cell and Tissue Research. 2022; 387: 225–247. https://doi.org/10.1007/s00441-021-03561-1.

[128] Seppala M, Xavier GM, Fan CM, Cobourne MT. Boc modifies the spectrum of holoprosencephaly in the absence of Gas1 function. Biology Open. 2014; 3: 728–740. https://doi.org/10.1242/bio.20147989.

[129] Seppala M, Depew MJ, Martinelli DC, Fan CM, Sharpe PT, Cobourne MT. Gas1 is a modifier for holoprosencephaly and genetically interacts with sonic hedgehog. The Journal of Clinical Investigation. 2007; 117: 1575–1584. https://doi.org/10.1172/JCI32032.

[130] Schiaffino S, Rossi AC, Smerdu V, Leinwand LA, Reggiani C. Developmental myosins: expression patterns and functional significance. Skeletal Muscle. 2015; 5: 22. https://doi.org/10.1186/s13395-015-0046-6.

[131] Sewry CA, Feng L, Chambers D, Matthews E, Phadke R. Importance of immunohistochemical evaluation of developmentally regulated myosin heavy chains in human muscle biopsies. Neuromuscular Disorders: NMD. 2021; 31: 371–384. https://doi.org/10.1016/j.nmd.2021.02.007.

[132] Rodriguez-Torres EE, Viveros-Rogel J, López-García K, Vázquez-Mendoza E, Chávez-Fragoso G, Quiroz-González S, et al. Chronic Undernutrition Differentially Changes Muscle Fiber Types Organization and Distribution in the EDL Muscle Fascicles. Frontiers in Physiology. 2020; 11: 777. https://doi.org/10.3389/fphys.2020.00777.

[133] Yang C, Li S, Li X, Li H, Li Y, Zhang C, et al. Effect of sonic hedgehog on motor neuron positioning in the spinal cord during chicken embryonic development. Journal of Cellular and Molecular Medicine. 2019; 23: 3549–3562. https://doi.org/10.1111/jcmm.14254.

[134] Ericson J, Morton S, Kawakami A, Roelink H, Jessell TM. Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell. 1996; 87: 661–673. https://doi.org/10.1016/s0092-8674(00)81386-0.

[135] Cai E, Barba MG, Ge X. Hedgehog Signaling in Cortical Development. Cells. 2024; 13: 21. https://doi.org/10.3390/cells13010021.

[136] Li X, Li Y, Li S, Li H, Yang C, Lin J. The role of Shh signalling pathway in central nervous system development and related diseases. Cell Biochemistry and Function. 2021; 39: 180–189. https://doi.org/10.1002/cbf.3582.

Funding

National Institutes of Health National Institute on Deafness and Other Communication Disorders(ECR R21 1R21DC017799)

Rowan-Virtua School of Osteopathic Medicine Seed Fund

Summer Medical Research Fellowship award