Re-orientation of the hallux during development

The cartilaginous precursor of Mt1 originates in the medial side of the foot, almost perpendicular to the limb’s main axis (HH30, fig. 2a, black arrowhead). A change in orientation of Mt1 begins at HH32, as the entire hallux becomes tilted, with its distal end slightly displaced towards ventral and rotates such that its ventral side comes to face the lateral side of the foot, much like a human thumb (see HH32, fig. 2b-d). This orientation is similar to that of most non-avian tetanurans10,11. At stage HH36, D1 is re-oriented such that the entire hallux becomes perpendicular to the main limb axis, in an elevated, spur-like orientation (fig. 2b-d). Also at HH36, the proximal contact of Mt1 to Mt2 moves markedly towards the ventral face of Mt2 (fig. 2b-d). However, this does not cause retroversion: The ventral side of the hallux continues pointing towards the lateral side of the limb, with Mt1 abducted almost perpendicular to the tarsometatarsus. At HH38 an opposable hallux has been attained: It is no longer spur-like, presenting an opposable orientation (i.e., pointing towards ventral, fig. 2b-d) and its lateral side faces the medial side of the foot. Figure 2e summarizes the changes in the orientation of Mt1 with respect to Mt2 in successive stages of development.

Figure 2 Re-orientation of the hallux. (a) Quail embryo stained with Alcian Blue, showing the small size and position of the hallux in the early stages of cartilage development (HH30); (b) Dorsal and medial views of Alcian Blue/Alizarin Red stained feet skeletons of quail embryos showing changes in the orientation of the hallux; (c) Details of hallux skeleton in dorsal view; (d) Details of hallux skeleton in medial view; (e) Diagram picturing the changing orientation of MT1 between HH32 and HH36 in relation to MT3 and MT2. Full size image

Maturation and ossification of Mt1 is delayed and begins at its proximal end

Embryonic cartilages are initially composed by immature chondrocytes, characterized by the production of collagen type IIA (COLII)12. Maturation of metatarsals 2,3 and 4 begins at mid-shaft as cells become flattened and start to secrete Indian hedgehog protein (IHH)13,14. Those cells later exit the cell cycle, begin to grow (hypertrophy), stop producing COLII and synthetize collagen type X (COLX)15. Eventually, the hypertrophic cells die by apoptosis, leaving their extracellular matrix for ossification and remodeling. This dynamic of expansion from the center towards the ends generates two fronts of differentiation – the growth plates16. This maturation pattern was also the ancestral condition for Mt1, as observed in crocodylians (bird’s closest living relatives) and other amniotes17. The ossification of Mt2-4 begins around HH34 and their diaphyses are well ossified at HH37. In contrast with the other three avian metatarsals, Mt1 is a small element since the beginning of chondrogenesis (fig. 2a). The size difference increases at HH32, when the other metatarsi elongate conspicuously and Mt1 keeps its small elliptic shape. Differentiation of Mt1 is greatly delayed and follows an unusual proximal-to-distal pattern. The presence of COLII as a marker of immature chondrocytes is detected in the entire element as late as HH38 (fig. 3a). In contrast, metatarsals 2, 3 and 4 exhibit large central regions with no COLII since HH34 (fig. 3f). IHH is clearly present in HH32 of other metatarsi but only becomes present in Mt1 at HH34, when its production starts in the proximal end (fig. 3b). Histological sections show that hypertrophic chondrocytes appear at the proximal end of the Mt1 at HH35 (fig. 3c). COLX synthesis begins in the proximal end of Mt1 at HH38 and advances towards distal in the next stages (fig. 3d). Calcification starts in the proximal end only at HH40 (SI, fig. S1) and the distal half of Mt1 is covered by collar bone at HH42 (fig. 3e)18,19,20. As a result of delayed differentiation from proximal to distal, Mt1 remains completely cartilaginous until exceptionally late (HH38) with a small diaphysis and just one growth zone in the distal end. Even after the beginning of ossification, the cartilaginous epiphysis continues to occupy about half its total length (HH42).

Figure 3 Arrested endochondral ossification of Mt1: (a) COLII expression during the development of Mt1 indicates the distribution of immature chondrocytes; (b) IHH expression during the development of Mt1 indicates the distribution of pre-hypertrophic chondrocytes; (c) Paraffin sections of Mt1 at HH34 and HH35 show the appearance of hypertrophic cells in the proximal side at HH35; (d) COLX expression during the development of Mt1 indicates the distribution of hypertrophic chondrocytes; (e) Calcein staining during the development of Mt1 showing the beginning of ossification; (f) Dorsal view of COLII (purple) and IHH (green) expression during the development of quail left metatarsi. Full size image

The torsion of Mt1

In quail and chicken embryos at HH35, the long axis of Mt1 is straight. At HH36 it becomes slightly twisted at its proximal end and at HH40 torsion is conspicuous along its entire axis (fig. 4a), similar to the completely ossified Mt1 of a juvenile chicken (fig. 4b). As described above, at HH36 the lateral side of the Mt1 is facing distal (figs. 2b-d and 4c, red line). The torsion of Mt1 is responsible for the change from this orientation to a completely opposable digit: as Mt1 twists, the lateral side of D1 changes from facing distal to facing medial (fig. 4c, red line). Throughout torsion (HH36-HH38) Mt1 is an immature, COLII-expressing cartilage (fig. 3a).

Figure 4 Torsion of MT1. (a) Alcian Blue/Alizarin Red stained Mt1 of chicken embryos showing the onset of its torsion. Arrows indicate the direction of torsion; (b) Alizarin stained Mt1 from a juvenile chicken in dorsal and ventral view; (c) Diagram picturing from proximal view the torsion orientation of Mt1. Full size image

Muscle development and activity in the hallux

Three muscles – two flexors and one extensor – control the avian hallux. The muscle flexor hallucis longus (FHL) is the only extrinsic muscle of the hallux – its belly is situated outside the foot, in the ventral shank; its tendon goes through the lateral hypotarsus and crosses obliquely to the ventral tarsometatarsus and its principal insertion is in the ventral base of the ungual phalanx (fig. 5c). Quail embryos exhibit a well-individualized FHL at HH33, as visualized by whole-mount immunofluorescence against Myosin (fig. 5a). Its insertion is discernible by Tenascin expression at HH35 (fig. 5b, white arrowhead).

Figure 5 The early development of hind limb muscles and tendons: (a) Whole-mount immunofluorescence against myosin in quail embryos between HH34 and HH36 reveals the development of musculus extensor hallucis longus (EHL), m. flexor hallucis longus (FHL) and m. flexor hallucis brevis (FHB); (b) Whole-mount immunofluorescence against tenascin in quail embryos reveals the insertions of EHL (whitearrow), FHB (white arrowhead) and FHL (red arrowhead) and the secondary insertion of the EHL (red arrow); (c) Schematic representation of the foot musculoskeletal system at HH36 and the muscular forces proposed to provoke the twisting of Mt1. Full size image

The other flexor of the hallux, the muscle flexor hallucis brevis (FHB), is located in the medio-ventral side of the foot; its tendon inserts in the ventral side of Mt1 (or at the base of the proximal phalanx, depending on the species)21. The muscle originates from a common mass of muscular fibre for all ventral intrinsic muscles. Its distal part is discernible since HH33 (fig. 5a); its insertion in the ventral Mt1 is visible at HH35 (fig. 5b, red arrowhead).

The musculus extensor hallucis longus (EHL) is the only extensor of the hallux. It arises from the dorso-medial border of Mt2 and its main insertion is in the base of the ungual phalanx. A secondary insertion in the base of the proximal phalanx is common. In quail embryos, it separates from the dorsal mass of intrinsic muscles at HH35 (fig. 5a), when its insertion is already visible (fig. 5b, white arrow); a secondary insertion is present at HH36 (fig. 5a, red arrow). Our data show that the musculoskeletal system is completely connected to the hallux at HH36, when torsion begins. The onset of digit movements occurs as soon as the muscular system is anatomically functional22. Digits are immobile until HH34, when the movements of the foot are restricted to the ankle joint. The first digit movements – synchronous extensions of all digits – appear at HH35, probably due to the action of the musculus extensor digiti longus. The flexion of the digits, including the hallux, starts at HH36 (see SI, Movie 1).

The effect of muscular paralysis on hallux retroversion

The temporal dynamics of muscle development and activity suggests their involvement in the torsion of Mt1. Pharmacological and genetic impairing of embryonic muscular activity have been widely used to show its importance in shaping the vertebrate skeleton23,24,25,26,27,28. We tested the hypothesis that muscular activity is influencing Mt1 torsion by paralyzing chick embryos before the twisting of Mt1 (HH34). Paralyzed embryos show normal ventral displacement of Mt1 (fig. 6b), indicating that the change of the articulation site to Mt2 from medial to ventral is not influenced by muscular activity. Nevertheless, in paralyzed embryos the ventral side of the hallux faces the medial side of the foot and at HH40 its long axis lays parallel to the long axis of Mt2, similar to control embryos before the torsion of the hallux (fig. 6a,b). Consistent with this, Mt1 of paralyzed embryos fails to twist and has a straight shape (fig. 6c). Importantly, the morphology of Mt1 in paralyzed chick embryos is remarkably similar to that of early tetanuran dinosaurs like Allosaurus29 (fig. 6d), having a straight shaft and a ventral side facing the medial side of Mt2 (See SI, Movie 2).