Meckel's cartilage breakdown offers clues to mammalian middle ear evolution

Abstract

A key transformation in mammalian ear evolution was incorporation of the primary jaw joint of premammalian synapsids into the definitive mammalian middle ear of living mammals. This evolutionary transition occurred in two-steps, starting with a partial or "transitional" mammalian middle ear in which the ectotympanic and malleus were still connected to the mandible by an ossified Meckel's Cartilage (MC), as observed in many Mesozoic mammals. This was followed by MC breakdown, freeing the ectotympanic and the malleus from the mandible and creating the definitive mammalian middle ear. Here we report novel findings on the role of chondroclasts in MC breakdown, shedding light on how therian mammals lost MC connecting the ear to the jaw. Genetic or pharmacological loss of clast cells in mice and opossums leads to persistence of embryonic MC beyond juvenile stages, with MC ossification in mutant mice. The persistent MC causes a distinctive postnatal groove on the mouse dentary. This morphology phenocopies the ossified MC and Meckelian groove observed in Mesozoic mammals. Clast cell recruitment to MC is not observed in reptiles, where MC persists as a cartilaginous structure. We hypothesize that ossification of MC is an ancestral feature of mammaliaforms, and that a shift in the timing of clast cell recruitment to MC prior to its ossification is a key developmental mechanism for the evolution of the definitive mammalian middle ear in extant therians.

Figure 1. cFos mutant mice mimic morphologies of PMME (“TMME”) in fossil mammaliaforms.

A: A generalized Partial (“Transitional”) Mammalian Middle Ear of eutriconodonts showing middle ear with ossified Meckel’s cartilage (reconstructed from refs3,6). B-C: Stained and cleared DMME middle ear from P3 wildtype (B) and cFos-/- mutant mice showing a persistence of Meckel’s cartilage, persistent cartilage in endochondrally ossifying elements such as the malleus and incus, and thickening of the tympanic ring and gonial bones. D-E: Picro-sirius red / alcian blue trichrome staining of horizontal section through malleus of postnatal day 6 (P6) wildtype (D) and cFos-/- (E) littermates. In wildtype mice the Meckel’s cartilage has undergone resorption and transformation to ligament connective tissue, whereas in the cFos-/- mutant mouse the Meckel’s cartilage is maintained (arrowhead in E). F-G: Col2a expression by in situ hybridization. Collagen type 2 (encoded by Col2a mRNA) is lost in Meckel’s derived ligament in wildtype mice (F), but continues to express during the prolonged retention of Meckel’s in cFos mutant. H-I Immunohistochemistry staining for the extracellular matrix protein Aggrecan (ACAN) shows weak expression in the ligament replacing the MC of the wildtype mouse (H) and strong expression MC of the cFos null (I) confirming a persistent cartilage (boxes in F and G show location of H and I). J-K: Picro-sirius red / alcian blue trichrome staining of horizontal sections of cFos mutant mouse at P26 (J) demonstrates that Meckel’s persists at this stage, and that calcified tissue is present. Furthermore, in situ hybridization for Osteocalcin (K) suggests that ossification occurs in the absence of Meckel’s breakdown. MC – Meckel’s Cartilage; M – Malleus; I – Incus; St – Stapes; Tymp – ectotympanic ring; G – Gonial; Cdy – Condylar process of dentary; Ang – Angular process of dentary; lig. – ligament. Scale bar in D-G,J and K = 100µm. Scale in H and I =50µm

Figure 2. Detachment of the malleus in mammals is due to recruitment c-Fos dependent chondroclasts,

A-D: Picro-sirius red / alcian blue trichome (A,D) tartrate-resistant acid phosphatase (TRAP) (B,E) , and Cathespsin K (CTSK) immunohistochemistry (C,F) staining of proximal Meckel’s cartilage in P4 wildtype (A-C) and cFos-/- (C-F) mice. TRAP and CTSK staining reveals chondroclast activity around the Meckel’s cartilage of wildtype mice, but not in cFos mutants. G. Stained and cleared mandible of new born corn snake reveals persistence of Meckel’s cartilage. H-J: Trichome staining of histological sections of 5 week old corn snake (Pantherophis guttatus). The Meckel’s cartilage persists (F), until it meets the ossified articular (I,J). K: TRAP staining in corn snakes demonstrates that chondroclasts and osteoclasts do not act upon Meckel’s cartilage, except where endochondral ossification has occurred at the articular. Box in F indicates approximate location of panels J and K. Meckel’s cartilage. MC – Meckel’s Cartilage; D – Dentary; SA – Surangular; A – Articular; Q – Quadrate Scale bar = 100µm.

Figure 3. Formation of Meckelian groove as evidence for presence of retained MC in mammaliaforms.

A: Mandible of the eutriconodont Repenomamus showing topographic relationship of Meckelian groove to surrounding structures on the lingual aspect of the dentary. The Meckelian groove (MG) distal section contains two thinner grooves; the Meckelian groove (MG) proximal extending below the mandibular foramen. B: 3D reconstruction of µCT scan of a P17 opossum (Monodelphis domestica). A Meckelian groove is present in the lingual surface of the dentary bone (red arrow). C: Picro-sirius red / alcian blue trichrome histological staining of a frontal section through the dentary Meckelian groove of P16 opossum. Meckel’s cartilage is located within the groove, indicating that the formation of the groove is dependent on the cartilage. D,E,D’,E’: 3D reconstruction of µCT scan of P21 wildtype and cFos-/- mouse mandibles. A groove is observed inferior to the mylohyoid ridge in the cFos-/- mouse (E,E’ red arrow), but not in the wildtype mouse (D,D’). F,G: Picro-sirius red / alcian blue trichrome histological staining of a frontal section through the dentary of P0 wildtype and cFos-/- mice, showing a groove formed by Meckel’s cartilage. MC – Meckel’s Cartilage; D – Dentary; mMHy – Mylohyoid muscle. Images A,B,D-E’ not shown to scale. Scale bar in C,F,G = 100µm.

Figure 4. Clast cell inhibition in the opossum Monodelphis domestica

µCT analysis of P22 opossum when treated with control carrier (A) or alendronate (B) injections. Breakdown of Meckel’s cartilage is reduced in treated pups when compared to control littermates (n=3 for each treatment). Distance between malleus (arrow x) and the Meckel’s cartilage (arrow y) was compared (n=3) indicating a significantly reduced separation of malleus and Meckel’s cartilage in treated opossums compared to controls (C). Error bars = 1 standard deviation.