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

Supplementary information Neal Anthwal, Daniel J. Urban, Zhe Xi-‐Luo, Karen E. Sears, Abigail S. Tucker

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONVOLUME: 1 | ARTICLE NUMBER: 0093

NATURE ECOLOGY & EVOLUTION | DOI: 10.1038/s41559-017-0093 | www.nature.com/natecolevol 1

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Supplementary Figure S1 – The presence of Meckel’s cartilage in postnatal stages of

reptiles. A -‐ Skeletal prep of juvenile chameleon. B -‐ Histological horizontal section of mandible

of adult gecko P.pictus.B’ Haematoxylin and eosin staining of boxed section in B to show

multinucleated clast cell are present in the articular bone, but absent in the Meckel’s cartilage. C -‐

Histological frontal section of mandible of adult green anole. A – Articular; D – Dentary; MC –

Meckel’s Cartilage; SA – Surangular; Q – Quadrate.



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Supplementary Figure S2-‐ Clast cell inhibition in the new born wildtype mice by

intraperitonael injection of Alendronate. A: At postnatal day 3 (P0) control treated pups

display a separation of the Meckel’s cartilage and malleus by wholemount alcian blue/alizarin

red staining. Histological staining shows that TRAP+ clast cells are present around the Meckel’s

cartilage, as well as in the alveolar bone. B: Clast cell inhibition by injection with alendronate at

P0 results in a prevention of Meckel’s cartilage breakdown by P3. Histological staining shows

that TRAP+ clast cells are absent from the Meckel’s region at P3 in treated pups, although a few

scantly distributed clast cells were observed in the alveolar bone. G – Gonial; I – Incus; M –

Malleus; MC – Meckel’s Cartilage; St – Stapes; Tymp – Ectotympanic Ring.




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Supplementary Figure S3 – Structural relationship of Meckel’s cartilage to the gonial

groove of malleus and the Meckelian groove of the dentary in P17 opossum. A-‐G: 3D

reconstruction of µCT showing the gonial trough of the malleus, and Meckelian groove in dentary

accommodating Meckel’s cartilage during pouch young P17 stage. H: frontal section through

gonial region of middle ear of P16 opossum showing the gonial trough supporting Meckel’s

cartilage. D -‐ Dentary; G – Gonial; M – Malleus; MC – Meckel’s Cartilage T – Tympanic ring.




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Supplementary Figure S4 -‐ Separate location of Meckel’s cartilage from mandibular

branch of the trigemal nerve in living placentals and marsupials. The mandibular canal that

carries the mandibular branch of the trigeminal nerve (V3), is separated from the groove/canal

that carries the Meckel’s cartilage. This corresponds to the pattern of the Meckelian groove and

the mandibular foramen in extinct mammaliaforms. A-‐C, Picro-‐sirius red / alcian blue trichrome

histological staining of a frontal section through the opossum (A), fruit bat (B) and mouse (C)

dentary. D – Dentary; MC – Meckel’s Cartilage; V3 – Mandibular (3rd) branch of the trigeminal

nerve (cranial verve V). Scale bar = 100µm.




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Supplementary Figure S5-‐ Variation in MC breakdown observed in Alendronate treated

opossum pups. Dorsal view of µCT reconstruction of middle ear region of control treated (A)

and alendronate treated (B) opossums. Distance between the distal edge of the malleus and the

proximal edge of Meckel’s cartilage was measured and presented here as “Gap=x”.




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Supplementary Figure S6 – Comparison in Meckel’s cartilage related structures of the

mandible among Mesozoic mammals, opossum, and the c-‐Fos mutant mice with prolonged

retention of ossified Meckel’s cartilage and Meckelian groove. A and B, Monodelphis

domestica (P17), composite image of both the jaw and the middle ear structure, with Meckel’s

cartilage reconstructed from the serial histological sections (not possible to image by CT but can

be seen in clear-‐stain and in serial histological sections) and the ossified incus, malleus and

ectotympanic and dentary (rendered from CT). The Meckel’s cartilage is nestled in the Meckelian

groove (A) and Meckelian cartilage is removed to show its corresponding Meckelian groove on

the mandible (B). C. c-‐Fos mutant mouse (P21) showing the Meckelian groove is retained well




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beyond its normal resorption immediately after P2 in wildtypes due to c-‐Fos-‐dependent clast

activities. D. Maotherium (stereophotos from Ref 8) with in situ preservation of the ossified

Meckel’s cartilage; it also shows a gracile Meckel’s cartilage corresponding to a shallow and

gracile Meckelian groove on the dentary, an example of a thinner cartilage and groove, among a

wide range of variation of the both structures. E. Eutriconodont Juchilestes: the mandible without

the ossified Meckel’s cartilage 37 and Meckel’s cartilage (image rendered from dataset published

by Ref. 37) reconstructed into the Meckelian groove. This shows an example of very deep

Meckelian groove and relatively stout Meckel’s cartilage. F. Repenomamus: example of an wide

and shallow Meckelian groove. The Meckelian groove and mandibular foramen (posterior

opening of mandibular canal) show similar topographic relationship in Mesozoic mammals as

their counterparts in opossum and the c-‐Fos mutant mouse.




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Supplementary Information

Comparative Morphology of the Meckelian Groove

Our new observations on the cellular and tissue-‐level mechanisms for MC ossification and

breakdown by clast cell activity in therians, alongside other genes and signaling pathways

revealed in the last decade (reviewed in Refs11,32,38) have built a compelling case that the

developmental potential of MC ossification is deeply conserved in extant mammals. This new

insight from molecular genetics of development, and the new fossils discovered since 200139

are corroborating each other, as an ossified Meckel’s cartilage has now been found in the two

major clades nested within the crown Mammalia. Nonetheless, the fossilized Meckel’s

cartilages of these Mesozoic mammals are different in shape from those in embryos and

neonates of extant mammals. Their unique morphologies 7,8,15,40,41 and their very wide range

of size variation has brought into question the identify of these features given the more

limited range of morphology of the transient Meckel’s cartilage in extant mammals. Here we

offer our comparative analyses of the historical alternative hypotheses.

Recently Maier and Ruf suggested an alternative interpretation of the ossified “Meckel’s

cartilage” could be an elongated and massive gonial, based partly upon the perceived size

discrepancy of the larger size of ossified Meckel’s in some Mesozoic mammals, for example

Repenomamus, and the relatively smaller Meckel’s cartilage of extant amniotes5.

There is a wide range of variation in the thickness of the Meckel’s element among Mesozoic

mammaliaforms. The thickness of Meckel’s cartilage is only about 0.3 mm thick (along the

widest dimension) for Yanoconodon and Jeholodens41, or about 0.3 to 0.35 mm in

Zhangeotherium and Maotherium8. The more robust Meckel’s cartilage can be 3 to 4 mm thick

(in the widest dimensions) for Repenomamus (the largest Mesozoic mammal known)7,39 and 1




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to 1.5 mm for Gobiconodon40. This element is also very thick in Spinolestes 26. The three

mammals with thicker Meckel’s all belong to the Gobiconodontidae. Their larger Meckel’s

cartilage is autapomorphic and unique to the gobiconodontid clade26, and is more of an

exception than a rule for the more inclusive clade of eutriconodonts, even more so for the

whole Mammalia. In the more basally placed eutriconodonts Yanoconodon and Jeholodens, the

Meckel’s cartilage is gracile, and thin enough to be within the size range of the transient

Meckel’s cartilage reported for embryos of extant mammals 5. Thus the perceived size

difference between the transient cartilage of extant mammals and Mesozoic mammaliaforms

is not conclusive.

Our histology data and µCT scans (Fig. 3 and Supplementary Fig S3) show that Meckel’s

cartilage itself becomes ossified in the c-‐fos null mouse and that in all cases Meckel’s is nestled

within, but is distinct from the gonial. Importantly the gonial in both the mouse and opossum

does not extend into the dentary and therefore does not make the groove in the c-‐fos mutants

or in P16 opossums. In the opossum the gonial appears to support MC posterior to the dentary

prior to function of the forming TMJ. Separation of an ossified Meckel’s cartilage and the gonial

part of the malleus is also preserved in Liaoconocon15. This rules out the possibility that the

entire ossified structure connecting the mandible to the middle ear in the Mesozoic mammals

would be a hypertrophied gonial.

An implicit assumption in considering the ossified Meckel’s cartilage to be a hypertrophied

and elongate gonial is that in normal development of extant mammals, the gonial ossifies but

the Meckel’s cartilage cannot. However, there are exceptions in extant mammals to this

pattern. For example there is evidence that in domestic dog Canis familiaris, the Meckel’s

cartilage section in the canine-‐premolar region is enclosed by and co-‐ossified with the




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dentary42, as is lateral incisor region of Meckel’s cartilage in humans43. Further more

developmental studies have shown that Meckel’s cartilage can ossify in mouse knock-‐outs32,

therefore, in agreement with our data, MC has the potential to form bone.

In the historical literature, the long and gracile distal extension of the mandibular middle ear

of Morganucodon was interpreted to be the prearticular44,45 and it has been widely accepted

that a part of the proximal extremity of the prearticular is homologous to the gonial element of

the malleus of extant mammals46. In the wake of better preserved middle ear structure in

many newer fossils of spalacotherioids (Zhangheotherium and Maotherium) and

eutriconodonts (Jeholodens, Yanoconodon, Repenomamus, and Gobiconodon), Kermack’s

prearticular has been re-‐interpreted to be the ossified Meckel’s cartilage3,6,15,26,41. The “gonial

interpretation” of the ossified Meckel’s cartilage in eutriconodonts5 would have been

consistent with Kermack’s interpretation of a long prearticular in Morganucodon44,45 but the

latter has not been supported by more recent studies (e.g., refs. 6,15).

Since the 1970’s, there has been no debate as to the identity of the Meckelian groove as

abundant fossils have revealed the so-‐called internal mandibular groove actually

accommodates the postdentary bony element connected to the middle ear45,47. But in the

historical literature, Simpson (1928)48 suggested an interpretation that the mylohyoid nerve

and artery bundle might be the main occupant of what is now universally accepted to be the

Meckelian groove. Some contemporary studies have used the Simpson’s non-‐committal

morphological term “internal mandibular groove”, but interpreted the primary occupant of

the groove to be the Meckel’s element. Simpson’s 48 proposal was based primarily on Homo

sapiens where a well-‐defined groove for the mylohyoid nerve and artery extends from the

mandibular foramen on the dentary. A similar groove is also present in the dasypodid




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xenarthran Priodontes48. To the contrary, our anatomical survey shows that the mylohyoid

groove seen in human is absent in all prosimians and some anthropoids; it is independently

derived within anthropoid primates. The mylohyoid groove in the dasypodid xenarthran

Priodontes48 is also independently derived within xenathrans, the majority of which lack this

feature. Our histological observation (Supplementary Fig S4) shows that Meckel’s cartilage is

the sole occupant of the Meckelian groove, and that the nerve and artery occupy a separate

compartment in the dentary in mouse, fruit bat, and opossum.

Additional References

36. Ealba, E. L. et al. Neural crest-‐mediated bone resorption is a determinant of species-‐

specific jaw length. Developmental Biology 408, 151–63 (2015).

37. Gao, C.-‐L. et al. A new mammal skull from the Lower Cretaceous of China with

implications for the evolution of obtuse-‐angled molars and ‘amphilestid’ eutriconodonts.

Proceedings of the Royal Society B: Biological Sciences 277, 237–246 (2010).

38. Oka, K. et al. The role of TGF-‐beta signaling in regulating chondrogenesis and

osteogenesis during mandibular development. Developmental Biology 303, 391–404 (2007).

39. Li, J., Wang, Y., Wang, Y. & Li, C. A new family of primitive mammal from the Mesozoic of

western Liaoning, China. Chinese Science Bulletin 46, 782–785 (2001).

40. Li, C., Wang, Y., Hu, Y. & Meng, J. A new species of Gobiconodon (Triconodonta,

Mammalia) and its implication for the age of Jehol Biota. Chinese Science Bulletin 48, 1129–

1134 (2003).

41. Luo, Z.-‐X., Chen, P., Li, G. & Chen, M. A new eutriconodont mammal and evolutionary

development in early mammals. Nature 446, 288–293 (2007).

42. Evans, H. E. & de Lahunta, A. Miller’s Anatomy of the Dog. (Elsevier, 2013).




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43. Shibata, S., Sakamoto, Y., Yokohama-‐Tamaki, T., Murakami, G. & Cho, B. H. Distribution

of matrix proteins in perichondrium and periosteum during the incorporation of Meckel’s

cartilage into ossifying mandible in midterm human fetuses: an immunohistochemical study.

The Anatomical Record 297, 1208–1217 (2014).

44. Kermack, K. A., Mussett, F. & Rigney, H. W. The skull of Morganucodon. Zoological

Journal of the Linnean Society 71, 1–158 (1981).

45. Kermack, K. A., Mussett, F. & Rigney, H. W. The lower jaw of Morganucodon. Zoological

Journal of the Linnean Society 53, 87–175 (1973).

46. Zeller, U. in Mammal Phylogeny; Mesozoic Differentiation, Multituberculates,

Monotremes, Early Therians, and Marsupials (eds. Szalay, F. S., Novacek, M. J. & McKenna, M. C.)

95–107 (Springer New York, 1993).

47. Allin, E. F. Evolution of the mammalian middle ear. Journal of Morphology 147, 403–

437 (1975).

48. Simpson, G. G. Mesozoic Mammalia, XII; the internal mandibular groove of Jurassic

mammals. American Journal of Science 15, 461–470 (1928).




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