The genesis of cartilage size and shape during development and evolution

Abstract

How do cartilaginous elements attain their characteristic size and shape? Two intimately coupled processes underlie the patterned growth of cartilage. The first is histogenesis, which entails the production of cartilage as a discrete tissue; the second is morphogenesis, which pertains to the origins of three-dimensional form. Histogenesis relies on cues that promote the chondrogenic differentiation of mesenchymal cells, whereas morphogenesis requires information that imbues cartilage with stage-specific (e.g. embryonic versus adult), region-specific (e.g. cranial versus appendicular) and species-specific size and shape. Previous experiments indicate that early programmatic events and subsequent signaling interactions enable chondrogenic mesenchyme to undergo histogenesis and morphogenesis, but precise molecular and cellular mechanisms that generate cartilage size and shape remain unclear. In the face and jaws, neural crest-derived mesenchyme clearly plays an important role, given that this embryonic population serves as the source of chondrocytes and of species-specific patterning information. To elucidate mechanisms through which neural crest-derived mesenchyme affects cartilage size and shape, we made chimeras using quail and duck embryos, which differ markedly in their craniofacial anatomy and rates of maturation. Transplanting neural crest cells from quail to duck demonstrates that mesenchyme imparts both stage-specific and species-specific size and shape to cartilage by controlling the timing of preceding and requisite molecular and histogenic events. In particular, we find that mesenchyme regulates FGF signaling and the expression of downstream effectors such as sox9 and col2a1. The capacity of neural crest-derived mesenchyme to orchestrate spatiotemporal programs for chondrogenesis autonomously, and to implement cartilage size and shape across embryonic stages and between species simultaneously, provides a novel mechanism linking ontogeny and phylogeny.

Fig. 1. Experimental design and methods

(A) Lower jaw skeletons of adult Japanese quail (Coturnix coturnix japonica) and white Pekin duck (Anas platyrhyncos). (B) Schematic of rostral neural tube at HH9.5, depicting the levels of neural crest cells grafted from quail to duck. (C) Schematic of a lower jaw skeleton at HH39, depicting the contributions of transplanted neural crest (red) to cartilage and bone (stippled). (D) Horizontal section through the mandibular primordium of a HH29 chimeric quck embryo (rostral at top), which will give rise to the lower jaw skeleton. Quail donor mesenchyme (black), as visualized by the quail-specific antibody Q¢PN, was found throughout the transplanted side, whereas few to no quail cells were observed on the contralateral duck host side. (E) Graph illustrating the distinct developmental trajectories of quail (red squares) versus duck (blue circles) after being stage-matched at HH9.5 for surgery (yellow triangle on y-axis). Control quail and duck embryos were separated by approximately three HH stages within 2 days of surgery, and throughout the initial stages of overt mandibular chondrogenesis (gray area).

Fig. 2. Mesenchyme determines the size and shape of Meckel’s cartilage

(A,B) Meckel’s cartilage in control quail and duck embryos was relatively short at HH28 (stained with Alcian Blue and shown in ventral view with distal towards the top). (C) In HH28 chimeric quck mandibles, the host Meckel’s cartilage was equivalent to an HH28 duck, but the quail donor side (right of broken line) resembled the size and shape of a control quail Meckel’s cartilage at HH31. (D,E) Meckel’s cartilage was slightly curved at HH31 in quail and duck. (F,G) At HH32, Meckel’s cartilage was S-shaped in quail and duck. (H) In HH32 quck, the host Meckel’s cartilage was like an HH32 control duck, but the quail donor side matched the size and shape of a quail Meckel’s cartilage at HH35. (I,J) By HH35, Meckel’s cartilage began to straighten, but some curvature persisted in duck. (K,L) This straightened morphology became augmented by HH38. (M) In HH38 quck mandibles, both the quail-derived Meckel’s cartilage and the contralateral duck Meckel’s cartilage were straightened, but the quail-derived Meckel’s cartilage was shorter than its duck-derived counterpart, and was more similar in size to control quail Meckel’s cartilage at HH41. (N,O) By HH41, the size and shape of Meckel’s cartilage was reflective of adult morphology.

Fig. 3. Landmark-based analysis of ontogenetic and phylogenetic size and shape

(A) Fifteen landmark points were selected along Meckel’s cartilage in quail, duck and/or quck embryos at HH28, HH31, HH38 and HH41. (B) X, Y coordinate data were analyzed using a Procrustes method, which removes the factor of size and reveals shape differences. (C) The average of the squared magnitudes of the vectors produced distance coefficients that were used in cluster analyses (unweighted pair group method using arithmetic averages). On the basis of overall shape similarity, duck at HH28 and HH31, and the duck host side of quck at HH28 were more alike than quail at HH31 and the quail donor side of quck at HH28; quail at HH38 and HH41, and the quail donor side of quck at HH38 were more alike than duck at HH38 and HH41, and the duck host side of quck at HH38. (D) When differences in size were included in the analysis, the groups clustered mostly by stage rather than by species. In addition, the relative amount of similarity was much less between early and late stages due to the vast differences in size between early and late stages (i.e. those associated with growth), and between quail and duck (i.e. those that are species specific).

Fig. 4. Mesenchyme regulates late histogenesis of Meckel’s cartilage

(A) Whole-mount Alcian Blue stained embryos at HH25 reveal that cartilage has yet to form in proximo-lateral regions of the avian mandible (arrow). (B) Duck host mesenchyme was negative for the anti-quail antibody Q¢PN, as shown in sagittal section. (C) By contrast, donor sides of HH25 chimeric quck mandibles contained abundant quail neural crest-derived mesenchyme. (D) Jaw cartilages became obvious by HH28 (arrow). (E) HBQ-stained histological sections through the jaw joint of control embryos at HH25 revealed diffuse Alcian Blue staining in mesenchyme with ill-defined borders. (F) Similar low diffuse levels of Alcian Blue were observed in host sides of HH25 quck mandibles. (G) In conjunction with the presence of relatively older quail donor cells, developing cartilages of quck chimeras stained strongly with Alcian Blue and exhibited a defined perichondrium. (H) Robust Alcian Blue staining and a clear perichondrium characterized developing cartilages at HH28. (I) Mesenchyme of the mandible was not immunoreactive for Collagen type II protein (Col2) in control embryos at HH25. (J) The host sides of chimeras were also negative for Col2 protein. (K) Quail-derived mesenchyme of HH25 quck mandibles demonstrated strong Col2-immunoreactivity. (L) Control HH28 mandibular cartilages were also positive for Col2 protein. (M) col2a1 expression appeared diffuse in developing mandibular cartilages in control HH25 embryos. (N) The host side of quck at HH25 also showed low levels of col2a1 expression. (O) Quail-derived mesenchyme of HH25 chimeric mandibles had more spatially resolved col2a1 domains, as well as higher col2a1 expression levels, when compared with contralateral duck host mesenchyme. (P) Similar expression domains were observed at HH28 in control quail.

Fig. 5. Mesenchyme regulates early histogenesis of Meckel’s cartilage

(A) Whole-mount Alcian Blue stained embryos at HH22 reveal that cartilage has yet to form in proximo-lateral regions of the avian mandible (arrow). (B) Duck host mesenchyme was negative for the anti-quail antibody Q¢PN as shown in sagittal section. (C) By contrast, donor sides of HH22 chimeric quck mandibles contained abundant quail neural crest-derived mesenchyme. (D) Jaw cartilages were still not present at HH25 (arrow). (E–H) Similarly, HBQ-stained histological sections through the jaw joint of control and chimeric embryos revealed diffuse Alcian Blue staining in mandibular mesenchyme. (I,J) The chondrogenic transcription factor sox9 was expressed broadly at low levels from the endodermal pouch across mandibular mesenchyme in control embryos, as well as the host side of chimeric quck at HH22. (K) On the donor side, coincident with Q¢PN-positive mesenchyme, sox9 expression was restricted at a distance from the endodermal pouch and levels were considerably higher than that observed on the contralateral host side. (L) Expression of sox9 in control quail embryos at HH25 was equivalent to the donor side of quck at HH22. (M,N) col2a1 was not detected in mandibular mesenchyme of control embryos or the host side of quck at HH22. (O,P) However, col2a1 was expressed in the pre-cartilaginous condensations on the donor side of quck and of control embryos at HH25. (Q–T) fgf4 was expressed continuously at HH25, HH22 and earlier in the epithelium of control and chimeric mandibles. (U,V) fgfr2, which encodes a receptor for FGF4, was not expressed in mandibular mesenchyme of control embryos or in the host side of chimeric quck at HH22. (W,X) fgfr2 transcripts were abundant in quail donor-derived mesenchyme of quck at HH22 like that observed in controls at HH25.

Fig. 6. FGF signaling regulates the timing of mandibular chondrogenesis

(A,C) Quail mandibles harvested at HH24 and cultured for 2 days show robust histological staining throughout Meckel’s cartilage (Alcian Blue). (B,D) Those mandibles treated biochemically with SU5402, which inhibits FGF signaling, lack cartilage matrix staining. (E) Collagen type II protein (Col2) is detected in control mandibles after 2 days of culture. (F) No Col2 protein is observed following treatment with SU5402.

Fig. 7. Mesenchymal regulation of chondrogenesis

Quail-duck chimeras reveal spatiotemporal plasticity in the molecular and histogenic programs underlying cartilage development. Bars represent stages when events are initiated in quail and duck, and the extent to which they are accelerated in quck chimeras.