Development and tissue origins of the mammalian cranial base

Abstract

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the "New Head".

Figure 1

Morphogenesis of the chondrocranium as shown by Alcian blue stained and cleared whole mount C57BL/6J embryos from Theiler Stages18 to 24 (E11 to E16). All images show the chondrocranium from a dorsal aspect (norma basalis interna) except for A, which shows a lateral perspective. (A,B) The parachordal cartilage is the first to appear caudally in the cranium at E11 in close association with the notochord (arrows). (C–G) Between E12 and E15, individual cartilages chondrify. (H) At E16, the chondrocranium is fully formed in the mouse; all cartilages have fused and ossification has just begun in the posterior chondrocranium. *=hypochiasmatic cartilage. Scale bar in H=1 mm. See key for abbreviations.

Figure 2

Dorsal view of endocranium in whole mounts showing development of cartilages and bones in the optic region. (A) At E14, the orbital, frontal, hypochiasmatic, and trabecular cartilages are present in the optic region of the chondrocranium. (B) At 1-day after birth (P1), ossification has begun in the body of the presphenoid bone, the frontal bone, and the greater wing (ala temporalis) of the basisphenoid bone. (C) By at least 6 months after birth, extensive ossification and remodeling has occurred around the optic foramen, with the hypochiasmatic and orbital cartilages forming the lesser wing of the prepshenoid bone. The arrow indicates the portion of the postoptic root of the lesser wing that derives from the hypochiasmatic cartilage. Laterally, the frontal bone and greater wing of the basisphenoid bone have formed interdigitations where they meet. Scale bars in A–C=0.5 mm. See key for abbreviations.

Figure 3

In situ hybridization of Col2 on sagittal sections of C57BL/6J embryos from E12 through E16. (A) At E12, Col2 expression can be seen in the parachordal cartilage, notochord (at tip of arrow), and some mesenchyme of the anterior cranial base. (B–D) Expression of Col2 increases in cartilages of the cranial base as they continue to chondrify from E13 to E15. (E) At E16, detection of Col2 decreases in the parachordal and hypophyseal anlagen where endochondral ossification is beginning. Normal fenestrae disrupt the continuity of cartilages in some sections (C & E); these are not present in the adult cranial base. See key for abbreviations.

Figure 4

Neural crest and mesodermal contributions to the E10.5 cranial base of X-Gal stained crania. (A) Ventral view of a Wnt1-Cre/R26R cranium cut transversely just inferior to the maxillary processes and through the fourth ventricle showing cells of the neural crest lineage inhabiting the ventral face. Neural crest cells are not present in the ectodermal invagination of Rathke’s pouch (arrow) or in tissue just caudal to it. (B) A section of a Wnt1-Cre/R26R embryo reveals neural crest-derived cells in the undifferentiated cranial mesenchyme of the developing anterior cranial base and face; an arrowhead indicates the caudal border of neural crest cell migration in the midline at this age, just rostral to Rathke’s pouch (arrow). (C) A Mesp1-Cre/R26R sagitally sectioned cranium shows mesoderm-derived cranial mesenchyme caudal to Rathke’s pouch; there is also a LacZ positive layer of undifferentiated cranial mesenchyme deep to a layer of LacZ negative ectoderm in the caudal presumptive vault. Dashed line in A indicates placement of section in B. See key for abbreviations.

Figure 5

Neural crest cell contributions to the rostral chondrocranium at E14 shown with Alcian blue stained C57BL/6J mouse sections and X-Gal/Hematoxylin stained Wnt1-Cre/R26 mouse sections. The whole mount insert identifies plane of section for A&B (midsagittal) and C&D (parasagittal). (A & B) The caudal border of neural crest cells is between the hypophyseal and parachordal cartilages. The future location of the acrochordal cartilage will form by E15 in X-Gal negative mesenchyme at the location of the asterisk (*). The arrow in (A) identifies a portion of the notochord superior to the parachordal cartilage. (B) In the midline, neural crest cells populate the hypophyseal cartilage and all rostral mesenchyme that will form the trabecular cartilage. (C & D) Laterally, the hypophyseal cartilage is still X-Gal positive, but the hypochiasmatic cartilage is negative, suggesting it has a mesodermal origin. See key for abbreviations.

Figure 6

X-Gal-stained sagittal sections of Wnt1-Cre/R26R (A,C,E, G) and Mesp1- Cre/R26R (B,D,F, H) mice at E17.5 showing the tissue origins of cranial base structures. A–D are midsagittal; E–H are parasagittal; rostral is to the right. Magnification of A & B is ×5; all others at ×10. See note in results section for this figure regarding endogenous ß-gal activity in bone (A,B). In the midline, the basal portion of the trabecular cartilage is neural crest cell-derived while the basioccipital bone shows only mesodermal contributions. (C,D) Although the basisphenoid bone develops from the completely neural crest-derived hypophyseal cartilage, its periosteum has both neural crest and mesodermal origins, with a clear boundary between the two components (arrows). Note that, due to glycogen deposition in the cytoplasm of hypertrophic chondrocytes (which is lost during tissue preparation) (Ross & Pawlina, 2006), the cytoplasm of hypertrophic chondrocytes was consistently LacZ-negative in both Wnt1-Cre/R26R and Mesp1-Cre/R26R mice, but nuclear staining indicating tissue lineage can still be observed. (E,F) The lateral neural crest-mesoderm boundary of the cranial base is found between the neural crest-derived alicochlear commissure of the basisphenoid bone and the mesoderm-derived auditory capsule. (G,H) The hypochiasmatic cartilage is entirely mesoderm-derived. See key for abbreviations.

Figure 7

Neural crest and mesoderm contributions to the cranial base at birth (P0) shown with X-Gal-stained crania of a Wnt1-Cre/R26R mouse (A & C) and a Mesp1-Cre/R26R mouse (B & D). In general, the anterior cranial base is neural crest-derived whereas the posterior cranial base is mesoderm-derived. The exception to this is the mesoderm-derived hypochiasmatic cartilages, which contribute to the postoptic root of the presphenoid bone. *hypochiasmatic cartilage. See key for abbreviations.

Figure 8

The developing spheno-occipital synchondrosis shows a mixed tissue origin in X-Gal stained whole mount crania of Wnt1-Cre/R26R (A,B,D,E) and Mesp1-Cre/R26R (C) mice. (A) At E10.5, a whole mount endocranium (dorsal view) shows that neural crest-derived mesenchyme has migrated as far caudally as Rathke’s pouch in the midline, which forms a keyhole shaped invagination (white arrow) surrounded by X-Gal positive tissue. (B) At E15, a whole mount endocranium (dorsal view) shows that the presumptive spheno-occipital synchondrosis (dashed outline) has two X-Gal positive projections that surround the same keyhole shape (white arrow), which is now filled with LacZ-negative, mesoderm-derived tissue. (C–E) Sagitally cut and X-Gal-stained whole mount transgenic newborn crania reveal that the dual tissue origin of the spheno-occipital synchondrosis changes postnatally. (C & D) The rostral half of the spheno-occipital synchondrosis has a dual origin whereas the caudal half appears to be wholly mesoderm-derived. (E) By P10, the spheno-occipital synchondrosis appears to have lost its neural crest contribution. (C–E ) The presphenoidal synchondrosis is completely neural crest-derived and remains so through P10. See key for abbreviations.

Figure 9

Schematic Summary of Results. (A) The individual cartilages of the mature chondrocranium (E16) are shown in different colors (the pterygoid cartilages appear on the ventral surface of the hypophyseal cartilage and are therefore not shown). (B) A newborn cranial base is shown here with bone in red, cartilage in blue. (C) The newborn cranial base is shown with colors corresponding to the cartilages from which each bone matures. Bones shown in grey ossify intramembranously and do not develop from the chondrocranium. (D) The tissue origins of the mature chondrocranium at E16 and (E) the newborn cranial base are shown with cartilages and bones derived from neural crest cells in blue and those derived from mesoderm in yellow. (F) A schematic drawing of the sagittal neural crest-mesoderm boundary in the cranial base at E16, P1, and P10. The notochord is shown in green above the parachordal cartilage at E16. The presence of non-neural crest-derived osteoblasts in the caudal portion of the basisphenoid bone at birth is indicated by circles. The mesoderm-derived hypochiasmatic cartilage is shown as a yellow triangle on the trabecular cartilage prior to birth and on the presphenoid bone after birth. Notice that the neural crest-mesoderm boundary moves rostrally out of the spheno-occipital synchondrosis and into the basisphenoid bone as development proceeds postnatally. See key for abbreviations.