Developmental Biology 322 (2008) 121–132

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Developmental Biology

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Development and tissue origins of the mammalian cranial base

B. McBratney-Owen a,⁎, S. Iseki b, S.D. Bamforth c, B.R. Olsen a, G.M. Morriss-Kay d

a Harvard School of Dental Medicine, Department of Developmental Biology, 190 Longwood Avenue, Boston, MA, 02115, USAb Molecular Craniofacial Embryology Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japanc Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UKd Le Gros Clark Building, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford, OX1 3QX, UK

Abbreviations: A, ala temporalis cartilage; AC, audicommissure; AR, acrochordal cartilage; AT, ala tempsphenoid bone; B, basitrabecular process; BF, basicranbone; BS, basisphenoid bone; CA, canalicular part ofmesenchyme; CO, cochlear part of auditory capsule; Dbone; EO, exoccipital bone (will eventually fuse tocartilage; FB, frontal bone; FV, fourth ventricle of hindtemporalis) of the basisphenoid bone; H, hypophysefenestra; LW, lesser wing of the presphenoid bone; Mmesencephalic vesicle of midbrain; MX, maxillary procesparanasal cartilage; O, orbital cartilage; OA, occipital arcON, optic nerve; OR, optic recess; P, parachordal cartideveloping pituitary gland; PN, paranasal cartilage;presphenoidal synchondrosis; S, supraoccipital cartilageeventually fuse to basioccipital bone); SOS, sphenosquamosal bone; T, trabecular cartilage (trabeculartrabecular plate; TG, trigeminal nerve; TN, nasal poTongue; TV, third ventricle of forebrain; Y, hypochiasma⁎ Corresponding author. Orthopedic Research Lab

Boston, Enders 910, 320 Longwood Avenue, Boston, MAE-mail address: bmcbratneyowen@post.harvard.edu

0012-1606/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.ydbio.2008.07.016

a b s t r a c t

a r t i c l e i n f o

Article history:

The vertebrate cranial base Received for publication 24 January 2008Revised 10 July 2008Accepted 10 July 2008Available online 22 July 2008

Keywords:Cranial baseChondrocraniumNeural crestMesodermWnt1Mesp1New Head HypothesisTissue originsCranial development

is a complex structure composed of bone, cartilage and other connective tissuesunderlying the brain; it is intimately connected with development of the face and cranial vault. Despite itscentral importance in craniofacial development, morphogenesis and tissue origins of the cranial base havenot been studied in detail in the mouse, an important model organism. We describe here the location andtime of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mousecranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cellmarker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning withdevelopment of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form thenasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of thevault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crestcells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with theexception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporalbones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derivedtissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derivedhypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissuesassociated with the visual apparatus may have evolved independently of the rest of the “New Head”.

© 2008 Elsevier Inc. All rights reserved.

tory capsule; ACO, alicochlearoralis (greater wing) of basi-ial fenestra; BO, basioccipitalauditory capsule; CM, cranialI, diencephalon; EB, ethmoidbasioccipital bone); F, frontalbrain; GW, greater wing (alaal cartilage; HF, hypophysealNP, medial nasal process; MV,s; NL, orbitonasal lamina of theh cartilage; OF, optic foramen;lage; PA, parietal cartilage; PI,PS, presphenoid bone; PSS,; SB, supraoccipital bone (will-occipital synchondrosis; SQ,plate); TB, basal portion ofrtion of trabecular plate; To,tic cartilage.oratories, Children's Hospital02115, USA.(B. McBratney-Owen).

l rights reserved.

Introduction

The skull is divided into two compartments: the viscerocranium,which comprises the facial skeleton (and some anterior neckstructures), and the neurocranium, which encases the brain andcranial sense organs. The neurocranium is formed from the endo-chondral bones of the cranial base and the intramembranous bones ofthe vault. The viscerocranium and the vault of the neurocranium arerelatively well studied; however, development of the cranial base hasnot been extensively investigated in the mouse. Because the cranialbase spans almost the entire rostro-caudal length of the head, fromthe foramen magnum to the tip of the cartilaginous nasal septum, itsproper development is crucial for subsequent normal development ofthe cranial vault and facial bones. In this paper we analyze thedevelopment of the mouse cranial base and determine the relativecontributions of neural crest-andmesoderm-derived cells bymeans ofin vivo permanent cell markers.

In the adult mouse, the cranial base is composed of the ethmoid,presphenoid, basisphenoid, and basioccipital bones along with theauditory capsules of the temporal bones. Early in development, thecranial base first appears as a sheet of undifferentiated mesenchymalcells that condense and chondrify to form the chondrocranium, a solidcartilaginous structure that spans the entire length of the skull and is

Fig.1.Morphogenesis of the chondrocraniumas shownbyAlcian blue stained and clearedwholemount C57BL/6J embryos fromTheiler Stages18 to24 (E11 to E16). All images show thechondrocranium from a dorsal aspect (norma basalis interna) except for panel A, which shows a lateral perspective. (A, B) The parachordal cartilage is the first to appear caudally in thecranium 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 themouse; all cartilages have fused and ossification has just begun in the posterior chondrocranium. Scale bar in panel H=1 mm. See key for abbreviations.

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formed by the fusion of numerous individual cartilages that appear atdistinct locations and times. The cartilages of the chondrocraniumthen undergo endochondral ossification to form the bones of cranialbase. Thus the structural origin of each bone can be traced from anumber of chondrocranial cartilages. Understanding chondrocranialdevelopment is a critical first step to understanding formation andgrowth of the mature cranial base.

In his seminal book, The Development of the Vertebrate Skull(1937), De Beer provided an in-depth analysis of chondrocranialdevelopment in a number of mammals. However, he notes that “nosystematic study has been made of the development of the skull ineither rats or mice”. Since that time, the mouse has become a popularmodel organism for studies of craniofacial development, and adetailed understanding of the normal development of all componentsof the skull is an essential foundation for experimental andpathogenic approaches. Most published studies that include thecranial base have examined late embryonic and postnatal endochon-dral ossification processes, particularly development and growth ofthe synchondroses (for example Isii-Suzuki et al., 1999; Chen et al.,1999; Gakunga et al., 2000; Eswarakumar et al., 2002; Shum et al.,2003; Olsen et al., 2005; Young et al., 2006; Koyama et al., 2007;Nagayama et al., 2008; Kolpakova-Hart et al., in press) or alteredcranial base morphology in mutant, knock-in, knock-out, or trans-genic mice (for example Jones and Roberts, 1988; Rintala et al., 1993;Lozanoff et al., 1994; Kaufman et al., 1995; Ma and Lozanoff, 1996;Chung et al., 1997; Belo et al., 1998; McBratney et al., 2003;Hallgrimsson et al., 2006). A minority of studies have focused onearly developmental aspects, from its origin as undifferentiatedmesenchyme through chondrogenesis of the chondrocranium (Woodet al., 1991; Savontaus et al., 2004; Nie et al., 2005a; Nie et al., 2005b;

Kettunen et al., 2006; Nie, 2006a; Nie, 2006b), yet understanding theearly developmental processes that create the cranial base will showus how the adult morphology arises. This insight is relevant not justto the cranial base itself but to the whole skull, since growth and formof the cranial base plays a central role in the covariation of othercraniofacial components (Lieberman et al., 2008).

Defining the tissue boundary between neural crest-derived andmesoderm-derived components of the cranial base may be a valuableendeavor as such a boundary has been shown to have cleardevelopmental significance in the vault (Jiang et al., 2002; Evansand Noden, 2006; Merrill et al., 2006; Yoshida et al., 2008; reviewed inGross and Hanken, 2008). Although the tissue origins of the murinecranial base have been neglected, the rest of the cranium has beeninvestigated. The skeleton of the mouse viscerocranium is derivedsolely from neural crest cells (Jiang et al., 2002). Most studies in thechicken and mouse report that the vault of the neurocranium isderived from both neural crest cells and mesoderm (Le Lievre, 1978;Noden, 1978, 1984, 1988; Jiang et al., 2002; Evans and Noden, 2006;Yoshida et al., 2008), although one study reports that the chickencranial vault is entirely neural crest-derived (Couly et al., 1993).Studies on the tissue origins of the cranial base in the chicken havedefined its relative neural crest-and mesoderm-derived components(Le Lievre, 1978; Noden, 1988; Couly et al., 1993; Le Douarin et al.,1993; Lengele et al., 1996; Le Douarin and Kalcheim, 1999). An in vivoanalysis of tissue origins of the mouse cranial base is required for adetermination of which cartilages of the chondrocranium are derivedfrom neural crest versus mesoderm in this mammal.

In this paper, we examine the development of the mouse cranialbase from E11 to E16. We show that the cartilages of the chondrocra-nium appear at specific locations and times, with chondrification

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beginning in the posterior (caudal) cranial base, followed by theanterior (rostral) cranial base, leading ultimately to fusion of the twoparts via a midline stem and lateral struts by E16. We also investigatethe tissue origins of themouse cranial base using in vivo permanent cellmarkers in Wnt1-Cre/R26R and Mesp1-R26R transgenic mice (Jiang etal., 2000, 2002; Yoshida et al., 2008). According to the New HeadHypothesis (NHH) proposed by Gans and Northcutt (1983; Northcuttand Gans,1983), the neural crest-mesoderm boundary should correlatewith the rostral-most tip of the notochord, thereby creating acoincident boundary with the prechordal-chordal boundary in thecranium. In the mouse, the prechordal-chordal boundary is betweenthe basisphenoid and basioccipital bones (Barteczko and Jacob, 1999),and our observations confirm that the major neural crest-mesodermboundary is located between the cartilages that contribute to these twobones. However, the mesoderm-derived hypochiasmatic cartilages arean exception to this generalization, as they are located among neuralcrest-derived cartilages in the anterior cranial base. Furthermore,although the hypophyseal cartilage is derived from neural crest cells,the bone that replaces it (the basisphenoid bone) has somemesodermalcell contributions in its caudal portion. The broader implications ofthese results to general bone development and evolution of themammalian cranium are discussed.

Materials and methods

Inbred mice and staging criteria

Chondrocranial development was examined in detail in C57BL/6Jembryos from timed pregnant females obtained from JacksonLaboratories. All embryos were first staged using criteria establishedby Theiler (1989) and Miyake et al. (1996) so that chondrocranialdevelopment could be observed relative to developmental stagerather than conception date. After being designated a specific stage(T18 to T24), embryos were labeled according to the generalcorresponding conception age (E11 to E16). Only C57BL/6J embryoswere used for results in Figs. 1 and 3. Fig. 2 includes embryos from A/J(Fig. 2A) and DBA/2J (Figs. 2B and C) inbred lines obtained from timedpregnant females from Jackson Laboratories.

Whole-mount staining

Some embryos were stained with Alcian blue alone in order tofocus on chondrocranial development, making a stain for bone (suchas alizarin red) unnecessary for this portion of the study. Wholeembryos from E11 to E16 were dissected from the uterus in PBS andstored in 70% EtOH. After being fixed in Bouin's fixative overnight,embryos were washed in 0.1% NH4OH, 70% EtOH until all traces of theyellow fixative were gone and embryos appeared bleached. Embryos

Fig. 2. Dorsal view of endocranium inwhole mounts showing development of cartilages andcartilages are present in the optic region of the chondrocranium. (B) At birth, ossification hastemporalis) of the basisphenoid bone. (C) By at least 6 months after birth, extensive ossificatioorbital cartilages forming the lesser wing of the prepshenoid bone. The arrow indicates thecartilage. Laterally, the frontal bone and greater wing of the basisphenoid bone have formabbreviations.

were then equilibrated in 5% acetic acid twice for 1 hour each time andleft overnight in 0.05% Alcian blue 8GX (Fisher) in fresh 5% acetic acid.This was followed by washing twice for 1 hour each in 5% acetic acid,soaking in methanol twice for 1 hour each and clearing in 1:2 benzylalcohol:benzyl benzoate (BABB). Crania were then removed from thebody and heads dissected to reveal the cranial base. Meckel's cartilagewas removed for photographs.

For combined alizarin red and Alcian blue staining of postnatalmouse crania, the vault and brainwas removedprior tofixing overnightin 100% EtOH. Cartilage was then stained overnight in 80 ml 95%EtOH,20ml glacial acetic acid,15mgAlcian blue (Sigma). Afterwashing twicein 95% EtOH and soaking in 2% KOH in water for 3 hours, bone wasstained in 1% KOH, 7.5 mg/ml alizarin red S (Sigma) inwater overnight.Specimens were destained in 20% glycerol, 1% KOH inwater for 2 days,and then put in 20% glycerol in water, changed daily for 5 days. Finally,craniawere placed in 20% glycerol, 20% EtOH overnight and then storedand photographed in 50% glycerol, 50% EtOH.

Microscopy

Whole embryos at E14 were dissected from the uterus in PBS andstored in 70% EtOH. Specimens were dehydrated , cleared in xylene, andmounted in paraffin. Wax blocks were sectioned at 10 μm. Slides werethen cleared in xylene, rehydrated, soaked in 1%Alcian blue (Sigma) in 3%acetic acid for 1 hour, followed by dehydration and a final clearing inxylene. Slidesweremountedwith coverslips andviewedonamicroscope.

In situ hybridization

C57BL/6J embryos from E12 to E16 obtained from timed pregnantfemales from Jackson Laboratories were fixed in 4% paraformaldehydeat 4°C overnight. Specimens were then dehydrated, soaked in xyleneat room temperature and placed in melted paraffin up to 65°C for nomore than 2 hours. Sections were cut at 10 μm.

Immediately prior to hybridization, slides were deparaffinized inxylene and rehydrated. Slides were then incubated in 0.2 M HCL for15 minutes and 10 mg/ml of Proteinase K in 10 mM Tris pH7.5/1 mMCaCl2 at 37°C for 15minutes, beingwashed in PBS at room temperaturebefore and after each of these steps. Slideswere then furtherfixed in 4%paraformaldehyde for 5 minutes at room temperature, washed in PBS,and then acetylated in 0.25% acetic acid for 15 minutes. After washingin PBS, slideswere dehydrated in an ascending alcohol series and left toair dry for 30 minutes. Col2a1 antisense probes synthesized to cover405 bp of cDNAwere labeled with digoxigenin-11-UTP as described inGong et al. (2001). Sox9 antisense probe was also used as described byHuang et al. (1997). Hybridization was carried out as described byBohme et al. (1995) and detected, counterstained, and mounted asdescribed by Gong et al. (2001).

bones in the optic region. (A) At E14, the orbital, frontal, hypochiasmatic, and trabecularbegun in the body of the presphenoid bone, the frontal bone, and the greater wing (alan and remodeling has occurred around the optic foramen, with the hypochiasmatic andportion of the postoptic root of the lesser wing that derives from the hypochiasmaticed interdigitations where they meet. Scale bars in panels A–C=0.5 mm. See key for

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Transgenic mice and X-Gal staining

The protocol for construction of Wnt1-Cre/R26R transgenic micehas previously been described by Jiang et al. (2000) and forMesp1-Cre/R26R by Saga et al. (1999). All cells and their progeny with an activeWnt1 or Mesp1 promoter stably maintain expression of the recom-bined R26R allele, thus driving the LacZ expression allele, allowing usto examine neural crest or mesodermal contributions to structures inboth pre-and postnatal mice by X-Gal staining (Jiang et al., 2002;Yoshida et al., 2008). Staining with X-Gal was carried out byconventional methods on whole embryos from E8.5 to E10.5 and onheads with the superior vault and brain removed prior to staining forE15.5 fetuses and postnatal mice. Sections of X-Gal stained E10.5embryos were dehydrated and embedded in paraffin prior tosectioning at 10 μm. E14 and E17.5micewere frozen in OCTcompound,cut at 10 μm on a cryostat, and then X-gal stained. Some sections werecounter-stained with eosin, nuclear-fast red, or hematoxylin. Between5 and 15 embryos or craniawere examined at each stage forWnt1-Cre/R26R mice. The cranial base of 1 to 2 Mesp1-Cre/R26R mice from alarger samplewas examined at each stage for this study. X-Gal-positivetissues and cells were consistent for all samples, with Wnt1-Cre/R26R

Fig. 3. In situ hybridization of Col2 on sagittal sections of C57BL/6J embryos from E12through 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 tochondrify from E13 to E15. (E) At E16, detection of Col2 decreases in the parachordal andhypophyseal anlagen where endochondral ossification is beginning. Normal fenestraedisrupt the continuity of cartilages in some sections (C & E); these are not present in theadult cranial base. See key for abbreviations.

samples staining positively in a complimentary pattern to that ofMesp1-Cre/R26R samples.

Results

Chondrocranial development in the mouse

Chondrocranial development in the mouse occurs in the followingsequence: first development of the caudal chondrocranium, followedby rostral chondrogenesis in the nasal capsule, and finally fusion ofthese two parts via the midline, central stem and the lateral struts ofthe vault cartilages (Fig. 1). Although the complete chondrocranium isa continuous cartilaginous structure, it develops from a number ofdistinct cartilages that appear at specific locations and times.Morphogenesis of the chondrocranium begins with the appearanceof the parachordal cartilage at T18.1 (E11) (Figs. 1A, B). Like manymidline cartilages in vertebrates, the parachordal cartilages likely firstappear as a pair that quickly fuse (DeBeer, 1937), but this fusion musttake place prior to our observations at T18.1 (E11) and may even occurat the prechondrogenic mesenchymal condensation stage. At T18.1,the notochord rests on the superior surface of the parachordalcartilage and extends rostrally as far as Rathke's pouch. By T20.32(E12), the occipital arch cartilages develop just caudal to theparachordal and the canalicular part of the auditory capsules developslaterally (Fig. 1C). At T21.22 (early E13), chondrogenesis of thetrabecular cartilage begins in the presumptive nasal capsule (Fig.1D). By the T21.31 stage (late E13), the hypophyseal cartilages appearin the center of the chondrocranium (just inferior to the developingpituitary gland), and the paranasal cartilages begin to form the lateralwalls of the nasal capsule (Fig. 1E). T22 (E14) is characterized by theappearance of 3 distinct pairs of cartilages in the optic region: theorbital, frontal, and hypochiasmatic cartilages (Fig. 1F). The hypophy-seal cartilages have also fused in the midline by this stage, while thetrabecular cartilage has continued chondrifying caudally. Laterally, theparietal cartilages have just begun to chondrify at the junction of thecranial base and presumptive vault. At stage T23 (E15), thebasitrabecular and ala temporalis cartilages have formed lateral tothe hypophyseal cartilages, while the acrochordal cartilages havefused to create a bar at the rostral end of the parachordal cartilages,forming the rostral border of the basicranial fenestra (Fig. 1G). Thecochlear part of the auditory capsules has also chondrified, and thesupraoccipital cartilages have developed caudally forming the super-ior border of the foramen magnum.

At stage T24 (E16), the chondrocranium is fully formed in themouse (Fig. 1H) as determined by the appearance and fusion of all ofthe cartilages that contribute to the chondrocranium. Key aspects ofthe mature chondrocranium are fusion of the central stem (trabecularplate) in the midline at the junction of the trabecular and hypophysealcartilages and fusion of the frontal and parietal cartilages laterally,thereby linking the rostral nasal capsule to the caudal cranial base.Remnants of the notochord are still visible superior to the parachordalcartilage. Also, the mouse chondrocranium can have a number ofmidline fenestrae, as clearly seen in the specimen in Fig. 1H. Thehypophyseal cartilage can have up to two hypophyseal fenestrae,while the rostral end of the parachordal cartilage often has onebasicranial fenestra. These fenestrae are not sites where nerves andblood vessels traverse the chondrocranium, and they disappear as anormal mouse matures. Finally, although the trabecular cartilage hasdeveloped as a single cartilaginous unit from the tip of the nasalcapsule to the center of the chondrocranium, it is worth distinguishingthe nasal portion from the basal portion of the trabecular plate at thisstage. Later in development, the nasal portion will remain cartilagi-nous as the nasal septum, while the more caudal basal portion willform the body of the presphenoid bone. Also, the pterygoid cartilagesappear on the ventral surface of the hypophyseal cartilage at this stage(not shown).

Fig. 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 themaxillary 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 ectodermalinvagination 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 cranialmesenchyme of the developing anterior cranial base and face; an arrowhead indicates the caudal border of neural crest cell migration in themidline at this age, just rostral to Rathke'spouch (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 ofundifferentiated cranial mesenchyme deep to a layer of LacZ negative ectoderm in the caudal presumptive vault. Dashed line in panel A indicates placement of section in panel B. Seekey for abbreviations.

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The optic region of the chondrocranium deserves specialattention for a number of reasons (Fig. 2). First the hypochiasmaticcartilages (ala hypochiasmatica) have been described by De Beer(1937, p.389) as “paired cartilages of independent origin”, which maydevelop in a variety of locations in the optic region of differentmammals. For example, they develop next to the preoptic root of theorbital cartilage in rabbits, attached to the postoptic root (pilametoptica) of the orbital cartilage in the armadillo, and attached to

Fig. 5. Neural crest cell contributions to the rostral chondrocranium at E14 shownwith Alciamouse sections. The whole mount insert identifies planes of sections for A&B (midsagittal)hypophyseal and parachordal cartilages. The acrochordal cartilage will form by E15 in X-Gal nportion of the notochord superior to the parachordal cartilage. (B) In the midline, neural crestrabecular cartilage. (C & D) Laterally, the hypophyseal cartilage is still X-Gal positive, but theabbreviations.

both roots in humans (De Beer, 1937). In the mouse, the hypochias-matic cartilages appear independently at E14 (Fig. 1F) and thenfurther develop in close association with the postoptic roots of theorbital cartilages (Fig. 2A). Although the hypochiasmatic cartilagesform a robust bridge linking the postoptic roots with the body of thepresphenoid bone just after birth (Fig. 2B), extensive remodelingduring ossification eventually diminishes them so that by 6 monthspost-natal they merely form the most medial portions of the

n blue stained C57BL/6J mouse sections and X-Gal/Hematoxylin stained Wnt1-Cre/R26Rand C&D (parasagittal). (A & B) The caudal border of neural crest cells is between theegative mesenchyme at the location of the asterisk (⁎). The arrow in panel A identifies at cells populate the hypophyseal cartilage and all rostral mesenchyme that will form thehypochiasmatic cartilage is negative, suggesting it has a mesodermal origin. See key for

Fig. 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. Panels A-D aremidsagittal; panels E-H are parasagittal; rostral is to the right. Magnification of A & B is x5; all others at x10. See note in results section for this figure regarding endogenous β-galactivity in bone. (A, B) In themidline, the basal portion of the trabecular cartilage is neural crest cell-derivedwhile the basioccipital bone shows onlymesodermal 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 clearboundary 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 indicatingtissue 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 thebasisphenoid bone and the mesoderm-derived auditory capsule. (G, H) The hypochiasmatic cartilage is entirely mesoderm-derived. See key for abbreviations.

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postoptic roots of the lesser (orbital) wings of the sphenoid bone(Fig. 2C).

There are also two distinct cartilages lateral to the optic foramen,the orbital cartilage and the frontal cartilage (Fig. 2A). De Beer (1937)identified cartilage immediately laterally to the optic foramen inmammals as simply the orbital cartilage and did not note a distinctcartilage in relation to the frontal bone. However, in the mouse, thereare two distinct centers of chondrification that contribute to twodifferent bones in the adult, thus we suggest giving each a distinct

name (Fig. 2). The orbital cartilage first appears in a semi-circlearound the optic foramen (Fig. 2A) and then chondrifies furtherlaterally (Fig. 2B). The orbital cartilage will eventually fuse with thehypochiasmatic cartilage, forming the lesser wing of the sphenoid,which is fused to the central body of the presphenoid bone (Fig. 2C).Meanwhile, the frontal cartilage forms from a separate chondrifica-tion center lateral to the orbital cartilage and later ossifies as part ofthe frontal bone (Fig. 2). Although it can be difficult to discernseparate orbital and frontal cartilages in some preparations (Fig. 1H),

Fig. 7. Neural crest and mesoderm contributions to the cranial base at birth shownwithX-Gal-stained crania of Wnt1-Cre/R26R and Mesp1-Cre/R26R mice. (A) In general, theanterior cranial base is neural crest-derived whereas the posterior cranial base ismesoderm-derived. The exception to this is the mesoderm-derived hypochiasmaticcartilages, which contribute to the postoptic root of the presphenoid bone.⁎hypochiasmatic cartilage. See key for abbreviations.

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other preparations show a clear border between these two cartilagesthat is maintained during ossification (Fig. 2).

Chondrocranial development can be observed in sections usingclassic Alcian blue staining or in situ hybridization with Col2a1 (Col2)and Sox9. All cartilages of the cranial base begin to express Col2a1 andSox9 and are positively stained by Alcian blue as they develop betweenE12 and E16 (data not shown). Midline Col2 labeled sections areshown here to illustrate key features of chondrocranial developmentfrom E12 to E16 (Fig. 3). Col2 expression can be observed in theparachordal cartilage at E11 (data not shown) and E12 (Fig. 3A).Wherethe notochord is present in sections, it is Col2 positive, along withsome mesenchyme in the anterior cranial base (Fig. 3A). At E13, Col2expression is seen in the chondrifying hypophyseal and trabecularcartilages and is more intense in the parachordal cartilage (Fig. 3B). Aschondrification continues at E14, expression is also seen where theacrochordal cartilage will develop, which creates the rostral border ofthe basicranial fenestra (Fig. 3C). Both hypophyseal and basicranialfenestrae divide the midline cartilages into separate pieces in somesections (Figs. 3C and E). Expression in the chondrocranium isstrongest at E15 (Fig. 3D). By E16, Col2 expression decreases in thehypophyseal and parachordal cartilages, where endochondral ossifi-cation has just begun (Fig. 3E).

The Col2 sections further reveal some unique features of themousecranial base (Fig. 3). The pituitary gland develops directly above thehypophyseal cartilage, which will eventually develop into the basi-sphenoid bone and spheno-occipital synchondrosis. A sella turcica, ordepression in the superior surface of the cranial base, is not prominentin the mouse. Instead, the pituitary is encased in connective tissuesthat suspends the gland above the dorsal surface of the cranial base.An acrochordal cartilage is present but, unlike other mammals, is flushwith the floor of the cranial base; this cartilage is homologouswith thedorsum sellae, which in many other mammals forms a verticalprojection that the pituitary rests against. Hypophyseal and basicra-nial fenestrae are normally present in the mouse chondrocranium(Fig. 3), but the exact number of fenestrae is variable (0-2 hypophysealfenestrae and 1 basicranial fenestra). The fenestrae normally dis-appear prior to birth but may persist postnatally in somemutant mice(B. McBratney-Owen, unpublished observations).

Tissue origins of the cranial base

Fate mapping of the cranial basewas accomplishedwithWnt1-Cre/R262R andMesp1-Cre/R26R transgenic mice. Previous research in micehas shown that neural crest cells migrate from the dorsal neural tubeto the frontonasal process between somite stages 5 and 23 (E8 to E9.5)(Jiang et al., 2002). We show here that by E10.5, neural crest cells havemigrated ventrally under the forebrain, populating the presumptivecranial base rostral to Rathke's pouch (Figs. 4A, B). A ventral view ofthe cranium reveals neural crest-derived tissues in the nasal processes,the maxillary process, and the trigeminal ganglion (Fig. 4A). Incontrast, Rathke's pouch can be distinctly identified as an X-Gal-negative invagination surrounded rostrally and laterally, but notcaudally, by X-Gal-positive tissue (Fig. 4A). Undifferentiated cranialmesenchyme rostral to Rathke's pouch is positive for the neural crestlineage marker (Fig. 4B), while cranial mesenchyme caudal to Rathke'spouch, and caudal to an equivalent level of the diencephalon dorsally,is mesoderm-derived (Fig. 4C). Scattered X-Gal-positive cells withinthe neural crest-derived mesenchyme of Mesp1-Cre-R26R embryoshave been identified as angioblasts and endothelial cells of developingblood vessels (Yoshida et al., 2008).

By E14, the relationship between the forming cartilages of thechondrocranium and their tissue origins can begin to be observed inmore detail in sectionedmaterial (Fig. 5). The midline border of neuralcrest-derived tissue is between the hypophyseal cartilages andcartilages of the posterior cranial base, directly below the formingpituitary gland (Fig. 5B). In the posterior cranial base, only the

parachordal cartilage has chondrified by E14, but the acrochordalcartilage will appear rostrally by the next day, creating thecartilaginous neural crest-mesoderm interface with the hypophysealcartilage. The mesenchyme rostral to the hypophyseal cartilage is alsoderived from neural crest cells and will form the trabecular cartilage(Fig. 5B). More laterally, however, not all cartilages rostral to thehypophyseal cartilage are formed from neural crest cells (Figs. 5C, D).The hypochiasmatic cartilages (Y) are X-Gal negative, suggesting amesodermal origin.

At E17.5, most of the anterior cranial base appears to be derivedfrom neural crest cells except for the hypochiasmatic cartilages, whichare derived from mesoderm (Fig. 6). The trabecular plate is derivedfrom neural crest cells and has not yet begun to ossify into thepresphenoid bone at this stage (Figs. 6A, B). The basisphenoid bone hasbegun to ossify from the hypophyseal cartilage; interestingly, thesesections show a mixture of mesoderm-and neural crest cell-derivedosteoblasts in the region of mineralizing matrix in the maturing bodyof this bone (Figs. 6C, D). Also, the periosteumof the basisphenoid boneis derived from both tissue sources; there appears to be a clear rostro-caudal boundary on both the ventral and dorsal surfaces of the bone,with the dorsal boundary being more rostrally placed than the ventralboundary (Figs. 6C, D). Both the fibrous and cellular layers of theperiosteum are derived from the same tissue source. Recent reportshave shown that there is endogenousβ-gal activity in bone, specificallyin osteoclasts (Odgren et al., 2006; Kopp et al., 2007), so we used TRAPstaining to identify osteoclasts in the cranial base of mice at E17.5 (datanot shown). Less than 10% of observed cells in the periosteum anddeveloping marrow cavity were TRAP positive. We can thereforeconclude that the majority of LacZ positive cells observed in oursections are not osteoclasts and are positive due to lineage-specific,promoter-driven β-gal expression. While the neural crest cell-derivedosteoblasts in the marrow cavity of the basisphenoid bone mustoriginate from the neural crest cell-derived periosteum, the meso-derm-derived osteoblasts could originate from the adjacent mesoder-mal periosteum or via invading blood vessels bringing cells from amore distant mesodermal source.

The spheno-occipital synchondrosis also has a unique tissue origin,being partially neural crest and partially mesoderm derived (Figs. 6A–D); there are some neural crest-derived cells in the rostral portion ofthis cartilaginous structure, but none in the caudal portion (Fig. 6C).Mesoderm-derived cells contribute to all parts of the spheno-occipitalsynchondrosis and basioccipital bone (which has ossified by this stagefrom the parachordal and acrochordal cartilages). More laterally, theneural crest-mesodermal boundary lies between the alicochlear

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commissures of the basisphenoid bone and auditory capsules (Figs. 6Eand F). The hypochiasmatic cartilage is entirely mesoderm-derived, incontrast with the orbital and paranasal cartilages (Figs. 6G and H).

At birth, whole mount cranial bases clearly illustrate the largelyneural crest-derived anterior cranial base relative to the mesoderm-derived posterior cranial base (Fig. 7). The nasal capsule and bonesassociated with the trabecular cartilage (the body of the presphenoidbone and the ethmoid bone) are neural crest-derived (Fig. 7). Thebasisphenoid bone and its associated greater wings (ala temporalis)appear to be largely neural crest derived, forming a border with themesoderm-derived basioccipital bone and auditory capsules (Figs. 7).However, the E17.5 sections reveal that the caudal portion of thebasisphenoid bone has a mesoderm-derived periosteum and somecells ofmesodermal originwithin the developing bone (Figs. 6B andD),making this midline neural crest-mesoderm border less discretehistologically in the bony cranial base than in the chondrocranium ofthe fetus (Fig. 5). Altogether, development of the mammalian cranialbase can be viewed as a dual dynamic process,withmigration of neuralcrest-derivedmesenchyme around the frontonasal process, eventuallyformingmost of the anterior cranial base, integrated with the buildingof the posterior cranial base from mesoderm-derived mesenchyme.

Development of the synchondroses

Synchondroses are similar to the epiphyseal growth plates of longbones except that they are double-faced, with a resting zone in themiddle sandwiched between pairs of proliferating and hypertrophicchondrocyte zones. Postnatally, they are important longitudinalgrowth centers of the cranial base. In the mouse, there are twosynchondroses in the midline cranial base: the spheno-occipital andpresphenoidal synchondroses. The mouse does not have a sphenoeth-moidal synchondrosis. Recent work by Wealthal and Herring (2006)found that endochondral ossification occurs at the junction betweenthe developing ethmoid and presphenoid bones via a briefly existing,single growth plate which disappears by E15, leaving interstitialgrowth as the major contributor to elongation of the septum at theend of the fetal period and postnatally.

Fig. 8. The developing spheno-occipital synchondrosis shows a mixed tissue origin in X-Gal(A) At E10.5, a whole mount endocranium (dorsal view) shows that neural crest-derived mekeyhole shaped invagination (white arrow) surrounded by X-Gal positive tissue. (B) At E15, asynchondrosis (dashed outline) has two X-Gal positive projections that surround the same kecut and X-Gal-stained whole mount transgenic newborn crania reveal that the dual tissue orithe spheno-occipital synchondrosis has a dual originwhereas the caudal half appears to bewlost its neural crest contribution. (C–E ) The presphenoidal synchondrosis is completely neu

As described above, we showed that neural crest cells migratearound the frontonasal process and into the cranial base as farcaudally as Rathke's pouch (Fig. 4). Rathke's pouch itself is formedfrom an invagination of ectoderm and is eventually separated from theoral cavity by the cranial base. At E10.5, neural crest cells havemigrated back to and partially around Rathke's pouch, creating anectodermal keyhole-shaped invagination at the caudal border ofneural crest-derived cranial base tissues (Fig. 8A). By E15, this samekeyhole shape can be observed in the mesenchyme of the developingspheno-occipital synchondrosis between the hypophyseal and acro-chordal cartilages (Fig. 8B). This suggests that as Rathke's pouch issealed off from the oral cavity, the opening of the pouch itself is filledin with mesenchyme from a non-neural crest source (possibly fromthe adjacent mesoderm-derived mesenchyme that forms the acro-chordal cartilage), conserving the keyhole-shaped pattern in an X-Galstained Wnt1-Cre/R26R fetus (Fig. 8B). Subsequently, the spheno-occipital synchondrosis will develop in that location from both neuralcrest and mesoderm-derived mesenchyme. The boundary betweenneural crest and mesoderm-derived chondrocytes observed inmidsagittal and parasagittal sections of this synchondrosis (Figs. 6A–D) indicates that the embryonic relationship is maintained during latefetal development.

However, the spheno-occipital synchondrosis does not retain itsdual tissue contributions as development proceeds postnatally (Figs.8C–E). At birth, part of this synchondrosis is mesoderm-derived(Fig. 8C), while the remainder consists of cells derived from the neuralcrest (Fig. 8D). By 10 days after birth, the spheno-occipital synchon-drosis no longer has any neural crest cell-derived chondrocytes (Fig.8E). Since chondrocytes in the proliferative zone eventually movethrough the hypertrophic zone, followed by apoptosis (Shapiro et al.,2005), neural crest cells populating the rostral zones of the spheno-occipital synchodrosis may disappear from this synchondrosis byfollowing this normal developmental route. In contrast, the presphe-noidal synchondrosis develops from neural crest cell-derived chon-drocytes at the caudal edge of the trabecular plate (Figs. 6A, B),remaining entirely composed of neural crest-derived cells at birth andup to at least 10 postnatal days (Figs. 8D, E).

stained whole mount crania of Wnt1-Cre/R26R (A, B, D, E) andMesp1-Cre/R26R (C) mice.senchyme has migrated as far caudally as Rathke's pouch in the midline, which forms awhole mount endocranium (dorsal view) shows that the presumptive spheno-occipitalyhole shape (white arrow), which is now filled with LacZ-negative tissue. (C–E) Sagitallygin of the spheno-occipital synchondrosis changes postnatally. (C & D) The rostral half ofhollymesoderm-derived. (E) By P10, the spheno-occipital synchondrosis appears to haveral crest-derived and remains so through P10. See key for abbreviations.

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Discussion

Mosaic development of the cranial base

The cranial base of the mouse develops from 14 pairs of cartilagesthat appear at distinct locations and times and are derived from eithermesoderm or neural crest cells. Fig. 9 summarizes our findings. Neuralcrest cells destined for the anterior cranial base leave the lateral edgesof the neural folds during cranial neurulation, beginning early at E8.5(Jiang et al., 2002). They migrate over the forebrain and around theeyes to form the frontonasal process, forming a boundary over therostral diencephalon and just caudal to the eyes. Between E11 and E14,these neural crest-derived cells chondrify as the trabecular, paranasal,frontal, orbital, hypophyseal, ala temporalis, and pterygoid cartilages(Figs. 9A and D). The mesoderm-derived hypochiasmatic cartilagesalso chondrify by E14 within the anterior cranial base. Meanwhile, theposterior cranial base develops wholly from mesoderm, with theauditory capsules and parachordal, acrochordal, exoccipital, andsupraoccipital cartilages chondrifying between E11 and E15 (Figs. 9Aand D). Formation of the mature chondrocranium is complete by E16,with a largely neural crest cell-derived anterior cranial base andmesoderm-derived posterior cranial base (Figs. 9A and D).

The bones and cartilages that form the mature cranial base areidentifiable at birth (Fig. 9B). Each bone is derived from a number of

Fig. 9. Schematic Summary of Results. (A) The individual cartilages of the mature chondrocrasurface of the hypophyseal cartilage and are therefore not shown). (B) A newborn cranial basewith colors corresponding to the cartilages from which each bone matures. Bones shown intissue origins of the mature chondrocranium at E16 and (E) the newborn cranial base are shfrommesoderm in yellow. (F) A schematic drawing of the sagittal neural crest-mesoderm bouparachordal cartilage at E16. The presence of non-neural crest-derived osteoblasts in the caderived hypochiasmatic cartilage is shown as a yellow triangle on the trabecular cartilagemesoderm boundary moves rostrally out of the spheno-occipital synchondrosis and into th

cartilages, revealing their mosaic origins (Figs. 9C and E). The ethmoidbone develops from the neural crest cell-derived paranasal andtrabecular cartilages. The presphenoid bone develops from the neuralcrest cell-derived orbital and trabecular cartilages and from themesoderm-derived hypochiasmatic cartilages. The basisphenoidbone develops from the entirely neural crest-derived hypophyseal,ala temporalis, and pterygoid cartilages, but endochondral ossificationalso introduces osteoblasts from a mesodermal source in the caudalportion of this bone. Inferior portions of the frontal and parietal bonesossify from the neural crest cell-derived frontal and mesoderm-derived parietal cartilages via a unique ossification process that isdistinct from normal endochondral or intramembranous ossification(Holmbeck et al., 2003). Finally, although the basioccipital, exoccipital,and supraoccipital bones develop from distinct acrochordal andparachordal, occipital arch, and supraoccipital cartilages, respectively,these bones eventually fuse to form the single, mature mesoderm-derived occipital bone.

Evolution of the vertebrate cranial base

In their New Head Hypothesis (NHH), Gans and Northcutt (1983;Northcutt and Gans, 1983) proposed that vertebrates evolved byadding a ‘new head' rostral to the notochord, made of ectodermallyderived sense organs and nervous structures, to aid in predatory

nium (E16) are shown in different colors (the pterygoid cartilages appear on the ventralis shown herewith bone in red, cartilage in blue. (C) The newborn cranial base is showngrey ossify intramembranously and do not develop from the chondrocranium. (D) Theownwith cartilages and bones derived from neural crest cells in blue and those derivedndary in the cranial base at E16, P1, and P10. The notochord is shown in green above theudal portion of the basisphenoid bone at birth is indicated by circles. The mesoderm-prior to birth and on the presphenoid bone after birth. Notice that the neural crest-e basisphenoid bone as development proceeds postnatally. See key for abbreviations.

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behavior. Subsequent work described this building of a new head asincluding, among other things, an “anterior extension of theconnective tissues providing a connection among the sensorycapsules” (Gans, 1993) and specifically posited that these rostralconnective tissues are neomorphic, developing from neural-crestderived mesenchyme (Northcutt, 2005). The NHH predicts that theprechordal-chordal boundary should be coincident with the neuralcrest-mesoderm boundary and that connective tissues of theprechordal head (including bone and cartilage) should be derivedfrom the neural crest. So far, the skeletal and connective tissues of therostral cranium of zebrafish, lungfish, axolotls, amphibians, and birdshave been shown to be derived from the neural crest while cranialmuscles are derived from prechordal and cephalic paraxial mesoderm(Schilling and Kimmel, 1994; Noden et al., 1999; Olsson et al., 2001;Ericsson et al., 2004; Hanken and Gross, 2005; Noden and Francis-West, 2006; Ericsson et al., 2008).

In this paper we show that there are coincident neural crest-mesoderm and prechordal-chordal boundaries between the hypo-physeal and acrochordal cartilages in mice (Fig. 9F), providingsupport for the NHH. However, these results do not completelyfulfill the predictions of the NHH due to the presence of themesoderm-derived hypochiasmatic cartilages rostral to the noto-chord. In other words, a small portion of connective tissue in theprechordal cranial base of the “New Head” in the mouse is notderived from neural crest. The presence of the mesoderm-derivedhypochiasmatic cartilages among neural crest cell-derived tissuessuggests that they may have an evolutionary origin independent ofthe origin of the “New Head”.

The hypochiasmatic cartilages have so far only been documentedin mammalian taxa (humans, monkeys, hedgehogs, rabbits, dogs,cats: De Beer, 1937); they appear as independent cartilages invariable places around the optic foramina, eventually contributing tobones in the sphenoid region (De Beer, 1937). These cartilagesprovide an attachment point for the extra-ocular muscles (Dierbach,1985), and, as these muscles are derived from prechordal andcephalic paraxial mesoderm (Wachtler et al., 1984; Noden et al.,1999; Noden and Francis-West, 2006), it is not unreasonable tohypothesize that the morphologically associated hypochiasmaticcartilages evolved neomorphically in situ from the same mesodermalsource. In vertebrates, the central nervous system and developingsensory organs can provide spatial programming cues to cranialconnective tissue precursors that, in turn, provide positionalinformation to developing muscles, blood vessels, and neurons(Noden and Trainor, 2005; Noden and Francis-West, 2006). Usingthat model, the developing optic epithelium may provide spatialprogramming cues to the mesenchymal cells of the presumptivehypochiasmatic cartilages, which then provide positional informa-tion to the extra-ocular muscles. Since the spatial position of thehypochiasmatic cartilages appears to be variable in mammals, theoptic epithelium could direct their location to a position that wouldbe optimal for eye function specific to each organism. Having aclosely associated mesenchymal cell source that is anatomicallyvariable may provide an evolutionary avenue for functional adapta-tion of the visual apparatus.

The hypochiasmatic cartilages may have evolved in the ancestor toall mammals, independently and repeatedly in different mammalianlineages, or in the ancestor of all tetrapods. Early chondrocranialdevelopment needs to be documented in other mammalian taxa tosee if these small and transient cartilages are present and common toall mammals. If so, they may have evolved at the origin of themammalian clade. However, if other mammalian taxa do not havethese cartilages, either they evolved in the ancestor of all mammalsbut were subsequently lost in some lineages or they evolvedindependently a number of times in only some mammalian clades.

A tetrapod origin for the hypochiasmatic cartilages may also besupported if cranial base element homology can be resolved more

completely among mammals, birds, and reptiles. Kuratani (1989)suggests that the supratrabecular cartilages of reptiles may behomologous with the hypochiasmatic cartilages of mammals. Inchickens, the supratrabecular cartilages serve as the origins of therectus muscles of the eyes (De Beer, 1937), supporting at least afunctional equivalence with the hypochiasmatic cartilages. A meso-derm-derived supratrabecular cartilage in the chicken (or otheramniote) is consistent with a homology with the mammalianhypochiasmatic cartilages. Unfortunately, the tissue origin of thesupratrabecular cartilages of birds is unclear since they fuse with thetrabecular and polar cartilages at the point where the neural crest-mesoderm boundary has been reported in the chicken chondrocra-nium (Couly et al., 1993). Examination of the tissue origin of thesupratrabecular cartilages of the chicken while they are still indepen-dent of other chondrocranial cartilages at the 7-day stage (De Beer,1937) is needed.

The tissue origin of the rest of the cranial base of the chicken is wellstudied, with the neural crest-mesoderm boundary formed at theprechordal-chordal boundary between the basi-presphenoid and basi-postsphenoid (Le Lievre, 1978; Noden, 1988; Couly et al., 1993; LeDouarin et al., 1993; Lengele et al., 1996; Le Douarin and Kalcheim,1999). However, it is difficult to compare these results with the mousedue to the confusing use of similar terms to describe structures that areprobably not homologous. For example, the basi-presphenoid identi-fied in the chicken chondrocranium by Couly et al. (1993) consists ofthe caudal end of the trabecular cartilage fused with the supratrabe-cular and polar cartilages (De Beer, 1937). While the trabecularcartilage is homologous with the structure of the same name in mice(and does develop into a portion of the presphenoid bone), the polarcartilages of chickens have been described as homologous to thealicochlear commissures of the mouse (De Beer, 1937). Although thereis also a neural crest-mesoderm boundary in the alicochlear commis-sures of the mouse, these are lateral structures not connected to thepresphenoid bone, suggesting that the basi-presphenoid of thechicken is not homologous to the presphenoid bone of the mouse.Regardless of the difficulties presented by homology of the cranialbase, the NHH is supported by the observations in the chicken andmouse, since the neural crest-mesoderm boundary is coincident withthe rostral tip of the notochord in both species.

Morphogenesis and the role of tissue origins

Although the tissue origin of each cranial base cartilage has beenshown to be discrete, being derived from either neural crest ormesoderm cells, some of the bones that develop from these cartilagesshowdual tissue origins. The presphenoid bone has a dual tissue origin,with the orbital and trabecular cartilages being derived from neuralcrest cells whereas the hypochiasmatic cartilages (which eventuallyform just the postoptic roots of the presphenoid bone) are derived frommesoderm. The basisphenoidbone also has a dual tissueorigin, but via adifferent mechanism. The hypophyseal cartilage, which develops intothe body of the basisphenoid bone, is derived solely from neural crestcells, but cells of both neural crest and mesodermal origin are found intheperiosteumandearlymarrowcavityof thedevelopingbasisphenoidbone. While the neural crest cell-derived osteoblasts likely came fromneural crest cell-derived periosteum, the mesoderm-derived osteo-blastsmay develop from the periosteumor an external osteoprogenitorsource that invades via blood vessels.

That some bones, but not the cartilages they develop from, havedual tissue contributions should draw our attention to an importantdistinction: although a mature bone may have osteogenic contribu-tions fromcells of different tissue origins, the cartilages fromwhich thebone develops are each derived from one tissue type, and it is thecartilages (and the mesenchyme fromwhich they condense) that firstestablish the morphological pattern. Rather than being potentiallydistracted by the source of osteogenic cells that arrive on the scene

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after much of the morphological pattern is well established, it may bemore interesting to focus on the fact thatmost of themesenchyme thatforms the anterior cranial base cartilages originates from frontonasaland post-optic streams of migrating neural crest cells. After enteringthe area beneath the telencephalon and rostral diencephalon, thesemesenchyme cells condense and chondrify in a specific spatiotemporalpattern to form cartilages that provide a framework for thecharacteristic adult morphology. It is with this in mind that we canask:what are the signals that establish the pattern in themesenchyme,and does it matter whether the cells are derived from neural crest ormesoderm in order to respond to specific patterning signals?

Research suggests that the notochord is amajor signaling center forpatterning the parachordal cartilage (Nie et al., 2005b; Young et al.,2006), but less is known about what provides the patterning cues forthe anterior cranial base. In gastrulating embryos, the mesendoderm-derived prechordal plate is rostral to the notochord and directly belowthe presumptive prosencephalon (Tam and Behringer, 1997). Anteriormidline tissues in this region have distinct and segmented molecularexpression, which appears to be involved in regionalizing the brain(Camus et al., 2000). Later, neural crest cells migrate to thepresumptive cranial base between the neuroepithelium and facialectoderm, and, by E10.5, neural crest-derived cells are the onlymesenchymal cells that populate the presumptive anterior cranialbase, although nearby prechordal plate mesodermal cells will latergive rise to the extrinsic ocular muscles and possibly the hypochias-matic cartilages. It is interesting to consider the patterning of theconnective tissues of the anterior cranial base in conjunction with themajor innovations of the ‘New Head', the expanded forebrain andsense organs. There is evidence that ectodermal signaling plays a rolein morphogenesis of the underlying mesenchyme. In zebrafish,morphogenesis of the trabecular cartilage and ethmoid plate relieson hedgehog signaling from the brain and facial ectoderm (Wada et al.,2005). In the chicken, Shh and Fgf8 signaling in the neural and facialectoderm is important for facial outgrowth (Schneider et al., 2001;Abzhanov and Tabin, 2004; Abzhanov et al., 2007). Future researchshould focus on the possible roles of the neuroepithelium and surfaceectoderm in determining where neural crest cell-derived cells migrateto and condense in the presumptive anterior cranial base andwhetherthey are restricted from entering the chordal region due to inhibitoryinteractions with regionally expressed proteins (such as from thebrain, surface ectoderm, mesoderm-derived mesenchyme and/or thenotochord itself). Once the influential signaling centers are known, itwill be interesting to determine whether all mesenchyme, regardlessof tissue origin, has the ability to respond to the ectodermal signals orwhether there are different responses to permissive and inhibitorysignals due to intrinsic capabilities born from their tissue history.

Acknowledgments

We are grateful to Dr. Jim Hanken for providing helpful commentson portions of a draft of this manuscript. B. M.-O. was generouslysupported by NIH grants AR 36819 and T32 DE017544 (to B.R. Olsen)and a graduate student fellowship from the Department of Anthro-pology at Harvard University.

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