The key problem we wish to address is the full distribution of skeletal post-otic neural crest (PONC). By using Wnt-1 17
recombinase mediated fate mapping we ask three questions: 1. Can we find evidence for post-otic neural crest (PONC) to form endochondral bones? This determines whether either the ‘ossification’ or the ‘scaffold model’ are applicable to the neck region. 2. Is the entire dermal skeleton behind the otic capsule neural crest derived, or is some of it mesodermal? This will test the validity of the ‘ossification model’ in the only species that is currently accessible to high-resolution lineage mapping: the mouse. 3. Does the distribution of neural crest and mesoderm correlate with muscle attachment points or with ossification types in the neck and shoulder skeleton? This will distinguish the explanatory value of the ‘ossification model’ from that of the ‘scaffold model’ as each model makes non-overlapping predictions about anterior shoulder girdle origins.
Neural crest proves to have an unexpectedly pervasive role in the mouse neck region, forming bone, cartilage and muscle connective tissue within two domains. First, an external, essentially tubular domain dominated by pharyngeal arch muscles that extends from the head to the entire ancestral shoulder girdle and incorporates its anterior part (). Secondly, a ventral internal domain comprising internal pharynx and larynx constrictors, tongue muscles, thyroid, cricoid and arytenoid cartilages and their respective muscle attachments at the oesophageal entry (,). Ensheathed between these two tubular domains lies a mesodermal domain centered on the somite-derived vertebral column, that reaches forward to the occipital region of the head.
Fig.3 Neural crest and somitic mesodermal origins of the neck. Green areas in schematic are neural crest-derived connective tissues, blue areas are mesodermal (somitic) connective tissues and muscle fibres. Neural crest-derived (left column a,b,c) (nuchal ligament (more ...)
Fig.4 Dual neural crest and mesodermal origins of the endochondral shoulder girdle. Green box PONC GFP+ green (a,b) components, turquoise box mesodermal LacZ+ blue (e) components of the scapular spine (box 1, a,b,e), the acromio-coracoid (box 2, c,f,) and sternum (more ...)
Fig.6 Pathological flexibility of post-otic neural crest differentiation. Changes in fate specification from bone (periosteum) into connective tissue (left half of figure) can explain localized cervical defects in cleidocranial dysplasia (b,c) and Chiari I.+II (more ...)
The largest component of the external crest domain is the trapezius muscle and its attachment regions (tra in ). This is a branchial muscle, innervated by the accessory nerve X/XI with a position that has remained remarkably conserved in all jawed vertebrates 5
. The cranial neural crest connectivity code revealed by our previous chick-quail work 13
led us to predict that the connective tissue of the trapezius and all its postotic attachments should be formed from PONC - even if they are endochondral. This indeed proves to be the case. At the anterodorsal end, a patch of LacZ+/GFP+ PONC endoskeleton forms the occipital protuberance inside otherwise mesodermal (occipital) territory (i.e. the trapezius attachment point in the skull, ). The labelling extends to the attached nuchal ligament (nl, ), the trapezius muscle connective tissues (), as well as their respective endoskeletal attachment region on the entire anterior margin and inside the scapular spine (), the coracoid, acromion (, box 2 in ) as well as the periosteal muscle attachment caps on spinous processes of all cervical vertebrae (pc in ,). Thus, the posterior neural crest boundary is found inside
the shoulder girdle endoskeleton where it forms an anterior attachment region of branchial muscles. PONC thus generates extensive areas of endochondral ossification in the shoulder girdle and cervical vertebral column, contradicting the traditional notion that these regions are wholly mesodermal4,11
. However, the crest contributions are morphologically cryptic: their only visible anatomical landmarks are the branchial muscle attachments. Tracing the posterior PONC boundary more ventrally we investigate the sternocleidomastoid muscle (N. XI, scm in ). This originates on the (postotic) mastoid process of the skull (m in ) and attaches onto the endoskeletal anterior sternum (st in ) as well as along the anterior margin of the dermal clavicle (cl in ). We find green LacZ+/GFP+ PONC cells inside all of these attachment sites. Our genetic labelling provides first detailed insights into cellular architecture and origins of the clavicle (black box, ). The clavicle bone (black box in ) forms from an anterior (buccal) dermal () as well as a posterior dermal ossification centre (Fig.5g), which later fuse and surround a cartilaginous core in mammals () 15,16
. The anterior dermal ossification is purely PONC derived. (green box, ). More medially, attachment regions of branchial muscles (sternomastoid, cleidomastoid, fascial sling of omohyoid ohs) and most of the cartilaginous core of the clavicle are also neural crest derived (). Thus, as inside the scapula, PONC inside the clavicle gives rise to endochondral bone.
Fig.5 The dual architecture of the clavicle: cell population boundaries coincide with muscle attachments and not with ossification modes. Black box surrounds data of the clavicle. a–e PONC-derived parts, f–g mesodermal parts. PONC connective (more ...)
The ventral shoulder girdle carries a series of muscles that connect it to ventral branchial elements (Box4, Co1, Co2 in , ): M.omohyoideus (connecting the anterior scapula next to the coracoid, and the internal clavicle, with the hyoid; oh, ,), M. sternohyoideus (connecting the manubrium sterni and clavicle to the hyoid; sth, ,) and M. sternothyroideus (sth, connecting the manubrium sterni with the thyroid cartilage). These are homologs of the coracobranchial muscles (Co1,2) present in all jawed vertebrates and effect rapid jaw opening and retraction of the branchial skeleton 3,5
. As these are in origin branchial muscles (innervated by cranial nerves 5
) we hypothesized that their connective tissue would be crest derived and this is indeed the case. Swallowing in all jawed vertebrates requires internal pharynx and larynx constrictors which constitute the internal tubular crest domain outlined above. These are connected to the mesodermal cranial base (the so-called pharyngeal tubercle) anterior to the foramen magnum via the pharyngobasilar fascia, as well as to the ventral neck vertebrae via the pharyngeal raphe (, ). Their branchial innervation (by N.IX, X.) suggests their attachments regions to be PONC-derived too in all vertebrates. Despite being at odds with the commonly held notion that ‘chordal’ cranial base is entirely mesodermal 4,11
, our PONC-labelled mice show constrictor muscle attachment points of neural crest origin focally inserted into the otherwise mesodermal endochondral cranial base (). More posteriorly, thyroid, cricoid and arytenoid cartilages and their respective muscle attachments at the oesophageal entry are also neural crest-derived with tracheal cartilages demarcating the anterior mesoderm boundary (data not shown). More surprisingly, the entire intrinsic tongue musculature that is attached to crest-derived branchial skeleton has crest-derived connective tissue, despite being innervated by N.XII and cervical spinal nerves (). This demonstrates that motor-innervation alone cannot serve as a reliable indicator of embryonic muscle connective tissue origins, but skeleton-muscular connectivity can. These genetic PONC-labelling experiments demonstrate that post-otic neural crest in mouse behaves in the same way as the pre-otic crest studied in our previous experiments 13
. Crest cells form not only the connective tissue of the muscle but all its attachment points, irrespective of how these attachment points ossify: endochondrally or dermally or – as inside the tongue ()- they do not ossify. This unveils a pervasive but anatomically cryptic ‘muscle scaffold system’ that is sharply defined at the single cell level.
Fig.7 Muscle scaffolds in fossils: the cleithrum (box 1) goes into hiding (C-) several times independently in evolution and ‘morphs’ into scapular spines. In all species with a cleithrum - be they fossil mammalian (Varanops ref38), amphibian (more ...)
Fig.2 Genetic lineage labeling of post-otic neural crest and somitic mesoderm in the neck. a. transgenics carrying 1.Wnt—Cre-17, 2.Sox10Cre- constructs were crossed into R26-RLacZ 45 and –GFP 46 reporters in order to permanently label all pre- (more ...)