Neural crest does not contribute to the neck and shoulder in the axolotl (Ambystoma mexicanum).

BACKGROUND: A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context. RESULTS: We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum) donor embryos into white (d/d) axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl. CONCLUSIONS: Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the "muscle scaffold theory," and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution

Relations of the shoulder girdle to the embryonic and adult anatomy.

<p>According to the fate map by Stocum and Fallon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052244#pone.0052244-Stocum1" target="_blank">[32]</a>, the shoulder girdle of the axolotl arises mainly from flank mesoderm as part of the embryonic limb field (left). The upper, scapular (sca), and the lower, coracoid (cor) parts of the shoulder girdle (right) originate from the specific areas of the limb field around the region, which gives rise to cartilage and connective tissues of the prospective free limb (fl) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052244#pone.0052244-Stocum1" target="_blank">[32]</a>. The shoulder girdle region is thus positioned just caudal to the branchial arches (ba), where the main streems of migrating neural crest cells pass. In adults, the coracoid plate of one side meets the contralateral counterpart along the ventral midline of the animal, while the upper scapular edge reaches the level of transverse processes of the thoracic vertebrae. These parts of the shoulder girdle are cartilaginous (grey) in the axolotl throughout life, while the middle of the shoulder girdle (both in the scapula and the coracoid plate), from where the limb emerges, are ossified in adults. The anterior, cranial edge of the scapula bears the attachment sites of muscles (m. cuccularis, m. opercularis), which connect the shoulder girdle to the occipital bones of the skull. Other abbreviations: e, eye; prn, pronephros; s, somite, tv, thoracic vertebrae. Not to scale.</p

Results of grafting one short left neural fold fragments.

<p><b>a,</b> Schematics demonstrating orthotopical grafting of a short left GFP+ neural fold fragment (including neural crest) into a white (d/d) host. The graft is extirpated from a GFP+ neurula (green, stage 16) and extends from a prospective posterior head to an anterior trunk region. It is implanted into a white host where a similarly sized fragment was extirpated previously. <b>b</b> and <b>c,</b> left flank of white hosts 1 day (<b>b</b>) and 3 days (<b>c</b>) after the operation. In vivo visualization of GFP+ neural crest cells at an anterior trunk level where they migrate laterally from the top of the neural tube; arrows show the main direction of migration. <b>d–h</b>, two months old juvenile carrying a short GFP+ neural fold fragment. No neural crest cells were present in the scapula, or elsewhere in the shoulder girdle. However, all other neural crest derivatives located at this level were GFP+. <b>d</b>, left side of operated juvenile where cranial and ventral margins of the GFP negative shoulder girdle are visible through the transparent skin. Girdle cartilage is outlined with a dashed line. <b>e,</b> ventral aspect of the juvenile. Gills, nerve fibres in the limb, pigment cells, heart and enteric ganglia are clearly GFP+, while the ventral halves of the cartilaginous coracoid plates (indicated with the dashed line) are GFP negative. <b>f</b>, enlarged area of the scapula framed in (<b>d</b>). Only spinal nerves of the brachial plexus appear GFP+. The cranial margin of the scapula is marked with white arrowheads. No GFP+ cells are detectable along its cranial margin, where muscles exist that attach it to the skull. <b>g, h</b>, transverse sections through the juvenile (sectioning planes see (<b>f</b>)) with GFP+ spinal nerves but GFP negative scapular cartilage and connective tissue. <b>i–l</b>, sagittal sections through the shoulder girdle region in a 1.5 month old juvenile from dorso-medial (<b>i</b>, scapula tip as in <b>h</b>) to ventro-lateral (<b>l</b>, glenoid region). Anti-Myosin heavy chain-rhodamine immunostaining only in <b>i</b>, for better visualization of GFP+ cells. Note GFP+ staining in all sections only in spinal nerves, but not in cartilage or muscle attachment sites of the shoulder girdle. Abbreviations: cor, coracoid; df, dorsal fin; drg, dorsal root ganglia; eg, enteric ganglia; g, gills; h, heart; ln, limb nerves; m.c., musculus cuccularis; m.d.s., musculus dorsalis scapulae; m.d.h., musculus dorsalis humeralis (latissimus dorsi); pc, pigment cells; sca, scapula; sn, spinal nerves. Scale bars: <b>b</b>–<b>f:</b> 1 mm; <b>g–l</b>: 100 µm.</p

Results of additional experiments.

<p><b>a</b>, Sagittal section through the neck epaxial muscles in between the scapular tip and occipital region of the skull; this region is devoid of any neural crest-derived connective tissue. Only intersegmental nerves are present along the intermuscular septae are GFP+ (green arrowheads). <b>b–e</b>, transverse sections through the shoulder girdle region of a juvenile (1 month) containing two GFP+ neural folds (see Fig. <b>3a</b>). The framed area in (<b>b</b>) is enlarged in (<b>c–e</b>). <b>c–e</b>, GFP+ spinal nerves close to the shoulder girdle cartilage (<b>c</b>) and Myelin Basic Protein+ cells (anti-MBP-Cy5 immunostaining) in (<b>d</b>) are co-localized (<b>e</b>) as indicated with white arrowheads. <b>f–h,</b> medial aspect of the right shoulder girdle (soft tissues included) of a mature axolotl (3 years) containing two short GFP+ neural fold fragments on either side (same experiment as in (Fig. <b>2a</b>), but with short double- sided graft). <b>f</b>, bright field micrograph of an isolated shoulder girdle whole mount with framed areas enlarged in (<b>g</b>) and (<b>h</b>). The dorsal border of the ossified part of the scapulo-coracoid is indicated with black arrows. <b>g</b>, GFP+ spinal nerves over the GPP-negative ossified scapulo-coracoid. <b>h</b>, nerve net in the muscles connecting to the scapula. GFP+ cells are not present in muscle attachment sites (empty white arrowheads) and the tip of the scapula of somitic origin (white asterisks). Abbreviations: tr2, transverse process of the second vertebra; occ, occipital bone; other abbr. as in Figs. <b>1</b>–<b>3</b>. Scale bars: <b>a–e :</b> 100 µm, <b>f–h :</b> 5 mm.</p