Figure 1.
Relations of the shoulder girdle to the embryonic and adult anatomy.
According to the fate map by Stocum and Fallon [32], 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) [32]. 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.
Figure 2.
Results of grafting one short left neural fold fragments.
a, 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 and c, left flank of white hosts 1 day (b) and 3 days (c) 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. d–h, 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+. d, 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. e, 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. f, enlarged area of the scapula framed in (d). 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. g, h, transverse sections through the juvenile (sectioning planes see (f)) with GFP+ spinal nerves but GFP negative scapular cartilage and connective tissue. i–l, sagittal sections through the shoulder girdle region in a 1.5 month old juvenile from dorso-medial (i, scapula tip as in h) to ventro-lateral (l, glenoid region). Anti-Myosin heavy chain-rhodamine immunostaining only in i, 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–f: 1 mm; g–l: 100 µm.
Figure 3.
Results of double-sided neural fold transplantations.
a, Schematics demonstrating grafting of both GFP+ neural folds (including neural crest) from a GFP+ neurula (green, stage 16) into a white (d/d) host. Both entire GFP+ neural folds were grafted into a white host in which the neural folds from both sides had been removed before. b–d, embryos containing 2 GFP+ neural folds 2 h, 1 day, and 5 days after the operation, respectively. e–g, 2 months old juvenile; all neural crest derivatives are GFP+. e, dorsal aspect of the juvenile; scapulae visible on both sides through the skin. f, enlargement of area framed in (e), the cranial margins of the dorsal scapulae are marked with arrowheads. g, the same larva viewed from the left side (head to the left). The scapula blade, visible through the skin between the spinal nerves of the brachial plexus, contains no GFP+ signal, neither within the cartilage nor along the cranial margin (arrowheads). h, transverse section through a three weeks old juvenile at the fore-limb bud level. Neural crest cells migrating in a kind of stream-like order are detected at the base of the forelimb bud where they might form sheaths of nerve fibres. i–j, transverse sections through the middle part of the scapulo-coracoid at two cranio-caudal levels on the left (i) and the right (j) sides of another 1.5 months old juvenile after double sided neural fold transplantation. Note GFP+ staining in all sections only in spinal nerves, but not in cartilage or connective tissue of the shoulder girdle. Abbreviations: lb, limb bud; other abbr. as in Fig. 1 and 2. Scale bars: b–g: 1 mm, h–j: 100 µm.
Figure 4.
Results of additional experiments.
a, 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–e, transverse sections through the shoulder girdle region of a juvenile (1 month) containing two GFP+ neural folds (see Fig. 3a). The framed area in (b) is enlarged in (c–e). c–e, GFP+ spinal nerves close to the shoulder girdle cartilage (c) and Myelin Basic Protein+ cells (anti-MBP-Cy5 immunostaining) in (d) are co-localized (e) as indicated with white arrowheads. f–h, 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. 2a), but with short double- sided graft). f, bright field micrograph of an isolated shoulder girdle whole mount with framed areas enlarged in (g) and (h). The dorsal border of the ossified part of the scapulo-coracoid is indicated with black arrows. g, GFP+ spinal nerves over the GPP-negative ossified scapulo-coracoid. h, 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. 1–3. Scale bars: a–e : 100 µm, f–h : 5 mm.