The right (j) sides of RE 640 another 1.5 months old juvenile after double sided neural fold transplantation. Note GFP+ staining in all sections only in Tramiprosate 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: 1326631 b : 1 mm, h : 100 mm. doi:10.1371/journal.pone.0052244.gLarvae carrying two grafted folds were carefully examined on ca. 300 transverse and sagittal cryostat sections (see below). There was no evidence of GFP silencing in neural crest derivatives, such asdorsal root ganglia. In addition we investigated both left and right halves of the shoulder girdle of two about 2.5 year old adult axolotls (when the scapulocoracoid is ossified) that had receivedLack of Neural Crest in the Axolotl ShoulderLack of Neural Crest in the Axolotl ShoulderFigure 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 , 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 ). c , 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 , 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?. Scale bars: a : 100 mm, f : 5 mm. doi:10.1371/journal.pone.0052244.gshort left and right neural fold fragments at the neurula stage as described [24].a secondary antibody conjugated with Cy5. All sections were stained with DAPI, embedded into glycerol-PBS (1:1) and analysed with epifluorescence microscopes.Sectioning and ImmunostainingTransverse cryosections (20?5 mm) were cut through the shoulder region of the anterior trunk in about 1.5?.5 month old juveniles that contained GFP+ tissues. Specimens were fixed with 4 paraformaldehyde at 4uC over night, washed in PBS, incubated in 30 sucrose overnight, infiltrated with 5 gelatine (Merck) overnight, embedded into 7.5 gelatine and frozen on dry ice. Cryosections were stained with primary antibodies against GFP (Invitrogen) to increase the visibility of transgenic donor cells. Alexa 488- conjugated secondary antibodies were used for detecting GFP. Additionally, we used 12926553 rhodamine-conjugated anti-Myosin heavy chain antibodies (clone 4A.1025, a kind gift from Simon Hughes, Kings College, London) to visualize skeletal muscles and anti-Myelin-basic-protein antibodies (GeneTex.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: 1326631 b : 1 mm, h : 100 mm. doi:10.1371/journal.pone.0052244.gLarvae carrying two grafted folds were carefully examined on ca. 300 transverse and sagittal cryostat sections (see below). There was no evidence of GFP silencing in neural crest derivatives, such asdorsal root ganglia. In addition we investigated both left and right halves of the shoulder girdle of two about 2.5 year old adult axolotls (when the scapulocoracoid is ossified) that had receivedLack of Neural Crest in the Axolotl ShoulderLack of Neural Crest in the Axolotl ShoulderFigure 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 , 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 ). c , 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 , 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?. Scale bars: a : 100 mm, f : 5 mm. doi:10.1371/journal.pone.0052244.gshort left and right neural fold fragments at the neurula stage as described [24].a secondary antibody conjugated with Cy5. All sections were stained with DAPI, embedded into glycerol-PBS (1:1) and analysed with epifluorescence microscopes.Sectioning and ImmunostainingTransverse cryosections (20?5 mm) were cut through the shoulder region of the anterior trunk in about 1.5?.5 month old juveniles that contained GFP+ tissues. Specimens were fixed with 4 paraformaldehyde at 4uC over night, washed in PBS, incubated in 30 sucrose overnight, infiltrated with 5 gelatine (Merck) overnight, embedded into 7.5 gelatine and frozen on dry ice. Cryosections were stained with primary antibodies against GFP (Invitrogen) to increase the visibility of transgenic donor cells. Alexa 488- conjugated secondary antibodies were used for detecting GFP. Additionally, we used 12926553 rhodamine-conjugated anti-Myosin heavy chain antibodies (clone 4A.1025, a kind gift from Simon Hughes, Kings College, London) to visualize skeletal muscles and anti-Myelin-basic-protein antibodies (GeneTex.
