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Figure 1. Topography of tissues used for fate mapping experiments in the axolotl neurula (stage 15).A, three defined neural plate areas (plate regions1â3) and neural fold areas (fold regions1â3) were grafted from GFP+ donors (stage 15) homo- or heterotopically into white (d/d) hosts (stage 15) for studying their potency to develop into striated tail muscle or fin mesenchyme. Experimental results were analyzed in larvae at stage 41 (1âcm length; details Figs. 2 and 3). Mapping was according to Bijtel20 who originally divided the neural plate of stage 16 neurulae along the cranio-caudal axis into 5 rectangular zones. We used stage 15 neurulae with a wider plate for better grafting. Here the length to width of each neural plate belt measures about 300âÃâ1000 (lxw) μm; anterior belts are wider than posterior ones. Neural fold areas on either side of the plate measure about 300âÃâ200â(lxw) μm. As only prospective trunk but no cranial plate was needed for grafting, we nominated the anterior trunk rectangle âregion 1â (plate region1), the middle one âregion 2â (plate region2) and the posterior one âregion 3â (plate region3). Trunk neural fold zones are called accordingly: âleft or right neural fold region 1, 2, and 3â (fold region1, -2 and -3).
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Figure 2. Results of fate mapping experiments of neural plate in the axolotl neurula (stage 15).Only posterior trunk neural plate regions contribute to posterior trunk/tail muscle and fin mesenchyme. AâC, homotopic transplantation of three defined GFP+ neural plate regions (accentuated in green) from a GFP+ donor (stage 15) to a white (d/d) host (stage 15) and visualization of graft-derived GFP+ cells in larvae at stage 41 (1âcm length). White arrowhead indicates position of cloaca in larvae. AââCâ, transverse cryosections through larvae shown in AâC containing GFP+ grafts; dashed lines in AâC indicate sectioning planes; sections are overlays of fluorescence images. Dapi, blue; anti-12/101 (muscle), red; anti-GFP, green. A and Aâ, GFP+ region3 plate gives rise to most myotome cells in the tail and posterior trunk and to mesenchymal cells of the dorsal and ventral tailfin (faintly visble). B and Bâ, GFP+ region2 plate gives rise to some cells in the spinal cord and tail myotomes. C and Câ, GFP+ region1 plate contributes to cells in the spinal cord of the anterior trunk. Aâ, enlargement of tailfin for visualizing mesenchymal cells (mes) in the dorsal (df) and ventral tailfin (vf); animal different from that in A. Number of experiments: A, 29; B, 16; C, 8. Abbreviations: df, dorsal fin; vf; ventral fin; mes; mesenchymal cell; my, myotome; spc, spinal cord; not, notochord; drg, dorsal root ganglia; spn, spinal nerve. Scale bars, 1âmm (AâC), 100âμm (AââCâ) and 500âμm (Aâ).
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Figure 3. Results of fate mapping experiments of neural fold in the axolotl neurula (stage 15).Only posterior trunk neural fold regions contribute to posterior trunk/tail muscle and fin mesenchyme. A-C, homotopic transplantation of three defined GFP+ neural fold regions (accentuated in green) from a GFP+ donor (stage 15) to a white (d/d) host (stage 15) and visualization of graft-derived GFP+ cells in larvae at stage 41 (1âcm length). White arrowheads indicates position of cloaca in larvae. AââCâ and Aâ, transverse cryosections through larvae shown in AâC containing GFP+ grafts; dashed lines in AâC indicate sectioning planes; sections are overlays of fluorescence images. Dapi, blue; anti-12/101 (muscle), red; anti-GFP, green. A, Aâ and Aâ, GFP+ region3 fold gives rise to few muscle cells in tail myotomes, to few mesenchymal cells in the dorsal and ventral tailfin and to some tail epidermis (upper and lower seam of tailfin). B and Bâ, GFP+ region2 fold contributes cells to the spinal cord, dorsal root ganglia and fin epidermis in the mid trunk. The labelling of the epidermis is not visible here in Bâ but optimal further posteriorly to the ganglia (see Fig. S1). C and Câ, GFP+ region1 gives rise to cells in the spinal cord, dorsal root ganglia, fin epidermis and to the middle lateral line nerve in the anterior trunk. DâF, higher enlargements of boxed area in Aâ. Presence of GFP+ mesenchymal cells in the dorsal tailfin after grafting GFP+ fold region3. Number of experiments: A, 30; B, 5; C, 5. Abbreviations: df, dorsal tail fin; vf: ventral fin; mes: mesenchymal cell; my, myotome; epi, epidermis; spc, spinal cord; lln, lateral line nerve; drg, dorsal root ganglia; spn, spinal nerve; not, notochord. Scale bars, 1âmm (AâC), 200âμm (Aâ), 20âμm (F) and 100âμm (Aâ, Bâ, Câ).
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Figure 4. Heterotopic grafting of plate region3 into plate region 1 area gives rise to an ectopic tailfin.A, operation schematic. B and C, an ectopic tail is formed from the GFP+ graft in the anterior trunk (C, enlarged); overlay of fluorescence and bright field images. D, schematic indicating transverse (EâH) sectioning planes through ectopic tail. E, distribution of GFP+ cells derived from grafted GFP+ plate region3. F, myotome cells detected with 12/101 antibody. Red dot in notochord is due to unspecific staining. G, merged images; the ectopic tail contains GFP+ cells in spinal cord, muscle and mesenchyme but not in notochord. Number of experiments: 10. Abbreviations: ant, anterior; post, posterior; spc, spinal cord; not, notchord; mu, muscle; df, dorsal fin; vf, ventral fin; ant, anterior; post, posterior. Scale bars, 1âmm (B), 500âmm (C and G).
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Figure 5. Central part of the posterior region 3 plate gives rise to fin mesenchyme and muscle.Homotopic transplantation (A) and heterotopic transplantation (B) of medial GFP+ neural plate tissue from the posterior region 3 of a GFP+ donor (stage 15) to a white (d/d) host (stage 15). A, visualization of graft-derived GFP+ cells in the myotomes (my) of the posterior trunk and anterior tail of a host at stage 41; dorsal fin mesenchyme (mes) is also GFP+; white arrowhead points to position of cloaca. BâF, visualization of GFP+ cells in the dorsal fin of the anterior trunk of a host at stage 41. DâF, demonstration of GFP+ fin mesenchyme (mes) and striated paraxial muscle on transverse sections (plane indicated in C). White lines in CâE help to see the outlines of dorsal parts of larvae. Number of experiments: A, 9; B, 3. Abbreviations: df, dorsal fin; vf, ventral fin; mes, mesenchyme; my, myotome; spc, spinal cord; not, notochord. Scale bars: 1âmm (A), 500âmm (B and C) and 200âmm (F).
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Figure 6. Expression of molecular markers for epidermis, mesoderm, and neural crest.In situ hybridization of axolotl neurulae (stage 15) with keratin (A, A’), sox2 (B, B’ and Bâ€), brachyury (C, C’ and Câ€) and tfap2a (D, D’ and Dâ€) riboprobes. A–D, dorsal views of whole embryos. A’–D’, posterior views of whole embryos. Aâ€â€“Dâ€, posterior aspects of anterior halves of bisected embryos. Sectioning planes are indicated by dashed lines and run through the middle of region 3 fold/plate (A’, C’ and D’) or through region 2 (B). Red double-dashed lines indicate neural folds; prospective epidermis is lateral and neural plate medial to the folds. E, fate of plate/fold region3 based on in situ hybridization with brachyury (bra), sox2 and tfap2a riboprobes (neurula stage 15). Brachyury: positive in the centre of plate region 3; tfap2a: positive.in cranial and trunk neural folds until the anterior part of fold region 3; sox2: positive in cranial and region 2 plate. F and G, transverse sections through neural fold/plate (stage 15) in region 1–2 (F) and middle of region3 (G). Axial differences of neural plate and neural crest potential become evident (neuroectoderm vs. mesoderm and neural crest vs. neural fold, respectively). These data and the indication of the distribution of the tfap2-, sox2- and bra-zones in E are based on in situ hybridization (see above). nc in F, prospective neural crest; nfo in G, tfap2a-negative neural fold tissue, probably mesoderm. Number of experiments: about 20 for each riboprobe. White arrowheads in A‘-D‘ point to blastopore. Abbreviations: not, notochord; nfo, neural fold; cr. nfo, cranial neurl fold; npl, neural plate; ax, axial mesoderm; pax, paraxial mesoderm. Scale bars, 500 μm (D’) and 200 μm (Dâ€).
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Figure 7. Grafted single GFP+ neural fold reg3 cells reveal mesodermal but no pigment cell traits in the host tailfin.A, Schematic of experimental procedure. Single neural fold cells were isolated from a dorsal neural fold explant (left middle to posterior region3) of a GFP+ donor (stage 15) and grafted homotopically into a white host (stage 15). B, summary of experimental results; frequency of GFP+ cells in tail tissues of host larvae (stage 41, 1âcm); tissues: mus, muscle; fin mes, fin mesenchyme; ct, connective tissue; epi, epidermis. CâG, types and frequency of GFP+ tissues that had developed from a single grafted neural fold region3 GFP+ cell in tail regions of living axolotl hosts (stage 41, 1âcm); bright field/FITC. CââGâ, higher enlargements of boxed areas in CâG. C and Câ, only muscle cells (39%); D and Dâ: muscle and fin mesenchyme cells (16%); E and Eâ: muscle and connective tissue (8%); as the connective tissue is located inside the larva it is dim and insharp in wholemount images; F and Fâ: only fin mesenchyme (12%); G and Gâ: epidermis (22%). H and L, Transverse sections through tails of larvae shown in D and E, respectively. IâK, higher enlargement of GFP+ fin mesenchymal and muscle cells from H. MâN, higher enlargement of connective tissue (M) and muscle cells (N) from L . HâL and N, merged images of GFP, 12/101 (red) and Dapi. M, merged image of GFP, bright field and dapi. Scale bars, 200âμm (H and L) and 50âμm (K and N). IâK and MâN have the same magnification.
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Figure 8. Descendants from blastomere C4 form fin mesenchyme in Xenopus laevis.A, Schematic of the experiment. At the 32-cell stage one C4 blastomere was labelled by microinjection of ruby-dextran. At stage 40 the labelling can be found in somites, in the posterior part of the intestine, in the blood and in the fin. The squares indicate the areas displayed in B,C,F,G. The vertical line indicates the cutting planes in D and E. B,B’C,C’, Groups of labelled cells migrate from the somites to the dorsal (B,B’) and ventral fin (C,C’). Squares in B and C indicate the regions displayed in B’ and C‘ at higher magnification. D, Transversal section through posterior trunk with labelled somite (som) and fin mesenchymal cells (arrows); overlay of dextran-fluorescence and phase-contrast image. E, Transversal vibratome section through posterior trunk with labelled somite (som) and with a labelled fin mesenchyme cell (arrow). DAB-peroxidase staining using the biotin-residues of the ruby-dextran tracer. F–G, Fin mesenchyme cells (arrows) at higher magnification; F, dextran fluorescence; G, after DAB-peroxidase staining. Abbreviations: A, animal; V, vegetal; DA, dorsoanterior; VP, ventroposterior; An, anterior; D, dorsal; Po, posterior; V, ventral; epi; epidermis; som, somite; Number of cases: 65
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