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Fate map of the chicken neural plate at stage 4

Pedro Fernández-Garre^1,*, Lucia Rodríguez-Gallardo^2,*, Victoria Gallego-Díaz², Ignacio S. Alvarez² and Luis Puelles^1,

¹ Department of Morphological Sciences, Faculty of Medicine, University of Murcia, 30100, Murcia, Spain
² Department of Cell Biology, Faculty of Sciences, University of Extremadura, 06071, Badajoz, Spain
^* These authors contributed equally to this work

View larger version (38K): [in a new window]   Fig. 1. Experimental design used in the present fate map. The epiblast at full-length primitive streak stage (stages 3d/4) was visualized through a grid centered upon the node (A) and plugs of labeled donor tissue were transplanted (circles in B; normally 125 µm in diameter; range 70-200 µm) at different radial locations ( in A). Positions of labeled grafts was recorded by fluorescence microscopy just after transplantation (C) and also after 24 hours survival. Control cases fixed and sectioned at 30 minute intervals revealed that most grafts appeared well integrated within 30 minutes (a small and a larger case are shown sectioned frontally in D), though occasionally some needed up to 2-2.5 hours for complete integration. For a more precise description see text. N, Hensen’s node; PS, primitive streak. Scale bars: 100 µm in C,D.

View larger version (148K): [in a new window]   Fig. 2. Three representative cases in which CFSE labeled grafts (thin arrows in A,C,E) contributed to non-neural ectoderm. (A,C,E) Fluorescence images at 0 hours (graft position relative to the node); (B,D,F) corresponding immunolabeled transverse sections of the same embryos fixed after 24 hours. Cells derived from the graft can be identified in non-neural ectoderm by the DAB label (arrowhead). Scale bars: 100 µm in B-G.

View larger version (30K): [in a new window]   Fig. 3. Examples of cases in which the grafted tissue contributed to both neural (arrowheads in B-D; unmarked in E-G) and non-neural ectoderm. The variously overlapping grafts that overstepped the neural border are represented in A. Cases illustrated in B-G are tagged and green in A; cross-sections in B-G show the locations of the transplanted cells, stained either with DAB (brown) or with AP (blue). Arrowheads in F show label in the otic placode; adjacent sections also had label in the dorsal neural tube. Scale bar: 100 µm.

View larger version (29K): [in a new window]   Fig. 4. (A) The overlap (brown) between the grafts labeling inclusively some neuroectoderm (orange), versus those labeling some non neural ectoderm (blue). Grey areas represent loci not sampled with grafts. (B) Topography and relative size of all studied cases at 0 hours, color-coded according to neural versus non-neural fate. Green identifies the grafts contributing exclusively to non-neural ectoderm and yellow corresponds to grafts producing exclusively neural ectoderm. The separating gap should contain the neural plate boundary, but is probably larger than the border itself. The border grafts contributing to both neural and non-neural regions are represented as empty circles (compare with this set isolated in Fig. 3A). The small empty circle highlighted by a darker outline represents the single case that selectively labeled the otic placode (compare with Fig. 3F). (C) Selection of cases which collectively allowed a more precise definition of the neural border (red line), with some extrapolation (see Results). N, Hensen’s node; PS, primitive streak.

View larger version (66K): [in a new window]   Fig. 5. (A-F) Schematic (A) and microphotographic examples (B-F) of whole-mount DAB or AP labeled graft derivatives obtained 24 hours post-operatively, illustrating different positions mapped along the anteroposterior dimension of the neural tube (insets in B-F show the positions of the five grafts at 0 hours). The relative position of the labeled cells at stage 10 is shown in A. Note that the circular grafts at stage 4 adopt an elongated configuration because of cell rearrangement along the neural axis (red areas in A) (Schoenwolf and Alvarez, 1989). Representative sections of these whole-mounts are presented in the right-hand column. (G,H) Optic vesicle (from B); (I,J) caudal forebrain and alar mesencephalon (from C). (K,L) rostral midbrain (from D); (M,N) caudal mesencephalon and rhombencephalon (from E); (O,P) spinal cord (from F). Scale bar: 100 µm.

View larger version (60K): [in a new window]   Fig. 6. (A) Graphic representation of the sets of grafts classified according to their main rostrocaudal derivatives. The arbitrary color code allows visualization of the areas of overlap between these sets (color summation), and thus tracing of the estimated prospective transverse boundaries (see Discussion). (B) Set of grafts found useful for characterizing the eye field (broken outline); six grafts shown in light blue ended just outside the eye vesicles, whereas seven yellow-colored grafts labeled partially the eye vesicle (see also Table 2). (C) Graphic superposition of color coded sets of grafts labeling the alar and basal plate regions; the overlap between these sets is highlighted by color summation (brown), roughly indicating where to trace the postulated longitudinal alar-basal boundary (thick black line). (D) Detailed fate map obtained, showing the longitudinal and transverse boundaries identified within the prospective neural territories at stages 3d/4. The floor plate territory was marked as well (see Results and Discussion). (E) The estimated main radial, longitudinal and transversal distances relative to the node are indicated for the stage 3d/4 neural plate fate map (yellow). F, forebrain; M, midbrain; H, hindbrain; S, spinal cord; MS, mesoderm; NNE, non-neural ectoderm; PS, primitive streak; OV, optic vesicle.

View larger version (53K): [in a new window]   Fig. 7. (A) Set of grafts that contributed to the alar plate. Tagged green circles in the drawing identify the representative examples of which cross-sections are presented in B-G. (B) underside of eye vesicle; (C,D) dorsal midbrain; (E-G) hindbrain. Scale bar: 100 µm.

View larger version (90K): [in a new window]   Fig. 8. (A) Grafts that contributed to the basal plate and/or floor plate. Tagged green circles identify the representative examples whose cross-sections are presented in B-L. (B-D) Graft overlapping alar chiasmatic region and basal hypothalamus, (E,F) hypothalamic basal plate and floor; (G,H) midbrain floor and basal plate; (I) basal hindbrain; (J,K) whole-mount view and section of hindbrain floor plate; (L) spinal cord floor plate. N, Hensen’s node. Scale bars: 100 µm in B-G.

View larger version (113K): [in a new window]   Fig. 9. Examples of experiments aimed to evaluate the relative final position of grafts made rostral to the node with respect to the anterior neural marker gene Ganf. (A) Initial Ganf expression pattern at stage 4. (B-D) Embryo transplanted just rostral to the node (0-125 µm) and allowed to develop until stage 8. There is no overlap between the grafted cells in the prospective hypothalamic floor (brown DAB reaction product; arrowhead in C) and the expression domain of Ganf (blue signal). (E-G) Embryo with a median graft located between 225-350 µm and allowed to develop until stage 9+. Most cells of the graft (in brown) appeared in the rostral head ectoderm (non-neural tissue), but the anterior region of Ganf expression (blue) at the neuropore in F shared some cells with the graft. Therefore, the anterior limit of the established fate map coincides with early and later Ganf expression at the neural canal boundary. Scale bars: 100 µm in B-L.

View larger version (54K): [in a new window]   Fig. 10. Whole-mount in situ hybridization reactions to show the expression of the genes Plato (A), Sox2 (B), Otx2 (C) and Dlx5 (D) at stage 4 and their relationship with the fate-mapped neural plate boundary obtained in the present study, after correcting for 3% retraction caused by the method used (neural plate outlined in each case). Scale bar: 100 µm.