Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

Abstract

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.

Figure 1. Fate map of the neural crest and the dorsal neural tube

(A, B) Method for plotting position of polyclones on the fate map. (A) For injections located caudal to the node, the position of the dye-labeled polyclones was placed on a standardized stage 4 embryo by determining for each embryo the position of the 30–70 µm spot of DiI relative to the height of the primitive streak and to the distance between the center of the streak and the AO/AP (Area Opaca/Area Pellucida) boundary at that particular rostrocaudal level. (B) For injections located rostral to the node, the mediolateral position was determined relative to an imaginary line between the area opaca and primitive streak; rostrocaudal position was determined relative to a line from Hensen’s node to the apex of the embryo. Plotting the positions using these percentages automatically compensates for variations in the size and shape of individual embryos. See Materials and Methods for additional details. (C) Color-coded fates of polyclones labeled at the depicted positions. Note that the neural crest and neural plate overlap at stage 4. For clarity, data from only a subset of the polyclones are depicted. In total, over 130 embryos were sectioned to validate the cellular identity of the labeled cells, 50 had perfect morphology and were used to define the fate map. (D) Color coded fate map of the neural crest showing rostrocaudal identity of the polyclones. Multiply colored spots show polyclones that contributed to more than one axial level. (E) Close-up of panel (C). (F) Close-up of panel (D).

Figure 2. The neural plate border region at stage 4 contributes to neural crest

Three representative embryos labeled with DiI at stage 4 in the neural plate border region of the fate map, cryosectioned and stained for Pax7, a marker of the dorsal neural tube, migrating neural crest and dorsal somite. Three different axial levels of injections into the neural plate border are presented: A–C” shows an embryo labeled in the neural plate border at the rostral hindbrain level; D–F” in the neural plate border at the level of the otic vesicle; G–I” in the neural plate border at the caudal hindbrain level. The “t=0” column shows images of the embryos at the time of injection; the “t=24hrs” column shows the same embryos after one day and the right three columns show sections through the labeled regions of those same embryos. Scale bars: 200 µm, except C-C”, F-F”, I-I” 50 µm.

Figure 3. Frames from a time-lapse sequence from stage 4 to stage 9

(A) Polyclones change shape and position. Note the change in individual polyclones—for example #4 and #14—between the beginning and the end of the time-lapse sequence. Unnumbered polyclones gave rise to extra-embryonic membrane, polyclones #6 and 7 labeled medial neural plate and became covered over by the fusing neural folds, #13 moved caudally and out of view on the computer screen during development. (B) Collage of images from z-stacks generated from this embryo at the end of the time-lapse, showing the different patterns of polyclones in the head versus trunk.

Figure 4. Analyses of convergent extension at the neural plate border

(A) Beginning frame from the time-lapse sequence of the embryo analyzed throughout this figure. The polyclones are numbered. (B) Image from a confocal z-stack generated at the end of the time-lapse sequence (embryonic stage 8+). Black arrows point to regions of mixed red and green cells. Note also how polyclones occupy different axial levels, testifying to the occurrence of reorientation. (C) Cell tracks of those polyclones, with the solid circle indicating the beginning of the cell path and the red tip indicating the end of the path. The tracks shown in black represent polyclones that did not give rise to neural fold. The green, peach and mauve tracks originate in the neural plate border and give rise to neural folds. P.S.: Primitive Streak. (D) To show the cell movements of the neural plate border, the polyclones were rendered as vertices of a grid. As each polyclone moved, the displacement of the corresponding vertex was followed to determine the distortion of the neural plate border, due to 1) extension, 2) reorientation, and 3) convergence towards the midline. (E) Frames from the time-lapse sequence of the embryo shown in (A), showing that extension progresses rostral to caudal. Polyclones are numbered at t=0hrs, and followed over time. Numbered asterisks show polyclones beginning extension and asterisks without numbers show those continuing extension. Note the rostral-to-caudal progression as polyclones #6 and #5 start extending before polyclones #9 and #8. (F) Frame from the same time-lapse as in (A) showing that formation of the neural folds progresses faster than the node regresses. The caudal extent of the forming neural folds lies caudal to the level of regressing node. Polyclones #9 and #8, both on the fold and both caudal to the node, have already extended considerably. CaudNF: caudal extent of neural fold. Frame at stage 6, 10h23min within the time-lapse sequence. Scale bars: 200 µm. P.S.: Primitive Streak; P.P.: Prechordal plate; Ntc: notochord; Hf: Head fold; Fb: forebrain; Mb: Midbrain; rHb: Rostral Hindbrain; cHb: caudal Hindbrain (level of somite 1 or 3 as specified).

Figure 5. Quantitative analysis of polyclone length and percent increase in length

(A, B) Polyclone lengths were measured at each stage, and show an increase in length over time in both the head (A) and in the hindbrain and trunk (B). (C, D) Although growth continues, the percent change in polyclone length shows a peak of extension occurs between stages 4 and 5.

Figure 6. Reorientation and deep movements

(A) In the cranial regions of the embryo, rows of polyclones reorient along the rostrocaudal axis. The angle (0º defined as the mediolateral axis) between originally mediolateral neighboring polyclone pairs at the beginning of the time-lapse (stage 4), the middle (stage 6) and the end point (stage 9) increases with development. (B) Representative polyclones initiated at stage 4 on different embryos, which were fixed at various stages. At stage 7, the polyclone labels the rising neural fold and the height of the labeled cell population has increased. At stage 9, the polyclone spreads both deep into the embryo and laterally, due to growth of the brain vesicle.

Figure 7. Time-lapse of DiI/DiO polyclones labeled at stage 5 and stage 6

(A) Time-lapse frames show that dye injection spots made at stage 5 undergo less rostrocaudal extension than spots made at stage 4 (e.g. Fig 3). (B) Time-lapse frames of dye injection spots made at stage 6 showing an even greater reduction in rostrocaudal extension than observed at earlier stages. CaudNF: caudal extent of neural fold. Scale bars: 200 µm.

Figure 8. Fate map of the neural plate border region at stage 7

(A) Fate map of the stage 7 embryo shows that the presumptive neural crest is well ordered along the rostrocaudal axis. The color code is indicated below the fate map. (B–J) Three representative embryos labeled at different axial levels with DiI at stage 7 in the neural fold, cryosectioned and stained for Pax7. In all three cases, the DiI injection spot labeled neural crest and dorsal neural tube cell populations. B–D” An embryo labeled in the neural plate border at the trunk level; E–G” An embryo labeled in the neural plate border at the level of the otic vesicle; H–J” An embryo labeled in the neural plate border at the caudal hindbrain level. The “t=0” column shows images of the embryos at the time of injection; the “t=18hrs” column shows the same embryos after one day and the right three columns show sections through the labeled regions of those same embryos. Scale bars: 200 µm, except D-D”, G-G”, J-J” 50 µm.

Figure 9. Reduced rostrocaudal rearrangements of polyclones labeled at stage 7

(A) Frames from a time-lapse sequence started at stage 7. The dye spots converge towards the midline as the neural folds come into apposition. These dye labeled polyclones do not elongate dramatically as the embryo develops, unlike polyclones labeled at earlier stages, denoting a decrease in extension after stage 7. (B) Polyclones (labeled with lower case letters) on the neural fold maintain their respective rostrocaudal position along the neural tube. This denotes an almost total absence of rostrocaudal reorientation along the axis. (C, D) Polyclones initiated at stage 7 are on the elevating neural fold (C). By stage 9, apposition and fusion of the folds brings the polyclones to the dorsal aspect of the neural tube. Scale bars: A and B 200 µm, C and D 50 µm.

Figure 10. Extension of the embryonic axis slows over time

The changes in the extension rate can be gauged by labeling different embryos at progressively older stages and generating time-lapse sequences to stage 9. Extension of polyclones marked at stage 4, 5, 6, or 7 and followed through stage 9 is decreased with the age of the embryo at labeling. Labels applied at stage 4 undergo the greatest amount of rostrocaudal extension; those applied at stage 7 show the least. The dye spots chosen to illustrate this point are each located in the caudal hindbrain/trunk area of the fate map (in the area of somite 1). Scale bar: 200 µm.

Figure 11. Correlation of positions at stage 4 and stage 7 with respect to stage 11

The position of individual injection spots at stages 4 or 7 is plotted along the y-axis. The position of the polyclone at the end of 24 hours re-incubation period is plotted along the x-axis. The correlation factor (r²) for each graph is presented to quantitatively assess the refinement of the fate map.

Figure 12. Pax7 and BMP4 expression overlap in the early embryo

(A, B, C) Stage 4, 6 and 7 embryos processed for mRNA in situ hybridization with a BMP4 probe. (D, E, F) A stage 7 embryo processed for double fluorescent in situ hybridization with BMP4 and Pax7 mRNA probes. The expression of transcripts overlaps in the neural folds. (G and I) Sections of embryos at the levels indicated in (D). (H, J) Higher magnification images of the boxed regions shown in G and I, respectively. Scale bars: 200 µm, except H, J 100 µm.

Figure 13. Fate map of the neural plate border superimposed on the expression of BMP4 at stage 4

The NC/dNT region of the fate map matches nicely with the expression of this gene in the neural plate border, indicating that presumptive neural crest cells arise in a region positive for BMP4. The fate map of the neural plate border shows an intermixing of rostrocaudal fates within the region of BMP4 expression. The large rostrocaudal expansion and mediolateral rearrangements of dye labeled polyclones from stage 4, and this intermixing of the fate map both support the hypothesis that presumptive neural crest cells have not acquired a definitive rostrocaudal identity at stage 4. Scale bar: 200 µm.