Pieper M et al. (2011), Origin and segregation of cranial placodes in X...

XB-ART-44157

Dev Biol 2011 Dec 15;3602:257-75. doi: 10.1016/j.ydbio.2011.09.024.

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Origin and segregation of cranial placodes in Xenopus laevis.

Pieper M , Eagleson GW , Wosniok W , Schlosser G .

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Cranial placodes are local thickenings of the vertebrate head ectoderm that contribute to the paired sense organs (olfactory epithelium, lens, inner ear, lateral line), cranial ganglia and the adenohypophysis. Here we use tissue grafting and dye injections to generated fate maps of the dorsal cranial part of the non-neural ectoderm for Xenopus embryos between neural plate and early tailbud stages. We show that all placodes arise from a crescent-shaped area located around the anterior neural plate, the pre-placodal ectoderm. In agreement with proposed roles of Six1 and Pax genes in the specification of a panplacodal primordium and different placodal areas, respectively, we show that Six1 is expressed uniformly throughout most of the pre-placodal ectoderm, while Pax6, Pax3, Pax8 and Pax2 each are confined to specific subregions encompassing the precursors of different subsets of placodes. However, the precursors of the vagal epibranchial and posterior lateral line placodes, which arise from the posteriormost pre-placodal ectoderm, upregulate Six1 and Pax8/Pax2 only at tailbud stages. Whereas our fate map suggests that regions of origin for different placodes overlap extensively with each other and with other ectodermal fates at neural plate stages, analysis of co-labeled placodes reveals that the actual degree of overlap is much smaller. Time lapse imaging of the pre-placodal ectoderm at single cell resolution demonstrates that no directed, large-scale cell rearrangements occur, when the pre-placodal region segregates into distinct placodes at subsequent stages. Our results indicate that individuation of placodes from the pre-placodal ectoderm does not involve large-scale cell sorting in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: frzb2 gad1.2 h2bc21 pax2 pax3 pax6 pax8 six1

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	Fig. 1. Schematic overview of placodal development in Xenopus laevis (lateral views). A: Neural plate stage embryo showing the approximate position of the panplacodal primordium giving rise to all placodes. B: After neural fold closure, placodal areas giving rise to different placodes are closely apposed. Otic, epibranchial and lateral line placodes will all arise from a posterior placodal area (turquoise; region of prospective otic placode shown in brown). Neural crest streams are shown as blue broken lines. C: At tailbud stages, individual placodes have segregated (same color code as in fate maps shown in Figs. 6–8). Abbreviations: VII: facial epibranchial placode; IX: glossopharyngeal epibranchial placode; X1: first vagal epibranchial placode; X2/3: second and third vagal epibranchial placodes (fused); AD: anterodorsal lateral line placode; AV: anteroventral lateral line placode; L: lens placode; LL/Ot/EB: posterior placodal area, from which lateral line (LL), otic (Ot) and epibranchial (EB) placodes develop; M: middle lateral line placode, Ot: otic placode; Ol: olfactory placode; P: posterior lateral line placode; Pr: profundal placode; V: trigeminal placode. Modified from Schlosser and Northcutt (2000) and Schlosser and Ahrens (2004).
	Fig. 2. Standardization of measurements for dye injections at neural plate stages (stage 13/14; A, A′, B, B′), at the end of neurulation (stage 19/20; C, C′, D, D′), and at early tail bud stages (stage 23/24; E, E′, F, F′). Lateral views in A, A′, C, C′, E, E′ and frontal views in B, B′, D, D′, F, F′. At the neural plate stage, outlines of neural folds are indicated (A′, B′). At neural fold and tailbud stages, double outlines are drawn for frontal views (D′, F′) to indicate that embryos are narrower rostral (inner outline) than further caudal (outer outline). Immediately after labeling with DiI (red) or DiO (green), embryos were photographed in a precisely oriented lateral (A, C, E) or frontal position (B, D, F). The position of labeled cells (green or red dots in A′–F′) along the anteroposterior axis (a–p) and their perpendicular distance from the dorsal edge of the neural folds (Nf-Inj.; A′) or from the dorsal edge of the neural tube (Nt-Inj.; C′, E′) were measured in lateral views. The position of the anteriormost labeled cells was determined in frontal views by measuring their position along the left–right axis (r–l) and from the inner neural folds (iNF-Inj.; stage 13/14; B′) or from the border of the cement gland (cg-Inj.; stages 19/20 and 23/24; D′, F′). All distances were expressed as a percentage of the total length (lateral views) or width (frontal views) of the embryo. Red and green arrows in A′–F′ illustrate the measured distances. Abbreviation: cg: cement gland.
	Fig. 3. Fate map of Xenopus non-neural ectoderm at neural plate stages using tissue grafts. A: Example of pigmented graft transplanted into albino host at stage 14 (left panel) and then followed throughout development until mid-tailbud stage (right panel). B: Example of tailbud stage embryo that had received a Hoechst labeled graft at the neural plate stage. C: Changes of shapes of three grafts (blue, yellow, red) between stage 14 (left), stage 22 (middle) and stage 24 (right) projected onto outlines of Xenopus embryos (Nieuwkoop and Faber, 1967). Note the wedge shaped expansion of the anterior graft. D: Summary of placodal fates adopted at tailbud stages (stage 39) by grafts from the indicated positions at neural plate stages. Data are compiled from pigmented and Hoechst-labeled grafts as indicated. Due to the close apposition of some placodes or their derivatives at tailbud stages – viz. the trigeminal and profundal placodes, the anterodorsal and anteroventral lateral line placodes, and the facial and glossopharyngeal epibranchial placodes – it was often not possible to determine whether one or both of these placodes were labeled by a graft and we, therefore, only noted whether any of them were labeled.
	Fig. 4. Examples of DiI or DiO labeled cells and their fates. The positions of DiI (red) or DiO (green) labeled cells in neural plate stage embryos (stage 13/14; A, B, C), at the end of neurulation (stage 19/20; D, E, F) and at early tail bud stage (stage 23/24; G, H, I) are shown. Lateral views in B, C, E, F, H and I, frontal view in A, D and G. Their fates were identified in transverse cryosections at late tail bud stage (stage 33/34) (A′–I′). Nuclei are stained with DAPI. Levels of sections are indicated in the diagram below. In neural plate stage embryos, cells at the border of the outer neural folds (A) give rise to cells of the olfactory placode (A′), whereas labeled cells in the outer neural folds (B) were later identified in the trigeminal ganglion (B′), and labeled cells located immediately ventral to the neural folds (C) were found in the ganglion of anterodorsal lateral line nerve (C′). At the end of neurulation, a group of cells labeled just dorsal to the cement gland (D), contributes to the olfactory placode (D′); cells labeled more posterior in a dorsal position (E) contribute to the middle lateral line and otic placodes (E′); whereas cells labeled more ventral (F) give rise to the posterior lateral line placode (F′). At early tail bud stage, cells located in the most anterior part of the non-neural ectoderm (G) give rise to the adenohypophysis (G′); labeled cells within the anteroventral part of the non-neural ectoderm (H) contribute to the lens (H′); while cells located more posterior and dorsal (I) developed into a part of the glossopharyngeal epibranchial placode (I′). Abbreviations: gAD: ganglion of anterodorsal lateral line placode; gV: trigeminal ganglion; n: notochord; nt: neural tube; pA: adenohypophyseal placode; pIX: glossopharyngeal epibranchial placode; pL: lens placode; pOL: olfactory placode; pOt: otic placode; pM: middle lateral line placode; pP: posterior lateral line placode; r: retina; s: somite; vOt: otic vesicle.
	Fig. 5. Overlap versus co-labeling of selected adjacent placodes at neural plate stages (A), at the end of neurulation (B) and at early tailbud stages (C). The degree of overlap (blue columns) was measured as percent of area of origin of first placode which overlapped with area of origin of second placode. Areas were defined by polygons enclosing all injection sites giving rise to a particular placode in fate maps and measured with ImageJ. Co-labeling (maroon columns) was determined as percent of all injection sites contributing to first placode (n) which also contributed to second placode. Columns marked with an asterisk in C are not very informative since the facial epibranchial placode was labeled by only a single injection site. Abbreviations: ADLL: anterodorsal lateral line; AVLL: anteroventral lateral line; EBVII: Facial epibranchial; EBIX: Glossopharyngeal epibranchial; EBX1: First vagal epibranchial; EBX2/3: Second and third vagal epibranchial; MLL: middle lateral line; PLL: posterior lateral line.
	Fig. 6. Fate map of Xenopus dorsal cranial non-neural ectoderm at the neural plate stage (stage 13/14). Each symbol represents one dye injection which contributes to a single placode or other ectodermal fates (circle) or to multiple placodes and/or other ectodermal fates (square). Black outlined circles and squares represent injections shown in frontal views as well as in lateral views. The inner gray colored line marks the ventral border of the outer neural folds. Frontal views are shown in A and C and lateral views in B, D–H. Note that only a subset of rostral injection sites mapped from a lateral view were also mapped from a frontal view. A, B: Positions of placodal precursors (red symbols) in relation to precursors of the neural tube (mint symbols), neural crest (blue symbols), epidermis (light gray symbols) and stomodeum (black symbols). All placodes arise from a crescent-shaped area around the anterior neural plate, the pre-placodal ectoderm. C, D: Positions of precursors for different types of placodes (color coded as indicated). There is extensive overlap and co-labeling of precursors for adjacent placodes. E–H: For clarity, these diagrams highlight injection sites contributing to particular subsets of placodes, while injections which contribute to other placodal fates are shown as light gray symbols. E: Adenohypophyseal, olfactory, lens and otic placodes. F: Trigeminal and profundal placodes. G: Lateral line placodes. H: Epibranchial placodes. For detailed explanation see text.
	ig. 7. Fate map of Xenopus dorsal cranial non-neural ectoderm at the end of neurulation (stage 19/20). Each symbol represents one dye injection, which contributes to precursors of a single placode or epidermis (circle) or to multiple placodes and/or epidermis (square). Black outlined circles and squares represent injections shown in frontal as well as in lateral views. Frontal views are shown in A and C, lateral views in B and D. A, B: Positions of placodal precursors (red symbols) within the non-neural ectoderm in relation to epidermal (light gray symbols) or stomodeal precursors (black symbols). C, D: Positions of precursors for different types of placodes (color coded as indicated). Overlap and co-labeling between anterior placodes has decreased but is still extensive for posterior placodes. Abbreviation: cg: cement gland. For detailed explanation see text.
	Fig. 8. Fate map of Xenopus dorsal cranial non-neural ectoderm at early tail bud stage (stage 23/24). Each symbol represents one dye injection, which contributes to precursors of a single placode or epidermis (circle) or to multiple placodes and/or epidermis (square). Black outlined circles and squares represent injections shown in frontal as well as in lateral views. Frontal views are shown in A and C, lateral views in B and D. A, B: Positions of placodal precursors (red symbols) within the non-neural ectoderm in relation to epidermal (light gray symbols) or stomodeal precursors (black symbols). C, D: Positions of precursors for different types of placodes (color coded as indicated). Placodes are now more separated from each other. Abbreviations: cg: cement gland. For detailed explanation see text.
	Fig. 9. Comparison of Six1 expression in Xenopus with placodal fate maps (lateral views). A, B: Embryo labeled with DiI at the neural plate stage, which was subsequently analyzed by in situ hybridization for Six1. DiI injections (white arrows in A, black circles and arrows in B) are localized within the expression domain of Six1 (outlined by green line) and map well within the pre-placodal ectoderm in the fate map of neural plate stage embryos. C–H: The expression domains of Six1 (C, E, G and green encircled areas in D, F and H) at neural plate stages (C, D), at the end of neurulation (E, F) and in early tail bud stage embryos (G, H) were measured and compared to our fate maps. Six1 expression corresponds overall well to the distribution of placodal precursors in fate maps. However, at early stages, Six1 expression excludes most injections which contribute to the posterior lateral line placode and second/third vagal epibranchial placodes (D, F). These are localized within the expression domain of Six1 only from early tail bud stage on, indicating delayed upregulation of Six1 in these precursors (H). Conversely, Six1 is downregulated in the future lens placode from late neural fold stage on (E–H). Abbreviation: cg: cement gland. For detailed explanation of fate maps, see Figs. 6–8.
	Fig. 10. Comparison of Pax gene expression in Xenopus with placodal fate maps (lateral views). The expression pattern of Pax genes as revealed by in situ hybridization (A–H) were measured at neural plate stages (shown for Pax6 and Pax8: A, E), at the end of neurulation (shown for Pax6 and Pax8: B, F) and in early tail bud stage embryos (shown for Pax6, Pax3, Pax8 and Pax2, C, D, G, H) and compared to fate maps (green encircled areas in A′–H′). A–C and A′–C′: Pax6 expression encompasses precursors for the olfactory, lens, and trigeminal placodes. D and D′: Pax3 expression is restricted to precursors of the profundal placode (placodal expression domain encircled by hatched green line in D). E–G and E′–G′: Pax8 expression is found in precursors of otic, lateral line and epibranchial placodes, but excludes precursors of the vagal epibranchial and posterior lateral line placode. H and H′: Pax2 expression in early tailbud stage embryos is distributed similar to Pax8. Abbreviation: cg: cement gland. For detailed explanation of fate maps, see Figs. 6–8.
	Fig. 11. Cell movements within the non-neural ectoderm (lateral views, anterior is to the right). A–D: Time lapse imaging of deep ectodermal layer in embryo expressing MEM-GFP (green membranes) and H2B-RFP (red nuclei, here shown in magenta false-color) from neural plate stage on until early tailbud stages (600 min). The superficial ectodermal layer was removed. A: Brightfield image of embryo at the beginning of the recording. Hatched line indicates ventral border of neural folds. Arrows indicate border of superficial ectodermal layer. B: Superimposition of both fluorescent channels for embryo at the beginning of the recording. A group of randomly selected cells were color coded (dots) to allow tracking of their positions with ImageJ. C: Magnified view of boxed area in B. Some of the tracked cells are connected by white lines to facilitate visualization of changes in their relative positions over time. D: Same region after 600 min. E, G: Tracking of movements of selected cells for the embryo depicted in A–D. Some tracked cells are connected by black lines. E: Position of tracked cells (grid) in relation to the neural plate stage fate map. F: Changes in positions of tracked cells over time due to cell division (red arrows) and local cell rearrangement. G, H: Similar analysis for time lapse movie of a different embryo. I–P: Time lapse imaging of deep ectodermal layer in embryos expressing EosFP from neural plate stage on for 500 min. I: Bright- field image of embryo after removal of the superficial ectodermal layer. Hatched line indicates ventral border of neural folds. J: Part of the embryo was covered with aluminum foil and UV irradiated for photoconversion of EosFP resulting in a sharp boundary between photoconverted (red; here shown in magenta false-color) and unconverted (green) fluorescent cells. K–P: Magnified view of anterior end of embryo depicted in I and J with superimposition of both fluorescent channels. The sharp border created by photoconversion of EosFP (dashed white lines) within the deep ectodermal layer largely persists until early tailbud stages. Because a large part of the neural plate and the neural crest was photoconverted in this embryo, the ventral migration of neural crest cells (magenta) underneath the deep layer of the ectoderm can be seen (indicated by white arrows in O). From 100 min on, a space begins to open up in the dorsal part of the embryo (asterisks), probably due to the delamination of neural crest cells (this was observed in several embryos after removal of the superficial ectodermal layer). Abbreviations: ba: branchial neural crest stream; hy: hyoid neural crest stream; ma: mandibular neural crest stream.
	Fig. 12. Analysis of cell neighborhood changes over time. A: Magnified view of rostral non-neural ectoderm of the neural plate stage embryo depicted in Figs. 11A–B (lateral view, anterior is to the right) showing MEM-GFP labeled membranes (green) and H2B-RFP labeled nuclei (red; here shown in magenta false-color). For eight target cells in the deep ectodermal layer (white dots), positions of all neighboring cells including all cells that leave and cease to be neighbors and all cells which become new neighbors were tracked over a time period of 600 min. B: Positions of these target cells (black dots) in relation to the neural plate stage fate map. C: Directions of cells moving away from or towards a target cell (black circle) were depicted as arrows pointing towards or away from it. Red arrows indicate the directions (0°–360°) into which old neighbors were lost, while black arrows indicate directions (0°–360°) from where new neighbors were gained. Directions were categorized into 45° sectors. The number R within the black circle indicates the number of neighbors that do not change over the recorded time period. D: Cell neighborhood changes for each of the eight cells depicted in A. In total, the neighborhood changes of 30 cells were analyzed.