Schlosser G and Ahrens K (2004), Molecular anatomy of placode development in Xen...

XB-ART-3353

Dev Biol 2004 Jul 15;2712:439-66. doi: 10.1016/j.ydbio.2004.04.013.

Show Gene links Show Anatomy links

Molecular anatomy of placode development in Xenopus laevis.

Schlosser G , Ahrens K .

???displayArticle.abstract???
We analyzed the spatiotemporal pattern of expression of 15 transcription factors (Six1, Six4, Eya1, Sox3, Sox2, Pax6, Pax3, Pax2, Pax8, Dlx3, Msx1, FoxI1c, Tbx2, Tbx3, Xiro1) during placode development in Xenopus laevis from neural plate to late tail bud stages. Out of all genes investigated, only the expression of Eya1, Six1, and Six4 is maintained in all types of placode (except the lens) throughout embryonic development, suggesting that they may promote generic placodal properties and that their crescent-shaped expression domain surrounding the neural plate defines a panplacodal primordium from which all types of placode originate. Double-labeling procedures were employed to reveal the precise position of this panplacodal primordium relative to neural plate, neural crest, and other placodal markers. Already at neural plate stages, the panplacodal primordium is subdivided into several subregions defined by particular combinations of transcription factors allowing us to identify the approximate regions of origin of various types of placode. Whereas some types of placode were already prefigured by molecularly distinct areas at neural plate stages, the epibranchial, otic, and lateral line placodes arise from a common posterior placodal area (characterized by Pax8 and Pax2 expression) and acquire differential molecular signatures only after neural tube closure. Our findings argue for a multistep mechanism of placode induction, support a combinatorial model of placode specification, and suggest that different placodes evolved from a common placodal primordium by successive recruitment of new inducers and target genes.

???displayArticle.pubmedLink??? 15223346
???displayArticle.link??? Dev Biol

Species referenced: Xenopus laevis
Genes referenced: bcl2l11 dlx3 eya1 foxd3 foxi4 frzb2 irx1 msx1 neurog2 nppa pax2 pax3 pax6 pax8 pnp six1 six4 snai2 sox2 sox3 tbx2 tbx3

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Placodal gene expression patterns in neural plate stage Xenopus embryos (stages 13–14) in dorsal view (anterior is to the left, all embryos are equally oriented). To facilitate comparison of the placodal expression domains of different genes, color-coded arrows and arrowheads corresponding to different placodal expression domains of Pax genes are used for the identification of comparable regions of gene expression (for more detailed labeling, see Fig. 12). Colors are explained in the schematic drawing, which depicts the approximate relative position of some gene expression domains and lists the placodes to which they will give rise subsequently. Green arrowheads identify placodal gene expression immediately rostral to the anterior neural plate border (prospective adenohypophysial placode medially and prospective olfactory placodes laterally), whereas green arrows identify placodal gene expression rostrolateral to the anterior neural plate (prospective lens and trigeminal placodes). Black arrows in D indicate the border of Dlx3 expression in superficial ectoderm, which extends further medial than its border of expression in the deep ectodermal layer (colored arrows). Black arrowheads in E indicate Msx1 expression in anterior neural plate. White asterisks in E and L indicate expression of Msx1 and Pax3, respectively, in a region encompassing neural crest and lateralmost neural plate. For detailed description, see text. Abbreviations: Ad/Ol, anterior placodal area, from which adenohypophysial (Ad) and olfactory (Ol) placodes develop; anp, anterior neural plate; cg, cement gland; L, lens placode; LL/Ot/EB, posterior placodal area, from which lateral line (LL), otic (Ot), and epibranchial (EB) placodes develop; np, neural plate; PN, pronephros; Pr, profundal placode; pnp, posterior neural plate; V, trigeminal placode.
	Fig. 2. Placodal gene expression patterns in neural plate stage Xenopus embryos (stages 13–14) in lateral view (anterior is to the left, all embryos are equally oriented). As in Fig. 1, color-coded arrows and arrowheads are used for the identification of comparable regions of gene expression as indicated in the schematic drawing. Green arrowhead corresponds to the lateral green arrowhead in Fig. 1 (prospective olfactory placode). Black asterisk in D indicates region where Dlx3 is expressed only in the superficial ectodermal layer. Black arrowheads in E indicate Msx1 expression in anterior neural plate. White asterisks in E and L indicate expression of Msx1 and Pax3, respectively, in a region encompassing neural crest and lateralmost neural plate. Insert in J shows incipient placodal Tbx2 expression at a slightly later (neural fold) stage. For detailed description, see text. For abbreviations, see Fig. 1.
	Fig. 3. Expression of Ngnr1 in neural plate stage Xenopus embryos. Green arrowheads identify placodal gene expression immediately rostral to the anterior neural plate border (medial arrowhead: prospective adenohypophysial placode, lateral arrowheads: prospective olfactory placodes), whereas green arrows identify placodal gene expression rostrolateral to the anterior neural plate (prospective lens and trigeminal placodes), and yellow arrows identify the prospective profundal placode. (A) Stage 12.5 embryo in dorsal view. Ngnr1 is expressed in an arc around the anterior neural plate. (B–C) Stage 14 embryo in dorsal (B; upper panel shows schematic drawing of various placodal gene expression domains for comparison) and lateral view (C). Ngnr1 is downregulated in the region of the prospective adenohypophysial placode (large asterisk) as well as in the region of the prospective lens placode (small asterisk).
	Fig. 4. Positioning of placodal Six1 and Eya1 expression domains relative to neural plate and neural crest as revealed by whole mount double-staining procedures. (A–H) Dorsal views of neural plate stage Xenopus embryos (anterior is to the left). (I–R) Lateral (I–M, anterior to the left) and frontal (N–R) views of neural fold stage Xenopus embryos. Expression of Sox3, Six1, Slug, and FoxD3 is shown individually (A–D, I–K, and N–P) and after double in situ hybridization (E–G, L–M, and Q–R) or combination of in situ hybridization for FoxD3 with Sox3 immunostaining (H). The crescent of Six1 expression is shown to be closely apposed to the neural plate domain of Sox3 rostrally (E, L, and Q) and to the FoxD3-positive neural crest domain laterally (F, M, and R). Slug- and FoxD3-expressing neural crest cells are located in the lateral outer neural folds adjacent to the Sox3-positive neural plate (C, D, G, and H). Asterisks in E, L, and Q indicate a gap between Sox3 and Six1 expression, corresponding to this Slug- and FoxD3-positive neural crest domain. Arrowheads in L, M, Q, and R emphasize the position of rostral Six1 expression, which is shown to be located between the neural plate domain and the lateral domain of Sox3 expression. However, posteriorly, Six1 expression overlaps broadly with the lateral domain of Sox3 expression as indicated by the arrow in L.
	Fig. 5. Positioning of gene expression domains at the anterior neural plate border as revealed by double-staining procedures in sagittal sections (anterior is to the left) of neural plate stage Xenopus embryos (stages 13–14). For each gene analyzed, a first panel depicts gene expression at the anterior neural plate border, a second panel shows the pattern of nuclear Sox3 immunostaining in the same section (apparent nonnuclear staining in the endoderm is an artifact due to autofluorescence of yolk granules), and a third panel presents a superposition of the first two panels. (A–C) Distribution of Sox3 expression (A) and Sox3 immunostaining (B). The anterior border of the neural plate is indicated by grey arrows. The superficial ectodermal layer is indicated by asterisks. Sox3 expression and immunostaining is found in the superficial (in the domain marked by a white asterisk) and deep ectodermal layers. The rostral limit of expression in the deep ectodermal layer (black arrowheads) extends beyond the neural plate border. Cells within this area of Sox3 expression rostral to the neural plate border are also immunopositive for Sox3 (black arrows). However, no Sox3-immunopositive cells are yet discernible in the lateral crescent-shaped domain of Sox3 expression (identified by blue arrowheads in A) as is particularly evident in the inserts of A–C depicting sections of the same specimen at a more lateral level. (D–R) Comparison of the expression pattern of Six1 (D–F), Eya1 (G–I), Dlx3 (J–L), Msx1 (M–O), and Pax6 (P–R) at the anterior neural plate border with Sox3 immunostaining. Inserts in D and G show Six1 and Eya1 expression patterns, respectively, in additional specimens. The borders of expression of each gene in the deep ectodermal layer are indicated by arrowheads. Double-labeled cells, which show expression of the respective gene as well as Sox3 immunostaining, are identified by black or white arrows. Because Pax6 expression and Sox3 immunoreactivity are largely coextensive, double-labeled cells are not specifically marked in P–R. Black asterisks indicate the superficial ectodermal layer (which is artifactually missing in D). White asterisks in L indicate cells in the superficial ectodermal layer that express Dlx3 and are Sox3 immunopositive (posterior border ofDlx3 expression is immediately to the right of panel). Red arrows in M–O indicate Sox3-immunopositive cells located rostral to the domain of Msx1 expression in the anteriormost neural plate. Scale bar in A: 100 μm (for all panels).
	Fig. 6. Positioning of gene expression domains at the lateral neural plate border as revealed by double-staining procedures in transverse sections through the cranial neural folds of neural fold stage Xenopus embryos (stages 16–17). (A and B) Double in situ hybridization reveals a gap between the neural plate domain of Sox3 expression and the lateral crescent of Six1 expression. Arrow in A indicates tip of the neural folds. Arrowheads in B (representing higher magnification of A) indicate medial border of Six1 expression in the deep ectodermal layer (superficial ectodermal layer marked by asterisk). (C–F) Double in situ hybridization for FoxD3 (red) and Six1 (blue; medial border of expression marked by arrowheads in D) combined with Sox3 immunohistochemistry (E) reveals the position of the FoxD3-expressing neural crest relative to the Sox3-immunopositive neural plate and the lateral, placodal region that expresses Six1 and is immunoreactive for Sox3. Arrow in C indicates the tip of the neural folds. Arrowheads in D (representing higher magnification of C) indicate the medial border of Six1 expression in the deep ectodermal layer (superficial ectodermal layer marked by asterisk). Green arrows in E and F (showing a superposition of a fluorescent image of FoxD3 expression with E) indicate that Sox3-immunopositive nuclei of the lateral neural plate are located within the domain of FoxD3 expression. Green arrowheads in E and F indicate Sox3-immunopositive nuclei in the deep ectodermal layer of the lateral, placodal domain, which also expresses Six1 (compare with D), but does not show clear overlap with FoxD3 expression. Scale bar in A: 100 μm (for A and C). Scale bar in B: 100 μm (for B and D–F).
	Fig. 7. Positioning of Pax gene expression domains relative to Sox3 as revealed by whole mount double in situ hybridization. Dorsal views (A, D, and G) of neural plate stage Xenopus embryos (stage 14; anterior to the left) and lateral (B, E, and H) and oblique dorsolateral (C, F, and I) views of neural fold stage Xenopus embryos (stage 16; anterior to the left) demonstrating the position of Pax6 (A–C), Pax3 (D–F), and Pax8 (G–I) expression domains relative to Sox3 expression. Inserts in A, D, and G show Sox3 expression individually for clarification. Asterisks indicate lateral domain of Sox3 expression. Arrows indicate anterior and posterior borders of placodal expression domains for Pax6 (green arrows: prospective lens and trigeminal placodes, A–C), Pax3 (yellow arrows: prospective profundal placode, D–F), and Pax8 (brown arrows: posterior placodal area, G–I). The green arrowhead in B and C indicates Pax6 expression in the lateral part of the anterior placodal area (prospective olfactory placode). Inserts in B, E, and H identify the corresponding subdomains of Six1 expression.
	Fig. 8. Changes in placodal gene expression patterns during neurulation in Xenopus embryos (stages 16–18). As in Fig. 1, color-coded arrows and arrowheads are used for the identification of comparable regions of gene expression indicated in the schematic drawing. Green arrowhead indicates the prospective olfactory placode. The green asterisk indicates an anterior subregion of the lateral Pax6-expressing domain, where panplacodal genes such as Eya1 and Six1 are downregulated during neurulation (note indentation in the red domain) and which will likely give rise to the lens placode. Green arrow indicates the posterior subregion of the lateral Pax6-expressing domain (prospective trigeminal placode). (A and B) Sagittal sections through the anterior neural folds reveal expression of Six1 (A) and Sox3 (B) in the deep ectodermal layer of the outer neural folds (black arrows). The outer ectodermal layer is indicated by black asterisks. (C–E) Frontal views of gene expression domains in neural fold stage Xenopus embryos. Note the separation of the anterior domain of Eya1 and Six1 expression (arrowheads) from the lateral domain (arrows) due to the elevation of the neural folds. (F–H) Lateral views of gene expression domains in neural fold stage Xenopus embryos. Note the downregulation of Eya1 (F), Six1 (G), and Msx1 (H) in a region that will likely give rise to the lens placode (green asterisk). The white asterisk in H indicates expression of Msx1 in a region encompassing neural crest and lateralmost neural plate. For detailed description, see text. Abbreviations: cg, cement gland; nep, anterior neuropore. For additional abbreviations, see Fig. 1.
	Fig. 9. Placodal gene expression patterns in early tail bud Xenopus embryos (stages 21–22) in lateral views (anterior is to the left) and right frontal views (inserts). Color-coded arrows and arrowheads are used for the identification of comparable regions of gene expression. Expression in the prospective lens placode (which is not yet thickened at this stage) is indicated by L. Colors are explained in the schematic drawing, which shows the distribution of placodes in a stage 21 embryo (modified after Schlosser and Northcutt, 2000; see Fig. 14 for detailed explanation). The unpaired adenohypophysial placode is located medial to the ventral part of the olfactory placode and is, thus, hidden behind the olfactory placode in this lateral perspective (indicated by light green arrow and Ad in brackets). Various shades of green identify placodes (including the prospective lens placode) expressing Pax6, yellow identifies the profundal placode expressing Pax3, and brown and pink jointly identify the posterior placodal area expressing Pax2 and Pax8, with pink being reserved for the subregion, which will form the otic vesicle. Blue–green asterisks indicate downregulation of the respective genes in the region of the prospective lens placode, whereas brown asterisks indicate downregulation of the respective genes in a region intervening between the anterior and posterior subregions of the posterior placodal area. The pink and brown asterisks in I indicate downregulation of FoxI1c expression in the dorsal part of the posterior placodal area. For detailed description, see text. For additional abbreviations, see Fig. 1.
	Fig. 10. Placodal gene expression patterns in mid-tail bud Xenopus embryos (stages 24–27) in lateral views (anterior is to the left). As in Fig. 9, color-coded arrows and arrowheads are used for the identification of comparable regions of gene expression. Expression in the lens placode is indicated by L. Colors are explained in the schematic drawings, which show the distribution of placodes in stages 24 and 27 embryos (modified after Schlosser and Northcutt, 2000; see Fig. 14 for detailed explanation). By stage 24, the posterior placodal area has extended caudally, the formation of the otic vesicle by invagination of the otic placode has begun, and epibranchial placodes (orange) begin to become recognizable in its ventral part (Schlosser and Northcutt, 2000). By stage 27, the posterior placodal area has broken up into distinct placodes, while invagination of the otic vesicle is continuing (Schlosser and Northcutt, 2000). The lens placode has appeared as a focal thickening. The orange and brown double arrows indicate expression of the respective genes in a region encompassing the facial epibranchial placode and anteroventral lateral line placode, because both placodes are closely apposed and, thus, cannot be differentiated from each other. White asterisks indicate gene expression in the pharyngeal pouches (which can be clearly distinguished from placodal expression domains in transverse sections). The brown asterisk in M indicates downregulation of Pax2 in the region of the anterodorsal lateral line placode. For detailed description, see text. Abbreviations: EB, epibranchial placodes; LL, lateral line placodes; Ot, otic placode or vesicle; vba, ventral branchial arch region (all three germ layers). For additional abbreviations, see Fig. 1.
	Fig. 11. Placodal gene expression patterns in late tail bud Xenopus embryos (stages 32–34) in lateral views (anterior is to the left). As in Fig. 9, color-coded arrows and arrowheads are used for the identification of comparable regions of gene expression. Expression in the lens is indicated by L. Colors are explained in the schematic drawing, depicting the distribution of placodes in a stage 33/34 embryo (modified after Schlosser and Northcutt, 2000; see Fig. 14 for detailed explanation). By stage 33/34, the profundal placode has disappeared, the trigeminal placode is shrinking rapidly, the lens placode and otic placode have completely invaginated to form the lens and the otic vesicle, respectively, and the lateral line primordia have begun to extend from the lateral line placodes (Schlosser and Northcutt, 2000). Again, orange and brown double arrows indicate expression of the respective genes in a region encompassing facial epibranchial placode and anteroventral lateral line placode. White asterisks indicate gene expression in the pharyngeal pouches. Green and yellow asterisks indicate gene expression in the trigeminal and profundal ganglia (which are fused proximately), respectively. For detailed description, see text. Abbreviations: bam, branchial arch mesenchyme; L, lens; Ot, otic vesicle; vba, ventral branchial arch region (all three germ layers). For additional abbreviations, see Fig. 1.
	dlx3 (distal-less homeobox 3) gene expression in Xenopus laevis embryo, NF stage 13-14, assayed by in situ hybridization, dorsal view, anterior left. Black arrows indicate dlx3 expression in superficial ectoderm; cg=cement gland primordium.
	dlx3 (distal-less homeobox 3) gene expression in Xenopus laevis embryo, NF stage 13-14, assayed by in situ hybridization, lateral view, anterior left, dorsal up. Black asterisk indicates region where dlx3 is expressed only in the superficial ectodermal layer.; cg=cement gland primordium; coloured arrows indicate other placodes with no dlx3 expresssion.