XB-ART-1139Dev Biol 2005 Dec 01;2881:40-59. doi: 10.1016/j.ydbio.2005.07.022.
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Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis.
Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.
PubMed ID: 16271713
Article link: Dev Biol
Species referenced: Xenopus laevis
Genes referenced: bmp4 eya1 fgf8 frzb2 nog six1 sox3
Antibodies: Myc Ab3
Morpholinos: fgf8 MO3
Article Images: [+] show captions
|Fig. 1. Six1, FGF8, and Sox3 expression during early Xenopus development. Embryos are shown in dorsal (A, E, I) or lateral (D,H,L) views (anterior to the left). (A) Weak Six1 expression (arrowheads) first appears in dorsoanterior ectoderm at stage 11.5 (A), intensifies at stage 12 (B), and becomes restricted to a crescent (arrowheads) around the anterior neural plate while being downregulated in the anterior neural plate at stage 12.5 (C,D). (E) In addition to circumblastoporal expression, a new domain of FGF8 expression in dorsoanterior ectoderm appears around stage 11.5 (E) and becomes stronger by stage 12 (F). By stage 12.5, expression of FGF8 is downregulated in dorsoanterior ectoderm except for the prospective midbrainindbrain boundary (MHB) and three nested arcs (1) (G,H). (I) Sox3, for comparison, is broadly expressed in dorsal and dorsoanterior ectoderm at stage 11.5 (I) but becomes restricted to the developing neural plate between stage 12 (J) and stage 12.5 (K). At stage 12.5, an additional crescent shaped domain of Sox3 expression (black arrowhead) appears around the anterior neural plate (white arrowhead) (K,L). (M) Sagittal sections through embryo depicted in (G,H) reveal that the two posterior arcs (1,2) of FGF8 expression (M,O) are situated within the anterior neural plate marked by Sox3-immunostaining, whereas the anteriormost arc (3) is located outside of the neural plate. Arrowheads indicate Sox3 immunopositive nuclei immediately rostral to the second arc of FGF8 expression. (P) Schematic summary of FGF8 expression relative to the expression of Sox3, Six1, and other markers based on these data and on our previous report (Schlosser and Ahrens, 2004). Blue line demarcates boundary of the lateral part of the pre-placodal region (LPR) used for grafts and extirpations. Yellow lines indicate boundaries of areas from which ectoderm was taken for anterior neural plate (ANP), posterior neural plate (PNP), lateral neural plate (LNP), and anterior neural ridge (ANR) grafts and extirpations. The green line demarcates the region removed in unilateral extirpations of the entire anterior neural plate (UNP).|
|Fig. 3. Role of organiser and endomesoderm in placodal Six1 induction. All embryos were analysed at early tailbud stages (stage 206). (A) Grafting a stage 11 organiser (dorsal blastopore lip) into stage 13 belly ectoderm induces Six1 expression (arrowhead) in host ectoderm next to anterior edge of graft. (B) After extirpation of axial mesoderm together with the overlying central neural plate at stage 13 bulging endoderm (asterisk) prevents regeneration of mesoderm and closure of the neural tube without resulting in any major deficits of placodal Six1 expression. (C) Grafting stage 13 cranial dorsolateral endomesoderm (green GFP label) into stage 13 belly ectoderm does not induce Six1 but placodal Six1 expression is lost in donors (D). Control side (CS) shown in insert for comparison.|
|Fig. 4. Role of neural plate in placodal Six1 induction. Embryos shown in lateral views (anterior to the left). Transplantations involved hosts and donors at early neural plate stage (stage 13) unless otherwise noted (see Fig. 1P for precise location of ectoderm grafted). All embryos were analysed at early tailbud stages (stage 206). (A) Grafts of anterior neural plate (ANP) in belly ectoderm strongly express Sox3 (A) and induce Eya1 (B, enlarged in insert) and Six1 (C) in surrounding ectoderm. (D) Cross-section of a neural plate graft (green GFP label) shows confinement of Six1 to host ectoderm. (G) Anterior neural plate grafts induce Six1 in surrounding belly ectoderm in 74% (28/38) when left untreated, but in only 22% (2/9) or 11% (1/9) when treated with the FGF inhibitor SU5402 or with BMP4 after transplantation, respectively. (H) Anterior neural ridge (ANR) grafts (enlarged in insert) in belly ectoderm express Six1 at their border (arrowhead) and induce it in adjacent ectoderm (arrow). Grafts of (I) posterior neural plate (PNP) or (J) stage 12 anterior neural plate do not induce Six1 in belly ectoderm. (K) ANR extirpation leads to reduction of anterior domains of placodal Six1 expression. (L) Unilateral extirpation of anterior neural plate (UNP) results in loss of placodal Six1 expression (white arrowheads; compare to Six1 expression on control side as indicated by black arrowheads) except for weak expression domain (arrow) next to remnants of neural folds (NF). (M) Six1 is expressed broadly (arrowhead) in belly ectoderm grafted unilaterally into anterior neural plate, sometimes extending into host ectoderm (arrow).|
|Fig. 5. Role of BMP inhibitors and FGF8 in placodal Six1 induction. Embryos shown in lateral views (anterior to the left). Transplantations involved hosts at early neural plate stage (stage 13) and were analysed at late neural fold or early tailbud stages (stage 186). (A,B) Implanting animal caps (pigmented) from BMP4-injected embryos (A) or beads (red asterisk; the bead itself is light blue) soaked with BMP4 (B) repress placodal Six1 expression (arrowheads) around grafts or beads, respectively. Insert in panel B shows control side (CS). (C,D) Animal caps from uninjected embryos grafted into the LPR strongly express Sox3 (C) but not Six1 despite normal Six1 expression in host ectoderm (D). (E) Animal caps (pigmented, green GFP label, arrows) from embryos injected with various constructs (E) or protein-soaked beads (K) were grafted into belly ectoderm. (E) noggin-injected grafts strongly express Sox3. (F) dnBMPR-injected grafts also express Sox3, but expression tends to be somewhat weaker and more patchy. (G) dnBMPR-injected grafts do not or only weakly affect placodal Six1 expression of host, whereas (H) noggin-injected grafts significantly broaden (arrowhead) placodal Six1 expression of host towards anterior graft border. (I) FGF8-injected grafts only weakly broaden (arrowhead) placodal Six1 expression in few cases. (J) noggin and FGF8 coinjected grafts broaden placodal Six1 expression and strongly induce Six1 (arrowhead) at anterior graft border. Beads (red asterisks) soaked with noggin (K) or FGF8 (L) alone do not, but beads soaked with noggin and FGF8 (M) do induce ectopic Six1 expression in ectoderm (beads enlarged in insert; Affi-Gel Blue beads used in panels K and M are light blue, while heparin acrylic beads used in panel L are colourless). (N) Anterior neural plate from stage 13 donors retains FGF8 expression after transplantation into belly ectoderm. Transverse sections (O,P) through embryo shown in panel N clearly show FGF8 expression (O) confined to GFP-labeled graft (P). (Q) In contrast, FGF8 expression is typically not maintained in LPR from stage 13 donors after transplanting into belly ectoderm.|
|Fig. 6. FGF signalling is necessary for placodal Six1 induction. Embryos shown in lateral (A,B,D,E; anterior to the left), dorsal (G; anterior to the top), or frontal views (J,K; midline in panel K indicated by dotted line). Embryos were analysed at early tailbud stages (stages 206: A, K) or neural plate stages (stage 14: G). (A) Placodal Six1 expression is reduced in embryos treated with SU5402 (stages 12/130) compared to controls (B). (C) Percentage of embryos with reduced Six1 expression after SU5402 treatment during different periods. (D,E) SU5402-soaked beads inhibit Six1 expression (arrowheads) after implantation at stage 13 into the region of the prospective ear placode (D) or of prospective trigeminal, lateral line, and epibranchial placodes (E). (F) Western blot demonstrating that the FGF8 MO but not an unspecific control MO specifically blocks in vitro translation of FGF8 but not of Eya1. (G) Embryos unilaterally injected with FGF8 MO (to the left on each panel) show strong reductions (arrowheads) of placodal Six1 (G,J,K), Sox3 (H), and FGF8 (I) expression, while neural plate domains of Sox3 or FGF8 expression are not reduced (asterisks). Both posterior (G,K) and anterior (J,K) subdomains of placodal Six1 expression are reduced in embryos analysed at neural plate (G,J) or tailbud stages (K; lower and upper arrowheads indicate olfactory and otic placode, respectively). The injected side was marked by coinjection of either GFP (G; not shown) or lacZ (J,K; light blue staining).|
|Fig. 2. Time course of placodal Six1 induction. (A,B) Specification of placodal Six1 expression. (A) Six1 expression persists in most LPR explants, explanted at stage 16. (B) Percentage of LPR explants expressing Six1 after explantation at different stages and analysed after control embryos had reached early tailbud stages (stage 206). (C,D) Commitment of placodal Six1 expression. (C) Six1 expression is maintained in LPR graft (arrowheads) after isochronic transplantation into belly ectoderm at stage 15. (D) Percentage of grafts expressing Six1 after transplantation of LPR at different stages into stage 135 belly ectoderm and survival of host until early tailbud stages (stage 206). Host ectoderm was not induced to express Six1 by grafts at any stage. (E) Ectodermal competence and availability of signals for placodal Six1 induction. All host embryos were analysed after survival until early tailbud stages (stage 206). After orthotopic transplantations (E,F), stage 13 donor LPR ectoderm expresses Six1 when grafted isochronically (E), but fails to do so when grafted into stage 16 hosts (F; insert shows control side of embryo). Heterotopic transplantations (G) reveal that belly ectoderm is competent to express Six1 when grafted into the LPR of stage 13 hosts from stage 13 (G) up to at least stage 22 (I), but does not express Six1 when grafted into stage 16 hosts (H; insert shows control side of embryo). Percentage of grafts expressing Six1 after transplantation of belly ectoderm at different stages into stage 13 LPR is indicated in (J). Light blue bars indicate percentage of grafts exhibiting a disturbed pattern of Six1 expression.|