Glavic A et al. (2004), Role of BMP signaling and the homeoprotein Iroq...

XB-ART-3310

Dev Biol 2004 Aug 01;2721:89-103. doi: 10.1016/j.ydbio.2004.04.020.

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Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field.

Glavic A, Maris Honoré S, Gloria Feijóo C, Bastidas F, Allende ML, Mayor R.

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Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.

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Species referenced: Xenopus laevis
Genes referenced: foxd3 irx1 myod1 pax2 six1 six4 snai1 snai2 sox2

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	Fig. 1. The iro genes are expressed in the placodal field of Xenopus and zebrafish embryos. In situ hybridization of iro1, preplacodal, placodal, and neural crest markers was performed at different stages of Xenopus (A-H, J, K, M-S) and zebrafish embryos (I, L). a: anterior; p: posterior; d: dorsal; star: neural plate expression of iro1; arrowhead: preplacodal expression of iro1. (A) Xiro1 expression in a stage 15 embryo. Lateral view. (B, C) Xiro1 expression in a stage 17 embryo, in an anterior (B) and lateral (C) view. (D) Six1 in situ hybridization of a stage 15 embryo. Lateral view. (E, F) Six1 expression in a stage 17 embryo, in an anterior (E) and lateral (F) view. (G, H) Xiro1 expression in a stage 19 embryo, in an anterior (G) and lateral (H) view. (I) ziro1 expression in a tail bud stage zebrafish embryo (10.5 h postfertilization, hpf). Dorsal view, arrow indicates the preplacodal domain of expression. (J, K) Xiro1 expression in a stage 19 embryo, in an anterior (J) and lateral (K) view. (L) Six4.1 expression in a tail bud stage zebrafish embryo (10.5 h postfertilization, hpf). Dorsal view, arrow indicates the preplacodal domain of expression. (M) Xiro1 expression in a stage 21 embryo, dorsal view, posterior to the top. Arrow indicates the gap of expression between the neural (star) and placodal (arrowhead) expression of Xiro1. (N) section of embryo shown in M. (O) Slug expression in a stage 20 embryo. Dorsoanterior view. (P) Double in situ hybridization of a stage 20 embryo for Six1 (purple) and Slug (green). Anterior view. (R) Section of embryo shown in P. Note the gap in the expression of both genes indicated by the arrowhead. (S) Double in situ hybridization of a stage 20 embryo for Slug (purple) and Sox2 (green). Anterior view. (T, U) Summary of neural plate, neural crest, and preplacodal markers, for early (T) and late (U) neurula stages.
	Fig. 2. Expression of MXiro1 at later stages. Anterior to the left, dorsal to the top: arrow, indicates placodal expression. (A, B) Xiro1 expression at stage 20 (A) and 33 (B). (C, D) Six1 expression at stage 20 (C) and 33 (D). (E) Double in situ hybridization for Xiro1 (green) and Six1 (purple) at stage 20. (F) Double in situ hybridization for Xiro1 (green) and Pax2 (purple) at stage 33. Arrow in F indicates the otic placode.
	Fig. 3. The preplacodal markers Six1 and Xiro1 are induced by an interaction between neural plate and epidermis. (A) A piece of anterior neural plate was dissected from a stage 13 embryo, previously labeled with FLDx at the one-cell stage, and grafted into the ventral epidermis of a normal stage 13 embryo. The grafted embryo was cultured until stage 25, when the expression of the preplacodal markers Six1 (B, D, F, G) or Xiro1 (C, E, H) was analyzed. Inset: control neural plate cultured in isolation, where no expression is seen. Arrow: induction of the marker; star: graft. Anterior to the left. (B) Induction of Six1 in the epidermis adjacent to the graft. (C) Induction of Xiro1 in the epidermis adjacent to the graft. (D) Higher magnification of the graft shown in B. (E) Higher magnification of the graft shown in C. (F–H) Section of different grafts, showing that the induced cells (arrow) are sometimes not adjacent to the graft (bracket in G). Forty-eight percent of the grafts showed Six1 induction, n = 29; 45% of the grafts showed Xiro1 induction, n = 28.
	Fig. 4. Specification of the preplacodal field by BMP in zebrafish embryos. Ten hours postfertilization embryos analyzed by whole-mount in situ hybridization for the expression ziro1 (AF), FoxD3 (GI), and Krox20/MyoD (J, L). A-C and J-L are shown in dorsal view with anterior to the top, while DI are shown in lateral view with anterior to the left. The placodal domains of ziro1 expression is indicated with arrow. Blue arrow: neural crest; white arrowhead: absence of ziro1 expression in the neural crest; black and red arrowhead: mesoderm. (A, D, G, J) Wild-type embryos. (B, E, H, K) Embryos injected at the one-cell stage with 100 pg of BMPR mRNA: note the expansion in the midbrain, hindbrain, and placodal field of ziro1 expression, and the moderate expansion of the neural crest and mesoderm domains. (C, F, I, L) Embryos injected at the one-cell stage with 100 pg of BMPR mRNA and a mixture of ntl and spt morpholinos to inhibit mesoderm formation. Note the expansion of the placodal domain of ziro1 (C, F), the moderate expansion of FoxD3 and Krox20 (I, L), and the absence of MyoD expression (L). Note that the embryo in K is slightly older than the embryos in J and L, this is why two bands of Krox20 expression are seen in K, but only one in J and L.
	Fig. 5. Induction of preplacodal markers by chordin expressing cells (A). Five hundred picograms of Chordin mRNA and 7 ng/μl of FLDx were injected at the one-cell stage and the embryo cultured until the blastula stage, when ectodermal cells were dissociated and grafted into the lateral ectoderm of a gastrula stage embryo (70–90% epiboly). The grafted embryos were cultured until the one- to two-somite stage, and the expression of preplacodal markers was analyzed by in situ hybridization. The grafted cells were visualized by alkaline phosphatase-mediated FITC inmunostaining and recognized by the blue color of the FLDx staining; they are indicated by a red arrowhead. (B–F) Analysis of Six4.1. (B) Dorsal view. Control (C) and grafted (D) sides of embryo shown in B. (E, F) Higher magnification of embryos shown in C and D. Note that the preplacodal marker is expanded a few cell diameters on the side that received the graft, but the border of expression is not as sharp as on the control side (E, F). (G–K) Analysis of ziro1. (G) Dorsal view. Control (H) and grafted (I) sides of the embryo shown in G. (J, K) Higher magnification of embryos shown in H and I. Note that the preplacodal marker is expanded a few cell diameters on the side that received the graft (E, F).
	Fig. 6. Specification of the preplacodal field and placodes by BMP in Xenopus embryos. The level of BMP activity was changed by injection of the mRNA that codes for the dominant negative of the BMP receptor (ΔBMPR) in Xenopus (A, C–E) or by implanting beads soaked with noggin in early neurula Xenopus embryos (B, F–I). The expression of the preplacodal (Xiro1) and specific placodal markers (Delta1, Pax2) was analyzed under these conditions. (C–E) An expansion in the preplacodal marker Xiro1 (C) or the placodal markers Delta1 (D) and Pax2 (E) was observed when 125 pg of ΔBMPR mRNA were injected in one blastomere of a two-cell stage embryo. Arrows: expansion of the markers in the injected side. (F–I) Implantation of noggin soaked beads (asterisks) leads to an expansion of the preplacodal marker Xiro1 (F) and the placodal markers Delta1 (G, grafted side; H, control side) and Pax2 (I). Arrow indicates the expansion of the markers in the experimental side.
	Fig. 7. Xiro1 is required for preplacodal and placodal development. Two-cell stage embryos were injected in one blastomere with 1 ng of the inducible forms of a repressor form of Xiro1 (HDEnGR) (A, C, E, G, I, L), or with a dominant negative form of Xiro1 (HDGR) (B, D, F, H, J, M). The embryos were treated with dexomethasone at stage 12 and the expression of Six1, Pax2, and Sox2 was analyzed by in situ hybridization. The injected side was visualized by alkaline phosphatase-mediated FITC inmunostaining. The uninjected (A, B, E, F, I, J) and injected (C, D, G, H, L, M) side of the same embryos are shown. (A, C, E, G, I, L). The injection of HDEnGR leads to a moderate enlargement of Six1 expression (A, C, arrow) and to a moderate expansion of the otic placodal marker Pax2 (E, G; arrow) and the epibranchial placode marker Sox2 (I, L; arrow). (B, D, F, H, J, M) The injection of HDGR leads to a complete inhibition of the preplacodal marker Six1 (B, D, arrow) and to an inhibition of the otic placodal marker Pax2 (F, H; arrow) and the lens and epibranchial placode marker Sox2 (J, M; arrows). (N, O) Rescue experiment. Embryos were injected with HDGR as described, showing the characteristic inhibition of Six1 in the injected side (arrow); however, this effect was rescued by coinjection of HDGR and HDEnGR, as is seen in the injected side (arrow). (P) Sox2 expression in stage 16 embryo injected with HDGR. No effect in the neural plate can be detected. Each experiment was performed at least twice with a minimum of 42 embryos. The percentage effect for each experiment was approximately 65%. For the rescue experiment, the percentage of effect was less than 10%.
	Fig. 8. Notch signaling on placode development. Two-cell stage embryos were injected in one blastomere with 0.7 ng of NICDGR (A, B, E, F) or 0.25 ng of Su(H)DBMGR (C, D, G, H). Injected embryos were cultured from stage 12 in medium containing dexamethasone and the expression Pax2 (A, E, C, G) and Sox2 (B, F, D, H) was analyzed by in situ hybridization. The injected side was recognized by alkaline phosphatase-mediated FITC immunostaining. The uninjected (A–D) and injected (E–H) sides of the same embryos are shown. The expression of markers in several placodes is indicated. O. otic; L: lens. (A, C, E, G). The injection of NICDGR does not produce any obvious effect in the expression of the otic placodal marker Pax2 (A, E) or the epibranchial placode marker Sox2 (B, F). The injection of Su(H)DBMGR does not have any effect on the expression of the otic placodal marker Pax2 (C, G) and the epibranchial placode marker Sox2 (D, H). Each experiment was done at least twice with a minimum of 45 embryos.