February 15, 2008;
Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis.
Rohon-Beard mechanosensory neurons (RBs
), neural crest
cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm
during anamniote vertebrate development. Neural crest
cells require BMP expressing non-neural ectoderm
for their induction. To determine if epidermal ectoderm
-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial
region of the neural plate
from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm
of host embryos at the same developmental stage. The neural plate border
were induced at the transplant sites, as shown by expression of Xblimp1
, and XHox11L2
, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue
. Because an intermediate level of BMP4
signaling is required to induce neural plate border
fates, we directly tested BMP4''s ability to induce RBs
; beads soaked in either 1 or 10 ng/ml were able to induce RBs
in cultured neural plate tissue
. Conversely, RBs
fail to form when neural plate tissue
from embryos with decreased BMP activity, either from injection of noggin
or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm
of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm
is capable of inducing RBs
, that BMP4
can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons.
[+] show captions
Fig. 2. Contact between neural plate and non-neural ectoderm induces RB sensory neurons. (A) Schematic of transplantation experiment. Intermediate neural plate tissue was transplanted into the ventral or ventrolateral non-neural ectoderm of same-stage embryos and allowed to develop until stages 14–28 (schematic of embryo described in Fig. 1; modified from Nieuwkoop and Faber, 1994). (B–I) In situ hybridization of embryos after transplantation of intermediate neural plate into non-neural ectoderm, lateral views, anterior to the left at 3.2× (B, D, F, H) or 6.6× (C, E, G, I). Endogenous gene expression is observed in all embryos dorsally (toward the top). (B, C) Several XHox11L2-positive RB sensory neurons were found surrounding the transplant site in a stage 26 embryo (arrows). The staining in these cells looks morphologically similar to the staining of endogenous RBs. (D, E) XN-tubulin-positive cells in a stage 27–28 embryo surround transplanted tissue. (F, G) XSox2 expression in the transplanted region indicating grafted tissue maintains its neural identity. (H, I) Xblimp1 positive cells were observed both at the region spanning the neural plate border (arrowhead) and at the site of the transplant (arrow). NP = neural plate; Epi = epidermal ectoderm.
Fig. 3. RB sensory neurons are induced in both donor neural and host epidermal tissue. (A–C) The location of ectopic XHox11L2 expression (A, B) compared to the location of lysinated rhodamine dextran (LRD, C) labeling donor neural plate tissue reveals that RB induction occurs at the border of the transplanted tissue. In embryos with pigmented neural plate tissue transplanted into the ventral ectoderm of an albino host, XHox11L2 appears within the region of the graft as seen in whole mount (D, 3.2×). (E–G) Sections of this tissue reveal the tissue contributions to ectopic XHox11L2-expressing cells. (E) Endogenous XHox11L2 stains cells in the dorsal neural tube bilaterally, shown at 40×. (F–F) Some ectopic XHox11L2 expression (arrow, F and F) was found in donor cells that contained pigment granules (arrowhead, F). Host tissue lacking pigment granules also expressed ectopic XHox11L2 (G–G). Blue Hoescht staining reveals the nucleus of each cell in the field in the sectioned tissue (F′–F, G′–G).
Figure 1. Competence of neural plate border ectoderm to form RB sensory neurons
(A) Schematic diagram of stage 11.5 Xenopus laevis embryo, vegetal view with the neural plate shaded in yellow, showing the region from which intermediate ( and lateral ( neural plate explants were taken to determine the competence of these tissues. (B, C) Intermediate (np and lateral (np neural plate explants were taken from stage 11.5−12 embryos and allowed to develop until stage-matched intact embryos reached stage 24−25. Intermediate neural plate explants stained negative for XHox11L2 expression (B), while lateral neural plate explants stained positive (C). (A) Modified from Nieuwkoop and Faber, 1994.
Figure 4. BMP4 can induce intermediate neural plate tissue to form RB sensory neurons
In situ hybridization of intermediate neural plate explants (A-D,F) or epidermal ectoderm (E) isolated at stage 11.5−12 and subsequently incubated with a heparin bead with or without hBMP4 (A-D,F) until stage-matched controls reached stage 24−25. (A) Intermediate explants incubated with a 0.5MBS-soaked bead never expressed the RB marker XHox11L2. Intermediate explants incubated with 1 ng/ml BMP4 stained positive for XHox11L2 (arrows in B) and XN-tubulin (arrows in C; white circle indicates position of the bead observed as an impression, since the bead was displaced during processing). (D) Expression of XSox2 in explants incubated with 1ng/ml BMP4 beads suggests that neural tissue is maintained in the area around the beads. (E) Epidermal explants stained positive for XKeratin, while neural plate explants incubated with 1 ng/ml BMP4 did not (F).