The position of the olfactory placodes suggests that these epithelial thickenings might provide morphogenetic information to the adjacent facial mesenchyme. To test this, we performed in ovo manipulations of the nasal placode in the avian embryo. Extirpation of placodal epithelium or placement of barriers on the lateral side of the placode revealed that the main influence is on the lateral nasal, not the frontonasal, mesenchyme. These early effects were consistent with the subsequent deletion of lateral nasal skeletal derivatives. We then showed in rescue experiments that FGFs are required for nasal capsule morphogenesis. The instructive capacity of the nasal pit epithelium was tested in a series of grafts to the face and trunk. Here, we showed for the first time that nasal pits are capable of inducing bone, cartilage and ectopic PAX7 expression, but these effects were only observed in the facial grafts. Facial mesenchyme also supported the initial projection of the olfactory nerve and differentiation of the olfactory epithelium. Thus, the nasal placode has two roles: as a signaling center for the lateral nasal skeleton and as a source of olfactory neurons and sensory epithelium.KEY WORDS: Chicken embryo, Placode, Nasal capsule, FGF8, Craniofacial, TuJ1, PAX7, Lateral nasal prominence Development 136, 219-229 (2009)
MATERIALS AND METHODS
EmbryosFertile White Leghorn chicken eggs (Gallus gallus; University of Alberta) and Japanese quail eggs (Coturnix coturnix japonica; Oregon State University, Corvalis) were used. Quail embryos were incubated ~12 hours after chicken embryos so that they reached the appropriate developmental stage (Schneider and Helms, 2003). Embryo work was approved by the UBC Animal Care Committee.
Extirpations of nasal ectoderm, foil barrier placement and bead implantsNile Blue sulfate (0.1% in phosphate-buffered saline) was painted on the nasal placode of stage 15-16 (25-28 somites) embryos or on nasal pits of stage 20 embryos. The epithelium was removed with a tungsten needle. Control embryos received Nile Blue and the epithelium was left intact. Embryos were collected at 0, 6, 16 and 24 hours following surgery and gene expression was analyzed. Other embryos were collected at stage 38 for analysis of skeletal phenotypes. A third set of extirpated embryos had either an all-trans-retinoic acid-soaked bead (0.01 mg/ml; AG1X2 beads, BioRad) or an FGF8b-soaked bead (1 mg/ml, Peprotech; Affigel beads, Biorad) stapled onto the exposed mesenchyme. For barrier experiments, aluminium foil was inserted on the medial or lateral side of the nasal placode of stage 15 embryos.
Grafting experiments Donor tissue preparationStage 20 or 26 donor (quail or chicken) frontonasal mass and lateral nasal prominences were collected in Hank's Balanced Salt Solution with Ca 2+ and Mg 2+ (HBSS). Frontonasal mass epithelium including the nasal pits was separated with 2% trypsin (see Fig. 1A) (Richman and Tickle, 1989). The surrounding surface epithelia were trimmed away from the nasal pit and 0.5% Neutral Red was adde...