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First published online 4 December 2008
doi: 10.1242/dev.023978

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Novel skeletogenic patterning roles for the olfactory pit

Heather L. Szabo-Rogers^*, Poongodi Geetha-Loganathan, Cheryl J. Whiting, Suresh Nimmagadda, Katherine Fu and Joy M. Richman

Department of Oral Health Sciences, Life Sciences Institute, The University of British Columbia, Vancouver BC, V6T 1Z3, Canada.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Grafting methods and the skeletal derivatives of facial prominences. (A) Nasal pits were dissected from stage 20 or stage 26 donor chicken embryos, trypsinized and epithelium was grafted to hosts. (B) Stage 20 nasal pit (i), stage 26 nasal slit (ii), or stage 26 frontonasal epithelia (iii), were stapled to the exposed mesenchyme of the stage 15 presumptive maxillary region. (C) Similar donor tissues to those in Bi-iii were grafted to maxillary mesenchyme of stage 20 hosts. (D) Skeletal derivatives of the facial prominences surrounding the nasal pit were determined by previous grafting and vital dye labeling experiments (Lee et al., 2004; MacDonald et al., 2004; Richman and Tickle, 1989) and are projected onto the skeleton at stage 38. fnm, frontonasal mass; ios, interorbital septum; j, jugal; lnp, lateral nasal prominence; md, mandibular prominence; mxb, maxillary bone; mxp, maxillary prominence; nb, nasal bone; nc, nasal conchae; np, nasal pit; npp, nasal process of premaxillary bone; p, palatine bone; pa, pharyngeal arch; pmx, premaxillary bone; pnc, prenasal cartilage; qj, quadratojugal.

View larger version (98K): [in this window] [in a new window]   Fig. 2. Effects of nasal placode extirpation on the expression of epithelial genes. Nile Blue sulfate removal of the right nasal placode at stage 15. Dashed lines in whole-mount hybridized chicken embryos show the plane of section. (A,A') Loss of band of FGF8 expression on the right side of the face (asterisk) as compared with the left side (arrow). (A'') Mesenchyme is thinner on the extirpated side (asterisk). Expression in the frontonasal mass ectoderm and telencephalon is visible. (B) FGF8 expression is present on the medial side of the treated nasal pit (arrow). (B') The nasal pit ectoderm is absent on the treated side (asterisk; compare with the untreated side, arrow). (C,C') DLX5 is eliminated on the treated side (arrowhead). (C'') Decreased thickness (double-headed arrow) of the mesenchyme near extirpation, and loss of expression (arrow). (D-D'') After 24 hours, some specimens retained the medial lip of the nasal placode as judged by DLX5 expression and thickened epithelium (arrowhead). (E-E'') Other specimens had no expression on the extirpated side. np, nasal placode or pit. Scale bars: 500 µm in A,A',B,C,C',D,D',E,E' and insets in D'' and E''; 200 µm in A'',B',C'',D'',E''.

View larger version (109K): [in this window] [in a new window]   Fig. 3. Lateral nasal prominence mesenchyme is affected following epithelial extirpation and placement of foil barriers. Lateral (A-E,A''-E'') and frontal (A'-E') views of treated chicken embryos. Black and white arrowheads indicate treated and control nasal pits, respectively. (A-B'') Embryos hybridized 24 hours after nasal pit extirpation show no change in frontonasal expression of TBX2 (A-A'') or TBX22 (B-B''). (C-C'') By contrast, PAX7 expression is ablated within the right lateral nasal prominence after 16 hours. (D-D'') Foil (arrow) placed on the lateral side of the nasal pit (bracket) greatly reduced PAX7 expression on the manipulated side after 24 hours. (E-E'') Foil placed on the medial side of the nasal pit (bracket) had no effect on PAX7. np, nasal pit. Scale bars: 500 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Effects of nasal placode extirpation and rescue of skeletal elements with FGF8 beads. Lateral (A-C) and frontal (A'-C') views of chicken embryo skulls fixed 11-12 days after manipulations carried out at stage 15. (A,A') Normal anatomy in a control embryo that had Nile Blue sulfate applied but no epithelium removed. (B,B') Nasal placode extirpation caused complete loss of the right nasal bone and nasal conchae (asterisk). The nasal process of the right premaxillary bone has expanded (white arrow). (C,C') FGF8-soaked bead applied at the time of extirpation rescues the right nasal bone and nasal conchae (white arrow, see adjacent camera lucida drawing). Inset shows the staple used to hold the bead. The nasal process of premaxillary bone is also normal on the treated side. The right nasal bone and nasal conchae are smaller than in the normal, untreated side. (D) Frontal section of FGF8-treated extirpated specimen fixed 24 hours after the manipulation showing increased BrdU labeling on the treated side (arrowheads). (D') Adjacent section showing TUNEL-positive cells (arrowheads) in the same area indicating high-level proliferation. L, left; R, right; b, bead; f, frontal bone; mxb, maxillary bone; nb, nasal bone; nc, nasal conchae; npp, nasal process of premaxillary bone; pmx, premaxilla. Scale bars: 5 mm in A-C'; 500 µm in C inset; 200 µm in D,D'; 100 µm in D' inset.

View larger version (97K): [in this window] [in a new window]   Fig. 5. Quail-chicken chimeras. (A) Twenty-four hours after grafting, only the graft (inset) is stained with quail-specific Q¢PN antibody. The staple that secures the graft is visible. (B) The donor nasal pit has incorporated into the host mesenchyme and comprises quail epithelium. There are no contaminating quail mesenchymal cells. (C-D'') An ectopic outgrowth is present on the side of the head under the eye. Alcian Blue stain shows cartilage is not present in the graft (C inset, arrowhead). The white line in the inset shows the plane of section in C and D. The graft is present within the outgrowth on the lateral side of the head (C,D, arrowhead). Differentiated neurons are observed near the graft (D', arrowhead). A near-adjacent section, staining with Q¢PN antibody indicates that epithelium and neurons (arrowhead) are quail derived (D''). bc, basii cranium; e, eye; en, entoglossum; mb, mandibular branch of the trigeminal nerve; mc, Meckel's cartilage; md, mandibular prominence; mxp, maxillary prominence; np, nasal pit; o, otic capsule; r, rhombencephalon; tg, trigeminal nerve body. Scale bars: 1 mm in A; 100 µm in A inset; 200 µm in C,D; 2 mm in C inset; 100 µm in B,D',D''.

View larger version (115K): [in this window] [in a new window]   Fig. 6. External and skeletal changes induced by ectopic nasal epithelia placed in the maxillary region of stage 15 embryos. Schematics show graft position. (A) External view of a typical graft-derived outgrowth (arrow) near the external auditory meatus. (A') A stage 20 nasal pit induced an ectopic process on the squamosal bone along with several other skeletal elements (box). (A'') The compound elements are dissected away from the skull to reveal at least five individual bones. (B) Minor changes in the nictating membrane (arrow). (B') An ectopic process on the proximal end of the quadratojugal was induced. (B'') This was accompanied by several ectopic bones directly underlying the changed nictating membrane (box in B'). The secondary cartilage of the normal quadratojugal is visible (white arrowhead). (C) A different embryo in which graft-derived bones have been dissected from the skull. Normal secondary cartilage is present on the quadratojugal (white arrowhead) and on the ectopic quadratojugal (black arrowhead). (D) A stage 26 graft formed ridges of epithelium surrounding an ectopic external nasal aperture (arrow and inset). (D',D'') More cartilage has formed in this graft than with stage 20 nasal pits. (E-E'') Embryos that had stage 26 frontonasal mass epithelium grafted to the maxillary prominence at stage 15 developed normally. epi, epithelium; fnm, frontonasal mass; g, graft; np, nasal pit; q, quadrate; qj, quadratojugal; p, palatine; pa, pharyngeal arch; s, squamosal. Scale bars: in A, 5 mm for A,B,D; 1 mm in D inset; in A', 5 mm for A',B',D',E'; 1 mm in A'',B'',C,D''; 2 mm in E''.

View larger version (91K): [in this window] [in a new window]   Fig. 7. Nasal epithelium identity is retained but mesenchyme is partly converted to lateral nasal mesenchyme. Coronal cryosections of stage 20 nasal pit grafts on stage 15 hosts grown for 6 days. (A,A') A lumen connected to the external surface of the head has formed (box, shown at higher magnification in A'). (B-B'') Adjacent sections contain TuJ1-positive cells and a nerve exiting the grafted epithelium. (C) PAX7 antibody stains only the lateral nasal but not the frontonasal mass or maxillary mesenchyme. (C') Induction of PAX7 staining in the maxillary mesenchyme (arrowheads). (D,D') Another example with PAX7 labeling of mesenchyme in several distinct locations surrounding the grafted epithelia (D', arrowheads). (D'',D''') TuJ1-positive neurons formed in one region of the grafted epithelium. This TuJ1-positive domain is likely to correspond to the olfactory epithelium, whereas the unlabeled region corresponds to the presumptive respiratory epithelium. e, eye; g, graft; n, nerve; nm, nasal mesenchyme; oe, presumptive olfactory epithelium; on, optic nerve; r, rhombencephalon; re, presumptive respiratory epithelium. Scale bars: 600 µm in A,B,C,D,D''; 300 µm in A',B',B'',C',D',D'''.

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