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First published online November 21, 2008
doi: 10.1242/10.1242/dev.026633

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Competence, specification and commitment to an olfactory placode fate

Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (110K): [in this window] [in a new window]   Fig. 1. Expression of Dlx3, Dlx5 and Pax6 in chick olfactory placode precursors. (A,A',D,D',G,G') Dlx3. (B-B'',E,E',H,H') Dlx5. (C,C',F,F',I,I') Pax6. (A,A') Ventral view of Dlx3 at HH8. Broken white lines trace outline of anterior neural fold region. Asterisks indicate olfactory precursor regions lacking Dlx3 expression in A and A'. Broken black line in A indicates transverse plane of section shown in A'. Low-level expression is seen in ectoderm adjacent to anterior neural folds (A', arrowheads). Broken line in A' outlines ectoderm. (D,D') Ventral view and transverse section, respectively, of Dlx3 at HH10 show strong expression in olfactory progenitors (arrowheads). (G) At HH13, Dlx3 refines ventromedially (arrowheads) within anterior ectoderm between eyes (outlined); midline of embryo denoted by a single white dotted line. (G') Lateral view of embryo shown in G. The eye is outlined and arrowheads indicate Dlx3 expression in nasal progenitors. (B) Dorsal view of Dlx5 at HH8. (B-B'') Low transcript levels within anterior neural folds (asterisks) with gradual posterior decline and exclusion from posterior neural folds (compare B' with B''). Dlx5 is expressed at high levels within the anterior ectoderm, but shows `salt and pepper' expression more posteriorly (compare B' with B''). Arrowheads indicate Dlx5 expression in adjacent ectoderm. (E,E') Ventral view and transverse section, respectively, of Dlx5 at HH11. Arrowheads indicate strong expression in olfactory precursors. HNK-1 staining in pink in E' shows distance of neural crest from placode precursors at this stage. (H,H') Dlx5 at HH15 is in migrating neural crest and olfactory placodes (arrow in H' section indicates Dlx5+ thickened ectoderm or ingressing placode cells). (C,C',F,F',I,I') Pax6 at HH8 (C, dorsal view; C', section), HH11 (F, ventral view; F', section) and HH14 (I, lateral view; I', section). (C,C') Abundant Pax6 in anterior neural folds and salt-and-peppered throughout anterior ectoderm (arrowheads in C'). (F,F') High levels in eye, but extinguished in olfactory precursors (between arrowheads, F,F'). (I) Robust Pax6 in eye and diencephalon at HH14. (I') Arrow indicates reappearance of Pax6 in presumptive nasal ectoderm. Broken white lines indicate planes of section in B-F,H,I. c, cornea; nt, neural tube; e, eye; fb, forebrain; op, olfactory placode. The schematic drawings on the left show the position of the olfactory precursors (blue) at the indicated stages.

View larger version (111K): [in this window] [in a new window]   Fig. 2. Dlx+ and GnRH+ neurons found in the nasal mesenchyme have a placodal origin. (A-C') Dlx3, Dlx5 and Pax6 in olfactory epithelium at HH19-20. Levels of Dlx3 are strong throughout the olfactory pit (A,A') and also in the otic pit (ot) and branchial arches. (B) Dlx5 is expressed in olfactory and otic placodes, broadly in branchial arches, in the neural crest-derived part of trigeminal ganglion (tg) and in ingressing olfactory placode cells (B', arrowhead). (C) Pax6 is expressed in olfactory pit, eye (e) and forebrain. (C') Section through olfactory placode. Broken black lines indicate planes of section. (D-f') Placodal origin of GnRH neurons. (D) Whole-mount view of HH18 embryo electroporated with pCIG-GFP at HH10. Broken black lines indicate transverse sections through the olfactory epithelium shown in d-d''. (d) Section through the olfactory pit. Arrows indicate multiple GFP+(green)/pan-Dlx+(red) cells. (d',d'') Sections through more basal regions of the olfactory system. Neurons (indicated by arrows, Hu+, blue) that ingress from the placode (GFP+,green) are also Dlx+. (E) Ventral whole-mount view of HH27 embryo electroporated with pCIG-GFP at HH10; inset shows schematic of ventral view of HH27 embryo, red box indicates area shown at high magnification. (e) Numerous GFP+ (green) cells are observed in a ganglion-like structure. Arrow indicates a GFP+ GnRH neuron (inset). (e') A nearby section has pan-Dlx+/GFP+ cells (arrows; insets). (f,f') Sections through a similar embryo. Arrows indicate GFP+/GnRH+ neurons (f) and GFP+/pan-Dlx+ neurons in an adjacent section (f'). Higher magnification views can be seen in insets. (G) Transverse section through the olfactory epithelium of a non-electroporated HH25 chick embryo. GnRH (red) Hu/neurofilament (green)-positive neurons (indicated by arrowheads) are seen within the olfactory epithelium and just ventral to it. oe, olfactory epithelium; m, maxillary epithelium; fb, forebrain; e, eye.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Dlx3 protein co-expresses with other Dlx family members, Pax6 and Hu in the olfactory epithelium. (A-C'') Specificity of Dlx3 antibody. (A) Section through r5 at HH11. Premigratory neural crest prominently expresses Dlx5 (broken white lines). Lower levels are also detected in otic placode (arrows). (A') HNK1+/Dlx3+/Dlx5+ (from A) otic placode (white arrowheads) and HNK+/Dlx5+ (from A) but Dlx3- (green) neural crest (white dotted lines). (B) In the trunk, Dlx5 transcripts are abundant in dorsal neural tube and emigrating neural crest. (B') No Dlx3 or HNK1 staining is noted in dorsal neural tube or neural crest. (C-C'') Cryosections of HH19-20 embryos co-stained for Dlx3 (green), pan-Dlx (red) and DAPI (blue) show overlap between two antibodies (C'', yellow) in olfactory, but not in forebrain, which lacks Dlx3, but stains with pan-Dlx (red). (D-D'') Dlx3 and Pax6 protein in forming olfactory placode (white arrowheads) at HH14. (D) Dlx3 (green) is in ectoderm proximal to forebrain (fb). (D') Pax6 (red) is in scattered cells but stronger in posterior ectoderm of future cornea. (D'') Overlay of Dlx3 and Pax6 shows some co-expression (yellow; D-D'', arrowheads). (E-E'') Dlx3 and Pax6 protein in olfactory epithelium at HH20. (E) Dlx3 (green) is found throughout the epithelium, but higher at margins. (E') Pax6 (red) is in cup-shaped olfactory epithelium. (E'') Dlx3 and Pax6 are co-expressed in many cells (yellow; E-E'', arrowheads). DAPI is in blue in D-E''. (F) By HH19, olfactory epithelium undergoes robust neurogenesis (pan-neuronal Hu is in red) and ingressing neurons are observed (arrowhead); some neurons within the epithelium are Pax6+ (green, arrows). (G) At HH21, a few neurons are also Dlx3+. (H) Nasal mesenchyme underlying olfactory pit at HH18-19 is Pax7+ (green). fb, forebrain; oe, olfactory epithelium; no, notochord.

View larger version (118K): [in this window] [in a new window]   Fig. 4. Isotopic/isochronic grafts and naïve epiblast grafts to presumptive olfactory placode ectoderm contribute to endogenous olfactory epithelium. (a-A'') After grafting anterior neural folds from HH8 quail, Pax6+ quail cells are in olfactory epithelium (A-A'', arrowheads) and telencephalon (A-A'', arrows). (b-B'') HH8 ectoderm grafted adjacent to anterior neural folds contributes to nasal pit and adjacent ectoderm (boxes in B,B', arrowhead indicates Dlx3+/QPCN+ cell). (c-D'') HH5 quail epiblast grafted to presumptive olfactory territory integrated into ectoderm adjacent to placode, thickened and expressed Dlx3 (box in C,C'; arrow indicates Dlx3+ host nasal pit; arrowhead indicates Dlx3+/QCPN+ cell). Hu staining (D-D'') shows extensive neuronal differentiation. In D,D', large box indicates neurons in grafted epithelium and small box indicates quail cells contributing to the olfactory nerve. Arrowhead indicates Hu+/QCPN+ cell (D''). Inset in D'' shows high magnification of neurons migrating from donor-derived olfactory placode. la, lateral; m, medial; op, olfactory placode; fb, forebrain.

View larger version (121K): [in this window] [in a new window]   Fig. 5. Head but not trunk ectoderm is competent to form olfactory placode. (a) HH8 hindbrain-level ectoderm expresses Dlx3 when grafted to presumptive nasal region (A-A''; arrowhead points to a Dlx3+/QCPN+ cell). (b) HH10 midbrain-level ectoderm expresses Pax6 when grafted to olfactory placode region (B-B''; arrowhead indicates an ingressing QCPN+ cell). (c-C'') Similar graft to b undergoes neuronal differentiation within olfactory pit (C-C'', arrowheads) and in ingressing cells lining olfactory nerve (C-C'', arrows). (d-D'') Grafts of HH8 trunk-level ectoderm fail to express Pax6. (e,E) Some head ectoderm grafts incorporate into nose, lens and cornea. (F) HH8 midbrain-level ectoderm expresses Pax6 in lens and cornea. (G) HH8+ hindbrain-level ectoderm is not competent to express Pax6 in cornea or olfactory placode (not shown), though three quail cells in the lens appear Pax6+ (arrowhead). la, lateral; m, medial; c, cornea; fb, forebrain; le, lens; op, olfactory placode; r, retina.

View larger version (112K): [in this window] [in a new window]   Fig. 6. Olfactory progenitors are specified to form neurons by HH10; placodal morphology is recapitulated in culture from HH13-14. (A-A'') Dlx3 (red) and Pax6 (green) are robustly expressed in HH8 explants; 1/3 cells co-express both (arrows, A'', yellow); non-overlapping Dlx3 (A,A'', arrowhead) or Pax6 (A',A'', arrowhead) domains appear distinct. (B) HH10 explant with neuronal differentiation; most neurons are Dlx3+/Hu+ (arrow), though some are Dlx3- (arrowhead). Insets show high-magnification views. (C) In HH10 explants, most cells are Pax6+ and few are Hu+ neurons. (C') Arrow indicates cluster of Dlx3+/Pax6+ cells. Dlx3+ and Pax6+ cells segregate from double-positive cells. (D) Olfactory placode explanted at HH13 undergoes thickening in culture and has some Dlx3+/Hu+ (red/blue) cells (arrows, insets). Pax7 (blue, distinguishable from Hu by nuclear localization) delineates nasal mesenchyme underlying olfactory placode. (E) Newly thickened placodal ectoderm shows robust neuronal differentiation (Hu+, blue); some neurons are Pax6+ (green; arrowheads; insets). (F) Location of olfactory precursors at HH8, HH10 and HH14 based on fate maps.

View larger version (108K): [in this window] [in a new window]   Fig. 7. Commitment towards olfactory fate occurs concomitant with acquisition of placodal morphology. (A) Schematic of HH13 quail embryo. Broken line indicates plane of section in A'. (A') Section through prospective olfactory placode; white lines indicate grafted ectoderm overlying lateral plate (see C). (B) Schematic of HH14-15 quail embryo; broken line indicates plane of section in B'. (B') Ultra-thin section through olfactory placode (courtesy of Dr John Sechrist, Caltech). (C) Red oval indicates placodal ectoderm grafted over lateral plate mesoderm. (C') In section, broken white lines outline host trunk ectoderm and the red line indicates transplanted ectoderm. no, notochord; da, dorsal aorta. (D) Grafted HH8 ectoderm expresses Pax6 but not Hu. (E) Grafted HH10 ectoderm expresses Pax6 and forms few neurons (arrowhead). (F,F') Grafts of HH13 ectoderm, thicken and differentiate as Hu+ neurons (cytoplasmic green); some are Dlx3+. Neurons delaminate and ingress, migrating in loose association (F,F', arrowheads), some reaching the dorsal aorta (da). Pax7+ (nuclear green, asterisk) marks quail mesenchymal cells grafted with the presumptive placode (F) and host dermamyotome (F'). Pax7 and Hu show no expression overlap in these sections. (G-G'') Grafts of thickened placodal ectoderm (HH14-15) express Dlx3 (blue) and Pax6 (not shown), invaginate and morphologically resemble an endogenous olfactory pit. Arrowheads indicate Hu+ neurons that ingress from the placode and migrate as a chain. No Pax7+ cells were detected in this graft.

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