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doi: 10.1242/10.1242/dev.00397

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A zone of frontonasal ectoderm regulates patterning and growth in the face

Diane Hu^*, Ralph S. Marcucio^* and Jill A. Helms

Department of Orthopaedic Surgery, 533 Parnassus Avenue, Suite U-453, University of California at San Francisco, San Francisco, CA 94143, USA

View larger version (89K): [in a new window]   Fig. 2. Transplantation of the FEZ creates an ectopic Fgf8/Shh boundary. (A) A region of stage 20 FNP ectoderm measuring 0.5-0.8 mm by 1.0-1.2 mm in width (indicated by the red striped box) was transplanted onto the dorsal FNP (D) of a stage 25 chick embryo and pinned into place. (B) Lateral schematic view of the FEZ donor site (broken red lines indicate cut sites). FEZ grafts encompassed the Fgf8 (green) ventral Shh (yellow) junction (indicated by a red arrow). (C) Lateral schematic view of the FEZ graft site in a stage 25 host FNP. The quail-derived FEZ graft is indicated by a darker green (dorsal) and a darker yellow (ventral) domain, and by red letters. Twelve hours after transplantation, (D) Shh expression persisted in the ventral FNP ectoderm (V) of stage-matched, unoperated embryos. (E) Ectopic Fgf8 and Shh in the dorsal FNP of chimeric embryos confirmed that the FEZ (red D, V) was appropriately positioned in the host FNP. (F) Higher magnification of the boxed region in E showed that only QCPN-positive quail ectoderm was transplanted. ps, palatal shelf; pe, pharyngeal endoderm; rp, Rathke's pouch; di, diencephalon; or, optic recess; t, telencephalon.

View larger version (93K): [in a new window]   Fig. 1. Defining morphogenetic movements and molecular boundaries in the upper beak. (A) A stage 20 embryo illustrating four primary sites of retroviral application. Broken red lines trace the border of the FNP and maxillary processes, and colored dots correspond to injections sites shown in B-E. (B) Infected cells were visualized after histochemical detection of alkaline phosphatase activity (blue precipitate). A frontal view of a stage 30 embryo showed that injections at site B labeled a patch of ectodermal cells that extended along the dorsal aspect of the FNP. The egg tooth is indicated by an asterisk. (C) Frontal view of a stage 28 embryo showed that injections at site C labeled ectodermal cells on the dorsal margin of the upper beak. (D) Frontal view of a stage 30 embryo showed that injections at site D produced labeled cells that resided along the dorsal margin of the upper beak. (E) Ventral view of a stage 30 upper beak, which demonstrated that injections at site E resulted in labeled cells along the midline of the ventral upper beak. The broken red line indicates the dorsoventral boundary, (F) which was demarcated by the Shh expression domain. (G) Whole-mount in situ hybridization demonstrated that by stage 20, Shh was expressed in ventral FNP ectoderm (V); transcripts were also evident in hyoid arch (hy) ectoderm. (H) Also at stage 20, Fgf8 was expressed in dorsal FNP ectoderm (D); transcripts were detectable around the nasal pit (np), in the maxilla (mx) and mandible (mn), and on the posterior aspect of the hyoid arch. (I) The juxtaposed (red arrowhead), but not overlapping boundary of Fgf8 (transcripts pseudocolored green) in dorsal FNP (D) and Shh (yellow) in ventral ectoderm (V) was confirmed by section in situ hybridization. ps, palatal shelf; pe, pharyngeal endoderm; rp, Rathke's pouch; di, diencephalon; or, optic recess; t, telencephalon. Scale bar: in B, 1 mm for B-F; in I, 0.5 mm.

View larger version (86K): [in a new window]   Fig. 3. Changes in gene expression patterns correspond to FEZ orientation. (A) Schematic lateral view of a stage 25 host FNP, indicating dorsal (green) and ventral (yellow) domains corresponding to sections shown in D,G,J,M,P,S. (B) Schematic view of a quail FEZ graft (dark green, dark yellow, red D, V) positioned on the dorsal surface of a stage 25 chick host FNP corresponding to sections shown in E,H,K,N,Q,T. (C) Schematic view of a FEZ graft that has been rotated 180° (red V,D) corresponding to sections shown in F,I,L,O,R,U. (D) In unoperated embryos, Shh (yellow) was restricted to ventral FNP ectoderm (V); Fgf8 was already down regulated in dorsal FNP ectoderm (D) but transcripts were still detectable in the optic recess (or). (E,F) Twenty-four hours after transplantation, Shh was detected in the ventral FEZ (red V) as well as in its endogenous, ventral domain (white V). Fgf8 was no longer detectable in the dorsal FEZ (red D). (G) Ptc1 transcripts were detected in ventral ectoderm and throughout FNP mesenchyme. (H,I) Ptc1 was ectopically induced in FNP mesenchyme beneath the ventral region of the graft. (J) Gli1 was expressed in ventral ectoderm and at low levels throughout FNP mesenchyme. (K,L) Gli1 was ectopically induced in mesenchyme beneath the ventral FEZ. (M) Msx1 was expressed in dorsal ectoderm and mesenchyme, corresponding to the site of outgrowth. (N,O) Msx1 was repressed beneath the ventral, and induced beneath the dorsal, FEZ; a small ectodermal patch of Msx1 was evident in dorsal host ectoderm but was absent in underlying mesenchyme (O, arrowhead). (P) AP2 was strongly expressed throughout the dorsal ectoderm and mesenchyme. (Q,R) AP2 transcripts were abundant beneath both the dorsal and ventral FEZ. (S) Lfng was expressed at low levels throughout the ectoderm and localized to the mesenchyme at the site of outgrowth. (T,U) The endogenous mesenchymal expression of Lfng was up regulated adjacent to the graft, as well as in the mesenchyme beneath the ventral graft, but was missing from the dorsal mesenchyme (arrowhead). Scale bars: 1 mm.

View larger version (94K): [in a new window]   Fig. 4. The FEZ induces outgrowth of an ectopic beak that exhibits dorsoventral polarity. (A) Schematic view of a stage 25 host FNP, indicating that the intersection of the dorsal (green) and ventral (yellow) compartments was the site of outgrowth (red arrow). (B) By stage 36, an egg tooth (*) was evident on the dorsal upper beak, while the ventral surface has no ectodermal elaborations. (C) Sagittal trichrome-stained section through the embryo shown in B demonstrates the dorsoventral relationships among structures in the upper beak. The egg tooth (asterisk) is located on the dorsal surface; beneath it is the premaxillary bone (pmx), and below this is the prenasal process (pnp). The ventral surface of the upper beak (V) lacks any elaborated ectodermal appendages. (D) Schematic view of a stage 25 host after transplantation of quail stage 25 FNP ectoderm (red DV). (E,F) This isotopic transplantation had no effect on facial morphology, despite the fact that the graft was derived from quail. (G) Schematic view of stage 25 host after FEZ transplantation. The graft is positioned in the normal, dorsoventral orientation. The expected outgrowths (arrowheads), with their predicted dorsoventral polarity are indicated. (H) This grafting scenario resulted in an outgrowth from the proximal region of the upper beak and contained two egg teeth. The autochthonous egg tooth (arrowhead) appears normally positioned. (I) The ectopic beak comprised two egg teeth (red asterisk and arrowhead) located above two premaxillae (red pmx) each surrounding a prenasal process (red pnp). These relationships indicated that the ectopic beak had dorsal-ventral/ventral-dorsal polarity (DVVD). The autochthonous beak contained an egg tooth (black asterisk), the premaxilla (black pmx), and the prenasal process (black pnp, dorsal ventral indicated by D and V). (J) Schematic view of stage A 25 host after transplantation of a rotated FEZ. The expected outgrowths (arrowheads), with their predicted dorsoventral polarity are indicated. (K) The ectopic upper beak had a single egg tooth (red asterisk). (L) The presence of a single egg tooth (red asterisk), a single premaxilla (red pmx), and a single prenasal process (red pnp) indicated the ectopic structure had ventral/dorsal polarity. (M) Schematic view of a stage 25 host after isotopic transplantation of a rotated FEZ. The expected outgrowths (arrows) and their predicted dorsoventral polarity are indicated. (N) An ectopic structure was evident and, (O) histological analysis indicated the presence of two egg teeth, a dorsal premaxilla and two prenasal processes. The relative positions of these structures indicated a DV/VDV polarity to the beaks. Scale bars: 1 mm.

View larger version (97K): [in a new window]   Fig. 5. QCPN staining indicates that FNP outgrowths occur at dorsoventral compartment boundaries. (A) Schematic view of a stage 25 host FNP illustrating two types of boundaries. One type of boundary is created between quail and chick tissues (i.e. a heterospecific junction); these are indicated with blue arrows. Another type of boundary is created by the juxtaposition of dorsal and ventral ectoderm; we refer to these as dorsoventral compartment boundaries and they are indicated with red arrowheads. (B) QCPN immunostaining identified ectodermal domains comprised of quail FEZ and chick FNP ectoderm. The colored lines indicate both the polarity and source of the ectoderm. Dorsal ectoderm is green and ventral is yellow, and quail tissues are indicated with darker lines. C, the transplanted FEZ; D, a new FEZ created by the juxtaposition of chick dorsal and quail ventral ectoderm (shown at higher magnification in C,D. Note that the junction of chick dorsal and quail dorsal ectoderm (blue arrowhead) did not result in an outgrowth. (C) Quail-positive ectoderm spanned the dorsoventral boundary of the ectopic outgrowth (quail nuclei are black; the broken red line demarcates the epithelial-mesenchymal interface). (D) The juxtaposition of quail ventral ectoderm and chick dorsal domains resulted in the creation of a chimeric FEZ. mes, mesenchyme; et, egg tooth.

View larger version (57K): [in a new window]   Fig. 6. The FEZ graft induces an ectopic outgrowth in the mandible. (A) Lateral, (B) ventral and (C) frontal views of chimeric embryos with ectopic outgrowths (red arrow) that formed 7 days after transplantation. No ectodermal appendages, such as the egg tooth (et), were present on the outgrowths. (D,E) Meckel's cartilage (mk) was bifurcated (arrow) in the ectopic outgrowth and was surrounded by ectopic bone that was continuous with the dentary bone (db). Broken line demarcates the bifurcation of Meckel's cartilage. The end of the ectopic element (arrowhead) resembled the mandibular symphysis. (F) Sagittal section through the head of chimeric embryo 24 hours after transplantation demonstrates that in the mandible, Shh (yellow) and Fgf8 (green) are normally expressed in adjacent domains in the oral ectoderm (arrows). By 24 hours after transplantation, Shh (yellow) and Fgf8 (green) transcripts were still detected in the FEZ (asterisks), which was grafted to the aboral side of the mandible. Fnp, FNP; mx, maxillary process; fg, foregut. Scale bars: 2 mm in A-C; 1 mm in D,E; 0.5 mm in F.

View larger version (95K): [in a new window]   Fig. 7. The FEZ graft does not alter hyoid arch pattern. (A) Side and (C) ventral view of normal, unoperated, and (B,D) chimeric embryo 5 days after transplantation of the FEZ to the hyoid arch demonstrates that no ectopic growth was observed after this transplantation. (C) The proximal region of the hyoid arch comprises the epibranchial (ec) and ceratobranchial cartilages (cc), while the more distal region of the hyoid arch comprises the basihyal (bh), the basibranchial (bb) and the entoglsossum (en). (D) After transplantation, these elements appear normal. mk, Meckel's cartilage; db, dentary bone; qu, quadrate; qj, qudratojugal; jb, jugal bar. Scale bar: 2 mm.

View larger version (88K): [in a new window]   Fig. 8. Fgf2 can restore FEZ activity to stage 25 FNP ectoderm. (A) Schematic illustration of a stage 25 ectoderm graft combined with an Fgf2 bead (green circle). (B) Seven days later, the upper beak exhibited an abnormal shape. (C) Trichrome stained sections demonstrated the presence of a bifurcated prenasal process and premaxilla in the ectopic outgrowth, which indicated a DV polarity. (D) Schematic view illustrating the placement of a Shh-N bead (yellow circle) underneath the dorsal ectoderm of a stage 25 host. (E,F) Exogenous Shh-N resulted in an overall increase in size of the FNP skeletal elements, but no patterning alterations were observed. (G) Schematic illustrating placement of an Fgf2 bead beneath the dorsal ectoderm of a stage 25 FNP. (H,I) Exogenous Fgf2 had no discernible effect on FNP development.