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First published online 4 December 2008
doi: 10.1242/dev.023978
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Department of Oral Health Sciences, Life Sciences Institute, The University of British Columbia, Vancouver BC, V6T 1Z3, Canada.
Author for correspondence (e-mail:
richman{at}interchange.ubc.ca)
Accepted 31 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Chicken embryo, Placode, Nasal capsule, FGF8, Craniofacial, TuJ1, PAX7, Lateral nasal prominence
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), within a pre-placodal domain
(Bailey et al., 2006;
Bhattacharyya et al., 2004;
Schlosser, 2005;
Schlosser and Ahrens, 2004)
where the olfactory and lens precursors are intermingled
(Bhattacharyya et al., 2004;
Streit, 2002). FGFs are
required for the earliest steps of olfactory placode specification
(Bailey et al., 2006;
Kawauchi et al., 2005) and
then later for formation of the nasal passages
(Kawauchi et al., 2005).
In the chicken, the olfactory placode is first visible at the end of
cranial neural crest cell migration, coincident with the appearance of the
pharyngeal arches [stage 15 (Hamburger and
Hamilton, 1951)]. Twenty-four hours later (stage 20), the nasal
pit has invaginated and, simultaneously, gonadotrophin-releasing neurons
(GnRH) begin migrating to the telencephalon
(Drapkin and Silverman, 1999).
Later, the nasal pit deepens to form a nasal slit and ultimately the nasal
passages that are lined by respiratory and olfactory epithelia
(Croucher and Tickle, 1989).
The olfactory placode gives rise to the olfactory nerve, which is composed of
several types of neurons, olfactory ensheathing cells and glia.
The timing of their formation and their position suggest that the olfactory
placodes might exert patterning influences on the adjacent facial mesenchyme,
a possibility that had not previously been addressed. The mesenchyme of the
frontonasal mass is medial to the placodes and gives rise to the midline
skeletal elements (prenasal cartilage and premaxillary bone), whereas lateral
nasal mesenchyme is lateral to the placodes and gives rise to the nasal
capsule (nasal conchae and nasal bone). Both mesenchymes are derived from the
same regions of neural crest and share similar migration pathways over the eye
primordia (Creuzet et al.,
2005). Cartilages from both frontonasal and lateral nasal
mesenchymes are initially patterned as a group by signals from the foregut
endoderm (Benouaiche et al.,
2008). However, later, when the placodes appear, there may be a
separation of the signals that pattern the nasal capsule from those that
pattern the prenasal cartilage and interorbital septum. We have shown, for
example, that Noggin and retinoic acid can induce the midline elements
(interorbital septum and prenasal cartilage) but not the nasal capsule in
pharyngula stage embryos (Lee et al.,
2001).
Several secreted signaling molecules are expressed around the olfactory
placode and pit in chicken and mouse embryos. The lateral edge expresses
RALDH2, a retinoic acid synthesizing enzyme
(Blentic et al., 2003;
LaMantia et al., 2000), and
the expression of FGF8 defines the medial edge of the nasal pit,
whereas expression of BMP4 and SHH is caudal to the nasal
pit (LaMantia et al., 2000;
Song et al., 2004). Previous
work in murine (LaMantia et al.,
2000) and chicken (Firnberg
and Neubuser, 2002) organ cultures has shown that frontonasal
epithelia, including the nasal pits, maintain gene expression in frontonasal
and lateral nasal mesenchyme.
Epithelial-mesenchymal recombination experiments have shown that by stage
20, frontonasal, maxillary and mandibular mesenchymes contain all the
necessary information for generating prominence-specific skeletal patterns
(Richman and Tickle, 1989).
However, prior to this stage, the nasal placode may direct some aspects of
skeletogenesis. Up until now, much of the work on nasal pit development has
focused on the interactions of the olfactory bulbs and the olfactory pit, as
well as on neuronal differentiation of the olfactory epithelium
(Kawauchi et al., 2005;
Lutz et al., 1994;
Wang et al., 2001).
To address the patterning abilities of the nasal epithelium on facial mesenchyme, we carried out a series of in ovo extirpation experiments and grafts of supernumerary nasal pits. Our data show a specific requirement for the olfactory placode in the patterning and differentiation of the lateral nasal skeleton. Grafts demonstrated skeletogenic capacity and the ability to form nasal passages and neuronal outgrowths when the nasal pit was placed in a competent, HOX-negative environment.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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 implants
Nile 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 preparation
Stage 20 or 26 donor (quail or chicken) frontonasal mass and lateral nasal
prominences were collected in Hank's Balanced Salt Solution with
Ca2+ and Mg2+ (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 added to the dissection medium to help visualize the
donor epithelium in the host.
Host embryo preparation
Stage 15 or 20 host embryos were stained with Neutral Red. A tungsten
needle was used to remove epithelium and expose the underlying mesenchyme. The
donor epithelium was placed with the basement membrane side in contact with
the graft-site mesenchyme and pinned in place with platinum staples
(Fig. 1B,C). Staples were
removed the following day to permit sectioning.
Bone and cartilage staining
To study bone and cartilage morphology, stage 37-39 embryos were fixed in
100% ethanol, permeabilized with acetone and then stained with Alcian Blue and
Alizarin Red (Plant et al.,
2000). For bone and cartilage staining on sections, Picrosirius
Red and Alcian Blue were used (Ashique et
al., 2002).
Whole-mount in situ hybridization
Whole-mount in situ hybridization (WISH) was performed in the Intavis
InsituPro Robot, with DIG-labeled antisense probes using previously published
protocols (Song et al., 2004).
A subset of embryos was embedded in 20% agarose and Vibratome sectioned at 50
µm. The following individuals provided avian probes for in situ analyses:
K. Patel, PAX7; A. Kispert, TBX22; C. Tabin, TBX2;
M. Kessel, DLX5; G. Eichele, HOXB1; A. Streit,
HOXB9; and O. Pourquie, exonic FGF8. We cloned a 920 bp
fragment of SP8 (bp 1776 to 2696) into pCRII-TOPO (Invitrogen).
Immunohistochemistry, BrdU labeling and TUNEL reaction
For immunohistochemistry with PAX7 and TuJ1, chicken embryos were fixed in
4% paraformaldehyde and chicken-quail chimeras in Carnoy's fixative. Undiluted
Q¢PN antibody supernatant (Developmental Studies Hybridoma Bank,
University of Iowa) was used to detect quail cells
(Creuzet et al., 2006). TuJ1
antibody (1:500; MMS-435P, Covance) was used on both wax and cryostat
sections. Antigen retrieval was performed in the cryostat sections with 0.1%
Triton X-100, whereas the wax sections were incubated in 0.05% trypsin at room
temperature. For PAX7 antibody, cryostat sections were incubated in ascites
diluted 1:1000 (Developmental Studies Hybridoma bank) and stained as described
(Kawakami et al., 1997).
Horseradish peroxidase-conjugated secondary antibody was used (1:100; Jackson
Labs) and sections were counterstained in 0.1% Methyl Green.
BrdU labeling and staining, as well as TUNEL, were carried out as described
on serial sections of paraffin-embedded embryos, 24 hours after the
manipulation (Szabo-Rogers et al.,
2008).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Richman and Tickle, 1989),
maxillary and mandibular prominences
(Richman and Tickle, 1989;
Richman et al., 1997;
MacDonald et al., 2004).
However, no one had studied the interactions between the nasal placode
epithelium and the adjacent facial prominences, which comprise the lateral
nasal prominence and the frontonasal mass. We first carried out
loss-of-function experiments to determine which parts of the beak skeleton
were dependent on the nasal placode.
Nasal placode extirpation decreases the expression of nasal pit and lateral nasal genes
We initially removed placodal epithelium and examined genes known to be
expressed in the epithelium itself and in the mesenchyme. Epithelial markers
were used to see whether the placode could be removed with mechanical
techniques and whether it would regenerate. FGF8 is expressed on the
medial side of the placode and in the frontonasal mass ectoderm from stage
15-22 (Song et al., 2004). The
transcription factor, DLX5, is expressed in the olfactory placode and
pit (Bhattacharyya et al.,
2004; Brown et al.,
2005; McLarren et al.,
2003). SP8 is an FGF8-responsive, buttonhead-like
transcription factor (Kawakami et al.,
2004; Sahara et al.,
2007). SP8 is also localized to the early placode
epithelium (see Fig. S1A',A'' in the supplementary material).
Chicken embryos fixed immediately after extirpation had lost the thin band of FGF8 expression that normally marks the medial side of the nasal pit (6/6 had no expression, data not shown). Expression did not return at 6 hours (4/4) (Fig. 2A-A''). By stage 20, 24 hours later, there is typically strong FGF8 expression in the frontonasal mass ectoderm between the nasal pits. In extirpated embryos, FGF8 extended laterally only as far as the presumptive location of the nasal pit (Fig. 2B', asterisk), illustrating that in the frontonasal mass, FGF8 was able to upregulate in the normal spatiotemporal manner without signals from the nasal pit.
DLX5 expression was also lost immediately following the surgery (0 hour, 6/6, data not shown; 6 hours, 5/5) (Fig. 2C-C''). A similar loss of expression was seen with SP8 (6 hours, 8/10) (see Fig. S1A-A''' in the supplementary material) However, by 24 hours, most of the specimens had some expression in what appeared to be a remnant of the medial edge of the nasal pit (DLX5, 12/19, Fig. 2D-D''; SP8, 4/8). The remaining specimens had almost no expression (DLX5, 7/19, Fig. 2E-E''; SP8, 4/8, see Fig. S1B-B''' in the supplementary material). In order to determine whether residual nasal pit was present in the 24-hour specimens, we sectioned the hybridized embryos. In the cohort of those embryos with some residual expression, DLX5 transcripts were found in medial lip epithelium of the remaining nasal pit (Fig. 2D''). The most likely explanation is that there was some residual nasal pit, rather than regeneration. The mesenchyme also appeared thinner on the treated side and the olfactory nerve was missing.
We assayed the mesenchymal response to extirpation of the nasal placode by
looking at the effects on TBX2 and TBX22, two T-box
transcription factors expressed in the frontonasal mass and lateral nasal
mesenchyme (Fig. 3A-B'')
(Firnberg and Neubuser, 2002;
Gibson-Brown et al., 1998;
Haenig et al., 2002). We also
examined PAX7, a specific marker of lateral nasal mesenchyme
(Firnberg and Neubuser, 2002;
Kawakami et al., 1997;
Otto et al., 2006).
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Extirpation leads to loss of nasal capsule and these defects are rescued by FGF8
The effects of epithelial extirpation on PAX7 and TBX2/22
expression were contradictory. Therefore, it was not clear whether the
morphology of the skull and jaw bones would be affected. In order to address
this, we examined the effects on the beak skeleton.
Extirpation caused significant loss of lateral nasal prominence-derived
structures (Fig. 1D) in the
majority of embryos, including complete deletion of the nasal bone (25/39) and
nasal conchae (28/39) (Fig.
4B,B'). Unlike the lateral nasal derivates, the skeleton
derived from the frontonasal mass was conspicuously unaffected, except for a
secondary overgrowth of the nasal process of the premaxilla: the larger bony
process was encroaching into the space left by the missing nasal conchae
(23/39) (Fig. 4B,B').
These data allow us to conclude that: (1) healing of the surface ectoderm or
any residual olfactory placode is insufficient to allow normal skeletal
patterning; (2) general outgrowth of the upper beak does not depend on the
nasal placode; and (3) the stage 15 olfactory placode is not required for lip
fusion. The effects of nasal placode ablations on the nasal cartilage agree
with those observed by others on slightly older, stage 16 embryos
(Wang et al., 2001). Although
the skeleton was not the focus of their study, an examination of their figures
reveals a unilateral absence of nasal cartilage on the ablated side.
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We then asked which signals the nasal pit was providing to promote
differentiation of the nasal capsule. Work in mouse and chicken has shown that
retinoids are important in lateral nasal development
(Bhasin et al., 2003;
Dupe et al., 2003;
LaMantia et al., 2000;
Song et al., 2004). FGFs are
also needed for olfactory epithelium differentiation and nasal capsule
morphogenesis (Kawauchi et al.,
2005). Furthermore, we have recently shown that FGFs are released
from the medial edge of the nasal slit to pattern the frontonasal mass
mesenchyme (Szabo-Rogers et al.,
2008). It is likely that the lateral side of the nasal slit is
also a source of FGFs because several FGF genes are expressed in this vicinity
(Karabagli et al., 2002). We
therefore attempted to replace the extirpated nasal placode with a bead soaked
in either FGF8 or retinoic acid (RA).
RA exacerbated the phenotype and caused reduction of the premaxilla and maxillary bone in addition to the lateral nasal derivatives (10/11, data not shown). RA beads placed in non-extirpated embryos had minimal effects on development (3/12 had mild nasal bone defects, data not shown). Therefore, in the absence of the placode, RA has complex actions that affect lateral nasal, frontonasal and maxillary morphogenesis. Rescue of lateral nasal development might not be possible owing to the effects on these other regions.
We found that FGF8 almost completely restored the nasal bone and nasal conchae on the treated side in the majority of specimens. In addition, the nasal process of the premaxillary bone was normal in width (18/21) (Fig. 4C,C'). To understand the mechanism underlying the FGF8 rescue, we analyzed cell death and proliferation. FGF8 increased cell proliferation in the mesenchyme compared with the contralateral side or non-rescued embryos (3/3) (Fig. 4D). As expected, we observed increased cell death in the extirpated specimens (3/3) and FGF8 did not prevent apoptosis at this time point (3/3) (Fig. 4D'). FGF8 therefore expands the progenitor population of mesenchyme cells that will give rise to the lateral nasal skeleton.
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;
Richman and Tickle, 1989) and
any induced ectopic cartilages will be obvious. The temporal constraints of
this experiment were determined by our previous work, which had shown that the
fate of the maxillary mesenchyme can be changed by exogenous factors at stage
15 (Lee et al., 2001). By
stage 20, the maxillary mesenchyme is determined to give rise to maxillary
bones (Richman et al., 1989) but still requires epithelial signals for
outgrowth. Facial mesenchyme at stage 26 no longer requires epithelium for
outgrowth or patterning (MacDonald et al.,
2004). We tested both stage 15 and stage 20 host embryos
(Fig. 1B,D). The interlimb
lateral plate mesoderm of the stage 15 embryos was used as the HOX-positive
region because this region forms ectopic limbs in response to FGFs
(Cohn et al., 1995;
Vogel et al., 1996), and we
had shown that the lateral nasal skeleton relies on FGFs for patterning.
Furthermore, grafts of the nasal region to the flank can induce ectopic
outgrowths in chicken embryos (Street,
1937). Stage 15 host embryos did not respond to grafts of stage 15 placodes, even though the placode is at the right stage to be inducing the nasal skeleton, as shown by our extirpation experiments (20/20, data not shown). By contrast, stage 20 nasal pits were more successful at altering the external appearance of the head and skeletal pattern, and even induced ectopic skeletal elements in the host embryo. The donor nasal pit integrated into the surrounding mesenchyme (Fig. 5A) and formed a lumen within 24 hours (5/5) (Fig. 5B). The graft epithelia were quail in origin, and the mesenchyme surrounding the grafts was derived from chicken (9/9 in whole-mount and 5/5 in sections) (Fig. 5A,B,D''). Six days after grafting there was an obvious outgrowth near the auditory meatus that, when sectioned, containing a lumen lined by thickened epithelium; however, no cartilage formed around the lumen (Fig. 5C; Fig. 7A,B,D).
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By the time host embryos had reached stage 38, ectopic outgrowths proximal
to the eye were often found (stage 20 nasal pit, 6/17,
Fig. 6A,B; stage 26 nasal pit,
12/22, Fig. 6D). There were
significant alterations in skeletal morphology. The stage 20 nasal pit grafts
induced skeletal changes mainly localized to the jaw joint region. The results
can be divided into two categories: one in which grafts consisted of a
collection of skeletal elements in close association with each other (compound
grafts, 4/14) (Fig.
6A',A'',C; Table
1A); and a second that includes ectopic, supernumerary elements
(10/14) (Fig.
6B',B''; Table
1A). The compound elements consisted of 4-6 intramembranous
bones that articulated with each other
(Fig. 6A'',C). In the
second, more common category, 1-2 isolated bones were formed at the proximal
end of the quadratojugal bone (7/10). The nasal pits also induced small pieces
of cartilage (6/10) (Fig. 6C)
and these were sometimes closely associated with membranous bones
(Fig. 6C, black arrowhead). In
general, few of the skeletal elements formed had recognizable features, with
one exception. The normal quadratojugal articulates with the quadrate bone,
and at the point of articulation a characteristic secondary cartilage forms.
We observed a rod of intramembranous bone with a similar secondary cartilage
in all of the compound grafts (4/4) (Fig.
6C). The remaining intramembranous bones could have been either
maxillary (jugal, maxillary or palatine) or lateral nasal (nasal bone).
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). The
induced bones and cartilages consisted of ectopic process (3/10) or isolated
combinations of ectopic rods of bone and/or cartilage (7/10, data not shown,
but similar to Fig. S4D,E in the supplementary material). Control grafts of
frontonasal mass epithelia, minus the caudal edge, developed normally (9/9)
(Fig. 6E-E'';
Table 1A). In older, stage 20 host embryos, ectopic bones and cartilages were not as frequently observed as in younger hosts. The most common phenotype induced by the stage 20 or stage 26 epithelium was the induction of ectopic processes on the quadratojugal or squamosal bone (29/36) (Table 1B; see Fig. S4A,A',C,C',E in the supplementary material). Out of the remaining cases, most had formed small pieces of ectopic bone (7/36) (Table 1B; see Fig. S4B,D,D' in the supplementary material). In general, cartilage was rarely observed in these grafts from stage 20 donors (3/12) (Table 1B), whereas the stage 26 donors induced large, lobular cartilages similar those seen in stage 15 hosts (10/24, data not shown). Controls in which only graft sites were prepared, or in which central frontonasal mass epithelium was placed, had normal morphology (Table 1B).
Therefore, we have shown that the stage 15 mesenchyme is able to respond to ectopic nasal pits, and that the inducing capacity increases with increased age of the epithelium. In general, there were no replacements or deletions of the normally occurring skeletal elements showing that there are no repressive effects on skeletal development. Instead, the nasal pits induced supernumerary structures.
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Similar to the effects of the maxillary bead implants, very few ectopic
elements were induced in the flank grafts (3/100). Therefore, the patterning
signals from the nasal pit are sufficient to pattern HOX-negative facial
mesenchyme, whereas the HOX-positive trunk mesenchyme is not competent to
respond. The nasal pit epithelium is clearly not sufficient to induce ectopic
structures in the same way that an FGF-soaked bead can
(Cohn et al., 1995;
Vogel et al., 1996). We tested
whether the nasal pit was able to locally change HOX gene expression in the
flank. We looked at the expression of several HOX genes and many had to be
excluded because they were downregulated in the lateral plate mesoderm at
stage 20. On this basis, two members of the HOXB cluster were selected,
HOXB1 and HOXB9. HOXB9 is responsive to FGFs and its
expression is altered in embryos at sites where ectopic limbs are induced
(Cohn et al., 1997), but
HOXB1 has not been examined in this context. We found no change in
either gene 16 hours after grafting (HOXB1, 4/4, see Fig. S5A,B in
the supplementary material; HOXB9, 3/3, see Fig. S5C,D in the
supplementary material), nor were SP8 and DLX5 present in
the graft (see Fig. S5A-D in the supplementary material). Thus, a lack of
change in gene expression correlated with the absence of ectopic limbs.
Nasal pits induce PAX7 and differentiate autonomously in facial mesenchyme
Since grafted nasal pits lacked SP8 and DLX5 expression
there was a possibility that the epithelium had lost the ability to
differentiate into olfactory epithelium. Furthermore, the grafting data were
suggestive of ectopic nasal structures but were not definitive. We therefore
investigated whether nasal passages lined with olfactory epithelium had formed
and whether these were adjacent to PAX7-expressing mesenchyme.
Initially after grafts were placed, we were unable to detect PAX7 in the surrounding mesenchyme (22/22 for 24-72 hours) (see Fig. S3C-D' in the supplementary material; data not shown). To determine whether there was a delay in the response of the host, we collected embryos 5-6 days after grafting. Indeed, after 5 days of growth we found that in all specimens (6/6) there was PAX7 antibody staining adjacent to the grafted epithelium in the mesenchyme (Fig. 7C-D'). The expression of PAX7 suggested that there was a partial conversion to a lateral nasal identity, while the remainder of the mesenchyme retained maxillary identity. This is consistent with the complex skeletal elements that were formed in fully differentiated grafts. We also examined sections to see whether the grafts were able to form neurons in an ectopic location. Differentiated neurons, as recognized by the TuJ1 (βIII tubulin) antibody, were present within and adjacent to the grafted epithelium (8/8) (Fig. 5D,D'; Fig. 7B',B'',D'',D'''). In addition, quail antibody staining showed that the neurons were graft in origin (Fig. 5D''; data not shown). In three cases, a distinct ectopic neural outgrowth close to the trigeminal ganglion was observed (Fig. 5D; Fig. 7B''), suggesting that the presence of the ganglion might have facilitated the ectopic outgrowth. There were regions of the grafted nasal pit epithelium that did not stain with the TuJ1 antibody and which we presume to be respiratory epithelium (Fig. 5D'; Fig. 7B',B'',D'',D'''). Thus, the nasal pit invaginated and differentiated into neuron-containing epithelium in the ectopic location.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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FGF8 is one of several signals required to make a nose
We showed that replacing the olfactory placodal epithelium with an
FGF8-soaked bead was sufficient to completely rescue the skeleton. We favor
the explanation that FGF8 stimulated the proliferation of mesenchyme and
restored some of the patterning cues provided by the nasal placode. A strong
proliferative response in facial mesenchyme induced by FGF was also seen in
another study from our laboratory
(Szabo-Rogers et al., 2008).
Similarly, FGF8 beads can promote proliferation of neural crest cells and
these cells can repopulate an extensive defect
(Creuzet et al., 2004). When
the placode ectoderm is removed, increased apoptosis is seen, depleting the
progenitor cells. Interestingly, FGF8 was not sufficient to rescue apoptosis.
Therefore, we might have removed additional, as yet unidentified cell survival
signals.
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),
to pattern the nasal skeleton.
The nasal placode can pattern skeletogenic mesenchyme but only in one direction
The in ovo extirpation experiments in our study have shown that signals
released from the placodal epithelium are secreted and are directional in
nature. The normal expression of TBX2/22 and FGF8 and normal
midline skeletal elements in extirpated embryos suggest that the nasal placode
does not affect frontonasal patterning. These data differ from those of our
previous study in which the medial side of the nasal slit epithelium was shown
to provide diffusible FGF signals to the frontonasal mass mesenchyme
(Szabo-Rogers et al., 2008).
The difference in our two studies is that the present study focused on stage
15 embryos, whereas the previous study investigated patterning in stage 26
embryos. This shows that new functions are acquired for nasal slit epithelium
as development proceeds. We also saw evidence of this in the induction of
skeletal elements by differently staged nasal epithelia in the present study.
The older nasal slit ectoderm was able to induce large cartilage elements,
whereas younger epithelia could not.
Other data also support the specific competence of lateral nasal mesenchyme
to respond to secreted signals, as reported in other studies
(Firnberg and Neubuser, 2002;
LaMantia et al., 2000). In
organ cultures of stage 18 chicken frontonasal mass and lateral nasal
prominences, replacement of epithelium by an FGF8-soaked bead maintained
PAX7 in the lateral nasal mesenchyme but did not ectopically induce
expression in frontonasal mesenchyme
(Firnberg and Neubuser, 2002).
Thus, we conclude that lateral nasal mesenchyme differs from frontonasal mass
in its sensitivity to patterning molecules.
The identity of host mesenchyme is influenced by the ectopic nasal pit
A directional patterning effect on mesenchyme was also demonstrated in the
grafting experiments. Although the formation of cartilage is suggestive of
lateral nasal-derived skeleton, the clearest evidence was provided by the
induction of PAX7 expression in the maxillary mesenchyme, a protein that is
exclusively found in lateral nasal mesenchyme. Although a specific frontonasal
mass marker awaits identification, we can nonetheless rule out the possibility
that skeletal elements were frontonasal mass in character by their morphology.
The frontonasal mass derivatives (prenasal cartilage, premaxilla or egg tooth)
can form in the stage 15 maxillary region when provided with the proper
signals (Lee et al., 2001);
however, here we have shown that nasal pit is incapable of inducing these
structures even when placed into competent mesenchyme.
The nasal pit is almost able to induce an ectopic nose, with nasal passages
and surrounding skeletal support, and this ability improves as development
progresses. However, some organizational information is clearly lacking. It is
possible that additional signals, perhaps from the adjacent tissues, would
give rise to a more complete nose. This idea is supported by the effect of
FGF4 beads placed in direct contact with nasal pits grafted onto frontonasal
mass mesenchyme (Firnberg and Neubuser,
2002). This experiment results in a broad induction of
FGF-responsive genes, an effect different to that of grafts of isolated nasal
pits (no induction of expression) or placement of FGF beads without epithelium
(local expression).
It is interesting to note that older studies on amphibians showed that the
nasal region is able to organize the growth of an ectopic limb in the flank
(Balinsky, 1933). By contrast,
we rarely saw induction of skeletal elements in the flank, nor was there a
change in HOX gene expression. We are unsure of the exact source of the donor
tissue in the studies by Balinsky, nor are we certain whether mesenchyme was
excluded. Perhaps tissue contamination or species differences between axolotl
and chicken contributed to the difference in results. The difference we
observed in the response of HOX-positive and HOX-negative mesenchyme is
consistent with a lack of skeletogenic capacity in neural crest cells cultured
from the trunk versus the head (Abzhanov et
al., 2003), and with the inhibitory effect on facial skeletal
formation if HOX genes are ectopically expressed
(Creuzet et al., 2002).
Autonomous differentiation of the nasal pit epithelium is supported in an ectopic location
We have shown that the nasal pit is irreversibly determined by stage 20,
coinciding with the onset of olfactory neuron production
(Drapkin and Silverman, 1999;
Wang et al., 2001). The
differentiation of the epithelium continued as it would have in situ, with the
formation of TuJ1-negative (presumptive respiratory) and TuJ1-positive
(presumptive olfactory) domains. However, the nasal pit grafts were unable to
maintain SP8 or DLX5 expression when grafted to an ectopic
location in the face. This could mean that the mesenchyme lacks the signals to
maintain olfactory epithelial gene expression, or that the maxillary
mesenchyme suppresses the site-specific gene expression in the nasal
epithelium. Nonetheless, even in the absence of stereotypic gene expression,
the nasal pit can form nasal passages and neurons in other areas of the
embryonic face.
The generation of neurons from our grafts is consistent with previous
studies on frogs in which ectopic nasal placodes were grafted in locations
close to the brain (Byrd and Burd,
1993; Koo and Graziadei,
1995; Stout and Graziadei,
1980). However, in these studies, not only did olfactory nerves
form, but in the case of the midline grafts the ectopic nerve made the correct
connection to the olfactory bulb (Byrd and
Burd, 1993; Stout and
Graziadei, 1980). These results are intriguing and in future
studies we will use our established grafting paradigm to determine whether the
olfactory nerve is capable of pathfinding and fully differentiating in facial
mesenchyme that is distant from the central nervous system.
Note added in proof
A recent study by Bhattacharyya and Bronner-Fraser
(Bhattacharyya and Bronner-Fraser,
2008) confirms our results that the olfactory placode is able to
form thickened epithelium and neurons in ectopic locations. Our data extend
their work by testing the competence of older mesenchyme. We show that
stereotypical gene expression is not a prerequisite for placode-derived
neurons to ingress into host mesenchyme.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/219/DC1
|
Footnotes |
|---|
* Present address: King's College London, Department of Craniofacial
Development, Floor 27, Guy's Tower, London SE1 9RT, UK 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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