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First published online November 21, 2008
doi: 10.1242/10.1242/dev.026633
Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA
* Author for correspondence (e-mail: mbronner{at}caltech.edu)
Accepted 17 October 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Olfactory placode, Nasal placode, Chick, Competence, Specification, Commitment, Induction, Ectoderm
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
von Kupffer, 1891) and
invaginate/ingress to form components of the nose, lens and inner ear
(Bailey and Streit, 2006;
Baker and Bronner-Fraser, 2001;
Schlosser, 2006). Of the three
sensory placodes, the lens and otic are relatively easy to distinguish and
manipulate early in development. Therefore, tissue interactions and signals
guiding their induction have been studied extensively (reviewed by
Brown et al., 2003;
Chow and Lang, 2001;
Donner et al., 2006;
Grainger, 1992;
Noramly and Grainger, 2002;
Riley and Phillips, 2003;
Saha et al., 1989;
Streit, 2001;
Torres and Giraldez,
1998).
By contrast, development of the olfactory sensory system has been primarily
investigated at later stages. An attractive model for studying neurogenesis,
its myriad of derivatives include the regenerative odorant sensing olfactory
neurons in the olfactory epithelium (Calof
et al., 1998; Graziadei and
Graziadei, 1979; Graziadei and
Metcalf, 1971; Graziadei and
Monti Graziadei, 1983), the ingressing gonadotropin-releasing
hormone neurons (reviewed by Parhar,
2002; Wray, 2002)
[for a contrary view see Whitlock
(Whitlock, 2005)], the
pheromone-detecting vomeronasal organ
(Dulac, 1997) and other
neuromodulatory and neuroendocrine cells
(Northcutt and Muske, 1994;
Tarozzo et al., 1995;
Yamamoto et al., 1996). It is
the only placode to give rise to glial cells that ingress and migrate along
the olfactory nerve towards the brain
(Chuah and West, 2002;
Ramon-Cueto and Avila, 1998).
Cues that guide differentiation along these different pathways and the
molecular mechanisms underlying the patterned wiring of olfactory sensory
neurons have been examined extensively (reviewed by
Baker and Margolis, 2002;
Balmer and LaMantia,
2005).
Contrasting with this wealth of data, inductive events that initiate
olfactory development remain ambiguous, partially because the placode is
morphologically invisible until relatively late (HH13+ in birds).
Transplantation studies show that precursors straddle the lateral anterior
neural folds and adjacent ectoderm in the neurula
(Couly and Le Douarin, 1985),
but their exact location at intervening stages was unclear. Recent cell
marking studies in zebrafish and chick have examined the origin of the
olfactory placode at higher resolution and at intervening stages
(Bhattacharyya et al., 2004;
Whitlock and Westerfield,
2000). Olfactory precursors are initially distributed in a broad
region spanning the anterior neural folds and overlapping with lens
precursors. Over time, they become progressively restricted to the most
anterior ectoderm, finally resolving to a discrete olfactory pit. Knowledge of
the location of precursors at different stages has facilitated manipulation of
their development.
To understand how and when the nasal epithelium is first induced, we
examined competence, specification and commitment of ectoderm toward an
olfactory fate. This information is a necessary prerequisite for interpreting
results of functional perturbations at the molecular level. First, we
correlated molecular markers with a more complete fate-map
(Bhattacharyya et al., 2004)
and found that Dlx3, Dlx5 and Pax6 can serve as molecular
guideposts for olfactory placode precursors at different stages. Although an
initially broad region of head ectoderm is competent to form olfactory
placode, this ability is restricted with time. In vitro explant assays suggest
that placode induction by adjacent tissues occurs by 
HH10, when olfactory
precursors have segregated from their neighbors. In vivo heterotopic
transplantation studies demonstrate that commitment occurs with a time lag of
12 hours. This suggests a period of developmental plasticity, perhaps
accounting for the regulative nature of early olfactory placode development.
This step-wise progression of placode formation favors flexibility in fate
until such time as the placode becomes morphologically apparent.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagen gel cultures
Quail embryos (HH8,10,14) were collected in Ringer's solution. Small pieces
of HH8 anterior ectoderm or HH10-14 presumptive olfactory placode were
dissected as above and stored in PB1 medium on ice until use. Collagen (90
µl; Collaborative Research) +10 µl of 10x DMEM (Invitrogen) in
0.375% sodium bicarbonate was solidified in four-well plates (Nunc); tissue
pieces were transferred, overlaid with 5 µl collagen mix, immersed in 0.5
ml of the defined medium, F12+N2 (Invitrogen), incubated for 24-48 hours at
37°C with 5% CO2, then fixed in 4% paraformaldehyde for 30
minutes at room temperature.
Electroporation of HH10 ventral ectoderm
pCIG-GFP plasmid DNA (Megason and
McMahon, 2002) was injected at a concentration of 2.5 µg/µl
in Ringer's solution with 50 ng/µl Fast Green between the embryo's ventrum
and extra-embryonic ectoderm at HH10-11. Platinum electrodes (4 cm long and 4
mm apart) and the electroporator were custom-built in our laboratory and by
the electronics shop, respectively. The positive electrode was placed on top
and the negative electrode under the anterior tip of the embryo and five
pulses of 8-10V for 50 ms at 100 ms intervals were applied. Embryos were
reincubated until HH18 or 27 (23/47 and 14/29 survivors, respectively),
collected, fixed in 4% paraformaldehyde overnight at 4°C and prepared for
cryosectioning.
Histology, immunocytochemistry and imaging
Embryos and collagen gels were embedded in 7.5% gelatin in 15% sucrose and
cryosectioned. Sections were degelatinized, stained with antibodies in
blocking solution, rinsed and mounted in Fluoromount-G (Southern
Biotechnology) or Permount containing 10 µg/ml DAPI. Primary antibodies
were: QCPN (DSHB; 1:1), QCPN conjugated to Alexa-568 (at the University of
Oregon; 1:50), Pax6 rabbit polyclonal (Covance; 1:100), Pax7 monoclonal (DSHB;
1:20), Dlx3 goat polyclonal (1:1500), pan-Dlx rabbit polyclonal (Dr Jhumku
Kohtz; 1:70), Hu monoclonal (Molecular Probes; 1:250), chick GnRH-I rabbit
polyclonal (Dr James Millam; 1:100) and mouse GnRH-I rabbit polyclonal (Abcam;
1:100). Antibodies against the mouse and chick GnRH-I peptides, which differ
at amino acid position 8, were used concurrently to maximize staining
efficiency. Secondary antibodies used include Molecular Probes Alexa 488 and
Alexa 568 (1:1000-1:2000), and Jackson ImmunoResearch AMCA and Cy5
(1:200-1:400).
Tri-color images were acquired on a Zeiss Axioskop2 using an Axiocam Mrm camera and Axiovision software or a Zeiss LSM 510 Meta confocal microscope.
Antibody generation and immunoblot analysis
GST-C-terminal chick Dlx3 fusion protein (amino acids 185-278) was produced
in BL-21 cells and purified using Glutathione Sepharose beads (Pharmacia)
(Sambrook and Russell, 2001).
Goats were injected with 5 mg Dlx3 protein (Alpha Diagnostic). Immunoblots and
immunostaining confirmed specificity of each bleed.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described
(Streit and Stern, 2001) at a
hybridization temperature of 70°C using cDNAs for Dlx3 (Dr Andy Groves),
Pax6 and Dlx5 (Dr Michael Kessel).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Their overlap is maintained until just before HH10, when Dlx5 is upregulated
and Pax6 is simultaneously downregulated in presumptive olfactory placode
cells (Fig. 1B,C,E,F)
(Bailey et al., 2006). At HH8,
both Pax6 and Dlx5 are expressed in a `salt and pepper' pattern in ectoderm
abutting the neural folds containing nasal precursors
(Fig. 1B,B'',C,C')
(Bhattacharyya et al., 2004);
anterior-most ectoderm expresses Dlx5 uniformly
(Fig. 1B'). Pax6
expression is also observed within anterior neural folds, which contribute to
olfactory epithelium and telencephalon
(Fig. 1C). Conversely, Dlx3 is
absent from anterior neural folds and low in adjacent ectoderm at HH8
(Fig. 1A,A'), but
strongly upregulated in olfactory placode precursors at HH10
(Fig. 1D,D')
(Pera and Kessel, 1999).
Expression refines over time to the medioventral region of the head ectoderm
between the forming eyes (Fig.
1G,G'). By HH11, olfactory precursors occupy a discrete
region in the ventral ectoderm
(Bhattacharyya et al., 2004),
where Dlx5 is expressed continuously (Fig.
1E,E'), but Pax6 is extinguished
(Fig. 1F,F').
Interestingly, Pax6 is re-expressed once the olfactory placode has formed at
HH14 (Fig. 1I,I'),
contiguous to corneal Pax6 expression (Fig.
1I', arrow) and subsequently spreads anteromedially. The
thickened placode shows continued expression of Dlx3 and Dlx5 transcripts at
HH14+ (data not shown) (Fig.
1H,H'). The first co-expression of all three transcripts is
coincident with morphological appearance of the olfactory placode.
As expected, no marker gene is expressed exclusively in the mature
olfactory placode/pit by HH19-20 (Fig.
2A-C'). While Dlx3 is found in olfactory and otic placodes
(Fig. 2A,A'), Dlx5 is
also expressed in the adenohypophysis and both are expressed in subpopulations
of neural crest (Fig. 2A,B;
data not shown). Dlx3 is expressed in cranial crest populating distal elements
of branchial arches 1-2 (Fig.
2A) (Depew et al.,
2005; Pera and Kessel,
1999), whereas Dlx5 is more widely expressed in branchial arches
1-4 and the trigeminal ganglion (Fig.
1H,H'; Fig.
2B) (Pera et al.,
1999). Dlx5 is also evident in newly emergent trunk neural crest
(Fig. 3B). Like the Dlx genes,
Pax6 is expressed at multiple sites, including lens and nasal placodes
(Fig. 2C,C'). However,
combined expression of Pax6 and Dlx genes by HH19 is unique to the olfactory
placode.
Unlike Dlx3 and Pax6, Dlx5 is expressed in a few cells in the mesenchyme as
the olfactory placode thickens (Fig.
1H', arrow), even continuing to HH20
(Fig. 2B', arrowhead).
This raises the intriguing possibility that it marks a subset of neuronal
cells ingressing from the olfactory placode. To test this, HH10 ventral
ectoderm was electroporated with a GFP construct (survivors n=37) and
examined for expression of Dlx and GnRH neuropeptide. At HH18, GFP+ cells are
Dlx+ within the olfactory epithelium (Fig.
2D,d). Moreover, ingressing GFP+/Hu+ neurons are also Dlx+
(Fig. 2d',d''). At
HH27, by which time GnRH neurons are detectable
(Fig. 2G)
(Mulrenin et al., 1999), some
GnRH+/GFP+ cells were located within a ganglion-like structure between the
epidermis and ventral forebrain (Fig.
2e,f), unequivocally demonstrating their placodal rather than
neural crest origin. Adjacent sections also had GFP+/Dlx+ cells
(Fig. 2e',f'),
leading to the conjecture that some GnRH cells may co-express Dlx; direct
double-labeling was not possible as both antibodies were raised in the same
species.
Dlx3-specific polyclonal antibody reveals Dlx3 protein distribution
To detail temporal expression of Dlx3 protein in the developing olfactory
placode, we generated a polyclonal antibody against the C terminus of chick
Dlx3, a region of minimal similarity between the six Dlx family members. The
polyclonal antibody recapitulates Dlx3 mRNA expression, though the protein has
more widespread expression in ectoderm than the mRNA (compare
Fig. 2A' with
Fig. 3C). To ensure against
crossreactivity with Dlx5, which has highest sequence homology and maximum
expression overlap, we compared localization of Dlx5 and Dlx3 transcripts and
protein. At HH11, the otic placode at the rhombomere (r) 5 level begins to
express Dlx5 weakly (Fig. 3A),
while strongly expressing Dlx3 (not shown). HNK1+ neural crest cells atop the
neural tube, however, express Dlx5 robustly (compare outlined domains in
Fig. 3A,A'). The Dlx3
antibody clearly recognizes otic placode cells (also HNK1+) but not dorsal
neural tube, conclusively demonstrating lack of crossreactivity with Dlx5
(compare Fig. 3A and
Fig. 3A'). Similarly,
emigrating trunk neural crest expresses Dlx5 but not Dlx3 (compare
Fig. 3B with
Fig. 3B'). To ensure
against crossreactivity with other Dlx family members, we co-labeled sections
through heads of 3-day-old chick embryos with Dlx3 and pan-Dlx antibodies
(Panganiban et al., 1995).
Dlx3 is the only family member not expressed in the CNS for the first three
days (data not shown) (Beanan and Sargent,
2000; Liu et al.,
1997; Panganiban and
Rubenstein, 2002; Pera and
Kessel, 1999; Price,
1993; Robinson and Mahon,
1994) (see also Dhawan et al.,
1997; Zhu and Bendall,
2006). However, it is expressed robustly in the olfactory
epithelium at HH18-19 (Fig.
3C). Other Dlx family members expressed in the olfactory
epithelium include Dlx5/6 (Brown et al.,
2005; Pera and Kessel,
1999). The pan-Dlx antibody recognizes all chick Dlx proteins;
therefore, strong co-localization of both the Dlx3 and pan-Dlx antibodies is
noted in the nasal pit (Fig.
3C'; Fig.
3C'', yellow). Dlx1, 2, 5 and 6 are expressed in the
telencephalon at various times in many vertebrates, including chick
(Eisenstat et al., 1999;
Liu et al., 1997;
Panganiban and Rubenstein,
2002), and are detected by the pan-Dlx but not the Dlx3-specific
antibody (Fig. 3C'').
These data indicate that the chick Dlx3 antibody is specific and does not
crossreact with closely related family members.
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The first signs of differentiation occur at HH14, when earliest
ingressing placode cells express the RNA-binding protein Hu, which marks
post-mitotic neurons (Fornaro et al.,
2001; Fornaro et al.,
2003). By pit stages, Hu labels abundant neurons within the
epithelium, which express Pax6 and/or Dlx3, as well as those migrating along
the olfactory nerve towards the telencephalon
(Fig. 3F,G). Ultimately, Pax6
expression is only maintained in a subpopulation of non-neuronal cells in the
nascent olfactory epithelium (Davis and
Reed, 1996). Dlx3+ neurons are unique to the olfactory system at
least at HH19, although Dlx3 expression has been reported in the ventral
forebrain at HH27-29 (Zhu and Bendall,
2006). Accordingly, we observed Dlx3+ but Hu-cells in the midline
between the two telencephalic vesicles at HH19 (not shown).
Interactions between placodal ectoderm and frontonasal mesenchyme, which is
largely neural crest derived
(Osumi-Yamashita et al., 1994;
Serbedzija et al., 1992), play
a pivotal role in orchestrating development and differentiation of the nasal
epithelium and capsule (Bailey et al.,
2006; Firnberg and
Neubüser, 2002; LaMantia
et al., 2000). To assess the contribution of neural crest cells to
olfactory placode specification and commitment, we used Pax7 as a marker for
neural crest and the lateral domain of frontonasal mesenchyme
(Fig. 3H)
(Firnberg and Neubüser,
2002).
Epiblast is competent to form olfactory placode
Fate maps (Couly et al.,
1985; Bhattacharyya et al.,
2004) suggest that the chick nasal placode arises from around the
anterior neural folds at HH8. Accordingly, isotopic and isochronic quail/chick
grafts of HH8 anterior neural fold (Fig.
4a) or adjacent ectoderm (Fig.
4b) gave rise to quail cells [distinguished using quail cell
peri-nuclear (QCPN) antigen] in the olfactory placode 2-3 days later
(Fig. 4A',B').
Donor cells expressed Dlx3, Pax3 (data not shown), Pax6 (n=6)
(Fig. 4A-A'',B-B'')
and differentiated as neurons. Because olfactory precursors closely associate
with both lens and forebrain progenitors
(Bhattacharyya et al., 2004),
quail cells were additionally found in the telencephalon
(Fig. 4A', arrow;
Fig. 4A'') or ectoderm
adjacent to the olfactory placode including parts of the cornea and lens
(Fig. 4B'; data not
shown).
|
; Garcia-Martinez et al.,
1993; Rosenquist,
1966; Schoenwolf and Alvarez,
1991), none of which co-express Pax6/Dlx3 and form Dlx3+ neurons.
However, this epiblast can express Pax6 and Dlx3 in the olfactory placode when
grafted to anterior neural fold region (n=3)
(Fig. 4c,C-C''; data not
shown). Interestingly, grafted tissue underwent thickening and invagination to
form another placode-like structure when incorporated aside the endogenous
olfactory placode (Fig.
4C', boxed area). A few graft-derived cells differentiated
into Dlx3+ olfactory neurons in the thickened epithelium (compare
Fig. 4C'' and
Fig. 4D''). Additionally,
we noted many quail Hu+ cells lining the olfactory nerve
(Fig. 4D'', inset). This
demonstrates that though epiblast tissue is regionally specified
(Wilson et al., 2001), it
nonetheless is plastic and retains competence to respond to diverse inducing
signals and differentiate accordingly.
Competence to form olfactory placode is restricted to head ectoderm
We determined the competence of ectoderm at different axial levels to
contribute to olfactory epithelium and express olfactory markers as a function
of time by grafting surface ectoderm from HH8-13 donor quails, to the
prospective nasal territory of HH7-9 chick hosts. Chimaeras were allowed to
survive until the olfactory pits were distinctly visible (HH18: 48-72
hours later).
Midbrain-level ectoderm
Midbrain-level ectoderm, which is fated to contribute to trigeminal
ganglion (D'Amico-Martel and Noden,
1983; Xu et al.,
2008), robustly expresses Pax3 from HH8
(Stark et al., 1997), as well
as low levels of Pax6 at HH8 (Fig.
1C) and Dlx3 at HH10 (not shown). However, their expression is not
maintained in the ophthalmic trigeminal (opV) placode
(Fig. 2A,C). Therefore, we
looked for prolonged expression of these markers and concomitant neurogenesis
in midbrain level ectoderm grafted adjacent to anterior neural folds
(Fig. 5b,c) (n=15).
HH8 presumptive opV placode ectoderm expresses both Dlx3 and Pax6 at high
levels following incorporation into the olfactory placode and robust
expression is maintained in grafted ectoderm taken from HH10 embryos
(Fig. 5B-B''). Similarly,
opV placode ectoderm that simultaneously incorporates into cornea and lens,
expresses high levels of Pax6 in these novel locations
(Fig. 5E,F). Additionally,
quail neurons contribute to the migratory mass lining the olfactory nerve
(Fig. 5C-C''). Shortly
after HH10, Pax6 expression is extinguished in grafted cells. In fact,
ectoderm grafts taken from HH12 and onwards show no Pax6 expression in
peripheral ectoderm that is ordinarily Pax6+. Weak expression is observed in a
few cells only if the graft incorporates into the placode (data not
shown).
|
;
Groves and Bronner-Fraser,
2000; Streit,
2002) was tested for its potential to contribute to olfactory
placode (n=16). Hindbrain-level ectoderm does not express Pax6 at
early stages in situ; however, Dlx3 is expressed at high levels by otic
ectoderm from HH9 onwards and at low levels in ectoderm rostral to otic
ectoderm (Pera and Kessel,
1999) (data not shown). Hindbrain-level ectoderm (HH8-) can
replace ablated nasal progenitors and express both Dlx3
(Fig. 5A-A'') and Pax6
plus generate placode-derived neurons that line the olfactory nerve. Shortly
thereafter, Pax6 expression is no longer induced in hindbrain-level ectoderm,
even when it spans the corneal ectoderm, which usually expresses Pax6 at high
levels (Fig. 5G). However,
three cells that contribute to the lens express Pax6 at this stage (HH8+)
(Fig. 5G, arrowhead). Dlx3 is
only weakly induced at these stages (data not shown). Neither gene is
expressed in grafts of pre-otic ectoderm from HH10-11 embryos; instead,
otic-derived ectoderm expresses Pax2 (an otic placode marker) in its new
location. Thus, hindbrain-level ectoderm loses capacity to respond to
olfactory placode-inducing signals with age more rapidly than midbrain level
ectoderm.
Trunk level ectoderm
Trunk level ectoderm does not express Dlx3 except in the flank that forms
the apical ectodermal ridge of the limbs. It never expresses Pax6 nor gives
rise to placodes. To test for placodal competence, trunk level ectoderm
(n=5) at the level of the 5th-9th somites was heterotopically
transplanted adjacent to the anterior neural folds of HH8 to HH8+ embryos.
Although the grafted ectoderm could not express Pax6 even at the earliest ages
tested (HH8-) (Fig.
5D-D''), it could express Dlx3 up until HH9-. Thus,
competence to express olfactory markers was particularly short-lived in trunk
ectoderm.
In conclusion, all levels of head ectoderm (which have some degree of placodal bias) are competent to form Dlx3+/Pax6+ cells and Hu+ neurons in olfactory epithelium and along the olfactory nerve. However, the level of competence in embryonic ectoderm is graded such that ectoderm closest to olfactory progenitors remains competent for the longest duration.
Nasal placode precursors are specified toward olfactory fate by HH10, prior to acquiring placodal morphology
Classically, specification is defined as the potential of tissue explanted
and grown without additional activating/inhibitory differentiation signals,
indicating their degree of progression along a particular developmental
pathway. To determine the time by which olfactory induction had occurred,
presumptive olfactory ectoderm was cultured without serum for 24-72 hours in
three-dimensional collagen gels. Expression of Pax6, Dlx3 and neuron formation
was assessed in isolates from HH8-14 embryos
(Fig. 6).
|
),
precluding isolation of a homogenous population of nasal progenitors. Bearing
this in mind, the explanted presumptive olfactory-lens region is both Pax6+
and Dlx3+ at 24 hours or 72 hours of culture (n=18/18)
(Fig. 6A-A'').
Interestingly, this ectoderm shows spatially segregated zones of gene
expression, with the Dlx3+ cell cluster at one end of the explant
(Fig. 6A) and Pax6+ cells at
the other, forming a lentoid-like structure
(Fig. 6A', arrowhead).
Pax6+/Dlx3+ cells are found at the intersection of the two domains
(Fig. 6A'', arrows). This
cell sorting is reminiscent of Dlx3 and Pax6 expression in the presumptive
placodal region at HH13 (Fig.
3D''). Consistent with the possibility that placodal
development in HH8 explants cannot progress beyond this stage in vitro,
neither thickening nor neuronal differentiation were observed in these
explants even after 3 days (Fig.
6A-A'') (Bailey et al.,
2006). Although this appears contradictory to the findings of
Sjodal et al. (Sjodal et al.,
2007) where over 10% of cells in HH8 olfactory explants were
keratin+/Hu+, these explants also expressed neural markers such as L5, HuC/D
and Nkx2.1 in a distinct region. Similarly, in our hands, 11% of
ectodermal cells in HH8 explants were Hu+ when cultured with the anterior
neural ridge (Bailey et al.,
2006). By HH10, after olfactory and lens precursors have separated
(Bhattacharyya et al., 2004),
nasal progenitors are specified to express Dlx3 and Pax6 (n=20/20)
and 20% of these explants differentiate to give rise to neurons
(Fig. 6B,C,C'). Although
Dlx3+ neurons can be unambiguously assigned to an olfactory fate
(Fig. 6B, arrow), Dlx3-neurons
(Fig. 6B, arrowhead) could
either be olfactory (Fig. 3G)
or forebrain-derived, as a few forebrain cells could have been explanted along
with prospective placode. The segregation of Dlx3+, Pax6+ and Dlx3+/Pax6+
zones continues (Fig.
6C'). Although some explants exhibit thickening
(Fig. 6C,C'), most
contain cell aggregates with no particular structure. However, prospective
nasal placode explanted at the cusp of thickening (HH13-14; n=25)
forms a thickened epithelium and undergoes significant neurogenesis
(Fig. 6D,E). Cumulatively,
these results suggest that nasal placode precursors are specified to express
Dlx3 and Pax6 as early as HH8; however, neuronal specification, as assayed by
Hu expression, is noted only once nasal precursors have completely segregated
from neighboring ectodermal populations and is complete just prior to
acquiring placodal identity.
Placodal precursors commit to olfactory fate concomitant with acquisition of placodal morphology at HH14
Operationally, a tissue is considered intrinsically committed to its fate
if it recapitulates that fate when challenged by transplantation to a novel
embryonic environment. As a rigorous test to determine irreversible commitment
of olfactory placode ectoderm, its developmental potential was challenged by
grafting it in place of non-placode forming trunk ectoderm. To this end,
presumptive olfactory placode ectoderm derived from HH8-16 was transplanted to
lateral plate ectoderm at the level of the most recently formed somites in
HH8-9 chicks (Fig.
7A-C'). Even at HH8, grafts were committed to express Pax6
and Dlx3, paralleling their specification status
(Fig. 7D). However, only a
single graft from this stage expressed Hu, suggesting that neurogenesis was
not yet fixed in most cases. At HH10, close apposition of the presumptive
olfactory placode ectoderm with the forebrain makes their separation
difficult. However, we cleanly dissected placodal ectoderm away from the
forebrain in a few cases. The results show that, by HH10, olfactory precursors
are not only capable of expressing Pax6/Dlx3 when grafted to the trunk, but a
few graft-derived cells even differentiate as Hu+ neurons
(Fig. 7E). We next
heterotopically grafted older ectoderm (HH13) just prior to its thickening to
form a morphologically distinct olfactory placode. In all cases, adherent
mesenchyme was grafted with the presumptive placode ectoderm to the ectoderm
overlying the lateral plate mesoderm. In two out of seven cases, the
presumptive placodal ectoderm (identified by both Pax6 and Dlx3 expression)
underwent thickening to acquire morphology characteristic of the olfactory
placode and neuronal differentiation as assayed by Hu staining
(Fig. 7F,F'). Isolated
neurons appeared to migrate towards the dorsal aorta
(Fig. 7F,F', arrowheads).
In the remaining five out of seven cases, no donor-derived neurons were
detected. In these cases, although some of the grafted quail cells looked
ectodermal and are Dlx3+/Pax6+, the majority of grafted cells appeared
mesenchymal.
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Cumulatively, our data suggest that once olfactory precursors segregate away from other ectodermal and placodal precursors, they are committed to express Dlx3 and Pax6 but have not yet received all signals for neuronal differentiation. Shortly before acquiring placodal morphology, transplanted olfactory ectoderm can express olfactory markers and differentiate; however, the neurons that ingress are not cohesive. Once the olfactory placode has acquired its characteristic morphology, differentiation occurs normally.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
We have previously demonstrated that olfactory induction requires inhibition
of lens fate by FGF at neurula stages and subsequently by signal(s) from the
neural crest (Bailey et al.,
2006). Here, we explore the spatiotemporal framework within which
olfactory progenitors are precisely and reproducibly generated from an
apparently homogenous field of anterior ectoderm. To this end, we define
intrinsic properties of cranial ectoderm precursors that result in eventual
irreversible acquisition of olfactory fate by a subset of these cells.
Molecular identity of developing olfactory placodes
Comparing fate maps and gene expression patterns enables identification of
molecular markers that foreshadow olfactory fate. Using this approach, we
identified three transcription factors, Dlx3, Dlx5 and Pax6, the combinatorial
expression of which uniquely demarcates progenitors of the chick olfactory
epithelium. Current literature suggests significant species-specific
differences in the co-expression of Dlx3, Dlx5 and Pax6 in the olfactory
epithelium. For example, Dlx3 expression in the mouse is undetectable at E8.5
and only weakly detectable by E9.5 (Quint
et al., 2000) or at E14.5
(Merlo et al., 2007), but
appears to be conserved between chick and zebrafish
(Akimenko et al., 1994;
Ellies et al., 1997). By
contrast, the expression pattern of Dlx5 is consistent between chick, mouse
and frog (Papalopulu and Kintner,
1993; Pera et al.,
1999; Yang et al.,
1998); interestingly, Dlx5 expression is not observed in the
gastrula stage zebrafish embryo and is detected only when olfactory precursors
coalesce to form the placode (Akimenko et
al., 1994; Ellies et al.,
1997; Quint et al.,
2000). Pax6 is expressed in the mouse
(Grindley et al., 1995;
Walther and Gruss, 1991),
zebrafish (Krauss et al.,
1991; Nornes et al.,
1998; Puschel et al.,
1992) and frog developing olfactory placodes
(Franco et al., 2001),
although a detailed timeline of its placodal expression is missing in the
zebrafish.
|
70% of the Dlx5+ cells become GnRH+
neurons that line the olfactory nerve
(Merlo et al., 2007). In Dlx5
knockout mice, GnRH neuroendocrine cells persist but are mislocalized and
express GnRH at reduced levels (Merlo et
al., 2007). Dlx5-null mice have greatly reduced olfactory
epithelia, with few sensory neurons therein, and rudimentary vomeronasal
organs in addition to grossly impaired otic vesicle derivatives
(Depew et al., 1999;
Long et al., 2003;
Merlo et al., 2007).
Similarly, nasal placodes and cavities are absent in small eye
(Sey) mice and rats lacking Pax6
(Fujiwara et al., 1994;
Grindley et al., 1995;
Hogan et al., 1986;
Matsuo et al., 1993). Chimeric
analysis shows a cell-autonomous requirement for Pax6 for full contribution to
the lens and nasal epithelium (Collinson
et al., 2000; Collinson et
al., 2003); Pax6 appears to be required for the transition from
nasal ectoderm to placode (Grindley et
al., 1995). Likewise, in the zebrafish deletion mutant, b380,
which lacks Dlx3a and Dlx3b, there is no induction of otic or olfactory
placodes (Solomon and Fritz,
2002).
Origin of GnRH neuroendocrine cells
Evidence from mouse and chick supports an olfactory placode origin for
basal forebrain localized GnRH neurons
(Wray, 2002). In both
organisms, detailed immunocytochemical analysis revealed GnRH+ cells in the
olfactory epithelium, later along the olfactory nerve and finally in the
forebrain (Murakami et al.,
1991; Norgren and Lehman,
1991; Sullivan and Silverman,
1993; Wray et al.,
1989a; Wray et al.,
1989b). Furthermore, birth-dating forebrain GnRH neurons suggests
they leave the mitotic cycle shortly before GnRH expression is seen in the
olfactory epithelium (Mulrenin et al.,
1999; Schwanzel-Fukuda and
Pfaff, 1989; Wray et al.,
1989a). More direct evidence for an olfactory placode origin for
these neurons have come from dye labeling
(Murakami and Arai, 1994),
transplantation (Yamamoto et al.,
1996) and ablation studies
(Akutsu et al., 1992;
Norgren and Gao, 1994).
However, it has been argued that cells with non-olfactory placode origins
could have mixed in with olfactory placode cells in these studies
(Whitlock, 2008). Additional
evidence in support of an olfactory placode origin has come from analysis of
the Sey mutant, which lacks olfactory placodes and has no GnRH
neurons (Dellovade et al.,
1998; Skynner et al.,
1999). Contrary to these studies, unilateral ablation of ectoderm
immediately adjacent to, but not including, the olfactory placode at an early
stage resulted in loss of forebrain-localized GnRH neurons
(el Amraoui and Dubois,
1993).
Advanced teleost fishes (atherinomorpha, percomorpha), including zebrafish,
have two distinct types of forebrain GnRH neurons: terminal nerve ganglia GnRH
neurons and hypothalamic GnRH neurons
(Parhar, 2002;
Whitlock et al., 2003). Of
these, the terminal nerve ganglia GnRH neurons were considered to originate
from olfactory placode (von Bartheld and
Baker, 2004). However, recent dye labeling and lineage analysis in
zebrafish suggest that these neurons are neural crest derived
(Whitlock et al., 2003).
Furthermore, a severe reduction in the number of hypothalamic GnRH neurons was
observed in the you-too mutant, which lacks most of the pituitary but
has normal olfactory placodes (Whitlock
et al., 2003). Our data are consistent with the hypothesis that at
least some GnRH neurons in the forebrain region are olfactory placode ectoderm
derived. However, we cannot rule out the possibility that other GnRH neurons
might originate from embryonic populations such as the adenohypophyseal
placode or the neural crest.
Spatiotemporal competence of ectoderm to form olfactory placode
Induction of structures at predetermined locations can occur by restricted
localization of inducing signals and/or strict delimitation of responding
tissue. For trigeminal placodes, dorsal neural tube at all axial levels have
inducing ability, but competence to respond is relegated to cranial ectoderm
(Baker et al., 1999). The
converse occurs for otic placodes where inducing signals are localized at r2-7
while competence to respond is broad
(Groves and Bronner-Fraser,
2000). Work from many laboratories suggests that growth-factors
such as Fgf8, Bmp4, Shh and retinoic acid are crucial for induction of the
olfactory placode (Bailey et al.,
2006; Bhasin et al.,
2003; Calof et al.,
2002; Kawauchi et al.,
2005; LaMantia et al.,
2000). Accordingly, competence to co-express Pax6/Dlx3 and
differentiate as olfactory cells is restricted and decreases steadily in an
anterior-posterior gradient, with trunk ectoderm possessing negligible
capacity to contribute to the nasal placode. Additionally, we find good
correlation between the loss in competence to express olfactory markers and
commitment to differentiate as other derivatives. For example, ability to
express Pax6 is lost in grafts of HH10+ hindbrain level ectoderm as it is
committed to an otic placode fate (Groves
and Bronner-Fraser, 2000).
In comparing chick trigeminal, otic and olfactory placode competence
(Baker et al., 1999;
Groves and Bronner-Fraser,
2000) (and this study), trunk ectoderm has either no capacity to
form placodes or only briefly retains this ability. Consistent with this,
cranial ectoderm expresses epibranchial placode markers in response to BMP7
from pharyngeal endoderm but trunk ectoderm does not do so
(Begbie et al., 1999).
Competence to form lens is also restricted to lateral and ventral head
ectoderm in amphibians and aves (Jacobson,
1966; Sullivan et al.,
2004). In Amblystoma punctatum, as in chick, competence
to form olfactory placode extends, at early stages, over a wide area of head
ectoderm, but not to anterior flank ectoderm after stage 15
(Haggis, 1956). Furthermore,
misexpression of signaling molecules or transcription factors involved in
placodal development usually results in a partial recapitulation of the
placode developmental program adjacent to the endogenous placodal territory or
placodes forming in conjunction with neural tissue
(Altmann et al., 1997;
Barembaum and Bronner-Fraser,
2007; Begbie et al.,
1999; Köster et al.,
2000; Ladher et al.,
2000; Taylor and Labonne,
2005; Vendrell et al.,
2000). No experimental manipulation has so far led to the de novo
generation of sensory or neurogenic placodes at all axial levels in the trunk
ectoderm.
Recently, Martin and Groves (Martin and
Groves, 2006) suggested a two-step model of placode induction
wherein cranial ectoderm must first acquire generic properties of the
pre-placodal region before responding to more specific placode-inducing
signals. Wnt signals from lateral/posterior mesoderm and within trunk ectoderm
have been hypothesized to restrict placode formation to a horseshoe-shaped
domain in cranial ectoderm; however, Wnt inhibition alone cannot induce
ectopic pre-placodal markers (Litsiou et
al., 2005). Instead, intermediate levels of Bmp and Wnt inhibition
in conjunction with Fgf signaling elicit pre-placodal markers such as Six1 and
Six4 (Ahrens and Schlosser,
2005; Brugmann et al.,
2004; Litsiou et al.,
2005). Thus, signaling pathways can prime cranial but not trunk
ectoderm to respond to placode-inducing cues.
Induction is complete before placodal morphogenesis
The capacity of presumptive olfactory ectoderm to express Pax6/Dlx3 and
form neurons was assayed in 3D collagen gels. We find that presumptive
olfactory placode ectoderm is specified to express Pax6 and Dlx3 by HH8
(Bailey et al., 2006) (this
paper). Neuronal specification begins around HH10-11, implying that ectoderm
has seen signals that will direct its fate even before it is morphologically
visible as a placode, analogous to trigeminal, otic and lens placode
specification (Baker et al.,
1999; Groves and
Bronner-Fraser, 2000;
Sullivan et al., 2004).
We also determined the time when presumptive olfactory placode ectoderm is
irreversibly committed by challenging its fate in a new environment via
grafting over lateral plate ectoderm of HH8-9 chick embryos. Distant from the
endogenous placode, this site probably lacks inducing signals. Interestingly,
commitment to express Pax6 and Dlx3 and form some neurons occurred by HH10,
before the placode is self-evident, consistent with early experiments showing
that grafts of the anterior tip of HH11 embryos to the chorioallantoic
membrane or trunk produced relatively normal olfactory organs in a small
percentage of grafts (Street,
1937). However, sustained olfactory development only occurred once
the placode acquired its characteristic morphology
(Street, 1937). In
Amblystoma punctatum, there is some discrepancy regarding when
olfactory determination occurs. Although one study corroborates our findings
(Haggis, 1956), another
observes commitment to an olfactory fate at neurula stages
(Carpenter, 1937). One
possibility is that neural tissue was present in grafts of olfactory
precursors taken at neurula stages, which would then continue to provide
signals necessary for growth and differentiation. Another possibility suggests
that the site of transplantation might have been in range of
olfactory-inducing stimuli (Haggis,
1956).
Conclusion
We show that co-expression of Dlx3, Dlx5 and Pax6 demarcates olfactory
precursors prior to acquiring placodal status. Competence to form olfactory
placode resides within head ectoderm up until HH9-10, but is largely absent
from trunk ectoderm. Olfactory fate specification and commitment are complete
just before acquiring overt placodal morphology and prior to differentiation
into olfactory epithelium. This may highlight a general trend in placodal
development.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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