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First published online 24 October 2007
doi: 10.1242/dev.02886
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Research Report |
1 MRC Centre for Developmental Neurobiology, King's College London, London SE1
1UL, UK.
2 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford
OX1 3QX, UK.
Author for correspondence (e-mail:
jo.begbie{at}dpag.ox.ac.uk)
Accepted 31 July 2007
SUMMARY
Neurogenic placodes are specialized regions of embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we examine in chick the mechanism underlying the delamination of cells from the epibranchial placodal ectoderm. We show that the placodal epithelium has a distinctive morphology, reflecting a change in cell shape, and is associated with a breach in the underlying basal lamina. Placodal cell delamination is distinct from neural crest cell delamination. In particular, exit of neuroblasts from the epithelium is not associated with the expression of Snail/Snail2 or of the Rho family GTPases required for the epithelial-to-mesenchymal transition seen in neural crest cell delamination. Indeed, cells leaving the placodes do not assume a mesenchymal morphology but migrate from the epithelium as neuronal cells. We further show that the placodal epithelium has a pseudostratified appearance. Examination of proliferation shows that the placodal epithelium is mitotically quiescent, with few phosphohistone H3-positive cells being identified. Where division does occur within the epithelium it is restricted to the apical surface. The neurogenic placodes thus represent specialized ectodermal niches that generate neuroblasts over a protracted period.
Key words: Placode, Delamination, Sensory neuron, Neural crest, EMT
INTRODUCTION
The neurogenic placodes generate the majority of neurons of the cranial
sensory ganglia, including all of those mediating the special senses of
hearing, balance and taste (D'Amico-Martel
and Noden, 1983
; Graham and
Begbie, 2000). These placodes arise at stereotypical positions in
the head of vertebrate embryos as the result of localized inductive signals
(Begbie and Graham, 2001a). The
neuronal precursors that are generated in each of the placodes express genes
indicative of their later sensory phenotype, even though the cells
delaminating from all of the placodes, except the ophthalmic trigeminal, are
mitotically active (Begbie et al.,
2002). The placodal neuroblasts are guided internally by neural
crest streams to the sites of ganglion formation where they terminally
differentiate (Begbie and Graham,
2001b). We have accumulated a body of knowledge about the
induction of epibranchial placodes and about how the migration of the
neuroblasts is organized, yet little is known about how these cells actually
leave the placodal epithelium.
The best-studied example of delamination in the vertebrate embryo is the
emergence of neural crest cells from the neural tube. This transient
population of multipotent cells is born in the dorsal aspect of the neural
tube, and then migrates into the periphery where they generate a wide range of
derivatives including sensory neurons. To leave the epithelium, the crest
cells alter their contact with the other cells in the dorsal neural tube and
assume a mesenchymal morphology, i.e. they undergo an
epithelial-to-mesenchymal transition (EMT)
(Le Douarin, 1999). The
emigration of these cells also involves a breach in the basal lamina around
the dorsal aspect of the neural tube, which subsequently seals. The process of
neural crest cell delamination has been shown to be transcriptionally
controlled by the Snail family of transcription factors
(Nieto, 2002), and to require
the small GTPase RhoB (Liu and Jessell,
1998). Importantly, there have been suggestions that the neural
crest and the neurogenic placodes exhibit a number of similarities. Both
tissues share an ectodermal origin and give rise to migratory cells, and the
likelihood of common developmental mechanisms has been suggested
(McCabe et al., 2004). We have
therefore carried out a detailed analysis of cell delamination from the
epibranchial placodes and compared this process with neural crest
delamination.
Here we present data that show, counter to expectations, that placodal delamination does not reflect neural crest delamination but is a distinct process. Crucially, we find that placodal delamination does not involve the assumption of a mesenchymal shape by the emigrating cells, and that the placodes do not express molecules associated with EMT. Furthermore, we show that the placodal epithelium is pseudostratified, mitotically quiescent and that when cell division does occur it is apically located.
MATERIALS AND METHODS
Immunohistochemistry
Fertile hen's eggs were incubated at 38°C in humidified atmosphere to
required stages (HH st) (Hamburger and
Hamilton, 1992). Embryos were fixed in MEMFA overnight at 4°C.
Whole-mount antibody staining was carried out as described previously
(Begbie et al., 1999). Primary
antibodies used were: mouse anti-neurofilament medium chain at 1:10000
(RMO-270, Zymed); rabbit anti-laminin at 1:100 (Sigma); mouse
anti-ß-catenin at 1:100 (Santa Cruz); mouse anti-Islet 1/2 at 1:1000
(Developmental Studies Hybridoma Bank); rabbit anti-phosphohistone H3 at 1:500
(Upstate). Secondary antibodies were Alexa 488-conjugated anti-mouse IgG and
Alexa 568-conjugated anti-rabbit IgG used at 1:1000 (Molecular Probes).
In situ hybridization
Whole-mount in situ hybridization was carried out as described previously
(Henrique et al., 1995).
BrdU labeling
BrdU (10 mg/ml) was applied to the surface of the embryo in ovo for 15
minutes at 37°C. Embryos were processed for whole-mount in situ
hybridization with Phox2a. Following color development, embryos were
processed for BrdU (anti-BrdU, 1:50, Biosciences Products) and cryosectioned
at 10 µm.
Electron microscopy
Embryos were processed and analyzed for transmission electron microscopy
(TEM) as previously described (Quinlan et
al., 2004).
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RESULTS AND DISCUSSION
Placodal neuroblasts emerge from a break in the basal lamina underlying distinctive placodal epithelium
Neurogenic placodes are focal thickenings of the cranial ectoderm in which
neuronal cells are born, as can be readily visualized by combined
immunostaining using an anti-neurofilament medium chain (NFM) antibody and an
anti-ß-catenin antibody (Fig.
1A). Such staining identified a number of important aspects of the
morphology of the cranial ectoderm. Firstly, it was clear that the
organization of the placodal ectoderm differs from that of the adjacent
ectoderm. The placodal ectoderm appeared as a region many cells thick, whereas
the adjacent surface ectoderm was squamosal. Secondly, the localization of the
ß-catenin staining in the placodal epithelium was distinct from that in
the non-placodal epithelium. In non-placodal ectoderm, ß-catenin was
localized to the cell membrane and there are discrete regions where
ß-catenin levels were high (Fig.
1C). In the placodal epithelium, the ß-catenin staining was
uniform throughout the cells (Fig.
1D).
Also notable is that whereas the basal lamina was continuous beneath the non-placodal ectoderm (Fig. 1E), it was absent beneath the placode itself. In cross-section, neuroblasts can be seen emigrating from the placode from a region showing a lack of laminin immunoreactivity (Fig. 1F-H). This strongly suggests that there is specific breakdown of the basal lamina at the site of neuroblast exit from the epithelium. That a breach in the basal lamina is seen in both placodal and neural crest delamination is to be expected as both processes involve the release of cells into the surrounding environment. It also suggests that the processes might share some of the same molecular mechanisms.
Delamination of placodal neuroblasts is distinct from neural crest delamination
The molecular control of neural crest cell production has been well studied
(Morales et al., 2005). These
studies have indicated roles for FoxD3, Ap2 and members of the SoxD and SoxE
families of transcription factors. In situ analysis shows no expression of
these genes in the placodal epithelium
(Perez-Alcala et al., 2004;
Veitch et al., 1999). However,
these genes are associated with neural crest induction in addition to
delamination. The process of delamination specifically has been shown to
involve Snail-family zinc-finger transcription factors
(Nieto, 2002) and members of
the Rho subfamily of GTPases (Liu and
Jessell, 1998).
RhoB
In the trunk, RhoB has been shown to be required for neural crest
cell delamination (Liu and Jessell,
1998). Correspondingly, our in situ analysis showed a high level
of RhoB expression in neural crest cells exiting the neural tube in
the trunk (Fig. 2C). Analysis
of the head in the same embryo showed that there is pronounced RhoB
expression within neural crest cells that adhere to the hindbrain
(Fig. 2A,B). By comparison, it
was clear that RhoB expression was not elevated in the epibranchial
placodes (Fig. 2B), suggesting
that RhoB is not upregulated in placodal delamination. Within the Rho
family of GTPases, RhoB forms a subfamily with RhoA and
RhoC (Wennerberg and Der,
2004). We therefore also analyzed the expression of RhoA
and RhoC, and found that they were likewise not expressed in the
placodes (data not shown).
Snail/Snail2
In neural crest delamination, Snail2 (formerly known as
Slug) is the key family member required in the chick and replaced by
Snail in the mouse. As with RhoB, we analyzed expression of
Snail2 at st16/17 when placodal cells are actively delaminating.
Where neural crest cells were being produced in the trunk, expression was seen
in the dorsal aspect of the neural tube
(Fig. 2F). However, expression
was confined to a small population of cells in the hindbrain and was absent
from the placodal epithelium (Fig.
2D,E). Analysis of Snail showed an expected lack of
expression in the presumptive trunk neural crest
(Fig. 2I). Expression was seen
throughout the cranial region, including the post-migratory neural crest;
however, it was absent from the ectoderm at both placodal and non-placodal
levels (Fig. 2G,H).
|
|
;
Thiery, 2002;
Tucker, 2004). The lack of
association of these molecules with placodal delamination suggests that cells
leaving the placode do not go through an EMT.
Placodal delamination involves the release of neuronal cells
It has long been assumed that cells leave neurogenic placodes as
mesenchymal cells (McCabe et al.,
2004; Schlosser,
2006; Webb and Noden,
1993). Therefore, to support the molecular data we went on to
analyze the cellular morphology of placodal cells within the epithelium and as
they exit. In longitudinal section through the pharyngeal arches at HH st17,
the thickened placodal epithelium and underlying breach in basal lamina were
clearly visible (Fig. 3A). In
an equivalent semi-thin section, the characteristic thickened placodal
epithelium could be seen adjacent to the endodermal pouch
(Fig. 3B). A clear distinction
could be made between the mesenchymal neural crest cells filling the
pharyngeal arch and the close-packed cells emerging from this placodal
epithelium (Fig. 3B). Electron
microscopy at this level showed that the cells emerging from the placode had a
distinctively neuronal morphology, with a round cell body and large nucleus
consistent with their migration as neuroblasts
(Fig. 3C). Thus, the placodal
cells do not assume a mesenchymal morphology and cannot be said to be
undergoing EMT.
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The pseudostratified appearance of the placodal ectoderm suggests that we
could compare the placode with the germinal neuroepithelium. Here, a
distinctive feature was that cell division occurred at the ventricular
surface. Double immunostaining with anti-phosphohistone H3 (PH3) and
anti-Islet 1/2 labels cells undergoing division in the context of the
epithelium and neuroblasts migrating away from the placode. Confocal analyses
of sections showed that where division does occur, it is restricted to the
apical surface of the epithelium (Fig.
4A-D); this can be compared with the ventricular zone (VZ) of the
neural tube in the same section (Fig.
4A,C). In the neural tube, release of cells from the VZ is usually
associated with cell division. Analysis of later placodal stages using both
PH3 and BrdU showed, however, that compared with the VZ, and with the
surrounding ectoderm, the placode is mitotically quiescent
(Fig. 4C-E). Furthermore,
neurons migrating away from the VZ are generally post-mitotic, whereas the
cells that leave the placode are mitotically active
(Begbie et al., 2002). The
absence of BrdU-positive cells at the point of delamination
(Fig. 4E) suggests that unlike
neural crest cell delamination, the exit of placodal cells from the epithelium
is not associated with S phase
(Burstyn-Cohen and Kalcheim,
2002).
Our results demonstrate that the delamination of neuronal cells from the
neurogenic placodes differs from neural crest delamination. Thus, whereas the
generation of neural crest involves an EMT, this is not so for the release of
cells from the placodes. This highlights the importance of considering the
accuracy of definitions: neural crest cell emigration is often considered
synonymously with delamination, and is cited as a parallel example in
situations of cells leaving an epithelium. However, for example in liver
formation (Bort et al., 2006),
this might not be accurate.
The observation that the cells leaving the placodes do not undergo an EMT
might in part reflect the fact that cells are produced from the placodes over
a protracted period: in the geniculate placode, neurogenesis is first seen at
st13 and is still ongoing at st20, a period of 
30 hours
(Begbie et al., 2002). By
contrast, the generation of neural crest cells from a particular axial level
of the neural tube is a fairly rapid process: at the level of rhombomere 2,
neural crest production initiates at st8 and is finished by st11, a period of
14 hours (Lumsden et al.,
1991).
ACKNOWLEDGMENTS
Thanks to Britta Eickholt (ß-catenin antibody), Jon Gilthorpe (chick ß-actin GFP), ARK-Genomics (RhoA and RhoC EST clones), Tom Jessell (RhoB) and Angela Neito (Snail and Snail2). Thanks also to Britta Eickholt and Richard Wingate for constructive discussion. This work was funded by The Medical Research Council UK and by The Wellcome Trust.
Footnotes
* Present address: GENETHON UMR CNRS 8115, 1 rue de l'Internationale, 91000
Evry, France 
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