Vertebrates differ from the other chordate subphylla by the
considerable expansion of their brain and sense organs. Those
structures have benefited from a crucial asset in evolutionary
transition, the neural crest, that has endowed cephalic structures
with skeletal protection and vascularization. In vertebrate embryos,
neural crest transiently consists in a neural fold bulging at the
margin of the neural epithelium in the early neurula. While the
neuroepithelium engages its dorsal closure to form the neural tube,
neural crest cells detache and become mesenchymal. Their
migrating properties lead them to spread and pervade different
regions of the embryo where they yield a broad range of derivatives
(from myofibroblast, melanocyte, endocrine cell, neurones, glial
cells to cartilage and bone). Investigations carried in the avian
embryos by the mean of the quail-chick chimeras were instrumental
in mapping their fate and emphasized the major role of neural crest
in head development (Le Douarin, 1982; Le Douarin and Kalcheim,
1999; Le Douarin et al., 2004).
At the cephalic level, neural crest cells provide the forepole of
vertebrate embryo with diverse mesenchymal derivatives that form
the mesectoderm (Kastschenko, 1888; Platt, 1893) and concur to
the emergence of the head by giving rise to the craniofacial
connective, dermal and skeletal tissues (Le Liиvre, 1974; Le Liиvre
and Le Douarin, 1975; Noden, 1983; Couly et al., 1993).
In the early neurula, previous fate maps performed the quailchick
chimera system have demonstrated that the association of
the ectoderm-derived components of the eye results from the
convergence of a medioventral retinal neurectoblast with the
dorsolateral ectoderm of the optic placode first identified lateral to
the diencephalic neural fold (Couly and Le Douarin, 1987). The
neuroectodermal origin of the eye lies at first in the diencephalic
neural plate, tightly interposed between the posthypophysis and
telencephalic vesicle anlagen, in their medial and lateral aspects,
respectively (Fig. 1A; Couly et al., 1985). The presumptive territory
of the retina accompanies the expansion of the diencephalic
vesicle, which grows out laterally until it faces the superficial
ectoderm of the optic placode (Fig. 1C). Concurrently with the
thickening of the ectoderm responsible for the differentiation of the
lens, the invagination of the diencephalic vesicle leads to the
delineation of an optic cup that will forms the optic vesicle (OV), the
margin of which encompasses the lenticular vesicle.
In the present study, we have revisited and illustrated the
contribution of the neural crest cells to the intra- and peri-ocular
structures using quail-chick chimeras. This paper discusses the
mesectodermal input as a contribution to optic physiology and tries
to provide a brief review of the molecular pathways the neural crest
relies on for its participation to the developing eye.
Fig. 2. Cephalic neural crest cell migration converging towards the optic vesicle. (A) Scanning electron micrograph (SEM) showing the forepole of a 5 ss chick embryo. The
of neural crest (NC) territory (delineated with dotted lines)
subjected for remplacement by its quail counterpart obtained from a
stage-matched chick involves the posterior diencephalic level as
schematically represented in (B). (C)Whole-mount immunodetection of
quail neural crest cells which have basically colonized the anterior half of the optic vesicle at E2, one day after grafting (arrows). (D) The same SEM as in A in which the mesencephalic and anterior metencephalic neural
fold is delineated (dotted lines). (E)Scheme representing the
interspecific transfer of this territory from a 5ss quail donor embryo to a stage-matched chick host. (F) Wholemount detection of the quail neural
crest cells that have spread from the graft into the host environment at
E2.5; laterally migrating neural crestderived cells have massively moved
(arrows) around the ocular region by encompassing the posterior three quarters of the optic vesicle (OV). Note that a large part of graft-derived cells which are destined to form the calvarium
(Cal) remains dorsally located thus covering the posterior mesencephalon and the isthmus (Is).
Discussion
The origin of the ocular mesenchyme has long been a matter of
debate tending to decipher the respective contribution of the cephalic
mesoderm and mesectoderm to the eye and periocular structures. In
this respect, construction of chimeras between quail and chick embryos has provided a reliable approach to trace and unravel the
long-term involvement of crest-derived cells in eye developement.
The present paper aims at reviewing and illustrating the contribution
of cephalic neural crest cells and the derivatives they give rise to the
intra- and extra-ocular structures.
Fig. 3. Localization of neural crest cells around the
developing optic vesicle. (A) At E3, quail neural crest
cells immunodetected with QCPN Mab have migrated
from the transplant implanted at the mesencephalic
level (Ms) and primarily form the perioptic mesenchyme.
(B) Higher magnification showing that crest-derived
cells are intimately associated with the ectoderm of the
optic vesicle (OV). (C) At E4, the quail crest-derived
mesenchyme is evenly distributed around the
presumptive eyeball filling the space between the optic
and diencephalic (Di) ectoderms. (D) Close-up picture
from framed area in C. (E) At E5.5, while the optic cup
neuroectoderm has evolved into a double-layered
structure consisting of the outer and inner retinal
epithelia (ORE and IRE, respectively), neural crestderived
cells are accumulated in the anterior segment
of the eye (at the margin of the optic cup where ORE
and IRE are linked) and form the chamber angle (F). (G)
Laterally, quail cells which have expanded from the
anterior chamber, occupy the interface between the
lenticular epithelium and the overlaying ectoderm both
of host origin; they give rise to the corneal endothelium
(arrows). (H) At E7, in the anterior segment closed by
the lens (L), the retinal margin (R) and the corneal
epithelium (CoEp), neural crest-derived cells form the
ciliary corpus (CC) and the corneal endothelium (CoEn)
thus lining the inner aspect of the anterior optic chamber.
From E2, the chick ocular region gains a vast mesenchymal
supply of neural crest cells which originate from the posterior
diencephalon, the mesencephalon and the metencephalon
(corresponding to the two first rhombomeres, r1r2). This territory
corresponds to the cephalic domain of the neural fold that yields the
formation of the facial skeleton (Couly et al., 2002; Le Douarin et al.,
2004 for a review). The optic cup is then invaded by the diencephalic
crest cells for its anterior and lateral aspects, by the mesencephalic
ones for its dorsal and medial parts and by the metencephalic crest
cells for its latero-ventral region. However, converging to the OV,
these different streams of crest cell migration extensively overlap,
precluding the accurate drawing of a fate map of the presumptive eye
colonization by neural crest cells.
Concurrently with the flux of crest cells reaching the optic cup, a
minor but essential progression of mesodermal cells occurs towards
the interface between the superficial ectoderm and the lenticular
vesicle leading to the differentiation of macrophage-like cells (Hay,
1980; Garcia-Porrero et al., 1984; Cuardos et al., 1991). These cells
clean up the debris released by the apoptotic events associated with
lens morphogenesis, a prerequisite for the deposition of a primary
corneal stroma permissive for neural crest cell migration into the lensectoderm interspace (Hay, 1980). At the posterior segment, neural
crest and mesodermal cells mix to synergize the development of a
functional vasculature as well as the extra-ocular musculature. By
replacing chick cephalic neural primordium by their quail counterparts,
Le Liиvre and Le Douarin (Le Liиvre and Le Douarin, 1975; Le Liиvre,
1978) first recorded the presence of Feulgen-positive nucleoli in the
connective tissues of the extraocular muscles while myofibers derived
from the host mesoderm (see Couly et al., 1992). In addition, the
musculo-connective wall of ocular vessels and pericytes of the
choroid membrane derived from the cephalic neural crest (Le Liиvre,
1978; Johnston et al., 1979; Etchevers et al., 1999). Furthermore, a
subset of neural crest-derived cells in the choroid membrane exhibited
a pigmented phenotype.
The mesectodermal differentiation at this level was further
documented by demonstrating that the neural crest is responsible for
the skeletal orbital and sclerotic structures housing the posterior
segments of the developing eyeballs (Le Liиvre and Le Douarin,
1975; Le Liиvre, 1978). That the neural crest yields a skeletal
protection for ocular structures is confirmed by experiments in which
the cephalic neural crest is surgically extirpated at 5-6ss. In this case,
embryos develop without sclera and, therefore, exhibit deformed
oblong eyes, due to the absence of a cartilaginous nest (Couly et al.,
2002). Surprisingly, when performed earlier in 3-4ss chick embryo,
neural crest extirpation results in cyclopia, the severity of which
appears to be stage-dependant (Etchevers et al., 1999; and our own
unpublished data). Such a phenotype likely points out to a transient
crest-dependent molecular requirement that has not been identified
so far. Taken together, these data emphasize the inability of the
cephalic mesoderm to substitute for the neural crest cells in building
mesectodermal skeletal elements of any kind (belonging to either
neurocranium or viscerocranium).
Furthermore, a great deal of regulation can occur within the
cephalic neural crest domain to yield sclerotic structures since only
a third of this territory, whatever its level of origin, can yield a normal
sclera (Couly et al., 2002). Additionally, as for the facial skeleton,
ocular structures turn out to be sensitive to ectopic Hox gene
expression to an extent that depends on the transfected gene:
Hoxa2 expression prevents sclera formation, Hoxb4 alike, while
Hoxa3 hinders crest cell differentiation into skeletogenic and
pericytic phenotypes all around the developing eye (Creuzet et al.,
2002). Thus, deficits resulting from Hox gene expression in the
forehead territory - as a consequence of either heterotopic
transplantation or transfection - accounts for axial difference in the
potential of neural crest cells to participate in posterior ocular
structures along the rostrocaudal axis. Deficits associated with
Hox gene expression also affect the other crest-derived structures
of the developing eye, as recently confirmed (Lwigale et al., 2004).
Once the contribution of neural crest cells to the posterior
segment of the eye established, most attention was paid to the
anterior segment of the eye due to its importance in the optic
physiology and its histological complexity. In the seminal study
from Johnston and coworkers (Johnston et al., 1979), interspecific
combinations involving either cephalic mesoderm or neural crest
ascertained the wide participation of mesectodermal cells at this
level. In the anterior segment, two main areas of neural crest
differentiation can be distinguished: an ocular one comprising the
iris, the ciliary process and the cornea that delineate the anterior
optic chamber and a periocular one including the nictitating
membrane and eyelids (see Table 1).
Taken together, these data conspicuously point to a key
contribution of the neural crest cells in endowing the anterior
segment with refractive structures and media. First, neural crest
cells ensure a mechanistic refracting function through the synthesis
of a collagen matrix by the crest-derived keratocytes of the corneal
stroma. Second, neural crest cells concur to the homeostasy of the
anterior chamber by being associated with the production of the
acqueous humor from the ciliary body, by collecting the anterior
chamber fluid that bathes and nourishes the lens epithelium and by
draining it out towards Schlemm’s canal. Third, neural crest cells
by giving rise to sclerotic ossicles exert tensile forces upon the
intraocular fluid pressure and constraint the proper corneal curvature
(Coulombre and Coulombre, 1958), thus directing light to the
retina. These views are in accordance with data that have dissected
the role of Pax6 in ocular epithelial structures in conjunction with
the development of ocular mesenchyme in mammals (see, Cvekl
and Tamm, 2004, for a review).
At the edge of both anterior and posterior eye segments, the
transition of neural crest fates does not occur by a clear-cut
boundary but through an intermediate zone corresponding to the
limbic area. Linking the margin of the sclerotic to the irido-corneal
angle, the limbus receives a massive contribution of neural crest
cells among which both pigment cells and smooth-actin expressing
cells are recorded. In this respect, this area displays an noteworthy
transition whereby crest-derived cells progressively diverge from
both pigmented and pericytic phenotypes in the choroid membrane
towards either pigmented cells for ciliary process, pericytes for
Schlemm’s canal, smooth muscle cells in ciliary muscle or striated
muscle cells in iris. This observation relates to another problem as
to whether the pigmented mesenchymal cells, abundantly found
both in ciliary process and ciliary corpus from stage HH37, are of
mesenchymal or of epithelial origin. It was of interest to determine
whether the peripheral retina would be able to generate pigmented
Fig. 4. Neural crest contribution to the morphogenesis of the ocular and periocular apparatus. (A) Feulgen-stained section of a chimeric eye
taken from an E12 chick that has been engrafted with quail diencephalic and mesencephalic neural crest at E1.5. (B) Enlargement from A focused
on the anterior optic chamber and (C) a close-up picture of the irido-corneal angle. (D) The iridial stroma (Ir) made up of quail neural crest cells (arrows) has accompanied the expansion of the host-derived pigmented epithelium from the retina; (E) quail specific labelling of the stromal cell nucleolus. (F) In the irido-corneal angle (open arrows), quail cells from the ciliary corpus have aggregated to form the ciliary muscle (CM) and line the endothelium of Schlemm’s canal (*). Note the presence of pigmented cells. (G,I) In the cornea, while the outer epithelium (CoEp) is of host origin, the stromal cells
(Co; arrows; G) as well as the inner epithelial cells (CoEn; arrows; I) are derived from the engrafted quail neural crest, as illustrated in (H,J) respectively. (K) In contrast, lens fibers are exclusively of host origin. (L) At the edge of the cornea and the periocular structures, quail cells form the sclero-corneal limbus (ScCl), linking the corneal stroma (Co) to the sclerotic cartilage (Sc). (M) In the vicinity of the latter, osteogenic foci corresponding to sclerotic ossicles have differentiated from crest cells (arrows) as well as periosteum. (N,P) Covering the nasal portion of the eye, the nictitating membrane (arrows in P) has a crest-derived mesenchyme (Ni; arrows in N). (O) Interposed between the sclera and the pigmented retinal epithelium of donor
and host origin respectively, quail mesectodermal cells give rise to pericytes and pigment cells in the choroid membrane (Ch). (P) The ectodermal folds which externally line the upper and lower aspects of eyeballs are filled with crest-derived mesenchymes; (Q,R) in the lower eyelid, the forming palpebral muscle (PaM in Q) consists of neural crest -derived cells (arrows in R) with a platismal insertion at the margin of the lid (S). (T,U) In hatched animals, the lower eyelid is the only motile one responsible for eye shutting. (V) Relative positions of the lower eyelid and the nictitating membrane (Ni) in which mesenchymal cells are of crest origin (W).
mesenchymal cells. This question has long been a matter of
dispute and recently re-addressed by Barrio-Asensio and coworkers
(Barrio-Asensio et al., 2002). Whilst re-evaluating the structure of
the ciliary muscle, the authors suggested that pigmented cells
arose from the delamination of the outer pigmented epithelium.
This assumption based on a descriptive account also led to the
confusing view that the pigmented ciliary process could give rise to
the internal portion of ciliary muscle meaning that retina could yield
smooth muscle cells. By using chimeras, we demonstrate here the
crest origin of the pigmented cells infiltrating the ciliary region.
However, due to their intimate connection, we cannot rule out that bulges of the pigmented retinal epithelium are involved in inserting the iridial muscle fibers.
Fig. 5. Fig. 5. Pigment and muscle fates of neural
crest cells in the anterior segment of the
eye. (A) Frontal section of the anterior chamber
taken from an E12-old chick embryo engrafted
with metencephalic neural crest at the 5ss. (B)
In the ciliary processes, the mesenchymal
pigmented cells are of quail origin (QCPN+)
(C), while the pigmented epithelium belongs
to the host. (D) Longitudinal section of iridial
muscle fibers in which myonuclei are quail
(QCPN+). (E) In the iridial stroma, a subset of
crest-derived cells express the myogenic factor,
MyoD. MyoD transcript detection is partly
correlated with the accumulation of either the
smooth-muscle actin protein (SMA) (F) and/or
a differentiating muscle cell-related antigen
(13F4 immunolabelling) (G). (H) In the lower
eyelid, quail cells detected by QCPN Mab form
the palpebral muscle consisting of smooth
muscle cells (I) at this stage. At the end of
incubation (i.e. E21), the plapebral muscle is a
combination smooth muscle- (J) and striated
muscle-type cells (K).
We also show here that neural crest cells evenly compose iridial stroma. At mid-incubation, the crest-derived stroma encompasses discrete domains characterized by i) the expression of the
myogenic factor MyoD at the periphery, ii) the accumulation of smooth muscle actin expanding from the periphery towards the pupillar margin and iii) the expression of the differentiating myoblast
antigen 13F4, superficially. For long, the coexistence of a smooth and striated musculature in the iris has been interpreted as resulting from distinct origins (see Gabella and Clarke, 1983 and references therein). Re-addressed by the use of quail-chick chimeras, ultrastructural studies yielded evidence that neural crest cells, which are responsible for the differentiation of dilatator (i.e. radial and oblique muscular components) and sphincter (i.e. circumferential component)
muscles, are able to generate both musculatures (Nakano and
Nakamura, 1985). Dissecting the fate of iridial musculature in
postnatal chicken until adulthood, Scapolo and coworkers provided
a refined and extensive analysis that fully documented the
differentiation of iridial muscle fibers according to their
histochemical, metabolic and functional traits (Scapolo et al.,
1988). Overall, these observations underline that, to a large
extent, mesectodermal-derived myogenesis shares similarities
with that of mesodermal origin. These similarities are consistent
with the myogenic capacity of cephalic neural crest cells that have been described as myonucleus donor cells for the skeletal
musculature of the jaw (Le Lievre, 1976).
It has been established that the anterior optic chamber
development is under the leading control of the lens (Genis-
Galvez, 1966). The lenticular vesicle, the morphogenesis of which
likely results from posterior retinal cues, in turn seems to specify
inner iridial and folded ciliary epithelia by both a prolonged contact
inhibition and gradient-dependent influences from the retina
(Coulombre and Coulombre, 1964; Stroeva, 1964). However,
while being early committed into a differentiated structure, the
iridial epithelium keeps a certain amount of plasticity until being
able to dedifferentiate and restore the lens morphogenesis from
the bulge of its pupillar margin (see Scheib, 1963 for a review); the
completion of such a regenerating process is potentiated in newt
compared to higher vertebrates. Supporting lens regeneration, a
molecular cascade initiated by both Shh and Ihh pathways has
recently highlighted this unique mechanism of retinal
transdifferentiation in amphibians (Tsonis et al., 2004). By contrast,
in chick, Shh signalling is essentially involved in retinal cell fate
decision and confers a pigment-cell-differentiating bias rather than
a neural-type fate to the retinal neuroepithelium (Zhang and Yang,
2001). These observations substantiate the influence of the local
signalling environment on the specification of the epithelial
components of the anterior optic chamber.
It turns out that environmental cues also locally coordinate the
differentiation of their mesenchymal components. In this respect,
Bmp expression from the inner ciliary epithelium is a crucial
signalling for the development of the ciliary body, the develoment
of which is thwarted by the production of the Bmp antagonist,
Noggin, from the pigmented retinal epithelium (Zhao et al., 2002).
If a source of Noggin is ectopically activates from the lens, the
formation of the ciliary epithelium ceases and, as a consequence,
the domain of neural retina is allowed to expand anteriorly. Moreover,
the myogenic differentiation of the iridial stromal cells is drastically
reduced (Zhao et al., 2002). Consistent with a crucial role of Tgf.
superfamily antagonists in this process, Follistatin, which is
expressed by the periocular mesenchyme (Feijen, et al., 1994;
Darland et al.,1995; Verschuren et al., 1995), has been shown to
orchestrate the transition from smooth-to-striated muscle in chick
iridial stroma (Link and Nishi, 1997; 1998). By contrast, in the
presence of an excess of Activin A that binds Follistatin, the
accumulation of «striated» myosin heavy chains stops and, in turn,
the development of smooth muscle characters is stimulated (Link
and Nishi, 1998).
TABLE 1
ORIGIN OF THE AVIAN OCULAR AND PERIOCULAR STRUCTURES
Another focus of neural crest-derived myogenesis concerns
eyelids. From E6, we show that the accumulation of crest-derived
mesenchyme at the lateral margin of the optic cup promotes and
supports the outgrowth of ectodermal appendages fated to form
the nictitating membrane and the eyelids. To date, little is known
about the development of these structures. According to recent
data, notable is the fact that eyelid mesenchyme relies on Egf
signalling since the dominant mutation of its receptor, Egfr, chiefly
hampers eyelid formation (Du et al., 2004). In addition, that the
craniofacial skeleton and periocular structures (i.e. eyelids and
nictitating membrane) share a common mesectodermal origin has
been recently strengthened by functional analyses of Osr genes
(Lan et al., 2004). Encoding a transcription factor akin to the
Drosophila odd -skipped, the Osr2 gene, if mutated, primarily
affects the morphogenesis of the palatal shelf and likely operates
through mesenchymal-epithelial interactions that imply Tgf.3
alteration in palatal epithelium. Surprisingly, this mutation also
results in the loss of eyelid along with conspicuous corneal defects
(Lan et al., 2004). Besides, using transgenic aproach, Dean and
coworkers have recently shown that the morphogen Bmp7 is
highly expressed in eyelid mesenchyme (Dean et al., 2004). As
evoked before, given the involvement of Tgf. family members and
their respective antagonists in specifying the anterior eye segment,
it is tempting to hypothesize that Bmp7 could partly determine the
fate of neural crest cells and/or regulate their myogenic potential at
this level. In this respect, investigating the neural crest-derived
myogenic processes that take place in subectodermal location
could possibly enlighten pathologies in human that relate to facial
myotonic deficiencies.
As a tribute to the quail-chick chimera model, the interspecific
combination has enabled a tremendous stride in the knowledge on
one of the most refined structures of the vertebrate head. In this
respect, the variety of cephalic neural crest-derived phenotypes
discovered in the intra- and peri-ocular region accounts for the
astonishing ability of the mesectodermal cells to fit and fine tune
their differentiation to microenvironmental requirements. Further
characterizations of the molecular pathways that govern neural
crest cell commitment in these processes will provide novel insights
into epithelio-mesenchymal cross-talks involved in eye
development.
CHRISTINE VINCENT and GERARD COULY
Fig. 1. Ectodermal origins of the ocular anlage. (A) Fate map of the anterior neural plate and neural fold in chick embryo at the 3 somite stage
(ss) as established by Le Douarin and coworkers (Couly and Le Douarin,
1987). The neuroectodermal territory fated to give rise to the retina (violet) lies in the diencephalic floor plate (pink),
interposed between the presumptive telencephalic neuroepithelium (yellow)
and the posthypophysis (beige). The optic placode (grey lattice) at the origin of the lens is lateral to the anterior diencephalic non-crest cell producing neural fold and is surrounded by the presumptive eyelid ectoderm (white). (B) Dorsal view of a scanning electron
micrograph (SEM) of the 3ss chick head in which the lateral ectoderm
has been extirpated thus showing the lateral aspect of the outgrowing optic vesicle (OV) surrounded by the cephalic mesoderm (Mes). (C) Inner aspect of the lefthand half of a 7ss-chick embryo sagittally cut; at this stage, the neuroectoderm of the optic vesicle expands laterally at the transverse level of the diencephalic floor plate (Di) to reach
and contact the ectodermal anlage of the optic placode. Note the position of the foregut endoderm (Fo; dotted line). 