hedgehog
Hedgehog roles in face and eye development There is growing evidence that implicates a role for Sonic
hedgehog (SHH) in morphogenesis of the craniofacial
complex. Mutations in human and murine SHH cause
midline patterning defects that are manifested in the head
as holoprosencephaly and cyclopia. In addition, teratogens
such as jervine, which inhibit the response of tissues to
SHH, also produce cyclopia. Thus, the loss of SHH
signaling during early stages of neural plate patterning has
a profound influence on craniofacial morphogenesis.
However, the severity of these defects precludes analyses of
SHH function during later stages of craniofacial
development. An embryonic chick system was used to
study the role of SHH during these later stages of
craniofacial development. Using a combination of surgical
and molecular experiments, it has been shown that SHH is
essential for morphogenesis of the frontonasal and
maxillary processes (FNP and MXPs), which give rise to
the mid- and upper-face. Transient loss of SHH signaling
in the embryonic face inhibits growth of the primordia and
results in defects analogous to hypotelorism and cleft
lip/palate, characteristics of the mild forms of
holoprosencephaly. In contrast, excess SHH leads to a
mediolateral widening of the FNP and a widening between
the eyes, a condition known as hypertelorism. In severe
cases, this widening is accompanied by facial duplications.
Collectively, these experiments demonstrate that SHH has
multiple and profound effects on the entire spectrum of
craniofacial development, and perturbations in SHH
signaling are likely to underlie a number of human
craniofacial anomalies (Hu, 1999).
In Drosophila, expression of hedgehog coordinates retinal development by propagating a wave of photoreceptor
differentiation across the eye primordium. Two vertebrate hh genes, sonic hedgehog (shh) and
tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed
in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands
outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. Expression of shh and twhh in the RPE commences
between 39 and 45 hours post-fertilization (hpf). At 45 hpf expression of both genes
in the RPE is restricted to a small region in the ventro-nasal
quadrant of the developing eye and therefore is visible
only in the few sections that passed through this region. Expression later spread
centrifugally such that sectioned 54-hpf embryos show
shh and twhh expression in a larger patch of RPE, but not
throughout the eye. By 81 hpf, embryos
show shh and twhh expression throughout the RPE. The spatiotemporal expression
patterns of shh and twhh in the RPE are similar, and
expression extends to, or slightly beyond, the limit of
photoreceptor lamination (where the photoreceptor layer
has not yet formed). Expression of shh and
twhh therefore occurs immediately prior to, or concomitant
with, the first morphological manifestation of photoreceptor
differentiation. A gene encoding a
receptor for the hedgehog protein, ptc-2, is also expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense
(ashh/atwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod
and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly
exhibit retardation of photoreceptor differentiation. It is proposed that hedgehog genes may play a role in propagating
photoreceptor differentiation across the developing eye of the zebrafish (Stenkamp, 2000).
Thus, both shh and twhh are expressed in a
restricted, ventronasal patch of RPE at 45-48 hpf, which
roughly coincides with the time of photoreceptor cell birth and is 2-6 h prior to the time of rod
opsin expression in the adjacent ventronasal patch of neural
retina. Expression of both shh and
twhh then spread centrifugally across the epithelial sheet,
in a manner that predicts the spatiotemporal pattern of
subsequent photoreceptor recruitment. The neurogenesis
and differentiation of retinal cells in teleost fish follows a
stereotyped pattern during eye development, with an initiation
site in ventronasal retina, followed by asymmetric
centrifugal spread, such that cells in nasal retina are born
and then differentiate prior to those in temporal retina. This consistent pattern
of retinal development suggests some degree of coordination
of retinal cell development in the teleost, either by
intraretinal cell-cell communication that propagates a
wave of differentiation or by extracellular signals originating
from outside the retina, but with a similar spatiotemporal
distribution. Zebrafish hh genes may be considered
candidates for involvement in either of these possible
mechanisms. However, it is unlikely that Hh signaling is
involved in the neurogenesis or differentiation of ganglion
cells, as these events begin well before hh genes are expressed
in the RPE (Stenkamp, 2000 and references therein).
While the function of hh genes in retinal development
may be similar in Drosophila and zebrafish, the tissue-specific
expression pattern is not. The RPE is a tissue layer
unique to the vertebrate eye, performing an array of metabolic
activities critical for retinal function. The RPE is a monolayer of cuboidal cells that lies in close association with the rod and cone photoreceptors. This epithelium functions as a barrier, protecting the neurosensory retina, and acts as well to absorb scattered light, improving visual resolution. The RPE functions in the phagocytosis of rod and cone outer segment fragments that are shed from their distal ends and regulate the uptake, processing, transport and release of retinoic acid and visual cycle intermediates (retinoids) and regulate ion transport in the subretinal space. Regeneration of the visual pigment
chromophore, 11-cis retinal, is performed by the RPE in
vertebrates, but takes place in the photoreceptor
cells in invertebrates. Expression
of hh genes for the regulation of retinal development may
be another example of an invertebrate photoreceptor function
assumed by the vertebrate RPE.
The importance of the RPE for photoreceptor development
and survival has been recognized for some time; indeed, the absence of RPE in vivo
results in failure of retinal development, followed by degeneration
and resorption of the retina.
A number of investigators have identified soluble
factors in RPE-conditioned medium, or in the interphoto-receptor
matrix, that have photoreceptor survival and/or
differentiation-promoting activities in in vitro models for
photoreceptor development. Hedgehog proteins
may now be considered candidates for some of these activities (Stenkamp, 2000 and references therein).
Production of retinal ganglion cells is in part regulated by inhibitory factors
secreted by ganglion cell themselves; however, the identities
of these molecules are not known. Recent studies have
demonstrated that the signaling molecule Sonic hedgehog
(Shh) secreted by differentiated retinal ganglion cells is
required to promote the progression of ganglion cell
differentiation wave front and to induce its own expression.
Evidence is presented that Shh signals negatively regulate ganglion cell genesis behind the differentiation wave front. Higher levels of Shh expression
are detected behind the wave front as ganglion cells
accumulate, while the Patched 1 receptor of Shh is
expressed in adjacent retinal progenitor cells. Retroviral-mediated
overexpression of Shh results in reduced ganglion
cell proportions in vivo and in vitro. Conversely, inhibiting
endogenous Shh activity by anti-Shh antibodies leads to
an increased production of ganglion cells. Shh signals
modulate ganglion cell production within the normal
period of ganglion cell genesis in vitro without significantly
affecting cell proliferation or cell death. Moreover, Shh
signaling affects progenitor cell specification toward the
ganglion cell fate during or soon after their last mitotic
cycle. Thus, Shh derived from differentiated ganglion cells
serves as a negative regulator behind the differentiation
wave front to control ganglion cell genesis from the competent progenitor pool. Based on these results and other recent findings, it is proposed that Shh signals secreted by early-differentiated retinal neurons play dual roles at distinct concentration thresholds to orchestrate the progression of retinal neurogenic wave and the emergence of new neurons. Likewise, in Drosophila, Hh signaling performs dual functions in eye neurogenesis, that is, advancing the MF and regulating the precise spacing of ommatidia. These effects are presumably achieved, at least in part, by the differential regulation of atonal expression by Hh at distinct thresholds (Zhang, 2001).
Appropriate interactions between the epithelium and adjacent neural crest-derived mesenchyme are necessary for normal pharyngeal arch development. Disruption of pharyngeal arch development in humans underlies many of the craniofacial defects observed in the 22q11.2 deletion syndrome (del22q11), but the genes responsible remain unknown. Tbx1 is a T-box transcription factor that lies in the 22q11.2 locus. Tbx1 transcripts were found to be localized to the pharyngeal endoderm and the mesodermal core of the pharyngeal arches, but are not present in the neural crest-derived mesenchyme of the pharyngeal arches. Sonic hedgehog is also expressed in the pharyngeal arches and is necessary for normal craniofacial development. Tbx1 expression is dependent upon Shh signaling in mouse embryos, consistent with their overlapping expression in the pharyngeal arches. Furthermore, Shh is sufficient to induce Tbx1 expression when misexpressed in selected regions of chick embryos. These studies reveal a Shh-mediated pathway that regulates Tbx1 during pharyngeal arch development (Garg, 2001).
During early formation of the eye, the optic vesicle becomes partitioned into a proximal domain that forms the optic nerve and a distal domain that forms the retina. In this study, the activities have been investigated of Nodal, Hedgehog (Hh) and Fgf signals and Vax family homeodomain proteins in this patterning event. Zebrafish vax1 and vax2 homologs of Drosophila ems are expressed in overlapping domains encompassing the ventral retina, optic stalks and preoptic area. Abrogation of Vax1 and Vax2 activity leads to a failure to close the choroid fissure and progressive expansion of retinal tissue into the optic nerve, finally resulting in a fusion of retinal neurons and pigment epithelium with forebrain tissue. Hh signals acting through Smoothened act downstream of the Nodal pathway to promote Vax gene expression. However, in the absence of both Nodal and Hh signals, Vax genes are expressed, revealing that other signals, which include Fgfs, contribute to Vax gene regulation. Pax2.1 and Vax1/Vax2 are likely to act in parallel downstream of Hh activity and the bel locus (yet to be cloned) mediates the ability of Hh-, and perhaps Fgf-, signals to induce Vax expression in the preoptic area. Taking all these results together, a model of the partitioning of the optic vesicle along its proximo-distal axis is presented (Take-uchi, 2003).
Loss of function studies in mouse and now in
zebrafish have revealed requirements for Vax proteins in several different
aspects of eye and forebrain midline development. The most conserved phenotype
following abrogation of Vax activity is a failure in fusion of the choroid
fissure. This phenotype is found in both mouse and fish lacking, or with
reduced, Vax1 or Vax2 activity (with genetic background-dependent penetrance
in mouse Vax2 mutants). The severity of this coloboma phenotype is
increased in fish embryos compromised in both Vax1 and Vax2 activity, strongly
suggesting that both Vax1 and Vax2 co-operate to regulate fusion of choroid
fissure. The proteins that mediate fusion of the retina at the choroid fissure
are unknown, but possible candidates include Eph receptors and their Ephrin
ligands. Members of this family of signalling proteins have been implicated in
fusion events in other epithelia, and several family members are expressed in the
ventral retina. Indeed, in mouse, changes in ephrinB1, ephrinB2
and EphB2 expression occur in the ventral retina of vax2 mutants, and
although these changes have primarily been considered in terms of retinal axon
pathfinding, they could potentially contribute to the choroid fissure
defects (Take-uchi, 2003),
A second conserved phenotype in Vax mutants is disruption to commissural
axon guidance and targeting of retinal axons. In Vax1 mouse mutants,
there is a severe disruption to midline development and
consequently, severe disruption in the pathfinding of axons as they approach
the midline. In vax2 mutants there are also variable defects in the
development of ipsilateral and contralateral retinal projections. These
may arise from incorrect assignment of identity to retinal neurons in the
Vax2 mutants, but the possibility that midline tissue is also disrupted
has not been excluded. Indeed, in fish, commissural axon pathfinding defects
are much more obvious in Vax1/Vax2 double morphants than in either single
morphant. This suggests, that at least in fish, Vax2 does co-operate with Vax1
to pattern midline tissue (Take-uchi, 2003),
The third conserved phenotype in animals compromised in Vax protein
activity is a failure to limit retinal development to the optic cup. The
initial indications of this phenotype came from analysis of mouse
Vax1 mutants in which expression of genes normally restricted to
retinal tissue (rx and pax6) was observed to encroach into
the optic nerve. This study provides dramatic confirmation of the requirement
for Vax protein activity to limit retinal development. In Vax1/Vax2 double
morphants, there is a progressive expansion of retinal tissue along the optic
nerve until by four days, neural and pigmented layers of the retina are in
direct continuity with diencephalic cells of the optic chiasm. Early
morphogenesis of the optic stalk and optic cup occurs relatively normally and
indeed, retinal axons navigate out of the eye and along the stalk or nerve to
the midline in the Vax1/Vax2 morphants. It is relatively late in development
that differentiating retinal tissue expands to the midline. This phenotype probably reflects a change in the fate of optic stalk or nerve cells to retinal tissue. The possibility that overproliferation and evagination of
retinal cells from the back of the eye may also contribute to the
phenotype has not been ruled out (Take-uchi, 2003).
Although there are clear similarities in the phenotypes following
abrogation of Vax function in mice and in fish, there are also differences.
For instance, midline defects are more severe in vax1 mutant mice
than in vax1/vax2 morphant fish. This of course could reflect true
differences in Vax protein function but there are other possibilities. Perhaps
the simplest would be that the vax1 MO does not remove all Vax1
function in fish. Although this possibility cannot be discounted, the severity
of the retinal expansion phenotype in the Vax1/Vax2 morphants (which is much
more severe than either vax1 or vax2 mutant mice) and the
penetrance of the coloboma phenotype suggest that the MOs probably severely
abrogate Vax function. A further possibility is the presence of other Vax
genes in the fish genome. Indeed, it appears that expression of the known Vax
genes is initiated a little later in fish than in other vertebrates, raising
the question of whether another Vax gene, perhaps with early expression and a
stronger role in midline development, might exist. The ongoing sequencing of
the fish genome should soon allow a resolution of this issue (Take-uchi, 2003),
The development of optic stalk neuroepithelial cells depends on Hedgehog (Hh) signaling, yet the source(s) of Hh protein in the optic stalk is unknown. Genetic evidence that Sonic hedgehog from retinal ganglion cells (RGCs) promotes the development of optic disc and stalk neuroepithelial cells. RGCs express Shh soon after differentiation, and cells at the optic disc in close proximity to the Shh-expressing RGCs upregulate Hh target genes, which suggests they are responding to RGC-derived Shh signaling. Conditional ablation of Shh in RGCs causes a complete loss of optic disc astrocyte precursor cells, resulting in defective axon guidance in the retina, as well as conversion of the neuroepithelial cells in the optic stalk to pigmented cells. Shh signaling modulates the size of the Pax2+ astrocyte precursor cell population at the optic disc in vitro. Together, these data provide a novel insight into the source of Hh that promotes neuroepithelial cell development in the mammalian optic disc and stalk (Dakubo, 2003).
Two distinct inductive events govern vertebrate eye development: an initial morphogenetic process results in regional specification of the various eye structures, and cells in these defined anatomic domains then differentiate to acquire their respective fates and functions in the mature eye. In mice, the morphogenetic phase begins at about embryonic day 8.5 (E8.5) with the lateral outgrowth of the prosencephalon to form the optic vesicles. By midgestation, the optic vesicle contacts and induces the formation of a lens placode from the overlying surface ectoderm, and simultaneously invaginates to form the bilayered optic cup, which is connected to the diencephalon by the optic stalk. The invagination of the optic vesicle extends proximally to include the ventral portion of the distal optic stalk, creating a transient opening (optic fissure) through which the hyaloid vessels gain access into the retina. All the cells of the optic stalk express the homeobox transcription factor Pax2, and some of these cells protrude into the retina and persist into late embryogenesis as a cuff of cells that form an annulus around exiting RGC axons. These cells separate the axons from the retinal neuroepithelium and the potential subretinal space. Subsequent differentiation of neuroepithelial cells in the optic vesicle depends on its interaction with other neural and non-neural tissues. For example, neuroretinal differentiation requires FGF signaling from the surface ectoderm, whereas pigment epithelial specification depends on sustained expression of the microphthalmia-associated transcription factor Mitf, which is maintained by activin-like signaling from the extra-ocular mesenchyme. In addition, the induction of Pax2 expression in neuroepithelial cells in the optic disc and stalk is necessary for their specification as glial cells (Dakubo, 2003).
Optic stalk neuroepithelial cell development as astroglia requires their
interaction with RGC axons. Classical embryological studies demonstrate that RGC axon
invasion of the optic stalk is associated with increased neuroepithelial cell proliferation, and survival and transformation into glial lineage precursor
cells. Moreover, the failure of axons to invade the optic stalk,
as observed in ZRDCT-An mice with inherited optic nerve aplasia and
ocular retardation mutant mice, as well as in Pax6-/- and Math5-/- mutant mice, results in abortive neuroepithelial cell
development in the optic stalk. These studies emphasize the critical
requirement of RGC axons in the induction and maintenance of gliogenesis in the optic stalk. The growth cones of RGCs have been shown to contain clusters
of small axoplasmic vesicles, which might contain factors that signal to cells in the
optic disc and stalk, with which they make tight contacts en route to the brain. Although it is well established that RGC axons are
necessary for the normal development of optic disc and stalk cells, the
signals that mediate this RGC axon-to-neuroepithelial cell interaction are unknown (Dakubo, 2003).
This study investigates the role of Shh from RGCs in optic disc and stalk
neuroepithelial cell development. Hh genes are important regulators of ocular morphogenesis and cellular diversification in several species examined. In the early somite stage mammalian embryo, Shh from the prechordal plate, and subsequently
from the ventral forebrain neuroepithelium, patterns
ventral forebrain structures including the hypothalamus and optic vesicles.
At later developmental stages, Shh and Ihh are expressed in
an overlapping temporal fashion but in distinct spatial domains of the rodent
eye. Although a group of peri-ocular mesenchymal cells express
Ihh at about E12, Shh is expressed in the emerging RGC layer. Shh signaling from RGCs regulates the proliferation, differentiation and organization of retinal neuroblasts, and, in
zebrafish, also drives neurogenesis across the retina (Dakubo, 2003).
Hh proteins are also axon-associated molecules in the visual systems of both invertebrate and vertebrate species. In the fly, Hh
transmitted along retinal axons induces neurogenesis and synaptic cartridge organization in the brain, whereas Shh from RGCs regulates astrocyte proliferation in the rodent optic nerve. Recent biochemical analysis of adult hamster ocular and brain tissues provides further support for a possible anterograde transport of Shh in the mammalian visual system. At about E12 of mouse development, neuroepithelial cells in the optic stalk express Ptch and Gli in the absence of Hh mRNA expression. The source of Hh in the optic nerve at this developmental stage is unclear. However, given that Hh proteins may be axonally transported, it is not inconceivable that Shh may be associated with the growth cones or axolema of RGCs and made accessible to neuroepithelial cells in the optic stalk. In addition, optic disc neuroepithelial cells express Hh target genes whereas differentiated RGCs express Shh, which suggests that RGC-derived Shh could signal to neuroepithelial cells at the optic disc. To investigate these two possibilities, a conditional gene ablation approach was used because Shh-knockout mice exhibit severe midline patterning defects and cyclopia. By successfully disrupting the Shh allele in regions of the CNS, including retinal precursor cells, prior to RGC differentiation, genetic evidence is provided for a requirement of RGC-derived Shh signaling in the differentiation of optic disc and stalk neuroepithelial cells (Dakubo, 2003).
Sonic hedgehog is involved in eye field separation along the proximodistal axis. As the optic vesicle and optic cup mature, Hh signalling continues to be important in defining aspects of the proximodistal axis. Two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. This zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, the pathway was activated by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium (Perron, 2003).
Hedgehog (Hh) signaling is required for eye development in vertebrates; known roles in the zebrafish include regulation of eye morphogenesis and ganglion cell and photoreceptor differentiation. A temporally selective Hh signaling knockdown strategy was used (either antisense morpholino oligonucleotides or the teratogenic alkaloid cyclopamine) in order to dissect the separate roles of Hh signaling arising from specific sources. Also, the eye phenotype was examined of zebrafish slow muscle-omitted (smu) mutants, which lack a functional smoothened gene which encodes a component of the Hh signal transduction pathway. Hh signaling from extraretinal sources is found to be required for the initiation of retinal differentiation, but this involvement may be independent of the effects of Hh signaling on optic stalk development. Hh signals from ganglion cells participate in propagating expression of ath5. It is suggested that the effects of Hh signals from the retinal pigmented epithelium on photoreceptor differentiation may be mediated by the transcription factor rx1 (Stenkamp, 2003).
The results of this study suggest that the effects of Hh signaling on photoreceptor development may involve the transcription factor rx1, and further confirm that Hh signaling from the retinal pigment epithelium (RPE) is primarily implicated. Antisense injections delivered at 51 hpf generated some of the same retinal phenotypes as antisense injections delivered at earlier time points, indicating that interference with Hh signaling rather late in development is sufficient to interfere with photoreceptor differentiation. Knockdown of Hh signaling with antisense-MO consistently resulted in failed rx1 expression in the outer nuclear layer (ONL), while crx expression was unaffected, further supporting the hypothesis that Hh signaling may influence photoreceptor differentiation via the transcription factor rx1. The rx gene product has been shown to participate in regulating photoreceptor-specific gene expression in cell-free systems. The chicken homolog of zebrafish rx1/2, RaxL, is involved in the early stages of photoreceptor differentiation. To confirm the proposed interaction in zebrafish it will be important to demonstrate that rx1 expression regulates photoreceptor differentiation (opsin expression) in vivo. One alternative to this hypothesis is that effects of reduced Hh signaling on rx and opsin genes are related manifestations of a photoreceptor maturation defect (Stenkamp, 2003).
The smu-/- embryos similarly show reduced expression of photoreceptor markers, and lack of rx1 in the photoreceptor layer. Interestingly, many of the smu-/- embryos develop normally laminated retinas. It is suspected that these mutants are those that had sufficient maternal smoothened expression to initiate retinal retinal differentiation, but lacked functional (zygotic) Smoothened at the time of Hh signaling from the RPE. In these mutants, it would be predicted that the only notable retinal defects would be those related to Hh signaling from ganglion cells and RPE. A fraction of the mutants showed a small patch of rx1 and rod opsin expression in the ventronasal ONL, suggesting that this region of retina may have requirements for cell differentiation that are distinct from the rest of the retina. This is consistent with the proposal that the ventral retina of the embryonic zebrafish comprises a discrete domain, influenced primarily by signals originating outside the eye, while the differentiation of the remainder of the retina requires the propagation of additional Hh, and other signals, from within the eye (Stenkamp, 2003).
The embryonic chick has the ability to regenerate its retina after it has been completely removed. A detailed characterization of retina regeneration in the embryonic chick has been carried out at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). Regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2. It can also be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE); ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, these results suggest that the hedgehog pathway is an important modulator of retina regeneration (Spence, 2004).
Neurogenesis in the zebrafish retina occurs in several waves of differentiation. The first neurogenic wave generates ganglion cells and depends on hedgehog (hh) signaling activity. Using transgenic zebrafish embryos that express GFP under the control of the sonic hedgehog (shh) promoter, the differentiation wave in the retina was imaged. In addition to the wave in the ganglion cell layer, shh expression also spreads in the inner nuclear layer. This second wave generates amacrine cells expressing shh, and although the second wave overlaps temporally with the first, it does not depend on it, as it occurs in the absence of ganglion cells. Differentiation of cell types found in the inner and outer nuclear layers, as well as lamination of the retina, depends on shh. By performing mosaic analysis, it has been demonstrated that Shh directs these events as a short-range signal within the neural retina (Shkumatava, 2004).
The work presented here indicates that shh signaling is required for the differentiation of all cell types found in the inner nuclear layer, including amacrine cells, bipolar cells, Mueller glia and horizontal cells. In fact, these cell types appear to be more sensitive to a reduction in shh signaling than are RGCs; the markers for differentiated bipolar cells (PKC) and Mueller glia (glutamine synthetase) are completely absent in shh mutant embryos, and a marker for differentiated amacrine cells (GAD67) is almost absent in shh mutant embryos. RGCs, however, are only partially depleted in shh mutant embryos and are completely lost only upon further reduction of Hh signaling with cyclopamine, probably due to the activity of twhh, which is also expressed in amacrine cells (Shkumatava, 2004).
Although shh is required for the differentiation of all major cell types in the retina, including glial cells, it does not appear to impart any information concerning which cell fate is adopted by the responding cells, and hence it appears to function simply as a differentiation promoting factor in the retina. The loss of differentiated cells is observed both in the central and peripheral regions of the retina in shh mutants, indicating that Shh is required in both of these domains (Shkumatava, 2004).
Taste papillae are ectodermal specializations that serve to house and distribute the taste buds and their renewing cell populations in specific locations on the tongue. Sonic hedgehog (Shh) has a major role in regulating the number and spatial pattern of fungiform taste papillae on embryonic rat tongue, during a specific period of papilla formation from the prepapilla placode. The Shh protein and the Patched receptor protein (Ptc) have been immunolocalized, and a potential role for Shh has been tested in formation of the tongue, emergence of papilla placodes, development of papilla number and size, and maintenance of papillae after morphogenesis is advanced. Cultures of entire embryonic mandible or tongues from gestational days 12 to 18 [gestational or embryonic days (E)12-E18] were used, in which tongues and papillae develop with native spatial, temporal, and molecular characteristics. The Shh signaling pathway was disrupted with addition of cyclopamine, jervine, or the 5E1 blocking antibody. Shh and Ptc proteins are diffuse in prelingual tissue and early tongue swellings, and are progressively restricted to papilla placodes and then to regions of developing papillae. Ptc encircles the dense Shh immunoproduct in papillae at various stages. When the Shh signal is disrupted in cultures of E12 mandible, tongue formation is completely prevented. At later stages of tongue culture initiation, Shh signal disruption alters development of tongue shape (E13) and results in a repatterned fungiform papilla distribution that does not respect normally papilla-free tongue regions (E13-E14). Only a few hours of Shh signal disruption can irreversibly alter number and location of fungiform papillae on anterior tongue and elicit papilla formation on the intermolar eminence. However, once papillae are well formed (E16-E18), Shh apparently does not have a clear role in papilla maintenance, nor does the tongue retain competency to add fungiform papillae in atypical locations. These data not only provide evidence for inductive and morphogenetic roles for Shh in tongue and fungiform papilla formation, but also suggest that Shh functions to maintain the interpapilla space and papilla-free lingual regions. A model is proposed for Shh function at high concentration to form and maintain papillae and, at low concentration, to activate between-papilla genes that maintain a papilla-free epithelium (Liu, 2004).
In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye (Masai, 2005).
Pulse treatment results suggest that Hh signalling between 24 and 29 hpf is required for the wave of atonal homolog ath5 expression. Furthermore, the introduction of shh and twhh morpholino-antisense oligonucleotides blocks neuronal production in the retina. These data suggest that Shh and Twhh regulate the progression of ath5 expression between 24 and 29 hpf. However, it is difficult to detect shh and twhh mRNA expression in the neural retina at this early stage, although it was reported that shh RNA is expressed in the retina at 28 hpf. One possibility is that Shh and Twhh expressed in the ventral forebrain may have a long-range action on progenitor cells of the optic cup. It was reported that Hh expressed in midline tissue is important for proliferation of the developing forebrain in chicks and mice, probably through its long-range actions. The most recent study on Hh signalling in the zebrafish retina also suggested that Hh signalling outside the optic cup regulates ath5 expression before 27 hpf. However, this is not considered as the most likely explanation, since the wave of ath5 expression normally occurs when the optic cups are dissected from the forebrain at 18 hpf, and cultured as an explant later, suggesting that a source of Hh signals is localised within the optic cup. Furthermore, when the dissected eye cup was divided into two (the nasal and temporal halves) only the nasal half expressed ath5, suggesting that short-range Hh signalling acts from the nasal to temporal regions across the neural retina. Transplantation of hh-MO-injected cells into wild-type host retinas demonstrated that ath5 expression is rarely observed in hh-MO-injected retinal columns, and that wild-type cells fail to express ath5 when they are located adjacent to the temporal side of Shh- and Twhh-deprived cells. These data suggest that a short-range action of Shh and Twhh expressed in the neural retina regulates the wave of ath5 expression and neuronal production. Low levels of Shh and Twhh expression may spread to the temporal retina up until 27 hpf and may be sufficient to regulate the wave of ath5 expression (Masai, 2005).
Does Hh function as a mitogen or an anti-mitogen in the vertebrate retina? In this study, it is proposed that Hh signals induce the cell-cycle exit of retinal progenitor cells. However, several studies have suggested that Hh functions as a mitogen for neural stem cells in the retina and in the brain, for astrocyte precursor cells in the optic stalk, and for granule cell precursors in the cerebellum. The observation of such an opposite phenotype may be due to the difference in cell types or the species used in the studies. Otherwise, the difference in the dose of Hh signals may cause the opposite behaviour of retinal progenitor cells. For example, a low dose of Hh signals as an anti-mitogen promotes neuronal production, whereas a high dose of Hh signals inhibits the differentiation of early-born cell types to maintain a pool of mitotic cells for the generation of late-born cell types. In Drosophila eyes, Hh regulates not only neuronal differentiation by inducing the proneural gene atonal, but also proliferation by inducing cyclin D/E. Such dual roles of Hh as a mitogen and an anti-mitogen may coordinate proliferation with cell differentiation in the vertebrate retina (Masai, 2005).
The mitogenic role of Hh has been proposed from in vitro experiments carried out using cell pellets or explant culture. Recent in vivo analyses of the role of Hh in the vertebrate retina showed different phenotypes of retinal neurogenesis. In conditional shh knock-out mice, Müller glial cells fail to differentiate properly and the outer photoreceptor layer shows a rosette structure. In frog retina treated with the Hh inhibitor cyclopamine, retinal progenitor cells are normal and only differentiation of the pigmented epithelium is perturbed. No defects were detected in the differentiation of the pigment epithelium in zebrafish embryos treated with forskolin or in smu–/– embryos. The roles of Hh signalling in retinal development may be diverse among different species of vertebrates. In the future, it will be important to elucidate why retinal cells show such different behaviours in response to Hh signals (Masai, 2005).
In the developing spinal cord and telencephalon, ventral patterning involves the interplay of Hedgehog (Hh), Retinoic Acid (RA) and Fibroblast Growth Factor (FGF) signaling. In the eye, ventral specification involves Hh signaling, but the roles of RA and FGF signaling are less clear. By overexpression assays in Xenopus embryos, it was found that both RA and FGF receptor (FGFR) signaling ventralize the eye, by expanding optic stalk and ventral retina, and repressing dorsal retina character. Co-overexpression experiments show that RA and FGFR can collaborate with Hh signaling and reinforce its ventralizing activity. In loss-of-function experiments, a strong eye dorsalization is observed after triple inhibition of Hh, RA and FGFR signaling, while weaker effects are obtained by inhibiting only one or two of these pathways. These results suggest that the ventral regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling. It is argued that similar mechanisms might control ventral neural patterning throughout the central nervous system (Lupo, 2005).
Dorsoventral (DV) patterning of the vertebrate eye underlies important properties of the visual system. First, it controls subdivision of the eye into optic stalk (OS) and retina, which form from the ventromedial and the dorsolateral parts of the optic vesicle, respectively. Second, the retina itself is patterned along the DV axis. A landmark of retina DV polarity is the choroid fissure at the ventral pole of the retina. Retinal neurogenesis is initiated in this region and then progresses to the dorsal retina (DR). DV asymmetries also exist in the distribution of differentiated cell types within the retina. Finally, ganglion cells located in the dorsal retina and the ventral retina (VR) send their axons to the lateral and medial optic tectum, respectively, creating an inverted topographic map of retinotectal projections (Lupo, 2005).
A model of ventral eye specification is proposed that involves interactions among RA, Hh and FGFR signaling pathways. According to this model, high levels of Hh and FGFR signaling interact with low levels of RA signaling to specify the OS by repressing retina-determination genes such as Pax6, and promoting the expression of Vax1 and Pax2. By contrast, high levels of RA act in concert with lower levels of Hh and FGFR signaling to specify the VR by repressing DR-specific genes such as ET and by inducing the expression of Vax2 in the presence of Pax6, but not Vax1 and Pax2. BMP signaling specifies DR regions by repressing Vax2 and inducing ET and other members of the Tbx gene family, such as Tbx5. In vivo, ventrodorsal (ventral high) gradients of Hh and FGFR signaling may be created by diffusion of Hh and FGF signals from their sources in the anteromedial neural plate. The regulation of RA gradients is more complex and the localization of different anabolic and catabolic enzymes needs to be considered. However, early expression of Raldh2 and Raldh3 appears to be localized close to the presumptive ventral eye, and the mediolateral gradient (lateral high) of Raldh2 expression in the ANR may contribute to create higher RA levels in the VR compared with the OS region (Lupo, 2005).
Recent studies have shown that ventral patterning in the spinal cord and the telencephalon involves interactions between Hh, RA and FGF signaling pathways. In the spinal cord, motoneurons and V3, V2 ventral interneurons originate from the ventral neural tube, while V1 and V0 ventral interneurons originate from more intermediate regions. Hh signals from the notochord and floorplate are thought to specify the progenitor domain of motoneurons, V3 and V2 interneurons, while RA signaling is crucial for the specification of V1 and V0 interneurons. In addition, although FGF signaling appears to function as a general repressor of ventral neural patterning, RA and FGF in combination can efficiently induce motoneuron progenitors both in explants and in vivo (Lupo, 2005).
In the telencephalon, the medial ganglionic eminence (MGE) originates from the ventral part of the telencephalic vesicle, while the lateral ganglionic eminence (LGE) originates from a more intermediate region. Hh signaling is involved in the specification of the MGE, while RA signaling appears to play a crucial role in the specification of the LGE. In addition, FGF signaling is involved in the specification of ventral, but not intermediate, telencephalic fates (Lupo, 2005).
In the developing eye, cells located more ventrally in the anlage give rise to the OS, while the VR originates from a more intermediate region. Hh and FGFR signaling play a crucial role in OS specification, although low levels of RA signaling may also be involved. Moreover, RA signaling could control specification of the VR in collaboration with low levels of Hh and possibly FGFR signaling (Lupo, 2005).
Similar mechanisms of ventral specification involving Hh, RA and FGFR signaling pathways appear to be at least partially conserved in different CNS regions. Several questions remain to be addressed concerning the precise role and the mechanism of action of these signaling systems. Clearly, DV patterning of the vertebrate CNS is a complex process, and the eye, because of its distinct regional composition, its finely graded topography and its experimental accessibility, is an exciting model with which to study how different signaling pathways interact to execute specific developmental programs (Lupo, 2005).
Ventral midline Sonic Hedgehog (Shh) signalling is crucial for growth and patterning of the embryonic forebrain. This study reports how enhanced Shh midline signalling affects the evolution of telencephalic and diencephalic neuronal patterning in the blind cavefish Astyanax mexicanus, a teleost fish closely related to zebrafish. A comparison between cave- and surface-dwelling forms of Astyanax shows that cavefish display larger Shh expression in all anterior midline domains throughout development. This does not affect global forebrain regional patterning, but has several important consequences on specific regions and neuronal populations. (1) Expanded Nkx2.1a expression and higher levels of cell proliferation are shown in the cavefish basal diencephalon and hypothalamus. (2) Nkx2.1b-Lhx6-GABA-positive migratory pathway from the subpallium to the olfactory bulb was uncovered that is increased in size in cavefish. (3) Heterochrony and enlarged Lhx7 expression was observed in the cavefish basal forebrain. These specific increases in olfactory and hypothalamic forebrain components are Shh-dependent and therefore place the telencephalic midline organisers in a crucial position to modulate forebrain evolution through developmental events, and to generate diversity in forebrain neuronal patterning (Menuet, 2007).
Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, it has now been determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain (Geng, 2008).
Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal-distal and dorsal-ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, the Smoothened (Smo) conditional knockout (CKO) mouse line was used. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. Expression was examined of various genes, such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. These data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology (Zhao, 2010).
In zebrafish, Hedgehog (Hh) signalling from ventral midline structures is necessary and sufficient to specify posterior otic identity. Loss of Hh signalling gives rise to mirror symmetric ears with double anterior character, whereas severe upregulation of Hh signalling leads to double posterior ears. By contrast, in mouse and chick, Hh is predominantly required for dorsoventral otic patterning. Whereas a loss of Hh function in zebrafish does not affect dorsoventral and mediolateral otic patterning, this study shows that a gain of Hh signalling activity causes ventromedial otic territories to expand at the expense of dorsolateral domains. In a panel of lines carrying mutations in Hh inhibitor genes, Hh pathway activity is increased throughout the embryo, and dorsolateral otic structures are lost or reduced. Even a modest increase in Hh signalling has consequences for patterning the ear. In ptc1-/- and ptc2-/- mutant embryos, in which Hh signalling is maximal throughout the embryo, the inner ear is severely ventralised and medialised, in addition to displaying the previously reported double posterior character. Transplantation experiments suggest that the effects of the loss of Hh pathway inhibition on the ear are mediated directly. These new data suggest that Hh signalling must be kept tightly repressed for the correct acquisition of dorsolateral cell fates in the zebrafish otic vesicle, revealing distinct similarities between the roles of Hh signalling in zebrafish and amniote inner ear patterning (Hammond, 2010).
The cavefish morph of the Mexican tetra (Astyanax mexicanus) is blind at adult stage, although an eye that includes a retina and a lens develops during embryogenesis. There are, however, two major defects in cavefish eye development. One is lens apoptosis, a phenomenon that is indirectly linked to the expansion of ventral midline sonic hedgehog (Shh) expression during gastrulation and that induces eye degeneration. The other is the lack of the ventral quadrant of the retina. This study shows that such ventralisation is not extended to the entire forebrain because fibroblast growth factor 8 (Fgf8), which is expressed in the forebrain rostral signalling centre, is activated 2 hours earlier in cavefish embryos than in their surface fish counterparts, in response to stronger Shh signalling in cavefish. It was also shown that neural plate patterning and morphogenesis are modified in cavefish, as assessed by Lhx2 and Lhx9 expression. Inhibition of Fgf receptor signalling in cavefish with SU5402 during gastrulation/early neurulation mimics the typical surface fish phenotype for both Shh and Lhx2/9 gene expression. Fate-mapping experiments show that posterior medial cells of the anterior neural plate, which lack Lhx2 expression in cavefish, contribute to the ventral quadrant of the retina in surface fish, whereas they contribute to the hypothalamus in cavefish. Furthermore, when Lhx2 expression is rescued in cavefish after SU5402 treatment, the ventral quadrant of the retina is also rescued. It is proposed that increased Shh signalling in cavefish causes earlier Fgf8 expression, a crucial heterochrony that is responsible for Lhx2 expression and retina morphogenesis defect (Pottin, 2011).
Specification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20 somite (early otic vesicle) stages. This study shows that Fgf signalling is both necessary and sufficient for anterior otic specification during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia-/- (fgf3-/-) mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear (Hammond, 2011).
Sonic Hedgehog and ear development Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its
surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. The localization of Sonic hedgehog (Shh) protein and expression of the Shh gene has been examined in the tissues of the developing mouse inner ear. In cultures of periotic mesenchyme Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. Inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. These results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis (Liu, 2002).
Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the
otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain
unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell
fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh-/- embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia fail to develop in the absence of
Shh. The origin of the inner ear defects in Shh-/- embryos can be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. Several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, these data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh (Riccomagno, 2002).
The failure in cochlear duct outgrowth in Shh-/- embryos is most likely mediated by the lack of Pax2, Otx1, and Otx2, genes previously ascribed with required roles in this process. Furthermore, the observations that Shh is both necessary and sufficient for the expression of Pax2 along the medial wall of the otic vesicle implicates Pax2 as a downstream effector of Shh signaling in the otocyst. The
regulation of Pax2 by Shh in inner ear development resembles the relationship between Pax2 and Shh in the formation of
another placode-derived sensory organ, the eye. In generating the
proximal-distal axis of the optic cup, Shh signaling from the ventral
forebrain promotes Pax2-expressing proximal fates (optic
fissure, optic stalk) at the expense of Pax6-expressing distal
fates (prospective retina, pigmented epithelium, and lens.
To maintain the border between proximal and distal lineages, Pax2 and
Pax6 antagonize each other by mutual transcriptional repression. The commonality in response by Pax genes to Hh signaling can be broadened to include Pax1 in the ventral somite and Pax6 in the ventral neural tube. In both of these cases, Pax family members with opposing
functions are expressed adjacent to sites of Pax1 and Pax6 activity. This is not a general rule, since Pax genes are not expressed complementary to Pax2 in the inner ear, although other transcription factors may be fulfilling an antagonistic role in this tissue. The observations thus add to the growing list of functions for Pax transcription factors in mediating
cellular responses to Shh signaling (Riccomagno, 2002).
Currently, few factors have been identified that provide the inductive
signals necessary to transform the simple otic placode into the complex
asymmetric structure of the adult vertebrate inner ear. Evidence
that Hedgehog signalling from ventral midline structures acts directly on the
zebrafish otic vesicle to induce posterior otic identity. Two strong Hedgehog pathway mutants, chameleon (contf18b) and slow muscle omitted (smub641) exhibit a striking partial mirror image
duplication of anterior otic structures, concomitant with a loss of posterior
otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of
the Hedgehog pathway, by injection of sonic hedgehog or
dominant-negative protein kinase A RNA, has the reverse effect: ears
lose anterior otic structures and show a mirror image duplication of posterior
regions. By using double mutants and antisense morpholino analysis, it is also
shown that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in
anteroposterior patterning of the zebrafish otic vesicle (Hammond, 2003).
Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Both gain- and loss-of-function approaches reveal that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus. Ectopic activation of Hedgehog signaling results in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles is also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1DeltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1DeltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine results in the formation of enlarged otic vesicles. In summary, these observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos (Koerbernick, 2003).
Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, embryos were examined with various combinations of mutant alleles for Shh, Gli2 and Gli3. This study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, these data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear (Bok, 2007).
Sonic Hedgehog and gustatory (taste) papillae Taste buds on the anterior part of the tongue develop in conjunction with epithelial-mesenchymal specializations in the form of gustatory
(taste) papillae. Sonic hedgehog and BMP4 are expressed in developing taste papillae, but the roles of these signaling molecules in specification of taste bud progenitors and in papillary morphogenesis are unclear. BMP4
is not expressed in the early tongue, but is precisely coexpressed with Shh in papillary placodes, which serve as a signaling center for both
gustatory and papillary development. To elucidate the role of Shh, an in vitro model of mouse fungiform papillary development was used to determine the effects of two functional inhibitors of Shh signaling: anti-Shh (5E1) antibody and cyclopamine. Cultured E11.5 tongue explants express Shh and BMP4LacZ in a pattern similar to that of intact embryos, localizing to developing papillary placodes after 2 days
in culture. Tongues cultured with 5E1 antibody continue to express these genes in papillary patterns but develop more papillae that are larger
and closer together than in controls. Tongues cultured with cyclopamine have a dose-dependent expansion of Shh and BMP4LacZ expression domains. Also, both antibody-treated and cyclopamine-treated tongue explants are smaller than controls. Taken together, these results suggest that, although Shh is not involved in the initial specification of papillary placodes, Shh does play two key roles during pmcry development:
(1) as a morphogen that directs cells toward a nonpapillary fate, and (2) as a mitogen, causing expansion of the interplacodal epithelium
and underlying mesenchyme (Hall, 2003).
From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue
locations. Furthermore, on the anterior tongue the fungiform papillae are patterned in rows. Among the many molecules that have potential
roles in regulating papilla location and pattern, Sonic hedgehog has been localized within early tongue and developing papillae. An embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla
morphogenesis and patterning was used to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when
papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine
and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium.
Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine
structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers
doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium
or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla,
regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue
cultures with standard medium or cyclopamine, and is conspicuously localized in the basement membrane region of the papillae. Ptc protein has a distribution similar to Shh, although the immunoproduct is more diffuse. Fungiform papillae do not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor is development of the single circumvallate
papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in
morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed (Mistretta, 2003).
Sonic Hedgehog and tooth morphogenesis The expression of genes involved in the Sonic Hedgehog signalling pathway, including Shh, Ptc, Smo, Gli1, Gli2 and Gli3, were
found to be expressed in temporal and spatial patterns during early murine tooth development, suggestive of a role in early tooth germ
initiation and subsequent epithelial-mesenchymal interactions. Of these, all but Shh (Ptc, Smo, Gli1, Gli2 and Gli3) are expressed in epithelium
and mesenchyme whereas Shh is only detected in epithelium. This suggests that Shh is involved in both lateral
(epithelial-mesenchymal) and planar (epithelial-epithelial) signaling in early tooth development. Ectopic application of Shh protein to
mandibular mesenchyme induces the expression of Ptc and Gli1. Addition of exogenous Shh protein directly into early tooth germs
and adjacent to tooth germs, results in abnormal epithelial invagination, indicative of a role for Shh in epithelial cell proliferation. In
order to assess the possible role of this pathway, tooth development in Gli2 and Gli3 mutant embryos was investigated. Gli2 mutants have abnormal development of maxillary incisors, probably resulting from a mild holoprosencephaly, whereas Gli3
mutants have no major tooth abnormalities. Gli2/Gli3 double homozygous mutants do not develop any normal teeth and do not
survive beyond embryonic day 14.5; however, Gli2(-/-); Gli3(+/-) mice survive until birth and have small molars and mandibular
incisors, whereas maxillary incisor development is arrested as a rudimentary epithelial thickening. These results show an essential
role for Shh signaling in tooth development that involves functional redundancy of downstream Gli genes (Hardcastle, 1998).
The murine tooth development is governed by sequential
and reciprocal epithelial-mesenchymal interactions.
Multiple signaling proteins are expressed in the
developing tooth germ and interact with one another to mediate
the inductive tissue interactions. Among these proteins are Sonic
hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2)
and Bone Morphogenetic Protein-4 (BMP4). The interactions among these signaling
proteins during early tooth development have been investigated.
Bmp4 is expressed in dental epithelium at the initiation stage (E11.5) and
then shifts to the dental mesenchyme shortly afterwards at the
early bud stage (E12.5). This shift of Bmp4
expression pattern coincides with the shift in tooth
developmental potential between tissue layers, indicating that
BMP4 may constitute one component of the odontogenic
potential. Indeed, BMP4 can induce in the dental mesenchyme
the morphological changes and expression of a number of
genes, including the transcription factors Msx1, Msx2, Lef1 and
Bmp4 itself, mimicking the effect of the early dental
epithelium. The mesenchymally expressed Bmp4 is believed to
exert its function upon the dental epithelium as a feedback
signal for further tooth development.
The expression of Shh and Bmp2 is downregulated at E12.5
and E13.5 in the dental epithelium of the Msx1 mutant
tooth germ and Bmp4 expression is significantly reduced
in the dental mesenchyme. Inhibition of BMP4 activity by
noggin results in repression of Shh and Bmp2 in wild-type
dental epithelium. When implanted in the dental
mesenchyme of Msx1 mutants, beads soaked with BMP4
protein are able to restore the expression of both Shh and
Bmp2 in the Msx1 mutant epithelium. These results
demonstrate that mesenchymal BMP4 represents one
component of the signal acting on the epithelium to
maintain Shh and Bmp2 expression. In contrast, BMP4-
soaked beads repress Shh and Bmp2 expression in the
wild-type dental epithelium. Ectopic expression of human Bmp4 in
the dental mesenchyme driven by the mouse Msx1
promoter restores Shh expression in the Msx1 mutant
dental epithelium but represses Shh in the wild-type tooth
germ in vivo. This regulation
of Shh expression by BMP4 is conserved in the mouse
developing limb bud. In addition, Shh expression is
unaffected in the developing limb buds of the transgenic
mice in which a constitutively active Bmpr-IB is ectopically
expressed in the forelimb posterior mesenchyme and
throughout the hindlimb mesenchyme, suggesting that the
repression of Shh expression by BMP4 may not be
mediated by BMP receptor-IB. These results provide
evidence for a new function of BMP4. BMP4 can act
upstream to Shh by regulating Shh expression in mouse
developing tooth germ and limb bud. Taken together, these
data provide insight into a new regulatory mechanism for
Shh expression, and suggest that this BMP4-mediated
pathway in Shh regulation may have a general implication
in vertebrate organogenesis (Zhang, 2000a).
During mammalian tooth development, the oral ectoderm
and mesenchyme coordinate their growth and
differentiation to give rise to organs with precise shapes,
sizes and functions. The initial ingrowth of the dental
epithelium and its associated dental mesenchyme gives rise
to the tooth bud. Next, the epithelial component folds to
give the tooth its shape. Coincident with this process,
adjacent epithelial and mesenchymal cells differentiate
into enamel-secreting ameloblasts and dentin-secreting
odontoblasts, respectively. Growth, morphogenesis and
differentiation of the epithelium and mesenchyme are
coordinated by secreted signaling proteins. Sonic hedgehog encodes a signaling peptide that is present in the
oral epithelium prior to invagination and in the tooth
epithelium throughout its development. The role of Shh in the developing tooth has been addressed in mouse by using a
conditional allele to remove Shh activity shortly after
ingrowth of the dental epithelium. Reduction and then loss
of Shh function results in a cap stage tooth rudiment in
which the morphology is severely disrupted. The overall
size of the tooth is reduced and both the lingual epithelial
invagination and the dental cord are absent. However, the
enamel knot, a putative organizer of crown formation, is
present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in
the wild type. At birth, the size and the shape of the teeth
are severely affected and the polarity and organization
of the ameloblast and odontoblast layers is disrupted.
However, both dentin- and enamel-specific markers are
expressed and a large amount of tooth-specific extracellular
matrix is produced. This observation was confirmed by
grafting studies in which tooth rudiments were cultured for
several days under kidney capsules. Under these
conditions, both enamel and dentin were deposited even
though the enamel and dentin layers remained
disorganized. These studies demonstrate that Shh regulates
growth and determines the shape of the tooth. However,
Shh signaling is not essential for differentiation of either
ameloblasts or odontoblasts (Dassule, 2000).
These data contribute to the accumulating body of evidence
that the hedgehogs act as growth factors during embryonic and
postnatal life. Previous studies have demonstrated the role of
Shh in growth and cell proliferation in the skin, lung and
cerebellum. Null mutations in
Ptch, which is a negative regulator of Shh signaling, also lead
to over-proliferation of the skin, to basal cell carcinomas and
to widespread growth anomalies throughout the embryos. Studies in hair, an organ that shares a set of early,
developmentally conserved steps with the tooth, show that
complete absence of Shh leads to a reduction in growth, but
not to a complete arrest in growth (Dassule, 2000 and references therein).
Interestingly, the growth defects in Shh null teeth are not
proportionate. Thus, by 14.5 days of development, in Shh mutants, the
development of the lingual side of the tooth (towards the tongue) is more severely
affected than the buccal side (towards the cheek). Very little outer enamel
epithelium on the lingual side is present, and the lingual inner
enamel epithelium has not invaginated. This disruption in
morphogenesis suggests that Shh signaling patterns the
development of the tooth crown.
In wild-type and heterozygote mice at 14.5 days of development, highest
levels of Ptch expression are found in the outer enamel
epithelium and the adjacent stellate reticulum on the lingual
side of the cap, indicating that the Shh pathway is normally
transduced at high levels in these cells. Considering the role of
Shh in proliferation, it is possible that lateral invagination is
driven by the proliferation of the outer enamel epithelium and
of the stellate reticulum, although no increase in mitotic index
has been reported for this population of cells. In addition, Shh may direct the growth of stellate
reticulum downwards. The absence of Shh-directed growth in
the mutants may result in the absence of the dental cord. Last,
Ptch expression observed in the lingual epithelial invagination
during normal development suggests that Shh also signals
directly to cells lingual to the enamel knot, resulting in
ingrowth of the lingual epithelial invagination (Dassule, 2000).
An alternative view, which cannot be ruled out on the basis
of available data, is that Shh controls the balance between
proliferative and non-proliferative cell fates. This hypothesis
receives some support from the fact that, in the inner enamel
epithelium of the mutant, non-proliferating cells extend
several cell diameters on either side of the zone of apoptotic
cells in the enamel knot and express several enamel knot
markers, including Wnt10b. Thus, a role for Shh may be to
restrict the enamel knot to a specific population of cells that
expands at the expense of proliferating cells in Shh mutants.
The observation that Shh is essential for the development of
the lingual side of the cusp is of particular interest because it
provides the first evidence that a signaling protein expressed
in the enamel knot is involved in tooth morphogenesis.
Although the cellular mechanisms remain to be clarified,
these results raise the possibility of directional signaling
within the tooth, particularly with respect to the buccal-lingual
axis (Dassule, 2000).
Sonic hedgehog plays a key role during embryogenesis and organogenesis. Tooth development (odontogenesis) is governed by sequential and reciprocal
epithelial-mesenchymal interactions. The first visible indication of the initiation of odontogenesis is the appearance of a local thickening of the oral ectoderm, which subsequently grows into the underlying neural crest-derived mesenchyme of the first branchial arch
to form epithelial buds. Ectomesenchymal cells condense around the epithelial buds to form the dental mesenchyme. The primary enamel knot, a tooth signaling center, becomes prominent at the cap stage. At the bell stage, the tooth consists of an epithelial enamel organ (EEO) and
a dental mesenchyme. The EEO components include proliferating preameloblasts and their progenitors, the cells of the inner dental epithelium (IDE),
the secondary enamel knots at the tip of nascent cusps, the stellate reticulum (SR), the outer dental epithelium (ODE) and the stratum intermedium
(SI). The latter consists of squamous cells adjacent to the IDE and preameloblasts. Later-arising secondary enamel knots, which form in the
developing molars, are thought to control cuspal morphogenesis and terminal differentiation of odontoblasts. Mesenchymal
cells of the dental papilla that lie adjacent to the IDE form the preodontoblast layer of proliferating cells; the rest of the dental papilla cells contribute to the later development of the dental pulp. Finally, dental mesenchyme that surrounds the tooth germ forms the dental sac, which gives rise to periodontal tissues (Gritli-Linde, 2002).
During the cytodifferentiation stage, the terminal differentiation of odontoblasts is accomplished by their withdrawal from the cell cycle, elongation, polarization and secretion of a predentin matrix. This, in turn, triggers terminal differentiation of ameloblasts. Differentiation of the ameloblast into a highly-polarized complex secretory cell involves considerable growth, elongation of the
cytoplasm, a change in nuclear polarity, a sequential development and change in polarity of organelles, and the appearance of a complex cytoskeleton. During this stage, there are other progressive changes within the enamel organ. Cells from the SI that are adjacent to polarizing
post-mitotic ameloblasts become cuboidal in shape, except in the future enamel-free areas in rodent molars. In addition, the SR is invaded by blood vessels and fibroblasts emanating from the dental sac (Gritli-Linde, 2002 and references therein).
The rodent incisor is unique in its tissue organization and consists of stem cells, differentiating cells and mature cells organized in defined regions along
its anteroposterior and labial-lingual axes. The rodent incisor is asymmetrical, as the labial or amelogenic IDE gives rise to ameloblasts and enamel, whereas the IDE on the lingual side does not produce enamel. The posterior-most aspect of the incisor has been postulated to contain stem cells
which give rise to the different dental cell populations. A posteroanterior gradient of cytodifferentiation is thus present in the rodent incisor throughout life, with the less differentiated cells located posteriorly and the most mature cells
anteriorly. Odontoblasts differentiate all along the epitheliomesenchymal interface of the incisor (Gritli-Linde, 2002 and references therein).
Like many organs, morphogenesis and cytodifferentiation of the tooth is governed by sequential and reciprocal epithelialmesenchymal interactions
mediated by several soluble bioactive proteins. In addition,
cell-matrix interactions and cell-cell junctional complexes and cytoskeletal components have been implicated in the regulation of histomorphogenesis
and proliferation. Sonic hedgehog signals are received
within a target tissue by the general Hedgehog receptor Patched 1 (Ptc1). Transduction of the signal within a responding cell absolutely requires the activity of a
second, multi-pass, membrane protein Smoothened (Smo). Smoothened activity leads to a conserved transcriptional response: up-regulation of Ptc1
and Gli1 in the target tissue (Gritli-Linde, 2002).
Shh is expressed exclusively in the epithelial component of the murine tooth from the dental lamina
stage until cytodifferentiation. At the cap stage, Shh is confined to the primary enamel knot. Expression spreads thereafter to the rest of the IDE laterally, the stratum intermedium and the stellate reticulum. At the cap stage, general transcriptional targets and effectors of Shh signaling, including Ptc1, Gli1 and
Smoothened (Smo) are, however, expressed in both dental epithelium and mesenchyme, but are excluded from the enamel knot. In contrast, Ptc2, while appearing to bind all mammalian Hedgehog proteins similarly to the related Hedgehog receptor Ptc1, is expressed in the enamel knot and IDE at the cap and early bell stages, respectively (Gritli-Linde, 2002).
Shh protein produced by the enamel knot and IDE moves many cell diameters to reach the rest of the dental
epithelium and the dental papilla, indicating that Shh has a long-range activity, consistent with the broad expression of Shh target genes. Together, the above observations suggest that Shh signaling may be operative intra-epithelially as well as in mediating epithelial-mesenchymal interactions during tooth development. Finally, genetic removal of Shh activity from the tooth leads to alterations in growth and morphogenesis and results in tissue disorganization, affecting both the dental epithelium and mesenchyme derivatives. It has not been possible to determine clearly whether the alterations in the dental mesenchyme and its derivatives
are solely generated by lack of Shh signaling by the dental epithelium, or whether they are secondary to the lack of proper signaling (via other
bioactive molecules) in the abnormal dental epithelium. Conversely, this approach has left unanswered the question of whether abnormal development
of the epithelial enamel organ in Shh mutant teeth is a result of a loss of intra-epithelial Shh signaling, or a secondary consequence of altered
signaling by the underlying dysplastic dental mesenchyme. In order to distinguish between these alternatives, and further define the roles of Shh in
regulating morphogenesis of the tooth, Shh signal transduction was abrogated by genetically removing the activity of Smo from the dental epithelium
and its derivatives while maintaining Shh responsiveness in the dental mesenchyme (Gritli-Linde, 2002).
Genetic removal of Shh activity from the dental epithelium, the sole source of
Shh during tooth development, alters tooth growth and cytological organization within both the dental epithelium and mesenchyme of the tooth. As explained, it has not been clear which aspects of the phenotype are the result of the
direct action of Shh on a target tissue and which are indirect effects due to deficiencies in reciprocal signalings between the epithelial and mesenchymal
components. To distinguish between these two alternatives and extend understanding of Shh's actions in odontogenesis, the
Cre-loxP system was used to remove Smoothened (Smo) activity in the dental epithelium. Smo, a seven-pass membrane protein is essential for the
transduction of all Hh signals. Hence, removal of Smo activity from the dental epithelium should block Shh signaling within dental epithelial derivatives
while preserving normal mesenchymal signaling. This study shows that Shh-dependent interactions occur within the dental epithelium itself. The dental
mesenchyme develops normally up until birth. In contrast, dental epithelial derivatives show altered proliferation, growth, differentiation and
polarization. This approach uncovers roles for Shh in controlling epithelial cell size, organelle development and polarization. Furthermore, evidence is provided that Shh signaling between ameloblasts and the overlying stratum intermedium may involve subcellular localization of Patched 2 and Gli1
mRNAs, both of which are targets of Shh signaling in these cells (Gritli-Linde, 2002).
The signalling peptide encoded by the sonic hedgehog gene is restricted to localised thickenings of oral epithelium, which mark the first morphological evidence of tooth development, and is known to play a crucial role during the initiation of odontogenesis. At these stages in the murine mandibular arch in the absence of epithelium, the Shh targets Ptc1 and Gli1 are upregulated in diastema mesenchyme, an edentulous region between the sites of molar and incisor tooth formation. This ectopic expression is not associated with Shh transcription but with the presence of ectopic Shh protein, undetectable in the presence of epithelium. These findings suggest that, in diastema mesenchyme, restriction of Shh activity is dependent upon the overlying epithelium. This inhibitory activity was demonstrated by the ability of transplanted diastema epithelium to downregulate Ptc1 in tooth explants, and for isolated diastema mesenchyme to express Ptc1. A candidate inhibitor in diastema mesenchyme is the glycosylphosphatidylinositol-linked membrane glycoprotein Gas1. Gas1 is normally expressed throughout mandibular arch mesenchyme; however, in the absence of epithelium this expression was downregulated specifically in the diastema where ectopic Shh protein was identified. Although Shh signalling has no effect upon Gas1 expression in mandibular arch mesenchyme, overexpression of Gas1 results in downregulation of ectopic Ptc1. Therefore, control of the position of tooth initiation in the mandibular arch involves a combination of Shh signalling at sites where teeth are required and antagonism in regions destined to remain edentulous (Cobourne, 2004).
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hedgehog
continued:
Biological Overview
| Regulation
| Targets of Activity
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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