Distal-less
Somitic segmentation provides the framework on which is established the segmental pattern of the vertebrae, some muscles and the peripheral nervous system. Recent evidence indicates that a molecular oscillator, the 'segmentation
clock', operates in the presomitic mesoderm (PSM) to
direct periodic expression of c-hairy1 and lunatic fringe (l-fng). The identification and
characterization of a second avian hairy-related gene, c-hairy2,
is reported, that also cycles in the PSM and whose sequence
is closely related to the mammalian HES1 gene, a
downstream target of Notch signaling in vertebrates. HES1 mRNA is also expressed in a cyclic fashion
in the mouse PSM, similar to that observed for c-hairy1 and
c-hairy2 in the chick. In HES1 mutant mouse embryos, the
periodic expression of l-fng is maintained, suggesting that
HES1 is not a critical component of the oscillator
mechanism. In contrast, dynamic HES1 expression is lost
in mice mutant for Delta1 that are defective for Notch
signaling. In order to investigate the relationship between the dynamic
HES1 expression in the PSM and the Notch signaling
pathway, HES1 expression was examined in Dll1
homozygous mutant mice in which Notch activation is
impaired in the PSM. Homozygous null mutants for the Dll1
gene exhibit strong segmentation defects and a severe down-regulation
of l-fng expression. The expression of HES1 at E10.5 was compared in wild type, heterozygous and
homozygous null mutants by in situ hybridization. The dynamic expression of HES1 in the PSM is
maintained in Dll1+/- embryos as shown by the different
expression patterns observed in the PSM. In contrast, all
Dll1-/- embryos show the same global downregulation of
HES1 expression in the PSM (n=7). This observation
suggests that HES1 expression in the PSM is dependent on
the Notch signaling pathway and suggests that Notch signaling is
required for hairy-like genes cyclic expression in the PSM (Jouve, 2000).
Thus HES1 and l-fng dynamic expression are lost in the PSM of Dll1 mutants, in which Notch signaling is disrupted. Various clock outputs appear, therefore, to be severely downregulated when Notch signaling
is disrupted. These observations raise the possibility that in
addition to being an output of the segmentation clock as
previously proposed, the Notch signaling pathway
might also be an important component of the oscillator.
Notch activation upon ligand binding involves a proteolytic
cleavage liberating the intracytoplasmic domain (NICD),
which translocates into the nucleus where together with
Su(H)/RBPjk it activates the transcription of genes such as
HES1 in vertebrates. The observations in the Dll1-/- mice
indicate that HES1 is downstream of the Notch pathway in the
PSM. Since c-hairy1 and c-hairy2 share a high similarity in
their sequence and in their expression patterns to HES1, they
are also likely targets of Notch signaling in the chick PSM. A
direct regulation of c-hairy1 and c-hairy2 expression by
oscillating Notch activation would explain why c-hairy1
expression is insensitive to cycloheximide, since protein
synthesis is not required for transduction of the Notch signal.
To achieve oscillations of Notch signaling, the activity of
the pathway would need to be modulated by a feedback
mechanism. However, known output events resulting from
Notch signaling are transcriptional regulation of target genes
and the clock is partly independent of protein synthesis. Notch1
and Delta1 are present along the whole presomitic mesoderm
and could generate constitutive activation of the pathway in the
tissue. The rhythmic modification of
this activation could, in principle, be achieved by the periodic
expression of l-fng (Jouve, 2000).
Dlx3 is a homeodomain transcription factor and a member of the vertebrate Distal-less family. Targeted
deletion of the mouse Dlx3 gene results in embryonic death between day 9.5 and day 10 because of
placental defects that alter the development of the labyrinthine layer. In situ hybridization reveals that the
Dlx3 gene is initially expressed in ectoplacental cone cells and chorionic plate, and later in the labyrinthine
trophoblast of the chorioallantoic placenta, where major defects are observed in the Dlx3 -/- embryos. The
expression of structural genes, such as 4311 and PL-1, which were used as markers to follow the fate of
different derivatives of the placenta, are not affected in the Dlx3-null embryos. However, by day 10.5 of
development, expression of the paired-like homeodomain gene Esx1 is strongly down-regulated in
affected placenta tissue, suggesting that Dlx3 is required for the maintenance of Esx1 expression, normal
placental morphogenesis, and embryonic survival (Morasso, 1999).
Ectodermal patterning (demarcation of the position of the ectoderm) of the chick embryo begins while it is still in the uterus and continues during gastrulation, when cells with a neural fate become restricted to the neural plate around the primitive streak, and cells fated to become the epidermis are restricted to the periphery. The prospective epidermis at early stages is characterized by the expression of the homeobox gene DLX5, which remains an epidermal marker during gastrulation and neurulation. Later, some DLX5-expressing cells become internalized into the ventral forebrain and the neural crest at the hindbrain level. The mechanism of ectodermal demarcation was studied by transplantation of Hensen's nodes and prechordal plates. The DLX5 marker indicates that not only a neural plate, but also a surrounding epidermis is induced in such operations. Similar effects can be obtained with neural plate grafts. These experiments demonstrate that the induction of a DLX5-positive epidermis is triggered
by the midline, and the effect is transferred via the neural plate to the periphery. By repeated extirpations of the endoderm, the formation of an endoderm/mesoderm layer under the epiblast was suppressed. This led to the generation
of epidermis, and to the inhibition of neuroepithelium in the naked ectoderm. This suggests a signal necessary for
neural, but inhibitory for epidermal development, normally coming from the lower layers. BMP4, as well as BMP2, is capable of inducing epidermal fate by distorting the epidermis-neural plate boundary.
This, however, does not happen independently within the neural plate or outside the normal DLX5 domain. In the area opaca, the co-transplantation of a BMP4 bead with a node graft leads to the induction of DLX5, thus indicating the
cooperation of two factors. It is concluded that ectodermal demarcation is achieved by signaling both from the midline and
from the periphery, within the upper but also from the lower layers. It is suggested that BMP2 is the vertical signal from the underlaying germ layers; BMP4 could act from the ectoderm itself. Induction of neural plate requires a postive signal from the midline. In the absence of this postive signal, neural markers (GSX, SOX2) are lost and epidermis develops, as evident by ectopic expression of DLX5 and BMP4. This neural inductive signal could be a BMP antagonizing protein such as Chordin, Noggin or Follistatin. These proteins are expressed in the node, and not in the endoderm, nor in the more lateral mesoderm. A DLX5 induction signal triggered by the midline, and transmitted via the neural plate is also required for ectodermal patterning (Pera, 1999).
Dlx (distal-less gene) homeogenes encode transcription factors that are involved in the patterning of orofacial skeleton derived
from cephalic neural crest cells. In order to study the role of DLX genes during embryonic development in humans, DLX5
expression pattern was investigated in 6- to 11-week-old human embryos. A DLX5 PCR fragment was amplified from a human
dental cDNA library subcloned and used for in situ hybridization investigations. DLX5 gene expression is primarily detected in
the mandible at 6 weeks and afterwards in the maxilla. DLX5 gene expression becomes restricted to progenitor cells of
developing tooth germs, bones and cartilages of mandible and maxilla. During odontogenesis from bud to late cap stages, DLX5
transcripts are present in both dental epithelium and mesenchyme tissues. DLX5 expression is restricted to few cells in the
vestibular aspect of the dental epithelium, while DLX5 mRNA signal is more widely distributed in dental mesenchyme. The
observed expression pattern of DLX5 homeogene extends the proposed site-specific combination of homeogene expression in
neural crest derived cells to human specific dentition. Furthermore, during the bud and cap stages of tooth morphogenesis, the
asymmetric expression of DLX5 in the dental epithelium and dental mesenchyme may contribute to the complex patterning of
human tooth shape (Davideau, 1999).
Dlx2, a member of the distal-less gene family, is expressed
in the first branchial arch, prior to the initiation of tooth
development, in distinct, non-overlapping domains in the
mesenchyme and the epithelium. In the mesenchyme Dlx2
is expressed proximally, whereas in oral epithelium it is
expressed distally. Dlx2 has been shown to be involved in
the patterning of the murine dentition, since loss of function
of Dlx1 and Dlx2 results in early failure of development of
upper molar teeth. The regulation of
Dlx2 expression has been investigated to determine how the early epithelial and
mesenchymal expression boundaries are maintained, and to
help to understand the role of these distinct expression
domains in patterning of the dentition. Transgenic mice produced with a lacZ reporter
construct, containing a 3.8 kb sequence upstreamof Dlx2, led
to the mapping of regulatory regions driving epithelial but
not mesenchymal expression in the first branchial arch. Epithelial expression of Dlx2 is regulated by
planar BMP4 signaling, which is coexpressed in distal
oral epithelium. BMP4 induction of Dlx2 expression in epithelium does not
require the presence of mesenchyme. Mesenchymal expression is regulated by a
different mechanism involving FGF8, which is expressed in
the overlying epithelium. FGF8 also inhibits expression
of Dlx2 in the epithelium by a signaling pathway that
requires the mesenchyme. Thus, the signaling molecules
BMP4, acting via an intra-epithelial signal, and FGF8 provide the mechanism for maintaining
the strict epithelial and mesenchymal expression domains
of Dlx2 in the first arch (Thomas, 2000).
Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily,
have been identified by their ability to induce cartilage and bone from nonskeletal cells and have been
shown to act as a ventral morphogen in Xenopus mesoderm. A murine
homeobox-containing gene, distal-less 5 (mDlx5), has been isolated as a BMP-inducible gene in osteoblastic MC3T3-E1
cells. Stable transfectants of MC3T3-E1 that overexpress mDlx5 mRNA show increases in various
osteogenic markers; a fourfold increase in alkaline phosphatase activity; a sixfold increase in
osteocalcin production, and the appearance of mineralization in the extracellular matrix. Furthermore, mDlx5
is induced orthotopically in mouse embryos treated with BMP-4 and in the fractured bone of adult mice.
Consistent with these observations, it has been found that injection of mDlx5 mRNA into dorsal
blastomeres enhances the ventralization of Xenopus embryos. These findings suggest that mDlx5 is a
target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and
dorsoventral patterning of embryonic axis (Miyama, 1999).
Murine Distal-less-related homeodomain gene Dlx3 is expressed in terminally differentiated epidermal cells. Ectopic expression of this gene in the basal cell layer of transgenic skin results in a severely abnormal epidermal phenotype and leads to perinatal lethality. Basal cell-specific keratins K5 and K14 are expressed at similar levels in transgenic and normal skin as are the supra-basal keratins K1 and K10. The basal cells of affected mice cease to proliferate, and express the profilaggrin and loricrin genes, two major markers for granular cells (residing in higher strata). Thus, misexpression of XDlx3 results in both downregulation of filaggrin and loricrin in granular layer cells and ectopic synthesis of these proteins in lower strata. The basal cells of affected mice cease to proliferate; all suprabasal cell types are diminished, and the stratum corneum is reduced to a single layer. These data indicated that Dlx3 misexpression results in transformation of basal cells into more differentiated keratinocytes (Morasso, 1996).
Treatments of zebrafish embryos with retinoic acid (RA), a substance known to cause
abnormal craniofacial cartilage development in other vertebrates, results in dose- and
stage-dependent losses of homeobox gene expression in several regions of the
embryo. dlx expression in neural crest cells migrating from the hindbrain and in the
visceral arch primordia is particularly sensitive to RA treatment. The strongest effects
are observed when RA is administered prior to or during crest cell migration but
effects can also be observed if RA is applied when the cells have entered the primordia of the arches. Losses of dlx expression correlate either with the loss of cartilage elements originating from hindbrain neural crest cells or with abnormal morphology of these elements. Cartilage elements that originate from midbrain neural crest cells, which do not express dlx genes, are less affected. Taken together with the observation that the normal patterns of visceral arch dlx expression just prior to cartilage condensation resemble the morphology of the cartilage elements that are about to differentiate, these results suggest that dlx genes are an important part of a multi-step process in the development of a subset of craniofacial cartilage elements (Ellies, 1997).
The Dlx homeobox gene family is expressed in a complex pattern within the embryonic craniofacial ectoderm and ectomesenchyme, tissues derived from the cranial neural crest. Dlx-2 is essential for development of proximal regions of the murine first and second branchial arches. Dlx-1 and -2 are closely linked on mouse chromosome 2. The skeletal and soft tissue analyses of mice with Dlx-1 and both Dlx-1 and -2 mutations provide additional evidence that the Dlx genes regulate proximodistal patterning of the branchial arches. This analysis also elucidates distinct and overlapping roles for Dlx-1 and Dlx-2 in craniofacial development. Mice lacking both Dlx-1 and -2 have unique abnormalities, including the absence of maxillary molars. Dlx-1 and -2 are expressed in the proximal and distal first and second arches, yet only the proximal regions are abnormal (Qui, 1997).
The nested expression patterns of Dlx-1, -2, -3, -5, and -6 provide evidence for a model that predicts the region-specific requirements for each gene. Analysis of Dlx-1, -2, -3, -5 and -6 expression proved information that may explain why the phenotypic effects of the Dlx-1 and -2 mutations are focused on proximal structures. While Dlx-1 and -2 are expressed along most or all of the proximal-distal axis, the expression of Dlx-3, -5 and -6 overlaps with Dlx-1 and -2 in more distal regions of arches 1 and 2. If Dlx-3, -5 and -6 are functionally redundant for Dlx-1 and -2, this could explain why these arches are not affected by these mutations. The Dlx-2 and Dlx-1 and -2 mutants also have ectopic skull components that resemble bones and cartilages found in phylogenetically more primitive vertebrates (Qui, 1997).
Hox genes appear to have a central role in specifying A-P positional information to the cranial neural crest up to rhombomere 3 and therefore may regulate the morphogenetic programs of the hyoid (branchial arch 2) and more posterior brachial arches. Mice lacking Hoxa-2 (Drosophila homolog: Proboscipedia) form skeletal structures in branchial arch 2 that resemble proximal first arch elements. Thus in B2, Hoxa-2 and perhaps Hoxb-2 may affect the expression of genes involved in P-D patterning (e.g., Dlx-1 and -2). This would be similar to the role of Drosophila Deformed regulating Distal-less during development of the maxillary segment. Otx-2 could regulate Dlx genes anterior to branchial arch 2 (Qui, 1997).
The generation and analysis of mice homozygous
for a targeted deletion of the Dlx5 homeobox gene is reported. Dlx5 mutant mice have multiple defects in craniofacial
structures, including their ears, noses, mandibles and
calvaria, and die shortly after birth. A subset (28%) exhibit
exencephaly. Ectodermal expression of Dlx5 is required for
the development of olfactory and otic placode-derived
epithelia and surrounding capsules. The nasal capsules are
hypoplastic (e.g. lacking turbinates) and, in most cases, the
right side is more severely affected than the left. Dorsal
otic vesicle derivatives (e.g. the semicircular canals and
endolymphatic duct) and the surrounding capsule, are
more severely affected than ventral (cochlear) structures.
Dlx5 is also required in mandibular arch ectomesenchyme,
as the proximal mandibular arch skeleton is dysmorphic.
Dlx5 may control craniofacial development in part through
the regulation of the goosecoid homeobox gene. goosecoid
expression is greatly reduced in Dlx5 mutants, and both
goosecoid and Dlx5 mutants share a number of similar
craniofacial malformations. Dlx5 may perform a general
role in skeletal differentiation, as exemplified by
hypomineralization within the calvaria. The distinct focal
defects within the branchial arches of the Dlx1, Dlx2 and
Dlx5 mutants, along with the nested expression of their
RNAs, support a model in which these genes have both
redundant and unique functions in the regulation of
regional patterning of the craniofacial ectomesenchyme
The evidence suggests that Dlx5 expression is required in specialized
epithelial tissues (placodes) for nose and ear development. An aberrant
epithelial forms, but it cannot support normal chondrogenesis
in the underlying mesenchyme.
Dlx5 is also required in the ectomesenchyme of the
mandibular arch for development of the proximal mandible.
Dlx5 further appears to exert an influence in the differentiation
of particular skeletal elements (Depew, 1999).
In the mouse embryo, Dlx5 is expressed in the otic placode and vesicle, and later in the semicircular canals of the inner ear.
In mice homozygous for a null Dlx5/LacZ allele, a severe dysmorphogenesis of the vestibular region is observed,
characterized by the absence of semicircular canals and the shortening of the endolymphatic duct. Minor defects are
observed in the cochlea, although Dlx5 is not expressed in this region. Cristae formation is severely impaired; however,
sensory epithelial cells, recognized by calretinin immunostaining, are present in the vestibular epithelium of Dlx5-/- mice.
The maculae of utricle and saccule are present but cells appear sparse and misplaced. The abnormal morphogenesis of the
semicircular canals is accompanied by an altered distribution of proliferating and apoptotic cells. In the Dlx5-/- embryos,
no changes in expression of Nkx5.1(Hmx3), Pax2, and Lfng have been seen, while expression of bone morphogenetic protein-4 (Bmp4) is drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. It is proposed that development of the semicircular canals and the vestibular inner ear
requires the independent control of several homeobox genes, which appear to exert their function via tight regulation of BMP4 expression and the regional organization of cell differentiation, proliferation, and apoptosis (Merlo, 2002).
The DLX gene family is a family of divergent homeobox genes, related to the Drosophila distal-less gene, expressed primarily in the forebrain and craniofacial structures. DLX-7 is expressed in normal hematopoietic cells and leukemia cell lines with erythroid
characteristics. An antisense oligonucleotide targeted against the translation start site of DLX-7 mRNA was used to inhibit its expression
in a human erythroleukemia cell line K562, which expresses DLX-7 at a high level. The antisense oligonucleotide efficiently reduces
the DLX-7 mRNA. Inhibition of DLX-7 expression decreases the plating efficiency by
approximately 70%, as compared with the control. The antisense treatment causes apoptosis. Down-regulation of DLX-7 expression by antisense treatment is associated with a reduction in GATA-1 (See Drosophila Serpent) and c-myc (See Drosophila Myc) mRNA levels. Thus, it is concluded that DLX-7 is expressed in hematopoietic cells and that the inhibition of its expression results in the decreased levels of GATA-1 and
c-myc genes, with an accompanying induction of apoptosis (Shimamoto, 1997).
The molecular events of odontogenic induction are beginning to be elucidated, but until now nothing
has been known about the molecular basis of the patterning of the dentition. A role for Dlx-1 and Dlx-2
genes in patterning of the dentition has been proposed with the genes envisaged as participating in an
'odontogenic homeobox gene code' by their specification of molar development. This proposal is based on the restricted expression of the genes in molar ectomesenchyme derived from cranial neural crest cells
prior to tooth initiation. Mice with targeted null mutations of both Dlx-1 and Dlx-2 homeobox genes do
not develop maxillary molar teeth, however, their incisors and mandibular molars are normal. With Dlx mutation, the epithelial-mesenchymal signaling pathways for Shh and Bmp4 are not disrupted, but there is a failure in the subsequent development of maxillary molar tooth germs. Heterologous recombinations were carried out between mutant and wild-type maxillary epithelium and mesenchyme. Under these conditions the ectomesenchyme underlying the maxillary molar epithelium loses its odontogenic potential. Mutant mesenchyme is unable to support tooth development. Using molecular markers for branchial arch neural crest (Barx1), and commitment to chondrogenic differentiation (Sox9), this population is shown to alter its fate from odontogenic to chondrogenic. These results provide evidence that a subpopulation of cranial neural crest is specified as odontogenic by Dlx-1 and Dlx-2 genes. Loss of function of these genes results in reprogramming of this population of ectomesenchyme cells into chondrocytes. This is the first indication that the development of differently shaped teeth at different positions in the jaw is determined by independent genetic pathways (Thomas !997).
During early tooth development, multiple signaling molecules are expressed in the dental lamina epithelium; these molecular signals induce the
dental mesenchyme. One signal, BMP4, has been shown to induce morphologic changes in dental mesenchyme and
mesenchymal gene expression via Msx1, but BMP4 cannot substitute for all the inductive functions of the dental epithelium.
To investigate the role of FGFs during early tooth development, the expression of epithelial and mesenchymal
Fgfs was examined in wild-type and Msx1 mutant tooth germs and the ability of FGFs to induce Fgf3 and Bmp4 expression was tested in
wild-type and Msx1 mutant dental mesenchymal explants. Fgf8 expression is preserved in Msx1 mutant epithelium while that
of Fgf3 is not detected in Msx1 mutant dental mesenchyme. Moreover, dental epithelium as well as beads soaked in FGF1,
FGF2 or FGF8 induce Fgf3 expression in dental mesenchyme in an Msx1-dependent manner. These results indicate that, like
BMP4, FGF8 constitutes an epithelial inductive signal capable of inducing the expression of downstream signaling molecules
in dental mesenchyme via Msx1. However, the BMP4 and FGF8 signaling pathways are distinct. BMP4 cannot induce Fgf3
nor can FGFs induce Bmp4 expression in dental mesenchyme, even though both signaling molecules can induce Msx1 and
Msx1 is necessary for Fgf3 and Bmp4 expression in dental mesenchyme. In addition, the effects of FGFs
and BMP4 were investigated on the distal-less homeobox genes Dlx1 and Dlx2 and the relationship between Msx and Dlx gene
function was clarified in the developing tooth. Dlx1,Dlx2 double mutants exhibit a lamina stage arrest in maxillary molar tooth development. Although the maintenance of molar mesenchymal Dlx2 expression at the bud stage is
Msx1-dependent, both the maintenance of Dlx1 expression and the initial activation of mesenchymal Dlx1 and Dlx2 expression
during the lamina stage are not. Moreover, in contrast to the tooth bud stage arrest observed in Msx1 mutants, Msx1,Msx2
double mutants exhibit an earlier phenotype closely resembling the lamina stage arrest observed in Dlx1,Dlx2 double mutants.
These results are consistent with functional redundancy between Msx1 and Msx2 in dental mesenchyme and support a model
whereby Msx and Dlx genes function in parallel within the dental mesenchyme during tooth initiation. Indeed, as predicted by
such a model, BMP4 and FGF8, epithelial signals that induce differential Msx1 and Msx2 expression in dental mesenchyme,
also differentially induce Dlx1 and Dlx2 expression, and do so in an Msx1-independent manner. These results integrate Dlx1,
Dlx2 and Fgf3 and Fgf8 into the odontogenic regulatory hierarchy along with Msx1, Msx2 and Bmp4, and provide a basis for
interpreting tooth induction in terms of transcription factors which, individually, are necessary but not sufficient for the
expression of downstream signals and therefore must act in specific combinations (Bei, 1998).
The Dlx5 gene encodes a Distal-less-related DNA-binding
homeobox protein first expressed during early embryonic
development in anterior regions of the mouse embryo. In
later developmental stages, it appears in the branchial
arches, the otic and olfactory placodes and their
derivatives, in restricted brain regions, in all extending
appendages and in all developing bones. A null allele of the mouse Dlx5 gene was created by replacing exons I
and II with the E. coli lacZ gene. Heterozygous mice appear
normal. beta-galactosidase activity in Dlx5+/- embryos and
newborn animals reproduces the known pattern of
expression of the gene. Homozygous mutants die shortly
after birth with a swollen abdomen. They present a
complex phenotype characterized by craniofacial
abnormalities affecting derivatives of the first four
branchial arches, severe malformations of the vestibular
organ, a delayed ossification of the roof of the skull and
abnormal osteogenesis. No obvious defect was observed in
the patterning of limbs and other appendages. The defects
observed in Dlx5-/- mutant animals suggest multiple and
independent roles of this gene in the patterning of the
branchial arches, in the morphogenesis of the vestibular
organ and in osteoblast differentiation (Acampora, 1999).
The Dlx5/lacZ allele is expressed in the otic pit and later in the
otic vesicle starting 8.0 dpc. Therefore, the development of the inner ear was studied in mutant animals.
In heterozygous animals, Dlx5/lacZ is initially expressed on
the dorsoposterior region of the otic vesicle and subsequently
in the semicircular canals and in the endolymphatic duct and
vesicle of the vestibular organ. In Dlx5-/- embryos, the
vestibulum is smaller in size and heavily deformed; the three
canals fail to form properly, the anterior and posterior canals
do not develop and are fused into one single large vesicle, and
the horizontal canal is also reduced. The
development of the endolymphatic duct is much less affected
by the mutation. The morphology of the sacculus and the
cochlea appears essentially normal. Although the lesion is
present with complete penetrance and is similar between
individuals, the severity of the dysgenesis varies among
individual mutant animals and between both ears within
individuals. The inner ear epithelium of mutant embryos and
newborn animals appears much thinner as compared to
normal and is composed of large flat cells. Within
this mutant epithelium, thickened regions are observed that
may represent remnants of the cristae ampullaris, which could
not be recognized. In contrast, the maculae of the utriculus and
sacculus appear normal.
Similar vestibular defects have been observed in mice
mutant for Nkx-5.1/Hmx3, an NK-related homeobox gene. In order to evaluate
whether Dlx5 might regulate or be regulated by Nkx-5.1, the expression of both genes was examined in Dlx5-/- and
Nkx-5.1-/- mice. Inactivation of either gene does not
abrogate or profoundly modify expression of the other,
excluding the possibility that they reciprocally control one another's
expression. Furthermore, while Dlx5 is strongly expressed in
the endolymphatic duct at all stages of development, Nkx-5.1
is never expressed in this structure, indicating a different
regulation (Acampora, 1999).
The data indicate that Dlx5 expression is elevated during
osteoblast differentiation and disappears in fully differentiated
osteocytes. Its expression is more evident in periosteal bone,
but is also seen in cells of the endosteal compartment, which
might represent osteoblasts at a specific stage of differentiation.
Dlx5-/- mice show a delayed ossification of dermatocranial
bones, which closely resemble that observed in mice in which
one copy of the Cbfa1 gene is inactivated.
The defect in osteogenesis observed in Dlx5-/- mice
suggests that this gene plays a role in osteoblast differentiation
and in bone formation; the data show an increased complexity
of the structure of woven bone and a reduction of the periosteal
bone lamina. The expression of Cbfa1, a key regulator of
osteoblast differentiation, is not affected in Dlx5 mutants.
However, an increased osteocalcin expression is observed in
the periosteum suggesting a lesion in osteoblast differentiation.
Unfortunately, since the mutant mice die at birth, the effect of Dlx5 in later phases of mineralization and during
the formation of compact bone could not be studied. The increased osteocalcin
expression in the periosteum observed in mutant animals could
corroborate the notion that Dlx5 can act as a repressor of the
osteocalcin gene; however other more
complex pathways of regulation cannot be ruled out (Acampora, 1999).
Neural crest cells play a key role in craniofacial development. The
endothelin family of secreted polypeptides regulates development of
several neural crest sublineages, including the branchial arch neural
crest. The basic helix-loop-helix transcription factor dHAND is also
required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the
branchial arches is down-regulated, implicating it as a transcriptional
effector of ET-1 action. To determine the mechanism that links ET-1
signaling to dHAND transcription, the dHAND
gene was analyzed for cis-regulatory elements that control transcription in
the branchial arches. An evolutionarily conserved
dHAND enhancer is described that requires ET-1 signaling for activity. This
enhancer contains four homeodomain binding sites that are required for
branchial arch expression. By comparing protein binding to these sites
in branchial arch extracts from endothelin receptor A
(EdnrA) mutant and wild-type mouse embryos, Dlx6,
a member of the Distal-less family of homeodomain proteins, was identified as an
ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis (Charite, 2001).
Human Dlx5 and Dlx6 homeobox genes have been identified as
possible candidate genes for the autosomal dominant form of the
split-hand/split-foot malformation (SHFM), a heterogeneous limb
disorder characterized by missing central digits and claw-like distal
extremities. Targeted inactivation of Dlx5 and Dlx6
genes in mice results in severe craniofacial, axial, and appendicular
skeletal abnormalities, leading to perinatal lethality. For the first
time, Dlx/Dll gene products have been shown to be critical regulators of mammalian limb development, since combined loss-of-function mutations phenocopy SHFM. Furthermore, spatiotemporal-specific transgenic overexpression of Dlx5, in the apical ectodermal ridge of Dlx5/6 null mice can fully rescue Dlx/Dll function in limb outgrowth (Robledo, 2002).
The vertebrate skull vault forms almost entirely by the direct mineralization of mesenchyme, without the formation of a cartilaginous template -- a mechanism called membranous ossification. Dlx5 gene mutation leads to cranial dismorphogenesis. In avians, little is known about the genetic regulation of cranial vault development. In this study, Dlx5 expression and regulation were analyzed during skull formation in the chick embryo. Dlx5 expression pattern is compared with that of several genes involved in mouse cranial suture regulation. This provides an initial description of the expression in the developing skull of the genes encoding the secreted molecules BMP 2, BMP 4, BMP 7, the transmembrane FGF receptors FGFR 1, FGFR 2, FGFR 4, the transcription factors Msx1, Msx2, and Twist, as well as Goosecoid and the early membranous bone differentiation marker osteopontin. Dlx5 is activated in proliferating osteoblast precursors, before osteoblast differentiation. High levels of Dlx5 transcripts are observed at the osteogenic fronts (OFs) and at the edges of the suture mesenchyme, but not in the suture itself. Dlx5 expression is initiated in areas where Bmp4 and Bmp7 genes become coexpressed. In a calvarial explant culture system, Dlx5 transcription is upregulated by BMPs and inhibited by the BMP-antagonist Noggin. In addition, FGF4 activates Bmp4 but not Bmp7 gene transcription and is not sufficient to induce ectopic Dlx5 expression in the immature calvarial mesenchyme (Holleville, 2003).
The vertebrate inner ear arises from an ectodermal thickening, the otic placode, that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to Fgf signals from adjacent tissues and express two highly related paired box transcription factors Pax2a and Pax8 in the developing placode. Compromising the functions of both Pax2a and Pax8 together blocks zebrafish ear development, leaving only a few residual otic cells. This suggests that Pax2a and Pax8 are the main effectors downstream of Fgf signals. The results further provide evidence that pax8 expression and pax2a expression are regulated by two independent factors, Foxi1 and Dlx3b, respectively. Combined loss of both factors eliminates all indications of otic specification. It is suggested that the Foxi1-Pax8 pathway provides an early 'jumpstart' of otic specification that is maintained by the Dlx3b-Pax2a pathway (Hans, 2004).
It is proposed that induction of otic fate by Fgf signals takes place only when cells are competent to respond, and that this competence is provided by Foxi1 and Dlx3b. A direct role for
Foxi1 and Dlx3b in competence needs to be demonstrated, for example by ectopic
expression and transplantation experiments. Foxi1 and Dlx3b function by
regulating pax8 and pax2a expression, respectively, in an
Fgf-dependent fashion. In Dlx3b-deficient embryos, expression of pax8
is indistinguishable from that in wild-type embryos, presumably owing to
normal Foxi1 and Fgf signaling. However, otic pax2a expression is
initiated only very late and weakly. By
contrast, otic pax8 expression fails and pax2a expression is
present although delayed in foxi1– mutants.
Inhibition of both factors, Foxi1 and Dlx3b, completely blocks otic
specification even in the presence of functional Fgf signaling. By activating Pax8,
Foxi1 thus provides competence to otic precursor cells to respond to early Fgf
signaling; Dlx3b and Pax2a subsequently maintain this competence (Hans, 2004).
Lower jaw development is a complex process in which multiple signaling cascades establish a proximal-distal organization. These cascades are regulated both spatially and temporally and are constantly refined through both induction of normal signals and inhibition of inappropriate signals. The connective tissue of the tongue arises from cranial neural crest cell-derived ectomesenchyme within the mandibular portion of the first pharyngeal arch and is likely to be impacted by this signaling. Although the developmental mechanisms behind later aspects of tongue development, including innervation and taste acquisition, have been elucidated, the early patterning signals driving ectomesenchyme into a tongue lineage are largely unknown. This study shows that the basic helix-loop-helix transcription factor Hand2 plays key roles in establishing the proximal-distal patterning of the mouse lower jaw, in part through establishing a negative-feedback loop in which Hand2 represses Dlx5 and Dlx6 expression in the distal arch ectomesenchyme following Dlx5- and Dlx6-mediated induction of Hand2 expression in the same region. Failure to repress distal Dlx5 and Dlx6 expression results in upregulation of Runx2 expression in the mandibular arch and the subsequent formation of aberrant bone in the lower jaw along with proximal-distal duplications. In addition, there is an absence of lateral lingual swelling expansion, from which the tongue arises, resulting in aglossia. Hand2 thus appears to establish a distal mandibular arch domain that is conducive for lower jaw development, including the initiation of tongue mesenchyme morphogenesis (Barron, 2011).
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