Dlx5 regulates regional development of the branchial arches and sensory capsules.

We report the generation and analysis of mice homozygous for a targeted deletion of the Dlx5 homeobox gene. 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. 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.     

Endothelin-A receptor-dependent and -independent signaling pathways in establishing mandibular identity

The lower jaw skeleton is derived from cephalic neural crest (CNC) cells that reside in the mandibular region of the first pharyngeal arch. Endothelin-A receptor (Ednra) signaling in crest cells is crucial for their development, as Ednra(-/-) mice are born with severe craniofacial defects resulting in neonatal lethality. In this study, we undertook a more detailed analysis of mandibular arch development in Ednra(-/-) embryos to better understand the cellular and molecular basis for these defects. We show that most lower jaw structures in Ednra(-/-) embryos undergo a homeotic transformation into maxillary-like structures similar to those observed in Dlx5/Dlx6(-/-) embryos, though lower incisors are still present in both mutant embryos. These structural changes are preceded by aberrant expansion of proximal first arch gene expression into the distal arch, in addition to the previously described loss of a Dlx6/Hand2 expression network. However, a small distal Hand2 expression domain remains. Although this distal expression is not dependent on either Ednra or Dlx5/Dlx6 function, it may require one or more GATA factors. Using fate analysis, we show that these distal Hand2-positive cells probably contribute to lower incisor formation. Together, our results suggest that the establishment of a 'mandibular identity' during lower jaw development requires both Ednra-dependent and -independent signaling pathways.     

Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5.

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. We have created a null allele of the mouse Dlx5 gene 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 characterised 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.     

Developmental regulation of EVF-1, a novel non-coding RNA transcribed upstream of the mouse Dlx6 gene

We previously reported that sonic hedgehog (Shh) induces the differentiation of rat ventral forebrain neurons expressing a novel marker, EVF-1 [Development 125 (1998) 5079]. In this report, we show that EVF-1 is a novel, developmentally regulated, non-coding RNA, with no homology to other known non-coding RNA sequences. Sequence analysis, in vitro translation, and comparison of the rat and mouse EVF-1 sequences suggest that EVF-1 contains no protein coding regions. Chromosomal location indicates that EVF-1 maps adjacent to the Dlx6 gene on mouse chromosome 6. RNA in situ hybridization of the embryonic rat forebrain shows that EVF-1 is expressed by immature neurons in the subventricular zone and its expression decreases during forebrain development. Whole mount in situ hybridization shows that EVF-1 is expressed at high levels in the branchial arches, ventral forebrain, olfactory bulb, and limbs. EVF-1 expression is linked to Shh and the Dlx family of proteins, genes with a demonstrated importance to ventral forebrain and craniofacial development.     

Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development

The endothelin (Edn) system comprises three ligands (Edn1, Edn2 and Edn3) and their G-protein-coupled type A (Ednra) and type B (Ednrb) receptors. During embryogenesis, the Edn1/Ednra signaling is thought to regulate the dorsoventral axis patterning of pharyngeal arches via Dlx5/Dlx6 upregulation. To further clarify the underlying mechanism, we have established mice in which gene cassettes can be efficiently knocked-in into the Ednra locus using recombinase-mediated cassette exchange (RMCE) based on the Cre-lox system. The first homologous recombination introducing mutant lox-flanked Neo resulted in homeotic transformation of the lower jaw to an upper jaw, as expected. Subsequent RMCE-mediated knock-in of lacZ targeted its expression to the cranial/cardiac neural crest derivatives as well as in mesoderm-derived head mesenchyme. Knock-in of Ednra cDNA resulted in a complete rescue of craniofacial defects of Ednra-null mutants. By contrast, Ednrb cDNA could not rescue them except for the most distal pharyngeal structures. At early stages, the expression of Dlx5, Dlx6 and their downstream genes was downregulated and apoptotic cells distributed distally in the mandible of Ednrb-knock-in embryos. These results, together with similarity in craniofacial defects between Ednrb-knock-in mice and neural-crest-specific Galpha(q)/Galpha(11)-deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G(q)/G(11)-dependent signaling, while the formation of the distal pharyngeal region is under the control of a G(q)/G(11)-independent signaling, which can be substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful tool for studies on gene functions in craniofacial development.     

Temporospatial cell interactions regulating mandibular and maxillary arch patterning

The cellular origin of the instructive information for hard tissue patterning of the jaws has been the subject of a long-standing controversy. Are the cranial neural crest cells prepatterned or does the epithelium pattern a developmentally uncommitted population of ectomesenchymal cells? In order to understand more about how orofacial patterning is controlled we have investigated the temporal signalling interactions and responses between epithelium and mesenchymal cells in the mandibular and maxillary primordia. We show that within the mandibular arch, homeobox genes that are expressed in different proximodistal spatial domains corresponding to presumptive molar and incisor ectomesenchymal cells are induced by signals from the oral epithelium. In mouse, prior to E10, all ectomesenchyme cells in the mandibular arch are equally responsive to epithelial signals such as Fgf8, indicating that there is no pre-specification of these cells into different populations and suggesting that patterning of the hard tissues of the mandible is instructed by the epithelium. By E10.5, ectomesenchymal cell gene expression domains are still dependent on epithelial signals but have become fixed and ectopic expression cannot be induced. At E11 expression becomes independent of epithelial signals such that removal of the epithelium does not affect spatial ectomesenchymal expression. Significantly, however, the response of ectomesenchyme cells to epithelial regulatory signals was found to be different in the mandibular and maxillary primordium. Thus, whereas both mandibular and maxillary arch epithelia could induce Dlx2 and Dlx5 expression in the mandible and Dlx2 expression in the maxilla, neither could induce Dlx5 expression in the maxilla. Reciprocal cell transplantations between mandibular and maxillary arch ectomesenchymal cells revealed intrinsic differences between these populations of cranial neural crest-derived cells. Research in odontogenesis has shown that the oral epithelium of the mandibular and maxillary primordia has unique instructive signaling properties required to direct odontogenesis, which are not found in other branchial arch epithelia. As a consequence, development of jaw-specific skeletal structures may require some prespecification of maxillary ectomesenchyme to restrict the instructive influence of the epithelial signals and allow development of maxillary structures distinct from mandibular structures.     

Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor-deficient mice

Most of the bone, cartilage and connective tissue of the lower jaw is derived from cranial neural crest cells (NCCs) arising from the posterior midbrain and hindbrain. Multiple factors direct the patterning of these NCCs, including endothelin-1-mediated endothelin A receptor (Edn1/Ednra) signaling. Loss of Ednra signaling results in multiple defects in lower jaw and neck structures, including homeotic transformation of lower jaw structures into upper jaw-like structures. However, since the Ednra gene is expressed by both migrating and post-migrating NCCs, the actual function of Ednra in cranial NCC development is not clear. Ednra signaling could be required for normal migration or guidance of NCCs to the pharyngeal arches or in subsequent events in post-migratory NCCs, including proliferation and survival. To address this question, we performed a fate analysis of cranial NCCs in Ednra-/- embryos using the R26R;Wnt1-Cre reporter system, in which Cre expression within NCCs results in permanent beta-galactosidase activity in NCCs and their derivatives. We find that loss of Ednra does not detectably alter either migration of most cranial NCCs into the mandibular first arch and second arch or their subsequent proliferation. However, mesenchymal cell apoptosis is increased two fold in both E9.5 and E10.5 Ednra-/- embryos, with apoptotic cells being present in and just proximal to the pharyngeal arches. Based on these studies, Ednra signaling appears to be required by most cranial NCCs after they reach the pharyngeal arches. However, a subset of NCCs appear to require Ednra signaling earlier, with loss of Ednra signaling likely leading to premature cessation of migration into or within the arches and subsequent cell death.     

Zebrafish foxi1 mediates otic placode formation and jaw development

The otic placode is a transient embryonic structure that gives rise to the inner ear. Although inductive signals for otic placode formation have been characterized, less is known about the molecules that respond to these signals within otic primordia. Here, we identify a mutation in zebrafish, hearsay, which disrupts the initiation of placode formation. We show that hearsay disrupts foxi1, a forkhead domain-containing gene, which is expressed in otic precursor cells before placodes become visible; foxi1 appears to be the earliest marker known for the otic anlage. We provide evidence that foxi1 regulates expression of pax8, indicating a very early role for this gene in placode formation. In addition, foxi1 is expressed in the developing branchial arches, and jaw formation is disrupted in hearsay mutant embryos.     

The mammalian twisted gastrulation gene functions in foregut and craniofacial development

Extracellular modulators of cell-cell signaling control numerous aspects of organismal development. The Twisted gastrulation (Twsg1) gene product is a small, secreted cysteine-rich protein that has the unusual property of being able to either enhance or inhibit signaling by the bone morphogenetic protein (BMP) subfamily of TGF-beta type factors in a context-dependent manner. In this report, we characterize the early embryonic and skeletal phenotypes associated with loss of Twsg1 function in mice. All Twsg1 mutant mice, irrespective of genetic background, exhibit deletions of neural arches in the cervical vertebrae. In a C57BL/6 background, we also observe pronounced forebrain defects including rostral truncations, holoprosencephaly, cyclopia, as well as alterations in the first branchial arch (BA1) leading to lack of jaw (agnathia). Characterization of marker expression suggests that these defects are attributable to loss of signaling from forebrain-organizing centers including Fgf8 from the anterior neural ridge (ANR) and Shh from the prechordal plate (PrCP). In addition, we find defects in the foregut endoderm and a reduction in Hex expression, which may contribute to both the forebrain and BA1 defects.     

Jaw transformation with gain of symmetry after Dlx5/Dlx6 inactivation: Mirror of the past?

In modern vertebrates upper and lower jaws are morphologically different. Both develop from the mandibular arch, which is colonized mostly by Hox-free neural crest cells. Here we show that simultaneous inactivation of the murine homeobox genes Dlx5 and Dlx6 results in the transformation of the lower jaw into an upper jaw and in symmetry of the snout. This is the first homeotic-like transformation found in this Hox-free region after gene inactivation. A suggestive parallel comes from the paleontological record, which shows that in primitive vertebrates both jaws are essentially mirror images of each other. Our finding supports the notion that Dlx genes are homeotic genes associated with morphological novelty in the vertebrate lineage.     

Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.     

Arch form,tooth size,and occlusomandibular kinesis in the Ceboidea

Correlations between dental morphology, arch configuration, and jaw movement patterns were quantitatively investigated in 23 ceboid species to elucidate integrative aspects of occlusal functional anatomy in an adaptive and evolutionary context. Differential maxillary-mandibular arch widths are primary in guiding lateral jaw movements. These movements are characterized according to their associated condylar shifts as either predominantly translatory or rotational. Predominantly translatory movements result from peripheral contact relationships between maxillary arches which are considerably wider posteriorly than their opposing mandibular arches. The greatest degree of mandibular movement is in the molar region in functional association with wide “primitive” maxillary molars, narrow mandibular molars, constricted maxillary intercanine widths, and narrow maxillary incisors. In contrast, predominantly rotational masticatory jaw movements result from differential arch widths which are greatest in the maxillary canine and incisor regions. Here most jaw movement is in the anterior segment and this is reflected in small maxillary-mandibular molar width differences, a high degree of premolarization, wide-set maxillary canine teeth, and wide maxillary incisors. Possible selectional factors in the putative evolution of rotational predominance in mastication from the more primitive translatory pattern are discussed.