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.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

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.     

Dlx5 regulates chondrocyte differentiation at multiple stages

Endochondral ossification, in which cartilaginous templates are progressively replaced by marrow and bone, represents the dominant mode of development of the axial and appendicular skeleton of vertebrates. Chondrocyte differentiation within the cartilaginous core of these skeletal elements is tightly regulated, both spatially and temporally. Here, we describe the expression of Dlx5 in the cartilaginous core of limb skeletal elements in chicken and mouse embryos. We find that Dlx5 is one of the earliest genes expressed in condensing limb mesenchyme that will give rise to the limb skeleton. Later, when proliferating and differentiating chondrocytes are found in spatially distinct regions of the cartilaginous model, Dlx5 is expressed in the zone of hypertrophy and in proliferating chondrocytes that are poised to differentiate. Consistent with this pattern of expression, we show that forced expression of Dlx5 potentiates early and late chondrocyte differentiation and inhibits proliferation in cultured cells. Examination of the limbs of mutant Dlx5 mouse embryos revealed that they displayed a delay in chondrocyte maturation compared with wild type littermates. Taken together, our data reveal a positive role for Dlx5 during multiple stages of chondrocyte differentiation and, along with previous studies of Dlx5 and osteogenesis, identify Dlx5 as a general regulator of differentiation in the mouse skeleton.     

Developmental origins of species-specific muscle pattern

Vertebrate jaw muscle anatomy is conspicuously diverse but developmental processes that generate such variation remain relatively obscure. To identify mechanisms that produce species-specific jaw muscle pattern we conducted transplant experiments using Japanese quail and White Pekin duck, which exhibit considerably different jaw morphologies in association with their particular modes of feeding. Previous work indicates that cranial muscle formation requires interactions with adjacent skeletal and muscular connective tissues, which arise from neural crest mesenchyme. We transplanted neural crest mesenchyme from quail to duck embryos, to test if quail donor-derived skeletal and muscular connective tissues could confer species-specific identity to duck host jaw muscles. Our results show that duck host jaw muscles acquire quail-like shape and attachment sites due to the presence of quail donor neural crest-derived skeletal and muscular connective tissues. Further, we find that these species-specific transformations are preceded by spatiotemporal changes in expression of genes within skeletal and muscular connective tissues including Sox9, Runx2, Scx, and Tcf4, but not by alterations to histogenic or molecular programs underlying muscle differentiation or specification. Thus, neural crest mesenchyme plays an essential role in generating species-specific jaw muscle pattern and in promoting structural and functional integration of the musculoskeletal system during evolution.     

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.     

Formation of the middle ear: recent progress on the developmental and molecular mechanisms

The middle ear allows animals to hear while moving in an aerial medium. It is composed of a cavity harbouring a chain of three ossicles that transmit vibrations produced by airborne sound in the tympanic membrane into the inner ear, where they are converted into neural impulses. The middle ear develops in the branchial arches, and this requires sequential interactions between the epithelia and the underlying mesenchyme. Gene-inactivation experiments have identified genes required for the formation of different middle ear components. Some encode for signalling molecules, including Endothelin1 and Fgf8, probable mediators of epithelial-mesenchymal interactions. Other genes, including Eya1, Prx1, Hoxa1, Hoxa2, Dlx1, Dlx2, Dlx5, and Gsc, are most likely involved in patterning and morphogenetic processes in the neural crest-derived mesenchyme. Mechanisms controlling formation of a functional tympanic membrane are also discussed. Basically, the tympanic ring, which serves as support for the tympanic membrane, directs invagination of the first pharyngeal cleft ectoderm to form the external acoustic meatus (EAM), which provides the outer layer of the membrane. Gsc and Prx1 are essential for tympanic ring development. While invaginating, the EAM controls skeletogenesis in the underlying mesenchyme to form the manubrium of the malleus, the link between the membrane and the middle ear ossicles.     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

Endothelin-1 regulates the dorsoventral branchial arch patterning in mice

Endothelin-1 (ET-1), a 21-amino acid peptide secreted by the epithelium and core mesenchyme in the branchial arches as well as vascular endothelium, is involved in craniofacial and cardiovascular development through endothelin receptor type-A (EdnrA) expressed in the neural crest-derived ectomesenchyme. Here we show that ET-1(-/-) mutant mice exhibit a homeotic-like transformation of the lower jaw to an upper jaw. Most of the maxillary arch-derived components are duplicated and replaced mandibular arch-derived structures, resulting in a mirror image of the upper and lower jaws in the ET-1(-/-) mutant. As for hyoid arch-derivatives, the ventral structures are severely affected in comparison to the dorsal ones in the ET-1(-/-) mutant. Correspondingly, the expression of Dlx5 and Dlx6, Distalless-related homeobox genes determining the ventral identity of the anterior branchial arches, and of the mandibular marker gene Pitx1 is significantly downregulated in the ET-1(-/-) mutant, whereas the expression of Dlx2 and the maxillary marker gene Prx2 is unaffected or rather upregulated. These findings indicate that the ET-1/EdnrA signaling may contribute to the dorsoventral axis patterning of the branchial arch system as a mediator of the regional intercellular interactions.     

Vertebrate head development: Segmentation,novelties, and homology

Vertebrate head development is a classical topic lately invigorated by methodological as well as conceptual advances. In contrast to the classical segmentalist views going back to idealistic morphology, the head is now seen not as simply an extension of the trunk, but as a structure patterned by different mechanisms and tissues. Whereas the trunk paraxial mesoderm imposes its segmental pattern on adjacent tissues such as the neural crest derivatives, in the head the neural crest cells carry pattern information needed for proper morphogenesis of mesodermal derivatives, such as the cranial muscles. Neural crest cells make connective tissue components which attach the muscle fiber to the skeletal elements. These crest cells take their origin from the same visceral arch as the muscle cells, even when the skeletal elements to which the muscle attaches are from another arch. The neural crest itself receives important patterning influences from the pharyngeal endoderm. The origin of jaws can be seen as an exaptation in which a heterotopic shift of the expression domains of regulatory genes was a necessary step that enabled this key innovation. The jaws are patterned by Dlx genes expressed in a nested pattern along the proximo-distal axis, analogous to the anterior–posterior specification governed by Hox genes. Knocking out Dlx 5 and 6 transforms the lower jaw homeotically into an upper jaw. New data indicate that both upper and lower jaw cartilages are derived from one, common anlage traditionally labelled the “mandibular” condensation, and that the “maxillary” condensation gives rise to other structures such as the trabecula. We propose that the main contribution from evolutionary developmental biology to solving homology questions lies in deepening our biological understanding of characters and character states.